CULT AM ata ih i HK cert ‘i Hh a nal i i tH a Mai ae i aa Ha ca Ha ata ee = -. 7 = @ , 4 =. as Annual Subscription, $2.00 Single Copies, 25 cents. Vol. VIII. No. 1 SATURDAY, JULY 1, 1933 THE USE OF PITUITARY IMPLANTS AND PITUITARY EXTRACTS FOR OBTAIN- ING AMPHIBIAN EGGS OUT OF SEASON Dr. L. G. Bartu Instructor Embryology, Columbia University A difficulty which confronts many embryolo- gists and experimental zoologists is that of obtain- ing material during the autumn and months. To be sure, one can always use the chick and eggs of certain fishes, but the ex- periments which can be carried out on these forms are limited. The amphibian egg which is RESEARCH FACILITIES AT THE SCRIPPS INSTITUTION OF OCEANOGRAPHY Dr. THomas WAYLAND VAUGHAN Director of the Institution The development of the Scripps Institution of Oceanography has been slow, having extended over fully forty years. The events which led to the establishment of the Scripps Institution for 3iological Research and the early history of that Institu- tion have been excellently pre- sented in a bulletin entitled, “The Marine Biological Sta- tion of San Diego, its history, winter M. B. . Calendar SATURDAY, JULY 1, M. B. L. Mixer. 8:30 P. M. ideal in many respects for em- bryological work is usually available only during a rela- tively short period of the year. By the use of the pituitary gland and extracts it is pos- sible, however, to induce frogs, newts and salamanders to lay their eggs from October until June. The technique is rela- tively simple and has been used by students in our labor- atory with amazing success. The effects of the pituitary have been examined by Wolf, 1929, who found that by implanting the pituitary body of the frog into the (Continued on page 7) WEDNESDAY, JULY 5, 8:00 P.M. Seminar: Dr. G. W. Prescott: “Some Effects of blue-green Al- gae on Lake Fish.” Dr. Hugh P. Bell: “Distribution and Ecology of the Marine Algae of the Maritime Provinces of Canada.” Dr. W. R. Taylor: “Distribution of Newfoundland Algae.” FRIDAY, July 7, 8:00 P. M. Lecture: Dr. G. H. Parker: Neuro- Humoralism and Nerve Trans- mission. present conditions, achieve- ments, and aims,” by Dr. W. E. Ritter: Subsequent to the publica- tion of the article by Dr. Rit- ter and prior to my succeeding him as director of the Institu- tion there were two notable improvements in the Institu- tion. One was the erection of a library-museum building, 60 ft. x 60 ft., which contains two floors and about a three- quarter basement. Two tiers of book-stacks can be installed on each of the two main floors of the building, giving a library capacity of about 50,000 volumes. TABLE OF CONTENTS Research Facilities at the Scripps Institution of Oceanography, Dr. Thomas Wayland A Simple Device for the Study of Small Opaque Objects, Dr. Anna R. Whiting... .11 Vaughan ........-.-se sees eee eee mie WeeRditorialwbar cheer wcrc eet nee 12 The Use of Pituitary Implants and Pituitary “ Extracts for Obtaining Amphibian Eggs Directory, LOVE NGS SS sever cmfels, castsireverty sens xctdueh suey o Me 13 Out of Season, Dr. L. G. Barth.............. 1 Chemical Room, Dr. Oscar W. Richards....... 16 Special Apparatus and Technical Services...... 8 M. B. L, Club, Past and Present, Dr. L. V. A Simple Apparatus for the Measurement of JK Uo) ybhatol Popo cere ey clo aos a naee Conto cio ces Gickoone 17 Radiation Intensities, Roberts Rugh........ TORR VOGUE ELGLE SISO Ra ierscehes sachs: a) «400 ¥cuaconahene ey atebehetee ate 22 2 THE COLLECTING NET [ Vor. VIII. No. 61 THE SCRIPPS INSTITUTION OF OCEANOGRAPHY View of the three principal buildings looking northwest. In the center is the Library-Museum building; at the right is part of the new laboratory, Ritter Hall; at the left behind the Museum-Library building is the old George H. Scripps Laboratory. At the extreme left may be seen part of the pier. The other improvement was the construction of a re-enforced concrete pier, 1,000 ft. long and 20 ft. wide. Therefore, at the time when I became di- rector of the Institution on the first of February, 1924, the land of the Institution consisted of a 177-acre tract on the sea front about two miles north of the village of La Jolla. There were, on this land, one laboratory building, two floors, 75 x 48 ft.; the library-museum building and the re- enforced concrete pier, which have been men- tioned ; a wooden aquarium, 24 x 48 feet, with 18 tanks; several service buildings and garages, tem- porary structures, and 24 wooden cottage resi- dences. In September, 1925, the Institution pur- chased a boat of the purse-seiner type, 64 ft. long, 18 foot beam, about 50 tons gross, and it was equipped to work to a depth of about 1,000 fath- oms. The boat is supposed to have a cruising radius of about 1,000 miles, but no attempt has been made to use it for cruises of more than about 200 miles radius. Nearly all of the expense of the acquisition of the land and the censtruction of the buildings on it was borne by Miss Ellen Browning Scripps. Mr. E. W. Scripps, however, helped defray the expenses of some of the construction, and he donated the boat to the Institution. The expenses of operation were divided be- tween the Scripps family and the State of Cali- fornia. Miss Scripps contributed $9,000, and Mr. E. W. Scripps, $5,000, per year. Revenue of about $7,500 per year was derived from the Insti- tution’s houses and its supply department. The University of California contributed $22,500 per year. The general set-up of the Institution when I became its director was as has been indicated. President Campbell proposed to Miss Scripps that, beginning July 1, 1925, she and the Univer- sity of California jointly make an annual step-up of $5,000 a year in the income of the Institution, each to contribute $2,500. This policy was con- tinued until there had been an increase of $35,000 per year in the Institution’s income. Besides the income above indicated the Institution has re- ceived a considerable number of special contribu- tions of significant size. The principal of these was a fund for the meteorological investigations, raised cooperatively by the organizations interest- ed in hydro-electric power development in the State. These contributions were from the De- partment of Water and Power, City of Los An- geles, and a number of commercial corporations. In 1929 it was decided to add a new laboratory building to the Institution’s equipment, and for this purpose $120,000 were raised by $40,000 con- tributions from each of the following: the State of California through the University, Miss Scripps, and the Rockefeller Foundation. Work on the building was begun in March, 1931, and during September of the same year it was ready for use. This building is a three-story fire-proof re-en- forced concrete structure. It is 100 ft. long and 46 ft. wide. For the biennium which began July Jjiwiexe My 7 THE COLLECTING NEE ; S 1, 1931, the State of Gaumeena made to the Uni- versity a special appropriation of $40,000 for the renovation and improvement of the old buildings and grounds of the Institution. The statements above made indicate the general nature of the physical set-up of the Institution at present, how the set-up was brought about, and what the present resources of the Institution are. But nothing has been said regarding the scientific purpose which was behind the development. The Institution at first was primarily one for biological research, but very soon after its estab- lishment important investigations in dynamical oceanography, particularly those of Dr. George F. McEwen, were undertaken, because it was recog- nized that knowledge of the physical features of the environment was necessary in order to under- stand the conditions of life of the various marine organisms. The lower floor of the old laboratory building, the George H. Scripps Laboratory, was supplied with running salt water, gas, and electri- city, and it was adapted for general morphological work and experimental work which did not re- quire controlled conditions. As is known by everyone who is familiar with the history of the Institution, numerous important scientific re- searches were conducted at the Institution during the incumbency of Dr. Ritter. Before Dr. Ritter’s retirement on June 30, 1923, the administrative officers and regents of the University of California and the interested members of the Scripps family decided to convert the Institution from one for biological research into one for oceanographic research. Therefore, when I came to the Institution on February 1, 1924, a change in the Institution’s policy was ini- tiated. An endeavor was made to develop a broad program of oceanographic research which would be in line with the generally recognized scope and functions of an oceanographic institution. In the lines of research which had been pursued under the incumbency of Dr. Ritter, it was decided to make only one significant change. Although it was recognized that the inv Beietion of geogra- phic races and heredity in the deer mice, Pero- myscus, by Dr. F. B. Sumner were of great im- portance, really an honor to the Institution with which Dr. Sumner was connected, the research was scarcely germane to an oceanographic institu- tion. Dr. Sumner himself suggested that, after his work on Peromyscus had been brought to a logical stopping place, he should transfer his ac- tivities to problems in fish biology, which had been subjects of investigation by him before he undertook the Peromyscus project. Dr. Sum- ner’s suggestion was adopted by the appropriate University officials. The investigations in dy- namical oceanography under Professor McEwen the studies of phytoplankton by Professor W. E. Allen, and the work on the zooplankton, especially copepods, by Professor Esterly, were continued. As soon as it was practicable to do so, the work in marine meteorology was expanded, and inves- tigations of the chemistry of sea water were es- tablished as one of the definite projects of the Institution, with Dr. E. G. Moberg in charge. The study of marine bottom deposits and the complex of associated physical and chemical problems in the ocean, in charge of T. W. Vaughan, was un- dertaken as soon as circumstances permitted. Since micro-organisms, bacteria and the lower forms of plant life, perform important roles in the metabolism of the sea, investigation of those organisms was initiated, first in charge of Dr. A ’ VIEW OF THE THREE PRINCIPAL BUILDINGS LOOKING SOUTHEAST FROM THE PIER At the left is the new building, Ritter Hall; in the center is the Museum-Library building; at the right behind the Museum-Library building is the George H. Scripps Laboratory. 4 THE COLLECTING NET [ Vor. VIII. No. 61 PLAN OF BUILDINGS AND GROUNDS H. Gee, later in charge of D. C. E. ZoBell. In- vestigations of the physiology of marine organ- isms with reference to their oceanic environment, in charge of Dr. D. L. Fox, were added. It is generally recognized that the adaptations of or- ganisms to their environment are fundamentally physiological. Therefore, in order to get at the more fundamental principles of such adaptation it is necessary to know the organism structurally, the environment in which it lives, and the organic processes which are necessary for the organism to continue its existence. The lines of research being prosecuted at the Institution may, therefore, be categorically stated as follows: dynamical oceanography and marine meteorology, including solar radiation and the penetration of radiant energy into sea-water ; the chemistry of sea-water; biology (bacteriology ; phytoplankton ; foraminifera, their life history and relation to marine sediments; biology of fishes, largely physiological; physiology of marine or- ganisms, with special reference to adaptation to PHOTOGRAPHIC LAB. SALT WATER LABORATORY CARPENTER Snop || \ = CORRIDOR: BOWERY MACH RM SALT WATER TANK RM REFRIGERATOR MACH. STORAGE ¥ FUTURE LABORATORY SPACE +6 TRANSFORMER RM: Room GROUND FLOOR PLAN, RITTER HALL jjronxe hy IE THE COLLECTING NET 5 the marine environment); geological processes, especially marine bottom deposits. The Insti- tution has on its grounds one of the seismo- logical stations established in connection with the study of seismology in southern California under the auspices of the Carnegie Institution and the California Institute of Technology. Each line of oceanographic work interlocks with other lines. Therefore, although it has been necessary more or less to sectionize the work of the Institution, be- cause of the need for skill in the use of special disciplines, the work of the Institution is really set up as a series of interlocking projects. In addition to what is done by the resident members of the staff of the Institution and by several non-resident assistants, the Institution de- rives great benefit from cooperative relations with a number of other institutions, especially various governmental organizations. The assistance re- There are also one rather large office, a drafting and computing room, two store rooms, and one room in which the temperature may be kept con- stant as desired, from 0° to 40° Centigrade. At the west end ot the top floor there are rooms for dynamical oceanography and marine meteorology, including a large computing room extending the whole length of that section of the building. In connection with the work in marine meteorology there are in the rooms on the top floor the record- ing devices of instruments, such as for solar ra- diation, which are exposed on the roof of the building. On the roof there is also a pent-house, in which the water distilling and ventilating fans are installed. On the second floor, east end, there are for the bacteriological investigations five rooms, in addition to a constant temperature room. Just west of the bacteriological suite there is a long room intended for spectrometric pur- PHYSIQLOCY Las: Puysics LAB section — —_—+ 460° sic CORRIDOR kcem UTMITY — Bacteriovocy SECTION BACTER! LOGY Las LaB PREPARATION AM OPTICAL FIRST FLOOR PLAN ceived from the United States Navy, the Coast and Geodetic Survey, and the Bureau of Light- houses, has been invaluable. It includes the col- lection of data and samples of water and bottom deposits froma large part of the northeastern Pa- cific. The Institution has also received aid from the Grace and Los Angeles Steamship Lines and from many other sources. The account of the purpose and organization of the Institution has been rather fully stated be- cause they have influenced the recent construction and other improvements of the Institution’s facili- ties for scientific research. The new laboratory building, which has been appropriately named Rit- ter Hall, has provisions in it for several different kinds of research. On the top floor, east end, there are three research chemical laboratories, a nitrogen room, an alcove for colorimetric work, and a small laboratory for routine determinations, such as salinity and. hydrogen ion concentration. poses, and a large laboratory which was intended as a special physics room. At present this room is being used as a biological laboratory, but such use is not intended to be permanent. At the west end of the second floor is the physiological suite. It comprises, besides the office, one large experi- mental laboratory supplied with salt water, a large chemical laboratory, and a large microscope room. There is also in this suite a dark room and a large constant temperature room. On the ground floor, there are two rooms in- tended for the large-scale cultures of marine or- ganisms; a photographic suite composed of four rooms; the. machinery room and_ transformer vault; and a room for the storage of oceanogra- phic equipment. On this floor, there remains con- siderable unassigned space, a part of which at present is in use as a receiving room and another part, as a carpenter shop. By means of the special State appropriation of 6 THE COLLECTING NET [ Vor. VIII. No. 61 OFFICE J LAB /* WATER ANALYSIS CHEMICAL / le CHEMISTRY CHEMISTRY Lal GLASS STORE Am: SECOND FLOOR PLAN $40,000, the George H. Scripps Laboratory, 75 x 48 ft., has been remodelled. A little less than half of the lower floor is devoted to fish biology, and about three and a half unit rooms are devoted to marine sediments. There are on the lower floor three unassigned rooms which may be used by visiting investigators or by staff members. These rooms are supplied with running salt and fresh water, compressed air, gas, and electricity. On the upper floor, there are the Director’s offices, the seminar room, and an unassigned room which may be used either as an office or as a laboratory for work which does not require experimentation. On this floor there are also the laboratories for phytoplankton, the offices of fish biology, and two laboratories for those studies on foraminifera which do not require experiments on living or- ganisms. As a part of the renovation and improvement made possible by the special State appropriation of $40,000, the old salt water system of the Insti- tution was replaced by a new system which con- sists of a 60,000 gallon re-enforced concrete tank, the concrete and covering cement being designed to prevent rusting of the re-enforcing. The water for the tank is brought from the west end of the pier, nearly 1,000 ft. from the beach line, through pure lead pipes, and from the tank water is dis- tributed through pure lead pipes to the physiologi- cal section of Ritter Hall, the aquarium, and the lower floor of George H. Scripps Laboratory. The system appears to be excellent. Ritter Hall is intended for investigations in dynamical oceanography, the physical properties of sea water, the chemistry of sea water, biochem- istry, bacteriology, and general physiology. The laboratory is primarily one for physics and chem- istry for those kinds of biological investigation which require either biochemical or biophysical methods. The equipment of George H. Scripps Laboratory is, for the kind of work which has been indicated, at present adequate. Since library facilities are essential for re- search, it will be said that the Institution’s library now contains 13,000 boand volumes, about 23,000 reprints, and many atlases and charts, An im- provement expected in the near future is the re- placement of the gasoline engine at present on the Institution’s beat Scripps by a Diesel engine. The funds needed for this change have been sub- scribed. The space available at the Scripps Institution purposely exceeds that which is needed by the In- stitution’s permanent staff which works through- out the year. The Institution can receive visiting scientists for virtually any kind of oceanographic research. There are special facilities for a num- ber of different kinds of biological work. The number of visitors who can be accommodated is approximately 25, that is, about 25 in addition to the residential members of the staff. The new equipment at the Institution first became partially available for use during the summer of 1932. The reconstruction and improvement program had not been completed at the beginning of the summer. Therefore, it is only at the end of the summer season of 1932 that full utilization of the facilities of the Institution has become possible. The In- stitute desires to be of service to the various _ branches of oceanographic research and, insofar as it has facilities, it will welcome those who are working on the biological, physical, chemical, and geological aspects of oceanography. 1 Univ. Calif. Publ. in Zool., vol. 9, pp. 137-248, pls. 18-24, two maps, March 9, 1912. veel e933) | THE COLLECTING NET THE USE OF PITUITARY IMPLANTS AND PITUITARY EXTRACTS FOR OBTAINING AMPHIBIAN EGGS OUT OF SEASON ( Continued from Page 1 ) lymph sac of the female ovulation could be stimulated. Noble, 1931, refers to the use of the pituitary for egg-laying in other amphibia. See also Buyse and Burns, 1931; Adams, 1930. RANA PIPIENS Starting from Wolf’s experiments the follow- ing technique has been worked out in our labora- tory. A male and female frog in healthy condi- tion are kept in an aquarium jar with a few inches of spring water and each is given two implants of frog pituitary daily for three or four days. A\l- most all frogs so treated go into amplexus, and normal ovulation and fertilization take place. The frogs used as recipients of the implants should be medium-sized frogs which have not been kept in the laboratory too long. When they have been kept two or three weeks at room temperature they do not respond so readily. Our frogs come from Vermont and can be obtained throughout the winter. The pituitary can easily be obtained by cutting off the head of a frog and cutting through the floor of the brain case on either side. The pitui- tary will usually adhere to the floor of the brain case and can be picked up with a pair of forceps and inserted through an incision in the skin of the host. We have found that by making a cut through the skin just hack of the foreleg the pi- tuitary can be pushed posteriorly into the lymph sac underneath the skin and will not slip out when the frog is returned to the aquarium. This in- cision remains open and all implants may be in- troduced through it. It is important to use two pituitaries for each operation if the eggs are to he fertilized. If only one is used, the eggs may be deposited without being fertilized by the male, and there is greater variability in the results. There is also a seasonal variation, and ovulation is more easily induced in the spring. Recently Mr. R. Rugh has simplified this pro- cedure by injecting into the abdomen of the fe- male an aqueous extract of the pituitary. The following account is from his unpublished experi- ments. The extract is prepared by grinding the pituitary with a glass rod in the bottom of a tube. _ Two pituitaries are used in about 2 ce. of distilled water. A single injection of this extract is usually sufficient to move the eggs into the uterus. After the injection the frogs are kept at room tempera- ture and after a period of from 24 to 48 hours the eggs are stripped into a large dish containing a thin layer of sperm suspension. In the fall and early winter the eggs appear after about 48 hours. In the spring from 3 to 24 hours after injection. The sperm suspension is prepared by macerat- ing the testes in spring water. The males do not have to be injected for active sperm. This sperm suspension is allowed to stand for about one half- hour before using. Two points should be em- phasized. First, that the sperm suspension should be allowed to stand for some time before using; and second, that the sperm suspension should form a thin layer and the eggs be stripped from the female directly into the suspension. If fresh sperm or large volumes of water be used, the jelly of the eggs swells before fertilization occurs. About 20 minutes after insemination the dish may be filled with spring water The jelly then swells and the fertilized eggs rotate within their membranes, After the female has been injected and the eggs accumulate in the uterus, the frog may be kept in a cold room at 9°-11° C. and the eggs may be used as needed. At room temperature the eggs are usually laid and then cannot be fertilized. However, the eggs remain fertile in the uterus for several days if the female is kept at a low temperature. The aqueous extract of the pituitary is stable and can be kept on ice for months. Pituitaries from females are more effective than those from males because they are larger. Many attempts have been made to substitute sheep's pituitary and Antuitrin-S for the frogs’ pituitary, but without success. On the other hand both sheep’s pituitary and Antuitrin-S are effec- tive in newts and salamanders. NEWTS In a similar manner ovulation may be stimu- lated in an American newt, 7riturus viridescens, and in a Japanese newt, Triturus (Molge) pyr- rhogaster. The female usually carries sperm, and if the newts be treated in the fall and winter, fertilized eggs are laid without implanting males. We have obtained fertilized eggs from the Japan- ese newt as late as May. Some females lay un- fertilized eggs, which may be inseminated arti- ficially with sperm from the male. The sperm are removed from the vasa deferentia and placed in a few drops of Ringer’s solution and the eggs are dipped into the concentrated sperm suspen- sion. The eges can he removed from the female by pressure on the abdomen or can be dissected out of the uterus. In either case they must be 8 THE COLEEGIRING NET [ Vor. VIII. No. 61 immediately dipped into the sperm suspension and then placed on the bottom of a moist dish. Later water may be added and the membranes then swell. Frogs’ pituitary, sheeps’ pituitary or Antuitrin- S may be used to obtain eggs from the newt. Single or double implants of frogs’ pituitary un- derneath the skin of the female for a few days are sufficient to cause ovulation. The animals are kept in a small aquarium with Elodea and the eggs are deposited on the leaves of this plant. Daily injections of extract of sheep’s pituitary as prepared by Parke, Davis and Company will cause the Japanese newt to lay. About 0.3-0.5 ce. is injected beneath the skin daily or into the abdo- men for three or four days. The newts, if well fed and healthy, invariably lay. The eggs will be fertilized or unfertilized, depending upon whether or not the female carries the functional sperm. Ogilvie, 1933, has used Antuitrin-S with the same success. Triturus pyrrhogaster is a very hardy newt which lives well in the laboratory and can be obtained in large quantities from New York aquarium supply houses. It is much larger than Triturus virides- cens and lays many more eggs. The eggs are very valuable for cleavage studies and, in later stages, for transplants. SALAMANDERS The Mexican axolotl, Amblystoma mexicanum, will also lay eggs after injection of sheep's pitui- tary or Antuitrin-S. If the females be injected with about 1 cc. of these extracts daily, they will deposit their eggs, but the eggs will not be fer- tilized. If fertilized eggs are desired, males only must be injected with about 1 cc. daily for three or four days. They will then deposit spermato- phores and the females will pick them up without injection, and lay fertilized eggs. The animals must be well fed. We have been using chopped beef liver a few times a week. The same females may lay more than once a year, by keeping several pairs eggs can be obtained from October until June. The eggs are particularly useful for experi- ments where it becomes necessary to remove the egg membranes and jelly before gastrulation. The jelly may be completely removed with two pairs of sharp forceps, leaving only the thin vitelline membra1e which need not be removed for experi- ments with vital stains. It is much more ditficult to work with the frog’s or newt’s egg before gas- trulation because of the large amount of jelly in the former and the tough membranes of the latter. All of the forms above-mentioned are easily ob- tainable and inexpensive except the Mexican axolotl. However, the latter lives well in the laboratory, and when once a stock has been ob- tained, they supply large numbers of eggs. The sheep pituitary extract was supplied by the re- search department of Parke, Davis and Company. Antuitrin-S can be obtained from the same com- pany or from local drug stores. Wolf, O. M.—1929—Proc. Soc. Exp. Biol. Med.XXVI, 692-693 Noble, G. K.—1931—The Biology of the Amphibia. New York, 299-300 Buyse, A. and Burns, R. K., Jr.—1931—Proc. Soc. Exp. Biol. Med.XXIX, 80 Adams, A. E.—1930—Anat. Rec.XLV, 250 Ogilvie, A. E.—1933—Proc. Soc. Exp. Biol. Med.XXX, 752 SPECIAL APPARATUS AND TECHNICAL SERVICES The mere expensive and delicate apparatus owned by the Marine Biological Laboratory has been segregated and is separately administered. It is especially cared for in the hope that individual problems of our investigators may be approached with the least delay and as directly as possible. Information concerning these devices and tech- nical supplies for research may be secured through the Apparatus Office, Room 216, in the Brick Building. The supervision of such special apparatus and technical supplies remains in charge oy IDiep staff of assistants. Samuel E. Pond, and_ his It is important to point out to investigators, particularly those arriving at the Marine Bio- logical Laboratory for the first time, that it is necessary to make arrangements for scientific ap- paratus and technical supplies in advance of their need. The Laboratory is only able to maintain an adequate technical service by being kept in To this end an “Ap- plication for Research Accommodations” has been touch with individual needs. sent to each investigator in which pro- visions was made for special preferences or re- quisitions for supplies of a technical nature. rom information supplied by these applications pre- liminary preparations for the season have been Such requests for special apparatus and general equipment have also been used as a guide made. to the location of the more permanent and semi- permanent laboratory devices. For certain spe- cial problems scientific apparatus has heen re- Joye ty 1933) THE COLEECRING NET 9 served on one of several plans which have been devised for the convenience of investigators. Those individuals who have not taken advantage of the preliminary notice or who have not in- quired about special provisions may have been handicapped upon their arrival and have thereby been delayed in starting their work. The Laboratory is in possession of a fairly complete equipment of physical instruments but with increasing demands and more technical re- quirements it is difficult to make the best dis- tribution of apparatus to fit peculiar and indi- vidual requirements. Since it is desirable to make the special apparatus generally available and to avoid restriction to a small number of persons, reservations for certain devices are arranged to suit particular periods of use. Where it is im- practical for investigators to bring with them cer- tain special apparatus the necessary equipment may be reserved for a required period but in such cases a fee will be charged for such use. Arrange- ments for EXCLUSIVE LOANS for a season or less must be arranged through the Apparatus Office, Room 216, in the Brick Building. Certain apparatus more generally available may also be rented where more or less personal use is required. Microscopes are reserved for the period of residence, the fee depending upon equipment and device. Special microscopes and many accessories are not usually available, in- cluding binocular, monobjective microscopes of the highly specialized or research type; aplanatic condensers; camera lucidas, demonstration ocu- lars, etc. These devices are customarily trans- ferred by investigators from their own institu- tions with personal supplies. Students in courses are supplied by the staff in charge of their instruction, Special apparatus cannot be loaned otherwise. Beginning investiga- tors may arrange for supplies through the investi- gator with whom they are working but must in such cases refer such special requests to the Ap- paratus Office as require technical assistance. As far as possible, use of special apparatus is fit- ted to individual requirements, but in most cases allowance for use with others must be made. The use of certain apparatus, such as analytical balances etc., continually requires the attention of investigators. Both individual assistance in the maintenance of such instruments and personal responsibility for care, cleanliness, and stability are essential to continue these devices in operating condition. Instruments of precision are located at advantageous points but weights and special supplies are not issued for general use. It has been found necessary to provide weights, or spe- cial supplies for such work only to individuals who it is expected will arrange for their return in good condition as soon as work permits. To those who use balances, scales and balance rooms particularly, a word of caution may not be amiss concerning the care necessary to avoid damage to delicate parts. An even greater care and more than usual cleanliness of such equipment is essen- tial not only to one’s own success but to that of others. TECH NICAL SERVICES Provision for part of this season has been made for certain technical services. Arrangements for these will be made through the Apparatus Office. Technical operation of X-ray equipment has been arranged after July 5th, continuing through August. Special problems involving X-radiation, radium, etc., will necessitate advance arrange- ments and some rotation or schedule in order to avoid loss of research time. Glassblowing, as in preceding summers may be arranged during the residence of Mr. James D. Graham, of the University of Pennsylvania, through July and August. Necessary repairs to certain kinds of glass can be done fairly promptly and a limited amount of new work may be ar- ranged. Engraving of glassware requires a great- er allowance of time since the work is partly done out of town. Technical work of this kind is done at cost of materials and labor. Photography may be done by individuals through loans of special apparatus. During part of the present season technical assistance may be ar- ranged and supplies secured if sufficient advance notice is received by the Apparatus Office. Lan- tern slides, the development of research films, plates and prints may be arranged at the cost of materials and labor, but amateur work cannot be undertaken. Dark room space is limited chiefly to research problems although provision for photographic dark rooms for limited periods may be arranged from time to time. Chemicals and photograph supplies cannot be supplied except to investigators who have made application for them. since local stocks are usually inadequate to meet other than technical needs. To those who contemplate returning subse- quently to the Marine Biological Laboratory it will be a material help to consider working con- ditions more or less peculiar to this Laboratory and to arrange with Dr. Pond any special allot- ments of apparatus or supplies for future pe- riods of residence for scientific work. 10 THE COLLECTING NET [ Vor. VIII. No. 61 A SIMPLE APPARATUS FOR THE MEASUREMENT OF RADIATION INTENSITIES Roperts Rucu Instructor in Zoology, Hunter College Non-selective radiation measurements are usu- ally made with a blackened thermopile and_gal- vanometer. With radiation measurements cover- ing a range of 10° C. as measured by ordinary thermometers, the thermopile method is open to considerable criticism. A simple instrument has been described by Hall (Ecology-13 :214) in which two parallel thermom- eters are exposed to light, one thermometer being blackened with black Duco enamel and the other whitened with zinc oxide. Hall reports that this apparatus compares favorably with the Macve.n Iiuminometer, having a correlation ccefficient of +0.9805 +0.0039. Using a Fahrenheit thermom- eter he reports that 1°F, equals 1,000 foot-can- dies, The disadvantage pointed out was in the measurement of low light intensities. With the aid of Mr. Lester Boss of the Marine 3iological Laboratory at Woods Hole, an appara- tus has been devised which eliminates some of the criticisms of Hall’s apparatus. A metal box measuring 51% x 314 x 214 inches was constructed to act as a water chamber containing within itself a smaller closed box of the same material. “lwo high precision thermometers which can be read to 0.1°C. accurately are placed with their mercury bulbs in the smaller air-tight box. The thermom- eters lie parallel to each other and about 1% inches apart. The thermometer blackened with optical black to a point above the mercury bulb is snielded from the untreated thermometer by a re- flecting metal surface and is exposed to the out- side through a window of Heat Resisting Clear Corex D (Corning) Glass. Quartz would be an improvement but the gain would hardly justify the additional cost. 3eckmann thermometers could be used to advantage if the temperature range considered is well within the limits of the 3eckmann scale. The inner, air-tight metal box measures 334 x 284 x 1% inches and is soldered into place. The inlet and outlet tubes for water circulation are located at one end of the box and at opposite corners. In using this apparatus it is necessary to allow the shielded thermometers to come to equilibrium in the running bath for a considerable period in order to determine the particular characteristics of the thermometers. If the bath temperature is quite different from the original temperature reading of the thermometers, more time should be allowed to attain equilibrium. All radiation meas- urements should take into account these thermom- eter characteristics. Radiation intensities within the range of 0.0-5.0°C. can be made within 10-15 minutes, but intensities comparable to sunlight re- quire haif an hour to attain equilibrium. ‘he ini- tial response of the exposed thermometer which is perceptible is a matter of 2-3 seconds at moderate inteasities. The temperature difference of the two ther- mometers with the apparatus exposed to direct sunlight at noon one da, last August was 32.20° C., waile exposure to the direct sun at a corre- sponding time of day during January gave 19.60° C. difference. The apparatus’ wil respond to roomlight radiations on sunny days particularly with beckmann thermometers, which can be read closer than 0.1° C. Numerous readings indicated the roomlight to be equal to about 0.1% of the direct sunlight. The radiation transmission characteristics of some Corning Glass iilters of different spectral values taken at Woods Hole last summer and the same filters re-calibrated with the same apparatus but a different pair of thermometers during Jan- uary, is given below to indicate how accurately tne apparatus can reproduce intensity measure- meats. In each case the apparatus was clamped into position beneath a G.E. Type Sl Sunlamp, and the filters were interposed at a constant dis- tance of six inches above the apparatus. The ra- diation intensity of the entire are of the Sunlamp varies tremendously with a shift of a few inches in any direction, so it was necessary to have both lamp and thermometers clamped into a permanent relationship to each other. The transmission characteristic of each glass filter was calculated in respect to the total transmission of the Sunlamp as 100%. It must be admitted that the spectral energies of the Sunlamp change with age, but this factor is negligible witnin the time limits of the observations made. ‘the distance from the Sun- lamp varied by 12 inches in August and January, hence the “100.0%” value of the Sunlamp was 15.00° C. in one case and 4.49° C. in the other. Nevertheless, the transmission characteristics of the various filters were surprisingly close as ex- pressed in percent. of total energy available. Jury 1, 1933 ] THE COLLECTING NET 11 August January mometers reversed, the temperature differences of Light Condition Transmis- eae Difference the two thermometers was only 6.5°C. This indi- GE) TypeSl (ard beast cated that blackening of the bulb increased the ef- Sunlamp 100% 100% 00.0% ficiency of the absorbing thermometer by approxi- Filter No. 774 91.59% 91.65% + =+0.06% mately 3007. Filter No. 970 92.59% 92.13% —0.46% The obvious disadvantage of this apparatus is Filter No. 980 85.99% 87.41% +1.42% its slow response as compared with the electrical Filter No. 385 80.20% 78.54% —1.66% methods. However, it must be remembered that Filter No. 349 61.92% 60.98% —0.94% with such great intensities and with isolated spec- Filter No. 254 59.60% 59.19% —O0.41% tral radiations, this non-selective method over- Filter No. 428 34.76% == 33.40% =—1.36% comes some of the criticisms aimed at thermopiles Filter No. 401 20.13% 20.04% = —0.09% and photo-electric cells. Filter No. 254 is an infra- Filter No. 597 11.51% 10.96% —0.55% red transmission filter; No. 401 is monochromatic Filter No. 396 10.73% 9.86% —0.87% To test tne advantage of blackening the radia- tion absorbing thermcmeter, some observations were made with the blackened thermometer shielded and the untreated thermometer exposed to the radiations. With radiations measuring 19.6°C. under normal conditions, with the ther- green; No. 597 eliminates all but the visible pur- ple and some ultra-violet and a little infra-red; while No. 970 transmits everything from about 2990 A® units deep into the infra-red. The total cost of the apparatus is negligible and the accur- acy with which results can be reproduced suggest its practical use. A SIMPLE DEVICE FOR THE STUDY OF SMALL OPAQUE OBJECTS Dr. ANNA R. WHITING Professor and Head of the Department of Biology, Pennsylvania College for Women The apparatus here described has been devised for the purpose of studying small opaque objects under high magnifications by reflected light with uniform illumination on all sides. Since the source of light is distant, little heat is present and even this can be filtered out between source and mirror if desired. It has the added advantages of be- ing simple, cheap, and durable. An old compound micro- scope without condenser but with good lenses was used (Fig 1). A large circular opening was cut in the stage so that only the rim (4) re- mained. On top of tlis was cemented with Duco a piece of glass (B) the exact size of the stage and a small area was painted black (C) on the under side beneath the objec- tive to shut out transmitted light. A large substage mirror (D) such as is used on bin- ocular microscopes was sub- stituted for the one of usual size. The concave metal re- flector (Fig. 1 E and Fig. 2) lite lamp, obtainable at many auto supply stores. In the edge of this on opposite sides shallow grooves one inch wide (F) were cut so that it would rest on the stage over a_ de- pression slide (G), the ends of which project through the grooves. The specimen is mounted in alcohol on the slide and placed over the painted black spot. The reflector is then dropped over the slide and the objective (H) focused down through the hole in its top. The mirror reflects a strong light from a distant source through the glass stage to the reflector which concentrates it on to the specimen uniformly from all sides. Minute opaque struc- tures (antennal segments, ocelli, ete.) have been mag- nified to +50 diameters, stud- ied, and camera lucida draw- ings made of them. This ap- paratus is in use in Room 3, Rockefeller Building and can be seen there by any one FIG. 1 was removed from a Strik-a- = interested, 12 DHE GOLERCLING NET { Vor. VIII. No. 61 The Collecting Net An independent publication devoted to the scientific work at Woods Hole Edited by Ware Cattell with the assistance of Margaret L. Goodson, Rita Guttman, Martin Bron- fenbrenner, Elizabeth Jenkins, Margaret Mast and Annaleida S. van’t Hoff Cattell. Copyright, 1933 THE COLLECTING NET IN 1933 The purpose of THE CoLLectiINnG NET is to as- semble material which is of especial interest to the workers in the biological institutions at Woods Hole. We want to record as fully as we can the research work and other activities of the members of the Marine Biological Laboratory, the Woods Hole Oceanographic Institution and the United In addition we want to seek relevant material outside of Woods Hole and to record local events of interest. The pro- jected editorial contents of our magazine can be divided into four parts: (1) Results of the scientific work reported during the summer at Woods Hole. (2) Items reporting the activities of members of the scientific institutions in Woods Hole. (3) World-wide news of the activities of in- stitutions and individuals working in the field of biology. (4) States Bureau of Fisheries. The more important local news. Tue Cottectine NET is an independent publi- cation. Its contents are based primarily on the three scientific institutions in Woods Hole, but it has no official connection with any one o1 chem. We believe that there is not only a place, but a real need for an informal magazine of biology which is prepared to include constructive discus- sion on any topic of interest to those persons working in the biological sciences. Sometimes material is submitted to an editor which he would like to print, but an_ editorial board makes its publication unwise or impossible. The fact that THe Cottectine NET is responsi- ble to no organization thus gives it a peculiar ad- vantage over many publications in the field of science, THE SCHOLARSHIP AWARDS Last summer from many cources we obtained enough money for THE CoLLectinG Net SCHOL- ArSHIP FuND to permit the award of six scholar- The sum of one hundred dollars was as- signed to each of the following five students in or- der to make it possible for them to work at Woods Hole during the present summer : ships. Name Course Iping Chao Physiology H. L. Eastlick Embryology J. J. Metzner Protozoology C. B. Havey Zoology Margaret Grierson Zoology The staff of the course in botany decided not to award a scholarship to one of its students. The firms that advertise in THe CoLLecTING Net make possible its publication. If they stopped buying space we would cease to function. who are in Woods Hole People can materially help if they will support our advertisers by purchasing from them. In its office on Main Street THE CoLLectina Net has a great many books for sale. They cover a wide range of subjects and the prices of many of them have been cut to one half or one third of their original cost. Money resulting from the sale of these books will be used this summer to help defray the cost of publishing Tie CoLLectine NET. CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the hole turns to run from Buzzards Bay to Vineyard Sound: Date ADM. PSM: Uudl\e also eee 4:50 5:21 Uibly Sos go dlat 5:48 6:16 ide WWio aioe 6:48 1/ gle cuit see 0S cuckoate o 7:50 8:10 nlivas ABiane sensi 8:5: 9:08 die (Janene eo 9:50 10:06 nalives AT éstenencr chen 10:46 11:02 iw iow fhe kero a 6 11:41 11:56 ile Gn ding oo 12:35 Mollie UOo cere sé 12:51 1:29 In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. Jury 1, 1933 ] THE COLLECTING NET 13 DIRECTORY FOR 1933 KEY Laboratories Residence aera Apartment <2. ing. These men include, in addition to Dr. Hugo Fricke, who is in charge of our laboratory for biophysics, Dr. Chambers, Dr. Gasser, Dr. Mac- Innes, Dr. Michaelis, Dr. Osterhout and Dr. Van Slyke. Their part in the program is indicated in the list of lectures and symposia which is pub- lished elsewhere in THE CoLtectinG Nev. This, then, is the putting into practice, this time in respect to the Laboratory’s summer work, of the second major step in our policy of fostering a closer relationship between biology and the basic sciences. The first step was the establishment in 1928 of our all-year laboratory for biophysics. In- cidentally, the fact that the laboratory for biophy- sics is functioning here throughout the year has been of very great practical aid in the i inaugura- Steer estes reo IS 7S THe AERIAL VIEW OF Woops Hote tion of our plan for group work and conference during the summer. The description of the plan, as it is operating this summer, is the best indication | can give of the way in which it will be developed in subse- quent summers. The subject will, of course, change from year to year, but the fundamental aim and the type of men chosen to aid in the reali- zation of that aim, will, in all probability, remain similar to those of this year. It is expected that many advantages will be se- cured through the operation of the plan. Out- standing among these is the value of the meetings to the men who form the group. Support of this assertion is to be found in the fact that, without exception, every man who was asked to become a member of the group, and who planned to be in this country during the summer, accepted. Another advantage lies in the fact that the preseace of such a group at Cold Spring Harbor each summer will aid the Laboratory in its pri- mary aim, namely that of being of as much sery- ice as it can be in advancing biology. Everyone at the Marine Biological Laboratory at Woods Hole is wholly familiar with the desire of the founders of that Laboratory and of those who es- tablished the Laboratory at Cold Spring Harbor, i.e., that these stations should not only provide op- portunities for summer work, but that they should be centers of growth and dissemination of new methods and ideas in biology. In my opinion, much of the ultimate value of the summer work of both institutions will always remain in the fulfillment of the basic desire which gave them birth. The opportunity to foster the development of promis- ing methods and ideas is greater now than when these laboratories were founded, and the number of relatively isolated colleges and universities in this country which can be ‘Lenefited by active, con- scious effort, on the part of summer laboratories, to stimulate interest in modern methods of re- search, is much larger than it was in the last quar- ter of the nineteenth century. The Laboratory’s plan to choose each year a group interested in an aspect of quantitative biology different from that of the year before, sures against our giving undue stress to any one part of modern biology, and at the same time brings a partially new mental environment to workers who, for one reason or another, prefer to spend every summer at the same laboratory. Parenthetically, there is, of course, the further consideration that the presence of a definite pro- gram of work within a summer laboratory seems to stimulate all of those in residence, and tends to eliminate the job-hunters and vacation-seekers who curtail the work of any institution at which ON THE Opposite PAGE Was TAKEN FOR THE COLLECTING Ner sy a Untrep Starrs Army Air Corrs PHOTOGRAPHER 32 THE COLLECTING NET [ Vor. VIII. No. 62 they congregate. In this connection we have found at Cold Spring Harbor that the all-year work of the Laboratory, with its definite program, provides a marked stimulus to summer workers. Finally, the Laboratory hopes to make available to all workers interested in the aspects which will be subjects of its group meetings, the tangible re- sults of these meetings. This will be done through the lectures and symposia, which assume, there- fore, a very great importance. These lectures and symposia should not be confused with the evening lectures and seminars which have been, and will continue to be, a part of the Laboratory’s summer work. The lectures and symposia connected with the group meetings will be given during the day, beginning at ten o'clock in the morning. Ample time will be allowed for discussion. Indeed lec- turers have been asked to give special considera- tion to theoretical and controversial aspects of the subject, in order that discussion may be significant and creative. Furthermore, in general, students and others not competent to take an active part in discussion will not be invited to attend the lectures and symposia. Attendance is not restricted, how- ever, to scientists in residence at the Laboratory. Moreover, it is planned to publish the lectures, to- gether with essential parts of the discussion, as a monograph, in order to make results of the meet- ings generally available. A small number of graduate students will be given the opportunity of working with members of the group who are in residence at the Labora- tory and who will further give special lectures for students from time to time, notably for those in the course in general physiology. The whole plan is obviously not the creation of any one man. All of the members of this year’s group have been helpful in offering suggestions, while Dr. Osterhout has been called upon fre- quently to give counsel. The other summer work of the Laboratory re- mains fundamentally unchanged. The number of students is at the reduced level which has been maintained during the past few years, namely about thirty-five. Prof. W. W. Swingle of Prince- ton University continues in charge of the course in Surgical Methods in Experimental Biology; Prof. S.-I. Kornhauser of the University of Louisville Medical School again leads the course in Field Zoology; Prof. I. R. Taylor, of Brown University, that of General Physiology, and Prof. H. S. Conard, of Grinnell College, that of Plant Sociology ; while Prof. Bert Cunningham of Duke University will give a series of lectures on the en- docrine system. Dr. A. J. Grout will again have a limited number of bryologists working under his direction at his personal laboratory at New- fane, Vermont. Provisions are made, as before, for independent investigation, and for young research workers to use the facilities of the Laboratory. The inauguration of the group work will not effect detrimentally the opportunities which have been traditionally extended to the “lone eagle” biologist. Indeed the present plan been brought into existence not to replace the previous work of the Laboratory, but rather to strengthen it and to aid it in fulfilling its destiny vis a vis with present and future advance in biology. has HEMOGLOBIN-RINGER: A NEW MAMMALIAN PERFUSION MEDIUM (Continued from Page 29) justified in our further studies, and we were en- abled to carry out a series of experiments on the permeability of the salivary glands for various or- ganic non-electrolytes, which have been described elsewhere. More recently my colleagues at Memphis and I have been interested to see whether “hemoglobin- Ringer” might be adapted to other mammalian problems. We were particularly concerned to know whether this fluid might be used to replace the blood in the whole animal, and are able to re- port that this may be done, with a continuation of most, if not all, vital functions, for a period of hours after the removal of the normal blood. The adequacy of the medium as a substitute for blood can be demonstrated in a variety of ways. We consider that our determinations of the oxy- gen consumption furnish the most convincing proof. Using an apparatus designed and described by Dr. Arthur G. Mulder we have been able to show that the oxygen consumption of the whole animal under veronal anesthesia continues with practically no change for several hours after the replacement of the normal blood by hemoglobin- Ringer. The hemoglobin in solution is able to ful- fill its respiratory role much as when it was en- closed within the red blood cells. Another convincing demonstration of the ade- quacy of the medium to support all vital functions is given by the return of consciousness in cats from which the blood has been removed under ether anesthesia. Such animals come out from ether in a manner hardly to be distinguished from the behavior of a normal animal under similar cir- cumstances. The respiration, however, is more rapid and deeper than normal, and respiratory dis- turbances of several types are observed during the succeeding hours. In spite of this abnormality, the animals are able to carry out a variety of ac- tivities. They are able to walk, run, see and hear. They are able to jump to the floor from a con- siderable height, judge distances correctly, and Jury 8, 1933 ] THE COLLECTING NET 33 make their way about in a normal manner. All postural and equilibratory reflexes are functional. When dropped upside down they land on their feet. They recognize a dog as an enemy and will attack it with swift blows from their forepaws. While swift movements are possible they tire quickly and spend much of the time in sleep. They may be readily aroused and show a high degree of activity, only to lapse rather suddenly into sleep. Such behavior is almost certainly connected with the initial low oxygen capacity of the hemo- globin-Ringer, and with the progressive loss of the hemoglobin from the blood stream as the ex- periment continues. Death finally comes to these animals, not because the hemoglobin in solution is unable to carry out its respiratory function, but because it leaves the blood stream, appearing particularly in the urine. It also may appear in the feces, and is to some extent taken up by cells of the reticulo-endothelial system. The conclusion that death results from asphyxia when the hemoglobin concentration in the blood has fallen below a critical value is strengthened by our observation that an increase in the amount of hemoglobin added to our solutions prolongs the life of the animals. In our earlier experiments we used the hemoglobin from 170 cc. of beef cells in each liter of solution. Our animals survived for five or six hours. More recently we have raised the concentration of hemoglobin about 50%, using 250 ce. of cells per liter. Our animals now live from ten to fifteen hours. We expect to make further increases in hemoglobin content in an attempt to get longer survivals, and, possibly, recovery, if the production of new red blood cor- puscles can occur rapidly enough to replace the hemoglobin which is leaving the body. In its present form, with 250 cc. of red cells used per liter, our solution has an oxygen capacity of 11 to 12 volumes per cent. Through the courtesy of Dr. Herbert Wells, of Vanderbilt, we have recently measured the colloidal osmotic pressure of the solution, by a modified Krogh method, and secured a value of 26 mm. mercury in a single determination. This value is quite within the range for the normal colloidal osmotic pressure of mammalian blood. One of the main considerations leading us to use the solution in the first place was the hope that the hemoglobin might supply a colloidal osmotic pressure which would regulate the water balance. This hope has been fully justified. At autopsy all tissues appear normal in size and color; edema is definitely not present. It might be added that, while hemo- globin passes into the urine with great ease, it does not leave the blood stream in most tissues, so is able in our solution to exert an osmotic pres- sure approximating that of the normal plasma colloids. Aside from the theoretical interest of our ob- servations, we believe that this solution may be employed in the study of many problems involv- ing the blood and circulation. For instance, blood volume may be easily and accurately determined, as in the Welcher method, with the assurance that all blood cells have been removed from the body. We have begun a study of the rate of replace- ment of various blood constituents. The leuco- cytes are rapidly replaced. Fibrinogen also re- turns rapidly, and has reached about 50 per cent. of the normal value in ten hours after the con- clusion of the bleeding. Fluid withdrawn at this time will form a clot, the hemoglobin-Ringer setting into a clear red jelly. We are led by these experiments to the con- clusion that a very major function of the verte- brate red blood corpuscles is to hold hemoglobin within membranes impermeable to it, so that it cannot leave the blood stream. In other respects, hemoglobin appears to be able to carry out its respiratory role in solution much as it does within the red cells, sustaining every vital func- tion, even the more complicated activities of con- scious life. COMMENTS ON THE PAPER. BY DR. AMBERSON Dr. AuGcust Krocit Professor of Zoophysiology, University of Copenhagen, Denmark The hemoglobin-Ringer of Dr. Amberson will undoubtedly prove a very valuable addition to the perfusion fluids already available, all of which have certain drawbacks. It is an interesting fact that an animal can retain or regain consciousness when supplied with this fluid instead of blood, and it is especially interesting because Barcroft has recently shown that when the environmental balance of the tissues is upset, the first organ to suffer is usually the brain and loss of conscious- ness is one of the first symptons. Even this perfusion fluid, however, is not in all respects tie ideal one. It seems to be quite toxic to the renal glomerul!, since the normal hemo- globin molecule is certainly large enough to be kept back by the normal glomerular epithelium. The potassium of the red cells must make the hemoglobin-Ringer comparatively high in potas- sium unless this is allowed for in the Ringer by leaving out some potassium or by adding a cor- responding surplus of calcium. I expect that the increase in viscosity will set a limit to the quantity of red cells which can be used to advantage. 34 THE COLLECTING NET [ Vor. VIIf. No. 62 Courses at the Marine Biological Laboratory THE PHYSIOLOGY COURSE Dr. R. W. GERARD Director of the Course, Associate Professor of Physiology, University of Chicago The physiology course got well under way this summer on June 20 and has already succeeded in shocking most of the community with the aid of a large electric ray. The eighteen students have a considerable choice of techniques and materials to study under the direction of the staff mem- bers. Besides the regular staff, including Drs. Amberson, Gerard, Irving, Michaelis, and Sum- walt, we are fortunate in having Drs. Chambers and Lucké contribute some laboratory instruction. Mr. A. L. Chute is continuing his able assistance with the apparatus. As in the past, the regular six weeks course is divided into three two-week periods. During the first two of these the various staff members offer selected experiments, largely along the line of their own research interests, which enable the students to acquire the appropriate experimental techniques and apply them to the study of familiar and novel materials. In the first period, work is being offered by Dr. Amberson on bio-electric phenomena, such as action and injury potentials, and on electro- kinetic behavior. He is also attempting for the first time a section on the physiology of secretion, using kidney and salivary glands. Dr. Irving is offering the Haldane and Van Slyke techniques in the study of carbon dioxide content, buffering power, acidity and the like, in tissues and bio- logical fluids. Dr. Michaelis is taking many of the students into his own laboratory to study electrometric and other means of determining pH and oxidation-reduction potentials. During the second two week period, Dr. Gerard will introduce the Warburg and other techniques for studying cell respiration. Dr. Sumwalt and Dr. Lucké will offer studies on the permeability of cells to water and dissolved materials, as in- fluenced by temperature acidity, and the like. Dr. Chambers will supervise micro-dissection work done during both periods. The last two week unit constitutes essentially an introduction to research, the students being en- couraged to select some small problem along the lines of their previous experience and work at it intensively under the guidance of a staff member. A few students who make a promising start may carry on their research throughout the summer. A lecture will be given at nine o’clock each weekday morning; at first by the regular staff members and later by other members of the scientific community whose research interests en- able them to speak with authority on various sub- jects of significance to the course. These lectures, as in the past, are open to all interested members of the institution. Notices will be posted at inter- vals announcing current lectures. MORPHOLOGY AND TAXONOMY OF THE ALGAE Dr. WILLIAM RANDOLPH ‘TAYLOR Director of the Course; Professor of Botany, University of Michigan Cut off from popular observation and knowl- edge by submersion in an aquatic the algae have long been a familiar and_ closely studied group of plants for but a small number of biologists. Their importance to the population of the waters is quite similar to that of land plants in the biological population of the higher ground, for in both cases the presence of animals is limited by the presence of the plants and by their activities in synthesizing carbohydrate and simple nitrogenous food substances. The im- portance of the algae extends from bodies of water to the land, for culturing of soil shows a very considerable and active algal flora in the up- per layers even when not so superficial as to be visable to the unaided eye. environment, - The ecological studies involving algal popu- lations have too often been taxonomically inac- curate, and have dealt vaguely with the presence of genera lone, even when a genus ranged widely in adaptability. Likewise in local listing of algae, far too little care has been exercised in securing a critical determination. In the algae course at Woods Hole an introduction to the group is first made by a consideration of the general classi- fication of these plants. With this to preserve or- der in ideas, the morphology and life history is considered in detail. At the present time active study of life-histories and sexual differentiation hoth in America and abroad has suggested many wide-reaching revisions of classification, which, even when incomplete and tentative, must be con- Jury 8, 1933 ] THE COLLECTING NET 35 sidered. It is possible also to call attention to the more marked physiological peculiarities of the various algal types. Every effort is made to se- cure living material to represent each family; in the case of the strictly tropical groups, a collection of preserved specimens of the more important genera is available. In order that the students may have expe- rience in the field, trips are taken at frequent in- tervals to collect material in a manner which would be suitable to an initial or exploratory sur- vey of the flora of the area. On returning, the material is searched and the genera present de- termined, this involving the use of introductory manuals. From those students who show suf- ficient critical ability, more accurate identi- fications are encouraged, involving the use of ori- ginal bibliographic sources. The smaller number of local marine algae enables all students to be- come familiar with these species in a detailed way. The excellent library facilities of the Marine Biological Laboratory give especial encourage- ment to students wishing to begin algal studies. With its aid, familiarity may be secured with all of the important publications needed to begin tax- onomic or morphological studies; for the student with a problem already begun, no better oppor- tunity could be secured to do the detailed library work which is the necessary accompaniment and often the most time-consuming part of modern critical research. Students with major interests in the physiological or biochemical phases of life will find the library even more complete in their lines and may pursue their work with the aid of the botanical and physiological research staffs. THE COURSE IN PROTOZOOLOGY Dr. Gary N. CALKINS Director of the Course, Professor of .Protozoology, Columbia University The course in protozoology is designed for graduate students wishing to begin research in protozoology or in other fields requiring a knowl- animals, free-living or edge of unicellular parasitic. A The lectures cover the principles of general biology; the morphology of the different classes of protozoa; the parasitic protozoa and_ the diseases caused by them; experimental proto- zoology and the theories of growth and cell divi- sion, of fertilization, of age and reorganization, of heredity and variation. ; The laboratory work gives an opportunity for the collection of material from natural sources; the study and identification of living organisms representing the main groups of the protozoa (seventy-five drawings) classified through genera, including ten classified through species, are re- quired) ; the cultivation of protozoa in artificial media; the determination of the pH of media and of water collected from ponds around Woods Hole. Opportunity is also given for special cytological studies of the protozoa. For this purpose, perm- anent preparations (ten) are required. In ad- dition to the ordinary techniques, osmic and sil- ver impregnation, the Feulgen nucleal reaction, and the chondriosome methods are employed. Fresh material stained by vital dyes is used to supplement fixed material when the identification of cytoplasmic elements is desired. EMBRYOLOGY COURSE Dr. H. B. Goopricu Director of the Course; Professor of Biology, Wesleyan University The embryology course opened on June 22. The chief objects of the course are to give oppor- tunities for studying the development of living eggs and embryos and to present various fields of embryological research. Fertilization cleavage and stages of development are studied in re- presentation of different phyla. It is not pos- sible to arrange material in logical order, because the schedule is dependent upon the breeding sea- sons rather than on zoological classification. Fish are studied first as their spawning season comes early. The eggs of the annelid Nereis can only be obtained between full moon and the new moon. The tentative schedule is given below. From time to time special lectures are given by various investigators. 36 THE COLLECTING NET [ Vor. VIII. No. 62 MECHANICS OF MATING IN LIMULUS CHARLES Marc POMERAT Department of Biology, Clark University Quantitative studies of mating behavior tend to support the view that the mating phenomenon does not rest upon chance pairing but that a con- siderable degree of assortative selection can be demonstrated. Mating of like with like is fre- quently designated as homogamy. The intensity of this tendency usually is measured by correla- tion coefficients derived from various physical characteristics of the species under consideration. Of the various possibilities (mechanical, chenii- cal, psychic, relative activity and physical vigor), the simplest explanation for the existence of homogamy in the majority of invertebrates studied thus far appears to rest upon purely mechanical factors involved in the mating act. On the basis of such an assumption, high homogamy correla- tions may be expected for organisms whose mat- ing involves exact juxtaposition of more than one point of contact. Moreover, the presence of a more or less inflexible cuticula or exoskeleton will tend to intensify the mechanical limitations. Such a theory is of value in an attempt to ex- plain the findings already reported and to suggest possible checks on the theory itself. Conjugation in the protozoans, Paramoecium and Chilodon; hermaphroditism in Chromodoris, and the me- chanical restrictions observed in mating gamma- rids suggests that the high correlations obtained for homogamy in these organisms may be ac- counted for on the basis that the mechanical pre- cision involved does not permit coupling of in- dividuals of widely divergent sizes. The low values obtained for the Japanese beetle, Popillia japonica Newm., do not tend to uphold this view unless it can be considered that a greater degree of adjustment at the contact points is possible than might a priori be expected. Copulation in Limulus polyphemus L. involves amplexus but without intromission, so that only one pair of contact points are involved, 1. e., clasp- ing without genital contact. Owing to this fact Limulus serves as an excellent contrast to the or- ganisms previously studied. In addition, the large size of this organism considerably reduces the technical difficulties usually involved in obtaining biometric data on invertebrates. With the aid of a CoLLecTING Net scholarship the writer studied the mating behavior of Limulus at Woods Hole during the summer of 1932. Approximately 1400 measurements were taken on 100 pairs of mated Limuli. A mean value of 0.10 ¢* 0.06 was ob- tained from 49 direct and cross correlation coeffi- cients for the seven characters studied. It was concluded that homogamy as regards size in Limulus is practically non-existent. A full ac- count of this study has been published in the Bio- logical Bulletin, LX1V, 2; 243-252, April 1933. In addition to the increased range of choice af- forded by the single-point type of clasping con- tact, a further analysis of the mechanics of mating in Limulus suggests that clasping in this animal depends upon a device which probably permits copulation between any male and female. The second pair of cephalothoracic appendages of the adult female Limulus is like the third. fourth and fifth in having a chela—that is to say the penultimate sclerite is produced so as to forn with the last sclerite a pair of nippers. In tht male this is not the case, the second pair being thicker and heavier than in the female and the penultimate joint not prolonged. It was observed long ago that the form of this appendage in Limu- lus polyphemus closely resembles the appendages of Arachnids such as Thelyphonus, though not of Scorpio. By means of this hook-like modified pincers the male attaches himself to the hinder half of the carapax of the female and trails along in shallow water. In all adult males observed the jaw of the claw has been found to be wider than the thickest carapax rim ever observed in the fe- male. On this basis the male may clasp any fe- male. It has been concluded that there is reason to be- lieve that there exists very little mechanical re- striction in the coupling of Limuli and that the range of choice is therefore markedly increased and the coefficient of homogamy proportionately lowered. The findings in Limulus polyphemus are in keeping with the notion that mating in cer- tain invertebrates rests at least in part upon a mechanical process. FURTHER STUDIES ON THE CONTROL OF ELASMOBRANCH MELANOPHORES HeLen M. Lunpstrom AND Purtip BARD Our previous work (Brot. Butt., 1932, 62, 1-9) has shown that a melanophore-expanding sub- stance is continually being secreted by the pos- terior lobe of the dogfish pituitary. Removal of this structure is followed by extreme cutaneous pallor, which is permanent. This change is unin- fluenced by the degree of illumination. Injection of post-pituitary extracts into such pale fish re- stores the normal dark coloration. This summer we have investigated the mechan- Jury 8, 1933 ] THE COLLECTING NET 37 ism governing the changes in pigmentation which occur with changes in illumination. When dogfish with normal eyes are placed in a brightly illumi- nated tank they slowly become paler. This pallor is never as great as that produced by hypophy- sectomy, and its development requires several hours. When fish, which have been made pale in this way, are restored to a dark tank they darken rapidly—within thirty minutes. The same rapid darkening to a maximum (complete expansion of melanophores) takes place when a pale illumi- nated fish remaining in the illuminated tank has been subjected to any one of the following pro- cedures: (a) covering the eyes with a light-proof material, (b) section of the optic nerves, (c) in- jury of the hypothalamus at any point between optic chiasm and the posterior hy pophysis. These same procedures carried out before exposure to light prevented paling. On the other hand, section of the spinal cord at any level or complete tran- section of the brain just behind the pituitary (de- cerebration) did not produce darkening of light individuals, and these operations left the animals able quite normally to alter their shade with changes in illumination. From these results we conclude that optic stim- ulation influences the secretion of melanophore- expanding substance from the hypophysis by a nervous process whose path involves optic nerves and hypothalamus. The final segment in this path must consist of fibers passing to the pituitary from the hypothalamic region. In these experiments temperature, an important factor in the pigmentary responses of amphibia, was controlled. The variation in temperature be- tween the illuminated and control tanks was never more than one degree Centigrade. Similarly care was taken to maintain the same degree of aera- tion in the two tanks. When a large part of the body of a fish was covered, leaving only the anterior end exposed, the entire fish paled when illuminated, but it was observed that the covered area was very slightly darker than the exposed region. The difference was only just perceptible. Whatever the explana- tion of the phenomenon may be, it seems quite correct to state that variations in the degree of cutaneous pigmentation of the dogfish are almost wholly due to variations in pituitary secretion, and that these variations are induced by changes in il- lumination of the eves acting through nervous channels on the secretory mechanism. THE “ATLANTIS” AND ITS OCEANOGRAPHIC WORK Co_umBus O. IsELtIn, 2nd Reseach Associate in Physical Oceanography Since last November the “Atlantis” has cruised about 10,000 miles and has occupied some 225 stations. She returned to Woods Hole at the end of May. The time in port until June 19 was utilized for a general spring overhaul so that she could be made shipshape before starting on the summer program. The first part of this is involving four short cruises to the Gulf of Maine, and then a longer trip northward is planned to begin in August. The summer pro- gram can be outlined briefly as follows. June 19-June 28. First half of the chemical survey of the Gulf of Maine; U.S. Bureau of Fisheries haddock spawning investigations. Mr. Herrington was in charge. June 29-July 8. Second half of the Gulf of Maine investigations and bacteriological samples for Dr. Waksman. Dr. Redfield is in charge. July 10-July 16. Light intensity and the verti- cal migration of plankton. Dr. Clarke will be in charge. July 20-July 25. deep water south of Woods Hole. be in charge. Aug. 12-Sept. 15. An investigation of the formation of bottom water in the North Atlantic. The deep layers will be followed northward un- til the region where they approach the surface is reached, Dr, Redfield will be in charge. Bottom samples from the Mr. Iselin will The main feature of the work done last winter was a thorough survey of the whole Caribbean Sea. In this program the Woods Hole Ocean- ographic Institution cooperated with the Bing- ham Oceanographic Foundation at Yale, which had made a good beginning the year before in the Gulf of Mexico. Dr. A. E. Parr was the scien- tist in charge. 3esides the routine temperature, salinity and oxygen observations, the majority of water sam- ples were analyzed for nitrates, phosphates and pH. stalled on Since echo sounding equipment in- “Atlantis” last autumn, soundings were taken during the course of the This should add greatly to our knowledge of the bottom contours along the many sections across the Caribbean Sea as well as in the passages between the islands. Another inno- vation was the use of a new type of trawel at mid- depths. This net, held open by three “boards,” is triangular at the mouth. It spreads fifty feet on a side at the mouth, and trails some two hundred feet behind the boards. It is by far the biggest net which has been tried out at depths below a thousand fathoms. Other work completed by the “Atlantis” during Was the hourly trip. (Continued on Page 388) 38 THE COLLECTING NET [ Von. VII. No. 62 The Collecting Net An independent publication devoted to the scientific work at Woods Hole Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Martin Bron- fenbrenner, Elizabeth Jenkins, Margaret Mast and Annaleida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford THE WOODS HOLE LOG The present number of THe CoLLectinG NET will ke the last one to contain The Woods Hole Log during the present summer. Circumstances make it possible to renew our venture initiated in 1929 of issuing the local news in a separate pub- lication. The first number of this off-shoot will appear next Wednesday or Thursday and it will be published weekly thereafter until the end of August. The Woods Hole Log will be edited by Miss Rita Guttman and Mr. Martin Bronfenbren- ner, published by THe CotLectinc Net and printed by the Darwin Press in New Bedford. The business affairs of this Woods Hole enter- prise will be in charge of Miss Margaret Mast. Removing the “Log Material” and the local ad- vertisements will leave more space for research reports, lectures and other material of more espe- cial interest to the biologist. In addition we hope soon to be able to further increase the number of pages in THE CoLLectinG NET so that we can de- vote still more space to scientific research. A few readers of THE CotiectinGc Net have thought it unwise to include the local news; others have enjoyed reading it. The former will now be able to escape it; the latter can now have more! Mntroducing Dr. Aucust Krocu, professor of zoophysiclogy at the University of Copenhagen is “‘visiting scientist’’ at the Woods Hole Oceanographic In- stitution this summer. In 1920 Professor Krogh was awarded the Nobel Prize in Physiology and Medicine for his outstanding work on the physiol- ogy of capillaries. He is the author of several books in physiology: “The Respiratory Exchange of Animals and Man,” one of the volumes in the English series of Monographs on Biochemistry ; a text-book for college students in human physiol- ogy which has been recently translated into the English language by Dr. Katherine R. Drinker; “The Physiology of the Capilaries.” The latter volume is based upon a series of lectures which Dr. Krogh delivered under the auspices of the Silliman Foundation at Yale University in 1922. Dr. Krogh came over to this country as one of the distinguished foreign guests of the Amer- ican Association for the Advancement of Science which has recently completed its meetings on the grounds of the Century of Progress Exposition at Chicago. He drove from New York to these meetings with Dr. Walter Mills who is professor of psychology at Yale University. Dr. and Mrs. Krogh will continue their studies at the Woods Hole Oceanographic Institution through most of August on the organic substances present in sea water and their possible role in the nutrition of animals. They plan to sail on August 24 on one of the boats of the Swedish American Line. Sometime during the summer Dr. Krogh will deliver an evening lecture, and his wife will present the results of their newer work in a semi- nar report. Mrs. Krogh is examiner in physiology at the University of Copenhagen and also lecturer in physiology and nutrition at the State Normal School for Teachers in Copenhagen. She is in- terested especially in medical physiology, and her primary problem is the study of the relation be- tween the thyroid and pituitary gland in the guinea pig. THE “ATLANTIS” AND ITS OCEANOGRAPHIC WORK (Continued from Page 37) the past winter can be summarized as follows: 1. Five coastal sections between Cape Hatteras and Cape Cod. 2. A section from the mouth of Chesepeake Bay to Bermuda. 3. A section from Bermuda to Bahamas. 4. Four sections extending from the coast out across the Gulf stream between the Straits of Florida and Cape Hatteras. - It can be seen that the ship has been kept busy and that she is fast completing a general chemi- cal and hydrographic survey of the western half of the North Atlantic. Nassau in the MM. B. L, Calendar (Continued from ist page) TUESDAY, JULY 11, 8:00 P. M. Lecture: Professor Svedberg, University of Upsala: “Ultracentrifugal and cataphoretic studies on respiratory proteins.” FRIDAY, JULY 14, 8:00 P. M. Seminar: Dr. M. M. Brooks: The effect of re- spiratory poisons and methylene blue on cleavage of certain eggs. Dr. Laurence Irving: Ionic changes during the development of fish eggs. Dr. E. Newton Harvey: The tension at the surface of egg cells. Dr. Robert Chambers: A peculiar feature of the cleavage furrow in Arbacia eggs shown in a motion picture film. - ‘Jury 8, 1933 ] THE COLLECTING NET DIRECTORY FOR 1933 KEY Laboratories Residence o= A Apartment .........+. A Botany RES ...-Bot Dormitory ........+.- D Brick Building....... Br prew House ........-Dr mecture Hall. ......... L Fisheries Residence...F Main Room in Fisheries aed a FERS any oe Bene ORY, coe es 3 Rie Kabler ns onoecees Ka Old Main Building...OM fKidder ...........--.- K Widely frot 50 Ane aoencmroc WwW Rockefeller Bldg.. ..Rock In the case of those individuals not living on laboratory property, the name of the landlord and the street are given. In the case of individuals liv- ing outside of Woods Hole, the place of residence is given in parentheses. MARINE BIOLOGICAL LABORATORY INVESTIGATORS Abramowitz, A. A. fel. biol. Harvard. Br 108. Ka 23. Adams, E. M. grad. biol. Cincinnati. Br. 8 Dr 2. Amberson, W. R. prof. phys. Tennessee. Br 109. Quisset. Anderson, R. S. res. assoc. phys. Princeton. Br 110. McInnis, Millfield. Armstrong, Louise 8. res. asst. anat. Cornell Med. L 22. D 315. Armstrong, P. B. asst. prof. anat. Cornell Med. Br 318. D 315. Atlas, M. asst. zool. Columbia. Br 314. Dr 5. Baden, V. univ. scholar zool. Pennsylvania. Rock 6. D 210. Bailey, R. J. instr. zool. Base. Stewart, School. Baitsell, G. A. prof. biol. Yale. Br 323. Brooks. Ball, E. G. assoc. physical chem. Hopkins Med. Br George Wasington. OM 110. D 309. Barron, E. S. G. asst. prof. biochem. Chicago. Br. 207. D 207. Barth, L. G. instr. emb. Columbia. Br 111. Hubbard, East. Beck, L. V. grad. asst. phys. Pittsburgh. Rock 7. Dr. Beckwith, Cora J. prof. zool. Vassar. A 209. Bell, H. P. prof. bot. Dalhousie. (Canada) Bot 23. D 216. Bissonnette, T. H. prof. biol. Trinity (Connecticut) OM 26. D 211. Bostian, C. H. asst. prof. zool. North Carolin State. Rock 3. Dr 5. Bowen, R. E. asst. prof. biol. Long Island. Br 126. Howes, Water. Bowling, Rachel instr. proto. Columbia. OM 21. A 307. Boyden, Louise E. ed. sec. “Biol. Bul.” Br 305. A 306. Bridges, J. C. instr. zool. Morehouse. L 33. A 106. Brinley. F. J. asst. prof. zool. North Dakota State. OM 38. D 201A. Brooks, Matilda M. res. assoc. biol. California. Br 233. Gosnold. Brooks, S. C. prof. zool. California. Br 233. Gosnold. Brown, W. R. grad, biochem, Cincinnati. Br 342. D 109. 539 Budington, R. A. prof. zool. Oberlin. Br 218. Or- chard. Butler, E. G. assoc. prof. biol. Princeton. Br 303. D 201. Butt, C. tech. Princeton. Br 116. Dr. Calkins, G. N. prof. proto. Columbia. Br 331. Buz- zards Bay. Cannan, R. K. prof. chem. N. Y. Univ. and Bellevue Hosp. Med. Br 310. Gardiner. Carleton, B. H. asst. phys. Rochester. Br 340. Dr. Cattell, W. assoc. ed. “Scientific Mo.” Br 344. North. Chambers, R. res. prof. biol. New York. Br 328. Gosnold. Chao, I. grad. phys. Chicago. Br 315. D 106. Cheney, R. H. prof. biol. Long Island. Br 126. D 308. Chesley, L. C. asst. biophysics. Memorial Hosp. Br 343. Chidester, F. E. prof. zool. West Virginia. Br 344. D 318. Chute, A. L. Toronto Med. (Canada) OM. 9. Ka. 3. Clark. Eleanor L. vol. invest. Pennsylvania Med. Br 117. West. Clark, E. R. prof. anat. Pennsylvania. Br 117. West. Clark, Frances Sec. to dir. Lilly Res. Labs. Br. 328 B. Howe, Main. Clark, Jean M. grad. Pennsylvania. Br 219. H 4. Clowes, G. H. A. dir. Lilly Res. Labs. Br 328 B. Shore. Coe, W. R. prof. biol. Yale. Br 323. A 201. Coghill, G. E. mem. Wistar Inst. Anat. and Biol. Br 220. Veeder, West. Coghill, Muriel grad. zool. Denison (Ohio) Br 220. Veeder, West. Cohen, Rose C. grad. asst. zool. Cincinnati. L 29. EG: Conklin, E. G. prof. biol. Princeton. Br 321. High. Corson, S. A. grad. res. asst. phys. Pennsylvania Med. Br 205. Young, West. Costello, D. P. instr. zool. Pennsylvania. Br 217n. Elliot, Center. Crampton, C. B. instr. biol. Wesleyan. Br 210. D 110. Croasdale. Hannah T. grad. bot. Pennsylvania. Bot. 22. W G. Dan, K. grad. zool. Pennsylvania. Br 111. Millfield. Day, Dorothy asst. prof. bot. Smith. Bot. A 205. Denny, Martha grad. Radcliffe. Br 312. Kittila, Bar Clark, Neck. Donaldson, G. C. prof. anat. Pittsburgh Med. Br 115. Crow Hill. Donaldson, H. H. Wistar Inst. Br 115. Belfry, Buz- zards Bay. Doyle, W. L. res. asst. phys. Hopkins. Br. 332. Dr 6. Duncan, P. M. grad. zool. Pennsylvania. Rock 6. K 9. Drumtra, Elizabeth asst. zool. Barnard. Br 314. K 3. Eastlick, H. L. asst. zool. Washington (St. Louis) Br 217g. Grave, High. Edwards, D. J. assoc. prof. phys. Cornell. Med. Br 214. Gosnold. Edwards, D. V. assoc. prof. phys. Cornell Med. Br 214. Gosnold. Edwards, T. I. instr. biol. Hopkins. L 21. Dr 7. Engel, F. L. Dartmouth. Br 109. D 111. Engel, G. L. Dartmouth. Br 309. D 111. Farrow, J. G. grad. zool. Pennsylvania, Rock, Pur- dam, Main, 40) THE COLLECTING NET [ Vor. VIII. No. 62 Finley, H. E. assoc. prof. biol. West Virginia State L 30. A 105. Fisher, K. C. fel. Toronto (Canada). Br. 107. Ka 1. Fleisher, M. 8. prof. bact. St. Louis Med. Br 304. D 112. Fowler, Virginia M. asst. bot. Barnard. Bot. K 3. Francis, Dorothy S. res. asst. biophysics. Memorial Hosp. Br 343. W F. Fry, H. J. prof. biol. New York. OM Base. Purdum, Woods Hole. Furtos, Norma C. fel. biol. Western Reserve. OM Base k. H 1. Garrey, W. E. prof. phys. Vanderbilt Med. Br 215. Gardiner. Gerard, R. W. assoc. prof. phys. Chicago. Om 3. D 303. Gilmore, Kathryn A. instr. bot. Pennsylvania Col. for Women. Rock 3. K 2. Goldforb, A. J. prof. biol. City N. Y. Br 122C. A 302. Goodrich, H. B. prof. biol. Wesleyan. Br 210. D 110. Grand, C. G. res. asst. biol. New York. Br 328. Mc- Leish. Grave, B. H. prof. zool. DePauw. Br 234. High. Grave, C. prof. zool. Washington (St. Louis). Br 327. High. Harnly, Marie L. asst. biol. New York. Br 1. A 102. Harnly, M. H. asst. prof. biol. New York. Br 1. A 102. Harvey, Ethel B. independ. invest. phys. Princeton. Br 116. Gosnold. Harvey, E. N. Osborn prof. biol. Princeton. Br 116. Gosnold. Harwood, EK. M. res. asst. Blue Hill Meteorol. 315. Young, Middle. Hayes, F. R. assoc. prof. zool. Dalhousie (Canada). OM 45. D 213. Hegnauer, A. H. asst. phys. Rochester Med. Br 340. Dr. Heilbrunn, L. V. assoc. prof. gen. sylvania. Br 219. Spaeth, Whitman. Henshaw, P. S. biophysics. Memorial Hosp. Br 3. D 206. Hetherington, W. A. fel. zool. Gray, High. Hotchkiss, Margaret instr. bact. N. Y. Homeopathic Med. 201. Wilde, Gardiner. Hibbard, Hope assoc. prof. zool. Oberlin. Br 218. K 12. Hicks, F. J. grad. zool. Pittsburgh. Rock 7. Ka 21. Obs. phys. Penn- Hopkins. Br 329A. Hill, E. S. res. asst. physical chem. Rockefeller Inst. Br 207. D 218. Hill. S. E. asst. phys. Rockefeller Inst. Br 209A. Veeder, West. Hoadley, L. prof. zool. Harvard. Br 312. D 315b. Hoijer, Dorothy J. Chicago Br 207. Neal, West. Hollaender, A. Nat. Res. fel. biol. Wisconsin. Br 225. Sylvia, Buzzards Bay. Hoppe, Ella N. res. biologist. Health. Br 122B. D 313. Howe, H. E. ed. “Ind. and Eng. Chem.” Br 203. Tinkham, West. Hunter, Laura N. grad. zool. Pennsylvania. Rock 6. Broderick, North. Hussey, Kathleen L. asst. zool. Base. W C. Hutner, S. H. grad. Cornell. OM Base, K 5. Hyde, Ida H. emer. prof. phys. Kansas State. L 34. Nickerson, Millfield. Irving, L. assoc. prof. phys. Toronto (Canada). Br. 107. A 202. Jacobs, M. H. prof. gen. phys. Pennsylvania. Br 102. Sippewissett. Jao, C, ©. grad, bot, Michigan, Bot 26. Dr 9, N. Y. State Dept. Connecticut OM Jenkins, G. B. prof. anat. George Washington. OM 34, Clapp, Gardiner. Johlin, J. M. assoc. prof. biochem. Vanderbilt Med. Br 206. Park. Johnson, Arline C. grad. asst. zool. Oberlin. OM Base g. H C. Jones, N. instr. sci. drawing. Swarthmore. Br 211. Hall, Main. Kaliss, N. Columbia. Br 314. McLeish, Millfield. Kekwick, R. A. London fel. phys. Princeton. Br 127. Minot. Kelly, T. L. prof. education. Harvard. OM 120. D 310. Keltch, Anna K. res. chem. Lilly Res. Labs. Br 319. McInnes, Millfield. Keil, Elsa instr. zool. N. J. Col. for Women. Br 8. W. D Kidder, G. W. tutor biol. City N. Y. Br 217. D 307. Kirkpatrick, T. B. assoc. prof. physical education. Columbia. L 27. Nickerson, Millfield. Korr, I. M. asst. instr. biol. Princeton. Br 110. Young, West. Krogh, Marie lect. phys. & nutrition. State Sch. for Teachers (Copenhagen). 105. D 301. Kyle, J. A. Br 107. D 108. Lancefield, D. E. assoc. prof. zool. Columbia. Br 333. Danchakoff, Gardiner. Lancefield, Rebecca C. assoc. bact. Rockefeller Inst. Br. 208. Danchakoff, Gardiner. Landowne, M. Harvard Med. Br. 108. Ka 24. Liedke, Kathe B. grad. zool. Columbia. Br 314. Syl- via, Buzzards Bay. Lillie, F. R. prof. zool. Chicago. Br 101. Gardiner. Lillie, R. S. prof. gen. phys. Chicago. Br 326. Gardiner. Lucke, B. prof. path. Pennsylvania. Br 311. Minot. Lynch, Ruth S. instr. zool. Hopkins. Br 336. A 101. MacDougall, Mary S. prof. zool. Agnes Scott. A 208. L 28. McLane, Kathryn E. instr. biol. Annapolis High School. phys. H 7. Magruder, S. A. grad asst. zool. Cincinnati. L 29. Kittila, Bar Neck. Manery, Jeanne F. res. asst. phys. Toronto (Can- ada). Br 107. H 2. Marsland, D. A. asst. prof. biol. New York. Br 339. A 102. Martin, E. A. chairman dept. biol. Brooklyn. OM 339. Newman, Prospect. Mast, S$. O. prof. zool. Hopkins. Br 332. Minot. Mathews, A. P. prof. biochem. Cincinnati. Br 342, Buzzards Bay. Maxwell, Jane. instr. biol. Carnegie Inst. Tech. Rock 3. K 2. Mazia, D. grad. zool. Pennsylvania. Br 219. Ka 24. Melampy, R. M. asst. animal nutrition. Cornell. OM Base. Dr 10. Metzner, J. J. grad. proto. Columbia. Br 331. White, Millfield. Michaelis, L. mem. Rockefeller Inst. Br 207. D 209. Miller, F. W. grad asst. zool. Pittsburgh. Rock 3. K 15. Moment, G. B. instr. biol. Goucher. Stewart, School. Moreland, F. B. res. asst. biochem. Vanderbilt Med. Br. 206. Dr 5. Morgan, Lilian V. independ. invest. genetics Calif. Inst. Tech. Br 320. Buzzards Bay. Morgan, T. H. prof. biol. Calif. Inst. Tech. Br 320. Buzzards Bay. Morrill, C. V. assoc. prof. anat. Cornell Med. L 24. Cape Codder, (Falmouth). Morris, S. grad. zool. Pennsylvania. Br 217m. D 305 Nelsen, O. E. instr. zool. Pennsylvania, OM 27. D 306. Jury 8, 1933 ] Newton, Helen K. ms. ed. “Ind. and Eng. Chem.” Br 203. Veeder, Millfield. Nichol, Margaret A. grad. gen. Pennsylvania Col. for Women. Rock 3. W A. Nonidez, J. F. asst. prof. anat. Cornell Med. Br 318. Whitman, Novikoff, A. B. fel. biol. Columbia. Br 314. Dr 1. Orbison, Agnes M. assoc. prof. biol. Elmira. OM 35. Nickerson, Millfield. Packard, C. asst. prof. zool. Columbia Inst. Cancer Res. OM 2. North. Palmer, A. H. instr. Univ. & Bellevue Hosp. Med. Br 310. Truslow, Gardiner. Palmer, Elizabeth T. instr. chem. Vassar. Br 110 g. Truslow, Gardiner. Parker, G. H. prof. zool. Harvard. Br 213. A 309. Parpart, A. K. asst. prof. phys. Princeton. Br 205. Minot. Pelluet, Dixie asst. prof. zool. Dalhousie (Canada). OM 45. D 103. Plough, H. H. prof. biol. Amherst. Br 204. Whitman. Pollister, Priscilla F. instr. Brooklyn. OM 44. D 314. Porter, Helen. asst. zool. Harvard. Br 213. Grinnell, Bar Neck. Prescott, G. W. asst. prof. biol. Albion. Bot 25. D 101. Rex, R. O. instr. anat. Pennsylvania. Br 117. Conk- lin, High. Richards, O. W. instr. biol. Yale. Br 8. A 303. Richardson, Margaret S. Brearley School. Br 318. Hubbard, Center. Root, W. S. assoc. prof. phys. Syracuse Med. Br 226. Oak, Park. Rubenstein, B. B. asst. phys. Chicago. Br. 309. D 106. Rugh, R. instr. zool. Hunter. Br 111. D 208. Rusch, Elizabeth res. asst. biophysics. Memorial Hosp. Br 343. Densmore, School. Russell, W. L. grad. genetics. Amherst. Br 204. Sauer, F. C. asst. prof. zool. Wichita. Br. Thompson, Main. 217. Schechter, V. instr. invert. zool. City N. Y. OM 1. Dr 2. Schweitzer, M. D. grad. zool. Br 333. McLeish, Mill- field. Scott, A. C. asst. zool. Columbia. Br. 314. Thompson, Main. Sell, J. P. grad. asst. biol. Yale. Rock 7. Ka 21. Shapiro, H. grad. biol. Princeton. Br 127. Edwards, School. Shaw, I. res. asst. biol. Long Island. Br 126. Shoup, C. S. asst. prof. biol. Vanderbilt. Br 315. D 301B. Sichel, F. J. M. asst. instr. biol. New York. Br 339. Dr. Slifer, Eleanor H. res. assoc. zool. Iowa. Br 217A. Kittila, Bar Neck. Sonneborn, T. M. res. assoc. zool. Hopkins. Br 336. D 204. Specht, H. grad. phys. Hopkins. OM Base. Dr 6. Speicher, B. R. grad. asst. zool. Pittsburgh. Rock 3. K 15. Speidel, C. C. prof. anat. Virginia. Br 106. D 104. Spek, J. prof. zool. Heidelberg (Germany). Br 223. D 316. Stabler, R. M. instr. zool. Pennsylvania. OM 22. D 210. Starkey, W. F. grad. zool. Pittsburgh. Rock 7. Ka 21. Stewart, Dorothy R. asst. prof. biol. Skidmore. Br. 222, Stokey, Gardiner. Stockard, C. R. prof. anat. Cornell Med. Br 317. Buzzards Bay. ‘THE COLLECTING NET # Strong, O. S. prof. neur. and neuro-hist. Columbia. Br 8. Elliot, Center. Stuart, Martha S. grad. genetics. Pennsylvania Col. for Women. Rock 3. W A. Summers, F. M. tutor biol. City N. Y. Br 2171. A 104. Sumwalt, Margaret asst. instr. phys. Pennsylvania. OM 3. W G. Tashiro, S. prof. biochem. Cincinnati. Br 341. Park. Taylor, G. W. Nat. Res. fel. phys. Princeton, Br 110. Cowey, School. Taylor, W. R. prof. bot. Michigan. Bot 24. Whit- man. Wade, Lucille W. grad. biol. Hopkins Sch. Hygiene. Br 319. W. I. Walker, P. A. fel. Water. Wallace, Edith M. scientific artist. Calif. Inst. Tech. Br 320. Main. Waterman, A. J. instr. biol. Brooklyn. OM 39. D. Weisman, M. N. tutor biol. City N. Y. Br 217J. McLeish, Millfield. Whiting, Anna R. prof. biol. Pennsylvania Col. for Women. Rock 3. Whitman. Whiting, P. W. prof. zool. Pittsburgh. Rock 3. Whitman. Wieman, H. L. prof. zool. Cincinnati. Br 334. D 311. Willey, C. H. asst. prof. biol. New York. Br 232. A 301. Wilson, E. B. DaCosta prof. emeritus zool. Colum- bia. Br 322. Buzzards Bay. Wilson, Hildegard, N. asst. biochem. Univ. & Belle- vue Hosp. Med. Br 310. Buzzards Bay. Winsor, C. P. grad. phys. Harvard. L 21. (Catau- met). Wolf, E. A. assoc. prof. zool. Pittsburgh. Rock. 7. Elliot, Center. Woodruff, L. L. prof. proto. Yale. Br 323. Gansett. Young, Roger A. asst. prof. zool. Howard. Br 315. Harvard. Br 312. Thompson, A 304. Young, W. C. asst. prof. biol. Brown. OM 34. Kit- tila, North. Zirkle, C. assoc. prof. bot. Pennsylvania. Bot 6. A 101. Zujko, A. J. asst. biol. Trinity (Connecticut) OM 26. Ka 23. STUDENTS Albaum, H. G. fel. biol. Brooklyn. emb. Dr 1. Alt, H. L. assoc. med. Northwestern Med. phys. D 217. Amidon, Elaine W. Syracuse. bot. H8. Armack, C. M. curator biol. Mus. Northern Arizo- na. emb. Crowell, Water. Bates, M. N. grad. asst. zool. Oberlin. emb. Dr 2.. Bechtel, W. R. instr. biol. Edinburg High Sch. (Ohio) proto. Bosworth, North. Bell, Ruth grad. asst. zool. Wellesley. emb. W B. Bengel, W. Z. asst. anat. & emb. DePauw. emb. Ka 2 e Bosworth, M. W. asst. biol. Wesleyan. bot. K 6. Botsford, E. Frances asst. prof. zool. Connecticut. phys. Stokey, Gardiner. Boyer, D. C. grad. biol. Columbia. proto. Hilton, Millfield. Campbell, Mildred F. teach. bot. Shortridge High Sch. (Indianapolis) bot. Hall, Main. Chen, Y. grad. zool. Pennsylvania. emb. Elliot, Cepter. 42 THE COLLECTING NET [ Vor. VIL. No. 62 Churney, L. grad. zool. Pennsylvania. emb. Ka 2. Cunniff, Hilda S. bot. H 9. Dennis, Nova N. teach. bicl. Portage County High Sch. (Ohio) proto. Clark, Millfield. Derrickson, Mary B. phys. W D. DeWolf, R. A. instr. zool. Rhode Island State. emb. (Hyannis. ) Foster, Edith F. Vassar. emb. Bosworth, North. Glassmeyer, E. J. grad. biochem. Cincinnati. phys. D 109. Godwin, M. C. asst. hist. & emb. Cornell. emb. K 7. Greco, F. M. Hunter. emb. Kittila, Bar Neck. Hamilton, Mary A. Elmira. emb. H 7. Havey, C. B. Acadia (Canada) phys. Dr 1. Hibbard, Jeanne Oberlin. phys. K 12. Hirschfield, N. B. Univ. and Bellevue Hosp. Med. proto. McLeish, Millfield. Hooper, Kathryn T. Wheaton. emb. Young, West. Howell, C. D. grad. biol. Hopkins. phys. Dr. Johnson, Edna L. assoc. prof. biol. Colorado. phys. A 305. Kagan, B. M. Washington & Jefferson. emb. K 6. Kriete, F. M. DePauw. emb. K 9. Lippman, R. W. Yale. emb. Hilton, Main. McAuley, A. A. DePauw. emb. K 9. McGehee, Elise grad. Newcomb. emb. Oak, Park. MacIntosh, F. C. dem. pharm. Dalhousie (Canada) phys. Thompson, Water. Mathews, R. S. Physicians & Surg., Columbia. phys. Dr 6. Melampy, R. M. asst. anim. nutrition Cornell. phys. Dr 10. Moreland, F. B. grad. res. asst. biochem. Vanderbilt Med. Phys. Dr 5. Moser, F. grad. zool. Pennsylvania. emb. D 214. Perkins, Irene T. grad. biol. Columbia. proto. A 204. Poris, Ethel Hunter. bot. Kittila, Bar Neck. Ramey, Sally Elmira. bot. Bosworth, North. Reot, Charlotte M. Ohio Wesleyan. emb. H 6. Rose, S. M. grad. asst. biol. Amherst. emb. K. Ross, E. grad. physico-chem. biol. California. phys. Ka 2. Rubidge, Karyl W. Vassar. emb. Bosworth, North. Solandt. D. Y. res. fel. phys. Toronto (Canada) phys. D 107. Solandt, O. M. res. asst. phys. Toronto (Canada) phys. D 107. Spangler, Betty A. Wheaton. emb. Young, West. Stricker, G. J. Yale. phys. Neal, West. Stubbs, T. H. instr. biol. & chem. Emory. proto. Sylvia, Buzzards Bay. Summers, F. M. grad. proto. Columbia. Br 217. A 104. Sweadner, W. R. grad. asst. Pittsburgh. Rock 3. Dr 8. Taylor, H. C. grad. asst. biol. Wesleyan. emb. K. Tukey, Gertrude R. Smith. emb. H 7. Turner, R. 'S. instr. biol. Dartmouth. emb. K. Urban, J. instr. biol. Randolph High Sch. (Ohio) proto. Bosworth, North. Vexler, D. E. grad. phys. Rutgers. phys. Ka 22. Ward, Mary Wellesley. proto. Grinnell, West. Wardwell, Judith S. grad. asst. zool. Wellesley. emb. W B. Webster, M. Dorothy grad. bot. Dalhousie. (Cana- da) phys. Young, West. Young, M. I. instr. biol. Junior Col. Augusta. protc Sylvia, Buzzards Bay. Zinn, D. J. Harvard. emb. D 108. ADMINISTRATION OFFICE Billings, Edith secretary. Millfield. Crowell, Polly L. asst. to bus. mgr. Main. Dillinger, Bessie R. secretary. Br 104 b. K 8. Karr, Dorothea secretary. W E. MacNaught, F. M. bus. mgr. School. LIBRARY. Endrejat, Doris assistant. W H. Lawrence, Deborah secretary. Locust (Falmouth). Montgomery, Pricilla B. librarian. Whitman. Rohan, Mary A. assistant. Millfield. SPECIAL APPARATUS AND TECHNICAL SERVICE Adams, E. M. grad. biol. Cincinnati. Chem. D 2. Apgar, A. R. Br 211. photographer. D 105. Boss, L. F. res. tech. Middle. Caliahan, J. Janitor. Ka. 3. Chute, A. L. Toronto Med. (Canada) asst. Ka. 3. Cornish, G. janitor. Br 1st floor. Dr 4. Densmore, S. gardener. School. Frew, Pauline. Bates. Chem. W. F. Googins, H. janitor. Quisset. Graham, J. D. glass-blowing. Veeder, Millfield. Hemenway, W. carpenter. Carpenter Shop. Haw- thorne. Johlin, Sally Wellesley. Chem. Gardiner. Kahler, R. MBL asst. Br 7. Glendon. Keil, Elsa instr. zool. N. J. Col. Women. Chem. W D. Keltch, R. janitor. Br 3rd floor. Millfield. Larkin, T. superintendent. Br 7. Woods Hole. Laug, E. P. instr. phys. Pennsylvania. Chem. D 302. Liijestrand, P. H. Ohio Wesleyan. asst. Dr 3. Liljestrand, R. S. Cazenovia Sem. night watch. Ka 4. McInnis, F. M. janitor. Bot & L. Millfield. McManus, J. janitor. Br 2nd floor. Ka 3. Mast, Louise grad. biol. Hopkins. Minot. Meier, O. Pennsylvania. night eng. Dr 15. Pond, S. E. asst. prof. phys. Pennsylvania. mgr. Queens (Falmouth). Richards, O. W. instr. biol. Yale. Chem. In charge. A 303. Sander, M. Pennsylvania. res. tech. Dr 14. Strong, O. S. prof. neur. & neuro-hist. emeritus. Elliot. Center. Swain, G. R. janitor. Br 3rd floor. Main (Quisset). Tawell, T. E. head janitor. Br Base. Thompson, Water. Tupper, Mary C. Swarthmore. Chem. W H. tech. Chemist SUPPLY DEPARTMENT Clarkson, W. collector. Water. Crowell, Ruth S. secretary. Main. Crowell, P. S. Harvard. collector. School. Erianger, H. Wisconsin. collector. Dr 3. Gray, G. M. Curator res. mus. Buzzards Bay. Gray, M. collector. (Teaticket). Hanau, R. collector. Dr 3. Hilton, A. M. collector. Millfield. Leathers, A. W. head shipper. Minot. Lehy, J. collector. Millfield. McInnis, J. resident mgr. Millfield. Neilsen, Anna M. secretary. Millfield. Pratt, M. collector. Dr 3. Sither, J. A. Wabash. collector. Dr. Smith, C. B. Syracuse. collector. Supply Dept. Thornley, W. Harvard. collector. Supply Dept. Wamsley, F. W. supervisor schools, Charleston. spec. preparator. Supply Dept. Jury 8, 1933 ] WOODS HOLE OCEANOGRAPHIC INSTITUTION INVESTIGATORS Beach, E. F. Brown. 109. Hilton, Water. Bigelow, H. B. prof. zool. Curator Oceanography. Harvard. 114. Luscombe, Main. Braarud, T. assoc. phytoplanktonol. Internat. Pass- amaquoddy Fish. Comm. 311. F. Brill, E. R. Harvard. 109. Neal, West. Bruce, W. F. Rockefeller Inst. 201. ‘Asterias.” Buck, W. B. Princeton. ‘‘Asterias.” Carey, Cornelia L. asst. prof. bot. Quisset. Church, P. E. res. asst. 315. Wilde, Gardiner. Clarke, G. L. instr. gen. phys. Harvard. 108. Mitchell, Barnard. 202. Orchard. Emmons, G. Mass. Inst. Tech. 208. Fish, C. J. exec. sec. Internat. Passamaquoddy Fish. Comm. 310. Buzzards Bay. Green, Arda A. res. fel. Fatigue Lab. Harvard. 101. D 202. Greenwood, T. S. tech. W. H. O. I. “Atlantis.” Hotchkiss, Margaret instr. bact. N. Y. Hom. Med. Col. & Flower Hosp. Wilde, Gardiner. Ingalls, Elizabeth N. res. asst. Harvard. 103. Young, West. Iselin, C. O. asst. curator oceanography. Harvard, 206. (Falmouth). Keys, A. A. Nat. Res. fel. phys. Cambridge (Eng- land) 106. Krogh, A. prof. zoophys. Lab. Zoophys. Copenhagen (Denmark). 105. D 301. Leavitt, B. B. 301*. Lichtblau, S. 209. Young, West. Mahncka, H. grad. Brown. 109. Oster, R. H. Harvard. 106. Rakestraw, N. W. assoc. prof. chem. Brown. 109. Redfield, A. C. prof. phys. Harvard. 103. Millfield. Redfield, J. H. 107. Breakwater. Bar Neck. Renn, C. E. asst. microbiol. Rutgers. 201. Young, Middle. Reuszer, H. W. instr. soil microbiol. Rutgers. 201. Young, Middle. Root, R. W. instr. biol. School. Rossby, C. G. meteorolgist. Mass. Inst. Tech. 207. Schalk, M. grad geol. Harvard. 211. Stewart, School. Sears, Mary. 301. Hilton, Water. Smith. grad. physical chem. Brown. 109. Stetson, H. C. asst. curator palaeon. Harvard. 212. (Falmouth. ) ‘ Taylor, Carola W. res. asst. geol. Radcliffe. 212., Cowey, Schcol. Turner, A. “Atlantis.” Waksman, S. A. prof. soil microbiol. Rutgers. 202. (Penzance. ) , Watson, E. E. hydrographer, Internat. maquoddy Fish. Comm. 310. Lakeview. Wheeler, C. Harvard. 108. Hatchfield. Wilson, C. B. 111. Clough, Millfield. Woodcock, A. tech. W. H. O. I. 207. Peck, Woods Hole. i City N. Y. 101. Cowey, Passa- *Further information refused. Enquire at Room 301. THE COLLECTING NET 43 OFFICE Schroeder, W. C. business manager. 118. W.H.O.I. Walker, Virginia B. secy; asst. bus. 112. Howe, Millfield. mer. BUILDINGS AND GROUNDS Condon, J. W. janitor. Millfield. Eldridge, S. N. carpenter. Woods Hole. Schroeder, W. superintendent. W. H. O. I. Sylvia, A. F. gardener. Millfield. “ATLANTIS” Bachus, H. ‘‘Atlantis’’. Carlson, ‘‘Atlantis’’. Cavalone, cook ‘‘Atlantis’’. Condon, J. G. ‘Atlantis’. Costa, W. mess boy “Atlantis”. Dwyer, E. mess boy ‘Atlantis’ Kehoe, T. capt. ‘Asterias’’. Kelly, T. 1st mate ‘‘Atlantis”’. Lindstrom, J. ‘“Atlantis’’. McLunin, “Atlantis”. McMurray, F. capt. ‘Atlantis’. Olsen, B. ‘Atlantis’. Potter, D. 2nd mate “Atlantis”. Turner, D. “Atlantis’’. U.S. BUREAU OF FISHERIES Bigelow, R. P. prof. zool. Mass. Inst. Tech. Center. Galtsoff, P. S. biol. U. S. B. F. (Washington) 122. F. Goffin, R. collector. U. S. B. F. 115. Millfield. Linton, E. fel. paras. Pennsylvania. M 5 West. Sette, O. E. director U. S. B. F. (W. H.) 118. F. Smith, R. O. asst. aquatic biol. U.S. B. F. M 124. F. SCIENTIFIC STAFF Bigelow, R. P. prof. zool. Mass. Inst. Tech. Center. Galtsoff, Eugenia assoc. zool. George Washington 122. F. Galtsoff, Paul S. biol. U. S. B. F. (Washington) 122. F. Goffin, R. collector. U. S. B. F. 115. Millfield. Linton, E. fel. paras. Pennsylvania M5. West. Sette, O. E. director U. S. B. F. 118. F. Smith, R. O. asst. aquatic biol. U.S. B. F. F. BUILDINGS AND GROUNDS Brown, G. guide. Hatchery. Conklin, Paul, fireman. Hatchery. Hoftses, G. R. superintendent 117. F. Howes, E. S. coxswain. Millfield. Howes, W. L. fish culturist. Millfield. Kristtan, M. apprentice fish culturist. Hatchery. Lowey, J. engineer. Glendon Road. Radel, A. H. apprentice fish culturist. Hatchery. Sanderson, A. apprentice fish culturist. Hatchery. Van Amberg, L. fireman. Hatchery. Webster, H. fireman. Hatchery. 44 < ies THE ‘COLLECTING NET sf Vor. VINEENG: 62 The A. B. C. of Woods Hole for 1933 All Schedules Set to Daylight Saving Time — Bold Type Indicates P. M. _ TRAIN SCHEDULE WOODS HOLE TO BOSTON Daily Daily Daily Sunday Sunday Woods Hole rhea Ws} 9:40 5:40 6:10 8:10 Falmouth M2 9:47 5:47 6:17 8:17 Boston 9:10 11:52 7:52 8:15 10:22 BOSTON TO WOODS HOLE ion Daily Daily Daily Sat. only and Sun. Boston Bes 1:30 4:47 1:03 4:03 Falmouth 10:32 3:35 6:48 3:00 6:02 Woods Hole 10:40 3:42 6:55 3:06 : 6:09 SEAPLANE SCHEDULE Eastbound Trip 1; Trip 3 Trip 5 Trips Trip 9 Leave New Bedford 7:00 10:30 12:45 3:30 5:55 Leave Woods Hole flag 10:50 flag 3:50 6:15 Leave Vineyard Haven 7:30 11:07 1:15 4:07 6:30 Arrive Nantucket 7:50 Ws 2 77; 1:35 4:27 6:50 Westbound No. 2+ No. 4 No. 6 No.8 No. 10* Leave Nantucket 8:50 10237 2:30 4:35 6:55 Leave Vineyard Haven 9:20 12:07 3:00 5:05 7:20 Leave Woods Hole 9:37 12:24 3:17 5:22 aor Arrive New Bedford 9:47 12:34 3:27 5:32 7:35 * Trip 10 runs Tues., Thurs., Sundays only. 7 Trips 1 and 2 do not run Sundays. BOAT SCHEDULE For New Bedford, Woods Hole, Oak Bluffs, Vineyard Haven and Nantucket Leave Daily Daily Daily Daily New Bedford 7:00 9:30 2:30 7:45 Woods Hole 8:20 10:50 4:00 9:00 Oak Bluffs 9:10 11:40 4:45 sets Vineyard Haven 9:30 12:00 5:00 9:45 Nantucket 11:30 2:00 7:15 aA Leave Daily Daily Ex. Sun. Sun. Daily Nantucket cee 6:30 2:30 3:00 5:00 Vineyard Haven 6:10 8:15 4:00 4:30 6:55 Oak Bluffs a Paens 9:00 4:30 5:00 7:15 Woods Hole 6:55 9:45 5:20 5:50 8:00 New Bedford 8:15 MBI 5; 6:45 7:30 9:25 eal Juty 8, 1933 | THE COLLECTING NET 47 CONTROL OF THE CAPE COD MOSQUITO The Cape Cod Mosquito Control Project, spon- sored by the Cape Cod Chamber of Commerce and financed largely by public subscription, has been in operation since May, 1930. It is now one of several projects under the State Reclamation 3oard. The work consisted chiefly of the elimina- tion of salt marsh breeding places by drainage and sometimes by dyking and filling. A relatively small amount of work has been done at fresh water breeding places, due to the fact that the salt marsh species of mosquito is much more numer- ous. In fact a survey of the mosquito situation made by R. W. Wales (Entomologist) and Per- cival M. Churchill (Consulting Engineer) for the State Reclamation Board showed that 90 to 95% of all Cape Cod mosquitoes were of species breed- ing in salt marshes. In addition to the type of work already men- tioned, a truck equipped for spraying oil has been in use since the season of 1931. This truck is used chiefly to combat the house mosquito which breeds in all sorts of places containing water near dwellings, and to oil salt marshes and other breed- ing places when conditions warrant it. It is now three years since the project went into operation and the progress made in combating mosquitoes can be measured fairly accurately by comparing the abundance of salt marsh mosqui- toes with the fresh water ones. From an esti- mated 90 to 95% in 1929, the salt marsh mosqui- toes have beeri reduced to a fraction of the num- ber of fresh water mosquitoes. At the present time there are large areas of the Cape where only fresh water species can be found. Formerly the salt marsh species covered every part of the Cape and sometimes cattle, horses and even people would be literally covered with hundreds of mos- quitoes. We no longer hear of hotels being closed and people being driven from the golf courses and beaches. With the salt marsh mosquitoes nearly under control the project will be able to turn its atten- tion more fully to the fresh water species. Un- fortunately these mosquitoes often breed in places where control measures are very difficult and ex- pensive. There is also the difficulty caused by the need of flowing water in cranberry bogs in the spring. Mosquitoes hatch in the bogs and in the adjacent flooded areas during the month of May. However, in spite of adverse conditions, if the most available breeding places are eliminated and some of the more important of the others con- trolled mosquitoes will be very few indeed. At present the house mosquito is one of the worst fresh water species. Its presence is mainly due to the carelessness of the people; in fact the pest is very rarely found away from human_ habitation. This mosquito breeds in water in barrels, tubs, cans and in all sorts of artificial containers. It is also found in flooded cellars, open cisterns, cess- pools, catchbasins and even in harmless pools if garbage or rubbish is thrown in. If people would be careful the Project would need only to oil the catch basins. The final degree of relief from mosquitoes will depend on how many of the fresh water breeding places can be eliminated, and this bears a direct relation to the amount of money available. At present the funds are rather limited. A small tax collected by the State provides for the up keep and maintenance of the ditching and oiling after the original work is done. The Commissioners of the Cape Cod Mosquito Control Project are: O. C. Nickerson, of Chat- ham; F. W. Norris, of Oyster Harbor; L. C. Weeks of Falmouth. —Cape Cod Mosquito Control Project. Important Mecting of the M. B. L. The annual meeting of the members of the M. B. L. Club will be held in the Clubhouse on Monday evening, July 10. All members are urg- ently requested to attend since officers are to be elected and important policies will be decided upon. A trip to New York in a fourteen-foot outboard motor boat was undertaken last fall by Victor Schechter and his brother. The trip took 414 days and stops were made at Palmer Island Light, Point Judith, Saybrook Point and Tavern Island. The navigators attended a breeches bouy drill at Point Judith and were escorted across Newport Harbor by a school of porpoises. The, Woods Hole fire station is inspected every fifth day by the Chief and Deputy Chief of the Falmouth Fire Department. The annual General inspection of all equipment and personnel took place on June 1. A Food Sale was held by the M. E. Woman’s League on Friday June 23rd from 3 to 5 P. M. in the M. E. church. The affair was a decided financial, social and gustatorial success. Mr. Gardner Handy, who tormerly druvé the bus for Mr. B. K. Nickerson is now working for Penzance Garage. 48 THE COLLECTING NET [ Vor. VIII. No. 62 THE SUPPLY DEPARTMENT OF THE MARINE BIOLOGICAL LABORATORY By the Staff of the Supply Department One of the main duties of the Supply Depart- ment during the summer season is to give the investigators and students the very best possible service. The available materials will be collected and delivered to all those who request them. Orders for material to be delivered the following day will be taken between 10:00 A. M. and noon-time. If the investigator who does not expect to be in his room between those hours will leave a notation of what he desires, it will greatly facilitate the service. This may be done by placing a slip on the door; then he may be sure that the boy will take it and the material be delivered. If there are any complaints about the material or service, it would be greatly appreciated if they were entered in the Supply Department office, in- stead of being given to the delivery boy or to a member of the crew. This department is maintained at a very great expense during the summer months. During the winter months, the Supply Department is main- tained as a Supply House, where students and teachers may order their needs for their class work. The all-year-round personnel is made up of six collectors, and in the summer this number is increased by eight additional collectors on the crew. Two people are on duty at the office at all times, and they will gladly give any information or adjust any complaints which may be entered. Few teachers realize the expense that is in- volved in the collecting and preparing of marine animals. Many, we are certain, believe that it is only necessary to walk along the beach, pick up the specimens and put them into formaldehyde. Nothing could be farther from the truth. The entire collecting region must be carefully explored RETRENCHMENT AT THE U. Governmental economy has hit the Bureau of Iisheries hard this year, and has resulted in a de- cided curtailment of services of the Woods Hole station. It is no longer possible to accommodate the twenty or thirty university investigators who formerly made use of the facilities, and therefore only two investigators, with their assistants, are occupying the laboratories and residence of the local station. Dr. Paul Galtsoff is continuing his work on oysters and Director Oscar Sette is in charge of the mackerel investigation. Both Fisheries boats, the Albatross IT and Phalarope IT are in tempor- ary retirement and are tied up at the wharf. The old Phalarope, in the Eel Pond, long a landmark —or should we say seamark—of Woods Hole, in order to find sources for the various forms, and at times it is necessary to take long trips to secure them. To do this exploring and collecting, boats costing several thousand dollars must be employed. These must be provided with pumps, so that the specimens may be kept in running sea-water while they are on board. Then, when they are brought to the laboratory, many of them must be put through long and complicated proces- ses to be properly narcotized, expanded and pre- served. The pumps and tanks needed to supply the laboratory with running sea-water are very expensive, and far beyond the means of any in- dividual who may be trying to collect without equipment. The Supply Department has a biology cata- logue, which will be given out upon request, and which lists the complete stock of preserved and living material. This may be obtained at the office. The prices of materials have been greatly reduced, and special attention is being called to the grading of the sizes in materials which have been arranged for the convenience of the customers. Should es- timates be desired, we will gladly give same, and all orders will have our careful attention. Our Department is, without doubt, the best equipped marine collecting station in the United States, if not in the world. Its collecting equip- ment, consisting of boats, fish traps, seines, dredges, tangles and laboratory facilities, are of the very best, and represent a great investment. Its staff of collectors and preparators has had many years of experience. It is these advantages in the collection and preparation of marine specimens which explain, to a great extent, the uniformly good quality of the preserved material furnished by the Supply Department. S. BUREAU OF FISHERIES has been sold, and has temporarily joined the army of the unemployed. The aquarium and other public exhibits are open only temporarily. Plans for next year are uncertain. During the winter, the exhibits were enlarged by the gift of the Vinal Edwards collection of stuffed birds from Woods Hole and vicinity. Dr. Fedwards was formerly director of the Bureau sta- tion at Woods Hole, and his heirs presented the collection to the station last year. It might be noted that all other permanent exhibits in the aquarium building were obtained during Dr. Ed- wards’ long regime as director, either as a result of his personal efforts or during the Biological Survey of the region, carried on independently from 1904 to 1911, Jury 8, 1933 } THE COLLECTING NET 49 LECTURES AND SYMPOSIA AT COLD SPRING HARBOR We have received from the Long Island Biological Laboratory the following an- nouncement of the lectures and symposia on “The Potential Difference at Interfaces and Its Bearing Upon Biological Phenomena” at Cold Spring Harbor: ~ SaturDAy, Juty 1: Hans Muller; The theory of the diffuse double-layer. Monpay, Juty 3: Hans Muller; The theory of cataphoretic migration. D. R. Briggs; Streaming potential measurements. Kenneth S. Cole; Sur- face conductance. WEDNESDAY, JULY 5: SyMPpostuM; OxIDA- TION-REDUCTION PorentTIALs. D. A. MacInnes; Meaning and calibration of the pH scale. L. Michaelis; The reversible two-step oxidation. Barnett Cohen; Reversible oxidation-reduction potentials in dye systems and their use in the ex- amination of cells and cell suspension. I. Wepnesbay, JuLty 12: Symposium; BIoELEc- TRICAL PHENOMENA. Hans Muller; The theory of ionic adsorption. W. J. V. Osterhout; Buio- electrical phenomena in large cells. Kenneth S. Cole; Electric excitation in nerve. Herbert S. Gasser; Axon action potentials in nerve. Wepnespay, JuLy 19: SympostuM; ELECTRI- CAL PROPERTIES OF SURFACES IN RELATION TO THE COAGULATION PRocess. Hans Muller; Sta- bility of colloids and the theory of rapid coagula- tion. Harold Abramson; The chemical constitu- tion of amphoteric surfaces. (Amino-acids; pro- teins). Stuart Mudd; Agglutination. Monpay, Juty 24: SyMPostumM; OSMOTIC PHENOMENA. D. R. Briggs; Electrosmosis and anomolous osmotic pressures. W. J. V. Oster- hout; Diffusion and osmosis in cells and models. Eric Ponder; Osmotic behavior of red cells. I. Robert Chambers; Intracellular oxidation-reduc- tion potentials. WeEDNESDAY, JULY 26: Symposium; ELEcrri- CAL PROPERTIES OF THE RED CorpuscLe. Hugo Fricke; Electric capacitance and conductance of red blood cells with an application to the study of hemolysis. Harold Abramson; The electrical charge of the blood cells of the horse and its rela- tion to the inflammatory process. D. D. Van Slyke; Factors controlling the electrolyte and water distribution in the blood. Eric Ponder ; Osmotic behavior of red cells. II. Fripay, Juty 28: Kenneth S. Cole; Electrical conductance of biological material. Barnett Cohen; Reversible oxidation-reduction potentials in dye systems and their use in the examination of cells and cell suspension. Il. Stuart Mudd; Pha- gocytosis. NOTES ON THE WORK OF THE M. B. L. CLUB Nearly two hundred people attended the “mixer” and dance last Saturday evening. A four- piece orchestra began playing at ten-thirty, and the floor was over-crowded with couples until much later in the evening when some of them went home. The refreshment part of the pro- gram was in charge of Miss Margaret Mast who had punch served to the thirsty group. Any worker at the three scientific institutions at Woods Hole is cordially invited to attend the Saturday-night dances. No charge will be made to them providing they are members of the Club. They may bring guests who are not connected with the laboratories, but guests of members will be classified in the “non-member” group and be subject to a charge of fifty cents. Thus anyone who has not contributed directly toward the sup- port of the Club will be expected to share in the expense of providing an orchestra. The weekly victrola-record concerts are ex- pected to begin next Wednesday. Notices sug- gesting that people loan records have been posted at the Laboratory and at various other points in the village. In order to eliminate the destruction of club property and the missuse of its facilities (which has been brought about almost entirely by non- members) Mrs. Morris has organized a number of investigators to serve as “host and hostess” during the evenings in July and August. Those which she has appointed for the coming week are : Monday, July 1O—Mr. and Mrs. Ware Cattell. Tuesday, July 11—Dr. and Mrs. Edwin Linton. Wednesday, July 12—Mr. and Mrs. Samuel Morris. Thursday, July 13—Dr. and Mrs. Moyer S. Fleisher. Friday, July 14—Dr. and Mrs. G. B. Jenkins. Saturday, July 15—Dr. and Mrs. R. M. Stab- ler. Sunday, July 16—Dr.and Mrs. Samuel Schoop. The primary duty of these individuals will be to make people feel at home in the Club, and to help the members meet each other. The next expansion of the activities of the M. B. L. Club is to be the acquistion of a ping- pong table. It will be the source of much amuse- ment, especially during evenings when the weather is poor. A ping-pong tournament is scheduled for later in the summer. 50 . THE COLLECTING NET [ Vou. VIII. No. 62 1 EAMES aoe FREDERICK L. GATES The sudden death of Dr. Frederick L. Gates, a worker at the Marine Biological Laboratory, shocked Woods Hole residents in June, especially those associated with him and his scientific work. A fall in his laboratory at Harvard University resulted in a skull fracture, and Dr. Gates died in 3oston on June 17. He was forty-six years of age. Dr. Gates was born in Minneapolis in 1886, and after completing a college course at Yale and tak- ing a medical degree at Johns Hopkins, he em- barked upon a career of biological research at the Rockefeller Institute, which his father had helped induce Mr. Rockefeller to endow. He re- mained at the Institute for seventeen years, be- coming an associate member before he accepted the lectureship in physiology at Harvard three years ago. At the time of his death Dr. Gates was investi- gating the effects of ultra-violet light of different wave lengths on living tissues of various kinds. Fle worked at the Rockefeller Institute with Dr. Peter Olitsky on several types of bacteria, with a view to discovering the causes of common colds and influenza. Dr. Gates’ home here was on Nobska Road. His family left at the end of June for Minnesota where they will spend the summer. Dr. Caswell Grave has left Woods Hole for six weeks in the Tortugas. Mr. Paul Nicoll is accom- panying him as an assistant. The tunicates of the region will be the objects of their study. Dr. and Mrs. W. J.°V. Osterhout (the former Miss Marian Irwin) have taken a house at Hale- site, Huntington, Long Island, for the summer. Dr. H. B. Bigelow, director of the Woods Hole Oceanographic Institution, attended the In- ternational Council for the Exploration of the Sea in Paris from May 8-13. He is now at Woods Hole. Dr. Henry Knower has been appointed Re- search Associate in Biology at the Osborn Zoolog- ical Laboratory at Yale. Miss Lois E. Te Winkel has been appointed instructor at Smith College for the coming aca- demic year. She will give a course in embryology and will assist in the mammalian anatomy course. IN TER Eisai THE COLLECTING NET SCHOLARSHIPS Dr. H. B. Goodrich, professor of biology at Wesleyan University and director of the course in embryology at the Marine Biological Laboratory, recently made the following statement concerning the value of THe CoLtectinG Net Scholarships: “In my opinion the CoLttectinG Net scholar- ships perform an exceedingly useful service. The students assembled at Woods Hole are carefully selected and are of unusual ability. Candidates from this group are certain to merit aid. The period of graduate study is often the most diffi- cult to finance in the student’s career. A scholar- ship of $100.00 will permit a careful student to pay board or room for ten weeks, or it may in part be applied to laboratory or tuition fees. It is undesirable for many to defray expenses by wait- ing on table at the ‘mess’ as this often demands four to five hours a day—or more than can be spared from their working time and energy. The purpose of the scholarships seems to me to be in every way admirable.” (Signed) H. B. Goopricu. Miss Elizabeth T. Kinney and Dr. Leonard Worley were married at South Hadley, Mass., on June 17th. Dr. and Mrs. Worley are motoring to Nebraska for their wedding trip. A daughter was born to Dr. O. E. Sette, direc- tor of the laboratory, U. S. Bureau of Fisheries, and Mrs. Sette during the month of March. New visitors and old residents of Woods Hole will be glad to hear that the Tennis Club has started its yearly activities. The courts are all in good playing order, and it is urged that everyone take note of the extensive repairs which have been made on backstops, nets and playing surfaces. The membership fee for this season is $4.00. Junior membership (for all those under sixteen) is $2.00. All dues are payable to Dr. Phil B. Armstrong. A North Carolina blacksnake in Dr. R. M. Stabler’s laboratory is in the process of laying eggs as the Log goes to press. The eggs have soft shells, and are white, a little over an inch long and a quarter of an inch high, much more flattened than hens’ eggs. Dr. Stabler expects to hatch the eggs and add seven more blacksnakes to his laboratory collection, which includes the blacksnake, a garter snake, and a six-foot gopher- snake from Florida, A rattler may arrive later this summer. : Annual Subscription, $2.00 Vol. VIII, No. 3 SATURDAY, JULY 15, 1933 Single Copies, 25 Cents. TRANSMISSION OF NEUROHUMORAL THE BERMUDA BIOLOGICAL STATION SUBSTANCES FOR RESEARCH Dr. G, H. PARKER ProFEssoR Epwin G. CONKLIN Professor of Zoology, Harvard University President of the Corporation The subject of my talk tonight is something The Bermuda Biological Station for Research, that grew out of a paper presented last summer Inc., is one of the newest of the biological stations and is a continuation of some work begun a num- as the Marine Biological Laboratory is one of the ber of years ago. It concerns oldest. In its organization the the transfer of substances in officers have had the advantage the body, the so-called neuro- of the experience of many humoral substances which are years at the Woods Hole lab- probably produced by the oratory and it is hoped that nervous system and may affect an account of the Bermuda local as well as distant parts Station may be of interest to of the body. As far as fluid ite meee of Imm Cote exchanges in the body are con- LECTING Net. The Sta- cerned, we ordinarily think tion is now located in one of simply of blood and lymph. the most beautiful sites in Ber- Blood carries food, oxygen, muda, and has thoroughly ade- and hormones and when it quate buildings and laborator- reaches the capillaries, the ies. It is the only station in the fluids pass through the walls west Atlantic that is open to and in this way the lymph receive research workers every thus formed reaches the cells. month of the year, and an in- The cells take up the nutri- creasing number of investiga- ment and these living units give tors will visit there throughout out excretory products which the winter months as well as are picked up again by the during the summer. lymph carried to the blood, and No courses of instruction MM. BR. LZ. Calendar TUESDAY, JULY 18, 8:00 P. M. Seminar: Dr. G. W. Kidder: ‘‘Chro- matin Extrusion in Certain Cili- ate Commensals of Mussels.” Dr. F..M. Summers: ‘The Reor- ganization Bands in the Macro- nucleus of Aspidisca.” Mr. H. E. Finley: ‘Comparative Studies on the Osmiophilic and Neutral-red-stainable Inclusions of the Genus Vorticella.” Mr. W. L. Doyle: ‘Experimental Cytology of Amoeba Proteus.” FRIDAY, JULY 21, 8:00 P. M. Lecture: Dr. Balduin Lucke: ‘The Zoological Distribution of Tu- mors.” Members of the M. B. L. Club (and those planning to join) are urged to come to the Clubhouse immediately after the lecture to attend the first weekly smoker. thus the circulation goes on. are offered at the Bermuda There is evidence, however, of transmission of Station, since it is exclusively a research substances from cell (Continued on Page 62) institution. Investigators in any field of TABLE OF CONTENTS Transmission of Neurohumoral Substances, Distribution of the Freshwater Algae _ of JO veg (EE CRM, IE ON aici e BANC OlGeeCRO Oeics Dera 57 Newfoundland, Dr. Wm. Randolf Taylor..... 68 The Bermuda Biological Station for Re- Diurnal Migration of Plankton in the Gulf Search, Prof. Edwin. G. Conklin... .........1.+ 57 of Maine and its Correlation with Changes Data from a Correlated Study of the Repro- in Submarine Irradiation, George L. Clarke. .69 ductive Cycle in the Female Guinea Pig, The Biological Laboratory at Cold Spring DES Wiliams Ce YOUN Fayre oo sin ehy euseneca a ened 64 ELAR BDO Mar telat hove ee ais Gael «ted stot Matera tay eens 70 Meron} Ole IWMEK CRIES lye tlunc ou Ce med nig ches cp Belted 67 Distribution and Ecology of the Marine The Role cf Bacteria in the Formation of Algae on Lake Fish, Dr. H. P. Ball......... 70 Nitrate in the Sea. Dr. Selman A. Waks- Epidermophytosis, Dr. David Cheever......... 72 man and Dr. Cornelia L. Carey............. 6S) Notesstrom ‘the Me By: Clubis ns. os esate 72 58 ASB COELECTING NET { Vor. VID. No: 63 SHORE HILLS FROM SOUTHEAST Showing Verandas and Sleeping Porches biology or oceanography are welcomed and are given all the facilities possible for carrying on their work. The regular charges for research room or table are $400 per year, $100 for three months, $25 for two weeks or less, but the president or director is authorized to remit these charges in whole or in part in special cases. It is hoped that many of the investigators at the Woods Hole laboratory may find it possible to utilize the fa- cilities offered at the Bermuda Biological Station. The history of the Station from its inception down to the beginning of the year, 1933, is con- tained in the following statements taken from the reports of the president and director, which are soon to appear in the Annual Reports of the offi- cers of the Station. The Bermuda Biological Station for Research was established in 1903 with the cooperation of the Bermuda Natural History Society, Harvard University and New York University, with Pro- fessor Edward L. Mark of Harvard as director, and Professor Charles L. Bristol of New York University as associate director. For three sum- mer sessions the work of the Station was carried on in buildings of the Hotel Frascati, near the present Government Aquarium. In 1907 the Ber- muda Natural History Society leased from the War Department Agar’s Island, converted its “magazine” into a public aquarium and _ invited Protessor Mark to continue the sessions of the Biological Station there. Summer sessions were held there from 1907 to 1930 inclusive, with the exception of the years 1917-18 when Agar’s Island was requisitioned for military purposes and the Station was transferred to an island near by. During all this time Dr. Mark served as director and in the years 1915-18 Dr. William J. Crozier was resident naturalist and the Station was kept open throughout those years. In the 28 years of the original station about 280 scien- tists were in attendance and nearly 170 articles were published as the result of work done there ; these articles have heen assembled by Dr. Mark in seven volumes of “Contributions from the Ber- muda Biological Station for Research.” Plans for the reorganization of the Station on a broader and more permanent basis had their inception at a meeting of interested persons at \Voods Hole, Massachusetts, in August, 1925. As an outcome of that meeting it was decided to fol- low in the main the plan of organization which had been so successful at the Marine Biological ory 15; 1933 ] Laboratory and also at the Plymouth Labor- atory of the Marine Biological Association of the United Kingdom, namely, the association into a Corporation of a large number of persons who were interested in the Station, and the subsequent election by this Corporation of a Board of Trus- tees to administer the affairs of the Corporation. This plan received the endorsement of the National Research Council of the United States, the Royal Society of London, the Royal Society of Edin- burgh, the Royal Society of Canada, the Biologi- cal Beard of Canada, and the Honorary Council for Research (Canada). One hundred and eighty persons joined the Corporation and a Committee on Reorganization was elected. This Committee drew up articles of incorporation and by-laws and nominated sixteen members of the Corporation to be trustees, the twelve receiving the highest number of votes to be declared elected. By the votes of more than 150 members of the Corpora- tion twelve trustees were elected. On June 28, 1926, the Bermuda Biological Station for Re- search was incorporated under the laws of the State of New York, and on April 26, 1930, the articles of incorporation were amended to permit the enlargement of the Board of Trustees to twenty-four. A more complete account of this period in the history of the Station was published in Nature, Jan. 22, 1927, and in Science, Feb. 4, 1927, Special meetings of the Trustees were held in August and October, 1926, and the first annual meeting of the Corporation and Trustees was held in New York, December 27, 1926. At that meeting offcers were elected, an Executive Com- mittee appointed and a report received from a committee of four Trustees who had visited Lermuda to select a site for a permanent station. After this committee had inspected more than twenty proposed sites they reported in favor of a tract of 12 acres in St. George’s Parish, knowa as the “Hunter Tract.” This report was approved hy the Trustees and Corporation and later a peti- tion was addressed to the Governor and Legisla- ture of Bermuda asking (1) that the Bermuda Biological Station for Research, Incorporated, be granted the privilege of holding real estate in the Islands of Bermuda; (2) that when the Trus- tees should satisfy the Governor-in-Council that not less than £50,000 endowment had been raised the Colonial Government should purchase and transfer to the Trustees the Hunter property ; (3) that all supplies and equipment imported for the purposes of the Station be exempted from cus- toms duties; (4) that an annual grant of £200 for a period of ten years be made by the Bermuda Government for the support of the Station. On June 24, 1927, “The Biological Station Act, 1927” articles. The Trustees then applied to the General Edu- cation Board of the Rockefeller Foundation for a grant to meet this conditional gift of the Bermuda Government and to provide for the development and maintenance of the Station, and on November 13, 1929 the Rockefeller Foundation appropri- ated £50,000 for the purpose. On March 29, 1930 the Hunter tract was purchased by the Govyern- meit of Bermuda and conveyed to the Trustees, and on April 4, 1930 the Rockefeller Foundation paid to the Trustees £50,000 ($243,265.63). After plans had been prepared for a labora- tory building but before construction had been started, the President and Treasurer of the Sta- tion were offered another near-by property known as the “Shore Hills Hotel and Sanitarium,” con- sisting of fourteen acres of land, a large hotel building and several smaller buildings, with pumping station, jetties, bath houses and well-kept grounds for a price which would involve a large saving as compared with the cost of developing the Hunter tract. Some leading citizens of VBer- muda suggested that the Trustees reconvey the Hunter tract to the Government on condition that the latter pay to the Trustees its purchase price of £5,500 to be used in acquiring the “Shore Hills’ property and converting it to the uses of the Station. On August 13, 1930 the Executive Committee at a meeting in Woods Hole approved this proposal, provided that the Legislature of Bermuda would arrange for the exchange, that the Rockefeller Foundation would consent to it. and that this action be approved and ratified by a majority of the entire Board of Trustees. Maps and photographs of the Shore Hills prop- erty and blue prints of the main building, show- ing the proposed alterations, together with bluc prints of the proposed new laboratory on the Hunter tract with a comparative statement of the advantages, disadvantages and estimated costs of each, were prepared by the President and sub- mitted to the Trustees, and in September 1930 they voted almost unanimously in favor of ex- changing the Hunter tract for the Shore Hills property, subject to the conditions specified by the Executive Committee, As a result of further expert examination of the Shore Hills buildings and more detailed esti- mates of the cost of repairs the Executive Com- mittee on November 29, 1930 asked the owners to reduce their price for the property. On Decem- ber 24th the purchase price was __ satisfactorily adjusted and steps were taken to secure the con- sent of the Rockefeller Foundation and of the Bermuda Government to the transfer from the Hunter tract to Shore Hills, 60 eae THE COLLECTING NET ary 3, 193i it was voted “that the Trustees ap- prove President Conklin’s action in offering $75,000 for the land and buildings of the Shore fills Hotel and an additional $5,000 for the furn- ishings, it being understood that the offer is con- ditional upon the Bermuda Government's being willing to resume ownership of the Hunter Tract and to pay £5,500 toward the purchase of the Shore Hills property.” This plan was submitted to the Rockefeller Foundation since it had paid to the Trustees of the Station £50,000 to meet the conditional grant of £5,500 by the Bermuda Goy- ernment for the purchase of the Hunter Tract, and on January 14, 1931 this exchange of prop- erty was approved by the Rockefeller Foundation. The “Biological Station Act, 1931” authorizing the payment of £5,500 to the Trustees of the Station, when the Trustees shall reconvey to the Bermuda Government the Hunter Tract, was passed by the Legislature and signed by the Goy- ernor of Bermuda on March 14th, and on March 18th the Hunter Tract was reconveyed to the Government of Bermuda and soon thereafter the sum of £5,500 was paid to our agent in Bermuda. On March 26th the President and Treasurer of the Station received the deed to the Shore Hills property, and paid to the former owners out of uninvested funds $80,000. A description of the new Station and announce- ment of the first session from June 15th to -\ugust 10th was published in the Scientific Monthly for June, 1931. A mess was opened in the main building. Dur- ing the first session twenty workers and guests were accomodated at approximately actual costs, which were $15 a week for room and board, just one-half the charge at the Grasmere Hotel for workers at the old Station on Agar’s Island. Ar- rangements have been made for accomodating workers and guests at the Station at any time throughout the year at this price when the mess includes six or more, and at $16 a week when less than six are present. Hereafter the Station will be open to workers throughout the entire year, and persons desiring to use its facilities should make application on a blank form which may be ob- tained from any of the officers or trustees. The list of workers and guests at the Station during the first session follows: Dr. T. C. Barnes, Yale; Dr. N. J. Berrill, McGill; Mrs. N. J. Ber- rill; Dr. R. E. Bowen, Harvard; Mrs. R. E. Bowen; Dr. EF. G, Conklin, Princeton; Mr. J. K. Donahue, Princeton; Miss Olive Earle, Artist, N. Y. City; Miss Jean Henderson, McGill; Dr. HH. S. Hopkins, N. Y. University; Mr. C. M. Lee, Jr., Univ. of Virginia; Dr. A. W. Lindsey, Deni- son University; Mrs. A. W. Lindsey; Mr. David = [ Vor. VIII. No. 63 Lloyd, McGill; Dr. E. L. Mark, Harvard; Dr. C. M. McFall, Univ. of Virginia; Dr. G. E. Nel- son, Col, €ity of New York; Mrs. Nelson; Dr. G. G. Scott, Col. City of New York; Dr. C. M. Yonge, Plymouth Lab., England. The Board of Trustees, at its meeting _ in Woods Hole on August 12, 1931, considered care- fully the question of the appointment of a resident director. It was felt very important that we should have a director on the ground as soon as possible, and after careful consideration of possi- ble candidates and of the present budget situation, it was decided te offer the directorship to Dr. John lrancis George Wheeler, age 32, B.Sc. and Se.D. of the University of Bristol, Student at the Marine Biological Laboratory at Plymouth. Investigator for the Ministry of Agriculture and Fisheries, Zoologist on the “Discovery” Ex- pedition 1924-’27 and 1929-'30, for the past year in charge of the scientific office of the ‘Discovery Il” in London and designated for the scientific leadership of the “Discovery Il” on her next commission. Dr. Wheeler accepted our offer and he and his wife have been in residence at the. Sta- tion since January Ist, 1932. A grant of $2,000 a year from the Woods Hole Oceanographic Institution is a very welcome addition to our income and is made as payment for the use of the Station and its facilities by members of the Woods Hole Oceanographic Institution; this use will be chiefly through the winter months when there are likely to be fewer visiting scientists at the Station than during the long vacation in summer. On November 13, 1931, the Executive Com- mittee of the Rockefeller Foundation appropriated the sum of $12,000 to the Bermuda Biological Station for Research, payable at the rate of £6,000 a year over a two-year period beginning January 1, 1932. Specific needs which can be met by the gener- ous cooperation of members of our Corporation and Board of Trustees are for microscopes, mi- crotomes, physiological and chemical apparatus, hooks, journals and reprints. Almost anything of this sort would be welcomed if in usuable condi- tion. The year 1932 began with the formal opening of the Station and the public induction of Dr. \Vheeler into the office of Director on January 6th. His Excellency, the Governor of Bermuda, pre- sided on this occasion and his presence as well as that of members of his family and his Official Staff lent especial dignity to the occasion. Six members of our Board of Trustees and about five hundred invited guests were present, and ad- dresses were made by His Excellency, the Goy- ernor, and by Mr. F. G. Gosling, Dr. Mark, Dr. Jury 15, 1933 ] Conklin and Dr. Wheeler. An account of these exercises was published in Science for January 29, 1932, and reprints were sent to members of the Corporation and Trustees, as well as to many others interested in the Station. The continued interest and support of the Goy- ernment of Bermuda is gratefully acknowledged ; its annual grant of £200 has been continued and free entry has been given for all apparatus and equipment, pilotage and port charges have been remitted for the research ship “Atlantis” of the Woods Hole Oceanographic Institution as well as for any other ship engaged in scientific work ex- clusively ; in many other respects officers of the Government, notably the Director of Agriculture, the Director of Public Works, the Superintendent of the Bermuda Aquarium, and other citizens of Bermuda have been very helpful in promoting the interests of the Station. The U. S. Coast and Geodetic Survey has don- ated to the Station a Standard Automatic Tide Gauge, with the understanding that it be suitably installed, together with certain bench marks, and that when in operation the tide roll be changed after each calendar month of record and __ for- warded to the Survey, where it would be tabulated and photostat copies would be furnished to our Station, to the Meteorological Station recently established at St. George’s, and to such others as we may designate. Our best thanks are exxtended to these governmental agencies for their coopera- tion. In view of the economic depression throughout the world n6 serious attempt has been made to increase our endowment or our regular income from cooperating institutions, although this must he undertaken at the earliest appropriate time. There has been no default of interest on our in- vestments and this income, together with the emergency grant of $6,000 from the Rockefeller Foundation and the grant of $2,000 from the Woods Hole Oceanographic Institution have en- abled us to close the year with a safe operating balance to our credit. During the past year five institutions have sub- scribed to the support of one or more research tables at the Station, viz., Harvard, Yale, Prince- ton, The Academy of Natural Sciences of Phila- delphia, and the New York Zooligical Society. Several other institutions have agreed to support tables but have not done so as yet. It is highly desirable that we should enlarge the list of co- operating institutions that make annual grants for the support of tables, whether they send repre- sentatives or not. In consideration of such contin- uing support the Station might well agree to ac- cept more than one representative from each of the cooperating institutions, as long as our facili- THE COLLECTING NET 6 ties permit. We gratefully acknowledge the re- ceipt of $500 from Mrs. J. J. Storrow, which gift has been used to provide a new launch to replace the old one which was unseaworthy. In addition to subscriptions of cooperating institutions four workers have contributed personally toward the support of their research tables. Scientific cooperation on the part of institutions is no less important than financial support and here success may depend to a considerable extent on scholarships or other forms of aid for worthy and needy investigators. Some excellent researc men were unable to work at the Station this past year because of lack of funds to pay traveling and living expenses. The Station has accepted all qualified applicants even if they were unable to pay tor the.r research tables, but it cannot afford to give free room and board, unless in return for services rendered. A system of scholarships or fellowships which would pay the living and travel- ing expenses of excellent workers who could not otherwise come to the Station would be very use- ful. Ihe Tortugas Laboratory of the Carnegie Institution of Washington makes such provision for all who are invited to work there. Some of our cooperating institutions provide funds to pay in whole or in part the living and traveling ex- penses to their representatives at the bermuda Station, in addition to the rent of a research table, and it would be well if all of them would do this. As soon as possible the Station should set aside a sum of say $1,000 a year to help pay the expenses of selected persons who would be invited to join the staff and carry on research at the Station for a limited period each year. Approximately twenty experienced investigators volunteer to serve on the staff at the Woods Hole Marine Biological Laboratory without pay, receiving at most free re- search rooms. It would be an excellent move for the Bermuda Station to make a similar arrange- ment with several experienced investigators, even at the additional cost of supplying some of them with free living accommodations. No more econ- omic method of furthering the scientific work of the Station could be devised than one which would thus bring to the Station and add to our staff at so small a cost investigators who would further the scientific work of the Station. For some time to come we shall need to depend largely on volunteer workers and it would be highly advantageous to have on hand a staff of such workers who could be counted on to help maintain the scientific esprit de corps. Indeed one of our most important needs at present is that the scientific world should become better acquainted w.th the Bermuda Station and that more students and investigators should make use of its facilities, The Station could readily 62 Ki THE COLLECTING NET [ Vor. VII. No. 63 accommodate 25 or 30 persons at one time. The Station is open throughout the entire year, but there are many months during the usual academic year when very few if any visiting workers are in residence. If the Corporation and Trustees were to make known to scientists generally, and not merely to biologists that the Station would welcome all scientists who could make use of its facilities, it would help to make the Station more widely useful. It must not be forgotten that the linancial as well as the scientific success of the Station will be greatly influenced by the extent to which it is used. In this connection it is highly important that the living conditions and __ social atmosphere of the Station should be as agreeable as possible and that our charges to workers should Le kept at the lowest figure practicable. The Trustees have decided that it is permissible and desirable, when facilities for room and board are available, to take in certain classes of paying guests, who are not actually doing scientific work, charging them a higher price than in the case of workers, and that cottages that are not otherwise assigned might be rented to such persons so as to augment the income of the Station. If desirable people can be thus attracted to the Station it will he a contribution to its social life as well as to its income. Of course such an arrangement should never be permitted to interfere with the scientific work of the Station, but with the excess rooms and cottages available, especially during the win- ter, it may well become a valuable source of in- come. Chief among our material needs is an ocean- going motor-boat with auxiliary sail, equipped with engine, power winch and wire rope, nets, thermometers, sampling bottles, refrigerator, etc., for work in deep water outside the reefs and around the islands. Plans and specifications for such a boat were carefully prepared after consul- tation with the Directors of the Bermuda Aquar- ium, the Woods Hole Oceanographic Institution, the PL mouth Laboratory and many others. Esti- mates were obtained from several boat builders and avencies for the sale of second-hand boats; some of these estimates are listed herewith. $4 000-£5,000. Built to specifications for Ler- mada conditions $1,000 more. b. Targer boat built to specifications, 50 ft. long, 13% ft. beam, 5 ft. draft, 40 h.p. gasoline eagine $7,000. Or 60 h.p. Diesel engine $8,000. c. Type of beat recommended by Ralph S. MeCallan, who is especially well acquainted with Permuda conditions and the Station’s needs, 55 ft. long, 15 ft. beam, 5 ft. draft, 60 h.p. Diese. engine $9 000-$10,000. d. Extra strong steel winch connected with engine $950-$1,000. e. About 1 mile of 14 in. steel rope, and 3 miles of 3-16 in. steel rope, probably not less than $1,000. f. Scientific and other equipment ca. $1,000. Total approximate cost of smaller boat $7,000- £10,000. Larger boat (MeCallan’s type) $12,000- $13,000. In making so large an outlay as is represented by any of these suggestions we should not be satisfied with makeshifts, but should seek to ob- tain a boat that will meet all our needs for many years to come. The cost of keeping such a boat in comnussion with a captain, a deckhand or boy, fuel, repairs and insurance would probably not be less than $2,500 a year and might be much more. (Continued Next Issue) TRANSMISSION OF NEUROHUMORAL SUBSTANCES (Continued from Page 57) to cell even in complicated animals, which is quite independent of other fluids ciren- lating in the body. I became interested in this through a study of the chromato- phores, and it is their response that I wish to call to your attention. The flat fishes are useful examples since they vary in color from light to dark with the background. Dr. Mast in his paper in Buil. U. S. Bur. Fish. shows the remarkable capacity for change in the exterior aspect of these fishes by the chromatophores. Pouchet has shown very interesting responses in turbots. These changes depend more or less on the nerves: If a nerve is cut the region does not change markedly at first but it takes place very slowly as a whole. In general | believe that these changes were con- nected with the nerves. When the eyes were covered the whole system failed to act; when one eye was left uncovered, however, it carried on just the same. In frogs, it does not matter how much you cut the nerves; the color is not in- fluenced. These animals change over the whole body rather than in parts of it. The pituitary gland is responsible in this case. When the stim- ulation is strong, the change is great; when the stimulation is light, the animal quickly relapses Jury 15, 1933 ] to its original condition. It is interesting to con- trast the reactions occurring from cutting nerves in flat fish and the color changes in frogs, the one connected with nerve secretions and the other with gland secretions. The experiments of Hogben and Mirvish on chameleons show that the part in which the ani- mal fails to make any change is the part that is brought out by cutting the spinal cord. When the cut is made at various levels the animal fails to change color posterior to the cut. The condition in these reproduces the condition in fish. It is quite evident that the nerves are the significant point. Last summer I called attention to the fact of these contrasts—a nerve control in flat fishes, hormones in frogs, and in reptiles nerves again. My explanation is that we were really dealing with one state of affairs manifested in these var- ious ways: In all cases the chromatophores res- pond to nerve secretions. In fishes the creature responds to a local secretion at the ends of the nerve fibers, causing the chromatophores to con- tract or expand. In frogs, on the other hand, the secretion comes from a local gland, is given out and passes through the blood a long distance to affect remote parts of the body. In fishes and reptiles the secretion is poured out at the tips of the nerves and there is a local reaction, although it may spread to give a general reaction. There is not a difference in principle but different appli- cations of the same general principle of nerve se- cretion. This secretory explanation has been sug- gested by Sherrington, Gerard, Giersberg, Koller, Meyer, Parkér, and Perkins and Kropp. This phenomena operates only in cold-blooded vertebrates. The higher vertebrates have no such systems that we are aware of—they change color by changing the hair or feathers. We have studied in particular the condition in Fundulus tails. If you place Fundulus in a light dish, in five or ten minutes they are quite light, or if put in a dark environment, they become dark, the changes taking place quite rapidly. Examination of the animals under these conditions indicates the mechanism that brings this about. For ex- ample, we have the fin rays with the chromato- phores in the light state, that is, they are reduced to the size of a dot, with the color material in the center of the cell. When the tail is in the dark state, the cells have sent the pigment out into their processes, and the chromatophores are then in a broad and extended condition. There is obvious cellular response produced by so-called expansion and contraction of the cells) The material is permanent and migrates out when the chromato- phores are in an extended state. If in an ordinary Fundulus you make a transverse cut in the tail 1 or 2 mm. in length, at THE COLLECTING NET 63 the end of thirty seconds a dark band appears across the tail. he nerve fibers in the cut region excite the cells and the dark area is formed in the otherwise light fish. We have studied this tail stripe with a good deal of care. In an hour or two the band reaches its maximum intensity, and then fades out in a day or two. At first there are fairly sharp edges then they gradually fade. This is explained by the assumption that sub- stances are produced which bring about these conditions. In cutting, the nerve fibers are stim- ulated in regions lying beyond the cut. When this stimulation dies down, there is no longer any differentiation. The nerve degenerates in 6 or 8 days, but in addition the reaction is probably influenced by introduction of material from the fibers into the band. ‘Contracting’ material could creep in at the edges, gradually causing the cells to contract, that is, they contract not simply because the nerves cease to act, but because a neurohumoral substance comes in from the fibers outside the streak and causes the melanophores to change. Evidence for that can be seen. The hand does not disappear as a whole, but it begins at the periphery and changes progressively. This leads me to conclude that the process is one in which something percolates in from the outside producing the contraction of the melanophores: I should like to say a few words about the de- tails of this work. Last year one of my students, Miss Mills, worked upon this portion of the fish and came upon a very interesting point. She was particularly interested in studying the edge of the band. She discovered that the edges of the dark and light bands did not coincide when produced on the tail of the same fish. I am unable to ex- plain this disagreement between the edges of the two bands unless there be two sets of nerve fibers. One set spreads across from one point, and the other from another. The melanophores have dif- ferent innervation, one set of nerves concerned with contraction and the other with expansion, that is, there is a contracting and an expanding substance acting alternately on the chromato- phores. It is not always possible to follow one initial cut, but these conditions can be produced in a recurrent way. If you cut a fish in the beginning and allow it to bleach out in a light dish, and then put it in a dark environment, the whole fish quickly becomes dark except the band. After a few. hours the band, too, becomes dark. The same thing occurs when you put a dark fish in a light environment. This lagging-behind exhibited by the band region can be explained again by the percolation of substances into the denervated region causing changes in the cells. There is further evidence in this later reaction to favor the idea that neurohumoral substances 64 THE COLLECTING NET _ [ Vor. VIII. No. 63 are active here. An initial stripe of 1 mm: dis- appears in 22—9%6 hours, these figures being mini- mum and maximum time limits. The average for 25 animals is about 29 hours. A 2 mm. stripe requires 34—143 hours, with an average of 78 hours for complete disappearance ; the time for disappearance therefore varies in proportion to the size of the initial stripe. It is a remarkable fact that if you take a fish in the dark condition, dark body and dark band, and inject adrenalin, the whole animal becomes light in ten to fifteen minutes. This, of course, is a question of change in blood and lymph which determines a response over the whole body. Neurohumoral changes, on the other hand, take place much more slowly. The substances are perhaps oil soluble and _ pass through the lipoid coverings of the cells from one to the other. What do we already know about cell transfers? There are a great many examples in cells in plants, for instance. Within the last year Kok published a paper on the transfer of caffeine through the tentacles of Drosera. These were immersed in a weak solution of caffeine which makes its way into the cells and produces a slight precipitate so that its progress can be detected. It can be seen to pass transversely through the cells at a very slow rate. It was estimated that the distal spread was at a rate of about 658 micra in 30 minutes, and the proximal, about 534 micra in 30 minutes. The spread in Fundulus is very much slower—9 micra in 30 minutes. Examples of this spreading of substances can be found in the human skin. Sir Thomas Lewis showed very striking evidence of it in his experi- ments with irradiating skin with ultra-violet. The skin of an arm was partly covered with an im- permeable substance so that the reddening ap- peared in a definite pattern. In the course of a day the edges of this reddening become less dis- tinct and spread out latterly to a distance of 2-3 mm., or if the treatment is strong, 4-5 mm. be- yond the pattern. Another instance can be found in erysipeloids, or sores to which fishermen and poultry men are peculiarly susceptible, and which appear isolated at first and then slowly spread to surrounding areas. Insect stings often react similarly, and the “piebald” skin that ap- pears sometimes in Caucasians and negroes is a further example. The latter consists of a loss of pigment which gradually spreads throughout the surface of the skin, the afflicted individual re- maining otherwise healthy and strong: It seems likely that this change is comparable to that oc- curring in the melanophores : There are naturally a great many examples in In Coelenterates it is impossi- ble to account for the transmission of nutriment the lower animals. from the digestive tract through the endoderm cells to the outside ectoderm cells, except by some such means as the passage of substances from cell to cell. It may be said that this manner of transmission is a much more primitive one than that by blood and lymph; it is found in all plants and animals from the lowest to the highest. It is probably operative in embryonic and regenerative tissue and in processes which are relatively slow. SOME PRELIMINARY DATA FROM A CORRELATED ANATOMICAL, PHYSIOLOGICAL AND BEHAVIORISTIC STUDY OF THE REPRODUCTIVE CYCLE IN THE FEMALE GUINEA PIG Dr. WILLIAM C. YounG clssistant Professor of Biology, Brown University Recent investigations on the oestrous cycle in our domestic and laboratory mammals have dealt very largely with special aspects of the subject, such as the cyclic changes in the genital tract, causal factors involved in the general phenome- non of oestrus, and to a lesser extent, with the rhythmic changes in behavior. There has been lacking, however, any comprehensive correlation of behavioristic changes with structural changes. For this reason, such a study has been undertaken and, because of the striking oestrous behavior manifested by the guinea pig, this animal has been used. With the help of Mr, Hugh I, Myers and Mr, Edward W. Dempsey, in conjunction with whom the investigation is being conducted, the normal behavior of ninety females was observed continu- ously over a period of more than two and a half months. During each pro-oestrum, oestrus and metoestrum, the animals were examined at half hourly intervals and data were recorded with re- spect to the condition of the vaginal closure mem- brane, the behavior of the females in the cages, and the assumption of the copulatory position when touched. During the period of observa- tion we were able to make 17 series of vaginal smears at half hourly intervals from the time of the first opening of the vaginal closure membrane Jury 15, 1933 ] WANE, (Cl ILLECTI NG NET 65 until its closure. We were able to determine ac- curately, on the basis of 231 individual cycles, the length of the oestrous or reproductive cycle and its variation in length during successive cycles. We were able to ascertain the length of the oes- trous period, that is, the period of sexual recep- tivity, by observing our animals continuously dur- ing 343 oestrous periods. This manner of ob- servation enabled us to ascertain the variation in length of successive oestrous periods in the same animal. We were able to determine the time of day when oestrus is most likely to begin and the time when most animals are likely to be found in heat simultaneously. Lastly, we were able to de- termine the sequence of events which characterize oestrous behavior. After this systematic study of the oestrous he- havior in normal females had been completed, ad- ditional data which would involve the sacrifice of the animals were obtained. At this time we un- dertook to make a study of the time of follicular development, to determine the number of follicles which develop and rupture at each oestrus, to cor- relate this number with the length of the oestrus, to determine the exact time of ovulation and to relate it to the other events of the cycle and per- iod, to determine the effect of copulation upon the length of oestrus and the time of ovulation, and finally, to determine the fertility of the female when mated at different times during the oestrous period. Finally, seventy-six animals were killed at twelve different stages in the established oestrous cycle and from each animal we removed and pre- served both ovaries, a section of one uterine horn, a section of the vagina, one mammary gland, and the hypophysis in addition to making a vaginal smear. It has not yet been possible to analyze all the data that have accumulated or to study all the material histologically. On the other hand, many of our data are complete and it is these which are being reported. It was learned, first of all, that the mean length of the oestrous period is almost exactly 8 hours. The range, however, was considerable. Two ani- mals which appeared perfectly normal in every other respect never did come into heat even though the vaginal closure membrane ruptured regularly. With the exception of these two ani- mals and an occasional “miss,” the shortest period was one of an hour’s duration, the longest 14.5 hours. One of the most definite events of the oestrous period is the time of ovulation. As nearly as could be ascertained from an examination of forty-two animals, ovulation occurs within an hour and a half of the end of oestrus whether oestrus lasted three, four or five hours, or twelve, thirteen or fourteen hours. From an examination of fifty animals it was found that no relationship exists between the length of oestrus and the number of developing follicles. Animals in which one follicle had de- veloped remained in heat 7 to 111% hours; animals in which four follicles had developed remained in heat approximately the same time or 54% to 10% hours. One of the most interesting characteristics of the oestrous period is the frequency with which oestrus occurs at night rather than during the daylight hours. Data have accumulated from 442 observations. Actually two-thirds of all the ani- mals came into heat between 6 P. M. and 6 A. M., while a large part of the remaining one-third came into heat between 4 P. M. and 6 P. M. A minor peak of activity at 6 P. M. was at first puzzling. However, after curves had been pre- pared from data for the two halves of the experi- mental period, February 15 through March 31, and April 1 through May 10, a possible explan- ation became clear. It was found, first, that ani- mals show an especial tendency to come into heat during either of two periods in the day, late in the afternoon or early in the evening, or if not then, later in the evening between about ten and two; secondly, that these maxima of oestrous activity shifted to the right as the days became longer, that is, toward the late night and early morning hours; and thirdly, that the hump to the left of the curve prepared for the entire period of ob- servation is really a second maximum which ex- presses the frequency of oestrous activity in the late afternoon and early evening and which has become somewhat leveled off because of the shift- ing of the time of oestrous activity with the lengthening day. Obviously, the occurrence of oestrus is subject to some nocturnal influence. What this influence can be is not understood. Nor are we able to ex- plain why there should be two maxima of activity unless in many animals threshold changes pre- paratory for heat are partially built up during the last night of the dioestrum and completed early the following evening. Both problems are being investigated. The entire study as far as we have progressed is, of course, preliminary. It has indicated, how- ever, that oestrus is not yet an exhausted subject and that factors may be involved, the existence of which has not hitherto been suspected. The data which have just been presented may or may not suggest what these factors are, but they have sug- gested ‘several interesting working hypotheses which have provided the basis for experiments which are now in progress. 66 THE COLLECTING NET [ Vor. VIII. No. 63 The Collecting Net An independent publication devoted to the scientific work at Woods Hole Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Martin Bron- fenbrenner, Margaret Mast and Annaleida S. van't Hoff Cattell. Printed by the Darwin Press, New Bedford THE M. B. L. CLUB Investigators and students alike are indebted to the out-going president for reviving the important work of the M. B. L. Club. Handicapped by a broken leg, and relatively inefficient co-officers, he has served as a catalizer so efficient that Pro- fessor Robert Chambers has consented to serve as president for the present year; Dr. Heilbrunn has stirred numbers of the scientific community to ap- preciate the important part that the club can play in their lives at Woods Hole. His contribution has been great. The M. B. L. Club can do much to add to the pleasure of laboratory life. But it can do more than that. It can make a detinite contribution to biological research. Gatherings in the club-house will serve as a medium for the in- terchange of ideas and act as a stimulus to the younger investigators. More than one prominent biologist has ex- pressed the opinion that they primarily benefit from the meetings of the American Society of Zoologists by the meeting old friends, the making of new ones, and by the consequent exchange of ideas. Zoologists have read, or can soon read, the substance of any paper on the program. This visual impression will mean more than the audi- tory one amidst many distractions. The situation at Woods Hole is analogous. The session here is not three days, but three months. The scientific program consists of the ijectures and evening meetings. Extensive discussion is an important adjunct to the evening programs and it should be fostered. The absence of a suitable environment has prevented the members of the laboratory from taking full advantage of their opportunity. That is why the club’s decision to hold a smoker after each Iriday evening lecture is a fundamental one. As soon as the lecture is completed, the speaker and all members of the club are asked to adjourn to the club-house. Refreshments and ‘‘smokes’’ will be served and on cool evenings an open log fire will radiate its warmth about the ‘‘smokers”’. The gatherings will be “co-educational,” SEAL SCHOLARSHIPS TO THE EDITOR: Why cannot there be a COLLECTING NET Scholarship to make it possible for two seals who would not otherwise be able to come, to spend the summer in Woods Hole? Due to the depression or the mackerel or something the Fisheries people seem unable to furnish the animals. For a number of years the seals have been universal after dinner favorites and their contributions to the good humor and friendliness of Woods Hole have been great. They have won the respect and admiration of the most distinguished visiters. Hans Spemann was proud to climb into the seal pen, and be photo- graphed with them, although he refused to be taken aboard the Cayadetta with members of the inverte- brate course. Is it too much to hope that the COL- LECTING NET will find it possible to tap its “many sources” and establish such a fund? G. B. MOMENT. CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the hole turns to run from Buzzards Bay to Vineyard Sound: Date EAS WE DER it JulyeU us niee 112s Jee July Gos lee OG 12:15 Vuk ye Geocaan isos 1:09 Jali Ge ene ele DS 2:03 Afi CY ecco, | ABE 2:49 Ariss 0) Soon he5 sedis) 3:35 Awl CS See a beth 4:19 Syncs eee 5:03 July $23) pp eee EBS 5:41 Onalliy (24 Necue exon LOM 6:23 In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. Jury 15, 1933 | THE COLLECTING NET _ ee MES 2©.F Dr. H. Boschma, professor of zoology and di- rector of the zoological laboratory at the Uni- versity of Leyden in Holland, visited at the Ocean- ographic Institution over last week-end. He came to this country to attend the Pacific Science Con- gress in Victoria, Canada, and is sailing for Holland the fifteenth on the Berengaria. Dr. Boschma is not a stranger to Woods Hole having spent six weeks in 1924 at the Marine Biological Laboratory working with the coral, Astrangia. A paper covering his work here was published in the Biological Bulletin for June, 1925, under the title “On the Color Changes in the Skin of the Lizard Ptychozo6n homalocephalum.” He ex- pects an article on the Dutch marine laboratory to Tue CoLtLtectinG Net later in season. Professor P. H. Mitchell of Brown University, with his family is visiting the Pacific coast this summer. He will be at the Scripps Institution of Oceanography at La Jolla, at the Hopkins Lab- oratory at Pacific Grove, and at the University of Washington in Seattle, for brief periods of work upon measurements of the alkalinity of Pacific coastal waters. This will be a continuation of similar work done at the Woods Hole Oceano- graphic Institution last summer. In a letter from Dr. Florence Peebles we learn that she attended the meetings of the American Association for the Advancement of Science in Chicago, and that she will visit her family in Vir- ginia. She hopes later to spend a few days both in Woods Hole and in Salisbury Cove. Her address until August 1 will be: c/o Dr. Eliz. McLaughry, The Overlook, New Wilmington, Pa. Dr. Esther Carpenter spent the past year at the Johns Hopkins Medical School as a_ research assistant to Dr. C. W. Metz of the Carnegie In- stitution of Washington. She will teach next year in the biology department of Albertus Magnus College in New Haven and continue with her research work at Yale University. Dr. B. H. Willier (instructor, assistant pro- fessor, associate professor and full professor of zoology at the University of Chicago from 1920 to 1933) has accepted the headship of the depart- ment of biology at the University of Rochester. Dr. Robert Payne Bigelow has been given the title of Professor Emeritus Professor of zoology and parisitology at the Massachusetts Institute of Technology, LNG Re oak, THE COLLECTING NET SCHOLARSHIPS Dr. E. C. Cole, director of the course in in- vertebrate zoology at the Marine Biological Lab- oratory recently made the following comments concerning Tur CoLttectinG Net Scholarships : Net proved to be of real value in stimulating qualified students, and enabling them to devote an addition- al summer to study or investigation at the Marine Biological Laboratory. Tue COLLECTING Scholarships have The courses may appro- priately be considered as “feeders” from which the Laboratory will secure individuals fitted to be- come investigators. Any factor which favors this ideal must be considered desirable, and that is precisely the role played by these scholarships. Awards are made on the basis of sound training, excellent work, and real evidence of future prom- ise as investigators. As instructor in charge of the Invertebrate Zoology Course, I am very glad to certify to the importance of these scholarships. It is highly desirable that they be placed upon a permanent basis. Certainly a permanent endowment for this purpose is by no means impossible. The achievement of this aim would be well worth the time and money necessary. Ina card to Mr. McNaught, Dr. A. R. Moore writes that he and Mrs. Moore are “greatly en- joying the surroundings and the wonderful sea fauna” in Misaka, Japan. Dr. Gardner B. Moment (who likes seals!) will return to Goucher College next year as in- structor in biology. Dr. George L. Clark of the Oceanographic Laboratory is occupying Dr. Mitchell’s cottage in Woods Hole during the present summer. Dr. Victor Hamburger, formerly of the Uni- versity of Freiburg, arrived at the Marine Bio- logical Laboratory. He is a Rockefeller Founda- tion Fellow from the University of Chicago. First reports from the Washington University- Woods Hole expedition to the Tortugas are be- ginning to filter through. The first day of the arrival was marked by a casualty, as Paul Nicoll, unused to the mysteries of coral reefs, stepped on one and was laid up for a couple of weeks with a cut foot, which limited his capacity for amuse- ment but apparently not for work, 68 THE COLLECTING NET [ Vou. VIII. No. 63 DISTRIBUTION OF THE FRESHWATER ALGAE OF NEWFOUNDLAND Dr. WM. RANboLpH TAYLOR Professor of Botany, University of Michigan In 1926 a collection of freshwater algae was made by Mr. J. M. Fogg, Jr., and in 1929 another by Mr. Bayard Long on expeditions directed by Prof. M. L. Fernald; in 1929 Miss Belle Burr also collected several samples on Newfoundland. The controlling features of the distribution of the flowering plants on Newfoundland as reported by kernald may be generalized as follows: The rocks east of the Long Range Mountains and south of the Northern Peninsula tend to develop an acid soil; the crests and western slopes of the range are predominantly calcareous, with some serpentine areas. The island in general escaped the main Pleistocene continental ice sheet, but had a local ice mass over the eastern part; the western summits underwent no considerable glaciation and probably were in considerable degree actually free of ice. At present the eastern and east central part of the island is cold and foggy under the in- fluence of the arctic Labrador current, while the western slopes and shores are relatively warm and sunny though the deep mountain valleys do retain heavy accumulations of snow. Many arctic, alpine and Cordilleran vascular plants appear in the flora of the western border strip; the soil was favorable to them and _ their colonies either antedated the Pleistocene ice and survived on unencumbered portions of the western ridge, or became established as it retreated. Many southern coastal plain (Carolinian) plants find northern limits in the south and southeast, because they are adapted to the sterile acid soil and reached that area by a land bridge after the Pleis- tocene ice disappeared. These two special classes of plants, and a number of endemic species, accent a flora the bulk of which is composed primarily of wide-ranging Boreal, and secondarily of Cana- dian-Alleghenian species, though it must be noted that the sterile soil appears to inhibit the growth of many species of these major elements which would otherwise be expected. In attempting an interpretation of the distribu- tion of the algae on Newfoundland, it was neces- sary to limit attention to the one large group which was known in sufficient detail to afford a basis for comparison with other countries, namely the Desmids, of which over 500 kinds are to be recorded. Of all other freshwater algae less than 200 are recorded (excluding diatoms). There may be selected from the 4 largest genera 130 species which fall into three groups: one with an arctic-alpine range, one wide-ranging but north- ern, and the third wide-ranging but southern. Comparing the individual station lists with these, we find that the last or southern-ranging types do not have a distinctive distribution among the New- foundland stations studied. The northern-rang- ing types are particularly abundant about Bonne Bay part-way up the west coast, and drop off about Ingornechoix Bay, a more northern station. At this place we find the maximum concentration of arctic-alpine species, the number being more than five times as great as at the other localities. Some confirmation of the distribution features may be secured by noting what species are found in one district to the exclusion of any other. In the Ingornechoix Bay district there is clearly a group of notable arctic-alpine species; in the southern district the proportion of wide-ranging types is very large, but in the residue we find a few notably southern types, including the two, Micrasterias arcuata and M. expansa, which first attracted attention to the Newfoundland study. The distribution of the desmids, although more generalized as appropriate to the wider ranges which desmid species cover upon the earth, was found comparable to that of the flowering plants. THE ROLE OF BACTERIA IN THE FORMATION OF NITRATE IN THE SEA Dr. SELMAN A. WAKSMAN AND Dr. CorneLIA L. CAREY Woods Hole Oceanographic Institution No other phase of marine bacteriology has at- tracted as much attention and has aroused greater interest than the process of nitrate formation in the sea, with the possible exception of nitrate re- duction. The formation of nitrate in the sea is usually considered as the final step in the trans- formation of nitrogen and as that form of nitro- gen which is most readily available to the phyto- plankton and other marine plants. Several theories have been suggested to explain the origin of the nitrate in the sea, the most im- portant of which are the following: (1) Nitrate does not originate in the sea itself, but is formed in land soils and brought into the sea by streams and by land drainage; nitrate may also be formed by electric discharges and thus introduced into the sea. This theory, originally proposed by Boussin- gault and Schlosing, has later found support in the work of Nathanson and Gran, (2) Nitrate is produced directly in the sea by bacteria which oxidize the ammonia formed in the decomposition of marine residues, first to nitrite and then to nitrate. This theory was first proposed by Vernon and by Brandt and later found support Jury 15, 1933 ] in the investigation of Baur, Thomsen, Issachen- co, Lipman and Harvey. (3) A third theory is that of photochemical oxidation of ammonia. This was propsed by Rao and Dhar for the oxidation of ammonia in solutions and in soils, in the pres- ence of proper catalysts, and was applied recently by ZoBell to sea water. The investigations reported in this paper have been limited to the study of the role of bacteria in the process of oxidation of ammonium com- pounds in the sea. The nature of the medium used for demonstrating the presence of these bacteria was found to be highly important. Be- cause of the specific physiological nature of these organisms and the difficulty of obtaining satis- factory growth on artificial substrates, special attention was paid first of all to the composition of the medium which would be favorable for development of these bacteria. By the use of a proper medium, one could easily establish the fol- THE COLLECTING NET 69 lowing facts: (1) Sea water, especially in the upper layers of the sea, is practically free from nitrifying bacteria or contains only very few cells of such organisms. (2) Nitrifying bacteria are limited entirely to the sea bottom. The presence of bacteria capable of oxidizing ammonium salts to nitrite in both sandy bottoms and mud bottoms could easily be established. In the case of the latter, these bacteria are limited largely to the very surface layers of the marine bottom. The oxidation of the nitrite to nitrate in the cultures could be demonstrated only after a long period of incubation, after all the ammonia has been oxidized to nitrite. Although these investigations have been lim- ited to the material obtained from various stations on George’s Bank and in the Gulf of Maine, they lead to the conclusion that nitrate is produced in the sea in the bottom by the action of specific bacteria; the nitrate then diffuses from the bot- tom upward into the water. DIURNAL MIGRATION OF PLANKTON IN THE GULF OF MAINE AND ITS CORRELATION WITH CHANGES IN SUBMARINE IRRADIATION 3y GEORGE L. CLARKE Instructor of General Physiology, Harvard University ABSTRACT distribution of copepods were made in the Gulf of Maine during a 12-hr. period, a 24-hr. period, and a 48-hr. period. Careful records of the light falling on deck were kept during these periods and measure- ments of the penetration of light into the sea were made at frequent intervals using the photo-electric method. These simultaneous observations enabled quantitative information to be procured on the importance of light in controlling the diurnal mi- gration of plankton, Five closing nets were towed simultaneously at different depths from a single verticle cable. A method was devised for sending the nets down in the closed position, opening them simultaneously and towing them all together horizontally for 10 minutes, and then closing them again before hoist- ing to the surface. This method made it possible to sample accurately the upper 50 meters of water at intervals of less than one hour. Ordinarily ten series were made each day and in some cases the series was repeated in the 50 to 100 meter stratum. The errors involved in the work at sea and in the sampling of the catch in the laboratory are at least as small as in other investigations of this type. Moreover, the methods used allow special dependence to be placed upon differences found among the hauls within each series. The general vertical distribution of the three Observations on the vertical species studied was as follows: Centropages typi- cus inhabited the stratum of water above the thermocline (10 to 20 meters, Calanus finmarchi- cus was irregularly distributed, and Metridia lucens occurred below the thermocline. The adult females of Metridia exhibited the most marked diurnal migration, the level of max- imum abundance rising in the afternoon and dur- ing the night and falling in the morning. These movements were found to coincide to a consider- able extent with changes in submarine irradiation. In the case of the 48-hr. station the behavior of these animals on the second day is very closely the same as on the first day. The changes in the vertical distribution of the other groups of cope- pods were slight or quite irregular. In some cases, however, there was a definite tendency for the maximum to occur at greater depths at noon than at other times. This investiagtion confirms the idea that light is the most important factor controlling diurnal migration. In addition the observations are shown to have a bearing on various of the theories regarding the manner in which — light exerts its effects. For example, the change of light intensity is shown to be probably not suf- ficiently rapid to reverse the sign of phototropism (as in Daphnia). Data on the rate of swimming of copepods indicate that these animals probably could keep pace with a given zone of light in- tensity as it changes its level during the course of the day. : 70 ; THE COLLECTING NET [ Vor. VIII. No. 63 THE BIOLOGICAL LABORATORY AT COLD SPRING HARBOR Evening lectures at the Biological Laboratory thus far this summer have been given as follows: June 23rd: Dr. Harold Abramson, College of Vhysicians and Surgeons: “ELecTRoKINETIC Po- TENTIALS IN BioLoGy AND MEDICINE”. June 28th: Dr. Charles B. Davenport, Director of the Department of Genetics, Carnegie Institu- tion of Washington: ““Waicn Came Frrsr IN Evolution, Form oR FUNCTION ?” July 6th: Dr. Felix Bernstein, of the Biological Laboratory; “Tie CONNECTION BETWEEN PRES- BYOPIA AND LENGTH OF LIFE.” ‘July 11th: Dr. W. W. Swingle, Princeton Uni- versity : “FUNCTIONAL STUDIES OF THE ADRENAL CorTex.” Dr. Felix Bernstein, director of the Mathema- tical Institute of the University of Goettingen previous to the present political upset in Germany, is working at the Laboratory this summer under a special grant from the Rockefeller Foundation. Tis research is concerned with the relationship existing between weakening of the accommodat- ing power of the lens of the eye and the length of life, and its inheritance. Prof. F. Botazzi and Signora Botazzi, of Naples, were guests of Dr, and Mrs. Osterhout at Huntington before returning to Italy. Prof. and Mrs. Botazzi visited the Laboratory and were pre- sented to those gathered for the symposium of July 5th on Oxidation-Reduction Potentials. ‘Two thousand five hundred dollars of a special grant from the Carnegie Corporation, to be used for equipment, has been received. Dr. Harold Abramson, who is in residence at the Laboratory this summer, was very recently married to Miss Barbara Smith. Dr. D. A. MacInnes, Dr. L. Michaelis, of the Rockefeller Institute, and Miss Elsa Michaelis, were guests of the Director of the Laboratory and Mrs. Harris on Tuesday and Wednesday of the week of the 3rd. Drs. MacInnes and Michaelis took part in the symposium of July 5th. Other recent visitors to the Laboratory include Dr. L. R. Blinks, of the Rockefeller Institute, and Dr. E. S. Guzman Barron, of the University of Chicago. DISTRIBUTION AND ECOLOGY OF THE MARINE ALGAE ON LAKE FISH Dri Ee Jey IBHoIHe, Professor of Botany, Dalhousie University The marine algae of the Atlantic coast of the Maritime Provinces of Canada were collected at The most intensive collecting was done at St. Andrews, New Halifax, Nova Scotia. A whole summer was spent collecting around Prince Edward Island. The report covers the work of more than seven years. The collecting was done chiefly during the summer, but regular collecting was also carried out for three winters. The species reported include thirty Chloro- phyceae, forty-one Phaeophyceae, and forty-nine Khodophyceae. The list of species, the regional distribution and the prevalence was given tabu- lar form. The coastal area of the Maritime Provinces is divided into three distinct geographical and eco- logical regions, namely, the Bay of Fundy, the Atlantic and the Prince Edward Island Regions. Each of these is distinctly different in regard to both marine flora and marine flora environment. The main features of the flora of each region are as follows: Bay of Fundy, generally dense and luxuriant ; Prince Edward Island, a barren littoral representative places all along the coast. Brunswick and at zone and a rich sub-littoral floral; Atlantic, inter- mediate in density and luxuriance with the pre- dominance of large linear forms in the surf. The dominant species are characteristic and constant for each region but they are quite different from region to region. The region that exhibits the greatest number of differences is that around Prince Edward Island. The physical factors varying throughout the area and associated with the floral differences are: water temperature, tides, wave action, clarity of the water as regards mud, structure and composition of the rocks along the shore, materials forming the ocean floor near the shore, slope of the region of growth, salinity and ice action. Each of these physical factors is associated with certain charac- teristic features of the marine flora. The growth is continuous throughout the year and consists of about five distinct crops. The barren period is in September or October and the most varied growth in March or April. The most im- portant result of the survey was the demonstra- tion of the wide ditferences existing between these three adjacent but sharply divided ecological regions. Jury te, 1933 4 : Ahan Announcement eee In its office on Main Street Tne CoLtectinc Nev has a great many books for sale. They cover a wide range of subjects and the prices of many of them have been cut to one half or one third of their original cost. Money resulting from the sale of these books will be used this summer to help defray the cost of publishing Tir Cottectina Net. JULY 17 TO JULY 29 roth Annual Sale of Wamsutta Percale Sheets & Pillow Cases at the Lowest Prices of all time Price List Sent on Request STAR STORE New Bediord, Mass. THE COLLECTING NET _ * 71 Non-Corrosive Non-Corrosive MICROSCOPIC SLIDES AND COVER GLASSES Do Not Fog At your dealer— or write (giving dealer’s name) to Cray-ApAms CompPANy 2S East 26th Street NEW YORK Church of the Messiah _- EPISCOPAL The Rev. James Bancroft Rector - 8:00 a. m. 11:00 a. m. Holy Communion = = - Morning Prayer - - - ben) - The Woods Hole Log A WEEKLY PAPER DEVOTED TO THE NEWS OF WOODS HOLE PUBLISHED EVERY WEDNESDAY For Sale Sixty Excellent Views of Woods Hole A. R. APGAR Brick 311 SCIENTIFIC DRAWINGS GRAPHS — CHARTS — ILLUSTRATIONS NORRIS JONES M. B. L. Rm. 211 Brick Bldg. BIOLOGICAL, PHYSIOLOGICAL, MEDICAL AND OTHER SCIENTIFIC MAGAZINES IN COMPLETE SETS Volumes and Back Copies For Sale B. LOGIN & SON, Inc. 29 East 21st Street Est. 1887 New York 72 THE /COLLECHING NET [ Vor. VIII. No. 63 EPIDERMOPHYTOSIS (Athlete’s Foot) Dr. Davin CHEEVER Harvard Medical School (The following account is the first of two articles by Dr. Cheever throwing light on the possible danger of the spread of skin diseases through con- gestion of the Bay Shore bathing beach.) “Athlete’s Foot” has become very common in the last few years, particularly among people of cleanly habits, which is explained by the fact that such people are able to make greater use of g,m- nasiums, athletic clubs, bath-houses, and the like, where people tend to congregate bare-footed. Bits of skin are constantly flaking off from our entire bodies and make up an appreciable part of the dust of our houses. From infected feet these particles of skin contain the germs of “athlete's foot’, and in spite of ordinary care of floors, es- pecially if their surfaces are rough, there is danger of foot infection. i:pidermophytosis is easily recognized on the webs of the toes , particularly between the fourth and fifth, and for some curious reason, more com- monly on the left foot. It is in the form of macerated surface layers of skin, containing mycelium and spores, which can be readily rubbed off, dropping as tiny skin flakes to the floor to be picked up by the feet of passers-by. Warm weather conditions make the skin more susceptible to infection by bacteria and fungi, and promote a more rapid and severe spread. For- tunately, there is an excellent check for those at the beach in the form of the extremely hot, dry sand. If the feet are wet for short periods only, and thoroughly baked in the hot sand during as much of the day as possible, a new infection may be kept from spreading and often a low-grade one may be largely, or quite, cleared up. Advertisements in the current magazines to the contrary notwithstanding, there is no certain cure for “‘athlete’s foot”. No general rules for treat- ment, except sunlight and dryness, can be laid down because the infection varies so in different people and at different times. Most applications which are of any value in this condition are rather strong and liable to irritate so it is usually best to get competent medical advice for each individ- ual case. Ina general way, one may say that the cases limited to maceration between the toes may be safely treated with mercurochrome, sulphur ointments, ammoniated mercury ointment, but very cautiously with iodine unless it is freshly purchased since evaporation causes it to strength- en dangerously with age. If the trouble becomes active and causes raw or blistered spots to develop about the toes, soles or sides of the feet, medical advice should be promptly sought as a pus infection (blood poison- ing) occasionally enters, producing a great deal of soreness and temporary crippling. Epidermo- phytosis occurs not infrequently on other parts of the body, but no rules for its recognition or treat- ment can be simply given. No entirely satisfactory methods of prevention have been devised, but around pools and shower- rooms various precautions are emplo,ed. In some instances all patrons are asked to wear rubber or paper shoes; in others, all are required to walk through a shallow tub containing sodium hypo- sulphite, or other solution; occasionally, all are requested to swab the webs of the toes with dilute iodine. In the home where a case of epidermo- phytosis exists, all members «f the family should wear inexpensive paper shoes rather than to walk barefooted, and the patient’s hose should be boiled each day. NOTES FROM THE M. B. L. CLUB At the annual meeting of the M. B. L. Club on Monday evening Dr. Robert Chambers was chosen president to succeed’ Dr. Louis V. Heil- brunn. Other officers elected at the same meet- ing were Dr. Samuel Shoup as Vice-President and Dr. Robert M. Stabler as Secretary-Treasurer. Miss Louise Mast was selected to serve as assist- ant Secretary-Treasurer. The weekly M. B. L. Club dance will take place this evening at the clubhouse, beginning at 9:00 M. The attendance is expected to equal that of the first two, two-hundred or thereabouts. A tentative program for next Wednesday's vic- trola concert, the second of the season includes Tschaikowsky’s “Nutcracker Suite” in its entirety and the four movements of the First Symphony of Brahms. The club is enlarging its facilities in line with its increased enrollment which is now one hun- dred and seventy, more than double that of last summer at the corresponding date. Beginning July 21, an informal smoker will be held after the Friday lectures. In line with the recently-adopted policy of pro- viding hosts and hostesses for each evening, the club has prepared the following list for the com- ing week : Sunday, July 16—Mr. and Mrs. Norris Jones. Monday, July 17—Reception for the new presi- dent and other officers—Dr. and Mrs. Robert Chambers. Tuesday, July 18—Choral club rehearsal—Dr. Edwin Linton and Mrs. G. B. Jenkins. Wednesday, July 19—Victrola Concert—Dr. and Mrs. P. B. Armstrong. Thursday, July 20—Dr. Mary S. MacDougall. Friday, July 21—Dr. and Mrs. G. B. Jenkins. Saturday, July 22—Dance—Dr. and Mrs. R. M. Stabler. Vol. VIII. No. 4 SOME EFFECTS OF THE BLUE-GREEN ALGAE, APHANIZOMENON FLOS-AQUA, ON LAKE FISH Dr. G. W. PREscotr Assistant Professor of Biology, Albion College During the past three summers opportunity was afforded to study the efficiency of copper sulphate as an algacide and to study the various biologi- cal effects of superabundant SATURDAY, JULY 22, 1933 MW. WB. UL. Calendar Annual Subscription, $2.00 Single Copies, 25 Cents. ULTRACENTRIFUGAL AND CATAPHOR- ETIC STUDIES ON RESPIRATORY PROTEINS Dr. THEODOR SVEDBERG Professor of Physical Chemistry, Umversity of Uppsala, Sweden An ultracentrifugal study of the blood pig- ments—or respiratory proteins—throughout the animal kingdom has shown that these proteins are surpris- ingly well defined with regard growths of blue-green algae in some lowa lakes. The investi- gations were carried on for the Iowa State Fish and Game Commission as part of a pro- gram to make the lakes suit- able for the stocking of game fish. Of late years there have been increasing* periodic wide- spread deaths of fish in alarm- ing numbers. The deaths have usually been associated with superabundant growths of blue-green algae. One of the many objectives of the inves- tigation was to determine a possible casual relationship between the deaths of the fish and the algal condition of the lakes. (Continued on Page 79) The most objection- Ultracentrifugal and Cataphoretic Studies on Respiratory Proteins, Dr. T. Svedberg...... cid Effects of the Blue-green Algae on Lake ERS Gre INV MOLES COD Dire ieee seveal Sie ple » Sie aie) oe Cts Cytology of Amoeba proteus, W. L. Doyle... .80 TUESDAY, JULY 25, 8:00 P. M. Seminar: H. B. Goodrich and C. B. Crampton: “One step in the de- velopment of hereditary pigmen- tation in the fish Oryzias latipes.” George D. Snell: “Translocations in the mouse and their effect on development.” D. E. Lancefield: ‘A series of prob- able mutations in Drosophila pseudo-obscura as compared with D. melanogaster.” P. W. Whiting: ‘Sex-determina- tion in Hymenoptera.” FRIDAY. JULY 28, 8:00 P. M. Lecture: Laurence Irving: ‘On the ability of mammals to survive without breathing.” Members of the M. B. L. Club are urged to attend the smoker fol- lowing the lecture at the club. helds, the molecular weight as sedimentation equilibrium determinations in cen- to sedimentation constant and molecular weight. Not only the mass and shape but also the chemical composition of their molecules as revealed by the electrophoretic behavior shows uncommon distinctness. A detailed investigation of the different kinds of respiratory proteins is therefore of great interest not only from a physi- ological but also from a physi- co-chemical point of Three different properties have been studied: the sedi- mentation constant as derived from measurements of the rate of settling of the mole- cules in centrifugal calculated from view. strong TABLE OF CONTENTS The Reaction of Kidney Tubules to Neutral Red and to Phenol Red, R. Chambers...... 94 Respiratory Poisons and Methylene Blue on Cleavage of Certain Eggs, M. M. Brooks.. .95 Fertilization Membranes of Centrifuged As- The Biological Laboratory: teriasyures, Db. PCostello. iiy-e see een 98 MLO GUCULOM Rec elerans\elte cya) eiccs cin kesclsie re Reve sues ce 82 Ionic Changes in Fish Eggs, L. Irving........ 98 The Theory of the Diffuse Double Layer, Comparative Studies on the Inclusions in Ep VUT Le reetetremsyauenalasdicsis scieicy-petetsster eke ee aca)’ estan 83 Vortitaila: Hiv eh, shinies spc -behe a ernseecyereete 99 The Theory of Electrophoretic Migration, Heredity and Environment, E. G. Conklin...100 H. Muller Impetigo; Dry Davidi@heever ja. seni se 103 eet 78 : THE COLLECHING NET [ Vor. VIII. No. 64 DR. ALFRED C. REDFIELD AND DR. THEODOR SVEDBERG This photograph was taken just before Dr. Svedberg boarded the evening train for Boston at the conclusion of his stay in Woods Hole. He was a guest of the Woods Hole Oceanographic Institution and carried on his research work in Dr, Redfield’s laboratory. trifugal fields of medium strength and the isoelec- tric point as measured by means of the migration of molecules in electric fields. The technique for the determination of sedi- mentation constant and molecular weight has been described in detail elsewhere. A small quantity of the solution enclosed in a sector-shaped cell is ex- posed to the influence of a strong centrifugal field in a special centrifugal instrument—the ultracen- trifuge—and the concentration gradient deter- mined by taking photographs of the solution dur- ing centrifuging. The pictures are then registered by means of a microphotometer and the curves obtained used for the calculations. A refined technique for the study of the move- ment of the boundary between solution and sol- vent in a homogenious electric field makes it pos- sible to determine the isoelectric point of the re- spiratory proteins with great precision, The meas- urements are made by taking photographs and registering the pictures in a microphotometer. The main results may be summarized as_fol- lows. Respiratory proteins contained in blood corpuscles have always low sedimentation con- stants. Hemoglobin, characterized by the sedi- mentation constant +.40 10°! and the molecular weight 69,000, only occurs in the higher classes of the vertebrates. The blood corpuscles of the low- est class of the vertebrates, Cyclostomata, as well as the blood corpuscles of the capitellide worms have a respiratory protein of much lower sedimen- tation constant, 2.0-2.3<107!*. The elyceride worms have corpuscle protein of sedimentation constant 3.51071, and probably a molecular weight equal to half that of hemoglobin. Respira- tory proteins dissolved in the blood plasma have Jury 22, 1933 ] as a rule high sedimentation constants and high molecular weights. The only exception is the blood pigment of the Chironomus larvae, which has a sedimentation constant almost identical with that of the cyclostomes and the capitellide worms. Within a well-defined animal group all the spe- cies have, as a rule, the same sedimentation con- stant. All the polychaete worms and hirudineans with the respiratory protein dissolved in the blood have the constant 57.110-!°. Some of these proteins are red (erythrocruorin) others are green (chlorocruorin). The oligochaete worms have a constant 60.8 10-!* very close to that of the other worms. Some of the crustacean families show the sedimentation constant 16.9 « 10°' (hemocyanin and erythrocruorin), others 23.4 10-71%, and one of them 34.1 & 10-18. The xipho- surans and scorpions have the same constant, viz. 34.1 & 10°. All the gastropods except Planor- bis and Arion have the constant 99.8 * 10-!. It is obvious from these regularities that biological kinship is usually accompanied by identity in the sedimentation constant. The determinations of, molecular weight by means of sedimentation equilibrium measurements have given the surprising result that a system of simple multiples seems to obtain among the mole- cules of the blood pigments. The molecule of the gastropod hemocyanin has a weight of 5,100,000. THE COLLECTING NET 79 The molecular weight of the erythrocruorin and chlorocruorin of the worms is about 14 of this figure. The erythrocruorin of Planorbis and the hemocyanin of the xiphosurans and scorpions is about 14 that of the worms. The hemocyanin molecule of sedimentation constant 23.4 « 10-14 is about 1% that of the Planorbis pigment, and the hemocyanin and erythrocruorin of constant 16.9 Sele 13 is about ie that of the former. This scheme of simple multiples for the mole- cular weights of the respiratory proteins is sup- ported by some observations about the reversible dissociation of their molecules into simple submul- tiples at the borders of the pH stability regions. It is further strengthened by previous observa- tions of simple relationships between the molecu- lar weights of the proteins of lower weight. The determinations of the isoelectric points of the respiratory proteins show that the blood pig- ments of the invertebrates are all much less alka- lin than the hemoglobin of the vertebrates. It is of great interest to notice that the isoelectric point varies from species to species. Even very nearly related forms have different isoelectric points. For a genus containing several subgenera the isoelec- tric points lie closer together within a subgenus. The situation of the isoelectric point is therefore to a certain degree a measure of the kinship. (This abstract is based on a lecture presented at the Marine Biological Laboratory on July 11.) SOME EFFECTS OF BLUE-GREEN ALGAE ON LAKE FISH (Continued from Page 77) able pest in certain Iowa lakes is the blue-green alga Aphanizomenon flos-aqua. It grows in such abundance as to make some entire lakes or parts of lakes a thick, green “soup” at various times during the summer months. This plant has the characteristic habit of readily concentrating by wave and wind action into great thick mats or blankets, collecting along the shores and in shal- low bays where the growth decays, thereby bring- ing about many objectionable conditions. It was found that such superabundant growths of algae suddenly bring about the death of many thousands of fish in three ways. First: The plants were so abundant that at night, when oxygen release by photosynthesis was not in progress, the oxygen demand for this great mass of organisms was so great that the dissolved oxygen content became lowered to a point that would not support fish life. In comparison with other aquatic plants, blue-green algae are, at best, poor oxygenators. Death of fish by this manner of suffocation was not found to be common. Second: When algae grew in such abundance the unbalanced condition brought about their sudden death and disintegration. Their death and subsequent bacterial decay occurred in periods of high temperature when the oxygen supply was normally low due to the inability of warm water to hold oxygen. The bacterial decay apparently rapidly depleted the oxygen supply necessary for small animals which died in prodigious numbers. In turn their decay further depleted the oxygen supply to the point that fish were suffocated. Small lakes or bays in certain larger lakes were observed to have scarcely one living thing in them but bacteria. Even bottom organisms were killed. After such a crisis, dead fish whiten thousands of feet of shore line. Third: It was noticed in areas where masses of algae were disintegrating, although not necessarily decomposing, that fish were dead or dying, ap- parently not from lack of oxygen. Dissolved oxygen readings showed sufficient oxygen to be present to support fish life (4.0 ppm) Such observations naturally raised the question as to the cause of the distress and death of the fish and the idea of a possible poisoning occurred. To test for this a number of simple experiments were carried on in a stripping shed laboratory. Large amounts of Aphanizomenon (20 to 50 80 THE COLLECTING NET [ Vor. VIII. No. 64 gallons) were collected and allowed to decay in closed receptacles. In some experiments open receptacles were used. Ten different species of fish were seined from the lake and placed in a holding tank. After the algae were thoroughly decayed the vats were emptied into a large tank. The tank was aerated by means of oxygen of pipe drilled with fine holes being fitted to the end of the inlet hose. This dispersed the gas in the water and increased the solubility. The fish were put into the tank with the de- cayed algae and their behavior observed. The D. O. at the time was 4.0 ppm. Intermittently oxygen was introduced and frequent D. O. read- ings were made to be sure that the supply was adequate for the fish. At the end of one hour the sheepshead and perch used in the experiment were behaving ab- normally. They dashed wildly about, driving into the sides of the tank and rolling on their sides. They showed no signs of being in distress for oxygen. There was no gulping at the surface nor did they gather about the oxygen inlet as they likely would have done had they been in need of oxygen. Soon other kinds of fish showed signs of dis- tress. The crappies, buffalo, perch, and sheeps- head died at the end of 90 minutes with the D. O. at 4.6 ppm. At the end of 6 hours all fish of all species were dead with the D. O. at 4.2 ppm. Similar experiments showed the same effects, in- dicating that the fish had not died from lack of oxygen and strongly suggesting that they had been poisoned. Decomposed algae were analyzed chemically in an attempt to determine whether or not poisons were present. Algae decomposed in closed con- tainers and masses decomposed in open recepta- cles in the sun were used in the analyses. Simple tests showed an abundance of inflammable gas or easses to be produced, at least when the plants decayed in closed receptacles. Since blue-green algae have an enormous protein content it was not surprising that large amounts of protein de- composition products were found. Among these was hydroxylamine. This is soluble in water and is very poisonous, at least for land animals and presumably for fish. While quantitative deter- minations were not successful it seems possible that sufficient amounts of hydroxylamine might be formed by decaying algae in restricted areas in the lake to bring about a poisoning of the fish. A search through the available literature has, to date, offered no information on the possible kill- ing of fish by hydroxyiamine or definite informa- tion concerning the poisoning of fish by blue- green algae at all. Hydrogen sulphide was also found to be given off in large amounts by decaying blue-greens which also might have had detrimental effects. While the experiments strongly suggest poison- ing as the cause of death further observations are needed, particularly on the factors involved in natural conditions. Although there is a strong feeling held by some that copper sulphate is objectionable as an alga- cide to inhibit such detrimental growths of the blue-greens, nevertheless it would seem to have its advantages. In this investigation fish were never found to be killed by copper sulphate nor were there significant detrimental effects on fish- food organisms. If some fish should be killed by improper use of the chemical, certainly many times more fish are killed directly and indirectly by the algae. As far as is known there has been no work on the by-products of blue-green algal decomposi- tion. The biological aspects and the possible commercial use of these products deserves study and consideration. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 95). EXPERIMENTAL CYTOLOGY OF AMOEBA PROTEUS LEIDY Wo. L. Doyte’ Research Assistant in Physiology, The Johns Hopkins University The granular material in the cytoplasm of Amoeba proteus contains the following struc- tures: (1) granules about .25 micron in diameter, called alpha granules by Mast (26); (2) spher- oidal vesicles about 1 micron in diameter, called beta granules by Mast (’26) and secretory gran- ules by Metcalf (710); (3) bodies with all essen- tial characters of mitochondria; (4) truncated bipyramidal crystals enclosed in vacuoles; (These crystals often have small blebs attached to them.) (5) fat globules; (6) highly refractive globular bodies, called Glanzkorper by Greef (’74), spher- ical bodies by Mast (’26), secondary nuclei by Calkins (05), and Monica Taylor (’24), Golgi bodies by Brown (’30), and nutritive spheres by Monica Taylor (7°32). By crushing the refractive bodies under the coverglass and by microchemical tests we have demonstrated that some of them contain a distinct fragile wall of carbohydrate material which sur- rounds a central plastic mass, and that the others are apparently homogeneous in structure. Mast Jury 22, 1933 ] DP COLEEeRING NET 81 and Doyle (732). Those with centers are scat- tered throughout the cytoplasm. In solubility and staining reactions they clearly resemble plastids. (Cf. Zirkle ‘29 and ’33). These will be referred to as spherical bodies A. The homogeneous bodies (spherical bodies B) are found in the food vacuoles. They have the characteristics of the structure called vacuome by Volkonsky ('33). The solubilities and staining reactions of spheri- cal bodies A and B and the blebs on the crystals show these elements to be similar. In attempting to trace the transformation of food into cytoplasm by a study of the origin and Crystals Sph. Bod. A Mito. Present Present Present ” —p » ae Absent ef Absent and cut 4 * out as formed. Present oe Present S. B. A.=Spherical bodies A. Mito. Chilo. = Mitochondria. =Chilomonas paramecium. The relation of mitochondria and crystals to spherical bodies A indicated in the table was con- firmed by three other distinct methods. One of these consisted of direct microscopical observation of operated specimens containing so few spherical bodies A and crystals that each was individually recognizable. It was found by this method that if the specimen under observation contained a Chilomonas food vacuole 16 to 30 hours old, numerous mitochondria were in contact with the wall of the vacuole and that some were leaving and others arriving. It was also found that in the vacuole there was a spherical body B which was decreasing in size. It was further found that the mitochondria which leave the walls of the vacuole come in contact eventually with either a function of these structures we made the follow- ing observations: If an amoeba is suspended in soluble starch and centrifuged, the fat globules aggregate at one end and the spherical bodies A at the other, and the remaining structures in fairly distinct regions between. By operating on chilled centrifuged amoebae some of the structures des- cribed can without serious injury be partially or entirely removed so as to ascertain their function. Such operated specimens were either starved or fed on selected food organisms. The results obtained are presented in the following table: Tood Result None S. B. A. reduced in size. Chilo. S. B. A. increased in size. Crystals arise in food vacuoles. Bacteria S. B. A. increase in size. Crystals decrease in num- ber followed by increased SeBieae Chilo. No S. B. A. formed. ty No S. B. A. formed. 2 S. B. A. formed. spherical body A or a crystal vacuole, that after a considerable number of mitochondria had been in contact with a spherical body A and_ the crystal vacuole, the former was increased in size, and a small bleb had appeared on the crystal, and that this bleb increased in size while the crystal decreased in size until the crystal disappeared and the bleb was indistinguishable from a spherical body A. It was found moreover that crystals arrive in the cytoplasm by subdivision of the food vacuole. (This article is based on a seminar report pre- sented at the Marine Biological Laboratory on July 18.) 1In collaboration with Prof. S. O. Mast. 82 THE COLLECTING NET [ Vor. VIII. No. 64 THE BIOLOGICAL EAbOR Ors. COLD SPRING HARBOR Dr. REGINALD G. HaArRIs Director of the Biological Laboratory The Biological Laboratory at Cold Spring Har- bor commences, with this issue, the experiment of using a definite section of THE CoLLectinG NET. wise to announce the beginning Experiments, like demonstra- tions, have a surprising ability of “going wrong.” The beginning of this particular experiment will be so obvious to all readers of THE COLLECTING Net, however, that we are left no choice but to announce it, and, hence, it seems desirable to give some of the reasons why it is being undertaken. Ever since the Biological Laboratory was trans- ferred from the Brooklyn Institute of Arts and Sciences to the Long Island Biological Associa- tion, some ten years ago, we have been giving sustained thought to the rdle which the Labora- tory should play in the progress of biology. We definitely took the point of view, stated in one of my “reports” published in THE CoLLectinG Net a few years ago, that, although useful ends could be accomplished by the development, at Cold Spring Harbor, of a small edition of the Marine Biological Laboratory of Woods Hole, still, a greater contribution to biology might be made by the development, at Cold Spring Harbor, of a laboratory employing somewhat different methods in the furtherance of the common cause. During the last several years certain steps which have thus far been taken, from time to time, to accom- plish this, have been reported in THe CoLLecTiNG Ner. This has been done with the hope of re- ceiving constructive criticism, as well as of mak- ing a report to additional numbers of biologists, for whom all independent biological laboratories should exist and must function. The latest distinctive move which we have made is concerned with the adoption, as part of our summer work, of a formal method of carry- ing into effect a portion of our policy of foster- ing a closer relationship between biology and the basic sciences, mathematics, physics and chemis- try. This method involves the calling together of a group conference, each summer, in which repre- sentatives of the sciences mentioned will carry on research, confer upon, and discuss, some one phase of modern biological research. The me- chanics of the method, as it is being used here, were described in some detail in the July 8th is- sue of Tue CotnectinG Net, It is not always of an experiment. At that time it was stated that an important part of the plan included the making available of certain results of a given conference, to the pro- fession at large, through the publication of a vol- ume containing the lectures and symposia deliy- ered at the conference, and such parts of the dis- cussion as seemed significant or creative. Since then, an offer to make use of a part of Tue CoLttectine Net, for the immediate publi- cation of papers and discussions, has been ac- cepted. There are certain possible disadvantages con- nected with our acceptance of this offer. Out- standing among these is the possibility that the sale of Volume I of Cold Spring Harbor Sum- mer Symposia in Quantitative Biology may be adversely affected Dy the availability of part of the papers in THe CoLLectine Net.. If this de- velops to be the case the possibility of our meet- ing the cost of publication through the sale of Violuene I, is, obviously, appreciably diminished. On the other hand, we hope that biologists in Woods Hole during the summer, and other read- ers of THe Cottectine Net, will find the papers delivered at conferences at Cold Spring Harbor, of more than enough yalue to counterbalance pos- sible financial loss to the B iological Laboratory. The Biological Laboratory exists for the ad- vancement of biology. The new idea of extended formal conferences, as outlined, has been inaugu- rated for the advancement of biology. We wel- come any practicable means of making the Biolog- ical Laboratory, and results of its work and con- ferences, more generally useful and available. At present THe CoLtLectinG Net seems to furnish a valuable opportunity to do this, particularly in respect to workers at the Marine Biological La- boratory of Woods Hole. At the same time we hope that more workers at Cold Spring Harbor will make use of Tuer CoLLecTING Net, and through its pages become better acquainted with research being conducted at Woods Hole. And finally that biologists, who do not make use of either station, will have available a source of information concerning work being conducted, and ideas being born, at both of these Laboratories. These are some of the reasons why making the experiment of using of Tue Coutecting NET, we are a definite section Jury 22, 1933 ] THE COLLECTING NET 83 THE THEORY OF THE DIFFUSE DOUBLE LAYER Dr. Hans MULLER The Massachusetts Institute of Technology It is a curious fact that the phenomenon which led to the discovery of electric charges, namely the production of electricity by friction, is evea now one of the least investigated and least under- stood fields of physics. We know practically nothing about the exact mechanism producing frictional electricity. We know, however, that friction is not at all required to produce these charges. The mere contact of two materials gives rise to a potential difference. Friction dces not create, but only increases, these potentials. Con- tact potentials exist on every boundary between two different phases, independent of whether one or both phases are electric conductors or insula- tors. The two phases need not even be chemical- ly different, they may only differ in tempe:ature or in cristallographic orientation. According to the laws of electrostatics a po- tential drop in a surface is always connected with the existence of an electric double layer. A po- tential drop creates an electric field, and, accord- ing to Gauss’s law, electric lines of force can only originate from positive electric charges, and they can only disappear at the seat of negative charges. Hence in big. la we must have positive charges where the potential begins to drop, and negative charges where the potential gradient vanishes. Mathematically this relation between potential and charge density p is expressed by Poisson’s equation d*¢ dip = (2) dx? D where x is the normal distance to the surface and D the dielectric constant. The body which is é a FA) = e a x vo z 2g al Bo @ 2 \ ® 2) al*y,e ~e»® ® cS) Ve ® Ns ey 8e 0 ° 3 Weele 2 5 6 G| Oo ®O 8 8 N heal Cao ao —A— AD H ! ' 1 i] nl 1 1 ! | 1 1 \ 1 1 t u a o c Fig. 1. Charge and Potential distribution in an electric double layer. a. General case. b. Helmholtz’s double layer. c. Diffuse double layer, RB — Rigidity boundary. at the higher potential carries, therefore, along the boundary a layer of positive charges, and the other body carries a layer of negative charges, the charge density of both layers being equal. The ensemble of the two layers is called an electric double layer. The distance between the two layers is about the same as the distance in which the entire po- tential drop occurs. In general this distance will be very small, perhaps of the order of magnitude 1 to 100 pp. Due to this small thickness, it is practically impossible to observe the course of the @ (xX) curve, and we must approach the prob- lem from pu-ely theoretical ground. Helmholtz, who first introduced the electric double layer, assumed that the potential drop. can be approximated by a straight line as in Fig. 1b, the eatire drop occurring within a definite region of thickness A. This assumption leads to the locating of each layer of charges within a geo- metrical surface. If the surface is: plane the two layers form a pa‘allel plate condensor, with a separation A between the plates, and we have.the well known equation for a plate condensor 4 TO = ——— (2) D Where € is the total potential drop, o the charge per cm.* on either layer, and D the dielectric con- stant of the medium. between the layers. This equation of Helmholtz gives the impor- tant information, that the contact potential does not only depend on the charges o accumulated on the surface, but also on the thickness A of the double la er. But this theory does not give any infcrmation concerning the magnitude of A. In all earlier investigations 4 was, therefore, con- sidered to he constant, and all changes of € were ascribed to changes of the charge o. Experimental and theoretical evidence points definitely to the fact that Helmholtz’s picture of the double layer is tco simple. Electric charges, whether they are electrens or ions, ave subject to temperature motion. If, therefore, one of the bodies is a liquid or a gas, the charges can not be located in a geometrical plane. A rigid layer is only possible on the surface of a solid where the (adsorption, chemical binding, lattice forces, Van der Waal forces) are so strong that the reduce the temperature motion to a mini- mum. In a liquid or gas, however, the charges are free to move, and the electric layer will be diffuse. The theory of the diffuse double layer was de- yeloped independently by Gouy “ for the case forces 84 THE COLLECTING NET { Vor. VIII. No. 64 in which the charges are electrolytic ions, and by Mie \) for the case in which the charges are electrons in gas or vacuum. For colloidal and biological problems only the first case is of importance. Let us consider, (Fig. lc), the boundary between a solid and an electro- lyte. We assume the solid to be at the higher potential, its surface is, therefore, positively charged. These charges are most probably ions attached rigidly to the surface by “adsorption” forces. Passing from the solid into the liquid we will pass a definite “rigidity boundary.” On the solid side of this boundary the temperature mo- tion is so small that it can be neglected. This boundary need not necessarily coincide with the surface of the solid or its adsorption range, it may lie outside the solid phase. We shall see that in the immediate neighborhood of the surface the electrostatic forces are so large that there the temperature influence is quite negligible. Around the solid there is, therefore, a film of liquid which is rigidly held by the solid. Our rigidity boundary is identical with Freundlich’s “> “Abreiss- schicht.”” The theory of the diffuse double layer is a theory of the electrokinetic potential € only and does not include the phase potential «. The total charge per cm.? on the rigid side of the boundary is denoted by o. This charge is not necessarily adsorbed, but part of the charge may be located between the adsorption range and the rigidity boundary. On the liquid side of the rigidity boundary the electrolytic ions can move freely. According to the law of equipartition of energy each ion has, on the average, an energy of translation equal to o/s ki —6) LOA rexe tive ions will be attracted by the positive surface charge o. The energy of this attraction is z e where z is the valency and e the electronic charge. If the energy of attraction were very large com- pared with the temperature energy, the negative ions would fall into the surface. But the electro- kinetic potentials are usually smaller than 100 Millivolts, hence Near the surface the nega- 0.1 —— = 16 107 erg 300 OC A77 1040 - is usually of the same order of CN A De If, therefore, a negative ion happens to come near the surface it has still enough energy to escape again from under the influence of the at- traction force. The attraction will, however, force it to linger relatively longer néar the surface than anywhere else. Consequently we will find, in the time average, more negative ions near the magnitude as surface than far away from it. The negative ions are ina situation quite analogous to the molecules of air in the atmosphere. In the atmosphere the gas molccules are attracted to the surface of the earth by gravity, but the gravity is not strong enough to force all the molecules to the ground. The consequence of the attraction is, that we have more air molecules, that is, a higher pressure at sea level than at higher altitudes. ‘ The positive ions, on the other hand, are re- pelled by the positively charged surface. While some positive ions will always penetrate near the surface, their number will, in the time average, be small, compared to the number far away from the surface. Far away from the surface the charge/em.® of all negative ions is equal to the charge of all posi- Since near the surface we have more negative and less positive ions than in the solu- tive ions. tion, we have there a surplus of negative charges. These charges form the negative layer of the electric double layer. This layer is not located im We have an ‘Sonic atmosphere” or a “diffuse double layer.” The irregularity of the Brownian motion makes it impossible to calculate at any instant the exact distribution of the ions, but we are able to give the time average of the distribution of the charges, and hence calculate the time average of the potential. The instantaneous potential differ- ence ¢ will fluctuate very little and very rapidly about this average value, and what we measure is this time average. a definite plane, but is distributed. Mathematically the problem is best formulated by using the procedure given by Mie and Debye 4) We use the analogy with the pressure distri- bution in the atmosphere. In the atmosphere we have the barometer equation p n Meh meh log —_ = log = = Po No IR a kT Where p is the pressure, n the number of mole- cules at the height h, and po, no their values at the elevation h=o. M is the molecular weight, g the gravitation constant, R the gas constant and T the absolute temperature. m=M/N, (N= Avo- gadro’s number) is the mass of oné molecule and k=R/N=1.37 107 is Boltzmann’s constant. In this equation gh is the potential of the gravita- tional field, and meh the potential energy of the molecule. In the case of ions we have the same equation, except that the gravitational potential is replaced by the electric potential ¢, and instead of the mass we have to introduce the charge of the oe Z 2599337) iuphse ion. Wor any arbitrary kind of ions of valency z, we have, therefore, Nj Zi Cb log = n;° Ike 10 or (3) ZC 7) in, = Tue kT This law can also be derived in a more general way from Maxwell-Boltzmann’s principle. nj is the number per cm.® of ions of valency z (7; with the sign corresponding to the charge of the ion) in a point where the average electric poten- tial # exists. nj,° is their number where ¢ = O, that means in the electrolyte far removed from the surface. 1,° is determined by the molar con- centration ¢, according to = (oH Gfe) (024 1; = ¢] The charge density p is then Zed p = 3nje z= n° ez, e ——- — ihe (4) Introducing this in Poisson’s equation (1) gives the fundamental. diffe srential equation of the po- tential distribution in the diffuse double layer Zed d2¢ fie: kT = Sime Ze dx? D If we replace d* die by the Laplacian A ¢ this differential equation holds for any kind of surface. ; Multiplying (5) by d@ and integrating from xX = to an arbitrary value x gives, d¢ Koo = Ova tcl 140) dx = FAS d clicsan= 47k T ah COLLECTING NET | 85 At the rigidity ijaunceee a= = ¢ ona Boeordiie to Gauss’s law d¢d tro dex S D hence (7) —4 e¢ Dik eT _— — = n° (e 2a where o has the same sign as ¢. This equation for the plane double layer permits one to calculate the surface charge o from the measured concen- tration ¢; and ihe electrokinetic potential ¢. There are two important cases for which this formula can be simplified. Case 1. The potential € is so small that e€ z,—— < 1 kT That is the case if z € is smaller than 25 Milli- volt. We can then develop FH ONG ke i en ef € = 1—7z,—— 4+ & (4,— = ket kT Introducing this in (7) and taking into account that ¥ ni z; = O (which expresses the fact that the total charge in the electrolyte is zero) gives 4 To 1 4 TO D k D where 1 47e? —— =) Ae (9) = SIDI AD We find again Helmholtz’s equation. In first ap- proximation a diffuse double layer is equivalent to a Helmholtz double layer, but the thickness of the double layer is now determined by (9). In- troducing the value for water at room tempera- ture one gets 4.32 10% cm Nice where yj; is the concentration expressed in micro- mols per liter. We observe that, for concentrations smaller than 1/10 molar, the thickness of the double layer is considerably larger than the diameter of an jon, = l | 86 THE COLLECTING NET [ Vor. VIII. No. 64 This fact justifies the statistical method employed. For higher concentrations, however, the calcula- tions are questionable. Introducing the series for ed Ie ah Zj e in equation (6) we get by a simple integration* the potential distributions in the double layer Reo pesh aca (10) The potential ¢@ decreases exponentially with the distance x from the rigidity boundary. At a dis- tance x = 2 the potential has the value ¢ = 6/2.718 “and at “the: distance x = 2X, o = 6/ (2-718)? ete. According to equation (9) the thickness of the double layer decreases with increasing concentra- tion, the decrease being the faster the higher the valency of the ions. Since the observed € poten- tials have these same properties, we believe that the variations of the € potentials are primarily due to the change of the double layer, and to a much less extent due to changes of the adsorbed charges. Introducing (8) in (3) we can finally deter- mine the distribution of the ions near the surface. Ze a te —Kx hear ny — tee * Introducing eg — Zi k T ed ed e iy yee Wg ey hea eae ed aay Ve {-—— 30 z+% ( ) Sze | kT ein J and since the first sum vanishes d¢ [Amerie F [aA Re, ce ee which leads directly to (8), (9) and (10). This double exponential gives a very rapid in- crease in the number of negative ions and a fast decrease in the number of positive ions near the positively charged surface. The first approxima- tion is, however, only valid if z € < 25 Milli- volt, and this condition is seldom satisfied. It is therefore, important to study the more general case where an accurate solution can he given. Case 2. If the electrolyte consists of only two kinds of ions of the same valency we have Vie ae Z = — Zs = 2 and equation (7) reduces to IDK at ze o = 2K — sinh c 4rez ANAL Using the abbreviation ze 3 e=Sr ear we can write this D sinh W/s C—— G Kee dr W/> Comparison with (8) shows that the first approx- imation leads always to too small values of o. In order to get the correct value of the charge the results of the first approximation have to he multiplied by a correction factor which depends on the value of W. Seme values of this correction factor are given in Table 1. TABLE 1. sinh W/s sinh W/» Ww a Ww aa V/s V/s 0.6 1.015 7 4.73 0.8 1.027 8 6.83 1.0 1.042 9 10.0 2.0 7S 10 14.84 3 1.42 7 33.62 + 1.81 14 78.33 5 2.42 16 186.31 6 3.34 18 450.17 20 1101.32 Contrary to the general belief the correction is quite appreciable as soon as ¢ is larger than 50 Millivolt for monovalent, and more than 25 Milli- Jury 22, 1933 ] THE COLLECTING NET 87 volt for bivalent, ions. The integration of equa- ed tion (6) is, in this case, also possible and gives* WwW (x) =z— kT 4kT hee ee then ‘ o= tangh-1 A e (11) % — 4 tangh 1 e —* (X—%o) (aul) Ze Where tanght is the inverse function of the hy- perbolic tangent and A is an integration constant determined by the € potential Z€ A = tangh é 4k T For a discussion of (11) it is better to substitute A = e*xx, and use again *With the assumption Hie lo =) 1, za = —- Zo = Z equation (2) is d¢ 4xrkTn Y, ( yr = z dx D ed ed + z7—— —2Zz -) k T k T (e —2+e But e¥Y—2+e¥ = (ety?—e-3/7)? — 4 sinh 7 ¥/. Hence d¢ kT ze =—2k sinh dx ez Z kali From which follows the result (10). Introducing ze v=— > gives kT dw -—— = — 2x sinh W/s = — 4« sinh W/, cosh W/, dx Whence d (U/, ) = «dx sinh ©/, + cosh @/, Or d (tangh W/,) = fe fall Ss tangh W/, Hence by integration log tangh W/, = — « x + const. Which leads directly to the above result. This function is plotted in Fig. 2 and compared with the first approximation (8) which in the same notation is (8’) Ui ermak (x—x, ) 13 10 7) / Esa a Fig. 2. Comparison of potential curves in a plane diffuse double layer using the first approximation and the accurate solu- tion for a Z - Z valent electrolyte. We observe that the approximation is only good as long as W < 2. For large values of © the potential drop is very much steeper than given by the first approximation. For instance the drop from w = 20 to = 10 occurs in the distance 1/100 A and the drop from ¥” = 40 to ¥ = 20 in less than '/so000 A. But usually A<10° cm. is of the size of the ionic radii. Consequently the theory of the diffuse double layer must break down for high potentials. We can interpret this fact in a more e- lucidating way. Let us assume that the theory of the diffuse double layer could be applied for high potentials W and that it did hold for the e-poten- tials. Weusethe reasonable values e=1%, Volt, v= 20,’ =10%cm. Since W drops to half its value within a distance 1/1900 4 = 10% cm we get near the surface of the adsorbed charges an elec- tric field strength of VA —— = 25 Million Volt/cm. 108 This result justifies our statement that the field near the surface of the particle is tremendous. It 88 THE COLLECTING NET [ Vor. VIII. No. 64 is so large that it annihilates any temperature in- fluence. We have no knowledge of the behavior of matter in such strong fields, but theoretical speculations lead us to believe that these fields not only will deform the ions, but will also act on the water molecules. The field will line up the elec- tric dipoles of the water and hold the molecules in a definite orientation. Connected with this ef- fect is a large hydrostatic pressure which will be discussed in another paper. In the region of these strong fields the liquid will, therefore, ad- here to the solid and the rigidity boundary is moved towards the liquid. The € potential is, therefore, not so large as the total potential drop e. The largest values € can assume is about 100 Millivolts, corresponding to maximum field strength of 100000 Volts/cm. If the surface is not plane, but spherical, as in the case of colloidal particles, or cylindrical, as in capillaries, the differential equation (5) has only been solved for the case Z.€& Breik, k T The result is in both cases the same: The diffuse double layer is equivalent to a Helmholtz double layer of thickness A='/x. For the sphere the two layers form a spherical condensor, and hence 4aro A © Spey: and for the cylinder 4ao0 ¢ = — r log (1+ A/r) D where r is the radius of the sphere or the cylin- der respectively. Where ¥>1 the problem of the spherical double layer has been investigated by La Mer, Gronwall and Sandved"®) and by the writer.'°) The first three authors gave a series development of W, while I developed a graphical method. It is found that the first approximation is not sufficient in quite the same way as for the plane double layer. Using this graphical method I was able to show, that measurements of the ¢ potential of an As» Sz colloid, carried out by Freundlich and Zeh, could be satisfactorily explained by assum- ing that the charge o remained constant. The theory does not only give the correct dependence of the concentration, but also of the valency of the ions. According to the approximate theory a four valent ion, for instance, should have twice as strong an influence as 4+ monovalent ions. Ex- perimentally one finds, however, that the influence of a 4 valent ion is about 2000 times stronger than a monovalent ion. The higher approxima- tion is able to explain this difference, and gives results in agreement with the observation. Fin- ally, the theory is also in good agreement with the observed temperature dependence of the € po- tential. Burton found that the cataphoretic mo- bility of colloids varies with temperature as the inverse of the viscosity. This indicates that the product € D must be constant. Assuming o« to be constant we have 47e? SS > n° 7 = |! V DT ok ¢D=47e and we find indeed that \/DT varies in water between O and 100° by less than the experimental error of Burton’s measurements. Since the theory of the diffuse double layer is based on generally accepted principles, and ex- plains the outstanding properties of the electro- kinetic potentials, we should accept it as correct. The theory, however, does not explain individual properties of the ¢ (c) curves, like maxima and isoelectric points. These properties must be ex- plained by changes of the adsorped charges. Discussion Dr, Fricke: Regarding your discussion of Freundlich and Zeh’s measurements, does your assumption, that the charge is independent of the electrolyte concentration, signify a belief that the charge is chiefly due to the adsorption of ions other than those in the solution, as, for instance, H_ ions. Dr. Miiller: The assumption of a constant charge is not necessarily correct. Freundiich and Zeh’s data agree with this assumption only, if the particle radius is 15.8. If the particles were smaller we must assume an increase, if they were larger we must assume a decrease of the charge, with increasing concentration. But since a radius of 15.8up leads to constant charges, independent of the valency of the ions, it is most probable that this assumption is here correct. The nature of the adsorbed ions is probably determined by the preparation of the colloid, they are not ions of the added electrolyte. Dr. Abramson: In general all surfaces can be divided into two groups. In the first group are the inert surfaces such as quartz and paraffin oil. The charge of these surfaces is particularly sensitive to changes in the salt concentration, and, as Dr. Muller and I have shown, the charge increases with increasing salt concentration reaching a lim- iting value. The charge-concentration curve is very much like the Langmuir adsorption isotherm. In the second group, the charge seems to be mainly determined by particular ions. That is, Jury 22, 1933 } THE COLLECTING NET 89 a protein surface has its charge determined by the activity of the hydrogen ions. Of course salts also shift the isoelectric point of proteins, and, consequently, the salts may also modify the charge of proteins. But in spite of the fact that there is no sharp line between these two groups, the description of the change in concentration curves, just given, is most useful. Dr. Fricke: What is the order of magnitude of the thickness of the adsorbed water layer? Dr. Miiller: This layer is probably monomole- cular, its thickness is of molecular dimensions, as calculations of Gyemant show. Dr. Fricke: What is the present state of the idea that the electrostatic image force is responsi- ble for adsorption ? Dr. Miller: For the theory of the ¢ potential, the image force does not have to be considered, since Poisson’s equation is used, and hence all electrostatic forces are taken into account. For the adsorption process, the image force is im- portant. It is the basis of the adsorption theory of Jaquard and Huckel. Dr. Cole: Since the image force depends on the dielectric constant of the solid, and decreases rapidly with the distance, it should be considered as a molecular force of the type of Van der Waal forces. Dr. Curtis: What value did you use for the dielectric constant of water, in view of the large fieldstrength at the double layer ? Dr. Miiller: li we neglect the hydration of the ions in the double layer, the use of the ordinary dielectric constant is probably justified in the entire range of the diffuse double layer, because the fieldstrength does not reach values above 100000 Volt/em. Within the rigidity boundary, however, the dielectric constant must be smaller, since we will get dielectric saturation. The cor- rect way to treat this problem is by using a molecular, rather than the classical, theory of dielectrics. This was done by Gyemant. Dr. Blinks: Is the rigidity boundary a definite surface, or is it also diffuse? Dr. Miiller: Since the electric fieldstrength diminishes very rapidly with the distance from the surface, the rigidity boundary is probably very sharp. Assuming a diffuse boundary, would create great theoretical difficulties. It is, however, feasible that the distance between solid and rigid- ity boundary changes with the electrolyte concen- tration. Dr. Cole: The existence of a rigid layer around the particle, and the large hydrostatic pressure in the rigidity boundary, would justify the use of the ordinary viscosity in the formula for the cataphoretic migration speed. Some ex- periments by Bond, on the apparent resistance of spheres of liquids, give the result that, above a certain radius, not only the viscosity of the ex- terior, but also the viscosity of the interior, plays a role. In the case of small particles the exis- tence of the rigid layer probably eliminates the influence of the viscosity of the interior. Dr. Abramson: Dr. Miller’s rigidity boundary is probably only acceptable for solid surfaces. I do not think that large oil droplets, such as those investigated by Mooney, can be treated in such a fashion. Internal rearrangements of the oil drop itself, incidental to the bodily movements of the drop, can certainly modify the chemical constitu- tion of the surface. Dr. Mudd: Does the theory of the diffuse double layer apply also to instances where the rigid part of the double layer is composed of ionogenic matter, such as protein? That is, are the surfaces of the material itself ionized ? Dr. Abramson: 1 would like to point out to Dr. Mudd that this theory actually has its best support in experiments with proteins. Using this theory 60 per cent. of the theoretical absolute value of the charge of protein molecules was cal- culated from electrophoresis measurements. Dr. Cole: Is there any justification for as- suming the e potential to be 44 Volt or larger? Dr. Miiller: Jf the measurements of e are correlated with the electrocapillary curve, values of this magnitude are found. It is, however, cor- rect that the use of the capillary electrometer is not free from objections. Dr. Fricke: tention to electric polarization as a I should like to direct your at- means of refer to polarization of the irreversible type, as observed studying the diffuse double layer. I at a metal electrode in an inactive salt solution. As you know, the polarization is equivalent to a capacity, the polarization capacity, in series with The po- larization capacity may be considered to be a a resistance, the polarization resistance. measure of the reciprocal of the thickness of the double layer which is built up by the electric cur- rent. Measurements of the polarization capacity for different frequencies of the electric current give the thickness of the double layer as a func- tion of time, and show a high value initially, which decreases within a fraction of a sec- 90 THE COLLECTING NET [ Vot. VIII. No. 64 ond to a constant value. Presumably, the decrease is due to adsorption, and it is in- teresting to note that the decrease over a considerable range of time takes place ac- cording to t*, where x is often about .30. This equation is similar to one used by Freundlich to account for adsorption as a function of time. These remarks must not be taken to mean that a complete theory of this type of polarization is available, but their purpose is to direct your at- tention to an experimental method which appears promising for the study of the diffuse double layer. LITERATURE 1. A. Gouy, J. de Phys. (4) 9, 457, 1910. 2. G. Mie, Handbuch der Radioaktivitat und Electronik. Vol. 6. 3. E. Freundlich, Kapillarchemie Vol. 1, Leip- zig 1932. 4. P. Debye and E. Huckel, Phys. Z, 24, 185, 1923. 5. V. LaMer, T. H. Gronwall and K. Sandved, Phys. Z., 29, 358, 1928. 6. H. Muller, Kolloidchem. Beihefte, 26, 1928. THE THEORY OF ELECTROPHORETIC MIGRATION Dr. Hans MULLER The Massachusetts Institute of Technology A rigorous test of the theory of the diffuse double layer requires the knowledge of the elec- trokinetic potential € as a function of the elec- trolyte concentration. Using this theory, it is then possible to calculate the charge of the colloidal particles. The ¢ potential can be measured with the help of any one of the electrokinetic phenom- ena. Frequently, however, the method of the cataphoretic migration is the only experimental method possible. This is the case in respect to many biological systems, such as bacteria or blood corpuscles. Electrophoretic or cataphoretic migration is the phenomenon of the migration of colloidal parti- cles in an electric field. All the theories developed so far give the result, that the velocity V of the particle is given by XDé Vv = ——_ (i) Kary where X is the strength of the applied field, D the dielectric constant of the liquid, and 7 its viscos- ity. According to this formula a particle moves towards the positive pole, if its potential, and hence its charge, is negative. Its velocity is pro- portional to the € potential. The variation of the velocity gives directly the change of ¢. For a quantitative investigation, however, it is of the greatest importance to find the absolute values of ¢. This requires the knowledge of the numeri- cal constant K introduced in equation (1). Un- fortunately, the various theories lead to different values of this constant. I propose, therefore, to compare, here, the various theories, their assump- tions, consequences and limitations. The purpose of this study is to make a decision concerning the numerical constant K. If a spherical particle of radius R and carrying a positive charge O is placed in a uniform electric field of strength X it will come under the influ- ence of a force OX. This force will produce an accelerated motion of the particle in the direction of the field. If the particle is suspended in a liquid, the motion will produce frictional forces proportional to the instantaneous velocity of the particle. The friction will decrease the accelera- tion, and finally cancel it entirely, when the fric- tional force is equal to the accelerating force. It can be shown that, for colloidal particles, this state is reached in an immeasurably short time. We observe, always, the unaccelerated state in which the particle has a steady velocity V. The frictional force on a spherical particle is, according to Stockes, By == Oana, Hence we have for the steady state OX — 67 7RV and x ao ———————— a (2) 677 R The potential ¢, of a sphere in a medium with the dielectric constant D is O ~ fo = (3) DR and the above equation (2) can, therefore, also be written XD& Ves 2 (4) Gen, This equation is correct for a sphere in a perfect- ly insolating medium. For a colloidal particle this equation is, however, not valid. Neither Stockes’ law, nor equation (3) for the potential, is appli- cable. This is due to the fact that a colloidal par- ticle is surrounded by an electric double layer. As discussed in the preceding article, the positive charges of the particle attract the negative ions of the electrolyte, and repel the positive ions. The Jury 22, 1933 ] THE COLLECTING NET oh liquid in the intermediate neighborhood of the surface is thus negatively charged. The electric field does not only act on the positive charge of the particle, but it acts also on the negatively charged liquid. The liquid will move in the di- rection opposite to the motion of the particle. The velocity of the particle, relative to the immediately surrounding liquid, is, therefore, increased. The friction force depends, evidently, on this relative velocity. Stockes’ force F, must, therefore, be replaced by a larger frictional force F. On the other hand, the electric double layer changes also the potential of the particle. In the first approximation we have Q l 1 =- (5) == b Dieeesyy e118) where A is the thickness of the double layer. The increased friction force will decrease the velocity V of the particle. Equation (2) can not hold. But according to equation (5) the potential also is decreased. There exists, therefore, the possibility that these two effects cancel cach other in such a way that the final result (4) 1s, never- theless, correct. This is certainly the case if the thickness of the double layer is very large com- pared with the radius of the particle. In this case the negative charges are far away from the par- ticle, and the velocity of the particle, relative to its immediate surrounding, is the same as in Stockes’ law. According to (5) the difference between ¢ and ¢, vanishes for A >> R. Equa- tion (4), giving K=6 is, therefore, correct, in the limiting case where the thickness of the double layer is much larger than the radius of the par- ticle. Any theory of the cataphoretic migration speed must, for this limiting case, give K=0. It is possible, but not necessarily correct, that equation (4) can hold for any arbitrary thickness of the double layer. This would be the case, if the electrophoretic motion of the liquid increases the friction force according to the law Pale S/n) (6) We would have then OX = 677 RV (1+*/A) and using (5) we would get again XUDIG V= —— 6 Tr 1) There are, however, no reasons which would justify equation (6). We can not even expect that the new friction force can be represented as the sum of Stockes’ force F, and an additional electrophoretic force, This is due to the circum- stance that the equations of hydrodynamics of a viscous fluid are not linear, and the principle of superposition is not valid. To give a theory of the cataphoretic migration speed, it is necessary to find a solution of an entirely new hydrodynam- ical problem different from Stockes’ law. One has to find a solution of the differential equation o Vx O° vx O° VX \ n( | + ) 352 éy- OAR dp + ie == ©) §* and two analogous equations for vy, v, satisfying the condition of incompressibility 8 Vx 8 Vy + | 3 x 6 We Oz ==(0) And the boundary conditions, that at imfinity Vs=V; ‘Vy=0; Vz==0, aiid at she ssumtace) (of tlie rigidity boundary vx=vy=v,=o0. In these equa- tions vx is the velocity of the liquid in the x direction, p the hydrostatic pressure, and F the force exerted on the liquid by the electric field. The first solution of the problem was given by von Smoluchowski!) in 1903. In this derivation he makes the following assumptions. 1. The presence of the particle produces a dis- tortion of the electric field in such a way, that the electric current passes tangentially along the sur- face of the sphere. 2. The double layer is so thin, that the electric field can be considered to be parallel to the double layer in the entire range of the latter. While Smoluchowski considered a Helmholtz double layer, this is not important. His derivation holds for any double layer satisfying the above condi- tion. This was shown later by Gyemant.* 3. The electric field does not deform the doubie layer. Smoluchowski finds the solution x Dé V = 4a He even could prove that this answer is quite in- dependent of the shape of the surface. Spherical, ellipsoidal, cylindrical or arbitrary shaped parti- cles should have the same migration speed. Smoluchowski’s formula is different from Stockes’ formula by the factor 47 instead of 67. It does not. therefore, satisfy the important con- dition of the limiting case. But this is not re- auived, since assumption 2 limits the formula to hold only for very thin double layers, It can not 02 THE, COLLECTING NE = Vow. Vil. No. 64 be extrapolated to thick double layers. Smolu- chowski realized this deficiency of his equation, but he could not explain it. It was, therefore, of great value that Debye and Huiickel® attacked the problem again in 1924. They solved anew the system of differential equa- tions, but without making use of assumption 2. They proved that, however thick the double layer might be, an equation of the type Dace Vc err is always valid, but they found that the constant K should vary with the shape of the particle. For the sphere, Hitickel calculated the value K=6: Hiuckel’s equation satisfies the limiting case, but his result is in definite disagreement with Smolu- chowski’s. It is not correct to explain this dis- crepancy, as Freundlich does, by saying the factor 4 is valid for a rigid, and 6 is valid for a diffuse, double layer. Htckel’s calculation should hold for any kind of double layer, and so should Smoluchowski’s result, provided the diffuse double layer is thin enough. This discrepancy gave rise to a long controver- sy. Eliminating the possibility that either calcu- lation contains a mathematical error—which is not the case—the question can only be settled in two ways. One way is the experimental method. This method, of course, can not tell which factor is the correct one, but it can definitely settle the ques- tion whether or not the factor changes its value with the form of the particle. The frst compari- son of cataphoretic migration speeds of different- ly shaped particles was performed by Van der Grinten.* He verified Debye’s contention. But Abramson® could show that the interpretation of his results is not correct, and Abramson gave definite experimental proof that Smoluchowsk1’s, and not Debye’s, conclusion is correct. The mi- gration speed does not vary with the shape of the surface. While this result indicates that the fac- tor 4 might be correct, it does not exactly dis- prove Htckel’s calculation, and it does not e@x- plain. why Smoluchowski’s result does not satisfy the limiting case. The second way to solve the dilemma is to com- pare the assumptions made in the two derivations. We have mentioned, already, that the change of assumption 2 can not be the reason for the dis- crepancy. Assumption 3 is also accepted in Debye’s and Htckel’s paper. However, assump- tion 1 is different in the two derivations, and this accounts for the different results. It is rather curious that this difference was only discovered in 1931 by D. C. Henry. Debye and Huckel as- sume, that the particle does not distort the electric field. If the particle is of a non-conductive ma- terial, a distortion certainly does exist. The rea- son why Debye and Huckel neglected this, is probably to be found in the fact, that they were primarily interested in the electrophoretic force acting on an ion. An ion is so small that it does not distort the outer field. The entire double layer around an ion is in a uniform field, and Hiickel’s result is unquestionably correct for this case. If the particle is, however, of colloidal size, then the question arises: is the distortion of the field large enough to change the result? The answer can evidently be given as follows: if the thickness of the double layer is so large that the greatest part of the double layer is so far away from the particle that the distortion of the field is negligible there, then Htickel’s result is _ still valid. Now, the larger the particle, the farther the distortion of the field will extend in the liquid. Consequently, Htickel’s result can he accepted only if the thickness, A, of the double layer is large, compared with the radius R of the parti- cle. If, however, the ratio A/R is small, then the distortion of the field is important. D. C. Henry has studied this case, and again solved the differ- ential equation, using a general double layer not subject to restriction 2, but taking into account assumption 1. He verifies Smoluchowski’s result if Nie el Henry finds, as we should expect, that Htickel’s result is valid if A/n is large. He eal- culates that A must he 600 times larger than the radius of the particle, in order that the factor 6 is justified. For values of A/R between 1 and 600 the numerical constant increases from 4 to 6, the variation being calculated by Henry. Henry’s paper solves the dilemma between Hiickel’s and Smoluchowski’s result. His work shows, _ that both results are correct in two different limiting cases, and his answer evidently satishes the con- dition of the limiting case. | wish to point out, however, that Henry’s werk should still be improved in two directions. In calculating the distortion of the electric field around the particle, Henry uses the classical mothod. He considers the liquid as a continuum with a uniform conductivity. This is evidently not quite correct. The concentration of the elec- trolytic ions is different in the double layer from that in the electrolyte. Hence the conductivity will change near the surface of the particle. The experiments on surface conductivity give definite evidence of this change. The distortion of the electric field can, therefore, be considerably dif- a Jury 22,1933] THE COLLECTING NET_ 93 ferent from the one assumed in Henry’s paper. This circumstance does not destroy the validity of the two limiting laws, but it will give a different change of the numerical factor with A/R. Even less satisfactory is Henry’s consideration of the case where the particle is an electric conductor. While Henry realizes, that this case is probably not in accord with his theory, I would like to point out that he neglects the fact, that real elec- tric charges of opposite sign are accumulated on the boundary where the current enters and leaves the particle. These charges would produce an asymmetric double layer. A second improvement is necessary, due to the third assumption, namely, that the outer field will not change the charge distribution in the double layer. fact that Henry accepts the This as- sumption is probably justified for very thin double layers. If the thickness of the double layer is small, the electric field in the double layer is large, (over 1000 Volt/cm.), and the small outer field, of a fraction of a Volt/cem., can have no appreci- able effect. If, however, the thickness of the double layer is large, a large region of the ionic atmosphere is under a weak field, and the outer field can pull the ions of different signs to op- posite sides of the particle. The double layer is then asymmetric. Debye and Hutckel have cal- culated this effect for ions. The asymmetry produces an additional force on the particle, de- creasing its velocity. Using Debye’s and Huckel’s result it can easily be shown that this effect is alsovof importance for colloidal particles. Mooney * reports a new formula for the electro- phoretic velocity, which takes into account this effect, but since he does not give its derivation, it is not possible to discuss his method. Since the asymmetry reduces the velocity, particularly where Huckel’s solution is otherwise valid, we must expect that the numerical factor can reach values even larger than 6. There is, finally, an additional difficulty, which will modify the electrophoretic migration speed. In the formulation of the hydrodynamical problem, it was supposed that the electric field acts on the liquid carrying the charges of the double layer. Actually the field acts on the ions. The question arises, therefore: is the entire force exerted on the ions, transferred to the liquid, or is only a cer- tain part of this force active. At present this question can not be answered. We have, how- ever, good reason to assume that, in a very thin double layer, the entire force is transferred from the ions to the liquid. Summarizing, we can, therefore, state: The classical formula of Smoluchowski is cor- rect, if the thickness of the double layer A, and the ratio A/,z, are small. Its exact range of va- lidity can not be given at present, and it is doubt- ful whether it holds for conductive particles. Out- side of this validity range, the numerical factor is larger than + and may reach values larger than 6. Within its validity range, Smoluchowski’s formu- la is probably applicable to any shape of surface, but outside this range the numerical factor will depend on the shape of the particle. Discussion Dr, Abramson: You brought out that the nu- merical factor may be as high as 10, if the theory is corrected. Using Henry’s approximation and Debye’s theory to calculate the charge, I get in- stead of a charge of 5 electrons on the egg albu- men only 3 at p H. 4.0. If the factor 10 were used perfect agreement would be obtained be- tween the charges calculated from electrokinetic, and thermodynamic, measurements Dr. Fricke: \Nould you like to discuss the theoretical case produced when the charge on a rather large particle, such as a protein molecule, consists of a few electrons only ? Dr. Miller: Vhe theory considers only the average value of the € potential. It is conceiv- able, that the potential varies along the surface, particularly if the charge consists of only a few ions on the surface of a large particle. But, due to the temperature motion, the particle will rotate, and only an average value can be observed. Dr. Mudd: Ft would be of interest to calcu- late the case, where different parts of the particle have a different € potential. Rabbit spermatozoa. for instance, dead or alive, migrate in a cataphor- esis cell tail foremost. When the direction of the current is reversed, they are slowly reoriented until they again move tail foremost. This must be due to the tails having higher € potentials than the heads. Dr, Abramson: 1 have confirmed Dr. Mudd’s explanation by studying amino-acid needles hav- ing small oil droplets attached to one end. Similar orientation phenomena were observed. Dr. Cole: There is one thing that is extreme- ly interesting in this apparent contradiction be- tween the case of the thin, and the case of the thick, double layer. The difference lies in the force on the double layer. If the double layer is thin, it is in the field existing near the particle. If it is thick, it 1s in the uniform field. If one has a nonconduective sphere in a continuous medium, one finds that the maximum field strength, which occurs on the equator normal to the field, is exactly 50% greater than the field at some dis- o4 _THE COLLECTING NET [ Vou. VIII. No. 64 tance from the surface. This difference of 50% accounts for the difference between 47 and 67. Dr. Abramson: Do you mind discussing the fact that the orientation of cylindrical particles does not influence their electric mobilities ? Dr. Miiller: If the long axis of the cylindri- cal particle is parallel to the electric field, it offers a small hydrodynamic resistance. If the axis forms a right angle with the field this resistance is much larger. On the other hand, the electric field will be less distorted in the first case than in the second case. The smaller the hydrodynamic resistance, the larger the mobility, and, as we have seen, the smaller the distortion of the electric field, the larger the mobility of the particle. In Smoluchowski’s case of a very thin double layer, these two effects just cancel each other, since an orientation with a small hydrodynamic resistance produces also a small distortion of the field. Dr. Abramson: In other words, if the factor 47 1s valid, you would expect that the electric mobility should be independent of the orientation. Dr. Miller: Yes. Tf, however, the double layer is thick, the factor will probably depend on the orientation. For a very large thickness of the double layer, the mobility will be the larger, the smaller the hydrodynamic resistance. Dr. Cole: In the matter of independence on size and shape, when one has a thin double layer, 1 should like to mention that, one of the simple ways of mapping hydrodynamic lines of flow con- sists in building up a model of an insulator, and then sending the electric current in at the place where the current of fluid would come in, and take it out where the fluid would come out. The hydrodynamic lines of flow coincide, then, with the flow of the electric current. The same differ- ential equations apply to the viscous flow, that ap- ply to the electric flow. The hydrodynamic re- sistance is, therefore, calculated with the help of the same picture as that used for the calculation of the electric stream lines. Consequently, one has no effect of the shape of the particle. As a corollary to that, there is no orienting effect on the particle, as long as the surface charge is uni- formly distributed. Dr, Miiller: This analogy is correct only out- side of the double layer, where the electrophore- tic force can be neglected. 1. M. Von Smoluchowski, Anz der Akad. der Wissensch. Krakau 1903, p. 182. 2. A. Gyemant, Grundzuge der Kolloidphysik, Vieweg, Braunschweig 1925. 3. P. Debye and E. MHuckel, Phys. Z. 25, 49 (1924) and E. Huckel, Phys. Z. 25, 204 (1924). 4. Van der Grinten, J. de Chem. Phys. 23 14 (1916). 5. H. Abramson, J. Phys. Chem. 35 289 (1931). 6. D.C. Henry, Proc. Roy. Soc. London, Series A, (1931) Vol. 133, 106. 7. Mooney, M., J. Phys. Chem. 35 331 (1931). THE REACTION OF KIDNEY TUBULES TO NEUTRAL RED AND TO PHENOL RED Dr. Ropert CHAMBERS Research Professor of Biology, New York University Irom observations on fragments of chick mesonephros in tissue culture, it has been shown definitely that phenol red, the salt of a highly dissociated sulphonated acid dye, is picked up by the cells of the proximal tubules, passed into the lumen of the tubules, and accumulated against a considerable concentration and pressure gradient. This action takes place in spite of varying con- ditions of acidity within and without the tubule as long as the metabolic activity of the renal cells is maintained. Respiratory poisons, such as KCN, CO, H2S and cold, upset the oneway passage of phenol red through the cells. This effect can he reversed when inhibiting conditions are removed. With neutral red, a hasic dye salt, the situation is quite different. It is a vital stain and behaves as such only under certain pll conditions, irres- pective of the metabolic activity of the renal cells. It penetrates and stains indiscriminately all the cells of the tubule, proximal, distal and of the collecting duct, and then only when the acidity of the environment is less than the acidity of the interior of the cells. It will not pass into the lumen of the tubule unless the acidity of the fluid in the lumen is more than that of the surrounding cells, a condition which obtains in tissue culture only in the case of the distal tubules. In brief the passage of phenol red into the tubule depends upon the metobolic activity of the renal cells while that of neutral red is controlled by differences in acidity irrespective of whether the cells are narcotised or not. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 14.) 22, JuLy 233 | THE COLLECTING NET : _ 95 THE EFFECT OF RESPIRATORY POISONS AND METHYLENE BLUE ON CLEAVAGE OF CERTAIN EGGS By M. M. Brooks Research Associate in Biology, University of California In these experiments, cyanide and carbon mon- oxide, which are well-known from Warburg’ work to poison the respiratory enzyme, were used. A brief account of the role of methylene blue in counteracting the effects of these poisons follows. That methylene blue is an antagonist for cya- nide was first shown by Thunberg in 1917, using succinic acid and a dehydrogenase. He found that the oxidation of succinic acid which was stopped by cyanide, was resumed when methylene blue was also present. In 1924 St. Gyorgy, using Thiinberg’s idea, showed that the O consumption of crushed muscle tissue could be restored after it was stopped by cyanide when methylene blue was added. In 1926 Sahlin used this idea in white mice, injecting them with a lethal dose of cyanide and then causing them to recover when methylene blue was injected. This is the first time that whole animals had been used in this connection. In 1928 Barron and Harrop showed in beautiful experiments using living cells in vitro, that the oxygen consumption of these cells could he restored by methylene blue after it had been stopped by cyanide. In 1931, Eddy again showed this antagonism in dogs. In 1932 I showed the same effect,in rats, and then suggested from all this evidence that methylene blue should be used in human cases. This suggestion was relayed by Dr. Hanzlik to Dr. Geiger in San Francisco five months later when an emergency case was brought in. In the case of carbon monoxide, omitting the early contributions which showed that CO stopped O consumption, Warburg showed in 1930 that methylene blue in certain concentrations antago- nizes the effects of CO in living cells in vitro. On the basis of these results, I injected rats with methylene blue after they had been poisoned with CO; immediate recovery followed. This was pub- lished in 1932. These were the first experiments using whole animals in this connection. I then suggested that this antagonism could be applied to human cases in monoxide poisoning, and asked Dr. Geiger to try it out in an emergency case which promptly appeared and very promptly re- covered on injection of this dye. Following these announcements, various writers have attacked the theory suggested in explanation of these experiments, the experiments themselves, and the clinical evidence. As to the theory, following Dr. Barron’s inter- some pretation that the action of methylene blue on cya- nide is that of a catalyst, and later, Warburg, who in 1930 discussed this action at some length, I ad- vanced as a working hypothesis, the idea that also in my experiments, methylene blue acted as a catalyst. However, Hug in Oct. 1932, and Wendel in April 1933, working independently, both came to the conclusion that the action of methylene blue in whole animals when injected into the blood stream in cyanide poisoning, is not one of a catalyst, but rather is due to the formation of methemoglobin which promptly unites with cyanide, taking it out of the blood stream. These authors also found that the nitrites had the same property, a finding which has been supported by recent work of Clowes and Chen. Since methemoglobin has no such affinity for CO as it has for cyanide, Henderson objected that there was no theoretical ground for using meth- ylene blue in monoxide cases, and also on the basis that it required a large concentration of CO to in- hibit oxidation in tissues as compared with the small concentrations fatal to whole animals. How- ever, the fact still remains that methylene blue is an antagonist for CO in whole animals and there- fore a theory must be found to fit the facts. In order to see if any light could be thrown on this problem, I studied the cleavage of sea urchin eggs and star-fish eggs, this being an activity of the cell closely related to or dependent upon cell oxidations. Furthermore, since these cells have no hemoglobin, the cyanmethemoglobin reaction postulated by Hug and Wendel need not be con- sidered. These eggs were subjected to KCN or CO or N, and also placed in these solutions plus various concentrations of methylene blue, fifteen minutes after fertilization. Although these exper- iments are only preliminary, it was found that cleavage is retarded by both CO and Cyanide. The effect with cyanide has been previously shown. Methylene blue acts as an antagonist for CO in the cleavage of Arbacia eggs if the proper concentration of dye is used; it increases the rate of cleavage, in low concentrations and inhibits it in higher concentrations; the antagonism in <1s- terias is not proven in these experiments; the dye antagonizes the effects of cyanide on cleavage in proper concentrations. (This article is based on a seminar report pre- sented at the Marine Biological Laboratory on July 14th.) 96 THE COLLECTING NET [ Vor. VIII. No. 64 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Martin Bron- fenbrenner, Margaret Mast and Annaleida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford PUBLICATION The question as to whether the printing of an article in THe CoLLectinG Net constitutes publi- cation is a pertinent one. Technically, it practically perhaps it does not. ‘Vypewritten or monographed copies of an article can be copy- righted and distributed. In the technical then, even this method is construed as publica- tion. The answer to the question depends upon the definition of the word “publication.” In the sense that it was used by Dr. Conklin the other day it does not constitute publication. He denned publication in a “recognized scientific magazine ’ as the accepted method of obtaining priority. We do not consider THE CoLLecTING NET a “recog- nized scientific magazine.’ We do not want it to become one. Tue CottectinG NET is glad to have the privi- lege of printing preliminary reports of research work, but that is not its only purpose. There are people who believe that we print a great deal of extraneous material which they feel it would be wiser to omit. One or two trustees of the Ma- rine Biological Laboratory have chosen not to submit their seminar reports to THE CoLLEctTInG Net because they do not think it is dignified to have them printed in a magazine which includes local news, expresses opinions, and is generally rather informal. Fortunately they are in the minority. That we do not aspire to mold Tur CouLectr- ING Net into an accepted medium for obtaining priority by the publication of research reports may seem puzzling to many people. We have long considered the possibility of attempting to become an accredited scientific magazine and in fact, only recently made the tentative proposal to the Marine Biological Laboratory that the maga- zine become affiliated with it in at least a semi- official way, and that it be edited under the di- rection of an editorial board appointed by the Laboratory. To all intents and purposes it would have then become an adjunct to THe BrotocicaL BuLLetin. Under these conditions it would per- haps not differ enough from existing publications to warrant its continuation. We have recently sought the advice of many people concerning the most useful way of con- ducting THe CoLtectine Net, and as a result does ; sense, have decided to make no radical change in our policies. It will remain independent for the pres- ent. It will continue to foster informality in its contents and to seek material of interest to the biologist even though it may not always be biolog- ical. It will intentionally avoid becoming a strictly scientific publication by including material which no recognized scientific publication would consider within its province. By fostering this spirit of informality authors will have freedom in the expression of opinion; they can make state- ments and review fields of work without append- ing long bibliographies. 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CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the hole turns to run from Buzzards Bay to Vineyard Sound: uly Z2ar as amen 4:56 5:03 uly 23ers ee Sasi 541 Sali 24 Re ae 6212 6:23 JtilyN 25) sere LOLSZ 7:05 ually (200s Scns sy este Hol 7 49 Jiulyt27 ere ors 8:35 Waly 2Ore tre cee 8:57 9:25 Tly<29 Us Senco at 10:21 Tulys30e eae oar LOESG Ss ele Weibel be 5.c% 506 11:34 In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. a PEEVES Or Dr. Robert Chambers will leave Woods Hole on Sunday and plans to return the following Thursday. On Wednesday he will take part in the symposium at Cold Spring Harbor on “Oxi- dation-reduction Potentials,” giving a paper on “Intra - cellular oxidation - reduction potentials.” Ikarler on the same day Dr. L. Michaelis will dis- cuss “The Reversible Two-step Oxidation.” These papers constitute a small part of an exten- sive program at the Biological Laboratory on “The Potential Difference at Interfaces and Its Bearing Upon Biological Phenomena.” Dr. Alfred C. Redfield is making plans for an extensive cruise ot the Atlantis, the purpose of which will this time be an investigation of the formation of bottomwater in the North Atlantic The deep layers will be followed northward until the region where they reach the surface is ap- proached This cruise is scheduled to begin on August 12 and it will extend over a period of at least a month. Several investigators from Woods Hole are planning to attend the Third International Con- gress for Experimental Cytology, which will con- vene in Cambridge, England, from August 21-20. Dr. L. Michaelis is chairman of the section on the “[lectrophysiology of the Cell.” He will read a paper entitled, “The Reduction Intensity of the Living Cells’ A paper entitled, “The Relation Between Ions and Potential Differences Across Plasma Membranes,” will be given during this session by Dr. S. C. Brooks. The Tenth Annual Meeting of the Long Island Jiological Association will be held at Blackford Hall, Cold Spring Harbor, on Tuesday, July 25, 1933, at 6:30 P. M. Agenda: Ratification of acts by the Board of Directors and Executive Com- mitte; report of the Laboratory Director; report of the Treasurer; election of seven directors of the class of 1937; such other business as may he brought before the meeting. The Michigan Academy of Sciences has under- taken to publish a 200-page treatise on the “Fresh Water Algae of Newfoundland” by Dr. Wm. R. Taylor who is director of the course in marine botany at the Marine Biological Lahbora- tory. Thirty plates accompany his manuscript. Dr. B. H. Grave left Woods Hole afternoon for his home at Greencastle, where he is professor of zoology at University. Monday Ind., DePauw _'THE COLLECTING NET ENE ST Dr. Brooks is planning to leave for England on Juiy 30 on the Britannic.. The meeting of the Physiological Society at Plymouth will be his first objective, and from there he will go on to the Congress at Cambridge. Mrs. Brooks 1s return- ing to the University of California to continue her investigations there. Mr. Robert A. Nesbit, assistant biologist in charge of starfish investigations at the Cambridge station of the U. S. Bureau of Fisheries, visited the Woods Hole station for three days last week, returning to Cambridge on Thursday evening. Miss Margaret Grierson, a member of last year’s invertebrate course, was married to Dr. E. C. Cole, professor of biology at Williams College and in charge of the invertebrate course here, at Maplewood, N. J., home of the bride, on July 20th. Mrs. Cole is Associate in Zoology at the University of California at Los Angeles. Mrs. Marie Laug has returned to Woods Hole from the Women’s Hospital in Boston where she underwent an operation for appendicitis. Mrs. Laug is the wife of Dr. E. P. Laug, instructor of physiology at the University of Pennsylvania. Dr. I. J. Kliger, Professor of Public Health at the Hebrew University in Jerusalem, is visiting Woods Hole this week with his wife and son, David, preparatory to leaving for Palestine. He has been spending the summer in Nantucket, hav- ing completed his work here in the interest of the University. Dr. Fred W. Stewart of New York, who has heen spending a month's vacation in Woods Hole, will leave Sunday to resume his duties at Memor- ial Hospital. Miss Edwina Morgulis has returned to Woods Hole from Paris. Since her graduation from Radcliffe she has heen studying French literature at the Sorbonne. She is the daughter of Profes- sor and Mrs. Sergius Morgulis. Some members of the Woods Hole younger set have embarked on a project known as The Sum- mer Club. The membership includes Molly Rugg, Constance Heilbrunn, Frank and Martha Lillie, Margaret and Samuel Morris Jr. Mrs. L. V. Helbrunn is sponsor of the club and Mrs. Sam- uel Morris, librarian. Plans for the summer in- clude picnics and the building of a shack on which construction has already begun. THE COLLECTING NET [ Vor. VIII. No. 64 FERTILIZATION MEMBRANES OF Donatcp P. CENTRIFUGED ASTERIAS EGGS COSTELLO Instructor in Zoology, University of Pennsylvania The pulling apart of the eggs of marine in- vertebrates by means of a strong centrifugal force was early observed by Lyon (1907) for Arbacia, by Lillie (1909) for Chaetopterus (also Wilson 1929, 1930), and by Morgan (1910) for Cere- bratulus. More recently Kk. N. Harvey (1931) has made use of this property to compute the tension at the surface of marine eggs, and E. B Harvey (1932) has studied the development ot the resulting cell fragments of the Arbacia egg Under ordinary conditions the fullgrown star- fish egg remains immature, with its germinal vesicle intact, until it is removed from the body cavity of the animal into sea-water. About ten minutes later the nuclear membrane begins to dis- appear, and in about sixty minutes the first polar body is separated. Accompanying the breakdown of the germinal vesicle, as Lyon (1907) has shown, there is a recrease in the viscosity of the protoplasm so that it is possible to stratify the egg by centrifuging. [he ae to be described are concerned only with the effects of centrifuging during the arly maturation period, that is, heraee nthe breakdown of the germinal vesicle and the sep- aration of the first polar body. Eggs were cent:i- fuged in sea-water with an isotonic sucrose layer at the bottom of the tube, with the tube tempera- ture at 20 to 25 degrees Centigrade in an electric centrifuge giving a force of about 6000 times gravity. ~ After the breakdown of the germinal vesicle, the egg is distinctly Stratined and slightly elongated by twelve minutes of centrifuging. A few minutes later, the same amount of centrifug- ing results in the pulling apart of the egg inside of the jelly hull. Two fragments are produced, be a centripetal light fragment containing the oil, hyaline zone, and some yolk spheres, and a smaller centrifugal fragment containing the re- mainder of the yolk. IONIC CHANGES DURING THE Dr. LAURENCE [RVING Associate Professor During the development of eggs of Fundulus heteroclitus the carbon dioxide content of the eggs increases. ‘lhe increase in carbon dioxide is greater than could he accounted for by changes in carbon dioxide tension and consequently repre- sents the development of an alkali reserve, which in turn indicates a gain in buffering power, as of Physi These fragmented eggs, contained in the jelly hull, were fertilized immediately upon being re- moved from the centrifuge tubes. Within sixty seconds after inseminating, a fertilization mem- brane can be seen to separate from the heavy fragment. A partial membrane, or none at all, separates from the lower pole of the light frag- ment. Even in those eggs in which the tragments are not in contact, both fragments are usually activated, presumably by different sperm, and subsequently cleave. During the late morula stage, the effects of the presence and absence of a complete fertilization membrane is particularly striking. The heavy fragment produces a spheri- cal morula, held together by a fertilization mem- brane. The light fragment produces a_ pear- shaped mass of cells which tends to bud off the blastomeres at the centripetal pole into the sur- rounding medium. If the ovoid stratified egg, centrifuged immed- lately upon the breakdown of the germinal vesicle, is inseminated, the fertilization membrane separ- ates in the normal manner from the heavy pole of the egg. Passing toward the light pole, the fertilization membrane is thinner and closer to the egg, and at the light pole, continuous with the ege surface. This is not merely an_ eccentric membrane, as the difference in membrane thick- ness demonstrates. During subsequent cleavage, a thin membrane may separate from the blaste meres at the light pole, but this membrane does not resemble, either in thickness or in proximity to the blastomeres, the fertilization membrane of the heavy pole. The evidence is believed to demonstrate that at the appropriate stage of maturation, the sub- stances responsible for membrane separation can he displaced into the heavy half of the egg by centrifugal force. (This article is based on a seminar report pre- sented at the Marine Biological Laboratory on July 14.) Cee er OF FISH EGGS AND JEANNE EF. MANERY ology, U ert of Toronto yolk material is synthesized into the embryo. In view of the significance of buffering power as a limitation upon the extent of vertebrate meta- holism, particularly of the anaerobic type, it is worth while to see if the same change occurs dur- ing the development of other eggs. The eggs of the speckled trout are more easily 2/0 Cee examined in this respect because during fifty days they develop in fresh water. During this time, the carbon dioxide content of the eggs increases from about 1.3 to 5.2 cc. per 100 grams. The construction of carbon dioxide dissociation curves showed that the increase is connected with a true increase in carbon dioxide capacity. Normally the carbon dioxide tension in the eggs is constant at between 2 and 4+ mm., so that the bicarbonate component is the one which gains. The propor- tion of bicarbonate increases from 2/3 to 9/10. This change represents an alteration in the av- erage pH of the egg components from about 6.6 to 7.5. A change in pH implies simultaneous altera- tions in other ionic components, which can be partially predicted if the ionic concentration of the egg remains constant, and from numerous ob- servations in the literature this assumption is cor- rect. As bicarbonate ions are gained, then other anions must be lost. It is natural to expect that loss of chloride ions would compensate for the bicarbonate increase. THE COLLECTING NET 99 Series of analyses of chloride contents of the eggs showed that the chloride concentration stead- ily diminishes. The chloride loss, however, is about six times the gain in bicarbonate, so that some other anions must replace the chloride. In view of the change toward alkalinity, the other anions must be those of weak acids. These might be supplied by the conversion of non-ionized phosphate compounds to ones which are ionized, a process occurring in the development of the hen’s egg. Since the probable phosphate changes are inadequate, it is considered that with increas- ing alkalinity the dissociation of anions by pro- teins may supply a sufficient amount. These alterations in the ionic components of fish eggs cannot be described in terms of the well known chloride shift of red blood corpuscles. The forces involved in the transfer of tons are, how- ever, common and seem in the eggs to be furn- ished by metabolic changes of growth. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 14.) COMPARATIVE STUDIES ON THE OSMIOPHILIC AND NEUTRAL-RED- STAINABLE INCLUSIONS OF THE GENUS VORTICELLA Harotp FE. FINLEY Associate in Biology, West Virginia State College This report is concerned with the morphology and physiological significance of osmiophilic, ar- gentophile and neutral-red-stainable inclusions in Vorticella convallaria, V. microstoma, and V. campanula. In vital staining, dilutions of 1:20 were found to be satisfactory. The dyes were made up in stock solutions of 1% absolute alcohol and diluted with the same solvent. Vorticellae were studied in culture medium placed on slides previously filmed with the dye solutions. The Da Fano method of silver impregnation, Mann-Kopsch (Weigl) and Kolatchey methods of osmic impregnation were used to prepare whole mounts and sectioned material. In each species studied, globular cytoplasmic inclusions were stained vitally with neutral red. Certain of these inclusions measured 0.5 to 1 micron in diameter and they seemed to follow the cytoplasmic streaming. After staining with a mixture of neutral red and Janus green B, (equal parts of a 1:20 solution) the rod-shaped mito- chondria could be distinguished from inclusions stained red. Material prepared according to osmic and silver methods revealed definitely blackened globules measuring 0.5 to 1 micron in diameter. After these methods food vacuoles, nucleus and con- tractile vacuole were occasionally blackened. Discrete globular cytoplasmic inclusions, de- monstrable by recognized Golgi techniques and also stainable with neutral red have been inter- preted by some investigators as “Protozoan Golgi material” and by others as the “vacuome.” It was pointed out that neutral red is not a spe- cific stain for the vacuome in Protozoa. In Vor- ticella it consistently stained food vacuoles and other cell structures as well. Therefore, one should be positively conservative in regard to the identification of the vacuome after the use of neu- tral red. Other sources of errors included ob- servations regarding the morphological peculiari- ties of the animals under consideration. In view of the fact that at the present time we have no definite methods for determining the phy- siological significance of the vacuome, in the Pro- tozoa, | must remain non-committal on that point. However, this investigation does not support the view that the vacuome is associated with food vacuoles in any way. This point was emphasized: If it can be de- monstrated that discrete osmiophilic to argento- phile globules are universally present in Protozoa, and that they are of physiological significance, then their homologues in the metazoa will be of secondary importance. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 18.) ; 100 THE COLLECTING NET [ Vor. VIII. No. 64 HEREDITY AND ENVIRONMENT* Dr. Epwin G. Conxiin, Emeritus Professor of Biology, Princeton University When I was asked to speak to you this eve- ning | agreed without any large amount of urg- ing because | am very glad to see any church take up work of the sort that is implied in an open forum for discussion, where thoughts of people can be brought out and where questions can be asked. Indeed I have often thought, since 1 have been a teacher and lecturer for very many years, that it would be highly desirable if public speakers, whether in church or on a platform or in a classroom, were to be subjected to a grilling questioning after they finished speaking. I have been announced to speak upon a rather hackneyed subject but one which is of the greatest importance to all of us, namely, which is the more important in the development of the individual, heredity or environment. There are no two persons in this audience who look alike to me—among all the people of Fal- mouth I should not find two persons who were identically alike. There are, of course, so-called “identical twins” but even these show slight dif- ferences. Recent studies have shown that such twins differ in minor details although they have identical heredity, since they come from a single egg. In the whole world there are estimated to be about one billion eight hundred million per- sons and it is safe to say that no two are identical. How is it possible then to have such great differ- ences in human beings who are still human? In the first place, all human beings, except for “iden- tical” twins, have a different inheritance. The fact is that the units of heredity, which are now called genes, get so sorted that in every genera- tion one-half of them that are carried by the father are thrown away, and one-half of those carried by the mother are thrown away. The remaining two halves unite to give rise to a new human being, but the units which are so united are never exactly the same. There is only one chance in several billions that two children of the same parents will have exactly the same genes. Nevertheless children of the same family have many genes in common and this accounts for certain family resemblances and traits. Besides physical resemblances there are also social and mental traits that are inherited, such as disposi- tion, degree of intelligence, type of personality. We must, however, also consider environment which plays an important part in the development of personality. A person may come from a fam- ily in which insanity is hereditary. This does not mean necessarily that he will become insane, but that he will be likely to do so under certain ad- verse conditions which a person with a different inheritance might be able to undergo unscathed. It is the same way with tuberculosis; the disease itself is infectious, but a person who has inherited slight resistance to tuberculosis would very prob- ably develop it, 7f infected, while someone with a stronger resistance would remain immune under the same conditions. Whether one is calm or excitable, conservative or radical, law-abiding or criminal, may have their beginnings in inheritance. So that we are not all equal by any means in the way in which we have been endowed by heredity. When Thomas Jefferson wrote in the Declaration of Independence, ‘“‘we hold these truths to be self-evident, that all men are created equal... ,” he did not mean that all men were qual as to color, or wisdom or ability ; he merely reterred to the fact that in a popular government, such as was being established in this country, all men were equal before the law. And yet men are not all equal before the law in responsibility. Children and insane persons are excepted, and even in the same individual, responsibility and capacity to resist temptation varies at different times. Responsibility rises and falls during the course of a day and lapses almost completely in sleep. What do we mean by responsibility? We mean the ability to respond to a situation in a reasonable, purposeful manner; the capacity, for example, to respond in a lawful manner rather than unlawfully. Some people maintain that heredity has made us what we are entirely, that we have no choice in doing what we do. They quote the Scriptural text, “Which of you by taking thought can add a cubit to his stature?” A cubit is about two feet and that is quite a bit to add to one’s stature, except by means of stilts; but we can, by taking thought, add or subtract two or three pounds of weight. A leopard cannot change its spots nor an Ethiopian his skin, but I have seen white people become very nearly the color of an Ethiop- ian on the beaches in the summer! We are not absolutely free—our heredity, our background, our early experiences all have an in- fluence over which we have no control. But we do have a large amount of control over our be- havior through the formation of habits. Educa- tion itself is very largely the establishment of good habits, “conditioned reflexes” the physiolo- gists call them, and a habit is something that has been learned or acquired. Good habits carry one along without very much effort, but habits are things which we are capable of modifying. We * Transcribed from shorthand notes of an extem- poranious talk which Professor Conklin gave last Sunday evening at the Methodist Episcopal Church in Falmouth. Jury 22, 1933 ] THE COLLECTING NET 101 do thus have a certain amount of power in shap- ing our own careers; we are not entirely bound by heredity nor by early environment, which we cannot change. You see therefore that both heredity and en- vironment are involved in the making of an in- dividual. Heredity determines the possibilities ; the realization of these possibilities has got to come about through their development under en- vironmental conditions. Every one of us could have been a different person from what he is; given the same heredity each of us might have turned out something different and possibly better or worse. I think it was John Wesley who, on see- ing a drunkard in the street, remarked, “There, but for the grace of God, goes John Wesley.” This realization of what we might have been gives us a sense of sympathy and understanding. As the French proverb puts it, “To know all is to pardon all.” If one “knew” more one could have more sympathy with offenders. They may not always need sympathy, but punishment should never be retributive, it should be made to fit not the crime but the criminal. Punishment should be inflicted for the training or reformation of the criminal or for the protection of society and not for any other reason. So I say, we should have in mind the fact that any one of us might have been different, might have been much better or much worse. Whittier wrote: “Of all sad words of tongue or pen, the saddest are these, ‘It might have been’.” I believe that even sadder than the words “It might have been” are these, “I might have been.” Heredity has been kind to most of us, the possibilities within us are great but they rarely come to full fruition. I used to tallk more enthusiastically of eugenics than I do now. The world is moving too fast to wait for the slow workings of eugenics; educa- tion is a much more rapid process in enabling human beings to realize their best possibilities. Heredity is difficult to control. Only when you choose a mate with wisdom and forsight can this be controlled. There is no other way by which bad heredity can be made good. But we can take the heredity that is given us and develop the better possibilities, suppressing the worse ones, and come to a better type of human personality. Question: What about Dr. Watson and the “School of Behaviorists”? Dr. Conklin: Dr. Watson is reported to have said that if you were to give him one hundred babies (no one, of course, would be likely to do it) he would agree to make out of any of them doctors, lawyers, writers, or anything that was desired. It is interesting fact that the less people know about babies the more sure they are of what they can do with them. When they have babies of their own they discover that babies have tendencies of their own. This claim of Dr. Wat- son’s is no more justified than an assertion that all babies are born of one color or one size. It might be possible to take a baby with the inher- ited qualities of a poet and bring him up as a lawyer, but you could be fairly sure that he would not be as successful or as happy as if he had fol- lowed his inherited bent. On the other hand, children who come of parents who cannot give them the proper opportunities are not able to develop their hereditary possibilities, but this does not change their fundamental endowments. In- telligence should be distinguished from education and knowledge; intelligence is the capacity to know, knowledge is the thing known. The capac- ity to know is inherited but not knowledge. Some people are born with one talent, others with ten; but none will develop unless used. Question: Is man more than simply an organ- ism? Is he free? Dr. Conklin: Man is more intelligent and free than any other organism. Intelligence is the ability to regulate behavior in the light of ex- perience, the ability to profit by past experience. Any individual, human being or animal, that can consciousl’ profit by past experience is to that extent intelligent. A horse that learns to open a gate is intelligent with respect to that one point. Any being that can thus profit by experience, that can learn to avoid mistakes and to repeat suc- cesses, 1s intelligent in that respect. Freedom is the ability to control behavior by means of in- telligence; the more intelligent we are the freer we are. A large amount of behavior is purely mechanistic and cannot be self controlled. There are many other processes which human beings can control, such as the formation of habits, by means of which they can free themselves from the mechanistic compulsion to which other creatures must submit. Wherever there is intelli- gence there comes this possibility of directing he- havior, halting hefore acting, and profiting by past experience of others as well as of one’s self. I do not want to be more free than that—free to discriminate and choose hetween given alterna- tives in the light of past experience. This is a mechanistic type of freedom for whatever the ul- timate nature of discrimination and choice may be they are causal phenomena. Science deals with phenomena that can be reduced to mechanistic principles and laws. Human behavior conforms to certain laws. There is therefore no such thing as absolute freedom. One can only choose be- tween alternatives that are open to him. Human beings sometimes choose the worse alternative, because they are not aware of it, because they think that it may be changed later to the better, or perhaps in a spirit of bravado, just to see how much they can “get away with.” 102 THE COLLECTING NED [ Vor. VIIT. No. 64 CONSTITUTION AND BY-LAWS OF THE M. B. L. CLUB Constitution shall be appointed by the President to serve for Article I. one year, subject to discharge by him at any time. The name of the club shall be the Marine Bio- logical Laboratory Club. Article II The object of the Club is to promote social inter- course among the scientific workers of the \WWoods Hole community and their friends. Article IIT.* The membership of the Club consists of two classes, active and associate. The scientific work- ers of the Woods Hole community and members of their families, eighteen years of age or over are eligible to active membership, and become members on payment of the annual dues. Other persons who are eighteen years of age or over may be elected to associate membership as pro- vided in the By-Laws. Only active members in good standing have the right to vote. With the exception of voting, the associate members have all the rights and privi- leges accorded to the active members. The annual dues for active and for associate membership are one dollar* for the fiscal year be- ginning with the annual meeting. The families of members are entitled to the use of the Club without further payment of dues, provided that this privilege is not extended to less than eighteen years of age. Article IV. The officers of the Club shall be the President, the Vice-president, and the Secretary-Treasurer™. Only dake active members who are in full stand- ing and who are also members of the Corpora- tion shall be eligible to these offices. The officers shall be elected at the annual meet- ing; they shall be subject to recall as provided in Article VII of the Constitution. Vacancies oc- curring at other times shall be filled as provided in the By-Laws. Article V. The annual meeting shall be held each year one week after the official opening of the courses, at which meeting the election of officers, the presen- tation of official and standing committee reports, and other stated business shall be transacted. Article VI. There shall be an Executive Committee of which the officers of the Club shall be ex officio members. Other members of this Committee Article VII. Special meetings of the Club for any purpose except that of amending the Constitution may be called at any time by any officer of the Club; they may also be called by petition by at least 15 ac- tive members who are in full standing; the pur- pose and date of such meeting shall be stated in a notice which shall be posted in a public place in each of the Laboratory buildings, and in the Club-House, at least five days before the proposed date of such meeting. VIII. Amendments fo this Constitution can be made by a two-thirds vote of those active members who are present at any meeting, provided that at least 30 such members are present, or provided that one-third of the total active membership below 90 is present, and provided that the proposed Amendment and the petition for the meeting to consider such Amendment or Amendments, and the date of such meeting, shall have been signed by at least 10 voting members and posted in a public place in each of the Laboratory buildings and in the Club-House at least 10 days before the date of the meeting. Article 3y-LAws 1.* The Executive Committee shall consist of nine members, and shall include the officers of the Club and the Chairmen of the House and Social Committees. The duties of this Committee shall be to attend of the Club as the running of the Club-House, and the appointment and dis- charge of a House Committee, a Social Commit- tee, a Membership Committee, and such special Committees as it may deem necessary. The Committee shall also have the power to fill vacancies in the offices of the Club occurring at other times than at the election at the annual meeting. The Committee shall further have power to elect those persons to associate membership who do not ipso facto, become members by the pay- ment of dues, as provided in Article III of the Constitution, but who have been proposed and seconded by active members of the Club who are not members of the Executive Committee. The to such general affairs * Altered by amendment. Jury 22, 1933 ] THE COLLECTING NET 103 Committee shall, further, have power to decide all doubtful cases of eligibility. II. A quorum of the Executive Committee shall consist of a majority of its members. AMENDMENTS I. Dues raised to $1.50 (Aug. 13, 1923). Il. Dues of Associate Members $3.00 (Sept. 1, 1924). III. (Insert in Article IV) and an Assistant Treasurer. This officer shall be a resident of Woods Hole. His function shall be to collect dues from members and to perform such other duties as may be delegated by the Treasurer. (June 30, 1930). IV. Employees of the M. B. L. shall be eli- gible to Associate membership in the M. B. L. Club, provided that their names are proposed and vouched for by two active members of the Club. Furthermore, such employees are allowed mem- bership at the reduced fee of $1.50, and such membe.s are allowed all the privileges of the Club, though they may not vote at its meetings. (By the Executive Committee, 1933). V. The Executive Committee shall consist of nine members and shall include the officers of the Club and the Chairmen of the House and Social Committees. The duties of this Committee shall be to at- tend to such general affairs of the Club as the running of the Club-House, and the appointment and discharge of a House Committee, a Social Committee, and such special Committees as it may deem necessary. (Aug. 4, 1916). IMPETIGO Dr. Davip CHEEVER i Associate Professor of Surgery, Harvard Medical School Uncleanliness is a term distinctly objectionable to people of refinement, and yet uncleanliness in the summertime is frequent among just this group —unintentionally so, of course. Summer weather demands a different type of hygiene from that required by winter conditions. All biologically inclined people know that there is a difference in the types and luxuriance of flora in culture test tubes depending on whether they are kept cooled or incubated ; and all housekeepers know how much more readily the contents of the bread box will mold in the summer than in the cooler months. These same principles apply to the care of the human skin, and yet mothers who will see that their children are bathed carefully at home in the winter will allow them to go for days in the summer, relying solely on the very doubt- ful cleansing effects of cold salt water without soap. These same children who have fresh cloth- ing every day in the city, wear playsuits at the beach for days without change, and bathing suits which are rarely thoroughly washed and are fre- quently borrowed more or less indiscriminately. In most cases these practices are not harmful, but now and then a wound in the skin will occur and, in an environment of perspiration and soiled clothing, a slight infection may start from organ- isms which ordinarily live harmlessly in and on our skins in small numbers, but under conditions favorable for rapid multiplication, they may cause a condition known as impetigo. Once this skin trouble has appeared it is a genuine problem, though rarely, if ever, a serious one, but very per- sistent unless correctly treated. It can spread rapidly to other parts of the same person and almost as readily to other persons. It occurs most commonly on the exposed parts of the body but may be transferred, especially by the fingers, to the covered parts. The first stage of’ impetigo is marked by tiny blisters, extremely superficial, which break almost immediately and form golden or brownish crusts under which serum is constantly poured out, spreading to form larger and thicker crusts. The conditions under these crusts are ideal for rapid growth of the organisms, and the application of ointments outside the crusts makes these condi- tions still more favorable and increases the sever- ity of the impetigo. The proper treatment consists of the careful removal of the crusts with soap and water on a bit of gauze or paper tissue which must he burned; then'the careful drying of the spots with fresh tissue to be immediately destroyed; finally, the application of very mild antiseptics. As a rule, ammoniated mercury ointment in the streneth of not over five per cent. will produce best results; strong ointments, strong soaps, and the use of alcohol will usually so stimulate oozing that further spreading will occur and delay heal- ing. The fingers must be kept absolutely away from the spots and fresh pillow-slips used every night, and the use of all towels and face cloths forbidden because of the easy spread to other surfaces. Ona man’s bearded face it is generally necessary to omit shaving for three or four days since the razor spreads the germs in spite of the greatest care. As is obvious, every person with impetigo should practice careful individual per- sonal hygiene. Isolation such as is required hy the “children’s diseases” is not necessary but is helpful and perhaps not unreasonable, inasmuch as proper treatment applied for three days should completely cure impetigo. 104 THE COLLECTING NET [ Vot. VIII. No. 64 To Present “You Never Can Tell,” Shaw Drama, at M.B.L. “You Never Can Tell”, an early four-act drama by Bernard Shaw dealing with that perennial issue of Shaw’s, the rights of women, will be presented in the Marine Biolo- gical Laboratory auditorium Monday evening by the Penzance Players, for the benefit of the Collecting Net scholarship fund. ‘The Players are to be remembered, especial- ly for their production of “The Queen’s Hus- band” by Robert E. Sherwood in the summer of 1931, and ‘‘Meet the Prince” by A. A. Milne last summer. These were given on Penzance Point at the residence of Mr. and Mrs. J. P. Warbasse, so this year’s Auditorium perfor- mance is a unique venture. The present pro- duction is under the direction of Mrs. Dorothy 3aitsell who has had many years experience in Little Theatre work in New Haven, Con- necticut. In the cast are several old favorites, besides some newcomers to Woods Hole drama. AlI- fred Compton, of Princeton University Theatre Group, who takes the Shavian role of the waiter, will be remembered for his excellent performance of the leading part in the “Queen’s Husband”, and his Mr. Battersby in “Meet The Prince’. Tommy Ratcliffe, of the Harvard Dramatic Association, who played the bombastic general in 1931, and the leading role of the imitation prince in last season’s production, will play Valentine, the Dentist “Duellist of Sex” in “You Never Can ‘Tell’. Vera Warbasse of the Connecticut College for Women, who has taken important roles in the preceding performances plays the part of Gloria—the “Woman of the Twentieth Century,” Mrs. Clandon, her mother, old fashioned champion of Women’s Rights move- ments, is to be played by Peggy Clark ot Smith College, who was seen last season in the character part of the overbearing Mrs. Faithful. Fred Copeland, of Williams, who has taken part in the other plays is the eminent Queen’s Councillor, Mr. Bohun who undertakes to straighten out all difficulties of plot. In addition to these old players, four new ones have appeared. Eric Warbasse will play Mr. Crampton, husband of Mrs. Clandon. Bol Giddings takes the part of McComas the solicitor, and Faith Adams of Vassar and Manton Copeland of Williams are the talk- ative twins—Dolly and Phil, Mrs. George A. Baitsell is the director, and Robert Chambers, the stage manager. Wister Meigs is in charge of the settings, of which there will be three in the course of the four acts. Maynard Riggs is property man, and James Sever electrician. Reserved seats are on sale at one dollar at the Collecting Net office, the M. B. L. office and Robinson’s Pharmacy in Falmouth, and other tickets, at fifty cents, will be purchase- able at the gate. St. Joseph’s Church Benefits From Musicale Last Monday The visiting Knights of Columbus choir from New Bedford, supplemented by local and visiting soloists, presented an evening of en- tertainment, mostly musical, for the benefit of St. Joseph’s church Monday evening at the Marine Biological Laboratory auditorium. The choir, the entire glee club of the Me- Mahon Council of the Knights of Columbus, opened the program with Houssian’s Messe en Vhonneur de St. Paul, in the conventional four parts, Kyrie, Sanctus, Benedictus, and Angus Dei, and contributed groups of English selee- tions, with the director, John T. Curry, Jr., leading. Miss Carolina Finni, lyric soprano sang a group of Italian and German selections, including an aria, ‘Donne Vaghe’ from Paisiel- lo’s opera, “La Serva Padronna,” and Miss Gertrude ‘Tripp, lyric soprano, several shorter Fnelish numbers. Male vocalists were Mr. James Evans and Mr. Leonard McDonneli. ‘The instrumental portion of the program fea- tured violin solos by Mr. Robert SanSouci, former Lawrence High School concertmaster, a member of the Boston Coilege quartet, and Miss Helen McKenzie, a flute solo by Mr. Robert L. McKenzie, and a group of piano numbers including Rubenstein’s famous “Kammenoi Ostrow,” by Mr. Harry Bowker. With such a varied program, numerous ac- companists were used, including Miss Mary Louise Stockard, Mrs. Maud Marceau Powers, Miss Joan Pecheux, and Mrs. Gladys Howard. The succession of musical selections was interrupted about the middle by a farcical playlet entitled ‘Just a Few Laughs,” with the following cast of characters: Piis) Hlomora tem) deere) nse James Evans euorolboil tj go nnoppomstuons Edward Doyle Mir); Gasey Bas iemitnts cscs eet Walter Considine Mrs. Casey, < nak spicy Mrs. James Evans Mr, Margolis vvverseeeses-. 0m MeConnell Vol. VIII. No. 5 THE BERMUDA BIOLOGICAL STATION FOR RESEARCH (Part II) J. F. G. WHEELER Director of the Station During 1932 there were forty-one visitors stay- ing at the Station for periods varying from a few days to three and four months. The following scientists car- ried out work there: Prof. Karl Sax, Bussey SATURDAY, JULY 29, 1933 MW. B. EU. Calendar Annual Subscription, $2.00 Single Copies, 25 Cents. SEX DETERMINATION IN HYMENOPTERA Dr. P. W. WHITING Professor of Zoology, University of Pittsburgh The problem of sex determination in the bee has excited interest from early times. Eventually the question seemed to be solved by the knowl- edge that a haploid set of chromosomes resulted in a male, a diploid set in a female. Inst. Harvard (Plant cytolo- gy). Prof. E. M. East, Bussey Inst. Harvard (Physiological work on Valonia & Halicys- tis). Prof. B. White, State Anti- toxin Lab. Mass. ( Physiologi- cal work on Valonia & Hali- cystis). Dr. H. R. Seiwell, Woods Hole Occanographic Research ship “Atlantis” (Phosphate content of sea water). Pierre Comte, Princeton (Geology ). Prof. J. P. McMurrich, Biol. Board of Canada, To- ronto (Actinia). Prot. B. R. Lutz, Boston Univ. (Physiological work on Ascidians). Mrs. B. R. Lutz, Boston (Physiological work on Bufo). TUESDAY, AUGUST 1, 8:00 P. M. Seminar: Dr. Conway Zirkle: “The effects of fat solvents upon the fixation of mitochondria.” Dr. A. W. Pollister: ‘The cytology of amphibian tissues.” Dr. B. M. Duggar and Dr. A. Hol- laender: “The irradiation of bio- logical suspensions by monochro- matic light.” Dr. C. C. Speidel: Motion pictures showing some varieties of nerve irritation, as seen in living frog tadpoles. WEDNESDAY, AUG. 2, 8:00 P. M. Lecture: Dr. O. E. Schotte: “Or- ganizers and inherent potencies in the embryonic development of Amphibians.” FRIDAY, AUGUST 4, 8:00 P. M. Lecture: Dr. August Krogh: ‘‘Con- ditions of Life in the Depths of the Ocean.” mated females. With the development of the idea of genic balance, how- ever, it became clear that this explanation was inadequate and in 1925 Bridges wrote, “sex determination in the bee is the outstanding unsolved puzzle, although before the de- velopment of the idea of genic balance it seemed one of the clearest and simplest of cases.” The inadequacy of the old ex- planation became very obvious when Bridges (1925b) report- ed that haploid tissue in Dro- sophlia appeared to be female and when the author discov- ered that diploid males in the parasitic wasp, Habrobracon, showing no traces of intersex- uality, occasionally appear among the progeny of Goldschmidt (1920) (Continued on Page 121) TABLE OF CONTENTS The Bermuda Biological Station (Part IJ), Translocations in the Mouse, Dr. G. Snell, J. F. Winceler Sere WE Cer SCR oir CEERI CI ee 113 Elsie Bodemann, and W. Hollander........ 141 Sex Determination in Hymenoptera, Dr. P. : WIEN ewe ee ter esc cthccpiee aces 113 Comments on the Seminar Report of Dr. The Biological Laboratory: Snell and Co-workers, Dr. P. Whiting..... 143 Streaming Potential Measurements, D. R. Chromatin Extrusion in Ciliate Commensals 115 22) gh oe Smee a ea aa aaa 123 of Molluscs, Dr. G. Kidder............--.. 143 Surface Conductance, K. S. Cole.........- 132 It Pummtevest 145 Ability of Mammals to Survive without (jek) se IOMUIRIS: Gaomomecooucadcodcocabonds Breathing, Dr. L. Irving ............-.-.- 138 News Items from Cold Spring Harbor....... 146 ne ea EEE EEE E ya ESE SIS ESSE ESE ERE RD REE Me THE COLLECTING NET _ [ Vor. VIL. No. 65 Photographs by Miss M. L. Russell HARBOR and TOWN OF ST. GEORGE, BERMUDA The Biological Station is on the horizon in the middle distance. Dr. M. Reid, Boston Univ. (Physiological work on Bufo). Ik. B. Benson, Boston work on Holothuria). Prof. L. V. Heilbrunn, Pennsylvania (Toxopneustes ). Dr. RK. L. Biddle, Coll. New York (Tunicates ). Dr. T. C. Barnes, Yale Univ. (Physiological work on Crustacea & Algae). Prof. E. G, Conklin, Princeton (Asymmetron ). Dr. R. Meader, Yale (Nerve tract degenera- tion studies in Fishes ). Prof. E. S. Goodrich, Oxford (General logy, Asymmetron and Polychaet worms ). Mrs. Goodrich, Oxford (Parasitic protozoa ). Univ. (Physiological Univ. ZOO- F. Gilchrist, Harvard (General zoology and physiological work on Ligia). F. Torrey, Harvard (Ecology of land Mol- lusca ). Prof. E. L. Mark, Harvard (General zoology, Odontosyllis ). Miss F. Felin, Seripps Coll. Calif. (General zoology, Fish eggs & larvae). Dr. H. Richards, U. S. Nat. Mus. (Coll, Ma- rine Mollusca ). Prof. R. B, Goldschmidt, Kaiser Wilhelm Inst. 3erlin (General Zoology ). Dire W. Seebe, N. ny; Zool. Soc. (Ecology & life history of shore and deep sea fishes). J. Tee Van, N. Y. Zool. Soc. (Ecology of fishes esp. shore fishes ). Mrs. Tee Van, (Artist). Miss G,. Hollister, N. Y. Zool. Soc. (Shore and deep sea fishes esp. osteology of tails). Miss J. Crane, N. Y. Zool. Soc. (Shore and deep sea fishes esp. scales). Miss E. Van der Paas, (Artist). V. Palmer, N. Y. Zool. Soc. (Shore fishes). Dr. G. M. Smith, Sch. Med. Yale (Lateral lines and regeneration of melanophores in fishes ). H. Antz, Sch. Med. Yale (Lateral lines and re- generation of melanophores in fishes ). Prof. E. Newton Harvey, Princeton (Physio- logical work on Echinoderm eggs with centrifuge microscope ). Mrs. E. B. Harvey, Princeton (Physiological work on marine eggs with centrifuge micro- scope ). GENERAL COLLECTING Many localities have been examined in the Reach, Castle Harbour, St. George’s Harbour and round the shores of the islands and islets in the vicinity to ascertain the nature of the fauna and flora and to gain knowledge of the best collecting grounds for the more abundant forms at different times of the year. A record has been kept of the trips of the collecting launch, with the forms ob- tained, notes on spawning animals and any pecu- liarities in distribution that have been noticed. This survey has, of course, been done mainly in connection with the work of the visiting scientists Jury 29, 1933 | who have in many cases identified specimens of the groups in which they were interested. Features of special interest were the wide dis- tribution of the alga Valonia macrophysa and the discovery of Halicystis growing in Castle Har- bour ; a good collecting ground for the white sea- urchin Hipponoe esculenta and the discovery (by Prof. E. N. Harvey) that eggs and sperm of this form are mature in December while those of Toxopneustes variegatus are 80 per cent. ripe in June (Prof. Heilbrunn) ; the recurrence of ripe specimens of Asymmetron during the first week in August (Prof. Goodrich) and the finding of the land Nemertine Geonemertes agricola under stones on the margin of the Parish Dump, St. George’s. lor Dr. Lutz’s investigations the giant toad, which does not occur on St. George’s Island, was collected from the ponds of the Aquarium at Flatts by kind permission of Mr. Mowbray and in the marshes near Hamilton. SCIENTIFIC CONSTRUCTION AND ADDITIONS TO EQUIPMENT A tide pool has been constructed on the point between Ferry Reach and Richardson’s Cove, a natural hollow being supplemented by a wall of stone and cement. This was originally intended as a fish pond, but mechanical difficulties in build- ‘ing the wall high enough to exclude the sea at all tides, together with the possibilities of the old fish pond, led to a change of plan. The pool forms a useful stock aquarium for invertebrates. THE COLLECTING NET 115 The old fish pond, which was used for many years as a convenient dump, has been cleaned and repaired, and is entirely successful. In September, as the result of a recommenda- tion of the International Hydrographic Confer- ence at Monaco a standard automatic tide record- ing machine was delivered to the Station from the U. S. Coast and Geodetic Survey, Washing- ton. A stone house was built to accommodate the machine on part of the sea-water supply pier. The tide pole was fastened near the house and three bench marks, set in cement beds for levelling pur- poses, were established. These were levelled to the Ordnance Survey mark on the far side of the Swing Bridge road by Mr. Cyril Smith of Ber- muda. The machine has been in continuous oper- ation from October Ist. In the chemical laboratory a small fume cham- ber has been built into one of the corners and a solid stone pier has been set up to carry the fine balance. Gas (Philgas) has been laid on in this labora- tory from a double cylinder installation placed on a cement bed outside the building near the eastern steps. Additions of apparatus include a Leitz binocu- lar microscope and a microtome from Prof. Conklin and a monocular microscope from Dr. G. G. Scott which fill a very great need. A series of plunger jars has been set up in the general laboratory for investigations upon small active organisms that would escape from the aquaria. These were used with success for post- SHELVING, ROCKY SHORE OF LONG-BIRD ISLAND One of the best collecting grounds near the Station. 116 THE COLLECTING NET [ Ve IL. VIII. No. 65 BERMUDA BIOLOGICAL STATION FOR RESEARCH Main Building, two cottages and part of grounds. There are seven additional buildings on the proper- ty. Long-Bird Isiand is beyond the Station and Castle Harbor is in the distance. larval fishes by Miss Felin. Another set in the cool room under the South Verandah was used by Prof. Goodrich in his work on the larvae of Asymmetron. A new pattern diving helmet made to the order of Prof. Conklin has proved very successful. LIBRARY More than two-thirds of the reprints and bound volumes in the library have been card-in- dexed under the author’s name. This work was done by Dr. and Mrs, Wheeler, Prof. McMurrich and later Miss Gallaudet. A sufficient number of the 1600 reprint cases devised and ordered by Dr. Mark have been made up to carry the reprints, and new shelves have been erected in the two library rooms to carry the present books and papers. The completion of the card index is at present in the hands of Mr. Cutter, temporary librarian. The library has grown rapidly thanks to Prof. Conklin, who has sent many papers, duplicate re- prints and bound works and to Dr. Mark who has made presentation of series from his library including the Proceedings of the American Aca- demy of Arts and Sciences, the Bulletin of the Museum of Comparative Zoology, Harvard, and the Publications of the International Council for Exploration of the Sea. Lately Dr. Mark has sent a great many reprints and works dealing with plankton for which I personally am very grateful since plankton literature was practically non-existent previously. The Station is indebted to the Smithsonian Institution for the Proceed- ings of the U. S. National Museum and_ other publications, to Prof. E. Newton Harvey for ar- ranging with the Wistar Institute of Anatomy and Biology for a year’s subscription to the Jour- nal of Cellular and Comparative Physiology, and to Dr. W. T. Porter who has presented back numbers to September, 1928 and the current is- sues as they appear of the American Journal of Physiology. Mr. Mowbray, Director of the Ber- muda Aquarium, has turned over to the Station a “BOILERS” OR DIMINUTIVE ATOLS OFF THE SOUTH SHORE, FRINGING REEF IN THE DISTANCE Jury 29, 1933 ] THE COLLECTING NET 117 complete set of the scientific results of the “Chal- lenger” Expedition which is a valuable acquisi- tion. The Trustees of the British Museum (Na- tural History) are presenting the “Great Barrier Reef” Reports as they are published. The Na- tional Academy of Sciences of the U. S. has pre- sented a set of all of its publications that are now available. Several workers have presented copies of their works and other gifts of this description have come from persons interested in the welfare of the Station. Purchases for the library include five general works on life in the sea, a complete set of the “Nordisches Plankton” and the ‘‘Treatise on Zoo- logy” edited by Sir. E. Ray Lankester in eight volumes. SALE AND SUPPLY OF SPECIMENS There has, of course, been no attempt at a sup- ply department, but specimens have been re- quested by scientific workers in other countries which I have been glad to collect seeing in this matter a means of widening the interest in the Station. A request for Halicystis was dealt with by Dr. Blinks of the Rockefeller Institute Staff in Hamilton who sent the material to me for pack- ing and shipment. T. Cunurrre Barnes, Yale University SUMMARIES OF THE WORK OF SOME OF THE VISITING SCIENTISTS DURING 1932 Series 1. The All-or-None-Law. Single fibre nerve-muscle preparations were made of a dozen species of Crabs to test the validity of the all-or- none law by electrical and natural stimulation of motor nerve fibres. It was found that this law does not hold for Crustacea. Paper to be pub- lished soon. Series 2. Origin of Kinesthetic Impulses in Crustacean Limbs. By means of an amplifier these impulses were located arising from the muscles themselves. Other sources of stimulation such as the bristles and integument were elimi- nated. Paper to be published soon. Series 3. Space Orientation and Salt Require- menis of Ligia. Approximately 1000 isopods were studied in salt solutions and in respect to their ability to orient towards the sea or survive in air. Paper to be published soon. Series 4. Influence of Ice-Water on Marine Algae. Valoma, Halicystis and Acetabulum were grown in sea water containing trihydrol. No definite results were secured with the first two forms. Further work is necessary to compensate for the dilution of the salt concentration. E. G. Conkiin, Princeton University BREEDING PERIOD AND BEHAVIOR OF ASYMMETRON LUCAYANUM Specimens of this interesting species occur in smali numbers in the clean, bottom sand under water 10-20 ft. deep in a small area near None- such Island. In 1931, between June Ist and Au- gust Ist, specimens were collected by dragging a bucket, weighted on one side, over this area and sifting the water and sand so taken through a fine wire screen. Animals were collected at various times during the day and evening and were kept for several days in dishes of clean sea water in the laboratory. They are hardy and will live for a week or more under such conditions. During the period mentioned no eggs were laid by the animals brought into the laboratory although the gonads were full and the spermatozoa active. At- tempts to artificially fertilize the eges failed, prob- ably because the eggs were not in the precise stage necessary. After my departure from the Station on August 5th, 1931, my assistant, Mr. J. K. Donahue, found on August 14th a lot of early gastrulae in a dish containing animals which were collected on August 12th and which must have laid during the evening of August 13th. Some of these gastrulae were reared to the early larval stage. In 1932 I collected specimens of Asymmetron between June 20th and July 11th but again was unable to obtain any embryological material dur- ing that peviod. Howeve:, Professor E. S. Good- rich found that eggs were laid and fertilized dur- ing the evening of August Ist, 1932, and many of these were reared to the larval stage with small, round mouth and first gill slit. Therefore, Asym- metron in Bermuda breeds early in August, al- though it is possible that it may breed also at other periods. The marked asymmetry of the adult suggested that the animals might occupy an asymmetrical position on or in the sand, and in order to test this those captured were placed in small aquaria on a deep layer of the sand in which they were taken, but owing to their small size and transpar- ency they could not be observed satisfactorily in such aquaria. Consequently a very narrow aqua- rium with a space of only about 14 inch between the two glass sides was made and filled with sand to a depth of 4-6 inches and with an equal depth of running sea water above the sand. Even in so narrow an aquarium it was difficult to see them when they were buried in the sand and according- ly a still narrower aquarium was made with the glass sides only about 1g inch apart, and in this 118 _THE COLLECTING NET [ Vor. VIII. No. 65 the animals could be seen and photographed when buried. Asymmetron, like Branchiostoma, remain inac- tive for long periods unless actively stimulated, when they race around violently for a short time only to relapse into a passive and apparently ex- hausted condition. Such passive individuals lie indifferently on their right or left sides, or when in narrow places on their dorsal or ventral sides, and when they were introduced into the narrow aquarium they would generally lie on the surface of the sand, unless they chanced to touch the sand head first or until the water was agitated, when they would burrow into the sand by an undulatory and more or less spiral movement. Once buried in the sand they would often lie passive for hours or even for days, but usually they would squirm to the surface of the sand and protrude the an- terior or posterior end or come out of the sand altogether; sometimes both anterior and posterior ends would protrude and in few instances did they stand vertically in the sand with the anterior end and mouth exposed, as in Willey’s (94) figure of the Amphioxus at Messina. On the contrary they took and held almost any possible position, some with head up, others with head down, some paral- lel with the surface and others at an angle with it. Usually the body was curved towards the ven- tral side and sometimes it was thrown into sinu- ous folds. A few photographs of animals in these various positions were taken. The asymmetrical organization of the adult Asymmetron is not as- sociated with any peculiar position which they as- sume when they are at rest either in the water or in sand. F. Gitcurist, Harvard University SALT REQUIREMENTS OF LIGIA Lover's Lake, one mile west of the Station, was explored, and found to have a considerable tide and to be nearly, though not quite, as saline as sea water. The lake is an excellent collecting spot for a small viviparous Synaptula. In collaboration with Dr. T. C. Barnes experi- ments were made on tidal-zone isopods, Ligia. These were found to be positively geotropic and phototropic and rheotropic. They will live in the laboratory on moist sand for periods up to twenty days, and will survive complete submergence in sea water for six days, but die quickly in a dry environment. They are remarkably tolerant of changes in the osmotic pressure of the fluids they are submerged in, surviving appreciable lengths of time in distilled water and sea water concen- trated by the addition of dry salts. In working with solutions containing single ions, it was found that potassium was the most toxic of the metallic ions commonly found in sea water. Sodium, cal- cium and magnesium were less toxic. Some meas- ure of the viability of the animals in the various solutions could be obtained before actual death by observing the rate of gill movements, which slowed down some time before actual death. In solutions containing only potassium ions there were no gill movements at all. BoRS Lurz, Boston University School of Medicine THE EFFECT OF ADRENALIN CHLORIDE AND TOAD VENOM ON THE BLOOD PRESSURE OF THE TROPICAL TOAD, BUFO MARINUS How Bufo marinus secretes and stores in its skin glands an enormous amount of venom con- taining powerful adrenalin and digitalis-like sub- stances without harm to itselt is not known. Vari- ous workers have determined the pharmacological action and the minimum lethal dose of toad toxin for animals other than the toad. They generally agree that the toad is relatively immune to its own venom. Abel and Macht (1912) found that both bufo-epinephrin and bufagin, isolated from the venom, when added to a Locke solution used to perfuse the blood vessels of Bufo marinus, caused vasoconstriction. Gunn (1930) found an adrena- lin-like substance in the skin secretion of the South African clawed toad, Xenopus laevus, which produced striking circulatory effects in the cat, rabbit and guinea-pig. Neither adrenalin nor the skin secretion had an effect on the circulatory system of X. laevus. No reference in the literature could be found concerning the effect of toad venom on the blood pressure of the toad, nor even concerning the measurement of the blood pressure in this amphi- bian. Beiter and Scott (1929) found that an in- travenous injection of 0.2 cc. of adrenalin chlor- ide, 1 in 10,000, gave a rise of blood pressure in the frog lasting one hour and a quarter. They found the systolic blood pressure in Rana cates- biana to be 32 mm. Hg. The present report con- cerns the effect of adrenalin chloride and_ the crude venom of Bufo marinus on the blood pres- sure in the same animal. A determination of the minimum amounts of these substances necessary to produce a rise in blood pressure on intravenous injection is also reported. The fore-brain of Bufo marinus was destroyed and the spinal cord posterior to the second verte- bra was pithed. The blood pressure was recorded by a mercury manometer from a cannula in the femoral artery. Injections were made through a cannula in the femoral vein. The average systolic blood pressure in twenty- five animals was 30 mm. Hg., and the average Jury 29, 1933 ] THE COLLECTING NET heart rate was 64 per minute. Adrenalin chloride, 0.2 cc. of 1 in 50,000 (0.004 mg.), caused a rise in systolic pressure from 60% to 100% of the original pressure and lasting from three to six minutes. Weaker doses, such as 0.2 cc. of 1 in 200,000 (0.001 mg.), gave up to 56% rise ac- companied by a 20% fall in heart rate. The threshold dose of adrenalin chloride for blood pressure was 0.1 cc. of 1 in 1 million (0.0001 mg. ). The crude venom was expressed from the large gland behind the ear, weighed, dissolved in Ring- er’s solution and injected immediately. This gave invariably a rise in systolic blood pressure, the ex- tent varying with the dose. For example, in one case 0.05 mg. of crude venom produced a 120% rise, and 0.13 mg. a 292% rise. The heart rate was usually decreased. The threshold crude venom for blood pressure was 0.0006 me. per 100 gms. of body weight. References. Abel, J. J., and D. I. Macht, Pharm. Exper. Therap., 3, 319. Bieter, R. N., and F. H. Scott, Journ. Physiol., 91, 265. ; Gunn, J. W. C., 1930, Quart. Journ, Exper. Physiol., 20, 1. dose of 1912, Journ. 1929, Am. FE. B. Benson, B. R. Lutz Boston University THE ACTION OF ADRENALIN AND CERTAIN DRUGS ON THE ISOLATED HOLOTHURIAN INTESTINE In vertebrates the action of adrenalin on smooth muscle is generally the same as the effect of stimulation of the sympathetic supply to the same muscle; consequently both excitatory and inhibitory effects are observed. In invertebrates only augmentor actions of adrenalin have been described, with one exception. Wyman and Lutz (1930) found that adrenalin caused inhibition of tone, amplitude and rate of beat of the cloacal muscle of the holothurians, Cucumaria frondosa and Stichopus badionotus, Selenka (S. moebti, Semper). The intestinal blood vessel of S. ba- dionotus however showed a marked acceleration of beat in adrenalin 1 in 100,000. By using other autonomic drugs Wyman and Lutz (1930) found that in the holothurian a fairly complicated schema of drug action exists similar to that which forms the basis of much of our: reasoning con- cerning the nature of autonomic innervation in higher and presumably more complex animals. The present report deals with the action of ad- renalin and other drugs on the intestinal muscle of S. badionotus. Adrenalin chloride solution (Parke, Davis & Co.) added to the sea water bath (to make 1 in 119 50,000) in which a ring of the holothurian intesti- nal muscle was suspended caused a rise in tonus and sometimes in amplitude. The threshold dose was 1 in 125,000. After atropine (1 in 8,000) the usual adrenalin response was reversed or pre- vented. Pilocarpine nitrate (Merck) 1 in 500 to 1 in 1000 gave a rise in tonus and an increase in am- plitude. Physostigmine salicylate (Merck) | in 10,000 to 1 in 1 million gave a marked rise in tone. The amplitude was generally increased or unaltered, but when the tonus effect was extreme, a decrease in amplitude occurred. Atropine sulphate (Merck) 1 in 8,000 or stronger caused inhibition of tone and amplitude and sometimes cessation of beat. Mechanical stimulation of the ring of intestinal muscle either by touching, or by squirting the fluid against it in the bath produced an immediate temporary contraction easily distinguished from the drug effect. Chloretone in a concentration present in adre- nalin chloride 1 in 50,000 was without effect. It appears, therefore, that the usual antagonism found in the vertebrate between atropine and the parasympathetic stimulants, physostigmine and pilocarpine, exists in the holothurian intestine. While the motor effect of adrenalin on the holo- thurian intestinal muscle,is the reverse of its ef- fect on the cloaca in the same animal, this is not without counterpart in the vertebrates, since Lutz (1931) found adrenalin to be motor to the stom- ach and inhibitory to the posterior end of the spiral valve and rectum in elasmobranchs. References. ee Wyman. L. C., and B. R. Lutz, 1930, Journ. Exp. Zool., 57, 441. Lutz, B. R., 1931, Biol. Bull., 61, 93. Marion A. Rep Boston University School of Medicine PILTYSIOLOGICAL PROPERTIES OF THE LYMPHATIC HEARTS OF BUFO MARINUS The lymph hearts are normally under the con- trol of spinal cord centers, but have been reported capable of beating after denervation since the time of this discovery (1796 or 1832). Many in- vestigators believed that this extra-spinal beating was due to accidental causes, drying or injury currents. Of course there was always the possi- bility that the nerve had regenerated. Last spring Dr. Pratt discovered that the lymph hearts, an- terior and posterior, of the same side were always synchronous. The same synchronism was found in the toad. In Bufo marinus I denervated an anterior lymph heart and then after a period of 120 THE COLLECTING NET [ Vor. VIII. No. 65 ten days took a record of its activity simultane- ously with that of the homolateral posterior or- gan. In the group of toads studied in Bermuda 1 always obtained synchronous and_ therefore neurogenic beating. 3ecause of the previous doubtful myogenic beating found in the frogs studied by the denerv- ated-in-situ method or by the use of isotonic salt solution baths, I tried transplantation. The re- trolingual lymph sac was chosen because of its transparent membrane and its easy accessibility. Within ten days most of these transplanted lymph hearts were beating. Furthermore, the organs were large enough to permit the taking of kymo- graph records. Such records supported previous observations to the effect that electrical stimuli, single or faradic, may increase the rate of the myogenic lymph heart contractions but cannot tetanize it. There had been a change from a skel- etal muscle type of response to a cardiac type. Two experiments showed that curare did not cause abrupt cessation of beat as in the normal, but rather an initial stimulation which was _ fol- lowed by continued activity when there was ample blood supply. To make this myogenicity unquestionable it was necessary to show that there were no nervous ele- ments present. I tried a few vital methylene blue preparations at the Station, but brought most of my transplants back here where I have used Rogers’ silver impregnation method (suggested by Dr. Meader). Apparently there are no gang- lia in either the normal or transplanted organs. The influence of possible vaso-motor nerves was obviated by the fact that one very active trans- plant floated absolutely free in the lymph sac. The manuscript of the paper that | have out- lined above will be finished soon. Other observa- tions which I have made on the normal neuro- genic beating of the lymph hearts, especially those on the location of the spinal centers, will probably be incorporated into another paper on synchro- nism. T. W. Torrey, Harvard University ECOLOGY OF LAND MOLLUSCA During a five week period, from late July through August, an ecological study of land mol- luscs was successfully carried on. This entailed extensive collections and detailed notes on all those factors believed to bear on the problem. The greater part of the work was intentionally limited to St. George’s Island. It was believed that a tre- mendous advantage could be derived from a de- tailed introductory study of a relatively small area. One thus obtains a real basis for compari- son with other more distant regions, but at the same time profits by shorter visits to them, the previous discipline furthering a rapid appreciation of the essential points involved. With this in mind, St. George’s Island was carefully examined, and then, as far as time permitted, the observa- tions were extended to smaller and more distant islands. The collection of forms, both snails and slugs, runs into the hundreds and includes probably all the living species. Of the ecological factors, vegetation, soil, other organisms, climate, topo- graphy, etc., special attention was centered on the first three. A report of the findings and conclusions de- rived therefrom will appear at a later date. Fol- lowing a careful check on the identifications, the shells will be turned over to the laboratory for its permanent collection. Kart Sax, Harvard University CYTOLOGICAL INVESTIGATIONS OF CERTAIN SEMI-TROPICAL PLANTS The work at the Bermuda Biological Labora- tory was confined largely to a study of chromo- some number and morphology in the plants avail- able on the island of Bermuda. The native species are not of much interest for such studies, but a number of introduced species were studied and provided some interesting material. The common Aloe was found to have four large pairs of chromosomes and three small pairs. This chromosome complex is exactly the same as found in the related genera Gasteria and Hawor- thia. In this case the cytological analysis is in harmony with the taxonomic classification. The work on Aloe will be included in Mr. Marshak’s study of this family. Last summer one of my students made a study of several species of Yucca and found that this genus has 5 large pairs of chromosomes and 25 very small pairs. The general appearance of Agave would indicate that it might be related to Yucca, but the taxonomic classification places Yucca in the family Liliaceae, while Agave is in the family Amaryllidaceae. A cytological study of Agave americana, which is abundant in Ber- muda, shows that the chromosome size and num- ber is exactly the same as in the genus Yucca. The cytological situation is so unusual in these plants that the same numbers and sizes in the two genera must mean that they have had a common origin and are rather closely related, even though the taxonomic characters have caused them to be placed in different families. It seems clear that the taxonomic grouping in this case is artificial and does not represent the phylogenetic relation- ships. The two genera are found only in the Jury 29, 1933 } THE COLLECTING NET 121 southern part of North America. A report of this work will be published in the Journal of the Ar- nold Arboretum. The chromosome number in Carica papaya was found to be 9. Although the sexes are separated in this genus, there was no evidence of hetero- morphic pairs or sex chromosomes. G. M. Smirr Yale University School of Medicine 1. Inflammatory reactions associated with healing of wounds were studied at various stages ina ntimber of Bermuda fishes. In these studies particular attention was given to the role of the pigmented cells, their development, function, fate and relation to other cells found in the inflamma- tory processes. 2. Along somewhat similar lines a study was made of the repair of wounds in Holothuria rathbunt. 3. Certain physiological experiments « on the lateral line of fishes, already begun at Yale Uni- versity and the New York Aquarium, were ex- tended by observations on a considerable number of Bermuda fishes. These experiments consisted in testing the intake into the canals and the sub- sequent outflow from canals of artificially colored fluids. The experiments confirmed the impres- sion that the lateral line canals of the head and body of fishes function in part at least as a test- ing mechanism for chemical or physical changes of surrounding water. SEX DETERMINATION IN HYMENOPTERA (Continued from Page postulated that the ege cytoplasm has a tendency toward maleness which dominates the female tendency of a haploid nucleus but which is domi- nated by the doubled female tendency of a diploid fertilized nucleus. Schrader and Sturtevant (1923) suggested an algebraic sum hypothesis with female tendencies of the haploid set inadequate to swing the bal- ance, while the genetically similar set, doubled by fertilization, results in a female. It is generally agreed, however, that none of the hypotheses is adequate and we must seek something more than mere quantitative or numer- ical difference between chromosomes of males and females in Hymenoptera. In Habrobracon, females heterozygous for one or more traits occasionally produce haploid mo- saic sons which show certain characters in one part of the body and allelomorphic ones in the other. In studying these males it has been found that several traits, in structure as well as in color, are not autonomous. In other words the recessive part of the mosaic may be influenced by the dom- inant allelomorph in adjoining tissue, as discoy- ered by Sturtevant for vermilion in Drosophila. One interesting combination of colors occur- ing in mosaic eye of Habrobracon will illustrate this. White and ivory are non-allelomorphic re- cessives, each causing the eye to be white. From a female heterozygous for both (Whwh-Oo') one section of the compound eye of the mosaic son may be genetically white, the other genetically ivory. Were these both autonomous we would expect such an eye to be uniformly white. Such is not the case however. The white non-ivory region remains white and is sharply marked off 113) from the ivory non-white region which is black at the border. The double dominant character is’ re- constituted in the region where the two recessives are in contact although there is no diploid tissue there. This appears to be accomplished by a dif- fusion of something from the white region into the ivory. Such a reaction has been discussed in some de- tail since a similar type of behavior in mosaic males has led to the formulation of the theory to be presented. Study of the external genitalia of mosaic males reveals that many show feminization close to the line where the genetically different tissues meet. A sensory appendage or even a sting may occur. These organs are characteristic of females only and are never found in non-mosaic males. The condition is particularly striking when the males are mosaic for honey body color. The fact that many of these males are from virgin mothers proves that the feminized region does not develop from a fertilized diploid nucleus. The female structures always occur on one side of the mid- line only, therein resembling the reconstituted dominant black eye color mentioned above, and suggest that some influence has been exerted by one type of haploid male tissue on another ad- joining. These feminized structures do not appear on all mosaics for in many the line of mosaicism does not pass through ‘the genitalia. Neither do they occur in ali mosaics where the line does pass through the genitalia. These facts led to the sup- position that there are in Habrobracon two kinds of males, genetically distinct for sex-determining factors but phenotypically similar. When tissues differing in these sex-determining factors adjoin 122 THE COLLECTING NET [ Vor, VIL. No. 65 in a haploid mosaic male, one influences the other and there result traits characteristic of diploid tissue, in other words, female. Of the two types of males postulated, one con- tains 1 X chromosome + 1 set of autosomes and, in its X chromosome, the genes F and g; the other contains 1 Y chromosome, + 1 set of auto- somes and, in its Y chromosome, the genes f and (Ge Females contain 1 X + 1 Y + two sets of au- tosomes, being heterozygous for factors F and G (F.¢/f.G). Segregating eggs would result in two kinds of males in equal numbers from a vir- gin mother. In the reduced egg there are four ootid nuclei, two being 1 X + 1 set of autosomes and two 1 Y + 1 set of autosomes. Normally three of these degenerate as polar nuclei in the peripheral cytoplasm. It is supposed that if an X-bearing sperm enters the egg there is selective fertiliza- tion, so that it fuses with a Y-bearing egg nu- cleus. Similarly if the sperm which has entered be Y-bearing, it will fuse with an X-bearing egg nucleus. Diploid males would result when an X sperm unites with an X egg or Y with Y. They would be 2 X + 2 sets of autosomes or 2 Y + 2 sets of autosomes and would have the same genic ha- lance as normal haploid males. It has been shown by genetic tests that maleness in fertilized eggs is determined at or shortly after fertilization. This hypothesis of sex-determination in Habro- bracon is consistent with results of genetic and cytological studies. It is, moreover, highly sug- gestive for explaining the known facts of the life histories of other Hymenoptera, such as alterna- tion of generations, polyembryony, and produc- tion of females parthenogenetically. Finally, it is consistent with the theory of genic balance on a ratio basis, and, whether it ultimately proves true or not, may serve as a working hypothesis stimu- lating new modes of attack. The facts and principles upon which the theory of sex-determination here reported is based have been brought to light by investigations which have been generously supported in recent years by grants from the Committee on Effects of Radia- tion on Living Organisms (National Research Council ). LITERATURE Bridges, C. B. 1925a. Sex in relation to chromo- somes and genes. Am. Nat., 59. 1925b. Haploidy in Drosophila melanogaster. Proc. Nat. Acad. Sci., II. Goldschmidt, R. 1920. Mechanismus und Physiolo- gie der Geschlechtsbestimmung. Gebr. Born- traeger, Berlin. Schrader, F., and Sturtevant, A. H. 1923. ¢D At equilibrium the applied hydrostatic pressure, P, would be partially neutralized by P,, the elec- Jury 29, 1933 ] THE COLLECTING NET tro-osmotic pressure, so that Ps, the effective pressure in determining the streaming potential, would be less than P by the amount P. That is, 3 (@ 1810) 4arndAE R= P;-- Ps = oe ca te é¢D The ratio P/P; would then be P/P; = From this equation it is seen that, as the radius of the tube becomes very small, P;, becomes in- creasingly important as compared with P. From the data of White, Urban, Krick'?8), and that of Bull and Gortner'*"), Bull calculates that for tubes having a radius of 1 p» the value for P, is equal to about 1% of P while for tubes of 0.1 » the value of P; is about equal to Ps, i. e., about 50% of P. Ettisch and Zwanzig'*!) have found an increase in the value of E/P (in a glass capillary) with increasing pressure up to a pressure of about 20 em. H,O, when aqueous solution of NaCl was the liquid being forced thru the tube. After this value of P was reached, the value of E/P became nearly constant with increased values of P. When alcohol-NaC1 solutions were used as the stream- ing liquid the value of P above which E/P be- came constant was much higher. Reichardt‘??? offers an explanation of these observations by picturing a difference between x, the distance apart of the double layers, and L, the distance from the wall at which maximum velocity of flow is attained and thru increments of which the viscosity is at variance (especially in immediate environment of wall) with the viscosity for the whole. Thus if x < L and 7 signified the vis- cosity effective thru distance x, and », that thru distance, L, then would = x x f iy d ¢, not i) d¢ = ¢i — da, as in Helmholtz’s equation. By assuming that the boundary viscosity 7 is a function of the applied pressure forcing the liquid thru the tube, which is in accord with the theory of Lamb”), it is possible to explain the results of Ettisch and Zwanzig. However, that such an explanation is needed has not been born out by results obtained by Bull'**) when repeating the work of Ettisch and Zwanzig. Bull used a pyrex tube and_ elimin- ated rubber connections in the system. He studied the streaming potential at pressures vary- ing from 9 cm, H2O to 90 em. H2O, and with mixtures of 10° normal NaCI and ethyl or iso- propyl alcohol, in which the alcohol concentration varied from O to 80%. He finds there is no evidence of a change in ¢ with pressure and in only two cases did the curve ,which is obtained by plotting E against P, fail to pass thru the origin. In all cases the curve was a straight line but in these two cases it failed to pass thru the origin. This was exactly what Ettisch and Zwan- zig’s data showed also, i. e., in their observations, E/P was not a constant but E/P + p was a constant, where p is the value of P when E = O. Bull found that in the cases of the two excep- tions observed, when the experiment was repeated after the system had stood for several hours, the discrepancies disappeared and values of E/P be- came constant thruout the whole range of pres- sure. The curves of E against P plotted from data obtained before and after standing were parallel (i. e., their slopes were identical) the difference being that the former curve failed to pass thru the origin while the latter did so. Bull feels that such results as those of Ettisch and Zwanzig may be due to failure to allow sufficient time for equilibrium to be attained in the interface before the readings are made and that when this is done E/P for a given tube will always be con- stant. that the streaming potential equation fails to hold for pore From these data it seems probable diameters which are very low. That it also fails for low values of P is not so likely . However, careful work is yet required to definitely settle these questions. The streaming potential method for estimating ¢ has been found useful in numerous cases. Freundlich and Rona‘) and Freundlich and Ettisch'**) used this means to determine the value of € for a sample of glass for which they also measured the e, or transverse, potential by use of the glass electrode of Haber and Klemensie- wicz'*8), They found that € was not a function of « and could vary entirely independently of it. Kruyt'?) used this method to demonstrate the lyotropic and valence effects of salts upon the ¢-potential at a glass-water interface. Lachs and Biezyk'8 have repeated part of this work. sriggs8) 9) using a cellulose diaphragm, studied the lyotropic and valence effects of vari- ous salts upon the €-potential and upon _ the surface conductance along the interface cellulose- water solution. He found, contrary to the theory of Smoluchowski™® which postulates that there 130 THE COLLECTING NET [ Vor. VIIL. No. 65 should exist a direct relationship between these two interfacial phenomena, that there was no ap- parent relationship between them. This relation- ship has been further studied by Bull and Gortner'?"). Bull and Gortner'") measured the temperature coefficient of ¢€ at water-cellulose alcohol- cellulose interfaces thru the temperature range of 20°C.-51°C. by means of the streaming potential method. They confirmed the earlier findings of Cruse’), who, working with a clay diaphragm, and and measuring € by the electro-osmosis method, found a maximum in the temperature-é-potential curve for the water-solid interface at about 37°C- 40°C. The temperature coefficient for the alco- hol-cellulose interface was positive thruout the range of temperature studied. The same au- thors'4#) demonstrated that no antagonistic action upon ¢ exists between ions of Na and Ca, K and Na, or Ca and M¢g at a cellulose-water interface, the effects of such ions being nearly additive in all cases. Martin and Gortner™® and Jensen and Gort- ner”) have studied the streaming potentials set up when pure organic liquids were forced thru cellulose and AlsO3 membranes. The calculated values of ¢ thereby obtained have heen shown to bear an interesting and fundamental relationship to the molecular structures of these liquids. Ina homologous series of alcohols, for instance, the value calculated for € varies regularly in a step- wise manner from one membrane of the series to another. That the streaming potentials ob- tained in such systems bear a definite relationship to the dipole moments of the molecules of the liquids being used, is indicated by the fact that those liquids which consist of symmetrical mole- cules, such as benzene and carbon tetrachloride, give no streaming-potential at all, while those which are known to have very high dipole mo- ments, such as nitrobenzene, show the highest streaming potentials. Bull and Gortner‘#*) have studied the streaming potential at a liquid-liquid interface. In these experiments a small stream of aqueous solution was forced from a jet containing one electrode thru a volume of white paraffin oil after which it impinged upon another electrode. They point out that while comparative values could be ob- tained for the streaming potential at the liquid- liquid interface in this manner, the values for € calculated therefrom on the basis of the stream- ing potential equation were not correct because of the fact that neither side of the interface was static. The term for slippage along the wall at the interface as introduced by Lamb“! could certainly not be neglected in such a case. REFERENCES 1. Beetz: Pogg. Ann., 146, 486 (1872). 2. Quincke: Pogg. Ann., 107, 1 (1859); 110, 38 (1860). 3. Zollner: Pogg. Ann., 148, 640 (1873). 4. Edlund: Pogg. Ann., 156, 251 (1875); Wied. Ann., 1, 161 (1877). 5. Haga: Wied. Ann., 2, 326 (1877); 5, 287, (1878). 6. Clark: Wied. Ann., 2, 335 (1877). 7. Dorn: Wied. Ann., 10, 46 (1880). 8. Elster: Wied. Ann., 6, 553 (1879). 9. H. Helmholtz: Wied. Ann., 7, 337 (1879). 10. J. Perrin: J. Chim. Phys., 2, 601-(1904); 3, 50 (1905). 11. H. Lamb: Phil. Mag., (5), 25. 32 (1888). 12. G. Gouy: J. Phys., (4), 9, 457 (1910); Ann. Phys., (9) 6, 5 (1916); 7%, 129; 1149) (1917); 13. J. J. Bikerman: Z. Phys. Chem., 378 (1933). 14. G. de Villemontee: J. Phys., (3) 6, 59 (1897). 15. D. R. Briggs: J. Pnys. Chem., 32, 641 (1928). 16. W. M. Martin and R. A. Gortner: J. Phys. Chem., 34, 1509 (1920). 17. O. G. Jensen and R. A. Gortner: J. Phys. Chem., 36, 3138 (1932). 18. Saxen: Wied. Ann., 47, 46 (1892). 19. K. Kanamaru: Cellulose Ind. (Tokyo) 29 (1931) (C. A. 25, 3895), 20. H. A. Abramson: J. Am. Chem. Soc., 50, 390 (1928). 21. D. R. Briggs: J. Am. Chem. Soc., 50, 2358 (1928). 22. H. A. Abramson and E. B. Grossman: J. Gen. Physiol., 14, 563 (1931). 23. H.R. Kruyt: Kolloid. Z., 22, 81 (1918). 24. H.R. Kruyt and P. C. v. d. Willigen: Koll. Z., 45, 307 (1928). 25. Grumbach: Ann. Chim. Phys., (8) (1911). 26. H.B. Bull and R. A. Gortner: J. Phys. Chem., 35, 309 (1931). 27. H. B. Bull and R. A. _Gortner: Chem., 36, 111, (1932). 28. H. L. White, F. Urban and E. T. Krick: J. Phys. Chem., 36, 120 (1932). 29. H. L. White, F. Urban and EH. A. Van Atta: J. Phys. Chem., 36, 3152 (1932). 30. H. B. Bull: Kolloid. Z., 60, 130 (1932). 31. G. Ettisch and A. Zwanzig: Z. Phys. Chem., (A) 147, 151 (1980); (A) 160, 385 (1932). 32. H. Reichardt: Z. Phys. Chem., (A) 154, 337 (1931). 33. H. B. Bull: Private communication. (A) 163, 7, 3, 24, 433 J. Phys. 34. H. Freundlich and P. Rona: Preuss. Akad. Wiss. (Berlin), 20, 397 (1920). 35. H. Freundlich and G. Ettisch: Z. Phys. Chem., 116, 401 (1925). 36. F. Haber and Z. Klemensiewicz: Z. Phys. Chem., 67, 3851 (1909). 37. H. Lachs and J. Biczyk: Roczinki, Chem., 11, 374 (1981). 38. D.R. Briggs: J. Phys. Chem., 32, 1642 (1928). Juty 29, 1933 ] THE COLLECTING NET 131 39. D. R. Briggs: Colloid. Symp. Monograph 6, 41 (1928). 40. M. v. Smoluchowski: Phys. Z., 6, 529 (1905). (1905). 41. H. B. Bull and R. A. Gortner: J. Phys. Chem., 35, 456 (1931). 42. A. Cruse: Phys. Z., 6, 201 (1905). 43. H.B. Bulland R. A. Gortner: J. Phys. Chem., 35, 700 (1931). 44. H. B. Bull and R. A. Gortner: Acad. Sci., 17, 288 (1931). 45. H. A. Abramson: J. Gen, Physiol. 15, 279, (1982). Proc. Nat. DIscussION Dr. Mudd: all electrodes come to a constant value and remain Has it been your experience that there as long as the pressure is constant? In some work which I did they did not come to a constant value. Generally the vaiues are quite Polarization of the electrodes dur- Dr. Briggs: reproducible. ing the measurement must be prevented. Using a quadrant electrometer, the circuit is open at all times and a negligible amount of current flow is needed to charge the electrometer. There is, therefore, no appreciable amount of polarization, and the potential difference measurement remains constant as long as the pressure remains so. Dr. Cohen: reproducible ? To what extent are the values Dr. Briggs: Using the same diaphragm and the same liquid the ratio E/P is constant. The value of E is read to an accuracy of about 1 mil- livolt, the percentage error in FE is, therefore, higher as the value of E decreases. The value of E/P is then less accurate at low values of E than at high. Dr. Fricke: Is it possible to obtain streaming potentials for tissues ? Dr. Briggs: By forcing a liquid of sufficient- ly low conductivity through a tissue membrane it should be possible to obtain a streaming potential. Surface conductance would be high, in all proba- bility, and the accuracy with which Zeta could be calculated would depend upon how accurately this quantity could be measured. Dr. Cole: I would think the difficulty there would be that of determining the cell constant of the tissue, because the size and shape of the pore enters implicitly though not explicitly. It is nec- essary to find out what the specific conductance of the liquid is when it is inside the pore. If one is trying to work with a live membrane, there would have to be doubt as to whether or not it was still alive throughout the whole course of the measurement. Dr. Briggs: Streaming potential measure- ments can not be made on systems in which the specific conductivity of the liquid is high, as would be the case with bathed in their normal fluids. The chief difficulty of malk- ing streaming potential measurements on living most tissues tissues is this,—that the liquid phases of such sys- tems are relatively concentrated electrolyte solu- tions. Dr. Miller: What is the order of change in Zeta potential with temperature, in the cases in- vestigated ? Dr. Briggs: For the cellulose-water interface the variation is within a range of 2 to 3 milli- volts within the temperature range studied (20° C — 51° C). At the cellulose-alcohol interface the Zeta potential varied through a range of about 30 millivolts for the same temperature change. Dr, Miiller: This is quite in agreement with what one would find using the theory of the dif- fuse double layer. In this comparison of your measurements with the electrophoresis measure- ments of Zeta is the factor 4 or 6 used in the electrophoresis equation ? Dr. Abramson: The factor 4 was used. The evidence which yields a good deal of justification for using the same factor in both equations will be presented in my paper on The Chemical Con- stitution of Amphoteric Surfaces. Dr. Fricke: To what extent could the devia- tion of E/P at low values of radius of the pore be accounted for by electro-osmosis ? Dr. . Briggs: Bull has made measurements which thus far indicate that not all the deviation observed can be accounted for by the electro-osmosis counterpressure, i. e. that at low values of pore radius there is an actual falling off of the Zeta potential. Dr. Cohen: Are these pores so small that the diffuse double layer would overlap in the center of the tube? Dr. Briggs: The effect begins to be apparent in tubes having diameters less than 60p. Dr. Miiller: It is hardly to be expected that the overlapping of the double layer would occur in tubes of this diameter although it might do so if the electrolyte concentration of the liquid were very low. 132 THE COLLECTING NET [ Vou. VIII. No. 65 SURFACE CONDUCTANCE KENNETH S. COLE Smoluchowski Concept The effect of a charged surface on the conduc- tivity of an electrolyte in contact with it was first recognized by Smoluchowski in 1905. He pos- tulated the “rigid,” or Helmholtz, double layer with a surface charge density, —o, for example, Figure 1, and an equal opposite charge o at a distance d in the electrolyte. When an electric x d— iS ’ Figure I field X is applied parallel to the surface, there is a uniform velocity gradient over the distance d, due to the force on the charge o, and there is a uniform velocity for x > d. This is the pheno- menon of electro-osmosis, and the velocity as given by Helmholtz is XDé Xo i = — -d (1) tary 1 where € is the potential difference across the double layer, D the dielectric constant, and 7 the viscosity. The charge o is then moving with the velocity u and as such constitutes a current den- sity per unit length of surface = Xue and the surface conductance o 1 di 7 7] d Diffuse Layer Concept 9 ID) IF fale 4m It is only recently that quantitative data on the phenomenon have been taken, but at the same time it was being generally realized that the Helmholtz double layer concept would have to be replaced by that of the diffuse ionic layer of Gouy, (1910), and Debye and Hiickel, (1923), : Figure II , Figure 2. Using the Debye approximation for low values of the € potential, the potential ¢ at a distance x from the surface is Sao Dr where « is probability thickness of the ion cloud. Then the charge density at x oKe Electro-osmosis takes place with the diffuse layer in exactly the same manner as for the Helmholtz layer except that the force of the field X on the part of the charge in a layer of thickness d x moves it a little faster than the adjacent layer nearer the surface and all of these increments of velocities have to be added together to give the velocity at any distance x. The force equation du 21D) -al2gh dx = 47 > when integrated gives, (4) XD Xo —kKx u= (¢—O)=— (hs Jury 29, 1933] | _THE COLLECTING NET 133 The current density due to the charge carried is then oe Xo s KX —Kx J=fupdx= fe (l—e ) dx O 4) ) and the conductance, o 2 Ay = ——— (5) 2 K 7 Thus we see that the consideration of a diffuse layer has not changed the surface conductance equation derived on the basis of a Helmholtz layer when d is replaced by 1% x. Tonic Mobility Concept There is, however, another factor, unknown to Helmholtz and not considered by Smoluchowski, that becomes obvious from our present knowledge of the structure of the diffuse layer. We have said, until the present, that the electro-osmosis arose from the force exerted by the electric field on the net charge in the water without consider- ing the manner in which this force is transmitted to the water. When the field X is applied, it exerts a force on each ion of X z,e (where e is the electronic charge and z; the valence) and in a very short time the ion reaches a velocity where the viscous friction of the water prevents a fur- ther increase and the velocity becomes constant. The reaction is then a force X ze that is applied by each ion to the water by its motion through the water. If there are equal numbers of the two ions of opposite charge, then the forces due to each are equal and opposite and there is no net force on the water. If there is an excess of posi- tive ions, they will exert more force on the water than the smaller number of negative ions which oppose the motion, and the water will tend to move with the positive ions, but the velocities of ions of both signs, relative to the water in their immediate vicinity, will remain unchanged in spite of whatever movement there may be of that water as a whole. This motion of the ions through the water is not of primary importance in other elec- trokinetic phenomena but should usually be re- sponsible for a considerable proportion of the surface conductance. Electrolytic conductivity de- pends upon the concentration of ions present, the velocity with which they move, and the charge they carry. If the measurement is made at a great distance from all boundaries, there are equal positive and negative ionic charges and the water does not move as a whole, so we have the normal or bulk conductance. If the presence of a charged surface alters the velocity or the concentration of any ions, then there is a change of conductance in that region. The difference between the bulk conductance and the observed conductance is their the surface conductance. Thus if a surface re- pelled ions of both signs (as at an air-water inter- face), the concentration of ions would be less, the conductance Jess than the bulk conductance. and this particular surface conductance would be negative. In a similar manner, if the concentra- tion of one ion is decreased from normal, the sur- rg al b els x \ : Figure If face conductance due to it is negative. But if, at the same time, the concentration of another ion is increased above normal, Figure 3, the surface conductance due to it will be positive, and the ef- fect of both ions, depending upon which predom- inates, may be positive, negative or zero. General Formulation In order to use these ideas we shall express the total conductance at any arbitrary point in the electrolyte. As has been said, the conductivity may, to a first approximation, be taken as the sum of the conductances due to the separate ions. The current density is then J; = X ujn,z;e where uj, is the total velocity of the i ion under unit field, nj and z; are the concentration and valence re- spectively. Then the total current density = She inne and the conductance NS neZ ne (6) The velocity u; is made up of velocity of the ion relative to water, which is the usual mobility u; plus the velocity of the water u. If we further say that the molecular concentration of salt in the center of a large body of electrolyte is n mole- cules per cc. and each molecule is completely dis- 134 THE COLLECTING NET { Vor. VIII. No. 65 sociated to produce v; ions of i type, and at the point under consideration there is a_ fractional change of the concentration of the i ion Aj, we have A= (u+ uy) ny (1 + Ai) xe Separating (7) A=uneXy74+ nes yy; Z; sS >) +unes Aj y z+ ne Ay uy Z; Since there is electric neutrality in the bulk of the liquid 3 uz; = O, while n e & u,v, z = A, is the bulk conductance. The difference in conduc- tance, which is the surface conductance, becomes A, = A—A,-=une Ss Aj yz+ o neXA; Uj vi Zj (8) Noting that nAj vj z; e = p; is the contribution of the i ion to the net charge density p Ay =U p sp > pi O (9) u, p and p; are functions of the distance from the surface, so we must integrate to get the surface conductance ea) 00 AN = Ay Ae=fupdx+ safind x ‘i oO (a) If we let ora) ao =fpdx oO since it is the contribution of the i ion to the total charge ive) Ag = ede ee nne tire (10) Debye Approximation Calculation In computing the diffuse layer analogue of the Smoluchowski conductance, we have already evaluated the first term with the Debye approxi- mation. This approximation for a salt which ionizes into only two ions per molecule gives 0) = — og = a f2 so ap o = of (uy Uz) (11) a 7 and if we let ez y= fj where f, is the friction coefficient of the ion then o” > Vi zi3/ hi a +oe (12) 2 2k U] > Vi Zy~ This formula, which has been derived on the Debye approximation, is applicable to ¢ potentials up to about 10 my. General Calculation In cases where € is larger it is necessary to use a more exact formula, which is somewhat more difficult to develop. In order to show how this is accomplished, let eS D do q =fpdx= x [DkTn 4x et 2a ° | Hed J kT V >> Vj (e — 1) Integrating by parts, oa) @) 9 Ai =fupdx =fudq= va] O qo qo (14) 18) oO —fqdu=—fqdu qo qo Substituting for q, and noting D i (¢ — €) 4a y from (3) we have A; = D |DkTn 4a iv 2a (15) A} to) hep —l)d¢ oO Sync é where ¢ is the potential at x = O or ¢& = £ Jury 29, 1933 ] THE COLLECTING NET 135 If vy = vo = V, Ay — zed zed = ee Zikeal Paka D ees ( ) ——_—_— e —e d¢ ey 2n oO zee zee ae es ae Dre Diketay ,-** ay = e —2 Pez 2r ( sae ) (16) For A» we must evaluate cO o =fpdx fa) er e hed a=neuaf(e —l1)dx oO D | 2m = ne vy Zz => 4x VDkTn zed kT fo) e —1 /| | aed d¢. ¢ k T Vsu(e —1) (17) li» =w=v —_ze€ | DkTny ( 2kT ) ee —1 S| er DkT (o1 iz a) =o Ui oi + Us oe 2ayze (18) This expression can be consolidated considerably since Oo VS ; —= 5 2 fe 2k T l+e BOEte 4 zet | [Dk T aw | CS Bere [ 2kT 2ilsals Vv 2a (= —e (19) 01 —oo—o0 ;j - ame | o1 + o2 = SSanEREEEEEE zet V2DkTn» | cosh Zikcaile and then 1 m1 Ae i= [: 3 2Ky ID) ke 1P 1 1 ze€ cosh iP OO —=-) 2kT tiie) te (20) Comparison with Other Derivations The relation above is, of course, the same as that of Smoluchowski when d = 1%,, the ionic mobilities are neglected and € is small, as has al- ready been shown. The next development is that of Komagata, (1929), for the case of a circular pore when € is small enough for the Debye ap- proximation to be used. The Smoluchowski con- ductance is neglected, and when x R (K== pore radius) is large, the Bessel function of the solu- tion may be approximated to give the ionic mo- bility term of equation (11). The other extreme, where the thickness of the ion atmosphere is large compared to the pore radius, has been indepen- dently derived and will be discussed elsewhere. It is difficult to appraise the formulation by Moon- ey, (1932),-since it differs from the above equa- tion (18) and the derivation has not been avail- able. With the exception of a single term, agree- ment is obtained if oy o»2 and o have the coeffi- cients ow 10} ow and eres ze€é where w = sinh 1 —o? 2kT 136 THE COLLECTING NET [ Vor. VIII. No. 65 Mooney states that he has considered the dis- tortion of the diffuse ionic layer by the electric field, and since this effect has not been considered here, it may well be the reason for the differences in the final result. The next derivation published was for a plane with small ¢, and both types of conductance were considered, giving equation (12), Cole, 1932. “It is difficult to discuss adequately the theory of Urban and White, (1932). They have as- sumed from Stern’s theory, (1924), of double layer, that there is a number of cations equal to Zi@G | DkTn 21m NI 27re? near the surface and an equal number of anions on the surface, both of which give rise to surface conductance by moving with their respective mo- bilities as found in free solution. The reasons for splitting the equation (19) for o and for taking a normal mobility for an adsorbed ion seem in- sufficient in view of the omission of the Smolu- chowski term. The recent work of Bikerman, (1933), seems accurate and complete, although the development is somewhat awkward due to the entire depend- ence upon the work of Gouy. The results are stated for a plane parallel slit, so the absolute values are for two square cm. of surface and therefore twice the above. The effect of the sep- aration of the surfaces is considered in some de- tail and has, of course, been neglected here. Bikerman also makes the most interesting sug- gestion that the Smoluchowski term should drop out at a comparatively low frequency of measur- ing current while the ionic term should remain constant over the range of practical frequencies. e Experimental Results After Smoluchowski’s theoretical prediction of surface conductance, it was first found experi- mentally by Stock, (1912). There have been other qualitative observations of it which are quoted by Briggs, (1928), but the value of ¢ has usually not been given so that it was not possible to make a quantitative check. The first satisfac- tory data that have been found are those of Briggs, (1928), on cellulose, and it is unfortun- ate that the surface area is not available for a determination of the absolute value of the con- ductance. Abramson, (1932), showed that for these data there was a qualitative agreement with Smoluchowski’s theory when the ionic strength of the solutions was corrected for the ionic strength of water. Using these same data for the uni- valent chlorides it was shown, (Cole, 1932), that the conductance was a linear function of the surf- ace charge, except at low surface charges, so that the ionic term was of much greater significance than the o® or Smoluchowski term. It was necessary, however, to ascribe an added conduc- tance to Cl~ and the question was left open as to whether this was due to a mobility of the adsorbed ions or due to the presence of molecular or Van der Waal forces which increased the concentra- tion of ions of both signs near the surface in pro- portion to the charge. * There has been considerable further work on packed cellulose done in Gortner’s laboratory, Bull and Gortner (1931), but it will not be con- sidered here, since, as in Briggs’ (1928) work, both the surface area and the pore diameters in- volved cannot be determined. The first attempt at the absolute value of the conductance was made by McBain and Peaker, (1929), but the € potential is not given, and White et al, (1932), have not: been able to re- produce the data. The conductance was consid- erably larger than calculated by Mooney from his formula, and he postulated a movement of ad- sorbed ions, (1932). Urban and White, (1932), have given data for two concentrations of KCI in pyrex capillaries, but state in an earlier paper, White, Urban and Van Atta (1932), that the potentials are the maximum observed with freshly prepared capil- laries and that € might decrease to the neighbor- hood of zero with but slight effect on the conduc- tance. However, the data are probably the best available, and a recomputation is worthwhile, TABLE 1. CALCULATION OF SURFACE CONDUCTANCE FROM Data oF URBAN AND WHITE Ago KCl Gs o Ay Ag Ago obs. 2.5-104 120° 2910 58: :62 -20) less 5.0-10* 124 4650) .92 .99) (19) 224 ee ee M my. esu x 10° mho. pay cm. (Table 1). The agreement is not as good as that obtained by the authors with their formula. It is, however, in line with the conclusions drawn from the data of Briggs, and agreement is ob- tained if it is assumed that the charge adsorbed is only Cl all of which moves with a velocity be- tween 25 per cent. and 30 per cent. of normal. The weight of experimental evidence which is promised will decide whether Urban and White, JuLy 22 NER Je THE COLLECTING NET 137 and Komagata or Smoluchowski, Mooney, Cole and Bikerman have a more nearly correct picture of the mechanism. Remarks The phenomenon of surface conductance has an apparently satisfactory and complete mathe- matical background that has had a rapid recent development. There is, at present, so little com- plete quantitative data that it is not possible to claim that the theory is well verified by experi- ment. ‘The indications are, however, that the con- ductance, due to the changes of ionic concentra- tions near the charged surface, is of the same or- der of magnitude as the electro-osmotic (Smolu- chowski) term in many cases, and that it may be necessary to supply a mechanism which allows ions of the sign of the adsorbed ions to play a more prominent role than the present theory al- lows. There will probably be need for an exten- sion of the theory in the direction of smaller pores which are more often encountered in bio- logical systems. It is to be hoped that the inves- tigation of surface conductance will give us a clearer insight into the mechanism of interfacial adsorption and will help to formulate possible membrane structures that will perform biological tasks. Discussion Dy. Fricke: Would you care to discuss the justification for omitting the conductivity within the rigidity» boundary ? Dr. Cole: It seems quite possible that the ions inside of the rigidity boundary may have a defi- nite mobility in a tangential electric field, but, since this mobility is not known, it can only be included in the theory in the form of an arbitrary con- stant. Dr. Miiller: Is it correct to assume that the electric field is the same everywhere ? Dr. Cole: This assumption seems reasonable for the case of an infinite plane, and a circular pore, when the solid is an insulator. Dr. Fricke: Serious errors might be commit- ted when the pores are not straight and when end phenomena are present. Is one justified in as- suming that the viscosity is the same in the dif- fuse layer as in the bulk of the liquid? Dr. Abramson: Janet Daniels has measured the electrophoretic mobility of protein in a series of alcohol-water mixtures up to 35% alcohol, where the charge density was constant. The di- electric constant changed from 80 to 68 while the bulk viscosity changed .01 to .02. The dielectric constant enters as the square root, and the viscos- ity directly, so that the changes were largely due to the latter. The agreement of the observed and computed mobilities indicates that the viscosity in the diffuse layer is the same as in bulk. What is found when the theory is arbitrarily made to agree with Briggs for KCI? ‘Dr. Cole: The formula will agree fairly well with the data on the univalent chlorides if it be assumed that all of the surface charge is due to C1 and that it can move with about one-third of the mobility in free solution. Dr. Cohen: The introduction of this arbitrary correction factor, which causes the above data to fall into a consistent series, is but a single in- stance, and may be unique, therefore it does not merit undue importance. However, an examina- tion of this ‘arbitrary’ correction may furnish a hint toward the solution of the problem. It is conceivable that the behavior of the ions involved in surface conductance may be modified by the forces peculiar to surfaces in such a manner that ordinary concepts of concentration, etc., do not apply without correction. Dr. Cole: One might expect the Smoluchow- ski effect to play a larger part than it does in Dr. Brigg’s measurements. This might be an exam- ple for Bikerman’s suggestion of the effect of the measuring frequency. Dr. Fricke: Is there a possibility that part of the observed resistance may be derived from a polarization resistance in the cellulose diaphragm ¢ If this were the case the resistance would depend on the frequency, and there should also be a capacity effect. A condenser would then be ne- cessary to obtain a good bridge balance. Dr. Briggs: Measurements were made at the frequency of one thousand cycles. A condenser was not used in bridge but the end-point was per- fectly satisfactory except at low concentrations of IGIE Dr. Mudd: 1 would like to suggest that there are a number of physiological problems that might conceivably find a solution in the applica- tion of this type of phenomena. There are the questions of the kidney and intestine secretion against osmotic pressure which are not explained by the ordinary laws of diffusion {and osmotic pressure. Dr. Abramson: Has the surface conductance at protein surfaces been measured ? Dr. Briggs: Jn my measurements of the streaming potentials of protein the surface con- ductance was so low that it was practically negli- gible. Bull has also found this to be the case. Dr. Cohen: It seems clear that experiment lags far behind theory in the study of surface conductance. Moreover, Dr. Cole’s formulation 138 THE COLLECTING NET { Vor. VIIt. No. 65 of the theory shows that surface conductance on a solid in contact with an electrolyte is beset with serious complicating factors that cannot be re- solved readily, if at all. The latter appear to be absent in the case of surface films between a sol- vent and its vapor phase, or an inert gas. Since films with measurable dimensions can be pre- pared from a variety of solvents and solutes, it would seem possible to obtain experimental data of sufficient variety to form a basis for test of the theoretical aspects. Dr.Cole: I think that Dr. Cohen’s suggestion is excellent. The conductivity vs. thickness data for films and € potential data from electrophore- sis of air bubbles should allow calculation of the surface conductance and indicate the faults in the theory. BIBLIOGRAPHY Abramson, H. A. 1932. J. Phys. Chem. 36, 2141. Bikerman, J. J. 1933. Z. f. phys. Chem. 163A, 378. Briggs, D. R. 1928. J. Phys. Chem. 32, 641. Bull, H. B. and Gortner, R. A. 1931, J. Phys. Chem. 35, 309. Cole, K. S. 1982. Physics 3, 114. Debye, P. and Huckel, E. 1923. Physik. Z. 24, 185. Gouy, L. 1910. Jr. de Phys. (4), 9, 457. Komagata, S. 1929. Bull. Chem. Soc. Japan. 4, 255. McBain, J. W. and Peaker. 1929. Proc. Roy. Soc. 125A, 394. McBain, J. W. and Peaker. 1930. J. Phys. Chem. 34, 1033. McBain, J. W., Peaker and King. Chem. Soc. 51, 3294. Mooney, M. 1932. J. Phys. Chem. 35, 331. Stern, O. 1924. Z. f. Elektrochem. 30, 508. Stock, J. 1912. Anz. Akad. Wiss. Krakau, (A) 635. Urban, F. and White, H. L. 1932. J. Phys. Chem. 36, 3157. White, H. L., Urban, F. and Krick. 1932. J. Phys. Chem. 36, 120. White, H. L., Urban, F. and Van Atta, E. A. 1932 (a) J. Phys. Chem. 36, 1371, and 1932 (b) J. Phys. Chem. 36, 3152. 1929. J. Am. ON THE ABILITY OF MAMMALS TO SURVIVE WITHOUT BREATHING Dr. LAURENCE IRVING Associate Professor of Physiology, Unversity of Toronto The subject about which I wish to speak to- night is the ability of warm-blooded animals to survive under circumstances in which respiration is temporarily impossible. In the case of man, the requirement of oxygen is one of the most in- sistent and will not tolerate any interruption for more than a minute or so. It is quite different from the other essential requirements. The period during which an individual may survive without respiration, being limited to such a short time, leaves only a very narrow margin separating him from the termination of existence from lack of oxygen. This narrow margin has had an extremely important influence on the habits and activities of human individuals. The termination of human life usually results from the interruption of respiratory activity as a whole. Or it may be that some essential link in the respiratory or circulatory system is affected and the organism ceases to function. The peril of death is always close in the matter of respiratory function, and realization of the imminent danger of asphyxia brings forth the most powerful protective re- sponses. The period during which one may reasonably hold his breath determines the timing of musical notation. It restricts the human environment and sets a definite limit as to time and depth to which man may penetrate the sea. If the respiration were temporarily arrested, the period to which any one of us could hold his breath would be about forty-five seconds. If, however, we made some previous preparation, such as forced deep breathing, that period might be extended to five or six minutes. If to that period of preparatory deep breathing, were added the respiration of oxygen, the time might be ex- tended to 10-15 minutes. This device was made use of in the preparation of sprint swimmers from one country in the last Olympic games. The rate of oxygen consumption is probably the factor which determines the time during which the breath may be held. For a person at rest we can take the oxygen consumption to be 250 cc. per minute; with only quite moderate activity, such as walking, 7-800 cc. per minute is required. For climbing or moderately vigorous exercise, the consumption may rise to 1200 cc. per minute. A Marathon runner, running seven- teen kilometers per hour may consume as much as 3500 cc. per minute. Against these require- ments we find that the ordinary individual has a certain capacity for the storage of oxygen. Stor- age in the lungs is one important factor to con- sider. The so-called “vital capacity’? amounts to about 10% of the body weight. A 70 kilogram man would thus contain 7 liters of air in his lungs. Of the seven liters of air, less than one- fifth would be oxygen available for respiratory requirements. The quantity of oxygen which would be present in the lungs would then not Furthermore, a certain amount of oxygen which would be stored in the blood would be available. Allowing ap- proximately 7% of body weight as made up of blood, with an average oxygen content of 16-17 amount to more than 1200 ccs. Jury 29, 1933 ] THE COLLECTING NET 139 cems. per 100 ccm. of blood, we should find that the total quantity of oxygen in the blood would be 800 ces. In addition to these more apparent stores of oxygen, we have other body fluids which are more or less saturated with oxygen. Assuming the tension of oxygen in tissue fluids to be about equal to atmospheric oxygen, there could be dis- solved in the tissues 1000 ces., making the total amount stored in the human body about three liters. This, of course, is a maximum figure. Actually it is quite impossible that the entire quantity would be available for use; there must always exist a certain gradient which serves to force the oxygen from the places where it is stored into the muscles and other tissues where it is to be consumed, Probably not more than one-half the total stored oxygen would be avail- able for use if respiration were arrested. This would amount to about 1500 ccs. Since the oxy- gen requirement at rest is 260 ccs. per minute, this would seem to allow for survival without respira- tion for about 6 minutes. That is just about the limit of human survival without previous prepar- ation, but it is not possible to hold out so long voluntarily. It is also true that in addition to the store of oxygen that is dissolved in tissues and blood there exists in the “oxygen debt” an important means for maintaining the muscle tissues during a period when oxygen is not available. It oper- ates by the transformation of glycogen into lactic acid without the intervention of oxygen. This reaction yields energy which can be applied to muscular activity. When oxygen is available, the lactic acid is removed by an oxidative process. The oxygen debt can be measured by determining an individual’s basal metabolic requirements, get- ting him to engage in violent muscular activity and measuring the oxygen consumption during recovery. More oxygen is invariably consumed during recovery than is needed for ordinary maintainance and this extra amount indicates to what extent the individual has gone beyond his means during the activity. A supply of energy becomes available in this way which would be equivalent to that furnished: by fifteen liters of oxygen in the case of a trained athlete. In contrast to the total stored oxygen of three liters, fifteen liters is quite a large amount, and one would be inclined to turn to it to provide means for survival when respiration is arrested. However, it is available only for muscular activ- ity, and is not used by any tissue other than muscle. So that while the oxygen debt may serve very well to carry on muscular activity during a short period of asphyxia, it does not solve the problem for the important non-muscular tissues, such as the heart and brain. Since the resources which have been examined do not provide the means for survival during ex- tended periods of asphyxia, we can scarcely see how it would be possible for man to adapt them to make longer survival possible. Many animals, on the other hand, are superior to man in this respect. The first systemic work on this subject was done seventy years ago by Paul Bert. He determined the period during which a number of animals survived forced submergence under water. Various terrestrial animals, such as the dog, cat, rabbit, and hen, survived for from 2 to 4 minutes under these conditions. Similar tests of animals with a partly aquatic habitat, such as gulls, indicated that they were not particularly superior to the others in this connection. But the ordinary domestic duck, which is usually not an actively aquatic animal, survived for a period of 10-15 minutes; a seal, (one which was not in very good condition) was still moving at the end of 15 minutes, and the heart continued to beat for 28 minutes after immersion. It is of interest to consider his observations on the peculiar ability of young animals to resist asphyxia. New born rats would survive thirty minutes; for adults the limit of survival was about two minutes. During development the period which they survived progressively dimin- ished. This remarkable capacity for resistance to asphyxia in new born animals is probably related to the necessity for sudden development at birth from a condition in which the respiratory appar- atus has not been in use. At the time of birth in mammals there must occur a period when respir- ation as such is quite impossible. Respiration starts somewhat hesitantly and only after some time does it become regularly and firmly estab- lished, but once established there is no further interruption. The above experiments would appear to be rather artificial, and would scarcely give the ani- nal a chance to demonstrate its full ability to resist asphyxiation. On looking through the liter- ature to ascertain the opinion of the various authors on the duration of the period under which diving animals can survive, we find that the in- formation is quite uncertain. Hill has said that the human limit was probably that of a pearl diver, who remained submerged for over 4 min- utes. Parker mentions that the regular respira- tory interval of the Florida manatee may be as long as 20 minutes. Beyond this the figures are not so certain. The largest mammal, which is most able in its capacity to dive, is the whale. On account of the conditions of observation and the somewhat ro- mantic flavor which attaches itself to whaling stories, we find that authors show a good deal of hesitation in committing themselves as to the 140 THE COLLECTING NET [Vor. VIII. No. 65 period during which a whale can remain sub- merged. ‘This hesitation is unfortunately not ap- parent with other authors—one reference states that a whale may remain submerged for as long as 10-12 hours. Most of the publications which one would consider fairly reliable, however, set the limit of submergence at about an hour or an hour and a quarter. It is difficult to make ac- curate observations of this kind. I tried myself one time to time the dive of a loon. The loon submerged and the next time I saw him was about 12 minutes later. Some people, and I am among them, are skeptical of this measurement, and others think it unfair—that the loon could actually do much better than that. There are re- ports that loons have been caught on set lines at 150 ft. depth, which shows its ability to travel under water. The “Old Squaw” is said to have been captured in nets in the Great Lakes at depths of 180 ft., which indicates a considerable diving ability. So we see that certain mammals have a capac- ity for resisting asphyxia which far exceeds the ability of man, and, as a matter of fact, exceeds the capacity which we would expect on the basis of the amount of oxygen stored. That being the case then, we might consider what possible modi- fications might occur which would adapt them to survival. There might be an increased vital ca-~ pacity, with an increase of air stored in the lungs. This capacity might possibly be doubled; on the other hand, if it were to be much more than doubled there would hardly be space available in the body to accomodate it. Birds in particular have made use of the larger capacity of their respiratory apparatus to increase to some extent their capacity for survival. Mr. Foster and I examined the vital capacity of the duck, and found that its vital capacity was actually double that which would be expected from a mammal of the same size. But this increase is not sufficient to account for the enlarged capacity for survival which has been observed in certain animals. An increase in the volume of blood might be considered as a possibility. So many authors have called attention to the vascular networks of many diving animals, the so-called retia miriabilia which are quite conspicuous and which would seem to give to the animal a greater capacity for blood and hence for oxygen storage in the blood. It is very difficult to see, however, where there would be space for the visceral and other organs if the vital capacity were to be doubled, and the blood content also. Doubling the vital capacity and blood volume would take up 35% of the en- tire body weight. If then we could give the ani- mal only a slight addition to its ability to survive, making the limit three minutes, possibly, instead of 1-2, it would not seem worth while to consider these modifications in the respiratory or circula- tory system as sufficient to adapt an animal like the whale to attain its outstanding submergence and under water activity. We should also consider the capacity for con- tracting a greater oxygen debt. That seems at first sight a tempting possibility to investigate. But if we were to increase the oxygen debt, it would only influence the capacity for maintaining muscular activity for a longer period of time. I do not think, therefore, that the difficulties of non-diving animals rests in the maintenance of the muscles ; the oxygen supply for an arm or leg may be cut off for 10-15 minutes without great discomfort, and for an hour without actual ser- ious damage to the part. The difficulty seems to arise in protecting the more sensitive tissues, such as the heart and brain, which are damaged at once by asphyxia. It usually happens that when we examine an animal part by part and then attempt to recreate the whole by the addition of parts, we find that the whole animal is quite different from the sum of its component parts. There are additional processes by which parts are made to cooperate together, a particular type of integration which completes the working organism. The mechanism for the control of this integra- tion we regard as the central nervous system. If we belong to one group of biologists,’ we are likely to call it behavior, or, examing it as physiologists, we are more apt to analyze _ this behavior into its component parts, and refer to these as reflexes. It seems possible that it might be pertinent to examine some of the reflexes of div- ing animals to see whether they may be respon- sible for some degree of protection from as- phyxia. The first information which appears was published again by Paul Bert on the behavior of the duck when forcibly submerged in water. It remained quite quiet for from 10 to 15 minutes. Just before the termination of its existence, it gave some convulsive movements, but remained otherwise very still. This is quite in contrast to terrestrial animals, which, when submerged, or when the trachea is clamped, immediately per- form convulsive movements which blindly attempt to bring relief from asphyxia. They are quite purposeless movements which actually serve to terminate their existence more quickly. The duck, on the other hand, conserves its energy by eliminating all muscular activitv. That process has been quite definitely worked out as a particu- Jar reflex reaction, evoked by postural stimuli which give this reaction even when the animal is Jury 29, 1933 ] THE COLLECTING NET _ 141 out of water. The stimulus is given by holding the head or neck in a certain position, which is similar to the one assumed swimming under water, with the head and neck extended and slightly depressed. When a duck is held in this position, all activity of the animal ceases even though it is out of water. The same sort of re- flex inhibition of muscular activity can also be seen in the muskrat under a stimulus of the same kind. Activity of the respiratory muscles is in- hibited as well, respiratory movements are at once abandoned; a duck or muskrat can be held in that position for 10-15 minutes and no attempt to breathe will be made. That type of reflex is ob- viously of adaptive nature in favoring certain animals for existence under water. Along with the depression of skeptical activity and the cessation of the working of the respira- tory system, the following observation has been made by Richet. Inhibition and retardation of the heart beat occurred during submergence. When the vagus nerve in a duck is cut it does not survive any longer than a hen would under the same conditions. This reflex mechanism then seems to have a definite positive effect in pro- tecting an animal during diving. Even so, while it may inhibit the activity of the animal, it would not abolish its basal meta- bolic requirements, and we are still faced with the problem of how these animals survive for so long a period. Gratiolet in 1860 made certain ob- servations on the vascular structure of the hippo- potamus. He claimed that there was a muscular band which passed about the vena cava about where it went through the diaphragm. He be- lieved it had the ability to contract and to prevent the remainder of the blood from returning to the heart. He thought that such a mechanism would be useful in preventing the engorgement of heart and brain which was supposed to occur during asphyxia. The idea of “engorgement” may be injected into the discussion by reason of one’s own sensations during asphyxiation, when the cerebral vessels seem to be engorged and the heart feels strained; but whether the mechanism works in just that way seems rather doubtful. On the other hand, if such a device shut off a large part of the posterior returning circulation and shut it down to just what was able to pass through the anterior venous return then it would be possible for such oxygen as was stored to be utilized by those organs which are _ particularly sensitive to oxygen want. Other tissues can get along with the help of the oxygen debt process, but the heart and brain have no such device to aid them. In recent years more and more attention has been paid to cerebral and coronary circulation, with the result that it is apparent that the condi- tion of control in those systems is quite unique from the type of control extended over ordinary systemic circulation. We find suggestions of this in the apparently opposite action of histamine and adrenalin on coronary circulation. We learn also from Lenox and Gibbs, that if 5% carbon dioxide is breathed and the cerebral circulation is judged by the amount of oxygen in the blood returning through the jugular vein, that the cerebral circu- lation will be accelerated by as much as 40%, while circulation returning through the femoral vein is even diminished. There is then a relation between the cerebral and systemic circulation, which under conditions os asphyxiation would serve to conserve the oxygen supply for the more sensitive tissues, leaving the others to develop an oxygen debt. I feel that when we cannot find either chemical or physical processes in the avian or mammal body which would adapt them for submergence, that we should turn our attention to reflex ad- justments. It might be pointed out that the physi- cal-chemical and muscular sysems in mammals are remarkably alike, for the blood and muscles apparently have the same characterists in all forms and the elements of the nervous system are so similar as to be practically identical. So we can see that throughout the operation of the forces of evolution the physical and chemical processes have remained extremely constant, maintained in an apparently rigid mold. On the other hand, the adaptation of various groups to different environments indicates a remarkable degree of plasticity in the nervous system in the integration of these essentially similar organs for very different habits of life. TRANSLOCATIONS IN THE MOUSE AND THEIR EFFECT ON DEVELOPMENT Dr. GeorGe D. SNELL, ELsieE BoDEMANN, AND, WILLARD HOLLANDER Department of Zoology, University: of Texas An experiment which has been in progress dur- ing the past two years has shown that when male mice are X-rayed with doses in the neighborhood of 600 Roentgen-units and mated to normal fe- males, approximately one third of their progeny show induced heritable variations. By far the commonest of these variations consists of a ten- dency to produce small litters, usually comprising four young or less, instead of the usual seven or eight. For convenience this tendency towards small litter size has been termed semi-sterility, and evidence will be presented that it is due to the 142 THE COLLECTING NET [Vor VIII, No. 65 presence of translocations induced by the x-ray treatment . Of 111 Fy individuals from x-rayed fathers, 33 were probably semi-sterile, as against none in the control. Of these 33, 10 were select- ed for intensive study, first, of the inheritance of of the trait, second, of the embryological impli- cations of the small litter size, and it is the re- sults of this study that are reported below. Up to the present time the genetic investigation has been confined to determining whether semi- sterile individuals transmit the trait to their pro- geny, and if so, to what proportion of their pro- geny. In the case of all ten of the semi-sterile stocks, it was found that the tendency to produce small litters was passed by the original semi-ster- ile individuals to a part of their descendants. Semi-sterile Male F,146 gave the largest num- her of offspring and therefere is selected for fur- ther discussion. In accordance with the practice followed throughout the experiment, Male F,146 was outcrossed to females of an untreated inbred stock. This rules out the possibility that his small litters were due to the segregation of recessive lethal genes. By these females he had 26 young in 7 litters, an average of 3.7 young per litter. This litter size differs significantly from the lit- ter size of about 8 which is characteristic of these females when mated to normal males. Eighteen of these Fy young were raised and again out- crossed to a normal stock to give the F3. Three of these F3’s by a semi- -sterile F. male were also raised and similarly outerossed to make up a to- tal of 21 individuals from the mating semi-sterile X normal. A number of litters, in most cases five or more, were raised from each of these 21 individuals to determine whether or not they had inherited, in whole or in part, the semi-sterility of their fathers. It was found that they fell into two groups, first, a group of 12 whose litters averaged 5.8 or less young, second, a group of 9 whose litters averaged 7.8 or more young. Twelve, therefore, were semi-sterile and nine nor- mal. While these figures are not sufficiently ex- tensive to establish the exact ratio of semi-steriles to normals occurring among the progeny of semi- sterile individuals, they are a satisfactory approxi- mation to the 1:1 ratio which is expected on the assumption that semi-sterility is due to the pres- ence of a translocation. Many additional data have been obtained which are in accord with the view that a semi-sterile individual transmits the trait to half its progeny, though no other one in- dividual has been tested as thoroughly as Male F146. Small litter size, proven by the above data to be a transmissible trait, has been shown in an embryological investigation to be due to the intra- uterine death of a portion of the embryos. In the case of most semi-sterile stocks the majority of nonviable embryos degenerate at about the time of implantation, leaving, however, swellings or solid moles which persist for six or more days and mark their location in the uterus. The_re- mainder of the nonviable embryos develop fur- ther, usually showing at 12 to 14 days gross ab- normalities in which the central nervous system is most conspicuously affected. The abnormali- ties can generally be classified as one of two types. The first of these is a type in which the neural tube, in whole or in part, fails to close over. Most commonly only the anterior end re- mains open, producing a monster in which that part of the brain normally forming the inner sur- face remains exposed to the outside. Less com- monly the whole neural tube fails to close, re- sulting in complete spina bifida. In the second type the neural tube closes throughout its length, but becomes distended so that it has to fold or kink to adapt itself to the available space. Cer- tain individuals of the first type occasionally come to term, but the majority die before term and are resorbed. Both of these types are produced by most of the semi-sterile stocks, and vary within wide limits according to the stock from which they come. Within any one stock, however, they show a certain amount of uniformity. Ot 971 embryos from 10 semi-sterile stocks, ranging in age from 10 to 14 days, 507, or 59%, were degenerate or abnormal, 67 being monsters. One semi-sterile stock, No. 109 produced 29 of these monsters out of a total of only 188 young, but all except two of the remaining nine stocks produced at least one monster. In a control group of 213 embryos, 30, or 13% were degener- ate. Only 3 were monsters, these 3 all being of the distended central nervous system tvpe. In some of the semi-sterile stocks there is rea- son to believe that two or more translocations are present. This is indicated by the high percentage of degenerate embryos, and by the fact that a greater or less degree of semi-sterility is trans- initted to considerably more than half the pro- geny. In the case of the others, however, less than one half of the zygotes are nonviable, about 38% in the case of stock 146, for example. This is in accord with the results obtained by Dobz- hansky and Sturtevant. and by Glass, for recipro- cal translocations involving the second and third chromosomes of Drosophila. These investigators have found that somewhat less than half of the gametes produced by an individual heterozygous for a translecation are of the types that give nonviable zygotes. It is noteworthy that many of the abnormal human embryos described by Mall and other in- vestigators are strikingly similar to the abnormal embryos produced by semi-sterile mice, and it ap- pears probable that some, at least, of these types Jury 29, 1933], THE COLLECTING NET 143 of abnormalities in man are due to translocations and other chromosome aberrations. To obtain further evidence as to whether or not semi-sterility in mice is due to the presence of translocations, cytological studies and linkage tests are at present in progress with the semi- sterile stocks. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 25.) COMMENTS ON THE SEMINAR REPORT BY GEORGE D. SNELL, ELSIE BODEMANN, AND WILLARD HOLLANDER P. W. WHITING Professor of Zoology, University of Pittsburgh Genetic evidence for chromosome transloca- tions was shown several years ago by Bridges in Drosophila and the brilliant X-radiation work re- ported by Muller in 1927 has made possible more rapid analysis of chromosome constitution. Many investigators have followed Miuller’s lead so that we now have abundant evidence for translocations and other chromosomal irregularities not only in Drosophila but in several insects and in numerous plants. To those who work with Drosophila, mammals seem painfully slow. For one who would attempt to demonstrate induced hereditary changes in a mammal, the mouse, however, is a happy choice. Small size requiring little space, rapid generations and large litters as well as the presence of several clear-cut genetic traits are assets which the thoughtful investigator may well prize. Treatment of the males might be expected to give dominant lethal genetic effects as was dem- onstrated by Miller for Drosophila in 1927, Dr. Snell and his co-workers have actually shown these by decreased litter size, correlated with the pres- ence of embryonic and fetal abnormalities. Dem- onstration of the hereditary tendency toward cer- tain non-viable types in different lines is of inter- est not only to genetics but to medical science as well. Dr. Snell appears to have done a very creditable piece of work with careful planning of experi- ments and accurate interpretation of results. Al- though we can hardly expect a demonstration of translocation as convincing as may be shown in Drosophila with its complete chromosome map, evidence that translocations lie at the basis of cer- tain embryonic defects closely parallel with human conditions is a distinct contribution. We shall be interested to hear more in detail of this work. CHROMATIN EXTRUSION IN CERTAIN CILIATE COMMENSALS OF MOLLUSCS Dr. G. W. Kipper Tutor in Biology, College of the City of New York Certain ciliate commensals of lamellobranch molluscs seem to offer excellent cytological ma- terial for the study of macronuclear chromatin. [ am going to describe briefly one phase of the macronuclear chromatin, that of the anlagen fol- lowing conjugation. First a review of the condition found in Con- chophthirius mytili from the common mussel 17 y- lilus edulis. This ciliate possesses in the vegeta- tive state one large macronucleus and from one to four micronuclei. After conjugation the amphinucleus divides rapidly four times resulting in sixteen apparently equal products. Of these sixteen, twelve to fif- teen become differentiated, by swelling, into the macronuclear anlagen, while the remaining four to one become the functional micronuclei. As the old macronucleus is degenerating a_ peculiar change takes place in each anlage. Dense chro- matin spheres are built up in the center and as the exconjugant prepares for its first cell division these spheres of chromatin mi- grate to the periphery and are cast out into the cytoplasm. Here they degenerate and are absorbed. The anlagen, in the mean- time, have been segregated into two groups and pass to each of the daughter cells. In order to ar- rive at the vegetative state the ciliate must, of course, undergo a number of segregating cell di- visions. At each division more chromatin is ex- truded into the cytoplasm, the amount decreasing at each successive division. Ancistruma isseli from the solitary mussel M/o- diola behaves in a very similar manner. This ci- liate always has one macronucleus and one micro- nucleus in the vegetative state. Only three am- phinuclear divisions occur before differentia- tion takes place, resulting in eight apparently equal products. Seven of these swell and become macronuclear anlagen while one becomes the functional micronucleus, dividing at each cell di- vision by typical mitosis. In this ciliate relative- (Continued on Page 146) 144 THE COLLECTING NET { Vor. VIII. No. 65 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Martin Bron- fenbrenner, Margaret Mast and Annaleida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford THE BIOLOGICAL LABORATORY AND THE COLLECTING NET We hope that our subscribers read the an- nouncement of Dr. Harris, director of the Bio- logical Laboratory at Cold Spring Harbor, intro- ducing his section of THE CoLLtectinGc Nev. The acceptance of our proposal for publishing the lectures and seminars in THe Cottectinc Net is a compliment which has made us happy. It is a partial realization of our plan that Tue Cot- LECTING Net might some day become the com- mon organ of the many marine stations for biolo- by in the United States. A bigger step toward complete realization could not have been taken. In his announcement Dr. Harris remarks that independent biological laboratories should exist and must function for all biologists, and that he welcomes the “valuable opportunity” of “any practicable means of making the Biological La- horatory, and the results of its work and confer- ences, more generally useful and available.” It is an especial privilege to be able to serve as a medium for the dissemination of the results of the important work of the Biological Laboratory. Dr. Harris adeptly emphasizes the importance of fostering a closer relationship between biology and the basic sciences: mathematics, physics and chemistry.” As science advances it becomes more exact. Biophysics is a new science; the phrase “quantitative biology” would have been out of place at the beginning of the present century. THe CoLLectinc Net began publication in 1926. For five years we were not called upon to use “‘z’’ or “\/"; seven years passed before our authors required exponents and calculus charac- ters. Is not this indicative of the trend of mod- ? erm biology f THE COLLECTING NET In our last issue we made some general com- ments on the editorial policies of Tie CoLLect- ING Net, emphasizing the fact that we wanted to retain our “informality” and that we did not as- pire to become an “accredited scientific maga- zine.” These remarks were cut short by limita- tions of space. It may be long before it becomes the custom for biologists to write informal articles about bio- logical problems; discuss freely the work of their colleagues and introduce some of their own phi- losophy into the articles that they contribute. General essays on the bearing of modern biology upon the other sciences, upon philosophy and re- ligion, indeed, upon any phase of human affairs, would be of great interest and perhaps of im- portance. The cynical will remark that we will publish a lot of nonsense. Probably we will! But after all one worthwhile contribution would make up for a good deal of “nonsense.” DISTRIBUTION AND ECOLOGY OF THE MARINE ALGAE ON LAKE FISH! Our increase in size has temporarily swamped our small printing plant in New Bedford, but we hope to return to our normal schedule soon, On more than one occasion we have discovered too late that galley proof has not been submitted to the author. We wish to tender apologies to authors to whom proof has not been submitted. Especially do we apologize to Dr. Bell. When his seminar report came to us it lacked its title which we added in New Bedford (just as the number was going to press) from the calendar in the previous number, the relevant portion ot which we reproduce here: Seminar: Dr. G. W. Prescott: “Some Effects of blue-green Al- gae on Lake Fish.” Dr. Hugh P. Bell: “Distribution and Ecology of the Marine Algae of the Maritime Provinces of Canada.” A glance at the lines will explain, though not ex~ cuse, the mistake. Almost simultaneously, so it seemed to us, with the delivery of the issue in question to Woods Hole, there suddenly appeared two posters—one at the laboratory and one at the mess hall. We confiscated them for publication purposes and one of them appears on the opposite page. The smoker held in the Clubhouse after Dr. Lucké’s lecture on “The Zoological Distribution of Tumors” was attended by more than two hun- dred people who exchanged ideas and opinions among themselves over punch and cigarettes. This gathering afforded an excellent opportunity for many of the audience to meet and speak with the lecturer. The M. B. L. Club will hold a similar smoker after each of the Friday evening lectures. All members of the club (and those planning to join) are urged to come to these informal gath- erings. Jury 29, 1933] PEE Ms. Or Introducing Dr. ZENON M. Baca, advance fellow of the Com- Relief Foundation who is spending July and August in Woods Hole. Dr. Baeq recently received the degree of “pro- mission for in Belgium Educational fesseur agrégé”™ in physiology from the Univer-~ sity of Liége for his studies on the humoral trans- His treatise the subject appeared in the Archives Internationales de Physiologie for April, 1933. He is investigating his problem from the view- mission of nerve impulses. on point of comparative physiology. At the momeni he is working on the humoral transmission o1 He is interested in the subject of the innervation of the nerve impulses in the heart of the squid. genital organs and is studying the reactions of the autonomic nervous system to drugs. When he leaves Woods Hole during the latter part ot August he will go to Harvard University where he will consult with Dr. Walter B. Cannon, pro- fessor of physiology at the Harvard Medica: Schooi. He will return to Belgium late in Octo- ber. A graduate of the University of Brussels in 1927, Dr. Bacq visited this country in 1929, again under the auspices of the Belgium Educationat Foundation, and worked with Dr. Cannon at Harvard during that year. He has recently been assisting Dr. Henri Fredericq, professor of physi- ology at the University of Liege, who worked at Woods Hole a few summers ago.—k. G. THE COLLECTING NET 145 LIN Ea an THE MIGRATION OF TUNNY FISH We recently received the following communica- tion from the Commissioner of Fisheries of the United States Department of Commerce: “The State Department has forwarded to the Department of Commerce a communication from the Portuguese Legation, advising of the con- tinued studies by the research ship Albacora with respect to the migration of the tunny fish in At- lantic waters, and requesting that proper publi- city be given to scientific and commercial institu- tions to the end that records of tagged fish taken in the North Atlantic might be obtained. The Bureau of Fisheries will appreciate the publication of the enclosed note in the columns of your journal.” The Notes The Portuguese research ship “Albacora,” em- ployed in oceanographic investigations, has re- sumed studies on the migrations of tunny fish in the North Atlantic by marking 60 fish with meta disks bearing the legend “Rk. P, AQUARIO— LISBOA—PORTUGAL.” The cooperation of the oceanographic institu~ tions and laboratories, and of the fishermen and fishing corporations has been requested by the Portuguese Legation in Washington to the ena that the disks when found may be returned to the AQUARIO VASCO DA GAMA, LISBOA, PORTUGAL, with appropriate information re- garding the day, hour and locality where the fish was caught. A reward is offered for such records of recapture. DISTRIBUTION AND ECOLOGY OF THE MARINE ALGAE ON LAKE FISH HP. Iie Professur of Hotany, Dalhousie University Salmon carrying dulce up Columbia River to sisters-in-law at Late Louise Trout bathing at Nobska jee to review Algae | aaa Professor B. of Bughousie Unversity explains distribution of the Rhodophyceae im Selkirk Mountains as a result of glacier front habitat —— 146 ‘THE COLLECTING NET [ Vor. VIIT. No. 65 CHROMATIN EXTRUSION IN CERTAIN CILIATE COMMENSALS OF MOLLUSCS (Continued from Page 143) ly huge amounts of chromatin are extruded at the lirst cell division of the exconjugant and smaller amounts at each of the subsequent divisions until the vegetative condition is reached. This summer I have been investigating the con- dition found in Conchophthirius anodontae from fresh water mussels. Although these observations are far from complete there seems to be a rather close agreement between the reorganizing forms and those I have just described. ‘Che vegetative individual has a single macronucleus and a par- tially imbedded micronucleus. After three amphi- nuclear divisions seven macronuclear anlagen and one micronucleus differentiate, as in Ancistruma isseli.. Spheres of chromatin form within each anlage, fuse and are extruded into the cytoplasm during the first exconjugant division. More ex- trusion chromatin is formed and thrown out in the next two divisions, as in the preceding species. The form of this later extrusion chromatin is slightly different but the result is the same. As to the meaning of this phenomenon we can only speculate. If we consider the macronu- cleus of ciliates to be trophic in function (in some way regulating the cell metabolism) and the micronucleus as germinal (functioning primarily during conjugation and endomixis) then this ex- truded chromatin may possibly be the germinal substance being cast out in the purification of a trophic cell element, the macronucleus. The mi- cronucleus must be considered as retaining the germinal substance and some trophic substance, or at least the potentiality for forming trophic sub- stance, as we know the new macronuclei are built up from micronuclear material following conjuga- tion or endomixis. It seems to me that we can compare this extru- sion chromatin to the degeneration products of amphinuclear divisions as reported in many other In those cases it may be that the germi- nal material is eliminated all at once. To bring this idea into line with those forms in which none of the amphinuclear products degenerate and there is apparently no extrusion chromatin, . we may suppose that the differentiating mitosis of the amphinucleus is at least qualitatively heteropolar, only trophic material going to the pole that will form the new macronucleus. ciliates. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on July 18.) News Items from Cold Spring Harbor Dr. Eric Ponder of the Department of Biology of Washington Square College, New York Uni- versity, has just returned from England, and is now in residence at the Laboratory. Dr. Robert Gaunt, Professor of Biology at the College of Charleston, recenily returned to the Laboratory, following his marriage to Joseph- ine Howland. Miss Howland, who 1s the daugh- ter of Dr. Howland of Schenectady, received the M. S. degree at Brown University in June. Both Prof. and Mrs. Gaunt are former students of the Laboratory. Dr. Hugo Fricke, of the Biological Laboratory, will sail for England and the continent on August 5th. Dr. Fricke will attend and present a paper before the Third Congress of Experimental Cytology at Cambridge, and will visit his parents in Copenhagen. Dr. Robert Chambers was here from Woods Hole for a few days, during which he presented a paper on “Intracellular Ovxidation-Reduction Potentials” in the symposium of July 24. Among those who have been visitors to the Laboratory since the last issue of THE CoLLEct- ING Net are: Dr. Robert H. Halsey of Post Graduate Medical College, Dr. L. R. Blinks of Rockefeller Institute, Dr. Cobum of the College of Physicians and Surgeons, Mrs, Janet Daniels, and Miss Capps of P. and S. —== a! ee ¢ Dr. Hans Miller of the Department of Physics of Massachusetts Institute of Technology will re- turn shortly to Cambridge. Prof. Muller has been a member of the group engaged in the con- ference on electrical potential differences at inter- faces and their bearing upon biological pheno- mena. He is to give a course at M. I. T. during the remainder of the summer. Three afl-day symposia, the last of the formal program of the conference this year, will be given this week; one each on Monday, Wednesday and Friday; for titles and speakers see the July 8th issue of THE CoLLecTING NET. dancing and card party was held in the re- Ad g¢ and 1 t) held in th creation room of Blackford Hall, Wednesday evening of last week. Vol. VIII. No. 6 SATURDAY, AUGUST 5, 1933 Annual Subscription, $2.00 Single Copies, 25 Cents. STUDIES ON THE CYTOLOGY OF CONDITIONS OF LIFE IN THE DEPTHS AMPHIBIA OF THE OCEAN Dr. ArtHUR W. POLLISTER Dr. Aucust KroGH Instructor in Zoology, Columbia University Professor of Zoophysiology, A study has been made of the cytoplasmic com- University of Copenhagen ponents in a variety of Amphibian tissues, nearly Having undertaken to speak on this subject [ all types being represented with the exception of | think it the safest plan to admit at once that I the striated muscle fibre and know next to nothing about it. the neurone. The Kull, Kola- What I hope to do is to show tchev, and Benda methods were QW. W. U. Calendar that there is a real and impor- chiefly employed. Every type tant problem and to create an of cell was found typically to THURSDAY, AUG. 10, 8:00 P. M. interest in it not only passing contain three types of formed Seminar: Dr. A. B. Dawson: “The | and academic but practical cytoplasmic structures, that are See eer cokte oc nigciiek Py since I am_ speaking at the sharply distinguishable from NGA AREID place which has the best op- one another in their morphol- Dr. V. Schechter: ‘Morphological portunities in the world for ogy, namely: chondriosomes, and electrophoretic effects of the | work of this kind and the best usually in the shape of separ- See cnn oe sone GEMM) |: Brains to itiltcer tea ate unbranched filaments ; Mr. K. Dan: “The electric charge I propose to speak only of Golgi material, always in the on the surface of sea-urchin the open ocean where > the form of thin lamellae; and, in eggs.” influence of the land is neg- of Dr. R. W. Gerard: ‘Electrical ac- licibl I ike sbyaRr ileal tivity of the brain.” igible, where the phytoplank- ton produces from the surface nearly every case, a pair centrioles, small granules closely adjacent to one an- FRIDAY, AUG. 11, 8:00 P. M. down to say 200 meters an ex- other. Decherd Osetspek.y sDie cess of organic material which = i Ser eae oe tac Protoplasmadifferenzierung der f Fy iaa AG SS. OE ea The tissue cells studied thus ‘Siicciilan wool coe GEion urnishes the basis o animal far fall into one or the other Entwicklung.” life through all depths. Be- of two schemes in the arrange- low 200 meters while some ment of these three cytoplas- assimilation may be going on, mic components. The first group contains cells the dissimilation is in excess and below 400 me- that are physiologically unpolarized in that all the ters there is dissimilation only, by animals and cell surface is similarly (Continued on Page 156) bacteria but no assimilation by plants. TABLE OF CONTENTS Studies on the Cytology of Amphibia, Dr. Agglutination, Dr. Stuart Mudd........... 174 Amur. Pollisters (airs says both scrunatdh et 153 ere ; - : Conditions of Life in the Depths of the [Diehimeyatyl Mea A home oi eno orto one wee 186 Oceanhe DOr AVSUSE IOP cc fete rete sete els MBE ~ (Chriseeuns) ital (oS ISOS Ra onoanvuokeooobdoncoec 186 The Biological Laboratory: 187 Stability of Colloids and the Theory of PEemSiOf (MCC eS Geer sere aie eter retraces ee ete Rapid Coagulation, Dr. Hans Mu'ler ....157 Penzance Players ...............+eueeeeeee 187 The Electric Potential and Charge of Dis- Di 183 solved and Adsorbed Proteins, Dr. Harold SEIDEL SEN TIGERS, SO Gon bition Someta AGEMA DEAIISOMN mist rerertesieucicea oie sie ese vis) ee 162% “Advertisings Sectionar < secemte ite « reterernae sls 189 154 THE COLLECTING NET [ Vor. VIII. No. 66 How does organic material and energy become available for the animals living below 400 meters ? One thing to consider at the very beginning is the question of intensity of animal life at the differ- ent levels. It appears that the intensity of life be- low the surface, that is, below 200 meters, de- creases greatly. The following figures obtained by Hentschel for nannoplankton organisms indi- cate the possible extent of this> Surface 10,000 per liter 50 meters 9,000 per liter 100 meters 2,700 per liter 400 meters 260 per liter 2000 meters 50 per liter 5000 meters 15 per liter This shows very clearly the decrease in number of these minute organisms with increasing depth. For the larger organisms there are no correspond- ing figures. But we ought to have them. As ] shall show later on they are necessary for the solution of the problem and it is undoubtedly now possible to determine the quantity of plankton at great depths. On and in the bottom itself a highly varied fauna exists. Let us consider for a mo- ment the conditions under which these animals live, confining ourselves for the present to the At- lantic Ocean. This ocean covers an area of 90 million square kilometers with a mean depth of about 4000 meters. The depth of over one-fourth of the area exceeds 5000 meters. The pressure increases One atmosphere for 10 meters—at the mean depth the pressure is 400 atmospheres. There is at this depth absolute darkness—except for a very faint light given off by organisms. The temperature is practically uniform —O0° —3° C. Oxygen is available in sufficient quantity 5-6 ce. per liter. Currents are flowing at a very slow rate estimated at 25 to 100 meters per hour. Let us now try to picture the effect of these conditions upon the fauna. In former days great stress was put upon the high pressure—it was supposed that this affected the organisms in many ways. Later investigations show that pressure does not matter very much. The water and the organisms themselves are slightly compressed—at 4000 meters about 2%. One might suppose that the pressure would increase the viscosity but there is no evidence of this. There is oxygen enough and light is not essential for most animal organ- isms. At the prevailing temperatures the meta- holism decreases to about 1/5 of the value at 10° C. This is of importance because it means that animals can subsist on much less food than would be necessary at higher temperatures. The density of population on the bottom is unknown. It is to be concluded from the dredgings and trawlings of the various expeditions that the den- sity must be low. When a trawl going over 10,000 square meters of bottom catches 200 specimens the haul is considered exceptionally successful and in such a haul 50 or more species may be repre- sented. I would strongly urge the adoption of quantitative methods. The Peterson grab for taking bottom samples comprising a known area has been successfully applied—depths down to 1100 meters and will probably work at any depth. The working of trawl and dredges at great depths is uncertain and they may take only a small frac- tion of the population actually present but when on similar bottom certain large areas give on average much fewer specimens per haul than others, it must be legitimate to conclude that the average density of population is actually lower in the first. It is therefore almost certainly signifi- cant that in the Challenger expedition the average trawling on the Globigerina ooze gave in the At- lantic 21 specimens, in the Pacific 56 and in the Southern ocean 97. This points to a correlation with the intensity of surface life and such a cor- relation has been definitely established by Hents- chel for the bathypelagic nannoplankton forms. When at the surface nannoplankton organisms number over 100,000, the number at 2000 meters is found to be over 100; if the number at the sur- face goes down to 5000, the population at 2000 meters will be less than ten. This relationship points to certain conclusions with regard to the food supply at great depths which we have now to consider. We have two possible alternatives. It is agreed that all the food comes ultimately from the sur- face, but the manner of its coming down, whether in the form of organisms and excreta from or- ganisms, or whether the organisms become dis- solved and are then utilized by some forms, at least, of those living at great depths of the sea, has not been certainly determined. The first of these possibilities, that animal life depends direct- ly upon organisms sinking down from above, 1s supported by those facts that were just cited. In spite of these, however, this assumption presents very great difficulties. When phytoplankton or- ganisms sink down through the water, they do be- come dissolved, so that when they are sought at great depths one finds only the empty shells— only in a very few cases is there a little proto- plasm left in the shells. At intermediate depths of 400-1000 meters, there is a comparatively large number of animals who feed directly or indirectly through smaller forms upon the phytoplankton, so that it seems impossible that there would be enough to support animal life at the bottom of the ocean. There is a possibility that we have animals from 200-400 meters living directly on phytoplankton sinking down, other animals feed upon these as they die and sink and so on down through the lower levels. This would involve a definite and Aueust 5, 1933 ] THE COLLECTING NET 155 very rapid falling off of the quantity of living material with depth, since by far the larger frac- tion of the energy and material available at each depth must be used up in the metabolic processes. Usually only the plankton organisms have been considered as a source of food for animals at great depths, but it might very well be that the bodies of large animals living at the surface could constitute a signicant source of supply. Large fishes or whales, will at least sink down to the bottom fairly rapidly, while the plankton organ- isms sink extremely slowly—it would be a year or so probably before the latter reach the bottom if ever they get so far, but a large fish would reach a depth of 4000 meters in a couple of days. Recent admittedly very rough, calculations by Hjort give the number of whales in the South Sea in an area of 8 million square kilometers as 300,000. Assuming a population in a state of equilibrium this would mean that about 50,000 whales would die and sink down to the bottom in this area every year. If that were so, it would mean one whale for each 160 square kilometers, corresponding to 50 square miles, each year. This does not sound like much but it would amount really to a great deal, about half a gram available organic material for each square meter. So it is possible that the bodies of large animals may con- stitute an amount of food for the animals at the bottom that is not at all insignificant. Many animals at great depths are adapted to have food only at rare intervals. Several deep sea fishes can swallow a prey of their own size or larger and if-such a fish had food once a year, that might be quite sufficient. Even at 15° C. many fish and other animals can live without food for six months, and at lower temperatures this period would be extended. There is not com- plete data at present to indicate whether animals sinking down to the bottom constitute a sufficient or insufficient food resource. Some years ago it was maintained by Putter that this source of food and excreta would be absolutely insufficient and must be supplemented by dissolved organic ma- terial. , There is no reason to believe now that for most animals the dissolved organic material is essential. Still, there is a possibility that for certain organ- isms, at least, dissolved organic material might constitute a very important item. Information as to the organic material present in solution in the ocean is admittedly incomplete and the older determinations are unreliable. I have made a series of determinations on samples taken in the Mid-Atlantic. These samples showed very little variation and an average amount of 244 mg. N per cubic meter, and of 2350 mg. C. per cubic meter. These figures are probably not quite right, since the water samples had been standing quite a while before they were analysed, thus causing a possible decrease. We are making new determinations now that should be more accurate. But whether the figures are a little high or low does not matter much, it is evident that the amount of organic material in solution is enor- mous. I have tried to figure out the amount pres- ent in organisms at any one time. It seems that this is only about 1/1000 of the total supply. This leaves a very large surplus available but it is a question whether it can be utilized. There is some reason to believe that a very large part of it can- not be utilized even by bacteria. If it were to constitute a source of supply for very many bac- teria, we should find them in far greater numbers than we now do. Asa matter of fact, these sub- stances, whatever they may be, are not very good food. It can be shown that all large animals, like all the fishes, crustacea, etc., are unable to take up organic material from solution in any significant amount. Bacteria, protozoa, and perhaps a few other forms have such a large surface compared to their volume that they would be more likely to manage to use it directly. Alexander Agassiz maintained fifty years ago that there must be at the bottom of the ocean a very large number of protozoa. And they may be there, but no one so far has seen them. But if they are there, they might live on dissolved ma- terial and themselves then constitute a source of food for the larger animals. It seems possible also that sponges might be able to utilize dissolved material to a significant extent. Sponges seem to be relatively abundant in certain places at great depths and it is not easy to see what else they can get. It is however quite certain that food must .be available in the bottom and just above the bottom in the form of minute particles, but where these particles come from is very difficult to say. Num- bers of animals at all depths live in mud. We have observed during the recent cruise of the At- lantis that even at the greatest depth the surface of the bottom is largely made up of fecal pellets, showing that animals eat mud and must be sup- posed to subsist on it. Even this conclusion is a little dangerous. I have made experiments on mud eating animals living at shallow depths where the mud contains a large amount of organic ma- terial. Samples of such mud were analyzed and the amount of organic material in it determined. Then different animals were kept in a known amount of the mud for a fortnight or a month. Considering the oxygen used by the animals, they should have used one-fourth to one-half of the organic material in the mud. As it turned out, there was exactly the same amount present in the mud as before. There is still reason to believe, however, that these animals do not in natural con- 156 ¢ THE COLLECTING NET [ Vor. VIII. No. 66 ditions eat the mud indiscriminately, but rather the material flowing along its surface. I think we must face the possibility that there may be at the bottom at great depths, micro-or- ganisms which live on dissolved substances, and which constitute an important source of ultintate food for higher animals. Personally I believe that other possibilities are perhaps more likely. It will be evident to all here that I have said very little—given very little definite information about what is going on at great depths. What I have tried to do was to present the problems that are still to be solved, which I hope may stimulate investigation in this direction. In conclusion I should like to give a quotation from a great countryman of mine, Niels Stensen or latinized Nicolaus Stenonius born just 300 years ago. In his inaugural address in the Copen- hagen Anatomical Theatre he said: “Beautiful are the things we see, More beautiful those we understand, By far the most beautiful are those of which We are still ignorant.” STUDIES ON THE CYTOLOGY OF AMPHIBIA (Continued from Page 153) exposed to the surrounding tissue fluid. An ex- ample of this is the leucocyte. Under these con- ditions the organization of the cytoplasm is very definitely focussed in the centrioles. These may be enclosed in a specialized area of the cytoplasm, the centrosphere, and with strong fixing fluids one can demonstrate in cells with a considerable vol- ume of cytoplasm, an extensive aster, the rays of which are radial to the region of the centrioles. The Golgi material is in the form of an irregular membrane surrounding the central apparatus. In cells with a prominent aster the chondriosomes in its vicinity are oriented with their long axes par- allel to the astral rays, i. e., radial to the centri- oles. In other regions of the leucocyte and in cells without an aster the chondrioconts are ori- ented quite at random. I have found that the following cells belong to the physiologically un- polarized type, in which the Golgi apparatus is closely aggregated about the centrioles: leuco- cytes, erythrocytes, mesenchyme cells, fibroblasts, gonial cells, peritoneal epithelial cells, endothelial cells, and smooth muscle fibres. The second cell type comprises the polarized epithelial cells, which have one surface in contact with a cavity or lumen, the opposite surface wholly or partly in contact with the tissue fluid, while the other sides are contiguous to adjacent epithelial cells. These external influences ceter- mine the orientation of the cytoplasmic compon- ents, replacing the possibly more fundamental or intrinsic tendency toward an organization about the centrioles. The most striking morphological difference between the polarized and unpolarized cell is the fact that in the former the Golgi ma- terial has no fixed close topographical relationship to the centrioles. The latter are usually located near the center of the distal end of the cell. The axis passing through the two is often nearly per- pendicular to the cell surface and, in some types, a flagellum is attached to the distal centriole. The Golgi material in most cases forms a thin irregu- lar belt that often surrounds the distal end of the nucleus. In some instances it is in the form of separate small lamellae in the same position as the other type. In glandular cells the Golgi ma- terial is localized at the site of synthesis of the secretory product. In all the polarized cells the chondrioconts are oriented so that their long axes are approximately parallel to the direction of the flow of material through the cell, i. e., between the base and the lumen. It is suggested that cytoplasmic currents are responsible both for this arrangement and for the radial orientation of chondriosomes in the unpolarized cells. (This article is based upon a_ seminar report which will be presented at the Marine Biological Laboratory on August 1.) Aueusr 5, 1933 ] THE COLLECTING NET 157 hae BIOLOGICAL LABORAMORY COLD SPRING HARBOR STABILITY OF COLLOIDS AND THE THEORY OF RAPID COAGULATION Hans MULLER The small degree of stability of certain colloi- dal solutions is well known to every experimen- ter. Small amounts of electrolyte may produce coagulation. Small changes in temperature or changes in the dielectric constant of the solvent may produce the same effect. Some colloids, like vanadiumpentoxyde, show “‘aging’’ effects; the properties of these colloids change with time. These facts raise serious doubts as to whether a colloid may be considered as in the state of ther- modynamical equilibrium. The colloidal state is, perhaps, only a state of transition, a pseu- doequilibrium with a relatively long life time. On the other hand, there exist col- loids which have a degree of stability as higi: as any chemical compound. We are, there- fore, at least in some cases, justified in consider- ing the colloidal state as a thermodynamical equi- librium. The laws of thermodynamics should, therefore, be able to explain why a colloid can exist, and furnish the conditions under which co- agulation occurs. Unfortunately, a satisfactory theory of the sta- bility of colloidal solutions encounters great diffi- culties. Let us consider a simple colloid, such as, for instance, a gold colloid. The micelles are small crystals of gold. The exact value of the surface tension , between gold and water is not known, but it must be of the order of magnitude -of 10 to 1000 erg/em?. Let us assume a cubical form of the particles, the edges of the cube hay- ing the length r. If two such particles grow to- gether along the sides of the cubes, two surfaces each of the area r?, vanish, and the surface ener- gy 2r*, becomes free. According to the second law of thermodynamics a system is in equilibrium if its free energy is a minimum. The surface tension acts, therefore, in such a way as to pro- duce coagulation. The temperature motion, how- ever, acts to prevent this process. Whether co- agulation takes place or not depends on whether the energy of the temperature motion is larger or smaller than the decrease of surface energy. Hence a colloid should be stable only if 2 ee = 4y, eT Introducing k = 1.37 107° erg, T = 300 and an average value of , = 100 gives alee Opsucrnte Even if we assume the surface energy to be 10 times smaller, we find that a gold colloid should only be stable if the particles consist of but a few gold atoms. To be conclusive, this elementary consideration requires, of course, a more rigorous derivation. One has to find the conditions for which the free energy of the colloid is a minimum. This was done in a paper by March “), and he comes to the same conclusion as that found above. From this theoretical point of view a stable colloid with a particle size larger than 10% cm. is thermody- namically impossible. Experimentally, however, we find colloids with particles of 10° cm. radius, which are apparently stable. There are two important points which have to be considered for an explanation of this discrep- ancy between theory and fact. The first point concerns the value of the free surface energy. The surface energy of a solid-liquid interphase is not determined by the surface energy of the liquid and the solid phase. The molecules on the surface of the solid exert forces which act on the molecules of the liquid. This leads to solvation and a smaller value of the surface tension. It is conceivable that in some colloids the hydration re- duces the surface tension to such a low value that very large particles are stable. But these cases are exceptions. In general the surface tension will have values of the magnitude assumed above. More important is the relation between stability and the electric potential of the micelles. The experiments point to the fact that coagulation oc- curs if the electrokinetic potential is diminished below a critical value. It is, therefore, evident that the factor determining the stability of a col- loid is primarily determined by the charge of the particle and by the constitution of the electric dou- ble layer. The reason for this correlation seems to be simple: if two particles approach each other they are repelled by the Coulomb forces acting between the charges of the double layer. These forces constitute a repulsion, since the charge of the outer layers have the same sign for the two particles. Unfortunately the calculation shows that these repulsive forces are not strong enough to balance the attraction due to the surface ten- sion. If the energy of the electric double layer is taken into account in March’s calculation, the 158 THE COLLECTING NET [| Vor. VIII. No. 66 stability of somewhat larger particles can be ex- plained, but the micelles should still be of ami- croscopic size in order to be theoretically stable. A similar calculation of Gyemant '*) also leads to the stability of particles with a radius of less than 10°° cm. only. Since stable colloids with ultramicroscopically and even microscopically small particles do exist, we must conclude that the present theories have neglected a source of negative free energy. This neglected energy must be connected with the elec- tric field around the micelles. The origin of this energy may possibly be ex- plained in the same way in which Zwicky ‘*) ex- plains the anomalous specific heat of strong elec- trolytes. While Debye’s and Hitckel’s theory of strong electrolytes gives an adequate explanation of the mobility and activity of the ions, it fails to explain the caloric properties of electrolytes. It has been observed that the specific heat of strong electrolytes is considerably smaller than the spe- cific heat of pure water. The interionic forces give rise to a small decrease of the specific heat, but they cannot account for the observed, large variation. In some cases the variation is so large that the heat necessary to change the temperature ot a solution containing | liter of water and 100 gr. of salt is even less than one calorie. According to Zwicky, this anomaly is due to the strong elcctric field existing in the neighbor- hood of the ions. This field polarises the water and gives rise to electrostrictive forces. The electrostriction produces a hydrostatic pressure of many thousand atmospheres. According to measurements of Bridgeman, the specific heat of water decreases with pressure. Around every ion there is, therefore, a shell of water, whose spe- cific heat is reduced by the electrostrictive pres- sure. Zwicky was able to show that this effect can account for the observed decrease of specific heat of the solution. The electric field existing near the surface of colloidal particles is of the same order of magnitude as the field around ions. The water in the double layer of a micelle is, therefore, under a large pressure produced by electrostriction. Since pressure reduces the spe- cific heat of water, the water in the double layer has a smaller free energy than the water in the solution. The calculation shows that this decrease of free energy is sufficient to explain the stability of large colloidal particles. This consideration verifies, to a certain extent, the conclusion of March. March states that one must assume the existence of a protecting skin around each micelle. This protective skin is here explained as a layer of water, under high pres- sure. If the thickness of the double layer is di- minished, by addition of electrolyte, the protective skin gradually disappears, and coagulation begins. It seems probable that this consideration may contribute to the understanding of the action of protective colloids, and the coagulation by non- electrolytes. If the electrolyte concentration of a colloid is gradually increased, until the € potential decreases to a critical value, slow coagulation he- gins. In this state only particles with a large kinetic energy are able to overcome the stabiliz- ing forces. If the € potential is made sufficiently small, all impacts between particles result in join- ing them. We have, then, the maximum rate of coagulation, or rapid coagulation. In his last paper before his death in 1917 y. Smoluchowski ‘4’ gave the mathematical theory of rapid coagulation. The results of this theory were repcatedly verified by Zsigmondi ‘*’, West- gren and Reitstotter '°’, Kruyt and van Arkel ‘” and Tuorila ‘*). Theory and experiment agree extremely well. Both lead to the conclusion that the rate of coagulation is independent of the size of the particles. This result seems, however, to be in contiadiction to measurements of Wiegner ! and Galecki ""’, who found, even before the theory was develcped, that small particles join, preferably, large particles. They observed, name- ly, that the amicroscopic particles in milk do not group. together to form large particles, but that they attach themselves to the larger particles in the solution. Following a suggestion of Dr. Wiegner, I studied this apparent contradiction to Smoluchow- ski’s theory, and I succeeded subsequently ‘!), not only in clearing up this question, but in giving a more generalized theory. It is now possible to calculate the rate of rapid coagulation of any col- loid with arbitrary numbers of arbitrarily large, and arbitrarily shaped, particles. The conclusions of this generalized theory have been experiment-_ ally verified by G. Wiegner “?), P. Tuorila [*) and C. E. Marshall “*, The theory of rapid coagulation is based on the following assumptions: (1) Every impact of any two particles results in joining them together. (2) The impacts are governed by the laws of Brownian motion. There are no other forces, besides the temperature energy, which produce, or prevent, impacts. The calculation proceeds in two steps. The first step consists in calculating the number of parti- cles of a certain type which collide during an in- finitesimal time dt with an arbitrarily chosen par- ticle. This number dn can be given by an equa- tion (1) where 1 is the number per cm.* of particles of the considered type in the coiloid at the time ¢, and p is a measure of the probability of occur- dn = 2nipidit Aucust 5, 1933 ] THE COLLECTING NET 159 rence of an impact. The main problem consists in calculating this probability p. The second step consists in counting the num- ber of disappearing and newly formed conglom- erates of particles. This leads toa system of infinitely many differential equations, with an infinite number of variables, but the solution of the equations is always simple, and leads to the result that the total number N of all particles de- creases with time according to the formula l dN = N (2) INGE dt Here P is an average value of the probabilities p of all possible impacts between all the different types of particles existing in the initial colloid, and all the possible conglomerates of particles formed during the coagulation. Since the distri- bution of sizes and shapes of the micelles changes during the course of the coagulation, the value of I’ changes with time. If P is considered as a con- stant, equation (2) can be integrated and gives No NG) === (3) t 1 + — z where 1 7 = —— (4) Nene and where NV, is the total number of particles be- fore the beginning of the coagulation. 7 is called the half-time of coagulation. 7 seconds after the beginning of coagulation, the total number of all particles has decreased to half its original value. After 27, 37, 47 . . . seconds the total number has fallen to 1/3, 1/4, 1/5... of its original value. The shorter the time r+, the faster the rate of coagulation. According to equa- tion (4) the rate of coagulation is the faster the larger the concentration of the colloid, and the larger the average probability P. It can be shown that the value of P changes in many cases very slightly. The results (3) and (4) are applicable for nearly all colloids. In order to calculate the probabilities Pp... . one has to find the number of particles of a cer- tain type 7 which collide during the time d¢ with a particle of another type k. For the sake of simplicity, we consider first particles of spherical shape with radii r; and 7;, respectively. A parti- cle of type 7 collides with the particle of type k if their centers are at a distance (rj + rx). Since we assume that all impacts are due to Brownian moyement, one has to solve a problem of diftu- sion, namely, one has to find a solution of the differential equation of diffusion dn; = DA ny (5) dt Satisfying the boundary condition n, = o for f= tj + ty, and ny = n,° for t = 0: Here is the sum of the diffusion constants of the two types of particles, and has, according to Einstein and Smoluchowski, the value ear 1 1 1D) = ( =F ) 6 71 Tj Tk (6) where k is Boltzmann’s constant, T the absolute temperature and » the viscosity of the colloid. It is easy to realize how the diffusion of the i particles will proceed. Shortly after the beginning of the coagulation all particles of type 7 in the neighborhood of the k-particle will have made an impact with it. This initial rush will slow down, and the number of impacts will assume a steady rate, determined by the rate with which the coagu- lating particles are replaced by diffusion of the i-particles further away from the k-particle. This result is verified by Smoluchowski’s calculation. One finds that the initial rush is completed after such a small time, that it is of no importance for the course of the coagulation. Consequently, one has to find only the steady rate of diffusion. The time element can, therefore, be eliminated, dn, — a0, dt and instead of (5) we have a much simpler dif- ferential equation iN oh = O (7) whose solution has to satisfy the boundary condi- OFOY vay = (0) Hove fe Sey SE ek, atalino seo Equation (7) is Laplace’s equation. The same equation holds for the electric potential distribu- tion around a charged conductor. The solution of the diffusion problem can, therefore, be found in the analogous problem of electrostatics. This an- alogy shows that the number of colliding particles is proportional to the number of electric lines of force in the corresponding electrostatic problem, and the probability p is given by = 2p IDC (8) where C is the electrostatic capacity of the sur- face, which the centers of the i-particles must reach in order to collide with the k-particle, 160 THE COLLECTING NET [ Vor. VIII. No. 66 For spherical particles this surface is the sphere of radius (rt; + rx). Since the capacity of a sphere is equal to its radius, we have for spheres (4 + tx)? Kei 4/3 (9) 4 Tj Tx y p= If r; = rx this reduces to Kean Po = 4/3 —— | If, therefore, a colloid contains spherical particles of uniform size, the probabality p, and hence also the average value P is independent of the size of the particles. This independence of size is due to the fact that a particle of large radius presents a large target for impacts, but has also a small velocity. These two influences of its size just cancel each other. Smoluchowski’s conclusions are based on this result. If however, r; > rx, then, according to (9) the probability, p is larger than p>. This fact explains the observation of Wiegner and Galecki. A small particle has a much greater chance to collide with a large particle, than it has with one of its own size. The large particles form nuclei of coagula- tion for the small ones. The rate of coagulation of a sol with small micelles can be increased by adding a small number of large particles. I have given the complete theory of the influence of large particles on the coagulation of small parti- cles. P. Tuorila has found that all conclusions of the theory are verified. Table I shows how the measured number of particles decreases ac- cording to my theory and not according to Smolu- chowski’s. (10) TABLE | Coagulation of a mixture of two gold sols, one having N, = 3.6 108 particles of radius 97pm, the other n, = 29282 particles of radius 2.91 pp, ac- cording to measurement of G. Wiegner and P. Tuorila. Z S S o 8 = A e's oc as Be —_ vo — WN) u +e cigs Bae Ex ZB lias Zia H ~—"O ~O i) 0 29282 120 AU yes (0) 4 15.2 240 So== 0:2 Be: 7.6 480 Galea Ont 19 3.8 The influence of different radii is only pro- nounced if the radii differ by more than a ratio 1:10. Furthermore, it is necessary that the large particles are just as numerous as the small ones. In an ordinary colloid the particles are usually not of a uniform size, but the radii vary between two limits, most particles having a radius near the average value. Such a colloid shall be called “practically” monodisperse. It can be shown that for such a colloid, f and P are practically equal to Po, and consequently they follow Smoluchowski’s law of coagulation No oe ——————— rT t No4kT Lae T The possible deviations are smaller than the error of observation. This is the reason why most measurements verify Smoluchowski’s curve, in spite of the fact that the assumptions of his theory are not justified for most of these investi- gated colloids. If, however, the particle sizes vary greatly, as, for instance, in a mixture of colloidal solutions, large deviation must he expected. The coagula- tion proceeds faster than according to Smolu- chowski’s theory. Using equation (8) it is possible to investigate the rate of coagulation of particles of arbitrary shape. It leads to the conclusion that particles of the shape of flakes coagulate in practically the same way as do spherical particles. The data on Kaolin verify this result. Rod-shaped_parti- cles, however, should have a much higher speed of coagulation than spherical particles. This con- clusion was verified by Wiegner and Marshal (4) who observed a rate of coagulation of a V» O; sol more than 30 times larger than Smolu- chowsk1’s theory predicts, The theory of rapid coagulation has also been extended to colloids in the state of sedimentation. If large particles drop under the influence of gravity, through a coagulating colloid of small particles, they collide with the small micelles and carry them along. This phenomenon has been studied theoretically and experimentally by P. Tuorila “). I have given a somewhat different derivation, but both theories give the same results and agree equally well with the observation. It is found that this effect increases with the size and the number of the sedimenting particles. But, however large these particles may be, they do not affect the coagilation of particles smaller than a definite size. Gold particles are never cleaned out by sedimenting large particles, if they have a ra- Avucust 5, 1933 ] THE COLLECTING NET 161 dius smaller than 300 pp. Quartz particles must 11. H. Muller, Kolloid Z. 38 1, (1926). have a radius of 500 pp. Koll. Chem. Beih. 26 257 (1927). ie ures Bp : 27 223 (1928). The same effect can also be produced by stir- 12. G. Wiegner &. P. Tuorila, Kolloidz. 38 3, ring, or centrifuging, the colloid. These opera- tions will increase the rate of coagulation if the colloid contains particles of different sizes; but the coagulation of particles smaller than a de‘- nite size will not be influenced by these mechan- ical means. In order to show this effect Uf ‘“or- thokinetic” coagulation the particles must be larger than 4]12kT r = ear: Tas where s is the density of the particle material, and a the acceleration produced by the mechanical operation. If the acceleration is very large, the coagulation may even still be produced if the electrolyte con- centration is so small that the colloid would other- wise be stable. Whether this mechanical coagu- lation is simply an accelerated slow coagulation, produced by the above effect, or an entirely new phenomon, is at present difficult to decide. Nearly all of the conclusions reached for rapid coagulation can be extended to the theory of slow coagulation. Slow coagulation differs from the rapid one insofar as only a certain percentage of impacts results in joining the particles. Conse- quently, the observed curves for slow coagulation are similar to those for rapid coagulation, but the coagulation time 7 is increased by a factor de- pending on the concentration of the electrolyte. The dependence on electrolyte concentration was given in a theory by H. Freundlich “®). The kinetics of the coagulation of colloids is, therefore, rather well understood. The problem, why and how a colloid is destroyed, is practically solved. The more important problem, however, why can a colloid exist, is still a matter of further investigations. LITERATURE 1. March, Ann. d. Physik. 2. A. Gyemant, Grundz. d. Kolloidphysik, View- eg (1925). 8. F. Zwicky, Physik. Z. 25, (1926). 4. M. v. Smoluchowski, Physik. Z. 17 557, 583 (1916). 5. R. Zsigmondi, Z. Phys. Chem. 92 545 (1917). 6. A. Westgren, &.J. Reitstotter, Z. Phys. Chem., 92, 750 (1917). 7. H.R. Kruyt, & J.v. Arkel, Rec. d. Trav. Chim d Pays Bas, 39 656 (1920). 8. BP. Tuorila, Kolloidchem. Beih. 22 192 (1926). 9. G. Wiegner, Kolloidz. 8 227 (1911). 10, A, Galecki, Z, anorg. Chem, 74 174 (1912), (1926). 13. T. Tuorila, Kolloid Chem. Beih. 22 (1926). 14. G. Wiegner & C. E. Marshall, Z. Phys. Chem. 140 1, (1929). 15. BP. Tuorila, Kolloidchem. Beih. 26 (1927. 16. H .Freundlich, Kapillarchemie. DiscussION Dr. Cole: You mentioned that the coagulation of small particles is not affected by the sedimen- tation of large particles. Is this an experimental fact alone, or does it also follow from the theory ? Dr. Miller: It is a consequence of the theory and is verified by observations. The small par- ticles make no impacts with the large ones, be- cause the hydrodynamic currents around the fall- ing particles carry the small particles away from the path of the large micelles. Dr. Chen: In the coagulation by mechanical means, such as shaking, stirring and bubbling air through the -solution, do you take into considera- tion the increased influence of the surface beside the increase of number of collisions among the particles? This kind of coagulation seems to de- pend upon the total surface of the colloidal solu- tion. Dr. Miiller: The present theory neglects the coagulation taking place on the surface. In the theory for systems in the state of sedimentation by gravitation or centrifuging, this effect can be neglected. In other methods of mechanical co- agulation, however, the surface effect can be much more important than the collision effect. Dr. Blinks: Wave any studies on coagulation of particles suspended in gases been made from this point of view ? Dr, Miiller: Smoluchowski’s theory has been used for the theory of the condensation of fog. Dr. Cole: According to your theory the sta- bility of a colloid depends largely on the radius of the particles. If, therefore, electrolyte is added to a sol one might expect that the larger particles should first become unstable, while the smaller ones might still be stable. Dr. Miiller: Investigations in Dr. Wiegner’s laboratory show that this is the case. It was fre- quently observed that the large particle of a poly- disperse sol underwent slow coagulation, while the small micelles wereenot flocculated. Dr. Cole: Can the electrostriction effect in- fluence the surface tension in the capillary elec- trometer ? Dr. Miiller: This effect is only appreciable if the surface has a large curvature. It should, there= fore, play no role in the capillary electrometer, 162 THE COLLECTING NET [ Vor. VIII. No. 66 THE ELECTRIC POTENTIAL AND CHARGE OF DISSOLVED AND ADSORBED PROTEINS Harotp A. ABRAMSON The study of the electrophoresis of dissolved proteins and of protein surfaces is of interest not only from the point of view of their role in bio- logical systems, but also because the theories of electrophoresis may be tested. I shall in this lecture attempt to review briefly some recent con- tributions to these aspects of the physical chemis- try of the proteins. Comparison of Dissolved and Adsorbed Protein Early data dealing with the electric mobility of the proteins included not only measurements on the moving boundaries of dissolved proteins but also on very fine suspensions of protein particles or of inert particles having adsorbed protein surfaces. Indeed it was apparent that the surfaces of denatured proteins behaved very much like the dissolved molecules themselves although no quantitative comparison was made for dissolved protein and the protein surfaces except at the isoelectric point'??. Loeb’), for example, performed numerous ex- periments showing that there was approximate Ecc Aveumin HOLS «105 30 ACID (BASE) PER GM. PROTEIN (c#oor cerrs) t=) j./sec/voLt/cm. (rors) _ ul FIG, 1. The open circles are values of electric mobility of dissolved egg albumin obtained by Tise- lius. The closed and half-closed circles are similar data for egg albumin studied under similar condi- tions but adsorbed 9n microscopically visible quartz particles. It is evident that the mobility and titra- tion curves belong to the same family, so that over this rauge of pH, mobility is proportional to the acid (base) bound. The dotted line indicates the very slight shift in electrophoretic mobility between adsorbed and dissolyed protein, agreement between the isoelectric points of gelatin and of egg albumin when in solution and when adsorbed on collodion. This identity of isoelectric points is a necessary condition, but it is not suffi- cient to establish the fact that protein adsorbed on Serum Aveunin MOLSx10° 50 150 & o M/sec/vort/em. ~“") + o uw to) 000 ACID (BASE) PER GM PROTEIN (oroorw cumre) no uw ' 9 a ° FIG. 2. The open circles are values of electric mobility of dissolved serum albumin (Tiselius). The other points are the mobilities of microscopically visible quartz particles covered with an adsorbed film of the same protein. There is no difference in mobility between the native dissolved protein mole- cules and the adsorbed protein. The heavy curve is the titration curve of a sample of serum albumin. inert surfaces apparently ionizes exactly the same as the dissolved protein. In Figures 1 and 2 the open circles are mobility data of Tiselius) on dissolved egg albumin and serum albumin in M/50 acetate buffer. All the other points have been obtained by the writer'*’ on microscopically visible quartz particles covered with protein and suspended in the same buffer. The quartz parti- cles are about 1000 times the size of the dissolved protein molecules but, when a surface layer of adsorbed protein has formed, the particles covered with protein move with practically the same speed as the individual molecules themselves. These data indicate then that the implication in Loeb’s experiments, that adsorbed protein had properties similar to dissolved protein, was justified. From this point on we shall accept the experi- mental fact that in the case of egg albumin and serum albumin the electric mobility of the single protein ion and an inert surface covered with the Aucust 5, 1933 } THE COLLECTING NET 163 protein have approximately the same ¢-potential. In consequence we can extend our knowledge of the behavior of these proteins by the relatively simple microscopic technic of electrophoresis and thereby analyze more minutely the changes under- gone by the protein ions incidental to adsorption. Denaturation and Flocculation The data on egg albumin and serum albumin can be used to interpret part of the process of denaturation in surface films. It is probable that a polymolecular film of protein is present at tne interface of quartz and liquid, first a monomole- cular layer and then successive layers being added. The data probably describe the behavior of the outermost layer. If denaturation occurs at the interface, except for the extremely small shift of the isoelectric point of about 0.05 of a pH, at the limits of the experimental error, no other im- portant change seems to have occurred in the total charge of the outermost molecules of egg albumin in contact with the liquid. Since measurements of mobility were made soon after the suspension of the quartz particles in the protein solutions, and since it is not impossible that in this type of denaturation the chemical process is a slow one, if a greater and sufficient length of time had been permitted to elapse, a more marked change might have been observed. The identity of the values for the electrophor- esis of dissolved native serum albumin and ad- sorbed albumin are of importance in connection with data of Pedersen'*). Pedersen has found that serum albumin, after heat denaturation, has an isoelectric point between pH 5.1 and pH 5.3. The mobility-pH curve was nearly parallel to that of the native protein. If the denaturation occurs at a surface incidental to adsorption, as in the experi- ments reported here, this change in the isoelectric point and the mobilities at different values of pH does not occur. There is then, a very great differ- ence between ‘‘surface denaturation” by inert par- ticles and heat denaturation of the type used by Pedersen, in terms of the charge of the protein. The Charge of Proteins by a Thermodynamic Method. Although yielding no information concerning the mechanism of charge and primarily investigat- ed for dissolved proteins rather than protein sur- faces, measurements of the activity of the ions in a solution of a protein in.an acid (base) can, under certain conditions, reveal the net charge of the protein. Given a very dilute protein solution in HC1 for example, [Ht Jere + [H*] = [C17] + [Clee ments of ag* and ag where the subscript Pr refers to the ions bound by the protein and the brackets represent concen- trations, If n is the time average of the net charge, n= [El py a LG and the charged protein molecule can be repre- sented as Pr"*. The activities of these ions when the solution is sufficiently dilute, say of egg al- bumin near its isoelectric point, can he taken equal to their concentrations so the measure- by the usual method be- fore and after addition of protein gives the re- duction in the number of ions of H+ and C1l— in the solution due to the presence of protein. St. Bugarsky and Lieberman, Manabe and Matula, Pauli and co-workers, Loeb, and Hitchcock early showed that, for proteins having isoelectric points close to pH 5.0, dissociation of protein chlorides (and other protein salts) was practically complete near the isoelectric point on the acid side‘®); that is [Cl~]pr > O, and that therefore, a) == [falar llign With this condition, the titration curve of the proteins portrays in the case of soluble proteins having known molecular weights the time average of the net charge per molecule. The problem at- tains a greater complexity at the ends of the ti- tration curves where the activities and concentra- tions of ions are no longer equal. Evidently, the expression, “Ht aC1— (Ce + [H*]p = YHt+ OKG A= 1C1= (Cito HCN measures the bound chlorine. By making use of this expression and assuming aq* = aq, and that the presence of highly charged protein ions can be neglected, an assumption which is some- what daring in solutions of proteins where the net charge is about 20, it can be demonstrated that proteins like some of the albumins and glob- ulins are at least 85% dissociated in more acid so- lutions where the concentration of salt and of acid and of protein is considerable. Failey has re- cently considered the problem from the point of view of complete dissociation. Failey measured the solubility of thallous chlo- ride in solutions of nitric acid containing varied amounts of edestin. Without assuming any com- bination of P"®*+ and C1~, he found that the mean activity coefficient of the ions of the salt is de- 164 THE COLLECT NG NET [{ Vor. VIII. No. 66 creased by the protein. Using these data and thereby correcting for the effect of the protein on the mean activity of the ions of HC1 in the presence of protein it seems likely that gelatin, casein and edestin have a definite maximal com- bining capacity for H+, with a vanishingly small combination of Cl~. By these thermodynamic means ‘it is possible to obtain values of the charge of protein molecules which may be used as references for testing the electrophoresis theory. Mobility aud Titration Curve of Proteins By combining the Smoluchowski-Henry theory l én D v= — — (1) On 7] with the Debye approximation, Ol/k 6¢= ———_ (2) Dr (r+1/k) where v = the electric mobility; ¢: = _ electro- kinetic potential; D = dielectric constant; 7 = coefficient of viscosity; Q = net charge; r = radus, when fe ae? i, kT e = 4.77x10 £. s. u. of charge, k = Boltz- man’s constant, and T the absolute temperature. Since « = 0.33 \/ Cx108 at 20° in a solution of a uni-univalent salt of ¢ moles per liter. In the ex- periments given in Figs. 1 and 2 the ionic strength was kept constant, varying aH, so that with this condition and combining equations (1) and (2), Q = 6rqvr ([Crx0.33x108] +1) (3) Equation (4) now states the conditions for which a protein ion has its charge, Q, proportion- al to its mobility, O=wi(e 4); (4) C’ and C” being constants. Making certain assumptions which are enumer- ated below equation (4) predicts the following rule: In solutions of the same ionic strength, the electric mobility of the same protein at different hydrogen ion activities should be directly propor- tional to the number of hydrogen (hydroxyl) ions bound. This statement includes the following assump- tions: (1) Complete dissociation of the protein salts or a constant fraction dissociated at different hy- drogen ion activities. (2) The hydrogen ions bound act as if they were at or very close to the surface or to the center and uniformly distributed. (3) and r do not change with pH. (4) The reaction of the protein with other than the H* (OH_) ion is negligible. ions (5) Only uni-univalent electrolytes are con- sidered. (6) D and 7 of the medium can be used for their unknown values in the double layer, the ef- fect of salts on D being unconsidered. With these assumptions in mind examine the smooth curves in Figs. 1 and 2. These smooth curves are not a “best” curve’ but the titration curve of the proteins in a region where the activ- ity coefficient of HCl is very nearly 1.00. Evi- dently, within the limits of error, the rule just stated is approximately followed and in the range investigated the electric mobility is proportional to the acid (base) bound. According to Svedberg and Nichols‘) the egg albumin molecule under the foregoing conditions is spherical and has a molecular weight of about 35,000. At pH = 4.0, by thermodynamic meth- ods, OQ = 25x10!" £. s. U. approximately, where- as using our approximation Q = 15x107 E.s. u. approximately. The general agreement as far as changes in vy with pH is indicative of the underly- ing soundness of the theory and justifies theory and experiments seeking a second approximation. It is possible that the fact that the titration was done in HCl and the mobilities measured in ace- tate buffers may account for part of the differ- ence between theory and experiment; or more likely the factor 67 is too small because of the distortion of the double layer. Using an empirical equation of the form of equation 3, the acid com- bining powers of Bence Jones protein, k-phyco- cyan and R-phycoerthyrin from the mobility data of Tiselius have been calculated and await ex- perimental test. Gelatin and Deaminized Gelatin The rule that mobilities are proportional to the number of hydrogen (hydroxyl) 1ons combined with a protein in solutions of the same ionic strength has been tested in another way. Hitch- cock!) showed that deaminized gelatin adsorbed on collodion particles had an isoelectric point at about pH 4.0, and that acid was bound by _ the deaminized protein. In Fig. 3 is plotted the titra- tion curve for “Cooper’s Gelatin” and for the same gelatin deaminized by acetic acid and so- dium nitrate. As before, the smooth curves, I and II, are the titration curves for gelatin and deaminized gelatin respectively. The closed cir- cles which follow Curve I are the electric mobil- ities of quartz particles covered with gelatine in Aueust 5, 1933 ] THE COLLECTING NET 165 N/150 acetate buffers. Curve I indicates that for gelatin itself, in solutions of the same _ ionic strength, the mobilities are proportional to the number of hydrogen (hydroxyl) ions combined. Let us assume (1) that after deaminization the average radii of cuvature of the surface of the deaminized gelatine is not appreciably changed by the loss of the amino groups; (2) that the disso- ciation of the deaminized gelatin salt in the range of pH studied is the same as for the gelatin it- self; (3) that the type of adsorption of both gel- atin and deaminized gelatin by quartz particles is the same, and that it represents a mean value of adsorption for a polydisperse system; (4) that the effective “molecular weight” is unchanged. Under these conditions, all.of which are rea- sonable, there should be obtained the following relationship : Combined (+ H*) gelatin Combined (+ H+) deaminized gelatin Mobility gelatin Mobility deaminized gelatin That is, in the same buffer, the ratio of acid (base) bound for the two proteins should be equal to the ratio of their mobilities. That this is true experimentally is shown beautifully by the open circles plotted in Fig. 3 along Curve II. These open circles are the mobility values of deaminized gelatin and, as predicted by theory, they fall along the smooth titration curve of deaminized gelatin. Casein Proteins like the albumins are soluble in the re- gion of the isoelectric point. For this reason the treatment of the relationship between combined GM. PROTEIN EQUIVALENTS AU/O(BASE) BOUND FER 2500 = ~ s 7 ESHOOTH CURVE TITRATION GELATIN XN 16 GELATIN ~o DEAMINIZED GELATIN: Ee-TInaATIONDG FIG. 3. In acetate buffer solutions of the same ionic strength, the ratio of the number of mols of hydrogen (hydroxyl) ions bound by gelatin and de- aminized gelatin at a given pH is equal to the ra- tio of their mobilities, ‘ FIG. 4. (half-closed circles) for the mobility of casein indi- cate that molecules of casein are highly charged on The open circles (Loeb) and our data both sides of the isoelectric point. Calculations of the base bound (closed circles) by casein lead to the postulation of a smooth curve of the sort passing through the closed circles going through the isoelec- tric point in a linear fashion as indicated in the figure and agreeing in slope with the mobilities. The inset gives a clear picture of the usual “titration” curve (dotted line) and the titration curve here postulated (smooth curve). acid and mobility has been uncomplicated by the insolubility exhibited by a protein like casein in the region of its isoelectric point. Fig. 4, Curve I-I-I, shows the “titration” curve of casein as or- dinarily plotted. The flat portions of the curve are in the zone of a heterogeneous system. Loeb", on the other hand, pointed out that casein particles are highly charged on either side of the isoelectric point. The slope of the v-pH curve is large and corresponds to those for the other proteins just discussed. Curve I-I-I in the figure represents acid bound for “total casein’ rather than for unit weight of protein dissolved. A serious discrepancy between our approximation and the relationship between combining power and mobility has been removed in the following simple fashion. Data in the literature have been recalculated so that values of hydrogen (hy- droxyl) ion bound per unit weight of casein dis= solved have been obtained. A straight line drawn through the mobility data for casein fits the new titration curve for dissolved casein reasonably well (Curve II-II-I). The slope of the titration curve of casein so plotted, agrees with the slope of the electric mobilities (plotted as before) of casein obtained by Loeb and by us (~ = 0.005) in this region and meets the other portions of the curve in a reasonable fashion. These data point to the validity of our rule in the case of casein, and indicate a rational: basis for the plotting of 166 THE COLLECTING NET [ Vor. VIII. No. 66 titration curves in heterogeneous systems. In this instance, that of an insoluble protein, the mobil- ity data give a much better index of the change in charge or ionization with pH than does the “titration” curve, wiless the titration curve is plotted as combined acid per unit weight of pro- tein, and the dissociation of the protein is known. The inset in Fig. 4 perhaps gives a clearer notion of the titration curve of casein as here postulated. Insulin: Amorphous and Crystalline In collaboration with Wintersteiner*) the writer has compared the electric mobilities of adsorbed (amorphous) insulin and insulin crystals. From measurements of v on adsorbed and amorphous insulin particles. (Fig. 5A) the isoelectric point is at pH 5.35 in M/30 acetate buffers in agree- ment with somewhat similar data obtained by Howitt and Prideaux''*), Compare the values of v obtained for crystalline surfaces of insulin (Fig. 5B, Curve II) with the amphorous surface (Curve I). It is reasonable to suppose that the differences in v obtained for amorphous and crystalline surface depend upon the changes in orientation of polar groups incidental to the for- mation of the lattice of the protein. This is sup- ported by the fact that when sufficient dissolved protein is present to form a complete protein film of adsorbed protein on the crystal, the crys- tal surface acquires the electrokinetic properties of the amorphous particle or quartz particle coy- ered with a protein film. FIG. 5, A. The electric mobility of quartz parti- cles covered with insulin in M/30 acetate buffers. The isoelectric point is between pH 5.30 and pH 5.35. The same data were obtained with particles of amorphous insulin. The ordinate units are in u per second per volt per cm. FIG. 5, B. The smooth curve (Curve I) gives the electric mobilities of adsorbed or of amorphous in- sulin. The lower curve (Curve II) gives the mo- bilities of insulin crystals or crystal fragments in the same medium. Curve II has been roughly fitted to the open circles (mobilities of crystals suspended in M/30 acetate buffer). For significance of the open and solid circles consult the text. The ordi- nate units are in + per second per volt per cm, Effect of Uni-univalent Salts Since the study of proteins by the moving boundary method is necessarily carried out in the presence of a considerable concentration of salt, it is difficult to obtain the effect of increasing salt-concentration on v by this method. Our in- formation at present comes from surfaces of pro- tein studied as the protein particle itself or ad- sorbed protein. Extensive studies of the effect of univalent ions on v have been made by Loeb"), the pH being held nearly constant. The measure- ments of pH in these experiments were usually made in the absence of salt. For this reason a slight error is introduced into the values of pH given, the salt error increasing with the valence of the ions. Collodion particles, covered with a film of egg albumin, denatured egg albumin par- ticles, casein and other proteins were among those investigated. The results of these numerous ex- periments may be stated briefly as follows: Small amounts of added salt, the pH held approximately constant, did not change v appreciably. Further addition of salt diminished v without the initial maximum in the vy-c curve observed usually for inert surfaces, a limiting value for v, apparently not equal to zero, being approached. This is readily understood on the basis of the following reasoning. Dissolved proteins and protein surfaces, like gelatin and egg albumin, differ in behavior in several important ways from “‘inert” surfaces. The charge of proteins in the absence of salts seems to depend mainly upon the pH, for at any given pH a certain number of hydrogen ions over a time average are attached to the protein mole- cule. In the special case under discussion, of a uni-univalent salt not shifting the isoelectric point*®), we can first for simplicity consider v to depend only upon x, if the pH is fixed. The sig- nificance of this result is evident when we con- sider the Debye-Henry approximation, for the potential, £, at the surface (equation 2), QO Dr (er+--1)_ Since v, is proportional to &, it will depend only upon « if all other terms are considered constant, giving, ¥ = v (x) pH = const. By assuming that QO remains constant we do not by any means imply that no change in Q occurs incidental to changes in x. It is merely postulated that the change in v with OQ due to « varying is very small compared with the change in y due to Aueust 5, 1933 } THE COLLECTING NET 167 explicit variation of «. Addition of salt, under these conditions, then, should cause only a diminution in v without a maximum in the curve. We have used out empirical form of equation O 6 = ———__, (2a) Dr («r+ 2.4) or Q Dr f(« r) to plot in Fig. 6, by evaluating f(kr) for r= 4x 107 cm, and various values of v for gelatin, the theoretical form of the v-c curves. Note the following points of interest in these curves. The curves should in reality not cut the ordi- nate at c = O, for, in order to fix Q, a certain U 05 10 15 Vo FIG. 6. Theoretical vm-c curves for gelatin at different values of pH, based upon single values given in Fig. 2. The two short vertical dotted lines at the lower left corner show the limits of extra- polation when the pH is sufficiently low to have appreciable amounts of acid present. amount of acid must be present even though the concentration of protein and of salt is vanishing- ly small. In other words v is always measured in the presence of finite value of « which is given for strong acids by the concentration, and not by the mean activity, of the acid. The dotted lines indicate, for example, the limiting position of the ordinate for c = 0.0025 M, and c = 0.01 M. In more concentrated salt solutions the valid- ity of equation 2 decreases; however, the curves indicate that v should be still quite large even in 4 M salt solutions. Technical difficulties at pres- ent prevent measurements of v in salts of this concentration; but values of the proper magni- tude™®) have been observed by Hitchcock in M/10 acetate buffers for gelatin and by the au- thor for serum proteins in solutions where c was equivalent to M/7. It would be most desirable to devise methods to discover if the available form of the theory is confirmed in that the prediction, v > O, is confirmed in concentrated salt solu- tions, FIG. 7. Data of Loeb on particles of denatured egg albumin in 0.0002 M NaOH. Loeb found no im- portant difference in the effect of the alkali halides. The smooth curve is calculated by means of the theory here proposed, based upon the highest value of v found by Loeb. In Fig. 7 are values of v (Loeb) for denatured egg albumin particles in M/5000 NaOH. At this pH, in the absence of salt, v is rather high, 2.8 p per sec. The smooth curve is the theoretical curve calculated by means of equation (2a) tak- ing r = 2.2x 107 cm. and making the usual as- sumptions in regard to 7 and D. It is noteworthy that the course of the theoretical and experimen- tal curves are almost identical. Fig. 8 gives the results of similar experiments and _ calculations for egg albumin in acetate buffers, a correction being needed for the shift in the isoelectric point with variation in x. Similar results were obtained for egg albumin in HCl and for gelatin in ace- tate buffers. To summarize: by assuming that the 11/1000 corrected for shift /' of Taoeleciic pou ‘ ‘ ro) FIG. 8a. The effect of the concentration of ace- tate buffers on the magnitude of vm (egg albumin) and on the position of the isoelectric point. FIG. 8b. By correcting for the shift in the iso- electric point, the effect of diluting the buffer on values of v is predicted. 168 THE COLLECTING NET [ Vot. VIII. No. 66 electric charge of proteins is primarily determined by the hydrogen ion activity of the medium and by making corrections when nevessary for saift of the isoelectric point, it is possible to aerive a simple relationship between v and the concentra- tion of uni-univalent electrolytes. This relation- ship, that v depends upon « in a way which indi- cates that the decrease in the electric mobility of a protein ion with increasing concentration of a simple salt can well be understood by a diminu- tion in the potential rather than in the charge, permits treatment of an electric mobility of an ion whose charge is given by a time average sim- ilar to that of an ion the charge of which is fixed. Effect of Polyvalent Tons Loeb"), in particular, has investigated the ef- fects of polyvalent ions on protein surfaces. In general, he found that ions of the same sign as f Effect of Ba**on Electric Mobility of Protein Sl. NNR aie 8s pea Corrected for I.P FIG. 9. Curves I and II are for egg albumin in Na— and Ba— acetate buffers. Note the shift in the isoelectric point and the change in shape of the curves. The dotted curve is Curve II corrected for the shift in the isoelectric point. (Data of Tiselius.) that of the protein had effects more or less sim- ilar to the addition of univalent ions. Addition of polyvalent ions of opposite sign resulted in the reduction of the ¢-potential to zero and sign re- versal. A consequence of this is that the isoelec- tric point of proteins should be shifted more by polyvalent than by univalent ions. That this is true for native proteins has been found by Tis- elius''®) who studied the moving boundary of egg albumin and phycoerythrin in barium acetate buf- fers. This isoelectric point of egg albumin was shifted to the alkaline side; the barium ions prob- ably reduce the net charge by reacting with the negatively charged albumin ions so that the iso- electric point is reached more quickly. The slight iacrease of v on the acid side is perhaps better understood if correction is made for the shift in the isoelectric point. If this is done (dotted line Fig. 9), note'™? that in accord with the viewpoint of Loeb there is hardly any effect of Ba * * on the acid side of the isoelectric point, but a no- ticeable change on the alkaline side. The ionic strength of M/50 barium acetate is slightly great- er than that of M/50 sodium acetate. Part of the diminution in v (corrected) on the acid side may be due to this difference. Tiselius and also Koe- nig and Pauli have performed some experiments in unbuffered solutions. EFFECTS OF ALCOHOL Electrophoretic Velocity and Field Strength The simple characterization .of particles by measurements of the electrophoretic velocity, V, depends upon the fact that V is proportional to the field strength, X, as is evident from equation (1) éD Vien = (C4 OK uy} This linear relationship has been found with few exceptions. Thus Ettisch and Zwanzig'?") inter- pret their data on streaming potentials as indica- tive of a complicated relationship between V and X, particularly in alcoholic solutions, V increas- ing and reaching a limit with increase of X. Also Kohler'?!) has reported that the volume velocity in electro-osmosis.is not proportional to X. Dan- iel has made a careful study of the electrophor- esis of gelatin, ghadin, egg albumin surfaces in various alcohols, Typical data are given in Fig. 10 20 - Gelatin 8 Alcohol per cent, a ° e 10 pu 38 Lio e (2) pi 38 o 660 pli 43 ° 48 4/1000 HQ ® 61 1/200 HCL e 67 ™/250 HCl ® 19 ry 200 phosphate 10 2 30 Volts /cm. FIG. 10. The electrophoretic velocities of gela- tin-covered quartz particles in media containing various percentages of ethyl alcohol are plotted against the field strength. In each medium the ve- locity is proportional to the field strength. Aueust 5, 1933 ] THE COLLECTING NET 169 e Gelatin in acetate buffer © Gelatin in acetate buffer} and 35 per cent alcohol 4 © Gelatin in acetate buffer § 08 and 60 per cent alcohol fp 3 0 “X02 04 06 400 600 pH FIG. 11. The electrophoretic mobility of gelatin- covered quartz particles is plotted against the pH of the medium for media containing different per- centages of ethyl alcohol. In the more acid regions NaCl-HCl mixtures were used in place of the ace- tate buffers. 3.00 6.00 for gelatin surfaces in ethyl alcohol. Between 2 and 30 volts per cm. V was proportional to X. Electric Mobilities of Gelatin in Alcohol- Water Mixtures Fig. 11 shows the mobilities of gelatin-coated quartz particles in N/150 sodium acetate buffer in O per cent, 35 per cent, and 60 per cent ethyl alcohol. Alcohol shifts the isoelectric point of the gelatin toward smaller hydrogen ion activities and lowers the maximum mobilities. This low- ering combined with the shift in isoelectric point causes the curves to intersect. The diminution in v produced by alcohol is not a simple phenom- enon. Alcohol changes at least both the dielec- tric constant and the viscosity of the medium and may also be expected to alter the electrokinetic potential '*?), Differences in the mobilities which were due to altered viscosity were eliminated by Daniel by calculating the mobility corrected for the viscos- ity of the medium, corrected mobility = vn/no This quantity has the significance that the dif fer- stant alone, the values being in some ways more representative of the effect of the alcohol itself on v. The data in Fig. 11 and lla give the mobilities uncorrected for 7. In Table I there are com- pared values obtained from smooth curves of v and v 7/ for equal charge (as determined by the amount of acid bound). The large differences in v disappear almost completely when the cor- rection for 7 is applied, only a slight decrease tak- ing place as the alcohol concentration increases. This result is similar to Walden’s results for ions. Mobility, Titration Curve and Charge 3y comparing the mobilities in the different media (differing in dielectric constant and viscos- ity) it is possible to test to some extent the ap- plicability of the viscosity and the dielectric con- stant of the bulk of the medium to the electro- e~4 10 2 : 3 a —_ ion a Qe a 10 60 50 40 D FIG. lla. Above, the change in pH of the iso- electric point of gelatin, caused by ethyl! alcohol, is plotted against the volumes per cent. alcohol in the solution. Below, the same data are replotted as change in pH of the isoelectric point against dielec- tric constant of the solution. phoresis equation for charge, equation (3). The simplest means if doing this is to calculate charge from mobility by means of equation (3), ences between curves of corrected mobilities using the viscosity and dielectric constant of the should be due to changes in the dielectric con- bulk of the medium. Note that a suitable correc- TABLE I. DS Vv Vv n/No t pea eee 35 percent. 60 percent. 0 per cent.’ 35 percent. 60 percent. 0.20 5 0.08 0.06 0.20 0.20 0.16 0.40 0.15 0.10 0.40 0.40 0.30 0.60 0.21 0.15 0.60 0.54 0.42 0.80 0.25 0.19 0.80 0.67 0.54 The figures in each horizontal row are for pH’s of equal charge as determined by the titration curves, 170 THE COLLECTING NET [ Vor. VIII. No. 66 tion must be made in « for the lowered dielectric constant. Since | tne $ ‘3 kK acl per If the acid bound (measured directly) is, in dif- ferent media, in the same ratio as the charge cal- culated from the mobility by equation (3), then within the limits of the experimental error equa- tion (3) may be used to predict changes in charge, using the viscosity and dielectric constant of the bulk of the medium. (See previous sections for other assumptions ). Fig. 12 shows the agreement between QO from equation (3) and titration curves in the middle pH region for O per cent and 35 per cent alcohol. This graph was made by drawing the O per cent alcohol titration curve and charge points to scales which made them coincide and then drawing the 35 per cent titration curve and charge points to the same scales. All the charge points calculated from mobilities determined in acetate buffer fall very well onto the titration curve. All of the data for acetate buffers and NaCl- HOI mixtures from pH 2 to pH 7 have been plotted in Fig. 13, titration curves in the upper half, mobility curves in the lower. The titration curves of gelatine in 0 per cent. and 35 per cent. alcohol have been compared by Daniel from pH 2 to pH 10. The curves are very much,of the same shape, the isoelectric point being shifted to a higher pH, the curves converging at the lim- its. The experiments of Daniel comprise one of the most striking examples of the fundamental Gelatin in acetate buffer © Charge |Gelatin in acetate buffer Oo from 4 and 35 percent alcohol o mobility] Getatin in HC1-NaCl © 2 +05 © and 85 per cent alcohol eS Titration curves & C3) * 00 <= 3 Ui 6.00 5 Se a Bis e -05 FIG. 12. The full circles show the charge of gelatin calculated from the mobility of gelatin-cov- ered quartz particles in acetate buffer. The open circles show the charge calculated from the mo- bility in acetate buffer and 35 per cent. ethyl alco- hol. The lines are titration curves of gelatin in 0 per cent. and in 35 per cent. ethyl alcohol. The figure is limited to a range fairly close to the iso- electric point. bound Gros Pe 6.00 © 0 per cent alcohol © 35 per cent alcohol © 60 per cent alcohol Charge rs mobility Tfols c=] FIG. 13. The upper curves are titration curves of gelatin in O per cent., 35 per cent., and 60 per cent. ethyl alcohol. The lower curves are the charge curves calculated from the mobility of gelatin-cov- ered quartz particles in 0 per cent., 25 per cent., and 60 per cent. ethyl alcohol, the circles being ex- perimental points. validity of the assumptions of the Smoluchowski theory of the double layer and of the usefulness of the modern theory of electrolytes in dealing with electrokinetic phenomena. Mechanism of Adsorption of Protein The fact that not only the isoelectric points but also the electric mobilities of quartz particles cov- ered with serum albumin or egg albumin are very nearly identical with the values of mobility found for the respective dissolved protein indicates that the protein molecules are available even after adsorption has occurred. To demonstrate this let us suppose that one of the hydrogen ions is lost incidental to the adsorption reaction. Near the isoelectric point one H* added to each protein molecule gives it a mobility of about 0.104 per sec. per volt per cm. This very small change can conceivably have oc- curred in the case of egg albumin, but it is not evident for serum albumin. Since the higher mo- bilities are practically identical, no change greater than the loss of one H+ is probable. In other words, adsorption of a large molecule such as a protein permits practically the full activity of the polar groups to be made manifest in spite of the adsorption. Fig. 14 illustrates schematically what can conceivably occur, the reaction between quartz and protein taking place possibly without appreciable loss of charge. Theoretically a change in the mobility of the protein-covered quartz par- ticles could have occurred also for the following reason. If we utilize the theory of the rigid double practically all the polar groups of Aueust 5, 1933 ] THE COLLECTING NET 171 layer to give a qualitative picture of what occurs, if d is the thickness of the double layer, I KY K «kr+1 Now r represents more strictly the effective radii of curvature of all points on the surface of the protein molecules or of the quartz particles. To have the protein-covered quartz particles possess mobilities identical with those of individual mole- cules, it seems necessary that (xr) remains un- changed, each molecule on adsorption taking ef- fectively its own (xr) along with it; for v = f («rand (xr) would vary sufficiently to affect v if any important change in r occurred. In calcu- lating Q for protein-covered quartz particles it is necessary to know the radius of the spherical molecules themselves. The bulk radius of the microscopically visible quartz particles is then probably not the mean radius of curvature of the surface. The calculation of © for blood cells, bacteria, and other microscopically visible par- ticles will always be complicated by the difficulty of ascertaining the effective values of r. If the mobilities are independent of size and shape of the particles, however, and if comparative meas- urements are made in solutions of the same ionic strength and species, the mobilities are propor- tional to the charges and a very good idea of the charge can be obtained by means of equation (2a). The reasoning in regard to («r) for sur- faces in general leads to the establishment of crit- eria which are necessary for the complete iden- tity of surfaces. It is necessary that not only the @ (°) Aosorsed_, ‘oo'o’e PROTEIN Quartz FIG. 14. Schema of proposed mechanism of ad- sorption of proteins like egg albumin and serum al- bumin. The protein molecule (central black filled circle) and the outer layer of the double layer (outer circle) are represented without their charges for convenience. Four molecules are adsorbed. Two are free in solution. According to the mechanism here postulated, (1) the adsorbed protein molecules adsorbed have their radii or equivalent radii un- changed. They do not “lie flat’ at the interface. (2) The effective thickness of the ion atmosphere about each molecule at the interface is the same thitkness as that found for molecules in solution. (3) The available charges are practically the same. (4) The protein molecules determine the nature of the ion atmosphere, the quartz surface playing a negligible role at the interface, ; Soruriov chemical (atomic) structures of two surfaces be identical and not only («r) but also « and r for each. Identical surface density of charge does not mean identity of surface properties. To illustrate this point imagine a protein molecule having r = 2.17 x 107% cm. and a smooth surface, grow- ing larger and larger to say, r = 1 x 10+ cm.,, its charge density remaining constant, and the sur- face still retaining its smoothness; for « = 0.33 x 10°, utilizing the theory of Henry it can be readily shown that the mobility of the larger par- ticle should be very much greater. Conversely, if the ¢-potential of the two different surfaces is the same, the effective radii of curvature of the surfaces may be producing changes bringing chemically different substances to the same ¢ -potential. Activity of Adsorbed Invertase The fact that adsorption need not involve cer- tain properties of the polar groups of large mole- cules simplifies the explanation of a phenomenon observed by Nelson and Griffin'?*), These investi- gators found that, under certain circumstances, adsorbed invertase did not lose a significant por- tion of its enzymatic activity. This is in complete harmony with the facts discovered relative to pro- tein adsorption. It is easily conceivable that en- zymes that are prctein-like in nature could be ad- sorbed or be active at an inert or living surface without diminishing either the number of the en- z, matically active groups or the activities of these groups qualitatively and quantitatively. The Validity of the Mass Law The fact that the same values have been ob- tained for mobilities of molecules dissolved in a homogeneous system and of molecules existing at a phase boundary indicates that the mechanism of adsorption per se need not change the proper- ties of the reactive groups. It could have been an- ticipated that the forces at a phase boundary would have disturbed the dissociation equilibria, yielding different apparent dissociation constants. This has not occurred. This idea has been de- veloped by Michaelis’*) in connection with enzymatic behavior of invertase. The Action of Immune Sera Shibley'??) has shown that certain bacteria treated with immune sera have _ electrophoretic velocities practically equal to that of serum globu- lin particles. The reaction of the bacteria with specific groups belonging to serum globulin can occur without disturbing the amphoteric proper- 172 THE COLLECTING NET [ Vor. VIII. No. 66 ties of the globulin as the simpler models here studied indicate. Further Experimentation The difficulties of the moving boundary meth- od, in particular the fact that it cannot be used for proteins in dilute salt solutions justifies the experimental extension of data of the type ob- tainable by the microscopic method employed here. This method can be used over practically the entire pH range usually studied with solutions from infinite dilution to solutions having the conductance of physiological salt solutions. By observance of the principle of having ionic strength and ionic types identical, the properties of the proteins possibly dependent upon their charge can readily be investigated and classified. This has been done for optical rotation else- where. BIBLIOGRAPHY Hardy, W. B., J. Physiol., 24, 288 (1899). Loeb, J., J. Gen. Physiol., 6, 307 (1924). Tiselius, A., Dissertation, Upsala, (1930). Abramson, H. A., J. Gen. Physiol. 15, 5 Pedersen, K. O., Nature, 128, 150 (1931). See Pauli, W., and Valko, E. “Elektrochemie der Kolloide,”’ Springer, 1929. 7. Failey, C., J. Am. Chem. Soc. 8. Henry, D. C., Proc. Roy. Soc., London, A, 133, 106 (1931). 9. Svedberg, T., and Nichols, Chem. Soc., 52, 5187 (1930). 10. Hitchcock, D. I., J. Gen. Physiol., 6, 95 (1923). 11. Loeb, J., J. Gen. Physiol 5, 395 (1923)., ibid, 6, 307 (1924). 12. Wintersteiner, O., and Abramson, H. A., J. Biol. Chem., 99, 741 (1933). 13. Howitt, F. O., and Prideaux, E. B. R., Proc. Roy. Soc. London, B, 112, 13 (1932). 14. Loeb, J., J. Gen. Physiol., 5, 395 (1923). ibid 6, 307 (1924). 15. Abramson, a 2. 3. 4, (1932). 5. 6. AS Jey dip /\ery H. A., J. Gen. Physiol., 16, 593 (1933). 16. Hitchcock, D. I, J.‘Gen. Physiol., 14, 685 (1931). Abramson, H. A., J. Gen. Physiol., 13, 160 (1929). 17. Loeb, J., Loc. cit. 18. Tiselius, A., Loc. cit. 19. Unpublished calculations. 20. Ettisch, G., and Zwanzig, A., Abhandl. des Kaiser Wilhelm Institute; Phys. Chem., 1930, p. 421. 21. Kohler, G., Z. Phys. Chem., 157, 113 (1931). 22. Daniel, J., J. Gen. Physiol., 16, 457 (1933). Abramson, H. A., and Daniel, J., Proc. Am. Physiol. Soc., 1931. Daniel, J.,. and Abramson, H. A., Proc. Am. Physiol. Soc., 1932. Abramson, H. A., and Daniel, J, Proc. Am. Physiol. Soc., 1933, 23. Nelson, J. M., and Griffin, E. G. J. Am. Chem. Soc., 38, 722 (1916). Nelson, J. M., and Wilke, J. Gen. Physiol., 1933. 24. Michaelis, L., Z. Physiol. (1926). 25. Shibley, G., J. Exp. Med., 44, 667 (1926). Chem., 152, 183 Discussion Dr. Cohen: Is there any information that the adsorbed protein is in crystalline form as against amorphous ? Dr, Abramson: Yes, there is some informa- tion. As noted, I have studied particles of crys- talline insulin, which is highly insoluble at its iso- electric point. I was able to study (1) adsorbed insulin on both sides of the iso-electric point, (2) amorphous insulin, and, near the iso-electric point, (3) crystalline insulin. The adsorbed insulin and the amorphous insulin had the same mobility-pH curves, whereas the particles of crystalline insulin had a different iso-electric point, but approached that of the adsorbed and amorphous insulin when sufficient quantity of the latter was present to cover the crystals themselves. The insulin crystals, thus covered, act just like a quartz particle with adsorbed insulin. Dr. Bates: Since addition of salt to a solu- tion of gelatin in HCl, changes the pH of the solution, will you tell the manner in which this fact is taken into account ? Dr. Abramson: J either measured pH, or cor- rected Loeb’s data so that the activity coefficients of the salts would give the correct value of pH. Dr. Miiller: In investigating the change of the mobility with x don’t you have to know the radius of the particles ? Dr. Abramson: I assumed that the mean radius of the curvature, at every point on the par- ticles, is unaffected by a change in x. Dr. Chen: Does the preparation of amorphous insulin involve processes which might have modi- fied its identity with the crystalline insulin? Do you have data, such as solubility, to show that the amorphous insulin and the crystalline insulin are the same? Can the amorphous insulin be crystal- lized ? Dr, Abramson: The amorphous insulin was prepared by merely changing the pH. It was used immediately. The amorphous insulin can be crystallized. Dr. Blinks: Have you found any cases where the iso-electric point does change simply by ad- sorption ? Dr. Abramson: Not as yet, but I expect that it will be found, since denaturation occurs when Aucust 5, 1933 } THE COLLECTING NET 173 serum globulins are “adsorbed” from immune serum. Dr. Cohen: Will you amplify your remarks about the iso-electric point of ampholytes with reference to dissolved amino acids and their crys- tals? Dr. Abramson: Historically the term isoelec- tric point was first used to designate a reference concentration at which the electric mobility of a particle of any sort was zero. It later became of importance in connection with dissolved ampho- lytes. On the basis of usage, therefore, a crystal of an amino acid, has an isoelectric point, as well as the dissolved amino acid. In the case of a particle of a crystal suspended in an acid, the iso- electric point of the crystal is the pH where the electric mobility is zero or, more formally where, 1 T eS fiat [ 3n (e) + 3n (—e) ] =0, To 1S SSer that is where time average of the total sum of all positive and negative ions in residence at the sur- face is rezo (7 is the time of residence of the ion having the longest period at the surface). This definition is quite general. The isoelectric point of a monobasic, monoacidic, dissolved amino acid, A, is the pH where, [At] = [A-], so that, in accordance with the preceding general equation, the time average of the net charge is zero, and, consequently, our general definition is applicable to both particulate and dissolved amino acid. Investigations on the isoelectric point of crystals of relatively insoluble amino acids have shown that the particles’ isoelectric point bears no simple relation to that of the dissolved substance, Dr. Cohen: material ? Dr. Miller: There is no sharp limit between the amorphous and crystalline state structure. Even liquids have to a certain degree a crystallo- graphic structure, and, according to Zeicky’s theo- ry of the mosaic structure of crystals, every crys- tal has amorphous regions. Usually the surface of a crystal has a distorted lattice. Would you define an amorphous 174 ELE, COLLECTING NET [ Vor. VIII. No. 66 AGGLUTINATION Sruart Mupp Three principle factors have come to be recog- nized as governing the stability of colloidal dis- persions, 1. e., the electrokinetic potential differ- ence of the individual particles, the solvation of the particles, and the force of cohesion between the particles when in contact. In this lecture the relation of these factors to the general problem of colloidal stability is discussed, and there is sug- gested a simpler treatment of colloidal stability in terms*of & 1) the probability of collision of par- ticles, and (2) the probability of cohesion of col- lided Bae Agglutination phenomena of bacterial and other cells represent special cases of colloidal aggrega- tion. Instances already studied show that bac- terial suspensions range from those owing their stability solely or principally to electrokinetic p.d. through intermediate cases to those owing their stability solely to hydration or to lack of cohesive- ness. Examination of these instances of bacterial agglutination may serve to illustrate and even to extend the principles of general colloidal aggre- gation. The Stability of Dispersions of Colloidal Particles in Water and Aqueous Solutions* There are two necessary conditions for the existence of a stable dispersion of colloidal parti- cles in an aqueous medium. The first is that the mass and size of the particles be small enough so that they remain suspended for the period under consideration against the force of gravity’, The second is that some factor must operate to prevent the aggregation of the particles to form larger masses which would no longer remain sus- pended.** Given a dispersion of particles in water whose mass and size are sufficiently small so that settling is slow, three general factors must be considered as determining whether or not appreciable aggre- gation of the ‘particles takes place within a given period of time. The first condition requisite to the aggregation of particles is that their Brown- ian motion must bring them into contact with one another. Therefore the first general factor which must be considered is the rate at which Brownian motion tends to bring this about. The major ex- perimental factors affecting this rate are the con- centration, or number of particles per unit volume ef the dispersion, the mass and size of the par- ticles, the viscosity and the temperature of the suspension’. Study of the effect of variation in these factors upon the rate of agglutination of bacteria is of interest“). However the observa- tions of the agglutination of bacteria which are of direct importance in bacteriology and immunology are made under fairly constant conditions, so far as these factors are concerned. These usual con- ditions are such that the expectation of collision due to Brownian motion in the absence of repel- ling force is sufficient to cause rapid, complete aggregation of the bacteria if each opportunity for collision results in contact of the particles, and each contact results in cohesion. For these two reasons further discussion of the effect of varia- tion in factors which affect the rate of opportun- ity for collision due to Brownian motion is un- necessary for the purposes of the present lecture. Discussion may be found in comprehensive treat- ises on colloid chemistry, and the most recent ela- boration of the kinetics of rapid coagulation has been given by Dr. Miller in the preceding lecture of this symposium. Northrop has discussed these factors in relation to bacterial agglutina- tion), The second general factor which must be con- sidered as determining whether or not appreciable aggregation of colloidal particles will take place within a given length of time is the probability of contact when opportunity for collision is provided in consequence of Brownian motion. It is clear that such contact must occur unless prevented by some repelling force acting between two particles tending to collide in virtue of their Brownian mo- tion. If this repelling force is sufficient to over- come the momentum of the particles contact will not occur. There must obviously be a value of any such repelling force at which it just balances the momentum of the particles. This may be called the critical value of the force in any case. The critical value described is strictly applicable only to a single pair of particles tending to collide under given conditions. In any system of dispersed particles, there is a statistical distribution of veloci- ties due to Brownian motion'®), Therefore a given repelling force may prevent certain colli- sions and not others in any given case. The criti- cal value of the repelling ‘force for a system of dispersed particles is therefore the value which is just sufficient to prevent a sufficient majority of contacts from taking place when opportunity is offered for collision, * The theoretical discussion which follows is essen- tially that of Mudd, Nugent and Bullock(1). ** The mass and specific gravity of bacteria is such that if no appreciable aggregation occurs, the amount of settling which takes place in eighteen hours is sufficiently small to be neglected. For this length of time, therefore, bacterial suspensions may be treated as suspensions of colloidal particles, Aveust 5, 1933 ] THE COLLECTING NET 1 5 I No The third general factor is the probability of cohesion after contact has been made. The inter- face between a dispersed particle and its disper- sion medium is the seat of free surface energy equal to the free interfacial energy per unit area multiplied by the surtace area of the particle. Contact of two particles results in a decrease in free surface energy* equal to twice the area of contact of the particles, times the interfacial ten- sion, or: AF=2S.yAB where A F is the decrease in free surface energy, S is the area of contact of the two particles in contact and y AB is the free interfacial energy per unit area. Following Harkins’) general treatmeni of work of cohesion, it is apparent that the work of cohesion between two such particles in contact is measured by the free energy increase necessarily attendant upon their separation under ideal con- ditions,* Or: Wie = 2) sy AB where We is the “work of cohesion” and the other symbols have the same significance as be- fore. In order to cause separation after contact has been made, the dispersive forces* must provide a minimum energy equal to We. If We is greater than the energy provided by the dispersive forces the particles will cohere after contact. If it is less the particles will separate again after collision. Obviously here too, as in the case of the repulsive force, theré must be a value of We which will just balance the dispersing tendency in any case. This may be called the critical value of We. As in the case of the concept of a critical repel- ling force, so also in the case of the concept of a critical work of cohesion, it is important to bear in mind the statistical distribution of kinetic ener- gies. The Brownian motion impulses tending to separate particles in contact vary in magnitude in a statistical manner. The critical work of cohe- sion for a system of dispersed particles is there- fore such that a sufficient majority of the im- pulses tending to separate particles in contact fail to do so. It is apparent from the foregoing considera- tions that if the repelling force is greater than its critical value, a dispersion will be stable. The same is true if the work of cohesion is less than its critical value. The question of the variation of the work of cohesion merits particular discussion at this point, remembering that the present discussion applies to dispersions in aqueous media. The work of cohesion has been defined as equal to 2 S. y AB. The variation of work of cohesion from case to case is therefore primarily a matter of variation in the free surface energy at the respective par- ticle-dispersion medium interfaces. In general, in accord with Harkins‘*), the more nearly similar the dispersion medium and the surface material of the particles, the lower the expected interfacial tension. In this connection, it 1s most important that certain colloidal particles have the property of associating themselves with large quantities ¢{f water from their dispersion medium. It is not necessary at this point to discuss the possible mechanisms involved in the taking up of the water. It is highly probable that different mechanisms are operative in different cases). It is however also highly probable that in some of these cases the combination of the particles with water results in a hydrous particle surface, which is much more similar to water in the Harkins’ sense than the surface of a particle of the same substance in the anhydrous condition. In such cases the inter- facial tension of the particles against their disper- sion mediums is presumably lowered and along with it the work of cohesion of the particles. It would seem that the lowering of work of cohe- sion due to the hydration of the surface of dis- * The free interfacial energy as considered here in- volves any effects due to the existence of an electri- cal double layer at the particle-dispersion medium interface. It is a composite result of the interac- tion of three sets of force fields, those of the par- ticle molecules, those of the dispersion medium mole- cules and ions and that due to the existence of the double-layer. No assumption is necessary here, and none is made as to the equality of the interfacial tension at the micro-particle-dispersion medium in- terface referred to and the interfacial tension at a macro-interface between the dispersion medium and the material of which the particles are composed. *In circumstances under which surface films have coalesced separation may be non-ideal and involve also work against viscosity. * Exact definition of these dispersive forces is diffi- cult or impossible. It seems, however, that at least three factors may be recognized in a qualitative way: (1) The Brownian motion itself. (2) Electrostatic repulsion. Hydrophilic col- loids which are ionogenic at least owe part of their electrokinetic p. d. to ionization at fixed points on the particle surface. All of these points obviously can not be in contact. It is probable therefore that there is some residual electrostatic repulsion even between particles coherent over a part of their sur- faces. (3) The tendency of the water molecules to wet hydrophilic substances in the surfaces of the coher- ent particles may tend to force these coherent sur- faces apart. It is also possible that statistical fluctuations in the internal energy of the molecules of colloidal particles in contact must be taken into account in a complete treatment of dispersive forces. (In this general connection see Burk: J. Phys. Chem., 35, 2446 (1931) ). 176 THE COLLECTING NET [ Vot. VIII. No. 66 persed particles must be considered as a poten- tial factor affecting the work of cohesion between them and thus the stability of dispersions of such particles in aqueous media. The relationship of the foregoing material to the well known experimental facts with regard to the stability of colloidal solutions is fairly obvi- ous. Colloidal particles dispersed in aqueous me- dia, may be conveniently considered in two classes for purposes of discussion, as hydrophobic parti- cles and hydrophilic particles. The first class have little or no affinity for water and the second a marked affinity. In the case of dispersions of hydrophobic par- ticles clear-cut experimental evidence!) has shown that the necessary condition for their sta- bility is that the electrokinetic potential difference at the surface of the particles exceed a certain limit- ing value known as the critical potential''*)*. When the electrokinetic potential falls below this value aggregation of the particles takes place. The electrokinetic potential difference between parti- cles and their dispersion medium results from the existence of an electrical double layer at the sur- face of the particles. The particles are positively or negatively charged with respect to the medium depending, respectively, upon whether the positive or negative side of the electrical double layers is associated with the particles. It has been believed that the repelling action of similarly charged par- ticles is responsible for the stability of suspen- sions of hydrophobic particles when the electro- kinetic potential exceeds the critical value''®). Dr. Miller has just given us a further interpre- tation of the nature of the stabilizing action of the electrical double layer. In terms of the general working theory of suspension stability which has been presented, this electrical double layer is the force which if sufh- cient can prevent the contact of particles when opportunity for their collision is provided in virtue of their Brownian motion, and thus stabil- ize the dispersion. Turning now to the question of the stability of hydrophilic colloidal particles, it is found that quite a different situation prevails. Kruyt and others working in his laboratory’) have clearly demonstrated that the condition of hydration of certain hydrophilic colloidal particles must be con- sidered as a stabilizing factor in dispersions of such particles in aqueous media. The first out- standing fact is that certain of the particles studied formed stable dispersions when their re- pelling force, as measured by their electrokinetic potential, was reduced to zero’), The addition of sufficient alcohol to such isoelectric dispersions caused them to precipitate. The alcohol in such cases is generally considered to act by dehydrating the particles. These facts indicate first that a stabilizing factor is active apart from electrokin- etic potential difference, and secondly that this stabilizing factor results from the hydrated con- dition of the particles. It is apparent that this second stability factor is capable of stabilizing a dispersion of these particles in complete absence of a repelling force. lt must therefore aci by de- creasing the work of cohesion ot fie particles below the critical value or by raising this critical value. Reasons for this stabilizing action asso- ciated with the hydrous condition of the particles then follow directly from the previous discussion. It was pointed out that increased surface hydra- tion sheuld accompany the union of hydrophilic particles with water of the dispersion medium. This increased surface hydration should cause the hydrated particles to have a much _ lower surface tension against the aqueous dispersion medium than would the same particles in a hypo- thetically anhydrous condition. Certain hydrous particles might well thus have very low surface tensions against aqueous media which in turn would cause them to have very low works of co- hesion, even possibly below the critical value. In this way the hydrous condition of particles in certain cases could be a stability factor which could result in the stability of a suspension of such particles even when their electrokinetic po- tential was reduced to zero. Moreover the tend- ency of the water to wet the hydrophilic surfaces might promote dispersion and therefore necessi- tate a high critical value of the work of cohesion. The mechanism outlined above is here offered as the one which is operative in the unquestioned stabilizing influence of the hydrous condition of the particles in hydrophilic suspensions. It is significant that the only dispersions of colloidol particles in aqueous media which are known to be stable in the absence of electrokinetic potential are those in which independent evidence points clearly to the hydrophilic nature of the particles. It should be pointed out that the concept of variation in the work of cohesion with surface hydration applies to variation in the state of hy- dration of a particular surface. In passing from one surface to another, as in the deposition of a protective or sensitizing film, no such relationship necessarily exists. Surface A may be less hy- drous than surface B and still have a lower work of cohesion. The point is that surface A for example presumably has a lower work of cohesion in a relatively hydrated state than in a relatively dehydrated state. In the sense of this lecture, changes in hydration of a particular surface such as may be brought about by the electrolyte con- *The critical potential referred to is the “first” critical potential (12). Aucust 5, 1933 ] THE COLLECTING NET 177 tent of the medium are considered as modifica- tions of an existing surface rather than the form- ation of a new surface. The critical potential as experimentally deter- mined for a system of dispersed particles is the minimum electrokinetic potential compatible with a stable condition of the dispersion under the de- fining conditions. Suppose that the work of co- hesion is sufficiently high so that practically every contact results in permanent coherence of parti- cles. The experimentally determined critical po- tential will then be such that it is just sufficient to prevent a sufficient majority of contacts when opportunity for collision is offered due to Brown- ian motion. If the work of cohesion is somewhat lower, that is if an appreciable number of con- tacts result in redispersion, the repelling force would not have to be quite so large, that is it would not have to prevent as many contacts as before in order to maintain stability. In_ this second case, the experimentally observed critical potential would be somewhat lower than in the first. Theoretically, therefore, in all cases in wuich the work of cohesion is insufficient to prevent all redispersion, experimentally deterinmed critical potentials should decrease with decrease in work of cohesion. When the work of cohesion is suffi- ciently small the suspension will be stable even at zero electrokinetic potential. Shibley"®) confirmed the critical potential value of Northrop and De Kruif") (+ 15 millivolts) with bacteria suspended in NaCl, ZnSO, and CeCly. In the presence of NasHPOs, however, the same microorganisms had a much _ higher critical potential (—34.6 millivolts in one experi- ment). It is possible that the higher critical po- tential in the presence of NasHPOy, was due to an increase in work of cohesion due to this parti- cular salt. Aggregation occurs when the electrokinetic po- tential difference is lower than its critical value in any system of dispersed particles, whose cohes- ive force is above its critical value. However the rate of aggregation within the critical potential zone varies with the residual p.d. upon the parti- cles. One reason is that the lower the p.d. the greater the majority of total opportunities for collision which result in contact, with all oppor- tunities for collision resulting in contact at the isoelectric point. Further, it has been suggested that residual potential difference is a factor aid- ing the redispersion of particles after contact, at least in some cases. In these cases, the greater the residual p.d., the greater the redispersion tendency and presumably the slower the rate of aggregation. The relationship of stabilizing and sensitizing surface films to the general question of the sta- bility of dispersions of colloidal particles in aqueous media is secondary to the factors which have been discussed. It will be treated in the section of the lecture dealing with the stability of suspensions of sensitized bacteria. The Stability of Suspensions of Unsensitized Bacteria The work of Northrop and De Kruif''1S) has been of great importance in the development of the theory of bacterial agglutination. The stability of the suspensions of the two types of bacteria which they studied varied markedly and regularly with the salt content of the dispersion medium. With salt concentrations below 0.001 molar both types regularly agglutinated when their electrokinetic potential was reduced below + 15 millivolts. Under these conditions + 15 mil- livolts was the critical potential. When the total salt concentration was raised to 0.1 molar, the sus- pensions were stable when the electrokinetic po- tential was reduced to much smaller values than + 15 millivolts, in some cases even when it was reduced to zero. Shortly after the work of Northrop and De Kruif, Loeb"®’ showed that the stability of gelatin solutions is influenced by salts in an ex- actly similar way, and further that the stability of suspensions of- collodion particles coated with surface films of gelatin also showed the same type of behaviour. Loeb pointed out the similarity of his results of this type to those obtained by Northrop and De Kruif with bacteria. He con- cluded from his experimental work that the in- creased stability of the suspensions of protein- coated collodion particles in the higher salt con- centrations was most probably due to increased affinity of their surfaces for water under these conditions. Later Oliver and Barnard'?°) and Netter'*!) also attributed the decrease in “cohe- sive force’’ of the surface of cells by salts to in- creased affinity of the surfaces for water. It would seem highly probable on this basis that the increased stability of the bacterial suspensions of Northrop and De Kruif may also have been due to an increase in the hydrous condition of the sur- faces of the bacteria in the higher salt concentra- tions. This probability also follows from the theo- retical considerations which have been presented in this paper. Since the suspensions were stable in some cases even when the electrokinetic potential was re- duced to zero, it follows that the work of cohe- sion of the bacteria must have been reduced below its critical value. It was shown that the most prob- able cause for the reduction of the work of co- hesion between particles dispersed in aqueous media, is an increase in the hydrous condition of the particle surfaces. Changes in salt concentra- 178 THE COLLECTING NET { Vor. VIII. No. 66 tion are well known to affect the state of hydra- tion of hydrophilic colloidal particles, and hence very probably to alter their state of surface hy- dration. Northrop and De Kruif clearly recognized that a decrease in “cohesive force’? must have taken place as between their bacteria in 0.001 and 0.1M salt solution. They devised and used an ingenious method for following changes in this value. They defined “cohesive force” by the values obtained by this method. There is some question as to whether the values obtained by them accurately expressed the value of the cohesive force as de- fined in this paper, because, for example, their method may well have involved work against vis- cosity in the separation of partially coalescent particles. Nevertheless, in any case, they definite- ly showed that a reduction in “cohesive force” as measured by their method always accompanied the phenomenon of suspension stability with elec- trokinetic potentials below + 15 millivolts. This parallelism is convincing evidence that they were able to measure true cohesive force with suffi- cient accuracy to arrange their suspensions in the proper order with regard to their value, and that was the most important object of their “cohesive force’? measurements. Northrop and De Kruif further showed that if the salt concentration is increased well beyond 0.1 molar their bacterial suspensions again became unstable. There seems to be no question but that they were correct in attributing this to a “salting out” mechanism, that is to a dehydration and precipitation in the presence of a high salt con- centration. The general conclusion from their re- sults is that both electrokinetic potential differ- ence and hydration are important factors in de- termining the stability of suspensions of the two types of bacteria studied by them, both being markedly affected by variations in the total salt content of the dispersing medium. According to Northrop and De Kruif, the low- est concentrations of salts acted to affect the elec- trokinetic potential difference, medium concen- trations to affect the “cohesive force” and still higher concentrations to affect the state of sur- face hydration. It appears that the effect of the intermediate concentrations on “cohesive force” may also be interpreted as resulting from an ef- fect on the hydration affinity of the bacterial sur- faces. 3acteria do not all have surfaces of this type, however. We have studied by the interfacial tech- nique'**) the relative ease of wetting by oil and water of the surfaces of a large number of dif- ferent types of bacteria. It has been found in this way that acid-fast bacteria are in general more readily wet by oil than by water where- as non-acid-fast bacteria in general are much more readily wet by water.'3) It would be expected on this basis that stability relations of suspensions of acid-fast bacteria would resemble those for hydrophobic colloidal particles rather than those for hydrophilic particles. In contrast to these acid-fast bacteria are cer- tain strains of aflagellate intestinal bacteria which have been studied in the writer’s laboratory. In their “smooth” form these bacteria have electro- kinetic potentials so small as not to be with cer- tainty measureable over a wide range of pH values and electrolyte concentrations.'*4) See Table 1. Yet these bacteria without measurable p. d. are stable in suspension, TABLE I. The electrophoretic mobility of Bact. flexneri “smooth” and “rough” as a function of pH. Smooth Rough Buffer pH p/sec/ p/sec/ volt/em. — volt/cm. Phosphate Zeal AORL 3.4 24 6.6 0.0 3.9 H 6.0 0.0 2.9 Acetate 57 0.1 3.9 o Dee 0.05 3.8 iy 4.8 0.1 3.6 ad 4.4 0.0 3.4 4) 4.0 0.0 Sal u Sei? 0.0 2.6 Phthalate BES 0.1 2.4 S 2.6 0.0 Ball The general contention of Northrop and De Kruif was that the stability of their bacteria with very low electrokinetic potentials when the salt content was increased to above 0.1 molar, was that the increased concentration depressed the co- hesive force of the bacteria. Since the above- mentioned strains of bacteria form suspen- sions which are stable in distilled water or very dilute electrolyte, with electrokinetic potentials of zero to a few millivolts, it is apparent that an ex- tension of the views of Northrop and De Kruif is necessary to account for the stability of these suspensions. According to the theoretical conclusions of the present paper it appears necessary that this type of stability is due to an extremely low work of cohesion due to surface hydration which is prob- ably in excess of that obtaining in the case of the bacteria of Northrop and De Kruif. It is at least quite definite that the primary stabilizing action of hvdration is operative over a wider range of conditions in the case of the strains de- scribed here. Acid-fast bacteria and the type just described represent extreme types selected from a large number of bacteria studied over a period Aueust 5, 1933 ] THE COLLECTING NET 179 Dysentery bucillus Colon bacillus Turtle bacillus Tubercle bocilus. 2010 5 2515 6 i) 2010 5 2512 6 o 20.10 5 2512 6 ° 20.0 5 25126 b} a ] - | 8 ” bo a eee ee Seeks a : SSS c 2 ” ir et c Li ——— = —4 9 Se a g 5 40 oe Sih & & -20 9 t au -25 o y -3.0) -40) {[. Fig. 1. Stability of hydrophilic bacilli and precipitation of hydrophobic bacilli in presence of acid. Washed bacteria suspended in solutions of HCl in distilled water. Abscissae, concentrations of HCl in millimols per liter. The hydrophilic dysentery and colon bacilli show little aggregation at any acidity. The hydrophobic turtle and avian tubercle bacilli show complete aggregation in acid concentrations which sufficiently reduce the electrokinetic p. d. To obtain electrokinetic p. d. in millivolts in this and subse- quent figures multiply /sec. per volt/cm. by 12.6. (In this connection see Northrop and Cullen: J. Gen. Physiol., 1921-22, 4, 638.) of years on the basis of their wetting properties and cataphoretic behavior. The two types seemed to offer splendid material for the exten- sion of the general theory of the stability of bac- terial suspensions. Accordingly experiments have been performed to test the hypothesis that the stability of suspen- sions of acid-fast bacteria depends upon conditions more closely resembling those for the stability of dispersions of hydrophobic colloidal particles ; and that the varieties with apparently markedly hy- drous surfaces form suspensions whose stability depends more definitely upon the hydration fac- tor, than do those of the suspensions of the two types of bacteria studied by Northrop and De Kruif. Fig. 1. records such an experiment. Washed suspensions in distilled water of two of the hy- drophilic bacteria, the dysentery and the colon bacillus, and two acid-fast hydrophobic bacteria, the turtle bacillus and the Arloing strain of avian tubercle bacillus, were mixed with water and with dilute HCl solutions. The concentrations of HCl after mixing, in millimols per liter, are given as abscissae. Agglutination was read after one hour and after 18 hours in the ice box. The electrophor- etic mobilities were determined in a microcatapho- resis cell(?*) following the 18 hours reading. Each suspension was examined in the cataphoresis cell in HCl of the same concentration as that in which the agglutination readings had been made. It 1s apparent that only a trace of agglutina- tion of the dysentery bacillus occurred in any acid concentration, although the electrokinetic p. d. was extremely low; in 0.6 millimolar HCl the p. d. for the dysentery bacillus was about 3 milli- volts. The colon bacillus showed very little ag- glutination although the p.d. was minimal; the colon bacillus was stable in distilled water with a p. d. of only about 3 millivolts. It is obvious that the stability of the colon bacillus in distilled water is attributable neither to reduction of the cohe- sive force by electrolytes nor to a high surface potential charge. The turtle and avian tubercle bacillus, on the other hand, which in an oil-water interface show marked preferential wetting by the oil,‘°?), are rapidly aggregated in concentrations of acid suf- ficient to redtice the p. d. below its critical value. In the case of the avian tubercle bacillus the value of this critical potential seems to be high, nearer that found by Powis‘? for oil drops than that found by Northrop and De Kruif for non- acid fast bacteria. 180 THE COLLECTING NET [ Vor. VIII. No. 66 Cataphoretic Mobility AL/Sec. per volVem. ° 8 3 o fey (2) [sO Zz a eo ee > = @ Fig. 2. Effect of salts on p. d. and lack of effect on agglutination of turtle bacillus and avian tubercle bacillus. Bacteria were suspended in 0.01 N HCl to which were added the amounts of NaCl indicated on the axis of abscissae. Circles, turtle bacillus. Crosses, avian tubercle bacillus. The upper broken line is the curve for typhoid bacillus in HCl and NaCl, redrawn from Northrop and De Kruif’s (17) Fig. 4, p. 647. Unbroken line, complete agglutation. Broken line, no agglutination. The high electrolyte content inhibited agglutination of the typhoid bacillus, but not that of the hydrophobic acid-fast bacteria. In Fig. 2 the same two acid-fast bacteria are set up in strongly acid solutions containing grad- uated concentrations of NaCl. The concentration of HCl after mixing was N/100 in each tube. The NaCl contents in the several tubes were 0.001, 0.01, 0.10 and 1.0 molar, respectively. These experimental conditions were chosen to du- plicate as nearly as possible those of Fig. 4 in the paper of Northrop and De Kruif.) In our ex- periment the acid reduced the potential below the critical value for the hydrophobic bacteria and agglutination occurred in all tubes in spite of the very high electrolyte concentration. The corre- sponding curve in Northrop and De Kruif’s Fig. 4 is renlotted for contract; with these bacteria no agglutination occurred until the very high “salting out’? concentration was reached. The “rough” variants of the intestinal bacteria differ in their stability relations both from these hydrophobic acid-fast bacteria and from the hy- drophilic smooth intestinal forms. The rough variants have electrokinetic p.ds. which are de- pressed by decreasing pH values and by increas- ing electrolyte concentrations. See Tables I. and II. In very dilute electrolytes these “rough” hac- teria form stable suspensions. They are usually readily aggregated, however, in solutions in which both acid-fast bacteria and the hydrophilic smooth forms are stable. The general conclusions of this section of the lecture may now be stated. Bacteria exist which display a wide range of surface types, from those which are markedly hydrophobic to those which are markedly hydrophilic. The factors gov- erning the stability of dispersions of the various types in aqueous media are the same as apply to the stability of colloidal particles with similar types of surfaces. The theoretical considerations are those which have been described in the previ- ous section, TABLE II. Precipitability by electrolyte of Bact. typhosum (strain 0 901) in sodium acetate-acetic acid buffer of pH=approx. 5.2. 0 901 Smooth 0 901 Rough 2s ~ ts a ts pe ear NS = Se: 0.2 0 0.0 ++++ —0.5 0.04 0) —0.1 atta —1.6 0.02 0 +0.1 ++ —2.4 0.01 0 0.0 ++ 3:2 0.004 0 -++0.1 ++ —3.6 0.002 0 -++0.5 +-++ —3.9 0.001 0 —0.1 + —3.9 3acteria with strongly hydrophobic surfaces are stabilized im aqueous media chiefly by elec- trokinetic potential difference. They agglutinate when their electrokinetic potential is reduced be- low a definite relatively high critical value. Others, of the type studied by Northrop and De * The sign of the bacterial charge is indicated by’ — or = before the mobility value. Precipitation readings made after 20 hours in refrigerator. Data in Tables I and II obtained by Miss Eleanore W. Joffe, Aveust 5, 1933 ] THE COLLECTING NET 181 Dilutions of Sensitizing Sera NaCl 4096 2048 1024 512 256 128 Gt 32 16 8 4 B 2 NaCl 1024 512 256 128 64 32 16 8 4 n 56 a pH of Isoelectric Points = Hemophil us Influenzae Br. abortus, Staphylococcus Salmonella pullorum Salmonella pullorum Pneumococcus, type I Fig. 3. Kruif, form suspensions in which both electro- kinetic potential difference and hydration are stabilizing factors of primary importance. This is not difficult to understand since evidence has re- cently been brought forward to indicate that the surfaces of many or most bacteria contain both hydrophobic and hydrophilic components.'°"’ Finally bacteria exist whose state of surface hy- dration is the primary stabilizing factor in their dispersions in aqueous media over a wide range of conditions. The Stability of Suspensions of Sensitized Bacteria 3acteria within the human or animal body are altered in their surface properties by the defen- sive mechanisms of the host. The globulins and albumin of the blood are adsorbed to a greater or lesser extent on bacteria with which they come in contact, and the intrinsic surface properties of the bacteria are thus masked by the adsorbed pro- tein. If the infection persists long enough new substances, known as antibodies, which appear to be globulins with physical-chemical differences from normal serum globulins'**), are elaborated. These antibodies possess specific chemical affini- ties for substances in the bacterial surfaces. The adsorption of the normal blood proteins and the specific chemical combination of antibody-proteins with the bacterial surface are known as serum “sensitization.” Sensitization results in marked changes in the physical properties of the bacterial surfaces, 1. e. the sensitized bacteria are more cohesive, their wetting properties are altered, their electrokinetic p.d. is, under the conditions of the usual sero- logical experiment, reduced, and their isoelectric point is shifted to a value near (but often not identical with) that of serum globulin.'** °°) These changes are consequent upon the formation of a surface deposit of antibody-globulin on the antigen. Since electrokinetic p.d., cohesion (and hydration), are the fundamental factors deter- mining stability, the stability of sensitized would 182 THE COLLECTING NET [ Vor. VIII. No. 66 be expected to differ from that of unsensitized bacteria. As a matter of fact agglutination is the most familiar consequence of combination with antibody. ; The remarkable specific chemical affinity be- tween antigen and antibody enables the antibody in exceedingly high dilution to form an effective surface deposit on the antigen. Thus antisera may be prepared which agglutinate typhoid bacilli in a dilution of one volume of serum in a hundred thousand volumes of dilutent. The surface de- posit once formed, however, has many points ot resemblance to deposits of serum proteins, eg, albumin or other proteins formed by non-speciiic adsorption on bacteria or other particles. The non-specific deposit of serum proteins, in addi- tion to requiring higher concentration of protein to form an equivalent deposit, is in general less firmly held than the specific deposit. In general with the progressive formation of a surface deposit the electrokinetic p.d. and isoelec- tric point of the particle approach those of the deposited substance. The effect of serum sensitization on the isoelec- tric points of various bacteria are shown in Fig. 3. (cf.Shibley°)). The bacteria were treated in the left hand side of the figure with serial dilu- tions of the sera of the patients from which they were isolated: on the right hand side of the figure the results of treatment with normal serum or serum from another disease are shown. A portion of the bacterial suspension was allowed to stand overnight in each serum dilution, the sensitized bacteria were then washed in 0.85% NaCl solu- tion, and their isoelectric points were determined in acetate buffer series with the aid of a North- rop-Kunitz microcataphoresis cell. Before sensi- tization the staphylococcus retained a negative potential even in N/100 HCl, the pneumococcus was isoelectric between pH 2.0 and 3.0, H. influenzae and Br. abortus between pH 3.0 and 4.0, and S. pullorum had little if any surface p.d. in any buffer used. With progressive sensitiza- tion the isoelectric points of all the bacteria con- verged progressively until values of pH=4.9 to 5.5 were reached after sensitization with the patient’s serum, and of pH=4.35 to 5.0 after sen- sitization with normal serum. The stability conditions in the case of gelatin adsorbed on collodion particles have been shown by Loeb closely to resemble these of gelatin solu- tions.“") Loeb showed on the other hand that collodion particles coated with egg albumin showed the stability relations of denatured albu- min rather than those of native albumin. Students of specific bacterial agglutination from Bordet on have been impressed with the fact that sensitized bacteria were aggregated by traces of cations which were alike incapable of precipitating the unsensitized (non-acid fast) bacteria or the serum globulins with which the antibodies are associated. The antibody-globulin combined with antigen has therefore been spoken of by Shibley'*°) and others as “denatured.” A given dispersion of particles is stable either if the repelling force (electrokinetic potential difference) is greater than its critical value, or if the cohesive force of the particles (2S. y AB) is below its critical value. It follows from this that for aggregation and precipitation to occur both the electrokinetic potential difference must be he- low its critical value and the work of cohesion must be above its critical value. A stabilizing or protective film forming sub- stance is one that, under the conditions of test, results in a surface such that either the electro- kinetic potential difference is above the critical value for that surface or that the work of cohe- sion of the surface is below its critical value, or both. A precipitating or sensitizing film forming substance is one such, that under the conditions of test, a surface results which is both below its critical potential and above its critical work of co- hesion. It is apparent that one and the same sub- stance may act either as a stabilizing or sensitiz- ing film forming substance depending upon the conditions of test. Since after combination with bacteria has oc- curred, the effect of antibody film on_ stability conditions is entirely analogous to that of other types of films, the above considerations apply to the agglutination of sensitized bacteria. Antibody films which cause agglutination of bacteria do so because they result in surfaces which are both below their critical potentials and above their critical works of cohesion under the conditions of test. Bacteria may also be agglutinated by tan- nin‘) and it may be shown that in this case also a surface deposit is formed whose electrokinetic p.d. is below and whose cohesion is above its critical value"). BIBLIOGRAPHY 1. Mudd, S., Nugent, R. L. and Bullock, L. T, J. Phys. Chem., 1932, 36, 229. 2. Bancroft, W. D., “Applied Colloid Chem- istry,’ New York, 2nd ed., 1926, 170; Freundlich, H., translated by Hatfield, H. S.: ‘Colloid and Capillary Chemistry,” New York, 1926, 370. 3. Kruyt, H. R., translated by van Klooster, H. S., “Colloids,” New York, 1927, 109. 4. Eagle, H., J. Immunol. 1932, 23, 153. 5. Northrop, J. H., in Jordan, E. O. and Falk, TI. S.: “The Newer Knowledge of Bacteriology and Immunology,” Chicago, 1928, Chapter LVIII. 6. Freundlich, H., translated by Hatfield, p. 443. Aucust 5, 1933 ] THE COLLECTING NET 183 7. Harkins, W. D. in Jordan and Falk: ‘The Newer Knowledge of Bacteriology and Immuno- logy,” 1928, 161. 8. Harkins, W. D., Brown, F. E., and Davies, E. C. H., J. Am. Chem. Soc., 1917, 39, 354. 9. Kruyt H. R., translated by van Klooster, H. S.: Chapter XII. 10. Seifriz, W. in Alexander, J. “Colloid Chem- istry,’’ New York, 1928, 2, 410; Gortner et al., Trans. Faraday Soc., 1930, 26, 678. 11. Gortner, R. A., “Outlines of Biochemistry,” New York, 1929, 190. 12. Freundlich, H., translated by Hatfield, H. S., 1926, 418. 13. Freundlicn, H. loc, cit. 432. 14. Kruyt, H. R., loc. cit., Chapter XIII. 15. Kruyt, H. R., loc. cit., 181. 16. Shibley, G. S., J. Exper. Med., 1924, 40, 453. 17. Northrop, J. H., and De Kruif, P. H., J. Gen. Physiol. 1921-2, 4, 639. 18. Northrop, J. H., and De Kruif, P. H., J. Gen. Physiol., 1921-2, 4, 655. 19. Loeb, J., ‘‘Proteins and the Theory of Col- loidal Behavior,”’ New York, 2nd ed., 1924, 327. 20. Oliver, J., and Barnard, L., Am. J. Physiol., 1925, 73, 401. 21. Netter, H., Pfluger’s Archiv ges. 1925, 208, 16. 22. Mudd, S. and Mudd, E. B. H., J. Exp. Med., 1924, 40, 647; 1927, 46, 167. 23. Cf. Reed, G. B., and Rice, C. E., J. Bacteriol., 1931, 22, 239. 24. Joffe, E. W., Hitchcock, C. H., and Mudd, S., J. Bacteriol., 1983, 25, 24. 25. Mudd, S., Lucke, B., McCutcheon, M. and Strumia, M., Colloid Symposium Monograph, 1928, 6, 131. 26. Powis, F., Z. Physik. Chem., 1915, 89, 186. 27. White, P. B., J. Path. and Bact., 1927, 30, 113; 1928, 31, 423. 28. Mudd, S., Lucke, B., McCutcheon, M., and Strumia, M., J. Exper. Med., 1930, 52, 313. 29. McCutcheon, M., Mudd, S., Strumia, M., and Lucke, B., J. Gen. Physiol. 1930, 13, 669. 30. Shibley, G.S., J. Exper. Med., 1926, 44, 667. 31. Loeb, J., loc. cit., p. 349. 82. Reiner, L., and Fischer, O.,Z., Immunitats, 1929, 61, 317. Freund. J., Proc., Soc. Exp. Biol. Med., 1929, 26, 876. J. Immunol., 1931, 21, 127. Neufeld, F., and Etinger-Tulczynska, R., Cen- tralbl., Bakt., Orig., 1929, 114, 252. Physiol., DiIscuUSssION Dr. Abramson: In discussing the interfacial technique, when you have bacteria partially coated with protein film, haven’t you also a pro- tein film at the aqueous-oil interface, and doesn’t that enter into the picture? Have you the right to call the interface oil-water in the presence of proteins or other adsorbable substances? A plastic film of some sort should be formed. Dr. Mudd: Undoubtedly the oil-water inter- face becomes contaminated with any adsorbable material present. We have had this fact in mind, however, and have taken care to reduce it to a minimum and so to arrange the experiments that it should not vitiate our results. Dr. Riddle: Do collisions between particles actually occur or do they not from the standpoint of actual visual observation ? Dr. Mudd: I presume you are referring to collisions without aggregation. I do not know of anyone who has written on that point. It would be difficult to tell, of course, because the distances involved would be of the order of 10° cm. which is considerably beyond the powers even of the ultramicroscope. I doubt if this question could be answered by direct observation. Dr. Cole: When bacterial agglutination takes place, how rapid is it? Dr. Mudd: Ordinarily of the order of slow coagulation rather than of rapid coagulation. It is possible, however, to get rapid coagulation of hy- drophobie bacteria with acid. Dr, Cole: This, I should think, would be a case where you would have practically a mono- dispersed system in which the Smoluchowski theory should follow. Is it followed ? Dr, Abramson: Oliver and Barnard showed that the Smoluchowski theory is followed per- fectly in rapid coagulation of red cells. Dr. Miiller: The coagulation of rod-shaped particles has a very much faster rate than for spherical particles. The deviations from Smoluch- owski’s curve are particularly large at the be- ginning of the coagulation. Later on the curves approach again the curve for spherical particles. Dr. Mudd: Bacterial suspensions offer a con- siderable range of shapes and surface properties favorable for the study of coagulation problems. Dr. Miiller: Would you prefer to assume that a colloid is not a system in thermodynamic equilibrium ? Dr. Mudd: The more usual assumption, which is implicit in the ordinary treatment, is that hydrophobic colloidal systems are metastable. If the particles could come in contact they would aggregate, but the double layer prevents the ap- proach of the micelles. Do you believe it necessary to assume that hydrophobic colloids are in ther- modynamic equilibrium ? Dr. Miiller: This assumption is not necessary. By giving it up, however, we can not. treat a col- loid with the’ help of the ordinary methods of ther- modynamics. If a colloid is only in metastable equi- librium, one can consider the problem in the fol- lowing way: If a colloidal particle approaches another one, it comes first under the influence of repulsive forces, created by the double layers. These repulsive forces can be represented by 184 THE COLLECTING NET [ Vor. VIII. No. 66 “potential-hill.” If the temperature energy of, the particle is large enough, the particle can traverse this hill and comes then under the influence of the attractive forces of the surface tension. These forces can be represented by a “potential-valley.” This valley is usually so deep, that the particles have not enough kinetic energy to come out of it —which means that coagulation takes place. Hy- dration decreases the depth of this valley, and it is possible that very strong hydration decreases the depth so much that a particle may again es- cape from the attraction of the surface forces. That is the case if the temperature motion is larg- er than the surface forces. Hydration may, there- fore, produce stability. If there is no or little hy- dration, the stability is due to the first potential hill. If this hill is high enough no particle has a large enough kinetic energy to overcome the re- pulsive forces. My contention is that the ordinary electrostatic forces are not strong enough to pre- sent a hill high enough. The stability of large part ticles is only assured if we take also into account the change of energy of the water due to electro- striction. This effect is similar to hydration; it differs from hydration insofar as it is produced by electrostatic forces, while hydration is due to molecular forces. My proposed theory is qualitatively in agree- ment with Dr. Mudd’s point of view. The difer- ence is only in the quantitative relationship. The consideration of the electrostriction is necessary to explain a sufficiently high potential-barrier. The height of the hill is again determined by the ¢-potential. The dependence of the stability on the properties of the double layer is, however, more complicated than if only electrostatic forces are considered. It seems probable that not only the ¢-potential, but also the thickness of the double layer is equally important for the deter- mination of stability. Kruyt has, for instance, found colloids which coagulated at a high ¢-poten- tial, and were stable at a low €¢-potential. Dr. Mudd: By treating our hydrophilic bac- teria with progressive concentrations of specific immune serum under the proper conditions, it is possible to get agglutination in parallel with eith- er an increasing negative or positive ¢-potential. In this case, however, we are progressively form- ing a new surface of hydrophobic protein upon the original hydrophilic bacterial surface. Dr. Cohen: What happens to the surface energy when particles aggregate? Dr. Miiller: 1 believe the energy is so small that you can not say very closely. Dr. Cohen: Since numerous particles are in- volved the aggregate effect may be appreciable. Dr, Miiller: One would expect that there would be a heating effect. Dr. Cohen: Dr .Mudd speaks of certain bac- teria as highly hydrated (hydrophilic) and cer- tain others as hydrophobic. The contrast between this view and Dr. Abramson’s discussion of pro- teins in water, which leaves water out of the pic- ture, is puzzling. Why was water ignored? Dr. Abramson: It might affect the surface as far as agglutination is concerned, but it appar- ently does not change the time average of the net charge. I can’t visualize the exact hydration me- chanism of proteins. Certainly very little is actu- ally known of the hydration of proteins. There is one interesting point I would like to follow up. This is Northrup’s curve for mobilities. The charge is not given by this curve. This curve gives the ¢-potential, and note that you get coagulation when the potential drops, say, from a relative value of 0.8 » per second to a value of 0.3 » per second. If you change the potential one-half you get coagulation. In the literature, you frequently see statements to the effect that the bacteria are discharged. This is not correct, because the charge is proportional to €e ike ab Actually where you get agglutination the charge is at a maximum or near its limit in value, where- as it is the potential which is depressed. Dr. Miiller: Nevertheless, the ¢-potential is the important thing for agglutination, not the charge, \/e sinh Dr. Abramson: But the charge is not de- creased as is frequently stated in the literature, nor need the €-potential be decreased ; it can also increase as in the case of sensitized bacteria. Dr, Blinks: Do these experiments work as well with dead bacteria ? Dr. Mudd: Yes, we use either living or dead bacteria. Dr. Briggs: In the case of the smooth strains of bacteria your observations show that they ex- hibit zero electrokinetic potential throughout a wide range of pH change. This is certainly an un- usual surface. Just what sort of a surface do you picture as existing there that could show such properties ? Dr. Mudd: Iwas in hope some of you would help us solve this perplexing question. However, many bacteria are known to contain polysac- charids in their surfaces, and it seems conceivable that these may neither themselves be ionogenic nor have a preferential adsorption affinity for ions. Dr. Briggs: But polysaccharids do show é-potentials and these change radically with pH, Avcust 5, 1933 ] THE COLLECTING NET 185 Tow do smooth and rough strains act at the in- terface between oil and water? Dr, Mudd: The smooth hydrophilic strains are preferentially wet by the water; they do not pass into the oil. A comparison of the correspond- ing rough strains in this respect would be very desirable but has not yet been made. Dr. Cohen: With reference to capsulated pacteria of the smooth type, is it not true that the capsules are largely composed of polysacchar- ids which are highly hydrated ? Dr. Mudd: 1 think this is true of the pneu- snococci and Friedlander’s bacilli at least. Their capsular material is quite soluble. Dr. Cohen: What I mean is that one can vften see on capsulated bacteria a capsule which is many diameters thicker than the cell itself. Re- cent evidence shows that this capsular material consists largely of polysaccharids. Under the microscope it often appears highly refractive, and there is much evidence that it is loaded with moisture. Having in mind also the chemical con- stitution and configuration of these carbohydrates, one may picture that the capsule is in essence a radially (not concentrically) stratified conglomer- ate of elongated polysaccharids micelles project- ing into the culture medium. Into this sort of radial structure water could penetrate some dis- tance ; in addition, the polysaccharid itself is high- ly hydrous, therefore the actual boundary be- tween this type of capsule and the surrounding liquid medium would be rather hard to define. Dr. Briggs: For the surface to exhibit no ¢-potential as a result of surface hydration would require that the double layer must be completely within the layer of adsorbed water. Is that pos- sible ? Dr. Miiller: It seems possible that if we have very strong hydration, the double layer is entirely inside the rigidity boundary. Since we must have a small dielectric constant in the adsorbed layer, this double layer is practically a rigid one. Dr. Blinks: What happens in the migration of a jell of agar? Dr. Abramson: It has a pretty high mobility. Dr. Briggs: Jf such a strongly adsorbed layer of water does exist at the surface of the particle, it should in reality form a phase, the properties of which would be different from that of the bulk of the water. Selective adsorption of the ions in solution should occur across this new boundary, and give rise to a ¢-potential. Dr. Abramson: water. Dr. Miiller: In ice the water molecules are not rigidly bound as in hydration. For low fre- quencies ice has the same large dielectric constant as water, which indicates that the HO molecules in ice can rotate. Ice shows electrophoresis in END OF COLD SPRING HARBOR SECTION 186 THE COLLECTING NET [ VoL. VIII. No. 66 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Jean M. Clark, Martin Bronfenbrenner, Margaret Mast and Anna- leida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford THE COLLECTING NET SCHOLARSHIPS The scholarships which we initiated in 1927 seem to have become a “permanent institution” although we never know for certain at the begin- ning of the season that we can accumulate five or six hundred dollars for award in September. Owing to our relations with the Biological La- boratory at Cold Spring Harbor it has been de- cided to have one scholarship of $100.00 awarded to a student working at that institution. The award will be made by Dr. Harris and a commit- tee appointed by him. Further, it has been de- cided that the scholarship winners from both in- stitutions may have the privilege of working at either Woods Hole or Cold Spring Harbor, pro- viding their application for work is accepted by the chosen laboratory. We look forward to the time when both institutions will grant scholarship holders a free research table, so that they can use the whole hundred dollars to meet their travelling and living expenses. We owe a debt of gratitude to Vera Warbasse, Alfred Compton and Thomas Ratcliffe who form the executive committee of the Penzance Playcrs, aid their staff, who presented George Bernard Shaw’s play “You Never Can Tell” in the inter- ests of our scholarship fund. We hope to be able to announce next week the sum of money which will be realized from this source, but we are con- fident that it will be enough to take care of one or two scholarships. Contributions—small or large, tiny or huge— will be greatly appreciated. It is our wish to accumulate a little more money each year than is needed for award at the end of the summer, in order to build up gradually a nucleus for our en- dowment fund. Introducing Dr. JosepH SPEK who is professor of zoology at the Zoological Institute, University of Heidelberg, and one of the editors of Protoplasma. Arriving in this country in the Fall of 1932, he worked at the laboratory of Dr. Robert Chambers at New York University during the Winter, injecting amoeba to determine the differences in pH of constituents of protoplasm. Since his arrival in Woods Hole on May 30 he has been carrying out vital staining experiments on the eggs of various organisms: Asterias, Nereis, Loligo, ete. Dr. Spek lectures on general zoology at the University of Heidelberg, and is interested prim- arily in the fields of cytology and experimental embryology. He is the author of the articles on experimental embryology in Gellhorn’s textbook : “Lehrbuch der allgemeinen Physiologie.” Pro- toplasma, which is published in Berlin, is edited by Drs. Spek and Weber of the University of Graz with the collaboration of Drs. Chambers and Seifriz in this country. Dr. Spek will deliver his lecture in German on Friday concerning protoplasmic differentiation in egg cells during early development. He was asked especially to speak in German, since it was thought that Laboratory members, beside being interested in the subject itself, would also appre- ciate the opportunity of hearing scientific Ger- man. Dr. Spek plans to sail for Germany from New York on the S. S. General Steuben on August 28. eae CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the hole turns to run from Buzzards Bay to Vineyard Sound: . . M. P. M. August 5 :20- 4:31 August 6 2 5625 August 7 59) Gr August 8 46 August 9 202 August 10 August 11 wm bd August 12 August 13 August 14 tS to tn 00 In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. Aucust 5, 1933 ] THE COLLECTING NET 187 PENZANCE PLAYERS PRESENT PLAY ITEMS OF INTEREST FOR SCHOLARSHIP FUND : civ j / F When the Third International Congress for The Penzance Players presented Shaw’s brilli- ant comedy, “You Never Can Tell” at the Marine iological Laboratory last Monday for the bene- fit of Tur Cottectinc Net Scholarship Fund. It was a gracious gesture on the part of these young people, and it was plain to observe that the large and friendly audience of summer residents, laboratory workers, and townspeople appreciated not only the high calibre of the production itself but also the co-operative and social spirit behind ihe The play was ably directed by Mrs. George A. 3aitsell, and the cast consisted of Vera Warbasse as Gloria Clandon, Thomas G. Ratcliffe as Mr. Valentine, Alfred Compton as the waiter, Peggy Clark as Mrs. Clandon, Faith Adams and Man- ton Copeland, Jr., as the twins Dolly and Phil, Eric Warbasse as the father, Mr. Crampton, Rob- ert Giddings as Mr. McComas, Frederick Cope- land as Mr. Bohun, Alice Cooper as the parlor- maid, and William Chambers as the second waiter. The production was staged by Alfred Compton, with Robert Chambers as stage manager, May- nard Riggs in charge of properties, and J. War- ren Sever in charge of lighting. Settings were constructed by J. Wister Meigs with the aid of Alice Gigger and J. Warren Sever. Charlotte litch was in charge of make-up. The business manager was Peggy Clark, who was assisted in her work by. Alice Gigger and Sebree Robbins. The executive committee of the Penzance Players group consists of three of the actors, Vera War- basse, Thomas Ratcliffe, and Alfred Compton. Selections by the Hawthorne String Quartette were heard between the acts. The action was of course laid in England and the set for the terrace of the Marine Hotel in Act II was especially pleasing to the eye. The plot concerns the uncon- ventional escapades of the offspring of a deter- mined feminist, Mrs. Clandon. Gloria, the eld- est, 1s at heart old-fashioned, and Shaw permits the love affair of Mr. Valentine and this daugh- ter to provide a happy ending for the play, but not before he has delivered himself of some typi- cally witty remarks on feminism, lawyers, love, etc. The Penzance Players entered into the spirit of the thing heartily, and a splendid time was had by them and by their delighted audience. Dr. Elmer J. Lund, professor of physiology at the University of Texas, is visiting for a few days in Woods Hole. Dr. Lund is at present in- vestigating the origin and function of bioelectric current. Experimental Cytology meets in Cambridge, Eng- land, from August 21 to 26, several investigators who have been working at Woods Hole during the earlier part of the summer will be present in Dr. Robert read a paper on “Some features of cell permea- bility in relation to kidney function,’ Dr. L. Michaelis, a paper on “The reduction intensity of the living cell,’ Dr. S. C. Brooks, one on “The relation between ions and potential differences across plasma membranes,” and Dr. C. C. Speidel will show his moving pictures of nerve growth and repair. an official capacity. Chambers will Dr. Clarence E. McClung, professor of zoology and director of the zoological laboratory at the University of Pennsylvania, left Philadelphia with Mrs. McClung on June 19 for Tokyo, Japan. He will spenr the next year there as professor of zoology at Keio University, under a grant from the Rockefeller Foundation. Although Tokyo is their ultimate destination, the McClungs will not arrive there until the be- ginning of the fall term, in September. On their way they visited relatives in Kansas and Wash- ington, and are now spending the rest of the sum- mer vacation in Hawaii. Miss Irene Corey, Dr. McClung’s secretary, joined them when they sailed from Seattle in the middle of July, and will also be in Tokyo this year. The grant under which Dr. McClung is work- ing has provided four previous professorships at Keio University. The other zoologists who have held the position are Dr. Pearce, of Duke Univer- sity, Dr. Tennent, of Bryn Mawr College, Dr. Jennings, of the Johns Hopkins University, and Dr. Curtis, of the University of Missouri. Dr. D. H. Wenrich, professor of zoology at the University of Pennsylvania, is spending the sum- mer with his family in Kansas. During his visit he has been working at the University of Kansas, collecting and studying parasitic protozoa. Dr. Arthur H. Compton, professor of physics at the University of Chicago, recently visited Woods Hole. While he was here he was con- cerned with the fate of the attempted balloon as- cension in Chicago since it contained some of his cosmic ray recording apparatus. Dr. Compton has been teaching in the summer school at Colum- bia University, and his trip to Woods Hole was motivated by an interest in the group of scientific workers, as well as by a desire to visit his friends here. 188 THE COLLECTING NET [ Vor. VIII. No. 66 Supplementary Directory for the Marine Biological Laboratory INVESTIGATORS Albaum, H. G. fel. biol. Brooklyn. OM 41. Dr 1. Bacq, Z. M. Nat. Res. Fel. Liege Br 114. D 317. Baron, H. asst. instr. biol. New York. Br 328. Dr. Beams, H. W. asst. prof. zool. Iowa. Br 7. Dr 1. Blinks, L. R. assoc. gen. phys. Rockefeller Inst. Br 209. D 203. Bradley, H. C. prof. phys. chem. Wisconsin. Br 122a. (Juniper Point). Carpenter, R. L, assoc. anat. Columbia. Br 217. A 203. Chen, T. T. instr. zool. Pennsylvania. Br 217. Elliot, Center. Cole, E. C. prof. biol. Williams. OM 28. D 315 B. Cole, Margaret G. assoc. zool. California. OM 28. D 315 b. Cowles, R. P. prof. zool. Hopkins. Br 329. D 215, Crane-Lillie, Margaret. investig. neuropath. Harvard. Br 227. Lillie, Gardiner. de Renyi, G. S. assoc. prof. anat. Pennsylvania. Br 118. D 112 B. Diller, W. F. instr. zool. Dartmouth. Br 217D. Lewis, Quisset. Gates, G. E. prof. biol. Judson (Burma) L. 26. Grin- nell, Bar Neck. Grand, C. G. res. asst. biol. McLeish, Millfield. Hadley, C. E. assoc. prof. biol. N. J. State Teachers Col. OM 32. D 202. New York. Br 328. Hall, E. K. instr. anat. Louisville Med. OM 31. D 209. Hamburger, V. Rockefeller fel. Chicago. Br 228. D 310. Haywood, Charlotte. assoc. prof. phys. Mount Holyoke. Br 222. A 206. Hill, S. E. asst. gen. phys. Rockefeller Inst. Br 209a. Veeder, West. Jasper, H. H. Nat. Res. fel. psy. Sorbonne (Paris) Br 110. Robinson, Quisset. Jennings, H. 8. prof. zool. Hopkins. Br 336. Whit- man. Kagan, B. M. Washington & Jefferson. OM 41. K 5. Katz, J. B. St. Andrews (Scotland) OM Base. Avery, Main. Knower, H. McE. res. assoc. Yale. Buzzards Bay. Knowlton, F. P. prof. phys. Syracuse Med. Br 226. Gardiner. Larrabee, M. G. grad. biophysics. Pennsylvania, Johnson Foundation. Br 311. Nickerson, Fal- mouth. Lawlor, Sister Anna C. instr. biol. Saint Elizabeth (New Jersey). 217. Goffin, Millfield. Lundstrom, Helen M. res. asst. phys. Pennsylvania. Br 233. W. E. Menkin, Miriam F. res. fel. path. Harvard Med. Br 108. Lahey, Millfield. Menkin, Valy. instr. path. Harvard Med. Br 108. Lahey, Millfield. Miller, J. A. grad. asst. zool. Chicago. Br 111. Mc- Leisch, Millfield. Moore, R. P. grad. West Virginia State, proto. K 14. Morgulis, S. prof. biochem. Nebraska Med. Br 122 D. White, Church. Moser, F. asst. instr. biol. Center. Pond, S. E. tech. mgr. M. B. L. Br 216 (Falmouth) Reznikoff, Paul asst. prof. med. Cornell. Br 123. Mc- Kenzie, Pleasant. Schotte, O. E. Sterling res. fel. Yale. Br 323. Avery, Main. Temple. OM 1. Elliot Scott, Sister Florence M. asst. prof. zool. Seton Hill. Goffin, Millfield. Smith, C. E. Pennsylvania Med. Br 118. Molstead, Depot. Smith, D. C. L 25, Lewis, Buzzards Bay. Snell, G. D. Nat. Res. Fel. biol. Texas. Br 315. Kit- tila, Bar Neck. Stein, M. H. Cornell Med. Br 122c. Dr 7. Stunkard, H. W. prof. biol. New York. Br A 301. Vicari, E. M. assoc. anat. Cornell Med. Br 317. A 207. Whedon, A. D. prof. zool. North Dakota State Col. OM 38. A 201. Wichterman, R. grad. zool. Pennsylvania. Rock 6. Elliot, Center. Wing, L. T. Harvard. 108. Gifford, Depot. STUDENTS in Invertebrate Zoology Bartholomew, Olive F. Radcliffe. K 10. Blades, Helen N. Michigan. Cowey, School. Bladd, Amy E. asst. prof. zool. Grinnell. H 3. Bosworth, M. W. grad. phys. Wesleyan. K 7. Bowman, Sarah B. asst. biol. Agnes Scott. H 7. Brooks, Virginia C. Wilson (Pennsylvania) H 4. Buck, J. B. asst. zool. Hopkins. Robinson, Quisset. Buell, Katherine M. Oberlin. Hilton, Water. Carmack, T. asst. zool. Wabash College. Ka 2. Clark, Frances J. Rochester. H 9. Coilings, W. D. asst. zool. DePauw. Hilton, Millfield. Cowles, Janet M. Hopkins. D 215. Denny, Martha. Radcliffe. Kittila, Bar Neck. Di Paola, Rose M. Hunter. K 10. Field, Mary F. Grinnell, Bar Neck. Gaw, H. Yale. Dr 7. Giddings, W. P. Amherst. (Quisset). Henderson, A. R. asst. zool. Yale. Ka 22. Horton, R. G. Williams. A 106. Hunter, F. R. grad. genetics Calif. Inst. Tech. D 107. Jones, L. M. asst. zool. De Pauw. Hilton, Millfield. Jones, R. W. assoc. prof. Central State. Okla. Kelly, Florence C. instr. bact. Simmons. H 3. Lagler, K. F. Rochester. Dr 5. Livingston, Mary C. American Cowey, School. Mast, Louise R. grad. zool. Hopkins. Minot. Metcalf, I. S. H. Oberlin. High. Miller, T. R. Hamilton. White, Millfield. Nichols, R. J. grad. zool. Illinois. Glendon. Odell, F. A. asst. biol. Osborn Zool. Lab. Ka 22. Padolsby, Sophia. Goucher. H 6. Painter, B. T. instr. biol. William and Mary. D 217. Parker, Rachel W. Goucher. H 7. Rogers, P. V. instr. biol. Hamilton. (Falmouth). Rohm, Pauline B. Oberlin. Hilton, Water. Shaw, Ruth K. asst. zool. Mount Holyoke. W B. Smith, S. D. Wabash. Ka 2. Spiegel, J. P. Dartmouth. Thompson, Water. Starrett, W. C. Illinois. Backus, Glendon. Stewart, J. T. instr. biol. Virginia. Ka 1. Stone, Faith. grad. zool. H 3. Stuart, M. S. Pennsylvania Col. for Women. W A. Taylor, H. C. grad. biol. Wesleyan. K 7. Todd, R. E. Moses Brown School. Wilde. Trezise, W. J. asst. zool. Hopkins. Robinson, Quisset. Van Deventer, W. C. grad. zool. Illinois. Backus, Glendon. Wharton, Marguerite. N. J. State Teachers. H 2. Whittinghill, M. instr. zool. Dartmouth. Clough, Millfield. Williams, Inez W. grad. entomology. Mass State. Williams, Marguerite. grad. zool. Iowa. W B. Wing. L. T. Harvard. Gifford, Millfield. Woodside, G. L. teach. fel. zool. Harvard. K 7. Vol. VIII. No. 7 SATURDAY, AUGUST 12, 1933 Annual Subscription, $2.00 Single Copies, 25 Cents. ORGANIZERS AND INHERENT POTEN- CIES IN THE EMBRYONIC DEVELOP- MENT OF AMPHIBIANS Dr. Oscar SCHOTTE Sterling Fellow in Zoology, Yale University Animal embryology has for its aim the de- scription of all the changes and transformations which the egg undergoes until it reaches the definite form and organization of its species. One division of this science, the morphology of develop- ment, has the task of describ- ing how from the initial sys- tem of a given complexity of structure, the egg, a new or- ganism arises, provided with regions and organs fund- amentally different from the primitive structure of the egg. This descriptive embryology has reached an amazing stage of perfection. The human mind, however, cannot be satisfied by the mere description of a phen- omena. The facts of develop- ment are too striking; their astonishing diversity brings up too many questions not to demand an explana- tion of how development occurs, and what are the reasons lying behind this amazing succession Organizers and Inherent Potencies WM. BW. Calendar TUESDAY, AUG. 15, 8:00 P.M. Seminar: Dr. A. H. Palmer: “The isolation of a crystalline globulin from the albumin fraction of cow’s milk.” Dr. Paul Reznikoff: ‘Studies in iron metabolism in humans.” Dr. Marie Krogh: “‘The hormonal connection between the pituitary and the thyroid.” Dr. F. E. Chidester: ‘Anterior pi- tuitary like hormone effects.” FRIDAY, AUG. 18, 8:00 P. M. Lecture: Dr. John H. Northrop: “Evidence of the protein nature of pepsin and trypsin.” TABLE OF CONTENTS in the THE EFFECT OF FAT SOLVENTS UPON THE FIXATION OF MITOCHONDRIA Dr. CoNWAY ZIRKLE Associate Professor of Botany, Uniuersity of Pennsylvania There are many uncontrolled variables in the usual cytological fixation. These rarely affect the preservation of chromatin, however, as the methods in general use have been derived empirically and have been carefully selected for the relia- bility with which they preserve the nuclear components. On the other hand, the cytoplasm and its inclusions have been relatively neglected, and it is still customary to the cytoplasm by a fixation image where its essential structure is destroyed and the inclusions, particularly the mitochondria, dissolved. A number of biological problems await dependable methods of fixing mitochondria, for at present the absence of mito- chondria in a cytological prep- aration is no evidence that represent they were not in the original specimen. ‘he presence in the fixing fluid of such fat solvents as the aliphatic (Continued on Page 202) Electrical Behavior of Large Plant Cells, Embryonic Development of Amphibians, \Wo ds Wo ORWeH@WinoHencoe copsoegoUOboUD 213 Dye, (OR CHS SOAC UWS CAE oS ocan ao nol geen oD 193 Meaning and Calibration of the pH Scale, Effect of Fat Solvents Upon the Fixation of IDR Ake, IMEC INES Ss bee dows oo ba omo Gb OOOn 219 Mitochrondria, Dr. Conway Zirkle......... 193 Irradiation of Biological Suspensions by Motion Pictures Showing Some Varieties of Nerve Irritation, Dr. Carl Caskey Speidel. .201 The Biological Laboratory: Influence of Salts on Electric Charge of Surfaces in Liquids, Harold A. Abramson and) Hans) Muller’ o2° - 0 Theory of Ionic Adsorption, Hans Miiller. ..208 Monochromatic Light, Dr. B .M. Duggar, Dr, Alexander Hollaender:. 0%... 2a. eee a a 224 Regional Differences in the Organization Cen- Items of Interest ter of the Amphibian Embryo, Dr. Edmund egies bb ave aaa ae Editorial Page ... { Vor. VIII. No. 67 3, (ClO) THI LLECTING NET ‘sdiog ary Auiry sazeyg peziuQ 3243 jo azaydei3o0j04d & Aq LAN DNILOATIOO AHL 10F 6Z6T ul ueHe, sem ydeisojoyd styL ATIOH SGOOM JO MAIA TVIYAV NV ‘punoiga10J 94} Ul JaSSouUIeUON JO }Iq B Zulmoys Aueust 12, 1933 } THE COLLEGTI NG N = i 195 of transformations which are so characteristic for all embryonic happenings. From the desire to un- derstand the mechanism of this phenomena and from the clash of opinions in the interpretation of facts of development, is born a new division of embryology, the physiology of development. Some problems dealing with the physiology of develop- ment of the Amphibian egg, exclusively, will be the object of our study this evening. Ii one uses the methods of vital staining to study the fate of different parts of a living Amphibian egg, one can observe during the later development how a stained spot moves, changes its form and travels often quite far from its ini- tial place to arrive eventually at the region in which it will stay to achieve its final evolution. By studying these movements systematically one can succeed in establishing a kind of a_geograph- ical map of the different parts of the egg which will build up the future animal. It was the merit of W. Vogt to establish thus the complete pat- tern of organ rudiments for both the Urodele and the Anuran eggs. This “map” is perfectly re- liable and allows us to make transplantations on definite parts of the young embryo, long before we can recognize on it the slightest trace of its future form or its future organs. The fact that we know perfectly that certain parts are going to become brain or eye or mouth region indicates that there is a mosaic in the egg, only defi- nite groups of cells being destined to become these organs. Another experiment of \W. Vogt seems to con- firm this mosaic tendency in the Amphibian egg. In this experiment Vogt places the egg in a dish in which, by a partition, a notable differeace of temperature is realised. The egg is placed directly under the partition so that one half of it is ex- posed to a temperature of 8° C and the other half to 15°. He could observe that the half of the egg exposed to a higher temperature had al- ready developed a spinal cord and the rudiment of a brain, while the half exposed to colder water was still in the blastula stage. This out- come of the experiment indicated that the parts of the egg can realise their common aims in per- fect independence of each other. Since the differ- ent regions of one half of the egg are here shown to be able to reach their definite stage without the interaction of the other half, this independence in development indicates a pronounced tendency to realise the pattern of the egg in a “mosaic” manner. The method of local injuries is another means of investigation, apparently confirming the mosaic interpretation of development for the Amphibian egg. Indeed, Brachet in a large series of experiments performed on Anuran eggs, and Suzuki on Urodele eggs, could show that each time a part of the embyro was destroyed, (be- sides some exceptions which could be explained ) the larva of the injured egg will show later a de- ficiency of structure corresponding to the area of presumptive organ primordiums which was de- stroyed. But the observation of this “embryonic segre- gation” (F. R. Lillie) by both Vogt’s methods or by following the fate of the embryo through the methods of local injuries, gives us only a “sug- gestive criterion,” as Professor F. Lillie expresses it, of the happenings during the development. What lies behind this segregation remains hidden from us. A second series of investigations representing a further attempt to give us some other informa- tion about facts of development, consists in sub- mitting parts of the egg to the method of inter- plantation or to the method of tissue culture. In the first place, pieces of presumptive ectoderm, entoderm or mesoderm are removed from the do- nor embryo and explanted into ectodermal vesicles (Bautzmann), into the orbital cavity of an elder larva (Durken, Kusche) or into the general cavity of larvae (Holtfreter). In all these cases, the presumptive fate of the explanted organ primor- diums is changed in the most bewildering manner : ectodermal formations giving birth even to chorda differentiations, entoderm to mesoderm, ete. It is not necessary to insist upon these forma- tions which Bautzmann called “bedeutungsfremde Selbstdifferenzierungen”. They are not “Selbst- dif ferenzierungen’”’—independent differentiations —because they are under a constant action of un- controllable factors, and so they lose their signifi- cance, The conditions of these experiments do not answer at all to the definitions of a “radical crit- erion,’ such as F, R. Lillie gave it. These latter conditions are fulfilled in a second series of explanations performed also by Holtfre- ter, in which he cultivated parts of the blastula or gastrula with known presumptive fate, in vitro, in a salt solution. There he obtained very differ- ent results:—ectoderm produces in vitro only ectodermal formations, presumptive’ medullary plate ectodermal formations and nervous tissue. Presumptive mesoderm in tissue culture will form chorda, connective tissue and muscles, but will never produce nervous system. We see now that from the one side we disting- uish in the Amphibian egg a pre-established pat- tern of organ primordiums, that this pattern can- not be destroyed without giving birth to deficient larvae, but on the other side we see already that the elements of this pattern of organ primor- diums, if submitted to different conditions out- side of the egg, manifest potencies which are dif- 196 DHE COLLECTING NE { Vor. VIII. No. 67 ferent from what their presumptive fate indi- cates. Up to this point, I have avoided bringing up any question of causality in this embryonic de- velopment. Yet, the physiologically minded em- bryologist must ask: What is the nature of fac- tors which in some cases make the egg develop like the unrolling of a pre-established and regu- lated-in-advance mechanism, while in other in- stances the same parts of the egg develop into very different organs from those they are sup- posed to, and in some further cases, they may go absolutely wild? Here a masterful piece of research, inaugurat- ed by Spemann, and continued in constant collab- oration with him by his followers, seems to have given, at least for the Amphibian egg, a very clear answer. Spemann invented in many years of patient ef- fort a remarkably simple method of operating on eggs of the European newt, Triton. By using in the beginning eggs of the same species, Triton taeniatus, but with marked shades of coloration, so as to make sure of the eventual fate of the transplanted piece, Spemann explored the surface of the young gastrula in many transplantation ex- periments. By transplanting, for instance, from one gastrula, presumptive abdominal skin in the region of the future medullary plate of another gastrula, the transformation of this presumptive epidermis into spinal cord, or brain, or an eye, has been observed by Spemann. On the other hand, presumptive brain could be transformed into simple belly skin, if transplanted into the ab- dominal region. There is one region, however, on the gastrula which behaves very differently from that de- scribed above—this is the region of the upper or dorsal lip of the blastopore. When Spemann grafted a piece of this region, he observed that it did not become assimilated to the new surround- ings, but that it behaved exactly as did the dorsal lip in the normal development; that is, it became tucked in beneath the ectoderm, underlying the latter, and induced the ventral ectoderm and the mesoderm to form a new medullary plate to- gether with the axial organs of a new embryo. The dorsal lip of the blastopore organizes by its direct action a secondary embryo, for which rea- son it is called an “organizer.” The action of the organizer seems to proceed with an elementary force—not only do we see a medullary plate arising at the expense of an ab- dominal skin, but eyes, ears, noses, a whole head with mouth and gills are formed; and if the em- bryo can be kept long enough, a secondary gut, even a functional heart will be observed. More than that, it is not necessary that one implant toa Triton gastrula, the organizer of a Triton; one can use for the same purpose the organizer of a frog or of a toad, or vice-versa. No wonder then that Spemann when he first saw these inductions thought that he had discov- ered the organizing principle of development it- self. Indeed, when we consider that in recent pa- pers, Fankhauser working on the Urodele egg and Pasteels, on the Anuran egg have been able to disclose the localization of the organizer in the unsegmented egg as early as a few minutes after fertilization, and, further, that these authors could actually prove that even in such early stages the presence of the organizer is an unavoidable condi- tion for the development of the totality of organs, then we will understand why the word “organ- izer’’ was for us wrapped up in a veil of mystery, which only recently has been dispelled by a new and sensational discovery, coming again from Freiburg, If I may recapitulate the previous results, we see that the various regions of the Amphibian gastrula manifest, when left alone to perform their normal development, a tendency to form from the given mass of cells always the same or- gan (Vogt); that these same regions, when de- tached from the embryo, develop : (a) according to the new environment if they are transplanted to another embryo of the same species (Spemann) ; (b) if submitted to a vigorous control of tis- sue culture (Holtfreter) they manifest larger but yet restricted tendencies ; (c) they will show up wild tendencies of dif- ferentiation in every possible direction if trans- planted into some cavities of older larvae (Dtr- ken, Kusche, Bautzmann and Holtfreter ) ; and lastly, (d) that these same parts of the em- bryo, when submitted to the vigorous action of the organizer are apparently compelled to develop into harmonious formations representing the essential features of a complete embryo (Spemann). Thus, the question arises, what are the factors which fix and limit the possibilities of segregation in the normal development and in some experi- mental conditions ; and also, what is the nature and the mode of action of these factors producing such an amazing reversal of potencies, as shown in Spemann’s experiments of induction. In other words, we shall have a perfect under- standing of the mechanism of development when we know the role of the inherent potencies of the egg, together with the nature and the role of the organizers. It was not my intention to speak to you about the intimate nature of the organizers, but two preliminary notes coming from Freiburg which Aueust 12, 1933 ] THE COLLECTING NET 197 reached us only some days ago demand special consideration, In several laboratories, an intensive study was carried on to establish the intimate nature of the organizer. Indeed, Spemann could obtain induc- tions with frozen and dried organizers; further- more Holtfreter could show that even non-organ- izing parts of the embryo can acquire this prop- erty after being killed by heat, for instance. A piece of presumptive ectoderm overlapping a dead piece, even of entoderm, showed obvious induc- tions into a medullary plate. These and some other facts made it likely that a substance of chemical nature was involved in the action of the organ- izer. The association with a skilled chemist of Freiburg brought the desired results and after a vast series of experiments, it was announced last month that the acting agent which produces in- ductions is glycogen. Indeed, glycogen dissolved into gelatine and introduced into the blastocoel of a gastrula produces the same medullary plate as does the upper blastoporal lip. Fischer and Weh- meier attribute this action of the glycogen to a glycolyse taking place on the points of contact of the overlapping ectoderm with this substance. If thus, at least a first, decisive step is made toward the knowledge of the intimate nature of the organizer, a series of problems dealing with the manner in which this “organizing sub- stance” acts still remains open. In a recent paper, Spemann could show that a part of the upper blastoporal lip which normally would become the underlying mes-entoderm of the head, so-called head organizer, can induce in the region of the trunk brain with eyes and ears. On the other hand, however, trunk organizer brought into the region of the head induces there also a head formation. Thus it becomes plain by these experiments that if the organizer can manifest some specificity in its action, an un- doubtful counteraction of the host cannot be denied. If head organizer can induce a head in the trunk region, whereas the trunk organizer in- duces in the trunk region only a spinal cord, there seems to be an indication that the organizer can actually direct the fate of the regions upon which it acts. Yet, in all these experiments, a clear an- swer about the action of organizers is always dis- turbed by the manifestation of factors due to the action of the inherent potencies of the host. In 1921 Spemann attempted an experiment which was intended to solve this question. In this heteroplastic experiment, he changed tissues he- tween two species of Tyiton, the incriminated species being 7. faeniatus acting as host and T. cristatus acting as donor. The result of trans- plantation of presumptive abdominal skin of cristatus into the brain region of taemiatus shows that while the belly skin of the donor is actually transformed into brain in the foreign host, it keeps its own specific characteristics. Belly skin of cristatus is transformed into brain, yet not into faematus brain, but into a typical cristatus brain. This shows that the organizing action of the host on the transplant is of a restricted na- ture, insofar as this action consists merely in transforming the foreign transplant into an organ which is fixed by its surroundings. But how this organ is constructed, its size, general shape and the disposition of its cells, is a task left to the donors tissues. While of extreme importance, this experiment could not give a very definite idea about the factors acting in the phenomena of in- duction. Both host and donor possessing in this case the same organ—brain for instance—they must possess also the same organizers to realize these common organs. Thus, only the different morphological tendencies of host and donor or- gans were revealed in the heteroplastic transplan- tation experiment. In order to discriminate be- tween the action, in the process of induction, of the organizer versus inherent potencies, it was necessary to find an experiment in which trans- plantations could be performed between embryos which do not possess common organs or in which these common organs are fundamentally differ- ent. These conditions are realized in transplan- tation, in the head region, between Anuran and Urodele embryos. Indeed, there are three organs of Anuran em- bryos which are not common to the Urodele larvae and which are all situated in the upper and lower head region. These are suckers, the opercu- lum and the mouth armament of a type not found in the Urodele mouth. As early as 1921, Spemann had foreseen some of the possibilities of the xenoplastic experiment ; at that time, however, the preliminary conditions for carrying out this investigation were not yet realized—there were some experimental and some theoretical difficulties which had to be overcome first. Since I was fortunate enough to work out a method of transplantation on young stages of Anuran eggs, and since I could show that the same conditions of only labile determination as previously shown on Urodele embryos were also realized in this type of egg, Professor Spemann suggested that I perform transplantations between frogs and salamanders. In these operations the donors were, always in the gastrula stage, while the host varied from the stage of young gastrula to late neurula. In a first series of experiments I performed xenoplastic inductions by transplanting the upper blastoporal lip from either Urodele or Anuran donor into the opposite host. Secondary embryos with a complete set of organs, as in a homeo: 198 THE COLLECTING NET [ Vor. VIII. No. 67 plastic experiment, could be obtained. As an inter- esting feature, the induction of suckers in the ven- tral region of a toad by a Urodele organizer could be ateeneil Furthermore, in both Rane and Urodele embryos, presumptive abdominal ecto- derm from gastrulae was observed to be trans- formed, if transplanted into the given region, into structures of corresponding nature, as xenoplas- tically induced lenses, ear vesicles, nasal placodes and pituitary glands. It was necessary to perform these experiments in order to show that tissues of Urodele and Anuran embryos can develop together well enough to succeed in forming even such a com- pound organ as an eye. Only now were the pre- liminary conditions for the second and most im- portant part of this research realized. In the lower ventral region of the head of young Anuran embryos, ectodermic cells become partly glandular and secrete a mucus by means of which larvae adhere to. various objects. The same transformation into a sucker is observed, always in the same region of the lower face, if one transplants presumptive abdominal skin of an Anuran gastrula into this region of a Triton or Amblystoma embryo, where evidently no such suckers can be found. These induced formations are not only histologically, morphologically and functionally typical suckers of Anuran tadpoles, but they reveal always the specific, often very striking features characteristic of the used donor. Indeed, the sucker induced in the Urodele at the expense of presumptive abdominal epidermis of Rana esculenta will be very different from the sucker obtained on the same host from Bombin- ator pachypus. The same observations have been made for the American species, a sucker induced on Amblystoma from Hyla crucifer tissues being very different from a sucker obtained from pre- sumptive abdominal ectoderm of Rana sylvatica. These induced suckers function perfectly, since the secretion of an abundant mucus has been ob- served in many instances. If the transplant from the same region of the frog egg happens to cover the upper side of the head of the Urodele, one sees about 6-7days later the formation of a cutaneous pocket at the expense of the transplanted tissue. As the form and position of this pocket is always the same and corres- ponds exactly to the position of an Anuran oper- culum, this formation is to be diagnosed as an induced Anuran operculum in a region of the Urodele head in which no such thing is expected and where it has no function. The histological study of such cases shows that this formation is situated in a region corresponding to the exact place in which the internal gills of the tadpole were to be covered, If now the presumptive abdominal skin is made to cover the mouth region of Amblystoma, very striking transformations of the ectoderm will be observed. In a first case, the upper and lower jaw from the right side of the mouth were covered by the transplant, and we observed consecutively the formation of two horny jaws and lips covered with numerous. horny teeth. In another case, I ob- served the formation of a real Anuran beak showing the characteristic conical proboscis with rows bearing horny teeth ,and on the free edge of the lips, especially in the corner, typical Anuran fleshy papillae, probably tactile in function, all in striking contrast to the dAmblystoma mouth. In another case only the upper jaw was covered with Anuran tissues and yet two horny jaws were found on the upper mandible of the host. Transverse sections through this animal showed on the lower jaw the formation of Urodele teeth, while on the upper jaw we noticed horny teeth, formed each time by the modification of a single epithelial cell in the shape of a hollow cone. It is, as in a typical Anuran jaw, a’ strong curved band of cornified epithelium. The cutting edge of such a jaw is formed by a row of horny teeth, very similar to those of the lips, but placed so closely side by side as to form a continuous blade. All the necessary precautions having been taken to avoid the transplantation of presumptive suck- er, operculum or mouth region of Anura to Uro- dele, the demonstration I have given you shows with sufficient evidence that any Urodele host, in the head region of which one transplants pre- sumptive abdominal skin of any Anuran gastrula, is capable of inducing there three organs which are typical for Anuran larvae, but which are lack- ing in the Urodele. How shall we explain these somewhat astonishing facts ? Al priori, it was not absolutely impossible to ad- mit the presence, in the Urodele organism, of a specific organizer for these foreign organs. But if one considers the matter more closely, this pre- sumption becomes highly improbable. If one could admit, for instance, that the balancer region could at the same time be bearer of the organizing fac- tors for suckers, then how shall we explain the case in which two balancers were observed togeth- er with two suckers, as shown in one of my ex- periments ? Furthermore, if the organizers for balancers and for suckers were common, then the trans- plantation of undetermined tissue of Anuran gas- trulae on the balancer, and of gastrula ectoderm of Urodele on suckers, must have been followed by the induction of the homologous organ. This is not what occurs if these conditions are realized. Indeed, in one case of transplantation of Triton gastrula ectoderm on a Bombinator, it was seen that half of the left sucker was covered by Uro- Aveust 12, 1933 | s _ THE COLLECTING NET 199 dele tissue. And yet, the transverse section through this region shows no sign of differentia- tion of the Urodele tissue into a balancer. The inverse condition is realized in the case of an Urodele having half of the balancer covered by Anuran tissue; here also the presumptive ab- dominal skin of the gastrula of the donor did not differentiate in anything else as simple skin and and not in suckers It would be very difficult also to admit the possibility of an operculum organizer in the upper head region of Amblystoma, while the gills are produced far away from this region and are never covered by it. And how can we sila that the Urodele, hay- ing already realized the mouth armament of his own, has still some organizers in reserve to form on the edge of its jaw some supplementary horny teeth and jaws, as is visible on one of the demon- strated cases ? Thus, we must exclude the possibility of or- ganizers for these foreign organs. The explanation of these strange inductions is already given by the analogy with Spemann’s case in 1921: just as we saw that the tissues of 7. cristatus are unable to form in the taentatus Drain region a faeniatus brain, but re- main characteristic for a brain of the donor’s species, so we can admit by analogy the assimilation of a piece of abdominal ectoderm into a face region in the Urodele. But, it re- mains Anuran material, and therefore the tissues have to develop organs belonging to the same re- gion of an Anuran larya—these are mouth arma- ment and suckers for the lower and middle face region and operculums for the upper and lateral head region. Spemann names this kind of induc- tion a “komplexer Situationsreiz.” That is very easy to say in German, but difficult to translate into English, though we could try to say “a com- plex stimulus of localization.” While this explanation seems to be rather ver- bal, it gives a satisfactory account of all the facts observed until now, and of some new experiments I performed this year and of which some aspects are still in course of investigation. I was very fortunate to find in this country two kinds of eggs which present such exceptional dif- ferences of size, that some very interesting prob- lems of development could be carried out on ac- count of this size difference. Indeed, the eggs of the common peeper, or tree-frog, Hyla crucifer, are quite exactly three times as small in diameter as the eggs of the common salamander, Ambly- stoma punctatum. In these experiments, which I will report very briefly, I used Amblystoma as host, and young gastrulae of Hyla as donor. To perform this operation, that is, in order to cover with the transplant approximately the face region of Amblystoma, it was necessary to re- move nearly the whole presumptive ectoderm, in- cluding the presumptive medullary plate. The re- sult of this operation is surprising; one ob- serves the formation of suckers which are typical Hyla suckers in their morphological characteristics and in their general shape, but about three times larger than their normal size on Hyla! The number of cells at the expense of which these suckers are formed is also about three times greater than is normally observed on Hyla embryos. Similar results were obtained also with other organs, like lenses, nasal placodes, ear vesicles and mouth organs. At first sight, this observation is contradictory to Harrison’s classical experiments on limb trans- plantation between A. punctatum and A. tigri- num, You recall that if Harrison transplants the limb bud from the big tigrinum on the small punc- tatum he obtains the formation of a huge limb on the small host. The reciprocal experiment gives corresponding results. There is a fundamental difference, however, between these two series of results: Harrison’s results are due to a phenom- ena of differential growth, while in my exper- iment, we observe a phenomena of induction of a piece of presumptive abdominal ectoderm into a face region on a large animal. Whether this donor comes from a large or from a small animal does not matter. What is important is that I had to remove nearly the whole body ectoderm in order to cover approximately the huge area of the Amblystoma face region. Under the organizing action of the host this large picce is transformed into a perfectly harmoniously adapted face region on the new host. On this face region it develops new organs, suckers and mouth armament, char- acteristic of the donor, but corresponding to the size of the host and not to the size of the donor. I have said that the aim of this series of ex- periments was to distinguish between the part of the inherent potencies and of the organizers in the Amphibian development. Was this expecta- tion justified? You remember that in order to distinguish between the acting factors of the or- ganizers and of the inherent potencies, we had to find conditions in which inductors and induced organs were different and that these conditions were realized in the xenoplastic experiment. The answer we obtained from this latter experiment was positive,: there is actually an induction of or- gans which are not present on the host acting as organizer, and yet the further investigation shows that there are no specific organizers present for these foreign organs. How then do the organizers act? We know now that these organizers do not pro- ceed by actions in detail and that the induction 200 THE COLLECTING NET [ Vor. VIII. No: 67 which occurs must be of a rather general nature such as a transformation of the traasplanted tis- sue into new regions as “upper”, “lower’ and “median” head region. In other words, the trans- planted tissues are transformed into new areas, but we shall never witness such a thing as, for example, a specific induction into one horny tooth, or one sucker cell. Once this transformation into a new region is realized, all the weight of the action relies upon the potencies of the reacting material and these potencies are limited by the stage of determina- tion already reached by the donor and by its hereditary constitution. Thus, we witness in each individual develop- ment a true creation of differentiation. Indeed, when we isolate parts of the gastrula, as Holt- freter did, by the methods of tissue culture, we bring out only the very slight inherent potencies of the egg which has already received some im- pulses of differentiation. In this case, the ex- planted parts manifest their potencies correspond- ing to the general pattern of organ rudiments. 3ut, by transplanting the same parts into the orbital cavity or into the general cavity of a larva, we submit these tissues to various and un- controllable factors—and so they are being in- duced to manifest their totally surprising and be- wildering tendencies of determination. If now we transplant parts cf the embryo into a definite region, this part is then submitted to very direct and precise actions of determination, thus explaining the complete rev ersal of the fate of the transplanted regions. There is evidently in the living embryo some- thing which gives to the parts of the egg an im- pulse to become a definite area and only then do the inherent potencies of the embryo become manifest and produce organs which belong to these regions. Induction seems chiefly to consist in an organization into regions. But there is a limit in this action of induction into new regions —sometimes the inertia of the reacting material is stronger than the inductor. This is shown by numerous cases I observed, in which the trans- plant was very large. As is visible on the demon- strated embryo, there is no trace of any differen- tiation into Anuran organs on the whole length of a very huge transplant, and yet it is visible how perfectly the Anuran skin was assimilated to the host. That a very large piece of presumptive ectoderm remains plain skin without manifesting the effects of induction into a new region is very significant. It shows that development is a very complicated result of an equilibrium between active forces which are the organizers and_ he- tween the inherent potencies of the reacting ma- terial, If we recapitulate the whole cycle of investiga- tions carried out by the means of homeoplastic, heteroplastic and xenoplastic transplantations, we will get a clearer idea about the respective actions of the organizers and of the inherent potencies in embryonic segregation and in induction. It is obvious that in the homeoplastic exper- iment performed by Spemann, the organizers and the inherent potencies are the same. It results from these experiments that the fate of organ primordiums is altered and will always corres- pond to the new environment. To find out what is the part of the organizer in this action of the environment, the hetero- plastic experiment was carried out by Spemann. One could legitimately presume that the strength of the organizer of one species might impose upon the donor tissue the morphological tenden- cies of its own species. This was proven not to be true. The result of the experiment shows indeed that the inherent potencies manifest their specific characteristics despite their submitting to the ac- tion of the foreign organizer. In the xenoplastic experiment, at last, for the first time we were able to separate the action of organizers from the action of the inherent poten- cies of the donor. Indeed, Anuran and Urodele larvae possess different organs in the head region and if there should exist organizers for these dif- ferent organs, their action would become manifest if brought into contact with the xenoplastic donor tissue. The outcome of this experiment shows that or- gans are induced at the expense of the transplant- ed tissue which are different from those the or- ganizers were supposed to form. Yet, closer ana- lysis shows that there are no specific organizers present for these organs. The final outcome of this experiment proves to be a restriction in the action of the organizers—these latter factors pro- ducing by their intervention merely a transforma- tion of the transplanted tissues from one region into another. It is the task left to the inherent po- tencies to form the necessary organs belonging to these regions independently of the nature of the host on which they are placed. There is one question left—and that is, how does the organizer cause the induction of new regions at the expense of material coming from another region? My experiments, however, are not appropriate to give an answer to this ques- tion, exactly as the very first Spemann transplan- tation experiment was incapable of doing so. We are coming back then to our starting point. In this eternal coming back of scientific prob- lems, we are, however, happy to know that the “dead” organizer will be of great help. Freed from its ties with living material, it will allow us to Aueust 12, 1933 ] THE COLLECTING NET 201 study more systematically and rationally the ef- fects of its action. We know the organizer is “dead’”—we can buy it in a drug store, and yet is it really dead? Spemann’s head and trunk organizer, and also the results of xenoplastic transplantations showing this strange induction into new regions cannot yet be explained by the mere presence of glycogen. Furthermore, would we really dare to assert, when a piece of transplant is too big and the host proves to be incapable of organizing the trans- plant into new regions, that there is not enough glycogen in the embryo to induce this mass of tissue? I believe, that like the legendary Phoenix, who each time he is burned, rises again from his ashes in new splendor, the dead organizer will still give us some hard problems to solve, in order to prove to us that the phenomena of embryonic segregation and of induction cannot yet be un- derstood by the sole action of one chemical sub- stance. (This article is based on a lecture presented at the Marine Biological Laboratory on August 2). MOTION PICTURES SHOWING SOME VARIETIES OF NERVE IRRITATION, AS SEEN IN LIVING FROG TADPOLES Dr. Cart CASKEY SPEIDEL Professor of Anatomy, University of Virgima Medical School The reactions of single internodal segments of myelinated nerve fibers in living frog tadpoles have been observed in minute detail following many varieties of experimental irritation of both acute and chronic types. Illustrative ciné-photo- micrographic records have been obtained. Below are specified the titles of some of the pictures : 1. Acute irritation and rapid metamorphosis of a myelin segment immediately after a hot water burn. .(A continuous picture showing mye- lin swelling, rippling, and ovoid formation. ) 2. Swelling and wrinkling of the myelin sheath following administration of a strong anes- thetic (chloretone). 3. The production of beaded myelin by alco- hol intoxication. 4. The swelling reaction shown by the same two myelin segments photographed immediately before and immediately after alcohol treatment. 5. Nerve irritation from low temperature (tadpole embedded in packed snow for several hours ). 6. Examples showing trophic (Wallerian) de- generation of a myelinated fiber after section. 7. Irritation and recovery of a myelinated fiber following adjacent wound infliction. 8. Traumatic irritation and recovery of a proximal stump myelin segment after nerve sec- tion. 9. Examples of myelin segments showing de- layed irritation, several days after exposure to x-rays. 10. Chronic irritation resulting from starva- tion. (Several examples of wrinkled and swollen nerves after six days’ total starvation. ) 11. Granular degeneration of nerve fibers and sheath cells associated with prolonged inanition (20 days’ starvation). 12. Effects on nerves of administration of thyroid gland extract. 13. Pressure irritation showing delicate con- necting strands between the axis cylinder and myelin sheath as these two structures undergo separation. 14. Temporary irritation and recovery of a myelin segment between two wounds, the nerve fiber remaining intact. 15. Polariscopic pictures showing the behav- ior of the anisotropic material of the myelin sheath during various stages of growth, irritation and degeneration. 16. Degeneration of sheath cells on normal nerves. 17. Leucocytes, chiefly macrophages, moving about in a degenerating mixed nerve. 18. A-macrophage leaving a blood capillary sprout (diapedesis ). 19. A macrophage containing ingested aniso- tropic granules moving about within a single de- generating myelin segment. (Pictures taken both with ordinary light and with polarized light. ) (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on Au- gust 1.) 202 THE COLLECTI NG NET [ Vor. VIII. No. 67 THE EFFECT OF FAT SOLVENTS UPON THE FIXATION OF MITOCHONDRIA (Continued from Page 193) acids, alcohol, ether, acetone and chloroform, prevents the fixation of all mitochondria irrespec- tive of the other constituents of the fixative, ex- cept in the case of certain complex mixtures which contain Og Oy. At present the chemical reactions involved in this type of fixation are not known. The fixing properties of several different groups of fat-soluble substances have never been investigated, the aliphatic aldehydes, for example, Formaldehyde is the only member of this group which has been used extensively. It preserves mitochondria as slender thread-like bodies, even when mixed with the fatty acids, provided it be given a head start so that it penetrates the tissue before the acids. Acetaldehyde, propionic alde- hyde and butyric aldehyde, on the contrary, not only do not fix mitochondria themselves but also prevent the fixation by such fluids as Miuller’s, which normally preserve them. Mixtures of pic- ric acid and any of these aldehydes fix mitochon- dria, although each substance by itself does not preserve them. The amines form a group of fat solvents, also, whose fixing properties have never been investi- gated. Those used in the present investigation are ethylene diamine, ethyl-, di-ethyl-, and _ tri- ethyl amine, di-methyl-, and tri-methyl amine, py- ridine and di-iso-amyl amine. These amines can- not be used alone as fixatives for they are so al- kaline that they distort the specimen to such an extent that it cannot be investigated critically. When added to a 1% solution of Hs Cr Oy until it reaches pH 5.0, the mixtures form bichromates which preserve mitochondria. In this they re- semble those inorganic salts of chromium previ- ously described (Zirkle 28, 33). Mitochondria are also preserved by the mixtures at pH 6.0 out here the other components of the ceil are badly distorted. When added to Ky Cre O; or Cu Cro Q;, these amines also improve the fixation of mi- tochondria. On the addition of fatty acids to the mixture, the images become acid (even at pH 5.0) and all mitochondria are destroyed. Unlike other fat solvents, the amines do not destroy The question naturally arises as to the fat solubility of the compounds The partition coefficients of these compounds between water and ether have not yet’ been determined. Certain of them, such as the compound formed by di-iso-amyl amine, however, are very soluble in most non-polar solvents. In addition, a propor- tion of the amines remains uncombined, even at pH 5.0. When the amines are given a head start by the specimen being placed in them from 1 to 5 minutes before it is put into the fixing mixture, the mitochondria are well preserved. At present, speculation as to the chemical com- position of mitochondria is hardly justified. It is necessary first to accumulate many more facts about their solubilities. mitochondria. formed by the amines plus chromic acid. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on Au- gust 1.) AuGusT 12, 1933 ] _THE COLLECTING NET 203 tie BIOLOGICAL LABORATORY COLD SPRING HARBOR THE INFLUENCE OF SALTS ON THE ELECTRIC CHARGE OF SURFACES IN LIQUIDS Harotp A, ABRAMSON AND Hans MULLER There have been a good many investigations of the electrokinetic potential, ¢, of flat surfaces (or large particles) in contact with liquids. In most cases ¢ is calculated from the theory of Helm- holtz-Smoluchowski"?, 4any So) — ay (1) D or 4arnE t= ———_ (la) DPR where the subscript, E, refers to calculations from electrophoretic mobility v; the subscript s refers to calculations from measurements of streaming potential, E; 7 = coefficient of viscosity; D = dielectric constant; P = pressure; R = specific resistance. All units are c. g. s. electrostatic, and properties of the liquid in the double layer are those of the solvent in bulk. In nearly every instance the inert surfaces have a value of ¢ which, in the case of electrolytes not reversing 7 5 3 i ~Log C of 1-1 Salt Fig. 1 H,0 the sign of charge, at first increases on addition of salt and which on subsequent addition of salt passes through a maximum (Fig. 1) then de- creasing in much the same way that the electric mobility of an ion decreases as the salt concen- tration increases. This decrease in £ on the addition of salt does not by any means indicate, as has been frequently assumed, that the particles are discharged. The density of net surface charge’) for large particles in contact with an electrolyte can be calculated for the case of an infinite plane surface from the Poisson equation: d?¢ dliexe D 4a p where ¢ is the potential at a distance x into a li- quid having a volume density of charge p, and the distribution of the charges in the liquid by the Boltzmann equation of ions of any valence z, of number per cm.*, n, (3) —z, pe + 22.¢e sana kT e)e é (i) == (Gh en ye where the subscripts 1 and 2 refer to cation and anion respectively, e = 4.77 X 10-! &. s, U., k = 1.37 * 10°18 ergs. per degree, and T is the absolute temperature. By multiplying equation (2) by e k T and substituting for p we obtain, letting pe ies) No 65 = NZ and ae ite at (4) dd? S82 Nze —21V + so W 2 _( ) (. ce +e ) (al 56 DkT The expression 87 Nze? eee Pee PY DkT where « is identical with the same expression in the Debye theory of electrolytes. Letting y = x x and irteg-ating once with respect to W, (5) dw \2 (ierecon= eve) Soha ( = e af e Speke dy 3 Fa 204 THE COLLECTING NET [ Vor. VIII. No. 67 dv for the concentration of ionized HzCO;). Note When - x = 0, ¥W = 0 and =. the regularity in all of the curves. In each dy instance regardless of the nature of the material o so that, (6) increases sharply and almost linearly at low con- 1 1 centrations rapidly changing the rate of charge, Combining equations (5) and (6), and substitut- ing N = 6.06 * 1073 molecules per gm. mole, ¢ = concentration in moles per liter and putting dw 4roe Any ssaDiceite where oa is the surface density of charge we obtain o as a function of the potential, ¢, of the plane surface, and of the concentrations c, with all the other teams constant or easily measureable, reaching a limiting value at about c = 0.01 M. The general shape of the curve is the same for all of the data on surfaces of this type known to the authors. It is evident that the descending part of the ¢ -c curve given in Fig. 1 instead of representing a discharge of the particles actually is accompanied by an mcreasing charge. It is unfortunate that no data are available in stronger uni-univalent salt solutions where o conceivably might decrease. The complicated course of the &c curve is due to the fact that € depends upon two variables, « and x, both of which depend upon c. We have here NDkT 1 C1(2) al ae 2000 x Ze nas Note that equation (7) is valid for ions of any valence and holds independent of the value of le kT which must be small in the Debye theory for par- ticles having small radii. In the case 2) = 22 equation (7) reduces to INDY ele AP? HO Ss —— (8) V 2000 x V ear The use of equation (8) for uni-univalent salts in aqueous solution is simple for it may be written when 31 = 25 = | =2« Je sinh (8a) B « = 17,650 and 8 = 0.025 volts at 18°, assum- ing the dielectric constant to be that of the solv- ent. Uni-univalent Salts There is much experimental work in the litera- ture which, for example, gives data for ¢ particu- larly on glass), quartz'*), paraffin oil'”), cellu- lose“, clay”), graphite'®’ and collodion™). By means of equation (8) the variation in o has been plotted from these data as a function of the added salt concentration in Figures 2-10, (Correcting fe =i 5 (7) kT e —1 = hellegel 71 done what is tantamount to assuming the validity of the theory of the diffuse double layer to obtain the dependence of « on c, permitting the calcula- tion of o (c). « and € presumably vary quite differently with c, As o increases 1/« decreases. When c¢ is small the increase in o is more im- portant, but as c increases the decrease in '/« be- comes more important flattening out the o -c curve’), It will have been noted by the reader that the form of all of the o -c curves bears a marked resemblance to the Langmuir adsorption isotherm. The smooth curves of Figures 3, 4 and 5 ete., have actually been plotted by an equation of the form XE 1+ BC which is for the simplest case of adsorption of type of particle, where oy f is the initial slope of the o -c curve and oy is equal to the limiting value of o within the concentration range. The agreement is very good indeed and indicates that selective adsorption of the negative ion takes place. The curves for the different alkali halogens and alkali earth halogens differ much less than the difference between the curves for potassium bromide, iodide and chloride. Especially large values of the charge are reached for the sulfides and ferro cyanides, The data indicate that for glass, graphite and col- (9) og — OL one Aueust 12, 1933 ] THE COLLECTING NET 205 5 —_‘([eaixanon] ¢ i Nagfe(Ch), ba. ee 1 0.00001 0.0001 0.0008 0.00! 0.01 Fig. 2a Fig. 3 GLASS _) CHLORIDES ©) Me Ba Ca CHLORIDES Ce Nisin mnKNONG hac, ri) 00s) 00,2 Fig. 2c Fig. 5 206 THE COLLECTING NET [ Vor. VIII. No. 67 COLLODION NaCt GRAPHITE O @) 0.002 = Fig. 6 é 400 * Acetate Propionate 200 Butyrate ea Canto Fig. 7 o Batts CHLORIDES 0.001 G o0001 Fig. 8 lodion the adsorption potential increases in the series : Claabiseals Li eNa Kee Riba Gsas- Mets, Batt, Carrs l=, (SOs> [Fe (EN )a]= and that in general for these surfaces the adsorp- tion potential of the positive ions are considera- bly smaller than those of the negative ions. The initial slope of the « (c) curves leads to values of the adsorption potentials of the same COLLODION O-Cone. NagFe(C), 4 2 SCALE . Na, 50, Ole C06 Fig. 9 GRAPHITE 0-Cone 5 © NayFe(CN), 4 scace NazoDy NaC SS Rens Care, Q CU TOOZ2T OOS DOE RAC OS Fig. 10 order of magnitude as found for the adsorption of gases. It is of interest to calculate the fraction of the area occupied by ions accounting for the limit- ing value of o. To take the case of glass, o, = 9000 FE. Ss. U., representing about 2 x 10" ions per cm*. If the area occupied by each ion is taken as 5 x 10°'§ cm?, the area occupied by the ions given by op is 0.01 em? or 1% of the surface. This is a rather higher value than that deduced by pre- vious investigators, but is compatible with the type of adsorption postulated by Langmuir’s theory. Aveust 12, 1933 ] THE COLLECTING NET 207 Polyvalent Ions In a future communication we shall give re- sults of calculations by means of equation (7) for the o -c curve for non-symmetrical cases of polyvalent ions not reversing the sign of charge. Using the Debye approximation for surfaces ge where is relatively small, we have ob- kT tained preliminary indications that the form of the curve is exactly the same as that obtained by the rigorous expression (equation 8) for uni-uni- valent salts. Some of the curves are given in Figs. 9 and 10 (collodion and graphite). Com- pare these curves with the o -c curves for the same substances using the full equation. There is obviously no difference in the shape of the curves. In previous communications the authors have shown that for certain inorganic solutions''®? and protein solutions!) the effect of salts on € could be explained by the effect of salt on x, with o taken as constant. The group of sub- stances here studied are more complicated in that both o and « depend more markedly on the salt. They all, nevertheless, show the remarkably con- stant shape of the o -c curve which can probably be taken as generally true for inert surfaces in so- lutions of salts not reversing the sign of charge. Summary It is proposed that an advance in the study of the constitution of the solid-liquid and liquid- liquid interfaces may be made by calculation of the surface density of electric charge o, in addi- tion to the analysis of the potential difference, €, between the movable phases. This calculation has been made for the negatively charged surfaces of graphite, glass, quartz, cellulose, “collodion” and paraffin oil, in the case of ions (valence 1:1; 1:2; 2:1; 1:4) not producing reversal of sign of charge. Although the € — concentration curves are rather complex, the « — concentration curve in every case yields a simple curve resembling typical adsorption with an initial steep slope and saturation at about 0.01 Molar. The character of the curves is determined by both ions. The slope at zero concentration varies markedly, particu- larly with value of the anion; e. g. for glass in the order, | > Br > Cl, the differences dimin- ishing as saturation is approached. The method of calculation employed here to ascertain the nature and magnitude of the forces involved in adsorp- tion of ions by “inert” surfaces at the liquid in- terface is justified by previous theory and exper- iment of the authors. LITERATURE 1. Smoluchowski, M., Anz. der Akad. der Wis- sensch., Krakau, 1903, p. 182. 2. Gouy, A., J. d. Phys. 9, 457 (1910). Stern, O., Z. Elektrochem., 30, 508 (1924). Gyemant, A. “Grundziige der Kolloidphysik,” Vieweg, Braunschweig, 1925. Miller, H., Kolloidchem, Beih. 26, 22 (1928). ell Mooney, M. J. Phys. Chem. 35, 331 (1931), and personal communication to one of us. Henry, D. C., Proc. Roy. Soc., London, A. 133, 106 (1931). 3. Lachs and Biczyk, Z. phys. Chem., 148, 441 (1930). 4. Glixelli, S., and Wiertelak, J., Koll, Z., 43. 85 (1927); ibid., 45, 197 (1928). 5. Powis, F., Z. phys. Chem., 89, 91 (1915). Mooney, M. J. Phys. Chem., 35, 331 (1931). 6. Briggs, D. R., J. Phys. Chem., 32, 1646 (1928); Colloid Symposium Monograph, 6, 41 (1928). 7. Tuorilla, P., Kolloidchem. Beih., 27, 44 (1928). 8. Loeb, J., “Theory of Proteins.” 9o%) Loeb; d:,, Loc} cit: 10. Miiller, H., Kolloidchem Beih. 26, 22 (1928). 11. Abramson, H. A., J. Gen. Physiol., 16, 593 (1933). DISCUSSION Discussion of this paper is included in that of Dr. Muller’s paper, “The Theory of Ionic Ad- sorption.” 208 THE COLLECTING NET [ Vor. VIII. No. 67 THE THEORY OF IONIC ADSORPTION Hans MULLER In the paper on the theory of the diffuse double layer it was pointed out that the existence of in- terphasial potentials leads necessarily to the con- clusion, that the surfaces are the seat of electric There are three possibilities to explain The first possibility charges. the origins of these charges. can only play a role if both phases are electrolytic conductors. The accumulation of the charges is then due to the difference of the ion activities in the two phases. If one phase is a solid, this pos- sibility is eliminated. A solid phase can acquire the charge by dissociation. If the material has the tendency for dissociation, it will send ions of one kind into the solution, and the charge is due to the ions or electrons whose charge is otherwise neutralized by the dissociated ions. The third possibility is adsorption. In this case the solid does not send ions into the solution, but its sur- face attracts electrolytic ions. They are bound to the surface by the unsaturated valencies of the atoms on the surface, or by the Van der Waals forces of the molecules of the solid. In general two or all three effects may be responsible for the surface charges. It is, therefore, in most cases, not possible to decide whether we are dealing with dissociation or adsorption. The theory of the diffuse double layer offers a means of calculating the surface charge o. In the first paper the following relation was derived: ee Fede = Il — Zz DKT — ——— 3 n,° e N25 This formula permits one to calculate the charge per cm.? from the measured electrokinetic po- tentials ¢, the concentration n,°, and the valencie z;, of the ions in the electrolyte. The observations on the change of the ¢ poten- tial with the electrolyte concentration c, give, for different surfaces and different solutions, a great variety of € (c) curves, and it seems, at first glance, to be quite impossible to recognize any kind of regularity. In general the ¢ (c) curves can be classified into three groups, which shall be discussed successively. (1) Group a. Very frequently the (c) curves (Fig. 1, a) start for very low concentration with a relatively large value of € and decrease continuously with increasing concentrations. This is, for instance, the case in the measurements of Freundlich and Zeh “) on As» S3 and Fes Os colloids. Assum- ing a reasonable value for the radius of the micelles, | was able to show ‘) that these curves can be explained without assuming any change of the charge o. The decrease of the £ (c) curve is only due to the change of the thickness of the double layer. Even if the actual radius of the particles (which is not known) would have been The three types of ¢ (c) curves and the corresponding o (c) curves. twice as large, or half as large, the data would have given a change of o by less than 10%. Similar verifications of this fact were given by Paine “). We are, therefore, justified in as- suming that in these cases no appreciable change of charge takes place. These examples furnish the best verification of the theory of the diffuse double layer. They justify the use of this theory for the more complicated cases. A similar course of the £ (c) curves is observed with the proteins; here, in general, they pass through the Zero axis. There exists an isoelec- tric point, where the ¢ potential changes its sign. ‘The isoelectric point is, however, not determined by the electrolyte concentration, but depends pri- marily on the hydrogen ion concentration. Also here there is no adsorption. The charge of the protein particles is determined by the dissociation equilibrium of the H and OH ions. The elec- August 12, 1933 ] THE COLLECTING NET 209 trolyte changes the charge only insofar as it pro- of Langmuir. It seems, therefore, that we are duces a slight change of this equilibrium. Group b. The € (c) curves of this group (Fig. 1, b) start with a small value of € for very low concen- tration. They increase first very rapidly and reach a maximum for concentrations of about 10-100 micromol per liter. For still higher con- centration they decrease again in a way similar to the curves in Group a. It is probable that many earlier investigations which gave ¢ (c) curves of Group a belong really in Group 0 since their failure to show the initial increase is only due to the fact that the colloids were not satis- factorily dyalized. This complicated course of the € (c) curves can, in my opinion, only be understood if one realizes that the ¢ potential is determined by two factors, namely, the charge of the double layer, and its thickness. If we accept the theory of the diffuse double layer, we can calculate the thick- ness of the double layer, and we are, therefore, able to calculate the charge o and its variation with the help of formula (1). Such calculations were recently carried out by Abramson, and are report- ed in the preceding paper. The following discus- sion refers to the figures of this paper. All € (c) curves show a linear increase of the charge o with small concentrations. For large concentrations the charge tends towards a saturation value. While the initial slopes of these curves and their satur- ation values change with the material of the solid phase and>the electrolyte, the type of the curves is the same in all cases studied. Before we begin the discussion of these results, I wish to describe briefly the course of the most general ¢ (c) curves. Group c. The € (c) curves of this group start the same way as those of Group > (Fig. 1. c). But, after reaching their maximum, they decrease faster, pass through an isoelectric point, and reach a minimum, which is usually much smaller and flat- ter than the first maximum. For still higher con- centration they approach again the value £ = O. To my knowledge, no calculations of the o values for this group of € (c) curves have been calcu- lated. It is easy to realize what the course of the o (c) must be. They will start (Fig. 1, c) at o = O and reach a maximum for about the same concentration where ¢ reaches its first maximum. At the isoelectric point we must have o = O and for higher concentrations the charge will ap- proach a saturation value of a sign opposite to the sign of the charge for small concentrations. The o (c) curves calculated by Abramson are very similar to the well known adsorption curves dealing here with adsorption, and that we are justified in using Langmuir’s theory of adsorp- tion. Gyemant'), and Stern) have repeatedly suggested that this theory may he applied to ionic adsorption. Abramson’s data lead to the same conclusion. The laws of adsorption can be derived by the following consideration. The adsorption is due to forces exerted by the surface. These are mole- cular forces. They fall off very rapidly with the distance from the surface. Their range of action is of molecular dimension. To simplify the cal- culation, one assumes that the adsorption range has a definite thickness d and that a molecule or ion within the adsorped layer has an average po- tential energy (-A). According to Maxwell- soltzmann’s principle the number n, of molecules per cm.? in the adsorption range is then A ke i Ny = nee Where n is the number of molecules in lec in the immediate neighborhood of. the adsorption range. If we are dealing with ions, the number of ions just outside of the adsorption range differs from their number in the electrolyte. This is due to the electrostatic forces in the diffuse double layer. We have ; F aie —Z eal 1 nee and hence A—zet k T ity, == he Since the adsorption range has a thickness d, the number of adsorbed ions per cm.” is then n,-d, and the adsorbed charge c= nedazae We assume that this consideration holds for each kind of ions. The adsorption potential A will, of course, depend not only on the nature of the surface. but also on the nature of the adsorped ion. We consider here the case where the elec- trolyte contains one kind of anions and cations of for valency z; and zy respectively. For smal! concentrations we have then c=nedzZ Ze Ai—z,e€ Ao+ ze€ kT eh 210 THE COLLECTING NET [ Vot. VIII. No. 67 where A, and Az are the adsorption potentials of the positive and negative ions respectively. For small concentrations o and € are small, and we can develop Ay Ay kT ke ¢ =n, e dz Ze e —e Ay No ef kel kT == Tay Ol va As me |- ze kT For sinall values of € we have, however, 4roa 1 ¢=— — D Kk where 47 n, e? ee = — Z1 Zz (“4 + zz) Dekel and hence Ay Az kT he aD n, e dz Ze Le e = Ay As 1 fede ep 1+ kd|/z7e + ze Z1 Z2 1/« is the thickness of the double layer. For small concentration 1/< = A is about 1000 times larger than d. Hence the denominator is practi- cally equal to unity, and we have A 1 As 2 k T (2) = sn, © Gl Ai 4y [Ue —e This equation states that for small concentrations the surface charge is proportional to the concen- tration. If the adsorption potentials A; and A» are markedly different, then o will have the sign of the stronger adsorped ion. Introducing this value of o in the equation of the double layer gives, for small concentration, a linear relation between £ and \/C. The data on glass, graphite and collodion, Figs. 2 to 8 of the preceding paper by Abramson and Miller, demonstrate the validity of this formula. Since all these surfaces are negatively charged, the halogen ions must have the higher adsorption po- tential. Consequently, the differences between the various alkali chlorides, and between the earthal- kali chlorides, are much smaller than between po- tassium-chloride, bromide and jodide. The valency z; of the positive ion is responsible for the fact that the earthalkali chlorides give a greater slope than the alkali chlorides, and accounts partly for the large charging action of the sulfides and fer- rocyanides. The o (c) for low concentrations are not reliable enough to give more than a quali- tative check of the theory. This is due to the fact that for these concentrations the formula for the electrophoretic migration speed is uncertain. The order of magnitude of the adsorbed charges is, however, quite reasonable. For KBr on glass we have, for instance, an initial slope Ai is da kT Ke 0 =10'’=ed /e —e 6. 1028 dc which gives with d = 3 10% Ay Ae kT Lo e — e = ilo leading to adsorption potentials of the order of magnitude of 10 k T. Langmuir’s data on the adsorption of molecules give values of A of the same magnitude’). The data on glass give some interesting information concerning the de- pendence of the adsorption potential on the nature of the ion. Since the curve for KI is higher than the one for KBr, and since this curve is again steeper than the one for KCl, we conclude that the adsorption potential of the halogen ion in- creases in the series Cl, Br, I. Similarily we con- clude that the adsorption potential increases in the series of ions, Li, Na, K, Rb, Cs, and Mg, Ba, Ca. Further data are required for a decision as to whether or not these series hold for any kind of surfaces. The o (c) curves for collodion and graphite indicate that this might be the case. In agreement with the data on glass, they give a very much larger adsorption potential for SO, than for Cl (in CaCly), and a still larger value for NasFe (CN) 5. The data even indicate a con- stant ratio of the initial slopes. The figures give approximately the following values of do - 10° for small concentrations. dc Electrolyte Collodion Graphite NaCl 4.0 2.4 CaCl. G2 50) NasSO, IS}S) 8.0 Aueust 12, 1933 ] THE COLLECTING NET 211 The values for collodion are in all three cases The observed values of omax vary between 100 about 70% higher than for graphite. This may be due to the fact that collodion has a wider ad- sorption range, or that all adsorption potentials on collodion are larger by the same amount than the potentials on graphite. The very limited amount of reliable data avail- able does not permit any definite conclusions. The above discussion will merely indicate how the measurements could furnish important informa- tion concerning the adsorption potentials and the range of the adsorption forces. For higher concentrations we have to take into account the fact that only a limited number of ions can be adsorped. Langmuir found that the entire course of the adsorption curve can be rep- resented by the formula xn n = ———— 1+£,8n To satisfy the limiting case of small concentra- tions we must have A—zeé ike ap Ge inl = alin, The saturation value of the number of adsorbed atoms is then «/B. This value shall be denoted by S, and we have A—zet k T oes ie Ny = A—zeé aby (Gl k T 1+ — ec S Gyemant and Stern have proposed that such an adsorption isotherm may be used for both kinds of ions. It is doubtful whether this is justified. Very little is known concerning the simultaneous adsorption of more than one kind of molecule. The data available point strongly to the fact that we have no superposition of the two adsorption curves, but that there is ‘‘selective” adsorption. One kind of molecule is preferred, and prevents the adsorption of the other kind of molecule. The well known exchange adsorption in colloids indicate that similar effects take place in ionic ad- sorption. Independent of the exact law, saturation will always occur, as is observed in the o (c) curves. The saturation value of the charge is given by Smax — € (Si “4 == Se Z2 ) and a few thousand E. S. U. Using omax = 1000 gives (Sy z; — Sv z2) = 5 10!. Since the ionic radit are of the order of 10% cm., S; and So could have values up to 10'° without contradicting the assumption that the adsorbed layer is mono- molecular. The Langmuir theory is, therefore, in principle quite sufficient for the explanation of the o (c) curves of Group 2. The more complicated o (c) curves of Group ¢ could be explained by assum- ing that a Langmuir isotherm holds for each kind of ion, and that we have Two such isotherms (Fig. 1,¢) would indeed re- sult in a o (c) curve of the type required, but it is questionable whether the two above conditions are compatible. So far it was assumed that the entire charge o, calculated from the £ (c) curves, is adsorbed. Actually this value of o is the charge within the rigidity boundary. We have seen that this bound- ary does not necessarily coincide with the surface of the adsorption range. There exists, therefore, the possibility that the calculated « must be con- sidered as the difference of two charges, namely the real adsorbed charge and the charge between adsorption range and rigidity boundary. It is evident that such an assumption introduces many uncontrollable factors and opens the possibility to explain almost any € (c) curve. O. Stern has given a theory of this type. He assumes a rigid double layer within the rigidity boundary, the outer layer of which does not entirely compensate the adsorbed charges. The compensation is com- pleted by the diffuse double layer. Stern's pic- ture of the charge distribution in a surface has the great advantage of being able to explain satis- factorily the difference between « and € potentials, the existence of isoelectric points and the experi- mental values of the electric capacity of the double layer, but it introduces so many variables, that it is quite impossible to decide whether or not the assumptions are correct. 3efore these questions can be satisfactorily set- tled, it is necessary to have many more reliable measurements of € (c) curves on well defined surfaces, using a large number of different elec- trolytes. LITERATURE 1. E. Freundlich and P. Zeh, Z. Phys. Chem. 114, 65, 1925. 2. H. Muller: Kolloidchem. Beihefte, 26, 8, 1928. 3. Paine, H. H.: Tr. Faraday Soc. 24 412 (1928). 4, A. Gyemant: Grundzuge der Kolloidphys. K. Vieweg, 1925. 5. O. Stern: Z. Electrochem. 30, 508, 1924. 6. E. Huckel: Adsorption, Monograph d. Kolloid wissenschaften, 1929. THE COLLECTING NET { Vor. VIII. No. 67 Discussion Dr. Shedlovsky: Has there been any attempt to deal with the question of adsorption on crystal- line surfaces? he crystallographic periodicity might produce periodicities of the adsorption po- tential and modify the charge density, particularly if the size of the adsorbed ions is smaller, or of the same magnitude, as the lattice constant. Dr. Miiller: Htickel has given a discussion of this question, but no experimental data are avail- able which could verify his conclusions. Dr. Shedlovsky: the adsorption potentials for the different ions ? Is there any indication that complicated ions, which might have dipolar characteristics, show a larger effect ? Is there a large variation of Dr, Abramson: The adsorption potentials, as determined from the initial slope of the o (c) curves, vary considerably. Accurate values are difficult to determine, because these data are to be obtained from measurements in solutions so dilute that one has to know exactly the carbon dioxide content of the water, and one must take into consideration the ionic strength of the water. Dr. Cohen: Is there anything that can be add- ed with reference to adsorption of ions on metal- lic electrodes, such as platinum or gold? Dr, Shedlovsky: There is some work by Frumkin on the adsorption of hydrogen ions on a platinum surface. If the solution is saturated with hydrogen, practically no adsorption of hydrogen ion on platinum is evident. Dr. Cohen: 1 was referring to blank metal. Dr. Shedlovsky: The same would hold there. Electrically there seems to be very little reason to suppose that the effect of a platinized electrode is more than to provide a very large surface. Dr, Cohen: It is perhaps also a matter of the crystal configuration of the surface. Dr, Mudd: Is there a known physical expla- nation of the fact that the forces of adsorption of ions seems to be of the same order as those of uncharged molecules ? Dr. Miiller: The adsorption forces are close range forces; according to the interpretation of Debye they depend on the electric dipole—or the quadrupole—moments, of the molecules, and should, therefore, be of the same magnitude for charged, or uncharged, atoms. Dr. Shedlovsky: The adsorption of ions dif- fers only insofar as the electric double layer forms an electrostatic screen, keeping one kind of ion away from the surface. Dr. Cole: Is there any possibility that the Van der Waals forces might act on the ions in the diffuse double layer ? Dr. Miller: This is very unlikely. The range of the adsorption forces is usually not larger than the diameter of the molecules. There are, how- ever, cases where the adsorped layer is definitely not monomolecular. In Stern’s theory, the ions, in the outer layer of the Helmholtz double layer, are held by Van der Waals forces. Avoust 12, 1933 ] THE COLLECTING NET THE ELECTRICAL BEHAVIOR OF LARGE PLANT CELLS") W. J. V. OstERHOUT The Rockefeller Institute for Medical Research The electrical phenomena observed in living cells might conceivably arise from phase bound- ary potentials, Donnan constraints, oxidation and reduction, or diffusion. Recent work indicates that phase boundary potentials are usually small and cannot at present be satisfactorily measured or calculated. Potentials in living tissues due to Donnan constraints are usually small, owing to relatively low concentrations of indiffusible ions, and can be calculated only at equilibrium, which seldom or never occurs in living, growing cells. xidation-reduction potentials could manifest themselves if metallic electrodes were brought into direct contact with the reactants (e. g. by inser- tion into the living cell), but it is difficult to see how they could manifest themselves in the experi- ments here discussed'?) where contact with the organism is made by means of salt bridges. Stud- ies of Nitella show that the most satisfactory calculations result when diffusion potentials alone are taken into account. This statement concerns only thermodynamic potentials to which the present discussion is lim- ited. These potential differences seem to reside mainly in the non-aqueous surface layers of the protoplasm. Diffusion potential in the aqueous phases seents to be unimportant compared with that in the non-aqueous layers at whose surfaces, moreover, phase boundary potentials may occur. These surface layers claim a special interest. They regulate the exit and entrance of materials and so determine metabolism. Their electrical behavior is a most delicate index of the condition and activities of the cell. Injury and death are indicated in this way long before any visible change occurs in the cell. Recovery from injury Fig. 1. Diagram of a cell of Valonia which is pierced by a glass capillary so that the protoplasm adheres to the glass and forms an electrical seal which prevents a short circuit through the cell wall. The arrow shows the direction in which the positive current tends to flow when cell sap is applied to the exterior of the cell. W, cell wall; P, protoplasm. can be traced as in no other way. The effect of all sorts of external agents can be followed by electrical measurements which in no way disturb the vital processes. This also applies to spontan- eous changes, such as action currents. Thus a fruitful field of investigation is open to the bi- ologist. Large cells are especially suitable for such studies. This is particularly true of certain multi- nucleate cells such as those of the marine plants Valonia and Halicystis and of the fresh-water plants Nitella and Chara. They can be obtained singly and are, therefore, free from certain com- plications attaching to bundles of cells, such as are found in muscle and nerve. In such cells the protoplasm appears to consist of an aqueous layer having an outer non-aqueous surface (next to the cell wall) and an inner non- aqueous surface (next to the large central vacu- ole, which is filled with a clear watery cell sap). The non-aqueous surface layers are unlike. In order to show this we need only pierce the cell with a capillary (Fig. 1) and lead off from the interior to the outside which is in contact with sap extracted from another cell. We then have the chain protoplasm sap M7 WE NE in which WV” is the aqueous layer of protoplasm, X and Y are the outer and inner non-aqueous surface layers. We should find no E. M. F. if X and Y were identical but this is never the case. In Valonia the observed E. M. F. is about 60 millivolts, in Nitella 15 millivolts (in both cases inwardly directed) ; in Halicystis it is about 50 millivolts (outwardly directed). It may be remarked that other evidence shows that X and Y are different, e.g. X secretes a cellulose wall but Y does not. One might be in- clined to say that this can hardly be due to the difference between the internal and external solu- tions since in Halicystis these are almost identical. 3ut we must remember that the sap is more acid, contains less oxygen, and more COs and organic matter than the external solution. What is the nature of the surface layers? They appear to be liquid since they round up in contact with aqueous solutions and show true surface tension. In respect to solubility they appear to act very much like chloroform toward dyes, and more like guaiacol in relation to such substances as potassium, sodium, magnesium and calcium. In sap 214 THE COLLECTING NET [ Vor. VIII. No. 67 the case of Valonia the potassium has apparently a greater partition coefficient in the surface layer than does sodium; those of calcium and magnes- ium are much less than that of sodium. Further- more that of sulfate is much less than that of chloride. All of this applies also to guaiacol. The apparent mobility of the potassium ion in the surface layer is much greater than that of sodium: this is especially clear in the case of Halicystis where the conditions are not compli- cated by differences in partition coefficients. Thanks to the unpublished work of physical chemists we now know that K* has a higher mobility than Nat in guaiacol which makes an- other point of resemblance to the protoplasmic surface. Let us now try to picture the bioelectrical situ- ation in Nitella. This may be done by means of a model'*’, Since the underlying principles can be illustrated as well by one non-aqueous layer as by two we employ a model consisting of a single non-aqueous layer B, with an exterior aqueous phase. 4, and an interior phase C, which may be called artificial sap. In our experiments B consisted of a mixture of guaiacol and p-cresol. When KOH is placed outside and COs, is bubbled inside (in C), to imitate the production of COz by the cell, potassium enters and combines in C to form KHCOsg: this reaction keeps the ionic activity product (K) (OH) lower inside than that outside so that potassium continues to enter until its concentration becomes much greater than outside. The osmotic pressure in C rises, and water enters. Eventually a steady state is reached in which water and electrolyte enter in the same ratio, and C increases in volume while its composition remains approximately constant. This seems to be analogous to what happens in living cells. When this has happened we find an outwardly directed E. M. F. of about 40 millivolts. This is evidently due to KHCOs in the artificial sap and may be most conveniently explained on the ground that K+ has a higher mobility than HCOs;~ or the guaiacol ion which can be proved experimentally (if phase boundary potentials en- ter in we may neglect them for the time being). It is clear that potassium enters and produces an outwardly directed E. M. F. against which it continues to enter. In other words it pene- trates against a constantly increasing potential gradient created by itself. We find, indeed, vhat at the start the E. M. F. is directed inwara and as potassium enters this is reversed. All this may be explained by saying that po- tassium enters in one form and leaves in another and that it sets up less P. D. in entering the arti- ficial cell than in leaving it. For example, if the cation and anion of the entering salt had the same mobility in the non-aqueous layer it would set up no diffusion potential in this layer. In this way we may explain the fact Nitella has an outwardly directed P. D. of a hundred millivolts or more. The non-aqueous layers are probably only a few molecules thick but supposing that taken together they are a micron in thickness, we have a potential drop of 100 millivolts or more across 1 micron, equivalent to 100 volts across a layer 1 millimeter in thick- ness. It thus appears that the electrical forces in the cell are of considerable magnitude. What do they accomplish ? Evidently there will be no flow of current as long as the P. D. is the same at all points on the surface of the cell. But if at any point local dif- ferences of metabolism alter the concentrations of ions and so change the P. D., there may be a flow of current between this and neighboring points and this may be accompanied by a flow of water). : If there be an outwardly directed P. D. of 100 millivolts or more it will give a corresponding current of injury. This can be measured by kill- ing one spot on the cell, 7. e. by substitutmg an injured area for the capillary shown in Fig. 1. Since the P. D. is outwardly directed the current flows in the external circuit toward the injured spot, 7. e. the current of injury is negative. This is stated in the literature as the invariable situa- tion, but this is an error. When we apply sap or 0.05 M KC1 to Nitella the P. D. becomes in- wardly directed and the current of injury _ be- comes positive. The same can be done in Hali- cystis by applying NHg3 (Blinks). In Valonia it is normally positive. When we reduce the P. D. at 4 (Fig. 2) by killing or by applying 0.01 M KC1, current begins to flow from surrounding regions to this spot. Suppose that with the outgoing current at B po- tassium moves outward from the sap until its concentration becomes as great outside as inside. The P. D. will then fall to zero. Current will then begin to flow from C to 8, along the cell wall and inwards to the sap, carrying potassium back into the sap at B. This movement of po- tassium will be assisted by the forces which normally cause potassium to diffuse into the sap that Cell walt Proto- plasm Fig. 2. denote P. D.: the feathered arrows show actual flow of current. Hypothetical diagram. The plain arrows Auvcusr 12, 1933 ] THE COLLECTING NET 215 and accumulate when no current is flowing. Asa result the potassium will resume its normal con- dition and the normal outwardly directed P. D. will return: this is called recovery. In the mean- time the outward current at C will cause its P. D. to fall to zero and this will be followed by recoy- ery. The same thing will then happen at D and later at E. (On this basis an outward electric current at B due to an external source should stimulate, as is actually the case.) In this way we can explain the action current and its propagation along the cell. There is con- siderable evidence in favor of this view but it must be regarded as merely a working hypothesis. A different explanation is offered by Blinks, based on his finding that internal changes of pH value greatly affect the P. D. of Halcystis®). He sug- gests that sudden increase of alkalinity in the cell caused by outward current flow, may cause a lowering of the P. D. and that recovery is due to subsequent production of acid'®). This is in har- mony with the theory of Bethe. These hypotheses are mentioned because they grow directly out of our experiments on large plant cells. Other explanations are possible but need not be discussed here. Action currents may be produced in Nitella, as in nerve and muscle, by electrical or chemical stimulation. Negative variations produced by mechanical stimulation are different since ney travel much faster and can pass a killed spot by the transmission of a wave of compression. Ordinary action currents cannot pass a_ killed spot except by the aid of a salt bridge as shown in Fig. 3. When a propagated variation coming from A reaches B the P. D. at B goes to zero and in consequence current flows from D through the salt bridge to B: this reduces the P. D. at D to zero and in consequence current flows from E to D and by a continuation of such processes the negative variation is propagated. When stimulated electrically Nitella responds like a skeletal muscle, giving a single action cur- rent: but when an area of the cell has its P. D. reduced for a period of minutes by applying 0.01 M KCl it gives a series of rhythmical responses like a heart muscle. This is to be expected on theoretical grounds. IGE Ne B/ 7a. CoN NDE Cell wall Proto- plesm Fig. 3. Arrangement of salt bridge to enable an action current to pass a dead spot. There is one very interesting feature of the negative variation which was described by Blinks. When the P. D. has fallen to zero the protoplasm no longer shows polarization, 7. e. it temporarily builds up no back E. M. F. when a current is sent through it from an external source. It seems to act as though it were completely per- meable to ions. The polarizability is regained as the P. D. returns. It is of interest to find that we can apparently remove certain substances from the non-aqueous surface layers and thereby change their electrical behavior™. After leaching with distilled water, or with acid or alkaline solutions, the cell can no longer be stimulated electrically but the irritability is restored when the cell is replaced in solutions containing calcium. Before treatment we find that potassium is very negative to sodium in Ni- tella but after treatment this is no longer the case. Evidently a marked modification of the properties of the surface has occurred. These experiments seem to show two things (a) that the non-aqueous surface is a mixture of substances, some of which can be taken away and still leave a non-aqueous layer, and (b) that anesthesia can be produced by removing certain substances from the cell. In conclusion it may be well to emphasize that electrical studies enable us to follow minute and rapid changes in the cell with little or no altera- tion of normal functions. This is the more im- portant since most attempts to investigate life processes introduce disturbing factors of which we are often unaware. LITERATURE 1. For the literature up to July 1931 see Oster- hout, W. J. V., Biol. Rev., 1931, 6, 369. 2. Oxidation can modify P. D. by changing the composition of the surface layers or the concentra- tion of ions. 3. Osterhout, W. J. V., J. Gen. Physiol., 1932-33, 16, 157. 4. Sollner, K., Kolloid-Z. 1933, 62, 31. 5. Blinks, L. R., J. Gen. Physiol., 1932-33, 16, 147. 6. Blinks, L. R., Proc. Soc. Exp. Biol. and Med., 1932-33, 30, 756. 7. Osterhout, W. J. V., and Hill, S. E., J. Gen. Physiol., 1933-34, 17, (Sept.) DISCUSSION Dr, Blinks: There is a point which possibly bears on the theory of the bioelectric potentials, namely, the amount of current which can be sup- plied by a living cell. When the circuit through a Halicystis cell is completed through a galvano- meter, a current of 5 to 10 micro-amperes will continue to flow for several days without greatly reducing the value of the potential difference, 216 THE COLLECTING NET [ Vor. VIII. No. 67 which remains close to 68 millivolts during this time. It seems unlikely that a phase boundary potential could supply such a current. Dr. Gasser: Is there any objection to the clas- sical view that the immediate source of the po- tential is a concentration cell ? Dr. Blinks: It is probably due largely to the concentration gradient of KCl in Nitella. In one species of Halicystis there are practically no gradients across the protoplasm, so it must be due to some gradient within the protoplasm itself. Finally in Valonia we have the potential in the wrong direction for the large KCl gradient, which shows that there must be some still larger potential directed inwardly. The question re- mains, what is this gradient. I think we are be- ginning to get some evidence. Obviously there must be something continuously generated by the cell to supply the energy for the continued cur- rent. This is probably derived from the process of respiration, but it seems unlikely that the po- tential is actually due to an oxidation-reduction system. Dr. Cole: It would look as though this cur- rent, produced in Halicystis, would, in a couple of days, discharge all of the ions of a 0.5 molar uni- univalent salt solution in the volume of the cell. Dr. Blinks: That is probably the right order of time. I once made a calculation for the time to do this in Valonia, where the potential and cur- rent is about one-tenth as large, and it came out to be about one month. That was assuming a sharp moving boundary, which, of course, would not occur. Dr. Fricke: The amount of electrical energy which is released from Halicystis, according to the data given by Dr. Blinks, is so large that a comparison with the energy which may be ex- pected to be available due to metabolism would appear to be of interest. A potential of 70 milli- volts and a current of 10 microamperes corre- spond to 144 - 10-8 gr. cal./hour. As a figure for the metabolistic rate we may use 1 gr. cal./hour, gr., and for the active mass of Hali- cystis 10-% gr. This gives 10~% gr. cal./hour, which is of the same order as the electrically re- leased energy. Dr. Cole: Has there been any attempt to measure the heat production in these cells when stimulated ? Dr. Blinks: No. It might be difficult on ac- count of the relatively small volume of the living protoplasm in these large cells. Dr. Cole: In this matter of discharging the cell continuously over a period of a relatively long time, since the potential remains so nearly con- stant there are comparatively few disturbing fac- tors entering in. The passage of the current it- self is not a disturbing factor in the sense that the hydrogen ion is? Dr. Blinks: Not with such currents as these (5 to 10 microamperes). If we apply progressive- ly higher voltages in the external circuit, so as to increase the current outward across the proto- plasm, there is, at first, a small decrease of the measured potential, due to polarization, then finally a point is reached where the potential goes through a complete reversal. It re- covers when the outward current is stopped. In- ward currents, up to very large values, only serve to increase the potential by polarization—up to 80 or 90 millivolts positive. Dr. Cohen: Is the reversal exactly the same effect as that produced by the internal increase of alkalinity ? Dr. Blinks: The effects are so much alike that I think the same mechanism is involved. Dr. Mudd: The assumption of a continuous non-aqueous layer at the cell surface is clearly of advantage from the point of view of simplicity. Is this assumption necessitated by the experimental data also? Dr. Blinks: Some other might do as well. It is easier to start with a simple substance. We do not know, of course, that the protoplasmic- surface 1s simple. Dr. Mudd: Is there anything to exclude the possibility that the protoplasmic surface should contain more than one component ? Dr. Blinks: It might contain a mixture of substances, or an amphoteric substance, to ac- count for the accumulation of both anions and cations. Dr. Gasser: About the mosaic notion’ of the plasma membrane—why should the surface form in mosaics unless there are many little cells at the surface of the main. cell ? Dr, Cohen: That would be all right if you make the assumption that the cell is a perfectly homogeneous system localized in the cell and, therefore, on the surface of the cell. Dr. Mudd: There are several classes of sur- face-active substances in protoplasm. It is diffi- cult for me to conceive of a mechanism by which one of these, exclusively, could form the proto- plasmic surface. White blood cells are perhaps instructive in this connection, These have been described by Kite, I believe correctly, as naked protoplasm. These cells may be observed, when studied with the cardioid dark-field condenser, to form long thread-like or veil-like processes. The formation of such processes may involve large spontaneous increase of surface. Such spontan- eous increase of surface, in the case of the myelin Aucust 12, 1933 ] THE COLLECTING NET 217 forms of lecithin, has been attributed, by Leathes, to the peculiarities of the lecithin molecule. These processes on white cells resemble the myelin forms of lecithin, and Fauré-Fremiet has found considerable data to indicate that they are, in fact, due to the presence of lecithin in the surface. The wetting and spreading properties of white blood cells, on the other hand, we have found sugges- tive of protein in their surfaces. At all events, it is difficult to conceive of so mobile a structure, which forms the limiting surface of the complex system protoplasm, and which undergoes rapid alterations of slope and surface area, and which spreads over and includes foreign particles, as composed of one homogeneous substance. Dr, Osterhout: The mosaic theory of the cell surface seems highly improbable, because a mosaic can exist only in a solid, and the proto- plasmic surface in these plant cells always acts like a liquid 7. e. it rounds up in contact with water, and in every way shows true surface tension. Dr. Blinks: No doubt all surface active sub- stances of the protoplasm would tend to migrate toward the surface and become concentrated there. But they would stay in the aqueous phase ; they would not produce a new phase on that sur- face unless altered in some way, as by denatura- tion of a protein, esterification of a soap, etc. Such an alteration might occur more readily with one of the surface active substances, than with the others, giving a single substance in the non aqueous phase. Dr. Gasser: Is the surface a constant thing at all? May it not be just a statistical condition de- pendent on the molecular species available at the moment for concentration in the surface? Dr. Blinks: Quite possibly. There might even be a definite alteration of properties in time, as suggested by G. E. Briggs (Cambridge Uni- versity,) with cationic permeable periods alternat- ing with anionic permeable ones. The effects of pH changes on the inner surface of protoplasm convince me that it may be extremely sensitive to metabolic changes. Dr. Abramson: The cell is fully permeable to water, is it not? Considering that fact, it is not necessary to go far to prove that the cell mem- brane is heterogeneous. If water goes in and out of the cell, water molecules must go in and out of the oil film, so that the oil film will always have some water areas, over a time average. Dr. Blinks: Dr. Shedlovsky’s measurements on the mobility of potassium and sodium ions in guaiacol were done with the latter nearly satu- rated with water, were they not? Dr. Shedlovsky: The water concentration in guaiacol amounted to about 4%. Dr. Cole: A membrane a fraction of a micron thick might allow considerable water transport. Dr. Gasser: 1 do not think that heterogeneity means the same as mosaic. The cell surface must be heterogeneous, but it was the mosiac structure I was discussing. Dr, Abramson: Please define what you meant by a mosaic. Is each mosaic combined with each neighboring mosaic with something like a chemi- cal bond, or is a Gibbs excess concentration con- sidered a mosaic ? Dr, Gasser: By mosaic I meant discrete areas of permeability, some having one nature, some another, the areas being arranged like the tiles in the floor. In this form the mosaic theory appeals to me as being a very artificial one. One thing that struck me is that, after all, the velocity of propagation of the action potent’al in Nitella is not very different from what it is in some nerves: 2 cm. per sec. for Nitella which may be compared with 30 cm. per sec. in the slowest frog nerves. Another thing, do you attach any importance to the fact that the action-potential has an_ initial spike-like form followed by a more prolonged por- tion rising to a second crest. The second part has the same relation to the spike as does the after- potential in nerve, but it is relatively very much larger. Do you think that the two crests may mean two processes occasioned by the presence of two surfaces? Dr. Blinks: Probably both are involved. For example, when KCI is applied to Nitella there is first a mobility effect on the outer surface, and then a secondary effect, possibly due to the en- trance and combination of KOH with some acid of the cell, increasing the pH at the inner surface. I should like to raise the question as to the possi- bility of such a high mobility of potassium ions in a non-aqueous phase as is postulated by Osterhout for Nitella—a mobility nearly 75 times that of chloride. Theoretically there may be grounds for doubting this. Do Dr. Shedlovsky’s measure- ments on guaiacol bear upon this ? Dr. Shedlovsky: I would like to mention one thing rather than the interpretation you asked for. It was very interesting to observe in work- ing on the conductivity of alkali guaiacolates in guaiacol that the conductivity of a certain concen- tration of potassium guaiacolate in anhydrous guaiacol was very much different from that of the saine concentration in guaiacol saturated in water. The conductance was 30 times greater in the water-saturated solvent than in the dry solvent. On the basis of dielectric constant alone you would not expect any such change in ionic mo- 218 THE COLLECTING NET [ Vor. VIII. No. 67 bilities, and the explanation for this tremendous change in conductance is probably due to the dit- ference in the size of ions rather than in the di- electric constant as such. The experiments of Kraus and Fuoss showed a similar tendency. In three expressions they derive, relating to the dis- sociation constant with the dielectric constant of the medium, and with the distance of closest ionic approach, the distance of closest approach 1s, at times, much more important than the dielectric constant. This is interesting in connection with speculations on the source of potentials arising from diffusions. It is possible that, in the living cell, there may be changes in the water content in the non-aqueous phase, running parallel with dif- ferent salt concentrations, so that, I think, one should be very careful in making the assumption that the mobility of the ions remains constant— it may or may not. Dr. Miiller: Is the change of mobility due to change of hydration? Dr. Shedlovsky: One must be careful to dis- tinguish between the mobility of ion as ion and the mobility of the ion as constituent. The signifi- cant thing is the mobility of the ion constituent, and that involves the mobility of the ion itself and the degree of dissociation. The mobility of the ion itself may not be changed very much, but the mobility of the ion constituent may be changed tremendously, if the degree of dissociation changes. Another thing in media of really low dielectric constant, around 8 or 10, the conduc- tivity curves frequently show a rather interesting nunimum, with increased concentration. Dr. Fuoss accounts for that on the assumption that there exists a possible equilibrium between the ions and undissociated molecules. By simply lo- cating the concentration corresponding to the minimum, and using these assumptions, he is able to reproduce the experimental curve from the theoretical curve, or vice versa. Dr, Blinks: You would say that the effects of the added water would be upon both anion and cation, increasing the mobilities of both and not exaggerating the mobility of one? Dr. Shedlovsky: The greater effect would be in the increased mobility of the ion constituents, by increasing the degree of dissociation. If we have any evidence that would point to a constant water content of the non-aqueous phase, I think that none of this material ought to be worried about. Dr. Blinks: 1 think there is not any evidence, but it seems probable that all parts of the cell are pretty well saturated with water. Dr, Shedlovsky: There may be an appreciable salting in, or out, of water. Aveust 12, 1933 ] THE COLLECTING NET 219 THE MEANING AND CALIBRATION OF THE pH SCALE D. A. MacINNES It is not necessary to emphasize the importance of the subject of this discussion. There is no bio- logical or chemical journal that does not make many references to hydrogen ion concentration, hydrogen ion activity and to pH values. This is, of course, natural since hydrogen and hydrogen ion are important constituents of water, and water constitutes a large part of ourselves and of our environment. Furthermore, the unhydrated hydrogen ion is the proton which is, at present reckoning, one of the three or four elementary particles of which our universe is constructed. The fundamental interest of the subject has also been shown by the investigations of Bronstead in Copenhagen and Conant and Norris in this coun- try. They have extended the ideas of acid and base from water to other solvents. According to these workers the relation between the generalized acid A and the generalized base B is A=B+ [+4] in which [+] is the proton. In this form the re- lation between an acid and a base closely resem- bles that between that of a reductant Re and an oxidant Ox in an oxidation reduction reaction. Such a reaction may be written Re= Ox + [—] in which [—] is the electron. Thus one of these fundamental types of reaction involves the proton and the other the electron. There is, however, not enough time to discuss the subject in its more generalized relations and we will confine our at- tention to water solutions. The gradations from acid on one hand through neutral to basic are now, almost univer- sally, stated in terms of Sorensen’s pH scale. In actual use this pH scale is established as follows. Galvanic cells, which may be schematically rep- resented by the typical cell: Ha, saturated KC1, O.1 N KC1, HgCl, Hg (A) are set up and their potentials, which we will de- note by E, are measured. The details of such a cell are as follows. The hydrogen electrode, which consists of a sheet of platinized platinum over which hydrogen gas is flowing, is inserted into “solution A,’ the pH of which is desired. This solution makes a liquid junction with saturated potassium chloride, and this in turn makes an- other liquid junction with a reference calomel half cell. This half cell consists of a mercury elec- trode which is in contact with a potassium chlo- solution A, ride solution (frequently tenth normal) which is saturated with mercurous chloride. Now the pH value is connected with the electromotive force E of this cell by the simple relation ee ei5 pH = (1) RT/F in which R, T and F are the gas constant, the ab- solute temperature, and the faraday respectively, and E, is a constant which I will have occasion to discuss fully later on. Physically it is the elec- tromotive force of the cell when it contains a so- lution which has a pH value of zero, which is ap- proximately that of normal hydrochloric acid. Such measurements as we have just described are either inconvenient or impossible in many cases, particularly with solutions of interest in biological work, and a number of alternative methods have been developed for determining pH values. The most useful of these are the colori- metric or indicator method, the quinhydrone method, and the glass electrode method. How- ever, these methods are considered trustworthy only in so far as they agree with the hydrogen electrode method briefly outlined above. The pit- falls encountered when an attempt is made to do accurate work with indicators are many. As you know there are “salt errors’ and “protein errors” and each indicator is a problem in itself. How- ever, I must mention with admiration the work ot Hastings and Sendroy as a beginning in the clear- ing up of this difficult field. The quinhydrone method has many of the difficulties of the hydro- gen electrode and has a limited range of applic- ability. The glass electrode is admirably suited to work with the solutions encountered in biological research. In our most recent forms the fragility inherent in the earlier types has been eliminated. It is, however, necessary to compare such elec- trodes with the standard hydrogen electrode. With electrodes of the best glass for the purpose at present available, such a comparison shows that the glass electrode is accurate within the pH range 1 to 8. At pH = 11, the error is about one tenth pH unit. If we regarded the pH simply as a variable that must be kept constant during a given set of experiments, it would only be necessary to agree upon an arbitrary value of E, in equation 1. However, pH values are fortunately much more useful than this. With them it is often possible to understand chemical reactions in solutions such as, for instance, the reaction between car- 220 THE COLLECTING NET [ Vor. VIII. No. 67 bon dioxide and other components of blood or plant sap. However, for this purpose it is neces~ sary to study the relation between the pH values and the hydrogen ion concentration, Cyt, or hydrogen ion activity, ay*. Two relations fre- quently given are pH = — log Cyt (2) (3) but the unforunate fact is that the first of these equations is not true and the second cannot be proved thermodynamically, for reasons to be out- lined below. If equation 2 were valid it would be possible to establish our pH scale by calibrat- ing with solutions for which Cy+ is known. Ac- cording to Arrhenius’ theory of dissociation, we could obtain the hydrogen ion concentration, of hydrochloric acid for instance, by means of the relation: Cy* = C A/Ag, in which C is the total concentration and A and A, are the equivalent conductances of the acid at the concentration C and at infinite dilution respectively. However, from a practical point of view, we would get a different value of E, if we used 0.1 N hydro- chloric acid than if we used say 0.001 N, for the calibration. Also we now know that the assump- tions upon which this computation is based are erroneous, though a discussion of the matter would take us too far afield at this point. This brings us to a discussion of equation 3. The activity is, as you know, a conception or- iginated by Professor G. N. Lewis. In the pres- ent case the activity of the hydrogen con- stituent ay* would be defined by the relation and pH = — log aqt ion RT E— EK, = log ay* (4) F if the potential at the hydrogen electrode were the only one that changed when the concentration of “solution A” is varied. E is once more the po- tential of a cell of the type represented by (A). The potential can, however, also change at the liquid junction between this solution and the sat- urated potassium chloride. By choosing saturated potassium chloride as one of the solutions, this effect is probably minimized, but, as we shall see, there is no way of telling, with certainty, whether such a technique is successful or not. To state the difficulty in other words, there is no way of deciding what portion of the measured potential of a cell is located at any particular point. One school of thought, which includes par- ticularly physicists, locates all the potential out- side the cell and places it at the points of contact of dissimilar metals. If, however, it were possible to obtain values of the activities of ion constitutents, such as that of the hydrogen ion ay*, it would, theoretically at least, be possible to compute values of the liq- uid junction, since its value, E;, can be found from the relation E; = RT/F if tid log a; (5) in which t; is the transference number of ion spe- cies 1, and a; is the corresponding ion activity. This evidently requires that we know all the in- dividual ion activities and the transference num- bers of all the ions in the solutions in contact at the liquid junction. The value of E; unfortunately also depends not only upon the solutions in con- tact, but upon the way they are mixed. Further- more the computation of the potentials of these liquid junctions taxed the ingenuity of such mas- ters of mathematical physics as Max Planck, even after he had made simplifying assumptions. We are thus in the position that we cannot obtain hy- drogen ion activities without correcting for the liquid junction, and we cannot correct for the liquid junction without hydrogen ion activities. Guggenheim, in a_ series of stimulating papers, has gone so far as to say that single ion activities have no meaning, largely because of this dilemma, It is also true that the ion activity depends not only upon the ion constituent itself, but also upon its environment, from which, of course, it cannot be separated. Although single ion activities cannot be meas- ured it is possible to determine ‘‘mean ion activ- ities’ by a number of thermodynamic methods in- volving no special assumptions. Two of these methods are the freezing point determinations and the measurement of potentials of concentration cells without liquid junctions. Though it is not possible here to demonstrate this, I will illustrate the ideas involved with a weak acid (say acetic acid) as an example. According to the Ostwald dilution law we have, [Ei | Aes a (6) [HAc] in which the brackets represent the concentrations of the substances enclosed and K’ is the ionization or mass law constant. We now know that this is true only at infinite dilution and that the correct thermodynamic expression is (H*) (Ac~) kK = —————_. (7) (HAc) in which K is the thermodynamic ionization con- stant, and the parentheses represent the activities THE COLLECTING NET 221 Aucust 12, 1933 ] of the substances enclosed. This can be put into the form: (EN Esce | YH+ ae [== (8) [HAc] Vitae in which the y values are the activity coefficients which convert concentrations into activities. Now although we are not able to assign definite values to the individual activity coefficients it is possible to tell, within limits, what values they approach for very dilute solutions. The activity coefficient for the undissociated acid Yyae is very nearly unity and will not be considered in what follows. The product of % 4 %4.— will be replaced by y? which is the square of the mean ion activity co- efficient. Professor Edwin J. Cohn”? has made the sug- gestion that if an accurate value of the thermody- namic ionization constant of a weak acid could be obtained, it could be used as a basis for the cal- ibration of the pH scale in terms of activities. Shortly before that, MacInnes?) and Sherrill and Noyes'*) had shown how the thermodynamic ion- ization constants could be found by the use of conductance measurements with the help of the Debye-Htckel theory. | MacInnes and Shedloy- sky) have recently obtained the necessary con- ductance data on acetic acid for the precise com- putation. In outline the computation is as fol- lows. Values of K’ as defined by equation 6 are first computed by a method in which a necessary correction is made. for “‘interionic attractions.” These are related to the thermodynamic constant by the relation. K=K'y (9) Now it is known from the Debye-Htckel theory that for low ion concentrations at 25° — log y = 0.506 VG, in which C; is the ion concentration. relations can be put into the form: log K = log K’— 2 X 0.506.\/G (11) Thus, if that theory is valid, for low ion con- centrations a plot of values of log K’ against \/C; should be a straight line with an intercept at log K. This has been found to be true and the method yields a value of 1.754 & 10~-° for the thermo- dynamic ionization constant of acetic acid. Re- cently Harned and Ehlers) by an entirely differ- ent method, involving concentration cells without liquid junctions, obtained the value —1.754 10° This agreement is most gratifying and makes it probable that we have here an accurate value of a constant of nature, A similar investigation for the (10) These two considerably stronger acid, chloro-acetic, also yielded results in accord with the Debye-Hutckel theory for the dilute solutions. It now remains to calibrate the pH scale with the aid of these thermodynamic constants. As- suming equation 3, the equation (alan)) (Cave=)) K => ——_—___—_ (7) (HAc) in which the parentheses represent the activities of the substances enclosed can be rewritten in the form [Ac™ ] pK = pH log log y (12) [HAc] in which pK is the negative logarithm of the thermodynamic ionization constant. Now if we measure the pH values of a series of buffer so- lutions in which the concentrations [Ac~ ] and [ HAc] are equal then, since y approaches unity and log y approaches zero for very dilute solu- tions, the pH value should approach pK if we have chosen the correct scale for the pH values. (A small correction for the shift of the ionic equilibrium is necessary.) If the Debye-Huckel theory is valid we should plot the observed pI1 values against the square root of the concentra- tion in order to make the extrapolation. The com- putation therefore consists in obtaining a series of values of pH corresponding to different values of E, in equation 1 until we find a value which will yield pK accurately on extrapolation. This has been done for both acetic and chloro-acetic acid buffers, using data recently obtained in, the laboratory of the Rockefeller Institute by Mr. Donald Belcher. The data for the former gives a value of 0.3358 for E, at 25° and the latter, the closely agreeing value 0.3357. The usually accept- ed value is 0.3376. As explained earlier in this pa- per, this difference would not be important if we were interested only in reproducible pH values, but becomes of interest if we intend to relate the pH values to chemical equilibria in the solutions under study. It is of importance that the slope of the lines obtained by the method of plotting just mentioned is very nearly that demanded by the Debye-Hiickel theory. There are, however, devia- tions from this simple theory, at the higher con- centrations, which might indicate that the liquid junction potentials change somewhat with the concentration. The question may be reasonably asked whether this procedure has any advantage over that of using hydrochloric acid for the standardization, since the ionization relations of that substance are presumably simpler than those of the weak acids. It is, howeyer, an unfortunate fact that the cali- 222 THE COLLECTING NET [ Vor. VII. No. 67 bration with a strong acid gives a different value for E, and the accuracy obtainable is much lower than that attained with the buffers described above. This difference is, undoubtedly, due to the larger potential at the liquid junction when a strong acid is used, and the greater dependence of the value of this potential upon the way the junc- tion is made. Furthermore, calibration with a strong acid means doing it at low pH values, where for biological and much other work the in- teresting field is roughly between the pH values 4 and 9. Some recent measurements with carbonate-bi- carbonate buffers at relatively high pH values cannot readily be explained by the current theo- ries, and must receive further study. The chief ideas in this paper may be roughly summarized as follows: The pH values of solu- tions are related by a simple formula to the po- tentials of certain galvanic cells containing the so- lutions. These pH values cannot be interpreted as measures of hydrogen ion concentrations. Fur- thermore, hydrogen ion activities cannot be de- fined thermodynamically, and may have no physi- cal meaning. Mean ion activities are, however, possible of measurement by thermodynamic meth- ods. By the proper choice of standards the pH scale can be adjusted so that the values in that scale will measure, as nearly as possible, ‘“‘the mean ion activities of the hydrogen ion con- stituent.” REFERENCES 1. Cohn, E. J., Heyroth, F. F., and Menkin, M. F., J. Am. Chem. Soc., 1928, 50, 696. 2. MaclInnes, D. A., J. Am. Chem. Soc., 1926, 48, 2068. 3. Sherrill, M. S., and Noyes, A. A., J. Am. Chem. Soc., 1926, 48, 1861. 4, Maclinnes, D. A., and Shedlovsky, T., J. Am. Chem, Soc., 1932, 54, 1429. 5. Harned, H. S., and Ehlers, R. W., J. Am. Chem. Soc., 1932, 54, 1350. DIscussIoNn Dr, Abramson: Does the dielectric constant of the solvent come into the theoretical slope ? Dr. MacInnes: three halves power. Yes. It enters as the inverse Dr. Abramson: We had similar success with the dielectric constant of the solvent in employing: the general theory of electrophoresis. Dr. Miiller: Are the deviations from the Debye-Hiickel equation connected with the fact that the hydrogen ion is small? The nature of the deviations which you observe is identical with what one would get by using the higher approxj- mations of the theory. : Dr, MacInnes: The hydrogen ion does not seem to be small. The data on hydrochloric acid fit the Debye-Htickel theory in its simple form. It behaves as if it had a “distance of closest ap- proach” of about six angstroms. If this distance gets below about three angstroms, corrections, for higher terms, such as you and Gronwall, La Mer and Sandved worked out, must be made. Dr. Michaelis: There are, of course, no naked protons in solution. Dr. MacInnes: Also the simplest compound of proton with water, that is H30*, is still too small, but it is probably hydrated extensively enough to increase its size so that the extended theory I have just mentioned is unnecessary. Dr. Fricke: The hydrogen ion is undoubtedly quite large. It may be well to remember that an explanation is necessary to account for the large velocity in view of the size, as we now under- stand it to be. Dr, MacInnes: Some people think it is due to the proton jumping from one ion to another. I am not completely convinced of this. Dr. Fricke: It is undoubtedly related to the fact that the hydrogen ion is a dissociation prod- uct of water. Dr. MacInnes: to do with it. That certainly has something Dr, Abramson: The constant K’ which you use in your equation for ion activity involves con- ductance measurement. You have a mobility re- lated explicitly to an activity. Dr. MacInnes: They are not related. We use conductance simply as an indicator of ion concen- tration and compute the activities by using the Debye-Huckel theory. Dr. Abramson: It is not a method of getting activity coef ficients ? Dr. MacInnes: No. My discussion simply shows that in a dilute solution, our use of con- ductance measurements gives the right concentra- tions if you accept the Debye-Htckel theory as valid for these dilute solutions. Dr, Fricke: Can you experimentally disting- uish between two ions kept together by electro- static force and an actual undissociated molecule ? Dr. MacInnes: No. It is a much discussed question. Bjerrum ascribes departures from the simple Debye-Hiickel theory to “ion associations” due to the action of intense electric fields when small ions get close together. Gronwall, La Mer and Sandved explained the same departures by extending the mathematics implicit in the simple Aveust 12, 1933 ] THE COLLECTING NET 223 theory. Recent commentators regard the two treatments as having formal differences only. Dr. Fricke: 1 presume that the formation of the real molecule would be distinguished by a definite change of energy. Dr. MacInnes: 1 do not know that there is any clear way of distinguishing between them. It is a question that is very much discussed. Dr. Cole: Another line of evidence on that has been published by Woodward, working in Leipzig on the Raman effect on strong electro- lytes. He has found in strong electrolytes no ev- idence of undissociated molecules. Dr. Mudd: Do amino-acids and proteins, at their isoelectric points, have the electric moments of doubly ionized particles ? Dr, MacInnes: Yes, you can raise the dielec- tric constant of water by adding certain amino- acids. Dr. Michaelis: constant changed 7 Dr. MacInnes: The work of Hedestrand shows that normal aqueous solutions of some amino acids have dielectric constants higher than water by nearly thirty percent. How much is the dielectric END OF COLD SPRING HARBOR SECTION 224 THE COLLECTING NET [ Vor. VIII. No. 67 THE IRRADIATION OF BIOLOGICAL SUSPENSIONS BY MONOCHROMATIC LIGHT (THE EFFECT OF ULTRA-VIOLET LIGHT ON A PLANT VIRUS AND BACTERIA) Dr. B. M. DuaGar! anp Dr. ALEXANDER HOLLAENDER? Laboratory of Plant Physiology, University of Wisconsin For the irradiation of certain biological sus- pensions with measured quantities of mono- chromatic light, special apparatus has been de- signed. Intense sources of monochromatic light have either been adapted or constructed. Special cells and stirrers have been built. The energy was measured by the usual thermopile-galvano- meter arrangement. The apparatus was _ built around a B, and L. quartz monochromator put at our disposal by the manufacturer, through the Radiation Committee of the National Research Council. The virus of typical tobacco mosaic, approxi- mately purified, was exposed in V/100 at about O° C. The virus suspension was made up after pasteurization in physiological salt solution. The bacteria used were Serratia marcescens, Bacillus subtilis, vegetative and spore stages, Bacillus megatherium spore form, For the determination, both of the lethal effects on bacteria and of in- activation of the virus, the two materials were combined in the same suspension, ~so that com- parative values might be obtained. Exposed ma- terials were accompanied by controls similarly treated, except as to protection from radiation. After exposure, dilutions of the bacteria were made in physiological salt solution of the irradi- ated cultures and the unirradiated controls, and by plating these on agar media, a definite quanti- tative comparison was possible. The percentage inactivation of the virus was determined by inoc- ulation of tobacco plants and by comparing the incidence of disease induced by the exposed virus as compared to that induced by unirradiated virus. Inactivation of the virus is confined to wave lengths shorter than about 43100 angstrom units. The energy required to produce perceptible effects at approximately A 3100 angstrom units is more than 100 times as much as is necessary at A 2650 angstrom units. The energy values of incident light, representing 100 per cent. killing of the bacteria are far below the values having any measurable effect on the virus. For both of these biological materials, using the range from A 2537 angstrom units to 3100 angstrom units, the great- est influence is at A 2650 angstrom units. The resistance ratio of virus to bacteria as represented by these results is about 200:1. Apparently there is very little relation between heat resistance and light resistance. B. subtilis (spore form) will survive extended boiling for fifteen minutes, the vegetative stage will scarcely withstand fifteen minutes at 65° C. The virus is inactivated with an exposure of ten minutes at SURG: There is only little difference in the energy necessary to kill the spore stage and the vegeta- tive stage of B. subtilis; but the energy to in- activate the virus in the same suspension is about 200 times the energy necessary to inactivate both forms of B. subtilis. In relation to wave length, bacteria and virus, as well as several other biological materials show the same sensitivity. 1Professor of Plant Physiology. 2National Research Fellow in Biological Science. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on Au- gust 1.) REGIONAL DIFFERENCES IN THE ORGANIZATION CENTER OF THE AMPHIBIAN EMBRYO Dr. Epmunp K. Hatt Instructor in Anatomy, University of Louisville School of Medicine Up to the present time, sufficient data have not yet been accumulated for a complete understand- ing of the inductive reaction which takes place be- tween the organization center and the ectoderm of the gastrula during the normal development of the Amphibian embryo, Evidently, there are sev- eral possibilities as to the manner in which this reaction occurs: Each part of the archenteron roof may induce specifically in the superjacent ectoderm the specialized structures which later develop there. Or, on the other hand, the archen- teron roof may give rise to a merely general neural stimulus ; the character of the organ which arises from any particular region of the medul- lary plate would then depend on factors present in the ectoderm, or perhaps on the “organism as a whole.” As a matter of fact, Spemann has found that both possibilities are realized when inductions oc- cur in the lateral and ventral ectoderm, “Head or- Aveust 12, 1933 ] THE COLLECTING NET 225 ganizer” induces a brain in all parts of this ecto- derm and so seems to exert a specific inductive stimulus for this organ. “Trunk organizer,” on the other hand, induces a spinal cord when act- ing on the posterior ectoderm, and a brain when acting on the anterior ectoderm; its stimulus therefore seems to be a more general one than that of head organizer, and the type of organ formed seems to depend on the level of the em- bryo at which the graft is acting. The results reported in last Tuesday’s seminar deal with the action of head organizer and trunk organizer when they are acting, not upon the lat- eral and ventral ectoderm of the gastrula, as in Spemann’s experiments, but upon the ectoderm of the future medullary plate. Trunk organizer, when it underlies the anterior end of the medul- lary plate, seems under these conditions to exert a specific stimulus, and not a general one, for the anterior end of the neural plate is much elon- gated, and is as narrow as it is posteriorly. In such animals, curious elongated heads develop, in which some of the head organs are either lacking, or very rudimentary. When head organizer un- derlies the posterior portion of the medullary plate, it seems to exert a merely general neural stimulus, and not a specific one, for the develop- ment of the spinal cord proceeds normally. In this case, of course, the level at which the graft is acting has in some way determined the type of reaction. Spemann’s results and those of this investiga- tion may therefore be summarized by stating that both head organizer and trunk organizer may either act to induce specific structures, or may give rise to a merely general inductive stimulus, the kind of organ produced depending on the level at which the graft acts. The type of reaction which occurs depends, of course, on the exper- imental conditions. (This article is based on a seminar report presented at the Marine Biological Laboratory on August 8). BOOK REVIEW The Freshwater Algae of the United States, Smith, Gilbert Morgan, XI—716 pp., 449 fig. McGraw-Hill Book Co., Inc. New York, N. Y. 1933. The appearance of Professor Smith’s text- book places American students at a great advan- tage in the study of freshwater algae. Dependent as they have been upon foreign texts for intro- ductory information, it is not surprising that many have written upon American algae with an inadequate general knowledge of the group. The British books, which have been our most available sources of instructional material, have become somewhat obsolete through the rapid increase in life-history studies, with their consequent revi- sions in classification. The book under considera- tion gives a most needed and acceptable resumé of current research. In general, after preliminary chapters on the nature and evolution of algae, their distribution and ecology, methods of collec- tion and study, the eight major groups recognized are discussed in chapters each prefaced by a general account of the struc- ture, types of reproduction, life . histories and evolutionary trends of the group. The book is clearly a textbook of morphology, life history, ecology and general systematics as they concern the algae. It is not intended to serve as a taxonomic manual, although much taxonomic information is included, and through literature citations the student is directed to suitable de- tailed sources. The major groups and genera are described and figured, but no attempt is made to list all the known species in the larger genera. In small genera all the American species are men- tioned, with their more obvious distinctions. The system of classification adopted is conservative, particularly in the Myxophyceae, which will aid its use by non-specialists. The illustrations, de- signed to show not only the morphology of each genus, but often the reproduction as well, are very clear and well executed. With some excep- tions, most frequent among the flagellate genera, they are original—_W. R. Taylor. Professor and Mrs. Charles D. Snyder with their son Thoma are planning to spend a couple of weeks in the White Mountains. At the end of that time they plan to come to Woods Hole for a visit before they return to Baltimore. Their daughter, Francina, is a guest of Professor and Mrs. Jennings in the Gansett Woods. Five of this year’s best sellers have been or- dered for the club, Hervey Allen’s “Anthony Ad- verse’, Gladys Carroll's “As the Earth Turns”, Robert Herrick’s “Sometime”, the two volumes of Theodore Dreiser’s “Gallery for Women”, and Pearl Buck’s latest novel, “The First Wife”. Last December, Dr. E. C. Cole was one of the ten of the faculty of Williams College to receive an annual grant for research of $400.00 from the “Williams 1900 Fund.” THE COLLECTING NET [ Vot. VIII. No. 67 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Jean M. Clark, Martin Bronfenbrenner, Margaret Mast and Anna- leida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford PAST AND FUTURE It gives us pleasure to announce that one hun- dred and sixty-six dollars and five cents have been turned over to THe CoLLectinG Net Scholarship bund by the Penzance Players which represents the proceeds from their play “You Never Can Yell,” so ably directed by Mrs. George A. Bait- sell. On Monday evening, August 28 TH CoLLectr- ING Ner will present a combination lecture and motion picture on “Whaling Lore” by the son of an old New England whaling captain in the audi- torium of the Marine Biological Laboratory. We sincerely hope that the community will gen- erously support this program, not only for the money that it will bring to the scholarship fund but because “Whaling Lore” deserves it on the basis of its interest and educational value. It has been much improved orally and pictorially since Dr. Conklin wrote of it: “Audience one of the largest ever assembled in our new auditori ; iti as you will ever address—lecture both interesting and profitable. I have heard only words of praise for your advan- tages of first hand knowledge.” THE VALUE OF SCHOLARSHIPS TO THE STUDENTS AT THE MARINE BIOLOGI- CAL LABORATORY Dr. R. W. Gerard, Associate Professor of Physiology, University of Chicago Having been asked to comment as to the value of the scholarships given worthy students each year for superior work in the Marine Biological Laboratory courses, and sponsored by THE Con LECTING Net, I take it that the question of the worth of scholarships in general need not be raised. Certainly, since the days when science changed from a pursuit of the wealthy amateur, as in the early days of the Royal Society of Eng- land, to the serious purpose of a corps of devotees of all resources and antecedents, it has clearly been impossible for the bulk of investigators to depend upon their own resources. Apparatus has become ever more complicated and expensive, techniques more intricate and training very pro- longed. To meet these growing requirements and in tacit recognition of the contribution made by research to society as a whole, endowments have been steadily increasing in the form of university reserves, special research budgets, and particular- ly, fellowships and scholarships to aid the student at all levels of development, from the time super- ior ability becomes evident until it has been fully nurtured and productive. The situation at Woods Hole, as the specific instance of these general conditions, is very in- teresting. Here, at one of the largest biological institutions in the world, at which gathers every summer an extraordinary variety and richness of scientific talent, there are given courses for the advanced training of some one hundred students a year. Speaking particularly for the physiology course, with which I am naturally best acquainted, the students are almost invariably college gradu- ates, often with years of additional training, and have come here largely at their own expense and initiative to’extend the training they have been able to obtain at the usual college and graduate school. A surprisingly large proportion of these students, probably well over half, have continued productive research in later years and, in fact, a very large number of the regular investigators who work here each summer have been recruited from the courses in years past. The students here constitute then, a selected group of embryonic in- vestigators assembled from the entire country. Most of them, of course, need money badly ; many eke out the summer’s expenses by waiting table at the Mess and the like, but such tasks do not mix well with the requirements of irregular re- search hours. All possible financial aid is wel- comed by the students, and, in fact, the number who have been able to return to Woods Hole in subsequent years and complete unfinished projects with the aid of CotLectinc Net scholarships must be very gratifying indeed to those who have contributed to their success. Iam happy, therefore, to lend the assurance of our experience in urging that these scholarships be generously supported. Dr. George deRenyi has arrived for his sum- mer’s work at the Laboratory. He spent the early part of the season at the Bermuda Biologi- cal Station. Dr. E. C. Cole, assistant and associate profes- sor of biology for nine years at Williams College has been advanced to the rank of full professor. Aucust 12, 1933 ] PT EVE SSO re At its Annual Meeting, held on the tenth of August, the Woods Hole Oceanographic Institu- tion re-elected its officers as well as five of the six trustees whose terms expired this summer. Lieutenant Commander E. H. Smith of the U. S. Coast Guard was elected to the Board to replace R. P. Seripps of Ridgefield, Connecticut, and the other members re-elected to serve until 1937 are: Newcomb Carlton of New York; Dr. T. H. Mor- gan of the California Institute of Technology; R. S. Patton of the U. S. Ceast and Geodetic Sur- vey; B. W. St. Clair of West Lynn; and the Hydrographer of the U. S. Navy Department. The re-elected officers are Lawrason Riggs, Jr., Treasurer, and Henry B. Bigelow, Clerk. Mr. C. H. Bostian and Mr. B. R. Speicher, graduate students at the University of Pittsburgh, are receiving the degree of Ph. D. this summer. They finished their theses during July, and took the final oral examinations in Woods Hole. The examining committees consisted of Dr. P. W. Whiting and E. A. Wolf of the Department of Zoology, Dr. John Donaldson of the Medical School of the University of Pittsburgh, Dr. Anna Rk. Whiting of Pennsylvania College for Women, and Dr. O. E. Nelson of the University of Penn- sylvania. Mr. Bostian, who is assistant professor of zoology at the North Carolina State College of Agriculture and Engineering, had as his thesis subject, “Biparental males and biparental ratios in Habrobracon.” Mr. Speicher’s subject was “A morphological study of the effective period of ‘Eyeless’ and ‘Glass’ in Habrobracon.” Dr. H. B. Steinbach, instructor in physiology at the University of Pennsylvania, who received his degree of Doctor of Philosophy this June, has been teaching physiology course in summer school. After a visit at his home in Michigan he will work at Woods Hole in September and in Chicago during the winter with Dr. Ralph Lillie under a National Research Fellowship. Dr. Maynard M. Metcalt, research associate in zoology at Johns Hopkins University, has come to Woods Hole with his family to spend several weeks at his cottage on Crow Hill. Dr. C. C. Speidel left Woods Hole on August 10 for England, where he will participate in the International Congress for Experimental Cytol- ogy which opens on August 21. Dr. Robert Chambers will leave on August 14 to attend the congress, THE COLLECTING NET Sey HON ese xs Sd Dr. Balduin Lucke left in the early part of last week for a vacation in Wyoming, where his wile has been spending the summer. Dr. Oscar Schotté is leaving Woods Hole very shortly for New Haven, where he holds a re- search fellowship at Yale University. Dr. Caswell Grave recently arrived at Woods Hole from the Tortugas Islands, where he, with the assistance of Mr. Paul Nicoll, has been studying the tunicates of the coral islands. Dr. Jenning’s son, Burridge, has come to Woods Hole for a part of his vacation. He has recently been taking a summer course at the Uni- versity of Michigan. Last week-end four Woods Hole investigators, Dr. Norma Furtos, Miss Arliner Young, Mr. Donald Costello, and Mr. Daniel Mazia, drove to Philadelphia. There Mr. Costello deserted the party and joined Dr. H. B. Steinbach, who was leaving for Detroit. Dr. William Young, of Brown University, took a short leave of absence from Woods Hole in order to climb Mount Washington. Mr. McInnes, of the Supply Department, took a baseball team composed of town people, collect- ing crew, and investigators to Vineyard Haven in the Nereis on Sunday afternoon, and brought back a 5-4 victory. Dr. Joseph Hale, now an instructor in chemis- try at Earlham College in Richmond, Indiana, re- turned to Woods Hole for the latter part of last week. He expects to spend a few weeks here later this month. Dr. Edgar “Tony” Hill is leaving Woods Hole on Wednesday to return to his home in Kentucky, where he will spend a short vacation before re- turning to the Rockefeller Institute in the Fall. Dr. Adam Boving, senior entomologist, Bu- reau of Entomology at the United States Depart- ment of Agriculture recently arrived in Woods Hole. He is an authority on the larval stages of beetles and is spending much of his time here with his old friend, Dr. August Krogh. 228 THE COLLECTING NET _[ Vo. VIII. No. 67 The Annual Meeting of the Trustees and Corporation of the Marine Biological Laboratory The annual meeting of the Corporation of the Marine Biological Laboratory and of its Trustees convened on August 8. The Trustees’ meeting, as usual, was divided into a morning and after- noon session. The question of finances, the rela- tion of the Laboratory to the National Industrial Recovery Act, and the appointment of the Com- mittee of Review were among the more important things discussed. The members of the Corporation met at 11:30 on Tuesday morning. The Clerk read the min- utes of the last meeting; and this was followed by a brief Treasurer’s report by Mr. Riggs. The major part of his report is available in the An- nual Report of the Laboratory published recently in the Biological Bulletin, Mr. Riggs emphasized the fact that the financial situation did not appear good in March, but that the gloominess prevalent then was not entirely warranted, and that the general financial condition of the Laboratory was better than that of many educational institutions. In the report of the Librarian, Mrs. Montgom- ery formally announced the gift of the scientific library of the late Professor William Patten by his son, Dr. Bradley M. Patten. She spoke at some length of the high cost of foreign scientific magazines, especially those in Germany, and was in favor of having people here protest against the excessive charges now being made by certain scientific publications in Germany. She mentioned that one or two German publishers had already come to realize that it was necessary to keep down the cost of these publications, and had taken steps to do so. In the Director’s report, Dr. Jacobs spoke espe- cially of the attendance and research productivity of the investigators at the Laboratory. He gave a series of figures showing that at the beginning of the season the attendance at the Laboratory was below that of the previous summer ; but later figures showed that as early as the end of June the daily attendance at the Laboratory was actu- ally greater than in 1932. The figure for August 7 of this year, compared to that for the same date last year, showed an increase of fourteen in the number of people working at the Laboratory. On Monday, August 14, 265 investigators had regis- tered during the summer, as against 251 for the same date last year. Dr. Jacobs emphasized the fact that the number of investigators present this summer comfortably uses all of the facilities of the Laboratory without unduly taxing any one of them, and that these conditions were optimum for comfort and research productivity. Dr, Jacobs exhibited three piles of reprints as indication of the research work accomplished dur- ing the past three years. The pile for 1932 was smaller than that for the previous two years, but he took pains to point out the fact that bulk is a poor measure of value and that the importance of the research work described in the publications for 1932 might be greater than in either of the two other years. He announced the retirement on a pension of the following three workers at the Laboratory: John J. Veeder, who first began work here in 1899; George M. Gray, who began in 1891, and Ellis M. Lewis, who began in 1897. The Director’s report was followed by the elec- tion of officers of the Corporation. The Treas- urer, Lawrason Riggs, and the Clerk, Dr. Charles Packard, were re-elected to their offices. Dr. W. R, Amberson and Dr. C. C. Speidel were elected to replace Dr. H. C. Bradley and Dr. C. E. Mc- Clung. The other six members of the group of eight whose terms automatically expired this \ear were re-elected to serve another four year term. They were: Drs. H. B. Goodrich, Wesleyan Uni- versity; I. F. Lewis, University of Virginia; R. S. Lillie, The University of Chicago; T. H. Mor- gan, California Institute of Technology; A. C. Redfield, Harvard University; D. H. Tennent, Bryn Mawr College. Under the head of new business, the only mat- ter which came up was the giving of a formal vote of thanks to Dr. Patten for the gift of his father’s library. The meeting adjourned at 12:20 A. M. CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the hole turns to run from Buzzards Bay to Vineyard Sound: A. M. P. M. FeNBEERBE MG g 6 cuslae EAU ILE) MMe WG Gagan 25 922i AN lis} Sabo 0 3:02) 3208 ENS ISE PLO eee 3:46) fates JeNoresi 740) 3G boas 4:26 4:37 Atioists Zils sere Gao sygilts) AMEUSE 220 ea 543 Sess In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. ene Vol. VIII. No. 8 COLOR CHANGES IN THE DOGFISH Dr. G. H. PARKER AND HELEN PorTER Department of Zoology, Harvard University Lundstrom and Bard have shown that the com- mon dogfish Mustelis canis can change from light In exper- to dark according to the background. iments on the pituitary gland they have further shown that dogfishes deprived of this or- gan become light. Moreover, when pituitary extracts are injected into these light ani- mals, they turn temporarily dark. The conclusion arrived at by these authors is to the effect that pituitary secretions control the color changes in this case, the animal being light when it is deficient in these secretions, and dark when it has an abundance of them. In our experiments we have found that wherever nerves are severed, light patches or streaks appear on the skin of the dogfish. If cuts are made in dark fish, these light marks R. E. Zirkle: X-rays.” tend to disappear in a few days; if they are made in a light fish, they appear to be indefinitely The marks (Continued on Page 25+) retained. SATURDAY, AUGUST 19, 1933 MW. KH. LH, Calendar TUESDAY, AUGUST 22, 8:00 P M. it has Seminar: R. Rugh: matic radiation and early am- phibian development.” “A non-linear rela- biological effect and ionizing power of alpha rays.” Leon C. Chesley: rays upon Cell oxidations.” P. S. Henshaw and D. S. Francis: “A response of Arbacia eggs to tion between THURSDAY, AUG. 24, 8:00 P. M. Dr. Robert Chambers and Mr. C. G. Grand, New York University: “Tissue culture technique and various aspects of the growth of normal and cancerous tissues.” FRIDAY, AUGUST 25, 8:00 P.M. Lecture: Edwin Grant Conklin: “Science and Progress.” Annual Subscription, $2.00 Single Copies, 25 Cents. EVIDENCE OF THE PROTEIN NATURE OF PEPSIN AND TRYPSIN Dr. Joun H. Norrurop Member, The Rockefeller Institute for Medical Research While the behavior of enzymes has been sys- tematically worked out in the last 40 or 50 years, very little advance has been made in the knowledge of their chemical nature so that frequently been as- sumed that they represent an unknown class of compounds. Indirect evidence has been ob- tained, however, that some, at any rate, are proteins. The rate at which they are de- stroyed by heat, for instance, is characteristic for the effect of temperature on proteins. The fact that they are ad- sorbed on finely divided par- ticles is also a property of pro- teins more than of many other classes of compounds. Pepsin, in particular, seems to have protein-1like characteristics, and in fact Peckelharing iso- lated an amorphous protein “Heterochro- “Effects of X- from gastric juice which was highly active and which he considered to be pepsin itself. He was unable, however, to show that the material was a TABLE OF Evidence of the Protein Nature of Pepsin and Trypsin, Dr. John H. Northrop............ 233 Color Changes in the Dogfish, Dr. G. H. Parkersand) Helen Portert cc vvccmcia ds sane te 233 The Biological Laboratory: Electric Excitation in Nerve, Kenneth S. COVER erncstnenioteccisnetoreiain spe intacase nisin watered aca 238 Axon Action Potentials in Nerve, Herbert BGS SEL amir saa creme felayaie cad ecasiel averse ea ais 245 CONTENTS Morphological and Electrophoretic Effects of the Galvanic Current on Griffithsia Cells, Victor Schechter The Absorption of Colloidal Carbon by the Mesonephric Epithelium of Necturus Ma- culosus,. Dr. Alden B. Dawson............. 254 The Austin Ornithological Research Station, DriOWMseAustin’g christie chess deletes 255 WM GifOriale Papen cvacyvo serene eer encores vos 256 Items of Interest 257 234 THE COLLECTING N [ Vou. VILL. No. 68 THE MARINE BIOLOGICAL LABORATORY Aveust 19, 1933 } THE COLLECTING NET 235 pure substance, and the view that this protein was really the enzyme was never accepted. The writer has repeated Pekelharing’s experiments several times in the last 15 years, but until re- cently had never been able to carry the purifica- tion any further. In the meantime Sumner re- ported the isolation of a crystalline protein from beans which appears to be the enzyme urease. Nearly all attempts to isolate enzymes have been done with relatively small quantities of material and in rather dilute solution. Absorption methods have also been extensively used. If enzymes really are proteins, these are not favor- able conditions for their isolation, since proteins are extremely unstable in dilute solution and are easily injured by adsorption on surfaces. The at- tempt to isolate pepsin was again undertaken three years ago from the point of view of protein chemistry, using only those conditions under which proteins are relatively stable, i.e., concen- trated solutions and low temperature. The method was based originally on that of Pekelharing. The last step in Pekelharing’s preparation consisted in dialyzing a protein fraction from gastric juice against dilute acid. Under these conditions a white precipitate is formed which is a protein and which contains most of the activity. This protein sometimes appears in a somewhat granular form and under the microscope looks as though it might be trying to crystallize. Many attempts were made to crystallize the protein without suc- cess. It was noticed finally that this precipitate dissolved if the suspension were warmed to 37° C. and reappeared again upon cooling. These are good conditions for the formation of crystals, and the experiment was repeated under varying conditions and especially with more concentrated solutions, since crystallization in general occurs more readily from concentrated than dilute solu- tions. A more concentrated suspension than usual was warmed to 37° C., and this solution was al- lowed to cool slowly to room temperature in a beaker. The next morning it was found to con- tain several grams Gf beautifully formed crystals in the form of double, six-sided pyramids. They were tested for activity and found to be highly active and also to be protein. The activity is about 5 times that of the most highly active commercial preparation and the quantity of protein which can be transformed by the enzyme is quite extraordinary. An ounce of the crystalline pepsin under favorable conditions would digest about 114 tons of boiled egg in 2 hours, or would clot about 600,000 gallons of milk, while it would liquefy about 10,000 gallons of gelatin in the same time. To imitate these re- actions by chemical means would require a great deal of work and violent methods, but the enzyme accomplishes it without any heat effects and, what is still more remarkable, without any- thing happening to itself. So far as can be de- termined it is present after it has done its work just as it was when the reaction was started. The next question was whether or not this di- gestive power was really a property of the pro- tein or whether it was due to the presence of more highly active molecules accompanying the protein. This question can be answered in two ways. If it can be shown that the material is a pure substance or, in other words, that it contains only one molecular species, then it follows that the protein-like properties and digestive proper- ties must both be attributes of the same molecule. Unfortunately, it is not possible to furnish defi- nite, positive proof of the purity of any sub- stance. It can only be stated that so far it has been impossible to separate it into two or more substances, and this statement may be made with respect to pepsin, The composition, optical activ- ity and digestive activity remain constant throughout 7 successive crystallizations, and this would usually be considered satisfactory proof of the purity of a substance. As a result of these experiments it can be said that no indication was found that the material was a mixture by the usual tests. Owing to the fact that it is a protein, however, it is quite pos- sible that the crystals are a solid solution of sey- eral related proteins. The relation between pro- tein and activity may, however, be tested in an- other way by comparing the loss in activity with the destruction of the protein. If the activity were due to some other molecule associated with the protein it seems probable that conditions could be found which would decompose or change the pro- tein molecule without affecting the activity, or vice versa, whereas if the activity were a prop- erty of the protein molecule itself it would be ex- pected that anything which affected the protein molecule would also affect the activity. It was found that this protein was denatured, that is to say, changed into an insoluble form, in very di- lute alkali. This is quite unusual for a protein. A careful study of this reaction was made, there- fore, and it was found that the loss in activity was just proportional to the amount of soluble protein transformed into insoluble protein when various amounts of alkali were added. If a solution of pepsin is allowed to stand-in dilute acid at 30° C. to 50° C. the protein hydro- lyzes slowly so that the quantity of protein in the solution becomes less and less. Under these con- ditions it was found again that the decrease in activity was just proportional to the decrease in the quantity of protein present. Finally, it was found that the denatured protein formed by the action of alkali could be changed back, at least to a small extent, to the soluble form by allowing it 236 THE COLLECTING NET [ Vor. VIII. No. 68 to stand for some time after the alkali solution had been partially neutralized. The soluble protein recovered in this wav has the same activity as the original protein, ‘These experiments, therefore, show that when the protein is denatured the ac- tivity is lost and when the protein is hydrolyzed the activity is also lost, and, furthermore, that none of the products originating from the hydro- lysis of the protein have any appreciable activity. They are very good evidence that the activity is really a property of the protein molecule. It is known that proteins are denatured by ultra-violet rays as well as by beta and gamma rays from radium, These reactions offer two ad- ditional ways of modifying the protein. Such ex- periments have been carried out by exposing pep- sin solutions to ultra-violet light or radium rays, and the change in activity compared to the loss of protein nitrogen. The loss in activity under these conditions is again proportional to the decrease in protein concentration. Dr. Herriott has prepared a crystalline acetyl derivative of pepsin by the action of ketene. There are six amino groups originally in pepsin so that it is possible to add at least six acetyl groups. As acetylation proceeds the activity be- comes progressively less until it is equal to about cne-half the original. A crystalline acetyl com- pound having about five acetyl groups and prob- ably one amino group may be isolated from this solution. If acetylation is carried further there is an additional drop in activity and an insoluble compound is formed. The results suggest that five of the amino groups of pepsin can be replaced without destroying the activity but that acetyla- tion of all the amino groups results in a total loss of activity. It has been known for a long time that insol- uble proteins take up pepsin and trypsin very readily from solution, and it has been suggested by Waldschmidt-Leitz that this reaction consists in the removal of the active group from the pep- sin protein. When the experiment was carried out, however, and the complex analyzed for pep- sin, the amount of activity present in the foreign protein is found to be just equivalent to the amount of pepsin protein present in the complex. This is analagous to the general reaction between proteins and nucleic acid and is dependent upon the pH. Such complexes may contain 60 to 70 per cent. pepsin. They can be quite easily separ- ated into pepsin and edestin. If the active edes- tin crystals are stirred in cold sulfuric acid at pH 1.0 the pepsin dissolves out and may be isolated from the solution. The edestin crystals remain in- soluble and are now inactive. The pepsin may also be recovered by allowing the edestin pepsin complex to autolyze at 37°C, and pH 2.0. Under these conditions the edestin is digested and the pepsin may be recovered from the solution. There seems reason to believe, then, that pepsin (and probably urease) are proteins; but evident- ly, since there are many hundreds of enzymes, it can not be concluded at once that all enzymes are proteins. There is some reason to believe that try- psin is also a protein, since it has been known since the time of Kuhne to be associated with the protein fraction. In fact, it had been supposed by some workers to be a nuclear protein, but Le- vene was able to show that this was not the case. An attempt was made to continue the methods used by the earlier workers and to isolate a crys- talline protein from pancreatic extracts. The problem turned out to be a difficult one, and a great deal of work was done before any encour- aging results in the way of either a crystalline product or a product of constant activity was ob- tained. The most hopeful method seemed to be a combination of fractionation with acid and salt, as was done in the case of pepsin, but with tryp- sin it was necessary to use ammonium sulphate. A protein fraction was eventually obtained which had a constant activity and gave some indication of crystallization. The work was made difficult by the very unstable nature of the protein. This un- fortunate property made it impossible to allow a solution to stand for more than a few hours, so that the usual process of crystallization, which consists in allowing a solution to concentrate or cool very slowly, could not be used. After a large number of unsuccessful attempts, Dr. Kunitz was able to secure definite, regular crystals by the very cautious addition of strong ammonium sul- phate to rather concentrated solutions of the pro- tein. The crystals are rather small and are of the cubic system. The proof that this material is a pure substance is still more difficult than in the case of pepsin, since it is more unstable. A large number of solubility experiments were carried out, but the results were not entirely satisfactory, as it was found impossible to complete the ex- periments quickly enough to avoid partial decom- position and corresponding loss in activity. The final solutions, therefore, always contained more or less inactive material formed during the prog- ress of the experiments themselves. Several se- ries of solubility measurements were carried out, nevertheless, as rapidly as possible and at 6° C. They were disappointing in that they indicated clearly that the preparation was a mixture. To confirm this result a study was made of the changes in activity when the protein is denatured, as was done with pepsin, except that in this case denaturation was carried out by heating in dilute acid. The trypsin protein when treated in this way becomes denatured and insoluble. This ex- periment showed clearly that the preparation, al- though crystalline, was undoubtedly still a mix- Aucust 19, 1933 | THE COLLECTING NET ture, since a considerable amount of the protein could be coagulated and removed from solution without decreasing the activity of the solution. As the heating was continued, however, and more and more insoluble protein was formed, it was found that the activity began to decrease about in proportion to the formation of insoluble protein. It appeared, therefore, that the original prepara- tion contained two proteins, one of which was easily coagulated by dilute acid and carried no activity with it, while the other one was much more resistant to acid and was associated, at least, with the activity. These results furnished also a further method of purification since, by heating the crystalline material in dilute acid, about one third of the protein could be removed without loss in activity. Considerable amounts of the preparation were treated in dilute acid in this way and a second preparation obtained which was about twice as active as the first one. It crystal- lizes more readily than the first preparation and the crystals are similar. The purity of this mate- rial was again tested by solubility measurements and the results were more satisfactory than with the first preparation but still not really convinc- ing, owing again to the very unstable nature of the substance. The loss in activity when a solu- tion of this substance was heated in acid was just proportional to the amount of native protein changed to denatured. The protein is rapidly digested by pepsin and several careful experiments were done in which the amount of trypsin protein digested by pepsin was comparéd with the loss in activity. They showed very clearly that digestion of the protein with pepsin resulted in the loss of a correspond- ing percentage of the activity, so that whenever a molecule of the protein is digested by pepsin it loses its tryptic power. There is, then, no evi- dence that the products resulting from the action of pepsin on trypsin have any tryptic power. The experiments were varied by allowing the prepara- tion to digest itself in dilute alkaline solution. Under these conditions also the decrease in the protein concentration is exactly parallel to the de- crease in the activity of the solution. It was found by Mellanby and Wolley that trypsin solutions possessed the remarkable prop- erty of retaining their activity after being heated nearly to boiling for a short time in dilute acid. The solutions of crystalline trypsin may also be heated for a short time nearly to boiling without “any loss in activity and, what is still more re- markable, without the formation of any dena- tured protein. This result is obtained only if a solution is allowed to cool before being tested for either denatured protein or activity. If the solu- tion is tested while still hot, it is found that the protein is all denatured and, in addition, that the solution is inactive. It is possible to show, there- aee7 fore, that the formation of denatured protein is accompanied by a loss in activity and, what is more significant, that the reformation of soluble, native protein from the denatured protein is ac- companied by recovery of the corresponding ac- tivity. As in the case of pepsin, therefore, it is found that whenever anything is done to the pro- tein molecule the activity is lost and that, on the other hand, when the denatured, inactive protein is changed back into soluble, native protein the activity is regained. If it be assumed that the ac- tivity is due to some special active molecule, then it must be assumed in addition that the condi- tions for inactivating these hypothetical molecules must be the same for denaturing the protein molecule and also that the conditions for render- ing the hypothetical molecule active again are precisely the same as those for forming native protein from the denatured protein. The behavior of proteins in general is so peculiar and charac- teristic that it is extremely unlikely that any other type of molecule would be affected in the same way and to the same extent so that the possibility that the activity is due to a non-protein molecular species present appears very remote. It is pos- sible, on the other hand, that the preparation is a mixture or solid solution of several closely relat- ed proteins and that only one of these is active. The general properties of pepsin and trypsin which have been determined by these experiments show that they are similar in many respects to hemoglobin, Their peculiar ability to digest pro- teins is lost as soon as any change, such as dena- turation, is made in the molecule. The denatura- tion of hemoglobin likewise results in complete loss of its characteristic property of combining reversibly with oxygen. On the other hand, some of the properties of hemoglobin, such as its combination with carbon‘ monoxide and its char- acteristic absorption spectrum, are retained by the denatured form and eyen to some extent by pieces of the molecule when it is hydrolyzed. In the case of pepsin and trypsin there is, at present, no indication that any of the pieces of. the mole- cule retain their digestive power; but it is quite possible that more careful search would show more or less activity associated with one of the decomposition products. The peculiar properties of hemoglobin are known to be due to the pres- ence in the molecule of a characteristic group which differentiates it from other proteins. It is quite possible that the enzyme proteins likewise contain a characteristic group, but so far no evi- dence has been found for its existence. They are, however, quite different from other known pro- teins in many respects and this difference must be due to some characteristic difference in chem- ical structure. (This article is based upon a lecture presented at the Marine Biological Laboratory on August 18), THE COLLECTING NET [ Vor. VIII. No. 68 THE BIOLOGICAL LABORA COLD SPRING HARBOR ELECTRIC EXCITATION IN NERVE KENNETH S. COLE When the external potential difference be- tween two points of a nerve is changed, the nerve may be stimulated and propagate an impulse. The magnitude of the electric field which is just sufficient to stimulate depends upon: (1) the angle between the axis of the nerve and the direction of the field, (2) the length of nerve which lies in the field, (3a) the length of time for which the field is applied, or in general, (3b) the manner in which varies as a function of time. Effect of Direction and Extent of Electric Field It has long been known that an electric field perpendicular to the nerve axis is very ineffi- cient as a stimulus but the early quantitative measurements of the dependence on the angle were not entirely satisfactory. The work has been repeated by Rushton, 1927, under carefully con- trolled conditions. The uniform field strength E (of a constant duration) which was required to excite a constant length of nerve in a large body of saline was measured as the angle @ between the direction of the field and the axis of the nerve was varied. Within experimental limits it was found that E = E,/cos 6 where E = E, when = O. This means that only the component of the field in the direction of the nerve is effec- tive for excitation. In the same paper, Rushton, 1927, a relation is derived between the exciting field strength E parallel to the nerve and the length of nerve x exposed to the field. For this purpose the nerve is considered as an insulated cable having a rel- atively well-conducting core in which the current is longitudinal and a relatively non-conducting sheath where the current is radial. The maximum current density is found under the electrodes and it is assumed that excitation occurs when this reaches a definite fixed value at the cathode. This is equivalent to a liminal potential differ- ence across the sheath since the resistances are assumed constant. It is then found that —x/d E = E,/(1 —e ) where E = E, when x is large and Q is the length of nerve for which the radial sheath resistance the field is equal to the longitudinal core resistance. Al- though the older results are open to question they follow the general form of this equation, and Rushton’s improved technique gives data which agree very well with the theory. As far as elec- tric fields of constant duration are concerned, we may feel justified in accepting the hypothe- sis that excitation occurs when a liminal potential difference is established across the sheath, or membrane, of the nerve. Effect of Duration of Electric Field When the duration of the electric field is varied, the problem becomes considerably more complicated. DuBois Reymond, 1848, stated that the excitation was a function of the time rate of change of the current density. Fick, 1864, showed that this was only partially true and that for rectangular pulses, in which the current rose and fell practically instantaneously, the duration was a factor. Since that time a relation between intensity and duration of stimulation of the type shown in Fig. 1 has been found for almost every known irritable tissue. Even for a very long duration, excitation will never occur when the intensity of stimulus is less than a certain value which Lapicque, 1926, has named the rheobase R. The duration of a just effective stimulus having twice the intensity of the rheobase has been called the chronaxie y, also by Lapicque. Fig. 1. tion t of an effective stimulus and its intensity FE. F is the rheobase and y is the chronaxie. Diagramatic relation between the dura- Aueust 19, 1933 ] THE COLLECTING NET 239 The first analytical expression for this curve given by Hoorweg, 1892, for a condenser dis- charge stimulus, and by Weiss, 1901, for a rec- tangular pulse, is E = R (1+y/t) (1) When the effective intensity is expressed as a current, the quantity of electricity delivered by an electrode is a linear function of the time. Ex- periments over an extended range of durations show systematic deviations from this simple law. Condenser Models, Rectangular Pulse In 1907, Lapicque considered the nerve sheath as a condenser C with a leak of resistance r; and represented the external and internal resistances by fo. If excitation occurs when the potential difference across the membrane reaches a definite value, the relation between the intensity E of the applied rectangular pulse, and the duration ¢ for excita- tion is given by _t/7 E=R/(1l1—e ) (2) where the rheobase is KR and the chronaxie y = .69 7. When t/z is not too large, this has the form of Weiss’s law, equation (1). Hoorweg’s post- ulate that the rate of increase of excitability dp —t/7 = we Ie dt gives the above result with a rectangular pulse if p = « 7 R for excitation. Recently *Ebbecke, 1927, and Hill, 1932, have extended Lapicque’s concept by considering specifically the inside and outside resistances and the sheath condensers under each electrode with a “resting” potential in series with the leakage resistance. This circuit can be reduced, Cole, 1928, Fig. 2. Equivalent circuit of condenser nerve model. 7» 7; are resistances and C is a condenser. (oe) (eo) 0 To To Ta 18 4 ol 0.2 O30 03 0.6 O9e ti Fig. 3. Plot of Rushton’s, 1932, data after Hill, 1932, on basis of condenser nerve model. Ordinates are log 1) (1—R/E) and abscissae are durations in o. Left is for warm nerve and right for cold. to that of Fig. 2 and gives equation (2) for ex- citation by a rectangular pulse. On the other hand, Blair, 1932, has disregard- ed structure and postulated the excitability p to be increased at a rate proportional to the stimulus and to be decreased at a rate proportional to the change in excitability or dp = KE — k p dt Excitation occurs when / reaches a certain value and for a rectangular pulse equation (2) is arrived at. Since these theories give the same equation, the data can at most only decide whether a con- denser nerve model is satisfactory or not. Blair, 1932, has analyzed a considerable amount of Lapicque’s, 1931, a, b, recent data from this point of view and finds that it fits rather well ex- cept for a constant which seems to depend upon the electrodes. Hill, 1932, has plotted log © (1 — R/E) vs. t from Rushton’s, 1932, data, as shown in Fig. 3, to support the theory. Here Blair’s constant term is assumed to be zero, Hill accounts qualitatively for the effect of electrode size, electrode separation, and fiber diameter, and calculates a membrane 2.2 p thick having a capac- ity of 1.6 - 10%» f. per sq. em. and a specific re- sistance of 10° ohm cm. Diffusion Polarization Models. Rectangular Pulse In 1908, Nernst published a theory of nerve excitation which has had a profound influence on the field. He assumed that the nerve sheath was a semi-permeable membrane which allowed only ions of one sign of charge to pass through. When 240 THE COLLECTING NET [ Vot. VIII. No. 68 Fig. 4. Equivalent circuit of general polariza- tion nerve model. 7, 7; are resistances and p the polarization element. an electric current flows, the permeating ions car- ry only their normal share of the current in the electrolyte on each side but must take the whole burden inside the membrane. This means that they must move into one side of the membrane faster than they are normally delivered up by the solution, while an excess is created in a similar manner on the exit side. Ions of both signs sent to or from the membrane surfaces by diffusion tend to keep the changes from proceeding too rapidly with the result that the concentration change at the surface Ac = Ki \/t where 7 is the current density and ¢ the time it has been flowing. For small concentration changes, the counter electromotive force produced is also pro- portional to i\/t. Nernst then assumed that when the concentration had changed by a certain amount (or the polarization had reached a certain value) excitation took place and E=K’/\/t. (3) For cells of small dimensions, this relation can only be expected to hold for short times, but Hill, 1910, extended the theory to include the interac- tion between two membranes so that it might be expected to hold for longer durations. The sim- plified expression for a rectangular pulse is E = R/(1—p6) (4) Strange as it may seem, p is nearly unity for many cases and then equation (4) the same as equation (2). Lapicque has met this failure of the Nernst theory at long durations and provided a rheobase by an empirically determined “canonical curve” which expresses a large amount of his data, | +e+ Ve— 0)? + 016 ne aa. 2t where 6 = 3.8 times the chronaxie. It should be pointed out that in order to obtain a rheobase it is only necessary to postulate that the diffusion polarization / is but one element of a circuit which may be reduced to that of Fig. 4, that is, it replaces the condenser of Fig. 2. The potential difference across p can never exceed r; E/(1 + 1.) and it is to be assumed that this must reach a liminal value for excitation to occur. Cremer has recalculated the data of Weiss, 1901, and finds that it agrees well with the Nernst theory when the duration is not too long. La- picque, 1926, has proposed the canonical curve as the best generalized expression of his data and it reduces to the Nernst equation for durations of a chronaxie or less. Rushton, 1932, on the other hand, finds that the canonical curve does not fit his data on the frog sciatic. In order to visualize this, Rushton’s data have been plotted in Fig. 5 in the form log E vs. log t. It is seen that for times less than a chronaxie the data are expressed by i S ikirs (except for the same point which Hill believes to be “obviously in error” in Fig. 2 above). « has the value 0.76 for warm nerve and 0.86 for cold, instead of 0.5 as required by Nernst and La- picque. Ninety-nine of Lapicque’s experiments show an average value of « = .656 with a “standard deviation” of .22, Wegel, 1932. It is thus impossible to make a catagorical de- cision between the condenser and the diffusion E 1OR 2R ! <> % SOS ~S ~ es Oho Oo t Fig. 5. Plot of Rushton’s, 1932, data on lo- garithmic coordinate scales. Ordinates rheobase units and abscissae are o. Aucust 19, 1933 ] THE COLLECTING NET polarization hypotheses on the basis of rectangu- lar pulse excitation. Some data fit one and some fit the other, and it is wise to look at other phe- nomena. Condenser Discharge Pulse Hoorweg, 1892, made extensive use of con- denser discharge stimuli and the method is so simple that it has had wide application. Since the form of the condenser pulse is so different from the rectangular pulse it might be thought that the predicted intensity-duration curves might be quite different for the condenser and diffusion polariza- tion nerve models. When A is the “time constant” of the condenser stimulus which corresponds to the duration of a rectangular stimulus ¢ of the same maximum intensity, it has been shown that for the condenser model t = 0.3466 A Blair,1932, while for the Nernst model t = 0.344 A Eucken and Miura, 1911. It seems quite impossible to determine this factor accurately by experiment—as is also: shown by both hypotheses—and Lapicque’s value 0.37 cer- tainly has no decisive value. Sub-threshold Excitability Before consideration of the other excitation phenomena which involve, in general, multiple - stimulation*it is well to discuss a very important result of Bishop, 1928, and the extended investi- gations of Erlanger and Blair, 1931, a, b. Bishop measured the excitability p of a nerve at different intervals ¢ after the application of a sub-threshold constant potential by means of a very short induc- tion test shocks, with the result shown diagram- matically in Fig. 6. If it be assumed that the ef- fect is linear and reversible, we can immediately explain most of the electrotonic effects by Fig. 7, t i ¢ a ar Fig. 6. Excitability p at cathode as a function of time ¢ during an inadequate direct current stimulus E (Bishop, 1928). cathode Pp anode E 7. Saar Sand ae — Ma ae. Fig. 7. Idealized excitabilities p at cathode and anode for make and break of inadequate stimulus E to be predicted from Fig. 6. See Er- langer and Blair, 1932. which are indeed the idealized results of Erlanger and Blair. As the duration of the stimulus is made very short we should find that the excita- bility d Po (t) dt which is again found by Erlanger and Blair. For linearly increasing strength of stimulus £ n= E, f Po (t) dt Oo which does not agree as well as might be hoped for with the results of Lucas, 1907, and Erlanger and Blair, 1931. Such treatment seems to be rather hard on the nerve and may involve still more complicating factors. It seems evident, however, that the explanation of Bishop’s curve will automatically solve many of the questions raised by the use of more than a single pulse for a stimulus, and furthermore that the initial rising portion of .this curve should explain the single pulse excitation. Bishop has proposed a complex resistance-capacity model, but Erlanger and Blair find that at low temperatures the maximum of the excitability curve is quite flat and starts down- ward at a finite rate instead of gradually. This suggests that an adaptation mechanism starts into action at what would be approximately the end of the absolute refractory phase for a stimulated 242 THE COLLECTING NET [ Vor. VIIL. No. 68 nerve,—when the propagated impulse is well un- der way. Since none of the excitation theories (with the possible exception of Hoorweg’s) allow of this decrease from the maximum of excitability for an inadequate constant potential stimulus, and since there are experimental indications that this de- crease may be due to another mechanism, it seems unwise to attempt to apply either the condenser or the Nernst model to other than single pulse ex- citation. Alternating Current Excitation Blair's, 1932, hypothesis and the condenser model both show that for alternating current (re- petitive) stimulation E? — R24 K? «2 where » = 27n and n is the frequency, while the simple Nernst model gives B= R We o There are no experiments which completely sub- stantiate either equation (Asher, 1923, Kruger, 1928, Renquist and Koch, 1930, Blair, 1932) over a wide frequency range. This is not surprising in view of encroachment into the relative and ab- solute refractory phases to be expected at high frequencies and the effect on the relative refrac- tory phase of a stimulus placed in the absolute phase as found by Erlanger and Blair. It should be suggested, however, that the rather abrupt changes in K and R found at different frequen- cies by Blair may well be due to the combined effects of the refractory periods resulting in both a change of apparent threshold, and excitation at less than the stimulating frequency. It now becomes reasonable to assume that only the excitation data for single pulses come within the scope of the two types of theory, and even then it seems impossible to generalize without in- dependent supporting evidence. A Generalized Hypothesis If it is allowable to assume that excitation oc- curs when a counter-electromotive force reaches a liminal value and that, without excitation, the magnitude of this counter e. m. f. p at a specified time, ¢, after the constant current, 7, has started to flow, is proportional to the current—then we may consider it in terms of a resistance r(t) which varies as a function of the time. That is, p = i - r(t). It is then only necessary to put fp in a network of the type of Fig. 4, or its equivalent, and compute its over-all resistance as a function of time. By comparison with experimental data it should be possible to determine r(¢). Unfor- tunately the mathematical problem in this form is not simple and it is only recently that measure- ments of this type have been made. There is, however, another possibility since a variable re- sistance of this type on direct current will be equivalent to a resistance and a reactance (capa- city, or inductance) when measured with alternat- ing current of a certain frequency. In general, both the resistance and reactance will change with frequency, for this is merely on alternating cur- rent application of the Fourier integral equation. When live tissues are measured with alternating current at various frequencies, it is found that they give the same results as a single element p having in series a resistance r, and a capacity Cp, both of which vary with the frequency n.. It is furthermore found, Cole, 1932, that for many tis- sues this element gives 1, Cp) = m where o = 2n and m is a constant, independent of frequen- cy. It is then said that the variable element ~ has a constant “phase angle’. By a Fourier in- tegral analysis which is the direct inverse of that employed by Fricke, 1932,—since we wish to cal- culate his assumption from his answer——it is found that iA (te) Se I ESS (5) where m = cot (az/2) and K’ is a constant. Thus if m is a constant and known we can im- mediately calculate «, which will lie between zero and one. From the data of Lullies, 1928, on frog sciatic nerve, it can be shown that m is constant over a considerable frequency range and has the value 0.49, Cole, 1932. Then « = 0.71 and we would suspect that if Lullies had determined the excita- tion curve he would have found E = Kt~%* when ¢ was less than a chronaxie. Since these data are not available, we note that Rushton’s ex- ponents were 0.76 and 0.86 for warm and cold nerve respectively, while over 60% of the expo- nents quoted by Wegel, 1932, lie between 0.6 and 0.8. The few biological materials for which the al- ternating current data have been computed give values of « ranging from 0.6 to 0.8, with the ex- ception of red blood cells where « = 0.9. The condenser model requires that « = 1.0 and the Nernst model that « = 0.5. Although the alter- nating current data are far from conclusive, the indications are that nerve lies in the region in- cluded by the tissues examined up to the present, and that this region is intermediate between the condenser and polarization membrane models. It is, therefore, suggested that many of the ac- tive and passive phenomena of living tissues may depend upon a membrane polarization having a Aveust 19, 1933 | THE COLLECTING NET 243 variable resistance to direct current of the form of equation (5). Summary. It is commonly assumed that a nerve fiber has a core of comparatively good electrical conductiv- ity with a sheath of much lower conductivity and that if electric excitation is to occur, a liminal po- tential difference across this sheath must be created. The relation of the threshold stimulus to (1), the angle between the nerve and the stim- ulating field, and (2), the length of nerve ex- posed to the field, may be explained on this basis. It has further been commonly assumed that the sheath undergoes a change in polarization as the result of current flow and this has been postulated as due to either (1), a pure static capacity, or (2), a diffusion polarization at a semi-permeable membrane. Neither of the theories developed on these two hypotheses has exclusively explained single pulse excitation data satisfactorily. While they both fail to explain the decrease of excitability in sub-threshold direct current stimulation, there is a_ possibility that this effect may result from a distinct recovery mech- anism. The theories should not then apply to more than the initial rise of excitability and it is not to be expected that multiple stimulation phe- nomena can lend support to either. Alternating current measurements of the resis- tance and reactance of nerve and other tissues suggest that the polarization of the sheath is neither that of a static capacity nor a simple diffu- sion, but intermediate between the two. Applied to the excitation of nerve, this leads to a strength- duration relation of the form Dai ts when the time is not too large, From alternating current data on one nerve « = 0.71 while from a recent set of excitation data « = 0.76. Thus alternating current measurements do not support either the condenser or the diffusion po- larization models but predict a type of polariza- tion which has some support from excitation data. BIBLIOGRAPHY Asher, L., 1923, Skand. Arch. Physiol., 43, 6. Bishop, G. H., 1928, Am. J. Physiol., 85, 417. Blair, H. A., 1932, (a) J. Gen. Physiol., 15, 709. (b) ibid. 15, 731. Cole, K. S., 1928, J. Gen. Physiol., 12, 29. Cole, K. S., 1932, J. Gen. Physiol., 15, 641. DuBois Reymond, E. 1848, Untersuchungen iiber tierische Elektrizitat, Berlin. Ebbecke, U., 1927, Arch. ges. Physiol., 216, 448. Erlanger, J., and Blair, E. A., 1931, (a) Am. J. Physiol., 99, 108. (b) ibid, 99, 129. Eucken and Miura, 1911, Arch. ges. Physiol., 14, 593. Fick, A., 1864, Untersuchungen iiber die elektrische Nervenreizung. Braunschweig. Fricke, H., 1932, Phil. Mag. (7), 14, 310. Hoorweg, J. L., 1892, Arch. ges. Physiol., 52, 87. Hill, A. V., 1932, Chemical Wave Transmission iu Nerve, Cambridge. Hill, A. V., 1910, J. Physiol., 40, 190. Kriiger, R., 1928, Arch. ges. Physiol., 219, 74. Lapicque, L., 1926, L’excitabilité en fonction du temps, Paris. Lapicque, L., 1907, J. Physiol. et Path. Gen., 9, 620. Lapicque, L., 1931, (a) J. Physiol., 73, 189. (b) wi 78, 219. Lucas, K., 1907, J. Physiol., 36, 253. Lullies, H., 1928, Arch. ges. Physiol., 221, 296. Nernst, W., 1908, Arch. ges. Physiol., 122, 275. Renquist, Y., and Koch, H., 1930, Skand. Arch. Physiol., 59, 266. Rushton W. A. H., 1932, J. Physiol., 74, 424. Rushton, W. A. H., 1927, J. Physiol., 68, 357. Wegel, R. L., 1932, Annals of Otology, Rhinology and Laryngology, 41, 740. Weiss, G., 1901, Arch. ital. Biol., 35, 1. Discussion Dr, Blinks: Would you introduce a chemical reaction or metabolic element—in addition to the purely physical effects such as polarization and diffusion—to account for the deviations of the excitability curve from the theoretical forms? There is apparently such a metabolic response in the plant cell which follows the production of al- kalinity by an external medium. When ammonium salts are applied to Halicystis there is a tendency for the potential to give a cusp and then recover as if the cell responded by increased acidity. The same can be observed with current flow. Of course, we can say nothing about the relations in nerve from the case of plant cells where it is a matter of seconds or even minutes in the case of ammonium application. Dr. Cole: I have no concept of the adapta- tion or recovery portion of the curve. The results of Erlanger and Blair at low temperature suggest that this phase may start in very abruptly for in- adequate stimuli and be linked with the relative refractory phase of an adequate stimulus. It would then seem reasonable—and considerably simpler—to ascribe it to a delayed process not directly connected with the rise of excitability which is under theoretical consideration. Dr. Gasser: There is one theoretical differ- ence between the Nernst and condenser hypo- theses which may be put to experimental test. According to the Nernst formula the energy of the current necessary for excitation is constant, whereas it follows from the exponential formula that at some value of the time the energy runs 244 THE” COLLECTING NET [ Vor. VIIT.“No: 68 through a minimum. In practice there is a current of minimum energy. Dr, Cole: I have not worked out the energy matter and the discussions which I have seen are not entirely convincing. For example, the energy has sometimes been computed for a constant cur- rent rectangular stimulus as proportional to i°t, which leads to the constant energy for the Nernst model and the minimum for the condenser model. We are concerned with the energy delivered to the nerve t W=f eidt which reduces to the above simple form when the resistance of the nerve is constant but may not do so otherwise. It is also possible that there is a dif- ference between constant current and constant voltage stimuli, so one must also be sure that the assumptions of the theories are satisfied by the experiments. Dy, Miiller: How large is the threshold value of an electric field of long duration ? Dr. Cole: The rheobase for frog sciatic as usually set up is less than 100 mv. Dy, Miller: Is it not possible that the current and not the voltage determines the excitation? The fact that a very slowly increasing voltage does not produce any effect might be due to the production of counter e m f s which can develop during a slow increase of current, but which have no time to establish themselves if the voltage in- crease is fast. Dr. Cole: It may be useful to take such a viewpoint—particularly for the falling portion of the excitability curve. The phenomena of linearly increasing and multiple stimuli are not handled by the theories in which it is assumed that the excitation is caused rather than hindered by the counter e m f. Dr. Gasser: Is there only one curve of excit- ation? The data which fit the canonical curve do not fit at all well to the exponential formula. Dr. Cole: It is probably too much to hope that all irritable tissues will fit the same formula but there seems to me to be a decided possibility that many tissues will polarize according to a law t9 and have values of 8 which lie in a limited range—say from 0.6 to 0.8. It may then be diffi- cult to distinguish the lower and upper ends of the range from the Nernst and the condenser formulae respectively. Dr. Blinks: How do you consider the evi- dence connecting chronaxie with the speed of propagation ? Dr, Cole: I know very little of the evidence, but I understand it has been found recently that the relationship is not as simple as had been pre- viously supposed. Dr. Shedlovsky: Over what frequency range has the phase angle been found constant? Dr. Cole: Lullies measured the resistance and reactance of nerve from 28.8 cycles per sec- ond to 332,000 cycles per second. His data indi- cate a constant phase angle element which pre- dominates except at the two highest frequencies, 48,500 cycles per second and 332,000 cycles per second. Dr. Fricke: In only very few cases do we find the polarization capacity of cells to vary as the inverse square root of the frequency. Usually the variation is less fast but increasing as the frequency is increased. Aucust 19, 1933 ] THE COLLECTING NET AXON ACTION POTENTIALS IN NERVE HERBERT S, GASSER Professor of Physiology, Cornell University Medical College Physiologists record the potentials generated in nerve during activity for a number of reasons. The primary one is to obtain the form of the po- tentials themselves, as the electrical sign of activ- ity is the only one which can be located with any precision. Heat measurements, ingenious as they are, are still under distinct limitations in their ability to reveal the times at which the heat is evolved ; for instance, they show very little of the cycle which must obtain in the course of a single impulse; and chemical studies only bring to light the stable end products of metabolism and _ tell nothing whatever about when the catabolites are formed. The potential curve being thus the one record of action which gives definite information as to when events occur, it becomes a frame of reference for possible correlations with other signs of activity and for comparison with irrita- bility changes (the time course of the latter may be determined with the precision necessary to make such a comparison possible). Furthermore, ac- tion-potentials may be used for the tracing of im- pulses through the nervous system and for an- alysis of nerve fibers into their components with the idea of associating the parts with the fune- tions they subserve. In all the foregoing enterprises the electro- physiologist is on solid ground, but he is not sat- isfied to confine himself within these limits of safety ; we often find him in the hazardous occu- pation of inferring from the potentials, them- selves, what the processes behind them may be. Just how far this is from sound practice follows from but a cursory consideration of the precau- tions taken by a physical chemist in the measure- ment of a potential. In the first place the physical chemist starts out with pure chemical substances of known concentration. If a chemical reaction be involved, its nature is known and the reaction it- self is chosen because it meets the necessary ex- actions imposed with respect to reversibility. The cells are set up in such a way as to avoid un- necessary diffusion potentials and the readings are made with the cell so balanced that it is sup- plying no appreciable current. None of these con- ditions is fulfilled when a lead is made from the surface of a nerve; the source of potential is un- known and no matter what precautions may be taken against the drawing of current into the cir- cuit in which the nerve is placed, the damage is already done. The seat of production of the po- tential is imbedded somewhere within the nerve and must set up currents of an undetermined nature in the inactive portions of the tissue, These can hardly fail to be without repercussion on the source; in fact, Lillie, in his theory, makes very definite use of the current in the local bio-electric circuit as an aid in restoring an active portion of a nerve to its normal resting state. Furthe..nore, in the absence of a knowledge of the resistance of either the source or the shunting resistance, it is quite impossible to know what relation the po- tential recorded has to the actual working poten- tial. On account of this unsatisfactory situation but little attention has been given to the absolute value of recorded potentials and all the interest has centered around the time at which they oc- cur. The magnitudes can be of use only in cum- parison of the same fibers in a given nerve uader two conditions of the environment in which the unknown and uncontrolled variables may be con- sidered to be sufficiently constant. ; From the foregoing remarks it can clearly be seen that it would be to the advantage of every electrophysiologist to nail this notice on an imaginary laboratory door—you cannot determine a process from a potential. It is not to be expect- ed that attempts to do so would be thereby pre- vented, but it would be done in the hope that the products of such attempts would at all times be recognized for what they are: a form of inspired guesswork. Now the general subject matter of this symposium indicates that the interest of the participants is focused primarily on the nature of the potentials rather than on their workaday ap- plications ; that is, on just that aspect of the sub- ject on which it is most difficult to supply infor- mation. \When confronted with a problem which has no definite answer, the only course open is to state the problem definitely so as to have clearly before one the phenomena which demand expla- nation; and this I shall proceed to do. Such theoretical considerations as suggest themselves will be left to the end. For the understanding of what is to follow it is necessary to mention two points in connection with the technique of making a lead. What is de- sired is the change of potential with respect to time at some point, as 4 (fig. 1), on the surface of the nerve and an appropriate electrode is lo- cated at that place. The difficulty comes in con- nection with the second lead which is necessary for the completing of the circuit. If the potential change is to be recorded without distortion, the potential at the second lead must not change. In practice it is of no avail to place the second lead off at a distance from the nerve, because that in 246 THE COLLECTING NET [ Vor. VII. No. 68 lead to the intact portion but that some twigs of STIMULUS LEAD current can run from the surface next to the ends Figure 1. Diagram of the position of the elec- trodes on a nerve when a “monophasic” lead is recorded. The theoretical movement of ions is indicated in the region polarized by the stimulat- ing current. the nerve’s surface and the sum of all the poten- tial-changes led off from the surface does not add up to zero. If the second lead be placed on the surface of the nerve the two leads are alike and one obtains only the momentary difference between them. So the routine procedure is to de- stroy the nerve under one electrode (6) by heat- ing or crushing and thus prevent the impulse from penetrating to this region. In theory the plasma membrane is now destroyed and the lead from electrode B is continued by way of the cytoplasm in the interior of the intact portion of the nerve to the inside of the plasma membrane in the region of A. In actual practice the proce- dure nearly always fails to achieve the desired result. Even after a fresh crush or heat coagulation some of the nerve’s activity is led off by way of electrode 6, producing what is known as the diphasic artifact, and, as the preparation stands, the lead becomes progressively greater. This is usually explanied as due to the re-establishment of some degree of polarization at the dead-live junction, so that the end of the nerve can undergo a_ potential change (at the line c-d) which can be effectively led off by the dead portion of the nerve which is now applied in the direction normal to the surface. The most effective methods of obtaining monophasic leads employ potassium salts or co- caine, the one method permitting a maximum demarcation potential, the other none at all or even a positive one’), It is difficult to see what these differently acting substances have in com- mon unless it is the prevention of formation of a surface film at the end of the nerve. Potassium salts destroy the surface film and by their con- tinued presence must prevent its reformation. Cocaine probably produces a block by an action at the surface of the fibers, and it is quite pos- sible that the interior is so little affected that there is no tendency of the protoplasm of the un- narcotized portion to wall itself off from the nar- cotized part. Even the last two methods often fail to free the nerve entirely from signs of diphas- icity. This may be due to the fact that a dead continuation of the nerve is not a perfect end-on by way of salt solution in the spaces between the fibers, in the sheath, and on the surface. Another method of obtaining a monophasic lead is to place the electrode A close to c-d so that the diphasic artifact is almost directly under the first phase. The result is then to depress the height of the latter rather than to distort its form. Only limited application of this method is possible, however, because the spike cannot be recorded beyond the point at which it still reaches full magnitude; and the method is useless for after-potentials. The second point in connection with the tech- nique of leading arises from the fact that nerves are not homogeneous in their composition. They are made up of fibers with and without myelin sheaths and having diameters ranging from 20u downward. If a nerve be stimulated with a strong very fast induction shock so as to cut the utiliza- tion and latent periods to a minimum, all the fibers will become active under the stimulation cathode at the same time, but they will be con- ducted along the nerve at very different rates, there being in the neighborhood of 100-fold varia- tion between the fastest fibers and the slowest. The result is that, 1f a lead be made at a distance from the stimulus, the impulses undergo consid- erable temporal dispersion and the potential pic- ture, which is the sum of the potentials in the in- dividual fibers, fails to show the form of the po- tential as it exists in any one of them (fig. 2). To obviate this difficulty several procedures are open. One of the best is to lead directly from the stimulating cathode with the employment of very fast induction shocks weak enough to stimulate only a portion of the more irritable alpha fibers. There is then no temporal dispersion and the con- ditions as to homogeneity of material, utilization period of the shock, and latency, introduce no se- rious difficulty. Another method is to use very weak shocks and considerable amplification. The fibers entering into the formation of the poten- tial are then so homogeneous in their qualities, and their velocities of conduction so much alike, that conduction may be permitted with the oc- currence of only negligible temporal dispersion. This method frees the front of the wave from the shock artifact but is not so useful for the low potentials which come at the end of the response. Carried to the limit the method can be made to reveal activity in single fibers. This is not neces- sary for many problems and when it is tried, new forms of distortion are encountered which are not inherent in the high amplification involved. In order to differentiate the action potential as it exists in any one fiber from the composite picture obtained from a mixed nerve it is designated as the axon action potential. Aucust 19, 1933 } THE COLLECTING NET : 247 Subthreshold phenomena. In the short inter- val between the start of a potential applied to a nerve for the purpose of stimulating it and the beginning of the actual disturbance which will be propagated, there is ample evidence that much is going on. The subject has been studied most re- cently and in greatest detail by Erlanger and Blair, the method being to apply a conditioning potential at an intensity below threshold and at various intervals thereafter to test the irritability with a fast induction shock. The value sought is that of the smallest shock which will bring the excitation process to threshold. When the condi- tioning shock is also an induction shock, the irritability immediately rises and for a period of about 0.50 (frog nerve at room temperature) ex- citation may be brought to threshold with a weak- er sheck than is necessary for resting nerve (sum- mation interval). Then the irritability falls below normal (depression phase). It reaches a minimum at about lo, after which it returns toward normal over a period of 4o or longer—a duration very similar to that of the refractory period which would have supervened had the excitation gone above threshold. The interesting feature of this cycle of irritabil- ities which is set up by a subthreshold shock is that there is nothing to parallel it in the electrical potential picture. If a lead be made from the stimulating cathode, all that is obtained is the well-known disturbance which is called the shock artifact. It is occasioned, among other things, by the polarization which is produced in the nerve structures and shows as a _ decremental curve which usually falls to half-value in well under 0.5e. Neither in form nor duration does it have Form of the action potential when conducted impulses are led from the sciatic nerve of the green frog. Single sweep recorded on a cathode ray oscillcegraph. Temperature 27° C. Con- duction distance 48 mm. The time is marked in o. Figure 2. The first deflection is the shock artifact; the time between it and the spike is the conduction time. The spike shows a, 8, and a small y wave. any relation to the irritability curve. If the temperature of the nerve he lowered the cycle of irritability changes is considerably prolonged, while the temperature coefficient of the artifact is so nearly unity that it seems to be so in actual measurements. At the anode the irritability curve is the mirror image of that at the cathode). If the conditioning shock be a rectangular cur- rent applied at a subrheobasic level, the irritabil- ity rises for a time, reaches a maximum which may be a plateau, then falls off again. The curve gives evidence that two processes are in opera- tion, one acting to increase irritability, the other to decrease it. The latter is brought out distinctly when the current is broken for the level of irritability then passes quickly into a depression phase"). As with the induction shock there is no potential sign of this irritability cycle. Even when the conditioning current is 95 per cent. of rheobasic strength, the electrical picture gives no hint of the potential change which would occur if only the remaining 5 per cent. of the threshold strength were added to the current. The spike. \Vhen the rectangular current is strong enough there appears quite abruptly, after a period of utilization which depends upon the strength of the current, a typical self-limited transient disturbance. This is the “action current” of the older literature, but it will be referred to as the “spike potential” to differentiate it from other potentials which also occur. The periods of utilization plotted against the strength of the cur- rent give the characteristic time-strength curve of excitation. Bishop'*’ has shown that the curve may be predicted, at least as a first approxima- tion, on a polarization basis, the times being de- termined as those at which a constant amount of polarization would occur at the several strengths of current. It is thus made apparent that a defi- nite amount of work must be performed upon the nerve before the spike is, in effect, released— we are dealing with a process which has the ap- pearance of trigger action. Once started the spike develops on an all-or-nothing basis; both its mag- nitude and time course are determined by the nerve itself and not by the manner in which the impulse is set up. The front of the spike is an S-shaped curve in which the maximum slope is attained very early. Following the spike crest (0.30 is a value for the crest time’ often seen in frog nerve), the decline of potential is slower than its rise. Restoration to within 5 per cent. of normal occurs in 3 cresi times; then the decline becomes more gradual, and what appears to be spike may he traced as long as 20-25 crest times'*’. It is at the bend in the curve at about 3 crest times that the nerve is first able to give a second response. The relative- THE COLLECTING NET [ Vor. VIII. No. 68 Figure 3. The parts of the axon action po- tential of the green frog sciatic nerve. The detlec- tion is upward when the active electrode (4 fig. 1) is negative. A. Spike. Record of the most irritable fibers only (a fibers) made after 4 mm. conduction; it gives approximately the form of the axon spike. Temperature 20°C. ; time marks, lo. B. Negative after-potential. Fresh nerve at 23°C. ; time marks, 100. Note that the potential ends by crossing the zero line. C. Positive after-potential in a fresh nerve with a very short negative after-potential (30c). The positive after-potential has a maximum value of 12 microvolts and amounts to 0.08 per cent. of the crest height. Time marks, 100c. ly refactory period begins at that point and is us- ually considered as having no potential sign, but it is possible that a correlation may be found with the tail of the spike. Throughout the general run of laboratory pro- cedure the spike maintains its form with great fidelity. Unbalancing the ionic environment of a nerve by the addition (or subtraction) of ions of the alkali metals or the alkaline earths (Graham) or of hydrogen ions only produces a change in height, usually a decrease. The same is true of asphyxia and drugs of the veratrine group. The one variable to which the spike is distinctly sus- ceptible is temperature ; cooling causes a prolong- ation accompanied by a considerable falling off in height. The Oy») of the duration is about 2 in the region of 20° C., and it increases progressively as the temperature is lowered. All the spikes in a mixed nerve are not alike. Three types can be readily identified: the A type which has already been described, the B type last- ing ten times as long, and the C type with a dura- tion more than seventeen times as great; but, in the light of the experiments recently reported by Blair and Erlanger, one can no longer consider the spike durations as completely described in three groups. These authors have examined the potentials in single fibers and found a continuous range of durations. The duration is nearly con- stant in most of the A range, as previously held; but it begins to increase in the slower A fibers and increases more rapidly below this point. From the new findings it follows that the existence of the major elevations in the action potential from mixed nerve are due to the predominance of fibers of certain types rather than ‘to the exis- tence of sharply differentiated kinds of fibers. After-potential. The disturbance which makes up the total of a single nerve response ends with a long negative potential of low magnitude, fol- lowed by another longer and lower potential with a positive sign (fig. 3, B and C). The negative potential, which is called the “after-potential,”” has been most studied. In contrast with the spike it is very variable both as to magnitude and duration. It practically disappears in cold frog nerve and is inconspicuous in mammalian nerve unless ex- aggerated by laboratory procedures. In a fresh frog nerve it may last for a period as short as 20c. After a period of stimulation its duration is in- creased, and when augmented by veratrine poison- ing it is to be measured in seconds rather than in sigmas. The after-potential can be brought into existence only following a spike, and it was first thought to be a long drawn-out continuation of the latter; a number of however, show that it is better to consider it as being caused by a separate process in the cycle of events which occurs in connection with a nerve impulse. In the first place the action-potential does not change form as a whole when a nerve is cooled ; the spike is prolonged while the after-potential becomes shorter. The constancy of the spike and the variability of the after-potential have already observations, Aveust 19, 1933 | THE COLLECTING NET 249 been mentioned; as would be expected from this observation, the two may be differentially modi- fied by changes in the environment of the fibers. Certain substances which in the necessary dosage have but relatively little tendency to modify the spike have the power of greatly prolonging and augmenting the after-potential. Chief among this group is veratrine and to a lesser extent its phar- macological allies, protoveratrine and aconitine 1) An analogous effect is also produced by an excess of the ions of the alkaline earths (Gra- ham), Ba++ is the most effective, then follow Cat* and Mgtt, while strontium falls a long way behind. In sufficient dosage all the fore- going substances can depress the spike, but this is not necessary for the effect on the after-potential to appear. Certain other substances which have a tendency to depress the spike have a much greater tendency to depress the after-potential. ‘hese are the univalent cations, particularly K*, Rb*, and NH,* ‘?; and the aliphatic narcotics. Asphyxia acts in a similar manner"). The anions are without effect (unless they are Cat + precipitants) as one would expect from the gen- eral lack of permeability of the nerve for anions. More important than the foregoing evidence in the differentiation of the after-potential from the spike is the fact that the after-potential does not always start out at a maximum. If the after-po- tential were merely a retardation in the decline of the spike, then whatever point we might select as the start of the after-potential should be at greater negativity than any subsequent point; but in numerous records this condition is not fulfilled, the after-potential shows clear signs of having a rising phase of its own. The best way to prepare a nerve for demonstration of a rising phase of the after-potential is to poison it with calcium or veratrine and then stimulate it rapidly. A re- sponse evoked in the period after stimulation may then show an after-potential which increases for 50 « or longer. It is one of the maxims of phar- macology that drug action is unable to create any new processes in cells; all that it can do is to make a quantitative change in existing ones. When this change is an increase, pharmacology may be of great service to physiology because it may bring to light properties which would other- wise be missed. This was the case in connection with the rising phase of the after-potential ; when it had once been found in poisoned nerve it was possible to demonstrate it in unpoisoned nerve. In this the usually bothersome diphasic artifact proved a help. It had been tacitly supposed that all the potential after the diphasic artifact was after-potential, but a close scrutiny of the diphasic notch revealed that in it there was a small devia- tion which was not accounted for (fig. 4, B). The explanation of this deviation was derived from a theoretical reconstruction of the diphasic artifact. For this purpose the most perfectly monophasic spike-curve available was employed. The second phase was placed in a position deter- mined by the conduction time between a pair of leading-off electrodes, assigned a magnitude which would lead to the imitation of the potential form as recorded in actual experiments, and the two phases added algebraically. The summation curve (fig. +, 4) led to the hitherto unsuspected result that there is a remnant of negative spike- potential after the diphasic notch. This was labeled the T wave in analogy with a_ similar event in the electrocardiogram. Its usefulness lies in the fact that when its crest is once identi- fied we know that from that time onward the spike potential will fall. If now the combined potential is still rising we have definite assurance that the after-potential must be rising, because the correction for the spike is a subtraction and not an addition. In many nerves the crest of the af- ter-potential cannot be identified, probably be- cause the crest is low and early and obscured by the end of the spike. The significance of the foregoing detailed anal- ysis lies in the fact that there are at one and the same time signs that one potential is rising while another is falling. A reconstruction from a Figure 4. phasic spike of a diphasic artifact having a poten- tial 10 per. cent. of the first phase. mono- B. Record of after-potential in a green frog nerve, 22°C., 3 mm. conduction, submaximal re- sponse. C. Shows composition of B. Curve from A summed with theoretical! form of after-potential to give the wave form recorded. All ordinates per- centage of crest height. 250 Positive after-potential. The final restoration of the resting potential to normal occurs from the positive side (fig. 3, C). In a fresh nerve, in which the negative after-potential might last 20c, the positive swing would be visible for about 200c; under other conditions the period of post- tivity may be very much longer and after a tet- anus of a minute, it may last a half hour. It is the only one of the nerve potentials which has a duration in any way comparable to the period of increased heat production after a tetanus. Considerable doubt has existed in the minds of many physiologists, including myself, as to whether the positive wave represents any real process in nerve, because of the possibility that it may be an artifact occasioned by negativity at the dead-live junction; but a recent re-evaluation of the subject has convinced me that one cannot rea- sonably reject the obvious interpretation that the potential is due to a change in the positive direc- tion under the active lead. One of the assumptions on which the possibil- ity of its being an artifact was based was that the negative after-potential is prolonged in the region of the junction with dead nerve, just as it is ina nerve which has stood for some time or has been much stimulated. It would then outlast the nega- tivity at the active lead and record as an artifact in the positive direction. An actual lead made in the vicinity of the junction showed, however, that on the contrary the after-potential is small and short just as it is in very deteriorated or potas- sium poisoned nerve. Another explanation of the positive wave which has been proposed is that it is a temporary increase of the demarcation poten- tial occasioned by a breakdown, as the result of activity, of a partially reformed plasma membrane at the end of the nerve. If this were the case the spikes recorded in the usual way should show a progressive decrease in their diphasic artifacts during a prolonged tetanus ; but tests of this point show that the decrease does not occur. Finally, if a lead be made under the most perfect mono- phasic conditions, such as are obtained by the utilization of potassium for the production of the indifferent lead or of Bishop’s cocaine method, the positive potential remains unabated, although the spikes do not show a trace of a diphasic ar- tifact. These are the potentials as they are known to occur in nerve. The spike-potential is the un- doubted sign of the nerve impulse itself; it has neyer been found to be absent when the end ef- fect has shown that an impulse has passed over a nerve. The after-potentials are very probably connected with restoration processes or the main- tenance of nerve in the proper state to conduct This interpretation follows from their long duration, the necessity of a continued supply spikes, _ THE COLLECTING NET [ Vor. VIII. No. 68 of available oxygen for the existence of at least the negative after-potential™ !°), and the greater metabolism per spike which occurs when the atter-potentials are large''*’. Further prog- ress in the interpretation of the after-potentials must come through their correlation with other signs of activity. For the theorist who would explain the origin of nerve potentials, the after-potentials have greatly complicated the problem, for three poten- tials now await elucidation instead of one. Nerve potentials and the membrane hypothesis. The starting point of all theories is the membrane hypothesis, which postulates a polarized surface at the boundary of the axon capable of under- going transient depolarization during activity. In its simple form the hypothesis is hardly more than a restatement of the facts and as such, af- fords a very convenient form in which to express the course of neural events. Historically the theory has been of much importance, but it has reached a stage in which there is danger of its be- coming a means of lulling the mind into a state of satisfaction rather than of serving the proper function of a theory: the suggestion of experi- ments. Also, one encounters many statements made under the egis of the hypothesis which will not withstand analysis. When a polarizing current is applied to a nerve, cations are carried to the inside of the membrane in the region of the cathode, and anions to the outside, in such a way as to oppose the polarized ionic distribution believed normally to obtain there (fig. 1). The implication is often encoun- tered that this decrease of depolarization is di- rectly responsible for the depolarization during activity; but a review of the subthreshold phe- nomena easily shows that such an interpretat.on is quite inadequate. The ionic migration pro- duced by a just subthreshold shock must be near- ly as great as it is at threshold, yet there is no trace locally of any effect resembling the spike. The depolarization which occurs during the spike must therefore be due to a process inaugurated by ioni¢ movement and not to be due to the move- ment itself. It is of much greater magnitude than the preparatory depolarization and amounts more nearly to a complete disintegration of the surface. What we need to know about nerve, more than anything else, is the immediate cause of the sud- den local breakdown in the surface film. This perplexing point is usually glossed over in theo- retical treatments by making the jump directly from the ionic concentration of the Nernst hypo- thesis to the depolarization idea of the membrane hypothesis, although Bernstein, in his pioneering formulation of the subject, proposed in no uncer- tain terms the intervention of a chemical link at this point. The importance of this stage in the Aucust 19, 1933 } THE COLLECTING NET 251 cycle of activity is illustrated by the beauty of the mechanism. For short periods at least, frog nerve at room temperature may produce 800 spikes per second; in theory this means 800 depolarizations per second and as many restorations (the de- polarizations are, of course, much smaller than normal at this frequency ). The language of the membrane hypothesis treats the membrane as though it had a separate existence. This is justifiable only on the ground that the language is figurative. It is well to keep in mind that the barrier at the surface of a cell is maintained only at the cost of a constant expendi- ture of energy and that it disintegrates if the cell be deprived of oxygen for any length of time. The composition of the cell-surface must vary with the cellular metabolism; that is, with the molecular species available at the moment for concentration in the surface, according to the Gibbs-Thomson principle. Conversely, if the sur- face be altered by external means it is no longer in equilibrium with the interior, and one would expect the metabolism to change. In other words, there is a continuous reciprocal relationship be- tween the surface and the cytoplasm. The sur- face changes may well be attended by potential changes, and by some such mechanism as this the after-potentials may be accounted for. Whether the spike, with its larger potential and different properties, can be accounted for on the same basis is more uncertain. In any case the idea that the surface potential is a reflection of several pro- cesses 1n a nerve makes it understandable that one potential may be rising while another is falling. The size of the spike has always been asso- ciated with the magnitude of the demarcation po- tential. If the propagated disturbance be a de- polarization phenomenon, the height of the spike is determined by the difference between the rest- ing potential and that at the point of maximum depolarization, from which it follows that the spike height could theoretically never be higher than the demarcation potential. This condition seems to have been satisfactorily met by experi- ment, but only after special precautions to obtain the demarcation potential at full value. In turn the demarcation potential is best explained on the basis of a concentration difference on the two sides of the plasma membrane. The idea was ori- ginally launched by Bernstein, with the proof that the potential is determined at the intact surface and that its size varies with the absolute tempera- ture; and the subsequent history has been such as to support the notion. The concentration dif- ference between the potassium inside and outside the fiber has been found to be sufficiently great to produce the potential (Cowan), and physical chemistry has provided two very satisfactory models which show ways in which a concentration .theory is of special interest. potential can be exerted without the presence of metals. One concentration-cell If increased per- meability, and therefore low resistance, be char- acteristic of an active ,region, current must flow through the active region from the inactive ones. When a concentration cell supplies current it gets the energy from its surroundings, therefore the nerve should tend to cool just as does an electric organ—particularly if the organ is caused to dis- charge through an external resistance in which the energy can be expended’). If, now, the energy of the local bioelectric currents be again all dissipated as heat, the net effect on a thermo- pile placed on the outside of a nerve should be zero. In an actual experiment, however, a ther- mopile records some initial heat. On account of the theoretical importance of the question of the existence of initial heat, A. V. Hill has ex- amined his thermal data to see whether they are susceptible of explanation on the basis that all the heat produced is recovery heat. He has come to the conclusion that a certain small portion of the heat must be considered as produced at the time of the impulse. This means that there must be some additional exothermic process; and it is quite possible that this process may enter into the mechanism at the stage in which the effects of ionic accumulation lead to the final opening of the membrane. While we speak of the spike as due to a de- polarization of the plasma membrane, it would be quite incorrect to think of it as a complete de- polarization. This follows from the behavior of the spike in cooled nerve. While the demarcation potential falls off as the absolute temperature the spike height falls off very sharply and at the same time becomes much longer. Thus the spread between the resting and activity potentials be- comes narrower. To account for the change we may make use of a possible increase in viscosity in the surface film, but we must also consider the effect of temperature on the final unknown event responsible for the depolarization; if that were understood we would be much farther on our way to the understanding of nerve. consequence of the REFERENCES 1. Amberson, Parpart, and Sanders. 1931. Am. J. Physiol., 97, 154. 2. Bernstein and Tschermak. 1906. Pfliiger’s Arch., 112, 531. 3. Bishop. 1928. Am. J. Physiol., 84, 417. 4. Bishop. 1932. J. Cell. and Comp. Physiol., 1, 177; 371. 5. Blair and Erlanger. 1933. Proc. Biol. and Med., 30, 728. 6. Cowan, Unpublished Soc. Exp. experiments quoted 252 THE COLLECTING NET [ Vor. VIII. No. 68 from “Chemical Wave Transmission in Nerve,” A. V. Hill, 1932. 7. Erlanger and Blair. 1931. Am. J. Physiol., 99, 108; 129. : 8. Gasser and Graham. 1932. Am. J. Physiol., 101, 316. 9. Graham. 1933. Am. J. Physiol., 104, 216. 10. Graham and Gasser. 1931. J. Pharm. and Exp. Therap., 43, 163. 11. Hill, A. V. 1932. Proc. Royal Soc., B111, 106. 12. Schmitt and Gasser. 1933. Am. J. Physiol., 104, 320. Discussion Dr. Irwin: J think that the temperature ef- fect on the spike potential may be a matter of viscosity. The temperature coefficient of viscos- ity in chloroform is very large. Dr, Abramson: \Vould that be a change in the sheath, something analogous to gel formation, or to an increase in the Newtonian coefficient of viscosity ? Dr. Gasser: Viscosity and perhaps conduc- tivity. The temperature coefficients of viscosity of some of the oils are very much like those of the nerve functions. Also specific conductance falls off very rapidly when a lipoid approaches its congealing point, as for instance tetraethylam- moniumbromide in a mixture of alcohol and lecithin. Dr. Abramson: It has to do primarily with the true viscosity, then, for there is very little change in the electrolytic conductance during the sol-gel transformation. Dr. Blinks: emulsion ? Dr. Gasser: Dr. Cole: As I remember Hill’s calculations, the energy of the initial heat would not be far from what you might compute the total electrosta- Was it a single phase lipoid or an Probably an emulsion. tic energy stored in a membrane to be, consider- ing it as being a condenser. Dr, Gasser: That is correct. Hill has also pro- posed the cycle; discharge of the condenser and recharge by means of a diffusion potential, with a net heat for the cycle of zero. Dr. Cohen: The single nerve fibers vary, of course, in diameter. Do the action potentials show any relation to the diameter? Dr. Gasser: Yes, the spikes are longer in small fibers, but the big medullated nerves have spikes that are very much alike in their duration. The fact that the latter have velocities ranging from 30 to 10 meters a second is not due to the duration of the spikes but must be connected with the size of the fibers—the variable which makes the difference in velocity is the diameter of the fiber not the cross section. The relationship is a practically linear one. Dr. Abramson: Can you reverse the demar- cation current in nerve? Dr. Gasser: G, H. Bishop has shown that treatment of a portion of a nerve with cocaine or one of the aliphatic narcotics causes that region to become slightly positive to an untreated por- tion. But if the nerve be cut across the demarca~ tion, current is always from the outside through the cut end to the inside. Dr, Blinks: When you suppress the after-po- tential what effect on heat production is ob- served? Would it tend to throw the recovery pro- cess closer into the spike or inhibit its recovery? Dr. Gasser: This subject has not been studied directly. As it is the opposite state from that ob- taining in the veratrine experiment, one would expect the oxygen consumption and heat produc- tion to be small. The interpretation might be ab- sence of the normal restitution, but other inter- pretations are possible. END OF COLD SPRING HARBOR SECTION Avueust 19, 1933 ] THE COLLECTING NET bt OT uw MORPHOLOGICAL AND ELECTROPHORETIC EFFECTS OF THE GALVANIC CURRENT ON GRIFFITHSIA CELLS Victor SCHECHTER Instructor in Biology, College of the City of New York Electrical energy in its application to living systems is of peculiar interest in that, unlike most other forms ot energy, organisms are not ordinar- ily subjected to it in nature. Yet, they are capable of response and, furthermore, they themselves produce electricity in amounts ranging from the distinctly perceptible voltage of the electric fish down to quantities so small that they are measur- able only with uncertainty. The latter magnitudes are, of course, the usual ones and some, workers have attached large significance to the fact that in no case where measurement is possible has the maintenance of potential difference been found to be absent. I wish to report here tonight, in abstract, some work with the red alga Griffithsia bornetiana, a form which I have found especially suitable for electrical experiments. The morphological polarity of Griffithsia is es- tablished very early. After many muclear divi- sions the cell divides once to form the rhizoid, and again to form the shoot. This takes place in a day or two. By continued rapid division the thallus is formed. Here we have branching chains of cells ranging in size from about 60 micra at the apex of the plant to 2000 or more micra at the base. The basal cells are attached by means of rhizoids. For the purpose of the experiments fragments of a thallus were placed in the experimental ap- paratus and oriented alternately with and against the current. The central part of the apparatus consists of a rectangular glass dish with sloping . bottom to provide a more or less uniformly graded current of known intensity. The rest of the attachments shown are for lighting, water ex- change and the introduction of the current with- out products of electrolysis. After two or three days of electrical treatment a very interesting pic- ture could be seen. In fragments which were oriented with base toward the anode, the rhizoids originated at the base ends of the cells, that is, toward the anode, and the appearance of the completed thalli was more or less normal. However, if the fragments were oriented with the apex toward the anode, the rhizoids were again produced toward this pole and thus at the apical ends of the cells. We have here a morphological indication of reversal of polarity. With the proper current strength every rhizoid formed, and there may be as many as 40 or 50 ina fragment consisting of 80 to 100 cells, originates at the apical end of the genera- tive cell when the apex is toward the anode; and in those fragments oriented the other way every rhizoid is in its natural position at the basal end of the cell. Accompanying the gross morphological effect just described are correlated intracellular phenomena of unusual interest. First, there can usually be seen in the thallus as a whole a grad- ient of color ranging from a deep pink in the cells toward the cathode, much more intense than the usual hue of the cells, to a pale tan in the cells toward the anode,—a shade rarely found in untreated cells. This color effect is, of course, in- dependent of the position of the fragment. A second intracellular effect is the aggregation of chromatophores and probably other cytoplasmic components toward the anode of each cell. It may be of significance in this connection that the first indication of rhizoid producton under nor- mal conditions is the formation of a visible pig- ment concentration, which then pushes out as. the cap protoplasm of the rhizoid To return to the color changes observed, it must be of some significance that rhizoid forma- tion in inhibited in the basal region or completely stopped in the apical region when these, by virtue of their orientation, assume the pale tan color. A pH change may be involved. This is supported by the fact that in acid the cell pigment approximates the deep pink color observed toward the cathode of the electric field, whereas in alkali it becomes pale and finally greenish in hue. Other evidence indicates that the electrical effect on color is not a pH change in the most usual sense; that is, it is not due to the production of acid or. alkali in the cells but rather to a migration of materials which themselves may produce relative acidity ar alkalinity by derived differences in concentration. This evidence was obtained by mounting the material in a miniature apparatus on the stage of a microscope. With high currents each cell becomes deep pink toward the cathode and green toward the anode. If the current direction is now reversed, after a few minutes the colors reverse their position. Ob- servations during the progress of this change show a fading of the pink at the anodal end be- fore any pink color appears at the cathode. In this way there is a slow shift of pink intensity 254 THE COLLECTING NET [ Vor. VIII. No. 68 from anode toward cathode, pointing to the prob- ability that the effect is not a pH change at the cell membrane. In the latter case we would prob- ably see the simultaneous appearance of pink and green at the respective cell membranes rather than a slow reversal of color. The possible integration of these morphological and intracellular effects must be delayed pending further data. I wish only to point out at this time the co-existence of the described intracellular phoretic phenomena with the determinative action of the electric current during regeneration. (This article is based upon a seminar presented at the Marine Biological Laboratory on August 10). THE ABSORPTION OF COLLOIDAL CARBON BY THE MESONEPHRIC EPITHELIUM OF NECTURUS MACULOSUS Dr. ALDEN B. Dawson Associate Professor of Zoology, Harvard University Colloidal carbon in the form of diluted Hig- gin’s waterproof India ink, when injected in- traperitoneally, reaches by way of the nephros- tomes and peritoneal canals the lumina of the primary and secondary tubules of the mesone- phros of Necturus. Usually it is absorbed and stored only by the brush-border cells of the prox- imal convoluted segment. When blockage of the distal segment occurs, the suspension is forced by the ciliary pressure of the peritoneal canal into the capsule; carbon is then also stored in the epithelium of the outer wall of the capsule. The amount of carbon stored by brush-border cells is very variable and could not be correlated with either the amount or duration of the injec- tions. Heavily laden epithelial cells may be des- quamated, and the epithelium is repaired by thin- ning of the cells followed by multiplication by mitosis. There is an extensive infiltration by carbon macrophages from the peritoneal cavity. These cells accumulate chiefly in the interstitial tissues but may infiltrate the renal epithelium and in many instances reach the lumina of the tubules and the capsular spaces. Ink injected intravascularly was found not to reach the mesonephric epithelium. Even the endothelium of the peritubular capillaries had not taken up any carbon after a forty-eight hour in- terval although the reticulo-endothelial cells of both spleen and liver contained large amounts, COLOR CHANGES IN THE DOGFISH (Continued from Page 233) represent the distribution areas on the skin of the We therefore believe that the light phase of the dogfish is not due simply to the nerves concerned, absence of pituitary secretions, but to the positive activity of the nerves. We agree entirely with Lundstrom and Bard that the darkening of the dogfish is due to the pituitary secretion, but we attribute the light phase of the animal to positive nerve action, In this sense, the dogfish is the first fish to show light responses on the severance of its nerves. It presents an unusual condition in that its dark phase of amphibians, depending upon a hormone; whereas its light phase is caused by direct nervous action as in most other fishes, (This article is based on an article presented at the Marine Biological Laboratory on August 8). Aucust 19, 1933 ] THE COLLECTING NET 255 THE AUSTIN ORNITHOLOGICAL RESEARCH STATION Dr. O. L. Austin, Jr., Director The Austin Ornithological Research Station at North Eastham, Cape Cod, while a protected sanctuary for all fauna whose presence is not seriously incompatible with bird life, is devoted to ornithological and closely related biological in- vestigations. Although owned. and financed _pri- vately it has a cooperative agreement with the U. S. Biological Survey. The Station’s six hundred acres on the bay side of the King’s Highway, comprises land of all types found on the Cape, affording a great variety of ecological associations. Salt and fresh water marshes, an extensive fresh water pond, two salt ponds, woods of large pitch pines, scrub oak and locust thickets, open meadows, an asparagus field and even old burned land, each attract different avian species. Cultivated fields of buckwheat and millet and planted berry-bearing bushes afford winter bird tood, while at all seasons grain of suitable sorts is broadcast in an amount sufficient to maintain all avian guests. Attention to their in- dividual preferences in the matters of shelter, food and environment attracts for study all spe- cies found on the lower Cape, affording abundant material for specific investigations, as well as in- dicating how greatly alterations in environment affect bird populations. An enlarged and remodelled old Cape Cod dwelling shelters in comfort the staff and its guests, houses the scientific records and an ornithological library of over four thousand vol- umes which is comprehensive in the bird litera- ture of Eastern North America. A commodious barn provides a workshop for the construction of traps and affords storage room for grain and necessary gear of all sorts. Automobiles of differ- ent types and equipment facilitate access to any desired locality from a traverse of scrub woods to a long trip over sand beaches. In project is the construction of a small building for research work where laboratory facilities will be better than they are at present in the Station home. Bird-work is carried on uninterruptedly the year round with always one, rarely less than two, scientifically trained and adequately experienced workers in residence. During migration and nest- ing seasons the staff is augmented so that at times six men are necessary for the work at hand. Aside from his part in the routine activities, each collaborator has at least one problem for inves- tigation and conclusion. Since the identification of individuals is essen- tal to the study of free birds, banding has been adopted as the major undertaking. By means of baited traps varied to suit the species and seasons, and by Italian bird nets, birds are captured, ringed with numbered and suitably inscribed bands and liberated immediately. In three years over 43,000 individuals of 162 species have been identified thus, while the recapture of these has resulted in the handling of over 88,000. Many of these birds return to the free food in the traps so regularly that it is possible to determine their dates of arrival and departure and to some de- gree the extent of their local wanderings. The large number of captures elsewhere, even so far as in South America, of birds banded at the Station and the reverse of this, gives clues to the times and routes of migration. Elaborate records are kept of all captures, bandings and ornitholog- ical observations. Card systems hold complete in- dividual histories of “repeaters.”” Thus there is being accumulated an enormous amount of data of value in the consideration of any problem. As extensively as time and opportunity permit, mo- tion picture film is made to record not only anatomical characteristics but more especially be- havior. Aside from the routine basic work already mentioned, material is being collected which will result in a comprehensive description of the Cape’s avifauna. This summer endeavor has been concentrated on the several large tern colonies. Last winter photoperiodism was studied with ducks and white-throated sparrows. The more academic biological investigations under way em- brace botulism, communism in terns, avian ma- laria and the etiology of the varying seasonal sur- vival rates of Tree Swallows. The Station has no museum and no caged liv- ing birds to entertain visitors. To laymen who drop in, the work on hand at the moment is de- scribed and illustrated in the field to a degree considered adequate to the stimulation of popular interest in ornithology and conservation. To or- nithologists and zoologists, the Station’s records, field material and assistance are available at all times. The School of Science held its Semi-Annual Meeting on Friday afternoon, August 18 at half past two. It decided that hereafter no child un- der seven could be permitted to attend the classes as it requires teachers especially trained in the handling of small children. The treasury, is not in a position where it could finance such an under- taking. bo or OV THE COLLECTING NET [ Vor. VIII. No. 68 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Jean M. Clark, Martin Bronfenbrenner, Margaret Mast and Anna- leida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford THE PUBLICATION OF THE SEMINAR RE- PORTS OF THE MARINE BIOLOGICAL LABORATORY, I. The Marine Biological Laboratory has an- nounced its plan of publishing abstracts of the papers presented at the evening meetings in the October number of The Biological Bulletin. Recently the following ‘unsigned memorandum was sent out to those persons scheduled to take part in the seminars : “The Biological Bulletin will publish in the October issue the titles appearing on the pro- gram of the evening scientific meetings and the general meeting to be held in the latter part of August. It will also publish abstracts of the papers presented, at the option of the authors. The Biological Bulletin does not wish to publish abstracts based on material which has already been published or which will be published be- fore the first of November in other scientific journals, but will gladly record the place of such publication along with the title of the communication. “Abstracts should be as brief as possible, and should in general not exceed three hundred words, including its equivalent in tabular mat- ter. When circumstances warrant it, abstracts not exceeding five hundred words will be ac- cepted. No illustrations will be published. Abstracts should be submitted to the Biological Bulletin, Room 305, prior to the time when the paper is presented.” Certain investigators have interpreted this note from the Director’s office to mean that prior pub- lication of seminar reports in THE COLLECTING Net would exclude abstracts of them in The Biological Bulletin. In view of this misunder- standing it seems worthwhile to quote part of a letter received from the editor of the latter publi- cation who writes under the date of July 5: “we do not feel that this policy! should interfere with your dealing with these papers in The Collecting 1 That of publication in “The Biological Bulletin.” Net as you have in the past.” Further he wrote that we might assure investigators that publica- tion of their seminar reports in THE COLLECTING Net would not prevent their later publication in The Biological Bulletin. Introducing Dr. Joun H. Norrurop, who was born in Yonk- ers, New York in 1891. He was graduated from Columbia in 1912 with degrees of B. S. and re- ceived the degree of M. A. in 1913 and Ph. D. in chemistry in 1915. He attended the course in Invertebrate Embryology in Woods Hole in 1910. He was appointed to the Cutting Travelling Fel- lowship and worked with Dr. Jacques Loeb at the Rockefeller Institute. He was appointed to the staff of the Rockefeller Institute in 1917 and part of that summer and the following one were spent in Dr. Loeb’s old laboratory in Woods Hole. He continued his work in the laboratory of the Rockefeller Institute and was made a member of the Institute in 1924. In 1926 he moved his labor- atory to the Princeton laboratories of the Rocke- feller Institute. Two years ago the Stevens prize of the College of Physicians and Surgeons of Co- lumbia University was awarded to Dr. Northrop. He has been interested primarily in physical chemistry of proteins and, enzymes with occasion- al work on the duration of life with Drosophila and the study of bacteriophage. He is one! of the editors of the Journal of General Physiology. Dr. Northrop is spending the summer in West Falmouth with his wife and two children, and is working at the Marine Biological Laboratory. CURRENTS IN THE HOLE At the following hours (Daylight Saving Time) the current in the Hole turns to run from Buzzards Bay to Vineyard Sound: Date P. M. August 6:40 August 24 7:24 August 8:11 August 9:02 August 27 9:56 10:57 August 28 August 29 In each case the current changes approxi- mately six hours later and runs from the Sound to the Bay. It must be remembered that the schedule printed above is dependent upon the wind. Prolonged winds sometimes cause the turning of the current to occur a half an hour earlier or later than the times given above. The average speed of the cur- rent in the hole at maximum is five knots per hour. Auecust 19, 1933 ] : THE COLLECTING NET 257 eM Si. O' F ENE hee Sali On the evening of August 21, a bust of Mathew Fontaine Maury was presented to the Woods Hole Oceanographic Institution by Mr. Charles R. Crane. Dr, Frank R. Lillie, president ot the Corporation, accepted the bust for the In- stitution. Mr. Crane gave a brief address, telling how he first became interested in the pioneer oceanographer and outlined the many things which Maury had done to establish the ground work upon which rests the present research work of the Oceanographic Institution. The dedicatory exercises were held in the chart room on the sec- ond floor where the bust now stands upon the mantel piece above the fireplace. The audience was a carefully selected one, consisting of the trustees and a few especially invited guests. Re- freshments were served in the chart room which was attractively decorated with flowers. Dr. David H. Tennent, professor of biology at Bryn Mawr, his wife and his son, David, who will be a sophomore at Yale next year, are spend- ing a month here in the small Danchakoff cottage. Mrs, Jaques Loeb, her son, Dr. Robert F. Loeb of the College of Physicians and Surgeons, Col- umbia University, her daughter, Mrs. Edward B. Osborne and her two grandchildren are spending the summer at Woods Hole. Dr. Edward B. Os- borne spends the week-ends with them. Dr. Philip Bard has been appointed professor of physiology at the Johns Hopkins University School of Medicine. The motion pictures of tissue culture technique which will be shown in the auditorium on Thurs- day were prepared by Dr. Robert Chambers and Mr. C. G, Grand for exhibition at the Cnicago World’s Fair. The pictures have been sponsored by the Society for the Control of Cancer, in order to acquaint the laymen with certain aspects of the cancer problem. Dr. Reid Hunt, professor of pharmacology at the Harvard University Medical School is visiting Woods Hole. Among those who have recently completed their work at Woods Hole for the summer are: Dr. and Mrs. Krogh, Dr. Coghill and his daughter, Dr, Schotté, and Dr. Gerard. Dr. S. A. Waksman recently received a letter from Hanover, Germany, which was opened by the censor before it left the country. It was re- sealed with a sticker bearing the words, “Zur Devisentuberwachung Zollamtlich geoffnet.” Professor Kenneth S. Rice, associate professor of biology at the University of Maine, is visiting Woods Hole with his family. They are living in the dormitory until their rented house in the Gansett Woods will be vacated early in Sep- tember. Dr. A. M. Reese, professor of zoology at West Virginia University, has written that he will not visit Woods Hole this summer. An article by him on the treatment of snake venom will appear in an early number of American Medicine. Julian P. Scott has returned to Woods Hole after an extended period of travel and is holding an exhibition of photographs of Woods Hole per- sonalities and other scientists at the Old Lecture Hall. Mr. Scott took a number of pictures of scientists at the recent International Entomologi- cal Congress held at Paris. Negatives of all the pictures in the collection are with the W. F. Roberts Co., 829 17th Street, Washington, D. C. The contributor of the two articles on ‘Impe- tigo” and “Epidermophytosis” were written for Tue Cotrectine Net by Dr. Austin W. Cheever of the department of dermatology at the Harvard Medical School. In error, the articles were cred- ited to another Dr. Cheever at the same institu- tion. The author’s father recently spent two summers at Woods Hole. Owing to the note which we printed recently concerning Dr. Elbert C. Cole’s book, “An In- troduction to Biology,” the author has written us a note correcting our statement by saying that this volume is intended primarily for high school students and the entire treatment of the subject was simplified as much as possible in order to bring it within the range of high school boys and girls. On Sunday evening, August 27th, there will be a concert by the Washington String Quartet for the benefit of the Woods Hole Public Library in the M. B. L. auditorium. Tickets at fifty cents and one dollar are on sale by Miss Polly Crowell at the M. B. L. office and at the Woods Hole Library. 258 THE COLLECTING NET [ Vor. VIII. No. 68 THE COLLECTING NET PRESENTS WHALING FILM FOR THE BENEFIT OF ITS SCHOLARSHIP FUND For those whose imaginations are stirred by brave tales of the sea and the men who follow it, a treat has been prepared by THE CoLLecTiNnG Ner. On Monday evening, Aug. 28th, Mr. Ches- ter Scott Howland, lecturer and son of an old New Bedford whaling captain, will give a lecture “Hunting Whales in the Seven Seas,” illustratea by moving pictures of his own making, for the benefit of THe Coitectinc Net Scholarship Fund in the Marine Biological Laboratory. The theme concerns those whalers of New Bedford and Nantucket whose vessels sailed the seven seas in search of fortune, the “praying deacons” who left their Cape Cod plowshares at the age ot fourteen to answer the call of the sea, of rigging and harpoons, of the toll of the sea, and the lore of whalers and whaling ways. The pictures are extremely interesting and show the methods ot whaling before the romance of the windjammer gave way to the progress of steel, and the whale- oil lamp to the incandescent bulb. Mr. Howland gave a similar illustrated lecture, entitled ““The Tale of an Ancient Mariner” for the benefit of the Scholarship Fund six years ago. At that time he was introduced by Dr. E. &. Conklin, who said he would give ten years of the peaceful years of his life to repeat the experience of being towed by a harpooned whale. In speak- ing of that lecture Dr. Conklin said: “The au- dience was one of the largest ever assembled in our new auditorium—and safe to say as critical as you will ever address. The lecture was both interesting and profitable. I have heard only words of praise for your presentation of the subject.” The lecture Mr. Howland will give on August 28th is an improved version of the old one, with a number of new reels of motion pictures and much new and interesting material added. Mr. Howland is a son of Capt. George L. Howland, for many years the skipper of the bark Canton. In 1890 Captain Howland was honored by the government of Great Britain for the heroic res- cue of sixteen members of the crew of the bark British Monarch burned at sea 700 miles off the coast of Africa. Among those warmly endorsing Mr. How land’s lectures, in addition to Dr. Conklin, are Professor Thomas N. Carver of Harvard Uni- versity, Professor Robert Chambers of New York University, George U. Denny of Columbia University, Professor Stanley E. Ball, Curator of the Peabody Museum, Yale University, and Wil- liam F. Macy, president of the Nantucket His- torical Association. THE CONGRESS FOR EXPERIMENTAL CYTOLOGY The Third International Congress for Experi- mental Cytology will convene at the beginning of next week in Cambridge. Among those persons who have recently been at Woods Hole, the fol- lowing will take part in its sessions: Dr. Robert Chambers, “Some features of cell permeability in relation to kidney function”; Dr. W. J. V. Oster- hout, ““The Electro-physiology of vegetable cells” ; Dr. L. Michaelis, “The reduction intensity of the living cell”; Dr. R. Beutner, “The vital battery system’; Dr. S. C. Brooks, “The relation between ions and potential differences across plasma mem- branes”; Dr. S. Gelfan, “The degree of independ- ence between the contractile and conductile mech- anism in the muscle fibre’; Dr. C. Speidel, “Growth and repair of nerves.” The program of the Congress is divided up into several all day symposia. Dr, E. Fauré-Fremiet, who recently spent a summer at Woods Hole, is chairman of the symposium on “Cell form and function as demonstrated by recent advances in tissue culture.” Dr. W. J. V. Osterhout leads the symposium on “The Electro-physiology of vegetable cells.” THE JAPANESE BEETLE During the summer at different times people have brought to us insects to be named, which they thought, or feared, might be the Japanese Beetle. So far, these have proved to be other forms of insects. We have felt, however, that it would be well to have a set of these beetles so that any one who desired to see them could call at the office of the Supply Department, or at the Museum, where someone would willingly show the set consisting of larva, pupa, and the adult male and female beetles. The beetles themselves are quite attractive, having a shiny copper-bronze colored body, and green head. The State is quarantined against these insects, but we would advise anyone finding the Japanese Beetle to notify Mr. Frank Bartley of Falmouth Heights, who is a landscape gardener and tree warden ; as we feel that all steps should be taken to eradicate this pest, if possible. GeEoRGE M. Gray, Curator of the Museum. There will be an informal exhibition of sculp- ture at the Breakwater Hotel showing the work of Bryant and Robert Baker. Among the busts that will be shown are several of people from Woods Hole. The exhibition will be open August 26, 27 and 28. Vol. THE DIFFERENTIATION VIII. No. 9 SATURDAY, AUGUST 26, Annual Subscription, $2.00 MEE Single Copies, 25 Cents. OF THE PRO- TOPLASM OF EGG CELLS DURING EARLY DEVELOPMENT Dr. JosEPH SPEK Professor of Zoology, University of Heidelberg A long series of experiments has been done on WAVE TRANSMISSION AS THE BASIS OF NERVE ACTIVITY IDS AX WW, Jeli, ICIS), Foulerton Professor of the Royal Society Wherever we look in the world of matter and events outside ourselves we find that oscillations various types of egg cells in order to study the origin of that organization of these ege cells which controls their further development. Many of these types of egg cells have a more or less con- centric organization before the formation of the polar bodies. Below the surface there is an accumulation of specific sub- stances which sometimes form a sharply separated ‘‘cortical layer.” A careful study shows that in the eggs of some ani- mals this cortical layer itself consists of several components arranged in a regular manner in several concentric layers. In the center of the eggs the large nucleus is situated ; around the nucleus, regularly distributed, are the microscopical compo- nents of the yolk, the different kinds of granules and droplets. arrangement of the components of these egg cells (Continued on Page 300) is transformed after Differentiation of the protoplasm of Egg MW. WH. L, Calendar TUESDAY, AUG. 29, 8:00 P. M. Seminar: Charles B. Wilson: ‘“‘The Copepod plankton.” D. E. S. Brown: ‘‘The pressure co- efficient of viscosity in the eggs of Arbacia.” Edwin P. Laug: ‘Observations on lactic acid, total CO,, and pH of venous blood.” Anna R. Whiting: ‘‘A study of eye color in the parasitic wasp, Habrobracon.” Marc A. Graubard: “The melanin reaction in races of Drosophila melanogaster.” THURSDAY, AUGUST 31st General Scientific Meeting. (See page 301). FRIDAY, SEPT. 1, 8:00 P. M. Lecture: Prof. Robert K. Cannan: “Studies in the amphoteric properties of proteins.” The concentric TABLE OF CONTENTS Osmotic Pressures and wave motion have a significant, often a dom- inant, role. It is not, therefore, astonishing to find that waves play an important part in our- selves also. Let us discuss the nature of these waves. Most of the well-known os- cillations with which physics is concerned are a consequence of the reaction with one an- other of properties analogous to inertia and elasticity. A moving or a changing system tends, on the one hand, to continue in its state of motion because it possesses, for ex- ample, inductance : even social, economic and in- tellectual changes are endowed with such characters of iner- tia, which keep them going when they have passed a true position of equilibrium. On mass or the other hand, such systems, if they are to con- tinue to exist, if they are not merely to be dissi- pated, must possess converse properties which in Relation to Per- Cells During Early Development, Dr. meability in Large Plant Cells and in dkoseioe Se 6 oonscugoocopaconadcaopusms 265 Models) WW: J: V. ‘Osterhout.. (2.25: .-06 287 Wave Transmission as the Basis of Nerve Osmotic Behavior of Red Cells. I. Eric PNgubprAy, Iie JX We deta cocooecocadabe a5 265 Te yer Cy Le RIOR ae Rarely 291 Workers of the M. B. L. Forty Years Ago.. .266 A Cataphoresis Chamber for Heavy Objects, C. B. Havey and Dr. W. R. Amberson..... 272 The Biological Laboratory: Anomolous Osmotic Pressures of Colloid Solutions at Equilibrium, D. R. Briggs. . .273 Effect of Wing Bud Extirpation and Trans- plantation in Chick Embryos, Dr. Viktor Jak healobtd={2) Untoineena rome mo Mcrae ae oe alco 299 General Scientific Meetingy. 3.0.5. ..5.. «ce es « 301 Wag ose WOW bo a So bode baDoOdoese onan oGoL. 303 “AVUL) “X) ‘SQANWNYG “D) “HY “avayy “CV NINNVY ‘IN “M ‘HSIY “V ‘d :mopuim ply} pup puoras “AaISMANG “Jf, “A ‘Peyyuepr jou ‘livig “q ‘[ ‘SHMAY “H ‘NVONOTY “HL “AsOT “[ “WaMavany “Ty : mor yymo-] ‘SANUA “YO ‘MOAMNAODNV] “OD ‘auoaMoIg “Y “Y ‘peyytepr jou ‘aay °S “y “TIraNOy “C “VY ‘A9G0q “A\\ “DO ‘peyuepr jou ‘rrayaay “EY “A\ ‘NITINNOD “*D “qf ‘(2 ) ‘AdTGAg “H “MA ‘SMAHLYIN ‘Gd “V ‘SNIMIVD “ND LVaLOg “[ MN : 04 pay “Waaay “Gq “H ‘NVNLIHA\ ‘OD ‘LOOF “SM ‘Hd IOGNVY “YH ‘Peyuapr jou ‘Horawayyoyy “q ‘[ ‘AVMMOONG ‘“[ “YA “AITITT “YY AVA “5D “Hs: 04 pulosas “NVWIIDNAOD “L,Y WANIGUVD “D “WA ‘INVNOD ‘S ‘“Yy ‘Adodvad “Y ‘[ ‘NASHID) NVA Va] ‘NOLONIMYVH “YON ‘NVH¥OD “qd “YW “ASVLV AA ‘S 241/14 07 3fa] ‘mor Ju01,7 OOV SUVHA ALYOA NAMVL AYOLVAOUVI TVOIDOTIOIN ANIYVW AHL HO SYHMAYOM HHL AO HdVADOLOHd V Avucust 26, 1933 } THE COLLECTING NET 267 tend to bring them back once they have overshot their equilibrium: such properties in physics are elasticity and electrical capacity, in finance and politics are fear and conservatism. These exercise a constraining force increasing with the displace- ment from equilibrium, and ultimately reverse the motion or change, and the same oscillation 1s repeated in the opposite direction. There is, however, another type of oscillation, less commonly discussed in physics or mechanics but none the less well known in every-day affairs ; that which depends upon a discharge taking place when some limiting potential or intensity is reached. For example, (1) water falling into a tank equipped with a siphon will come out in rushes whenever it rises to a certain level. Or again, (2) a population in which an epidemic of measles can not start because of the number of people in it who are immune, having had the dis- ease already, will gradually become less immune as time goes on, and finally an epidemic, a wave, of measles will sweep through it. (3) A neon lamp with a parallel condenser, in series with a resistance and a source of electromotive force, will discharge at regular intervals, namely, when- ever the potential difference across the condenser reaches a certain critical value. This type of oscillation does not depend upon inertia reacting with elasticity. Its essential nature is (a) that some state, some potential, some intensity, is built up by a continuous pro- cess, and the condition becomes less and less stable until one is reached in which discharge must take place, and (b) this discharge, once started, forms a path for itself by which (as ina siphon or an electric arc) further discharge is facilitated until what has been built up gradually has been broken down and the process begins again. This type of oscillator (sometimes referred to as a relaxation oscillator) is the one with which alone we are concerned in physiology. Waves may be transmitted on the same prin- ciple in a system extended in space. An unstable state is gradually built up until at some point, either through an external agency or by some in- trinsic process, discharge is begun, which starts and facilitates a discharge in neighboring regions which themselves discharge, and so a wave is propagated. Such waves will occur periodically if at some region the potential at which discharge begins is less than that finally attained by the con- tinuous process of charging: they will require, however, an external agency (a “‘stimulus’’) to start them if the unstable condition, the limiting potential, is not attained spontaneously. Models of such waves will occur to all of you: their prin- ciple is obvious. I have emphasized it because the waves on which nervous activity is based appear to be of this type. All detailed theories of nervous transmission may well be wrong, but this general idea of it, involving building up and discharge, is almost certainly right. One of the chief characteristics of living sys- tems, particularly of animal ones, is “excitability” —the property of reacting to a small change in the environment, or even in the system itself, by a greater response. This excitability is zero imme- diately after a response has occurred: the cell or organ passes through a completely refractory stage: its potential is at first not yet high enough for any further discharge to take place. After the completely refractory phase, it passes into one in which its excitability is lower tnan normal, in which stronger provocation than usual is required to produce a discharge. Finally it returns to its initial state. This state may be one in which spon- taneous discharge occurs, or, on the other hand, discharge may require what is known as a stim- ulus to start a path for it. In a single organ the final level may be above or below that of spon- taneous instability, according to the circum- stances. The greater the constraint on it the more quickly, in general, it reaches the level of dis- charge, the greater the frequency of the so-called “response.” The time for the complete cycle from the absolute refractoriness following a response to complete recovery ready for further discharge varies greatly from one organ or cell to another. It may be measured in thousandths or in fractions of a thousand or a second, it may be measurcd in seconds or even minutes. An ordinary nerve trunk is a bundle of separ- ate fibers along which messages are sent. The unit, the nerve fiber, is not normally spontaneous- ly unstable, but it can be excited by various agen- cies, particularly by an electric shock, after which a wave starts off from the point excited and travels to the end of the nerve. Once the dis- charge has been started it persists and travels in space. If a nerve be injured persistent spontan- eous discharge may occur. An ordinary muscle, which is a bundle of separate fibers which con- tract and do mechanical work, is not spontan- eously excitable, though it also can be stimulated by various agencies; it is easy, however, by a slight change in the salt content of its environ- ment to render its state unstable, so that regular oscillatory discharges take place in it; these can be recorded electrical ly and result in visible twitchings of the fibers. The heart muscle, on the other hand, has naturally an inherent rhythm of its own; after a beat it is at first completely re- fractory, then for a period it can be excited, e.g., by an electric shock, finally it beats again of itself. Something i in it is gradually restored, until a po- tential is reached at which discharge must occur. This restoration is quicker in one region than in another, and since the discharge once started is 268 THE COLLECTING NET [ Vor. VIII. No. 69 propagated as a wave, the rhythm of the heart as a whole is set by that of the region in which res- toration is quickest. All parts of it, however, have the same property of spontaneous discharge, and even an isolated portion of heart muscle will beat of itself, though more slowly than when attached to its natural pace-maker. If its mechanical ten- sion be diminished enough the spontaneous beats may disappear; the lack “of mechanical constraint somehow raises the level at which instability is reached and spontaneous discharge occurs. It is necessary sometimes for a physiologist, as Rutherford was once heard to say, to make a noise like a physicist. Physics is not indeed the only, or even the chief, way of approaching phys- iological problems, but it has among its advan- tages that of providing at such a conference as this a language by which other people, for ex- ample, engineers, physicists and chemists, may be introduced to physiology. Through no fault of their own, no doubt, many people, people of high scientific standing, have never had any experience at all of that subject. They often have very ab- surd ideas about it. They do not perhaps go so far as the lady who, to a doctor trying to explain to her what was wrong with her, made the ap- veal, “Don’t, Doctor, don’t—I like to think 1 am lined with pink satin.” But they are apc never- theless to picture the inside of the body, if not as pink satin, at least as beyond the range of rea- sonable scientific method. If, as I hope, there are a few engineers, physicists and chemists here, I would reassure them: physiology, and in particu- lar that of the nervous system, is an experimental science like any other: no doubt it requires great experimental skill, but that makes it the more amusing: it is complex, but not beyond the wit of man to investigate: its complexity depends partly upon the difficult nature of its experimental unit, the single living cell, partly upon the fact that most of the effects which can be observed are due to the action and interaction of very many of these units. The nerve fiber in which the waves run is part of a nerve cell: the central part of the nerve cell usually lies in or near the nervous system: the fiber—the axon—runs out to the organ, mus- cle, gland or sensory ending, with which it is connected. The fiber is a fine thread of protoplasm, a few thousandths to a _ few hundredths of a millimeter in diameter but often of considerable length. The velocity with which waves are propagated in these fibers may be anything from 100 meters to a few centimeters per second. In our own motor or sensory nerves the speed is somewhere near the upper of these limits. The messages which pass in nerve fibers can be detected by various means apart from the re- sponse, ¢.g., movement or sensation—which they provoke. The chief of ‘these depends upon the fact that each impulse has an electrical accom- paniment, which, owing to recent improvements in electrical technique, can be easily and accurate- ly recorded. Not only, indeed, can we see nerve waves chasing each other along the screen of an oscillograph, not only can single impulses in sin- gle fibers be recorded photographically, but we can even listen on a loud speaker to sensory im- pulses caused, for example, by gentle pressure on the toe of a cat. As Adrian says, there would be no particular difficulty in demonstrating the po- tential change in a frog’s nerve fiber as an audible signal on the radio. The power in the input circuit would be of the order of 10 watt, the transmit- ter might radiate 50 kilowatts, five million mil- lion million times as much. It would, as he adds, be a sad confession of failure if with these re- sources we had learned nothing fresh about the working of the nervous system. As a matter of fact, we have. In, and in connection with, the living body it- self as part of the complex telephone arrange- ments of the central nervous system, this elec- trical sign of nerve activity is the chief means by which in the last few years the subject has grown so fast. Other signs, however, there are, and the investigation of these has led to considerable ad- vance in knowledge of the physical nature of the nerve wave itself. For example, when a single impulse travels down a medullated nerve there is an immediate rise of temperature of the order of Ose (one ten millionth of a degree). This represents a liberation of energy, small, indeed, but perfectly definite, of about 4 ergs per gram of nerve. A single fiber one hundredth of a milli- meter in diameter, if it weighed one gram, would need to stretch about 10 kilometers. Thus, in sending a single nerve impulse 10 kilometers, the amount of energy immediately set free would be about 4 ergs. One gram-calorie would send it 10:5 kilometers, about half the distance to the sun. Clearly, communication by nerve fiber is not very expensive ! Let us remember, however, the number and variety of nerve waves involved even in ordinary activity. Nothing perhaps can better illustrate nervous action than a short discussion of the na- ture of muscular skill. What does a skilful mus- cular movement feel like to the performer him- self: how does he control it as he proceeds: how does he learn it: how does he remember it: how does he reproduce it? We know that when any particular task is undertaken, any particular movement made, a stream of messages is sent out in appropriate sequence, most accurately adjust- ed, along tens of thousands of nerve fibers that carry it out. We may imagine as a first and rough Aueust 26, 1933 ] THE COLLECTING NET 269 approximation that the brain and nervous system contain a carefully catalogued set of gramophone records, each ready to be taken out and turned on when required. If we come to hurdle the high jump record is required: when we come to a ditch, the long jump record. Ina sense this simple idea is true. Our behavior is largely composed of ready prepared, gramophone records called up and set going by the appropriate stimuli, and our skill in movement depends very largely on the degree to which we have learned to make it au- tomatic. Nearly everything we do is partly un- conscious. When we walk down the road we just give our nervous system general instructions to walk. We don’t set out in detail how every muscle shall move, how every unevenness is to be over- come, we probably don’t even know. Our move- ments, therefore, are largely automatic, they con- sist largely of gramophone records, prepared be- fore hand by instinct or by training and ready in- stantly to be turned on. On the number of the actions which we have learned to make automatic, on their coordination one with another, on the fineness and accuracy with which they are adjust- ed to the stimuli which evoke them, depend large- ly the skill and efficiency with which we work. True as this is, however, it represents only a very limited aspect of the truth, because it neglects one half of the nervous system, the half that tells us what we are doing and enables us to adjust it as we go, the half by which we really remember what muscular movement is like. Take the simplest possible movement. Try to bend your finger slowly at a uniform constant speed. You will find that it does not really move uniformly but in a rapid series of jerks. A record may be made in some way, for example, by con- necting your finger to a lever writing on a smoked drum, or by photographing a beam of light reflected from a little mirror stuck upon the knuckle. The record allows the jerks to be count- ed. If the jerks be few they will be large and the finger will seem shaky; if they be many they will be small and the finger will seem steady; the most skilful person, and the one with the stead- iest hand, is he who checks and controls his movements most frequently and rapidly. In all our movements the mechanism “hunts” to find the right adjustment. The smaller the amplitude of the “hunting” the more skilful we are. How is this done? From all the moving parts of the body, muscles, tendons and joints, a sys- tem of nerves runs inwards carrying information about what is happening in those moving parts. These messages are started off by end organs, ex- cited by movements in the tissues where they lie. Muscles are arranged in pairs or groups, and any given movement is due to the cooperation or an- tagonism of a whole set of different muscles. When you sail a boat you don’t just set the sails and tie up the rudder: you watch the wind, you adjust the sheet, you keep your hand upon the tiller, there is a continual interplay between the wind, the sea, the sails and the steering. In the case of bodily movement the nervous system is the steersman, who has to compound all the mes- sages—the nerve waves—he receives to form one general impression on which to act. When a given muscle shortens, its antagonist has to lengthen. 3ut notice: the antagonist does not let go atl at once: if it did, the result would be like letting the sail out with a crash: it pays out gradually, in little jerks, each element in the contraction of one muscle provoking reflexly an element in the relaxation of its antagonist. This interplay is going on continually, one muscle hauling in a little, another paying out, so guiding and con- trolling the movement, keeping it as smooth, as accurate and as well coordinated as may be. This continual reaction between muscles, nerves, end- organs and central nervous system is the phystol- ogical basis of muscular skill, and on its smooth and efficient working depend many of the things that mankind finds worthy of accomplishment. I will not venture inside the nervous system itself. That would require not a lecture but a se- ries of lectures to discuss. You must think of the nervous system as a vast exchange, in which in- coming messages from all corners of the body are sorted, assessed, correlated, recorded, in which the response most necessary in view of all the circumstances is worked out. The automatic tele- phone exchange, the control post of the automatic trafic signal, both carry out this function in re- spect of a single purpose: the central nervous system does it in respect of all possible purposes. I can do little more in this lecture than refer to the messages themselves, those which come in, those which go out, those which—in all probabil- ity—circulate within the exchange itself. These messages are all of one kind, and before we can begin to understand the nature of nervous action it is necessary that we should know something about the unit on which the whole of the mechan- ism is based, the wave which travels in the thread of living protoplasm. The ending of every sensory nerve fiber has a specific kind of sensitivity, to touch, to pressure, to heat, to cold, to light, to vibration, as the case may be, and its manner of reacting also is spec- ific, Certain properties, however, are common to all. Of these the most significant is the cycle of refractoriness and returning excitability which follow. All these organs are “relaxation oscilla- tors” of the type I referred to earlier. The spec- ific stimulus is the external cause or constraint which alters the level at which spontaneous dis- charge takes place: the stronger the stimulus, the COLLECTING NET [ Vou. VIII. No. 69 270 TSDe, lower the ee the more frequent the discharge. Some of these organs “adapt” to a constraint or stimulus. The hand put in warm water feels it warm, but in a very short time the organs sen- sitive to warmth adapt and no sensation is ex- perienced. Similarly with touch. With sight there is some adaptation but not to the extent of com- plete lack of sensation: with the organs which t-ll us the state of tension in our tendons there is lit- tle: with sound there is practically none. The mechanism of this adaptation, this change of level of touch, is not understood, but its existence is an essential part of our being. The level of dis- charge of some of our oscillators is not constant under a constraint but gradually rises; with these the frequency of discharge diminishes, with oth- ers the level remains constant for long periods and so the same frequency persists. One limit to the nerve fiber and the wave that travels in it as a means of reporting to the ner- vous system events occurring outside is that it can not react (in the case of man) more than about 1,000 to 2,000 times per second and at that speed only for a short time. Two of the most important stimuli are those of light and sound: light waves have a frequency of the nerve fiber; to limit ap- preciation of sound below 1,000 to 2,000 waves per second would greatly interfere with its equal- ity. Now if a single nerve fiber, with no sp ae d R Fh * at O'S foe kn) Tarot een. ar ee ES ~ tae Y- (0+ Ws & 3 ‘ou0d UO] +H jo [OA [e}I0L ° ="00 0ZE UL ‘INVLISNOO GQYASSANd “LINVEISNOO [10] IVLOL ‘Hd ONIANVA “III AIAVL [ Vor. VIIT. 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In this case a mixture of 5 parts NaCl and 1 part HCl was added to the individual experiments in varying amounts so as to change the total Cl. The HCl was added in order to keep the pH on the acid side of neutrality and minimize COs ab- sorption. In this table the activity coefficients of NaCl were used to calculate the concentra- tion of Nat and Cl— ions present, 1. e., 100% dissociation was not assumed. This use of the activity coefficients of NaCl is not entirely cor- rect (not all the electrolyte was NaCl) but serves to give a somewhat closer approximation to the actual conditions than if 100% ionization were assumed. Experiments in which the K and Li salts of arabic acid were used in place of the Na salt, and in which KCI and LiCl were used in place of NaCl, respectively, showed no lyotropic effect for monovalent cations, the values obtained being identical (within a few per cent) with those given in Table V for the Na salts. While not shown in the table, the Cl~ ion concentrations were determined electrometrically with silver— silver chloride electrode in the external solution and were found to agree closely with the calcu- lated values given. The agreement for the inside solutions was not good, especially at low Cl con- centrations,the electrode measurements indicating CO {c'); vary ing o ~Alcohel conc. vary ing ’ -pl varying + = vary ng 3 + s 6 concentrations which were somewhat higher than the calculated values given in the table. In Table VI are shown the results obtained when the Ca salt of arabic acid and CaCl» were used in an experiment otherwise similar to that of ‘able V. In this case the Cl~ ion concentration outside the membrane at equilibrium was de- termined electrometrically and the equivalent Ca** ion concentration outside taken to be equal to [Cl-],—[Ht],. From the value of [Ca** ],, could be calculated [Ca* +]; from the equation [Cat] =R4 x) [Cats], The value of [Cl~]; was obtained from the equation [Cl~ Jo [a>], = —— R [H*]s where R was the measured value of — [ai In this table the values of [Ca*+], are not very dependable when R is high but values obtained from the last five experiments shown in the table are dependable, becoming more so the small- er the values of R become. The value of Px is re- versed in its direction of action across the mem- brane at the higher concentrations of CaCl». The calculated concentration of ions present inside the membrane becomes lower than that necessary to exert the observed osmotic pressure, P,, against the external solution. Analysis of these data shows, empirically, that Ea is a constant in those cases the factor Px (Table II, 11] and IV) in which the foreign salt concentration (as measured in terms of [—]j, the total negative ion concentration inside the membrane other than the colloid ion itself) in each table of experiments is nearly constant. The factor E is the membrane potential as obtained from the pH measurements, and Px = P,. — P, and is a measure of the anomalous pressure found. The factor, a, signifies the degree of ion- ization of the colloid. Knowing in each case the gram concentration of gum arabic present and its equivalent weight, it is possible to calculate its equivalent concentration. The equivalent concen- trations of [H+];+ [Nat],— [Cl—]; give the total equivalent concentration of positive ions which must be derived from the colloid. This latter value divided by the former gives the per- centage ionization, a, of the colloid. (Continued on Page 285) [ Vor. VIII. No. 69 THE COLLECTING NET N ‘UJOS [BIOL “09 OZE UT o'9c0¢ 962° 8°6Eh 96°60 o TOE&T 0800°¢ O19z's o0cé6’z 2260 00z°€ , i F ‘ L96°F oce’ T 00Z0ZL 0267 LT FES 210°¢ oes: O89 LLS'€ POCr €2°60€ o POET ==> OFZTS O9LEs% 0160°% 9€6°0 ara : , " LOGS 008° T O'GLOL CIs 6 66F S9T'OL lore 00¢" LecEe OSt'r 0°80¢ c0'L0¢ (9) | ¢ FOET O9eT T CesT t 02260 0960 0960 | 4 : i A e6r'9 Coa" T } 0°SO6T 0cL's 8'POF 66F ZL 9cF'9 ezz" | 16°ZT 1838'S o'e6¢ 08'Z08 ) i OTEL 0g6¢'0 9909°0 8cTE'0 1160 0ce'0 5 , ‘ 81¢°8 00a" T 0'°SES 629'F @8TgS F08'9T 9Z9'R 00z" Orme L 9sT'9 1109 C6 'e0E o ost O9LF'O TRSF'O * 20220 €860 $220 A ‘ A 3 F961 GLT T OTLT LOL'F @ 96S eOL'cT 680'8 Or L93°T TEL'9 O'et9 €L° FOE 1) Cc Tét Otee'0 oce¢'0 8600 8860 960'°0 A é “6 a TOOL ost’ T Ol PrL OfT's o8tS OL6'FT 699") ocr’ G1Z8'0— Z80'L 1899 PL'e08 ) 80ST 9612'0 LT82°0 s8TE0'0 £66'0 ce0°0 7 ; d ‘ cers ocl T 02°29 9020'S L3c¢c 696'ST LETS ocr’ ¢ZL1c'0 Gta L 0'eL19 9c ¢0¢ o e Test 0g¢2'0 oscz'0 | | ( fy c ' cea's8 oct” T | | CS'6P cos's 9°8c¢G CPL OL 10Z'8 oer’ 3 | 10 X “AU O°H ‘wo ys ae “ws 201 x “ba s0OTxX"be = s0Tx"ba = [OBN JO e0T x "ba = aurea F, ‘0u09 BN ‘9u09 [DO ‘yeo0o -Wway [+H] Hd ANA d A a Vv Xk ‘ou0d BN XA ow ‘9u09 [QO «Jo apis ‘9u09 UCI +H (0 +1) } } ‘NOILWO-+*®N “INWEISNOOD GQYOSSaud “ONIAUVA [10] ‘A GTA L 285 r ola 4 COLLECTING NI ) THE Aueust 26, 1933 ] oc96sT 0006 00906 o T#TEO’ O9ZTS’ = OFZ = 86GB ~—-SOOT'0 TS9 - @6T = OOZLL SIE ace 8 £620 028992 osc6s — 00geTz : osecsr 0019 — og9c9 0 z90g0" OZ6LZ—ss«E9'Z~=—SiOVS'O~—-260T'0 998 O11 ozsgs g68% TLS —L I 6re'0 00zTZz Ostsz 00F06T : 0809 016 09E08 ° s9Tgo 0080%~—s«SFZ'E = LTS'O~—€zeT'0 O'8FE F8L% OLTOTT ees wer — 9 Zz 0S90LT OFSG = OOZEOT t ; 08002 00s0T 0116 ) PITEO © OZ8ZT SFB CIQ'|D ~—OORFZO G19% 868 OZLLST Zrst wL —S I £180 OOSLLT 89g OOOLLT ~ : OZEFT OSL 0602 o cizeo’«O8ZIT’ «OTS SSF sSSz'0 L0SZ 6S — 0ZOZST Ov Cee ye 928°0 OFE99T 608 Oc6c9T : OFeL o9¢ =s«OTTS~S—Ss«éCE o oFzE0 0c280° 9809 r1eF0 TOLEO ooze Ice ozr6e> oe6s St6 —E rf T 628°0 O9F9FE «= TPT )©= LL = OOS 9T ; PELE 098 oor _—«T98T o Te0go $990" 09'S ~=—ssaTHE'O TOGO SeIZ Thre e9r9e4r Erey, COL —~ \! c18°0 L886EI SFIT FEET O0086ET 0008 00ST 00st ) cotgo 0FZ90" GGL" = SOSOSLEF'O O68t Goze s9s9zr ov9s FFIT —T T Z28'0 cosézt = FLT 00862T : "ai *d O°H wo OTH WO 20T x LOL | LOL Opes OT “AuL d 2 ea os ec —. 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OITs 008" i p89l 6S G6IIT &Z0T OSs’ GLF9 82z9 cere o 009T" 9°88 O'9TS O6zFOZ ST'6I 8°CL PLZT 8880 Z80°0 O6T8" ZLGGOZ O8G0Z OOLITH FES eZF9 Z6I'S O'ZSS Gro, ce ust ! OFS OST" — (pauinss®) 2% “062— O0&F 6&8 0 ZZUT 0969 seco 608 0 EeLT’ 000'L O'T9F O06 oOGEEz 888% 8°08 6821 oreo 0 ; he or OTS 008" I Pez OSLPEZ OOGE9 0 zz9z Ise ocLe 66Z'0T f o0ste e3r'S 008" Ma) >} 3 feo ; : ‘ , Gix s ‘ : F s01 x be g0L x ‘ba Sm x4 Be Pe remem clin J 59 101 X "bo s0T x AuL 10TX AW O H wo *99 ws ‘ws “QUOD BD “09 [D aS | > FS) Gees ee tort Ol Ol vee tae Lari Ces sone idl TA eee ey 30 ae = 4 a : Be a ac) S S S ‘Quod UOl +H Sy Onn ee 5 “NOILV)O — ++®) “INVISNOOD GHoOSsSdud “ONIAUMVWA [IOI TA AIEV Ausust 26, 1933 ] _THE COLLECTING NET 285 Ee Bik hal Bares. When, however, the foreign salt concentration, [—]i, is changed radically, as in Table V, the Ea value is no longer a constant. Figure 2 Re Ea shows that the value varies with the factor Be [—J];i in a log-log ratio. Graphic solution of the curve so obtained gives the equation Ea log = — 0.211 log [—]; — 1.52 ibe a [—]io-222 Thus, it appears that the value ———————— is a Px constant when, other than the colloid, only monovalent cations and anions are present in the system. This constant does not vary with concen- tration of colloid, salt, or pH. When the divalent Ca* * ion replaces the mono- valent cations in the system, it is seen from Fig. Ea 3 that the linear relationship between log pe and log [—]; still holds (at higher concentrations of CaCl.) but that the slope of the curve is much steeper and that as CaCls increases in concentra- tion there is a reversal of the effect. At concen- trations below this reversal concentration, P, acts counter to the osmotic gradient, while above this concentration P, acts in the same di- rection as the osmotic gradient across the mem- brane. While this analysis of these data is empirical and no attempt will be made to devise an expla- nation for the existence of such a relationship, it may be worth while to point out that the factors. a and [—]i, probably determine the density of charge carried by the colloid particle. It seems possible that the observed anomalies in osmotic pressures of the colloid solutions may through some mechanism be brought about by an inter- action of two potentials, one, the electrical poten- tial existing across the membrane, and the other, that determined by the charge carried on the col- loid micelle. The reversal of the effect with CaCl. would seem to indicate that the electrokinetic potential or the sign of charge carried by the colloid is a determining factor in the phenomena. LITERATURE 1. Detrochet—Ann. de chim. et phys., 60, 337, (1835). 2. Graham, Thos.—Phil. Trans. Roy. Soc., Lon- don, 144, 177, (1854). 3. Flusin—Ann. de chim. et phys., (8) 13, 480, (1908). 4. Girard, P.—Compt. rend., 146, 927, 159, 99, (1914). 5. Loeb, J.—J. Gen. Physiol. 1, 717, (1919); 2, 173, 255, 387, 563, 577, 659, 673, (1920). 6. Bartell, F. E—Colloid Symp. Monograph, 1, 120, (1923). 7. Duclaux,J.—Compt. rend., 140, (1905), J. Chim. Phys., 5, 40, (1907). 8. Loeb, J.—J. Gen. Physiol., 3, 691, (1920-21). Proteins and Theory of Colloidal Behavior, New York, (1922). 9. Loeb, J—J. Am. Chem. Soc., 44, 1930, (1922). (1908); 1468, 1544 10. Hammarsten, E.—Biochem. Z., 144, 383, (1924) 11. Hammarsten, H.—Biochem. Z., 147, 481, (1925) 12. Donnan, F. G. and Harris, A. B.—J. Chem. Soc., 99, 1554, (1911). 13. Bayliss, W. M.—Proc. (1909). 14. Zsigmondy, R.—Z. f. phys. Chem., 111, 211, (1924). 15. Bjerrum, N.—Z. f. phys. Chem., 110, 656, (1924). 16. Samac, M., Knop, L. and Pankovic, Z.—Koll. Z., 59, 266-78, (1932). 17. Adair. F. S—Proc. Roy. Soc., A, 108, 627; 109, 292, (1925); 120, 573, (1928). 18. Barcroft, J. and Hill, A. V.,—J. Physiol., 39, 411, (1910) 19. Thomas, A. W. and Murray, H. A.—J. Phys. Chem., 32, 676, (1928). Roy. Soc., 81, 269, Discussion Dr. Abramson: Did you say that this devia- tion from the thermodynamically predictable values of osmotic pressures has always been ob- served, even with proteins ? Dr. Briggs: Loeb found that he could explain the osmotic pressure of casein chloride entirely in terms of the Donnan theory. He could not do so for gelatin salts, however. Others have found that, assuming a definite molecular weight for the colloid, within certain ranges of salt, H+ ion, and colloid concentrations, the observed osmotic pres- sure could be completely defined as equal to the sum of the osmotic pressure exerted by the col- 286 THE COLLECTING NET [ Vou. VIII, No. 69 loid micelle, and that exerted by the diffusible ions present in unequal concentrations on the two sides of the membrane. Those who have studied osmotic pressures of colloids of a non-amphoteric nature have, in general, found the observed os- motic pressures to be less than that required from the unequal concentrations across the membrane of the diffusible ions alone. Dr. Abramson: How do you account for the fact that in some cases the Donnan theory pre- dicts the osmotic pressure, and in other cases it doesn’t? You certainly have the charge changing in the case of proteins, and you haven't the diffi- culty in respect to a. Alpha, in your case, rep- resents a measure of the net charge per average particle weight, unless the solution is mono-dis- perse, in which case, a would be the net charge per particle. Do you think that the fact that the gum arabic system may be hetero-dispersed might have anything to do with it? Dr. Briggs: 1 am not attempting to give an explanation of the anomaly. I am only present- ing the facts, first, that so far as the distributions of ions across the membrane is concerned, they are entirely equal to that which is to be expected upon the basis of the Donnan equilibrium theory, as based on measurement of the ratio of distribu- tion of one ionic species (i. e. H* ion), and on the assumption of nearly 100% dissociation of all Na and Cl ions present in the system, and second, that the osmotic pressure calculated from such distribution is greater (in cases of monovalent cation salts) than that observed. Whether this may result because the ions, in presence of the colloid, lose some of their osmotic activity, as some suggest, or whether a force, definable in terms of the factors E, a, and [—] ;, is working at the membrane, must be left to further ex- periment to decide. Heterogeneity of the colloid phase, I feel, is of little importance, because in the solution every point of possible ionization is exposed to the solution, i. e., the neutralization equivalent of the arabic acid is independent of the degree of dispersion, at least it is independent of dilution of the solution of the acid. Also, in the analysis of the data, the colloid has been assumed to consist of such large micellae that no osmotic pressure arises from the colloid ion. Dr, Reiner: Did you determine the activity coefficients of the Cl— ion in the presence of the gum arabic ? Dr. Briggs: The chloride ion concentration inside of the membrane in these experiments was very low, and no attempt was made to analyse it for total chlorine content at equilibrium. Cl~ ion measurements, made with the silver-silver chlor- ide electrode in these solutions, were very unde- pendable because of the low concentrations. Only in those cases where the [Cl~]; became fairly high, i. e., in those cases where total Cl in the system was high, and the ratio of distribution, R, was low, did values of R as determined by the hydrogen ion measurements and those made with the Ag — Ag Cl electrode become equal. Dr. Mudd: It is not possible to explain your results in terms of an electro-osmotic pressure, acting across the membrane, and giving rise to the anomalous pressure observed ? Dr. Briggs: 1 must confess that I have thought such an explanation possible. I am, how- ever, convinced that a membrane potential, E, can not function to give rise to electro-osmotic flow of liquid through the pores at equilibrium. While the value of Px does happen to be definable in terms of this membrane potential, E, and the fac- tors a + [—];, which could easily be pictured as the determining factors in the electrokinetic po- tential of the arabic particles, of which the pore walls of the effective membrane could conceivably consist, [ think that some other mechanism must be sought out to explain the anomalies observed. Aucust 26, 1933 ] | THE COLLECTING NET 287 OSMOTIC PRESSURES IN RELATION TO PERMEABILITY IN LARGE PLANT CELLS AND IN MODELS W. J. V. OsteRHOUT Plant cells are especially suited to the studies which here concern us!. As a rule they consist of a thin protoplasmic sack with cell sap inside, and a cell wall outside. When the osmotic pressure in the sap is higher than in the external solution, water enters and distends the protoplasmic sack until it presses against the cell wall (somewhat as the inner tire of an automobile is pressed against the outer shell). When the osmotic pressure in external solution becomes greater than in the sap, water goes out, and the protoplasmic sack shrinks away from the cell wall: this is called plasmolysis. This process affords a means of measuring the osmotic pressure of the sap, by finding what osmotic pressure in the external solution causes water to move out. To be a perfect osmometer, the protoplasm should be permeable only to water and not to dis- solved substances. This is never the case, but it is approximated with certain substances which enter very slowly. By ascertaining how rapidly various substances enter, we can gain an idea of the nature of the protoplasmic surface. Overton formulated the hypothesis that the surface is lipoidal, and that only those substances which are soluble in lipoid can enter. We can agree to this if we widen the conception by saying that the sur- face consists of a non-aqueous layer, and if we also take into account the relation between this layer and the aqueous protoplasm, or sap, as formulated by Irwin in the hypothesis of multiple partition coefficients. This hypothesis has furnished a useful means of approach to the central problem which here concerns us, namely, how can the cell absorb water and increase its volume indefinitely, and still maintain a higher osmotic pressure than ex- ists in the surrounding solution? If it did this by manufacturng substances like sugar, the problem would be simple. But, in such a cell as lalonia, we find osmotic pressure in the sap to be almost wholly due to KCl and NaCl. The concentration of KCl is about forty times as great as in the sea water outside. By what mechanism is this main- tained ? Since the sap is more acid than the sea water, it seems possible that potassium enters chiefly as KOH which becomes neutralized in the sap. This 1. Large multinucleate cells such as those of Valonia are especially suitable for these studies. For the literature up to July, 1931, dealing with such cells see Osterhout, W. J. V., Biol. Rev., 1931, 6, 369, may come about in much the same manner as in certain artificial cell models!. In these, potassium passes from an aqueous alkaline solution, 1, through a nonaqueous layer B (representing the protoplasm), into an aqueous phase C (represent- ing the cell sap). At the start, C consists of dis- tilled water: CO, is bubbled through C, during the experiment, to imitate the production of CO. in the cell. In 6 we place a mixture of guaiacol and p-cresol: these may be collectively designated as HG. When we place KOH in A, it at once reacts to form KG, according to the scheme KOH + HG = KG + HOH. On arriving at C, KG reacts with CO. thus KG + HsCO; = KHCO;3 + HG. This process tends to go on as long as the ionic activity product (K) (OH) is greater outside than inside. Since the concentration of KOH in Cis kept constant, and since the bubbling of CO. keeps down the concentration of OH in C, potas- sium continues to enter C, until its concentration is many times as great as in 4. The osmotic pres- sure in C exceeds that in A, so that water moves from 4 to C. Eventually a steady state is reached, in which water and electrolyte enter C in a fixed ratio, so that the volume in C increases, while its composition remains approximately constant, with a higher osmotic pressure than in 4. Something like this seems to take place in living cells. It is of interest to note that in this model we derive no energy from the formation of COs, but employ COs after it has been formed, using what is ordinarily regarded as a waste product of the living cell. We find that the order of penetration is the same as in Valoma, i.e. K > Na > Ca > Mg. To explain the difference in rates of entrance, e. g. between those of sodium and potassium, we must consider conditions in B, where the rate of diffusion is so slow that it controls the whole process of penetration. Although the diffusion constants of the two salts in B are about the same, the partition coefficient of the potassium salt is so much higher that its concentration gradient in B is much steeper, in consequence, more potassium than sodium moves through B, in unit time”. 1. Osterhout, W. J. V., and Stanley, W. M., J. Gen. Physiol., 1931-32, 15, 667. 2. Osterhout, W. J. V., J. Gen, Physiol., 1932-33, 16, 529, 288 THE COLLECTING NET [Movs VEE avs There is an important difference between the model and Valonia, since in Valonia potassium accumulates as KCI: this might be due to the fact that as bicarbonate increases in the sap, it tends to go out in exchange for chloride which comes in from the sea water. It should be remembered, however, that in many cells! where oxalic, malic, citric, tartaric and other acids are produced in considerable quantity, potassium may accumulate as the salts of these acids. This would be quite comparable to the accumulation of KHCOs in the model. We have set up models with formic and citric acids in place of carbonic, and have found that potassium readily accumulates as formate and citrate. It is worth while to consider the manner in which electrolytes pass through the non-aqueous layer. In the model, KOH reacts to form KG in b, and this reacts to form KHCOs: in C. The net result is that 4 loses a potassium ion, and C loses a hydrogen ion, and from a thermodynamic standpoint it amounts to an ionic exchange, 7. ¢. K* passes from 4 to C, and H+ passes from C to A. But when we consider rates, we see that this picture is wrong. On. the basis of ionic ex- change, it would make no difference whether we placed in 4 0.01 m KG or 0.01 m KCl. But from a kinetic standpoint, it makes an enormous difference, since KG penetrates very much faster than KCl, because its partition coefficient, and consequently its concentration gradient, in B is so much greater. The relatively rapid movement of KG in B is almost wholly in molecular form, since it is a very weak electrolyte in B. The model illustrates a possible mechanism by which the living cell can absorb water and expand indefinitely, and yet maintain a higher osmotic pressure than exists in the external solution. To maintain such a difference evidently requires an expenditure of energy. This takes place in the model in an interesting way which is hest seen when we substitute HCl for COs, and leave the system to itself, instead of renewing certain sub- stances. We then have KOH on one side of the non- aqueous layer, and HCI on the other. They tend to mix by passing through this layer, thus form- ing KCl in 4 and in C. But KOH moves so much faster that most of the KCl is formed in C, where the concentration of potassium becomes much greater than in 4. This, however, is only tem- porary, for when the system comes to equilibrium the composition of A and C will be identical, 1. Czapek, F., Biochemie der Pflanzen, Jena, Gustay Fischer, 3rd edition, 1922-25. For potassium oxalate see Vol. 3 p. 69: for other organic acids consult the following pages. See also Evans, H., Biol. Rev., 1932, 7, 181, since all the substances present are able to move through the non-aqueous layer. If we designate the difference in thermodyna- mic potential between KOH in A and C as Axon it is evident that A xon steadily falls, and eventually equals zero. This is also true of A nei. The case is quite different for the accumulating substance’, KCl. We find that Axe, starting from zero, rises to a high value, and then falls to zero at equilibrium. In a medel with KOH and HCl constantly re- newed?, A xe: would rise steadily until it reached an approximately constant value. This constant value would be maintained in the same way as that of KHCOs, in the model previously men- tioned. The same principles may apply to living cells, particularly where potassium accumulates, com- bined with organic acids (e.g. as potassium oxalate). Such cells closely resemble the model. These experiments serve to illustrate some of the methods and conceptions which have been found useful in a field which has many attractive problems. Discussion Dr. Chambers: Do either narcotics or oxida- tion-inhibiting agents interfere with the selective passage of cations into the vacuole of Valonia? And do they interfere with the working of the model ? Would it be possible to stop the liberation of COs into the vacuole and to determine whether this interferes with the accumulation of HCl in the living cell? If the continual evolution of COs on one side of the membrane is necessary for the passage of materials through the membrane, how would you explain conditions of plant cells with chloroplasts in bright sunlight when there exists a COs lack in the protoplasm to the extent that COz, is being consumed from without ? Also, what is the source of carbon with which the cell must be continually supplied in order to produce the CO? The supply would have to be enormous. Dr. Osterhout: Experiments with narcotics have not yet been made with Valonia but cells were placed in a refrigerator to check the produc- tion of COs. We might then expect the entrance of potassium to fall off more than that of sodium because as the pH value of the sap rises the dif- ference between the ionic product inside and out- side approaches zero much more rapidly in the 1. Unpublished results. 2. Here the movement of HCl would have com- paratively little effect on the composition of A. AuGUST 26, 1933 | THE COLLECTING NET | 289 case of potassium. l'or example, if we raise the pH of the sap to 6.3 the ionic activity product (IX) (OH) inside becomes approximately equal to that outside and we should, therefore, expect the entrance of potassium to stop but sodium should continue to enter because the ionic activity product (Na) (OH) remains much greater out- side. We found that this actually happened whea the pH of the sap was raised by the entrance of NH, and we surmised that in the refrigerator something of the same sort occurred but that the entrance of potassium was merely slowed down instead of being stopped altogether. At any rate in the refrigerator the ratio K + Na in the sap fell off, as would be expected. But unfortunately no determinations of the pH value of the sap were made in the refrigerator experiment. In daylight the cells quickly raise the pH of the sea water just outside the protoplasm from about 8 to about 9.6 but the pH of the sap re- mains practically unchanged. Hence the difference of pH between inside and outside increases in the light, and the entrance of potassium and sodium is hastened. The cell maintains the pH of the sap at a much lower value than that of sea water. It averages about 5.8, that of sea water being about 8.0. The difference seems to be due mostly to COz since the sap contains very little organic matter (about 1.4 parts per thousand ). In the model there is no oxidation. We make no use of the energy set free in producing CO, but start with it already formed. Hence experi- ments with narcotics have no bearing on the op- eration of the model. But we can slow down the rate of bubbling of COs and the entrance of elec- trolytes then slows down. Applying substances intended to lessen produc- tion of CO» might have a variety of effects. For _example, in applying KCN to Valonia we should have to do with the entrance of KOH and of HCN and probably with changes in the pH of the sap. Injury might result in increasing per- meability so that potassium would come out and sodium go in. Experiments of this sort may, therefore, be difficult to interpret. Dr. Fricke: What is the water content of the guaiacol ? Dr. Osterhout: Dr. Shedlovsky found that 100 gm. guaiacol dissolves 4.63 grams of water. Dr. Fricke: \Vould you discuss the actual use which the plant can make of these various differ- ences (in osmotic pressure, electric potential, chemical composition) between the plant interior and the surrounding fluid ? Dr. Osterhout: The difference between the inside and outside is so characteristic of living things that it seems essential to the conception of life. A simple experimental test is to place Valonia in its own sap in which it quickly dies. To be more specific the higher osmotic pres- sure inside the cell causes water to enter and thus brings about growth. Even when the cell is not actually expanding the higher osmotic pressure inside keeps the cell wall from collapsing and maintains the form of the cell. In the higher plants the lack of such pressure produces wilting. The potential difference across the protoplasm is a necessary condition for action currents. It may also cause movements of water and of elec- trolytes where local differences of potential per- mit a flow of current. To what extent such cur- rent flow may be of use in correlating the activ- ities of various parts of the organism is an open question. The difference in chemical composition between the protoplasm and the outside is, of course, of fundamental importance, but that between the vacuole and the outside depends on circumstances. In Halicystis the difference is not great aside from the fact that the sap has a higher acidity (which is probably necessary for the entrance of electrolytes), more organic matter and less oxy- gen: part of the time at least the content of free COs is higher. In most organisms there are other ditferences to which I am unable to assign any teleological significance. Dr. Fricke: Can you say anything about the reason why potassium should go through more than sodium? Dr. Osterhout: The essential reason is that potassium has the higher partition coefficient. Dr. Fricke: As soon as you stop carbon diox- ide production does the process immediately re- verse and the potassium begin to diffuse out? Dr. Osterhout: When the bubbling of COs ceases in the model potassium begins to move out, but we have not followed the process to equilibrium since it takes a long time. Dr. Fricke: If Valonia is not completely im- permeable to sodium would you not expect that sodium would in time come into equilibrium in- side and outside? Or is such an equilibrum not reached within the life of the plant? The red corpuscle presents a similar difficulty. Dr. Osterhout: No such equilibrium is reached in Valonia. Ordinary equilibrium would mean identity inside and outside. Donnan equil- ibrium would mean that the activity of the hydro- gen ion inside should stand in the same relation to that outside as in the case of potassium and sodium, This is very far from being true. What we apparently have in the model and in Valoma is a steady state in which water and elec- trolyte enter in a fixed ratio so that as the volume THE COLLECTING NET [ Vor. VIII. No. 69 increases the composition remains approximately constant. Dr. Abramson: Is there evidence that this model proposed for Valonia may also be applied to the red blood cell which has in some cases been considered as in thermodynamic equilibrium ? Dr. Fricke: he red cell comes into thermo- dynamic equilibrium if it is actually impermeable to sodium and potassium. But that is the question. Is there anything that is actually impermeable ? Dr. Cohen: If you asked the proponents of the viewpoint that there was thermodynamic equilibrium, | think they would agree that abso- Jute impermeability to sodium was never intended. Dr. Osterhout: The scheme proposed for Valonia is one for actively growing cells and hence does not seem to apply to the erythrocyte. It may be said, however, that in the case of pot- assium in Valonia two processes go on simultan- eously, (1) the entrance of potassium, probably as KOH, and (2) the exit of potassium, as KCI. As the cell grows older and produces less CO. the first process will fall off, but the second will not. When the exit becomes equal to the entrance we might have a steady state without growth, but this would be very different from equilibrium. Dr. Fricke: Is the membrane distinct from the protoplasm, or is it just the surface of the protoplasm in this particular case ? Dr. Chambers : plasm. Dr. Fricke: Is there direct evidence that it is? Dr. Chambers: There is indirect evidence. Non-penetrating dyes injected into the interior do not pass out, or if you put them on the outside they never go in, so there must be something on the surface that keeps them from going in, or out. Dr. Mudd: Would it not be interesting to in- ject with a micropipette droplets of various oils and aqueous solutions between the outer wall and the protoplasm of Valonia and other plant cells ? Direct evidence concerning the wetting properties of the protoplasmic surface might thus be ob- tained. It is the surface of the proto- Dr. Chambers: Oil drops can be applied to the external surface of plasmolysed protoplasts. This has been done with the epidermal cells of the onion bulb scale. Plasmolysis causes the proto- plast to shrink and draw away from the cellulose wall leaving a space between the wall and the protoplast. When a strip of plasmolysed tissue is chopped into bits at right angles to the long axes of the cells one may find many cells on the bor- der of the cut with thin end walls cut off but with their protoplasts still intact. If a relatively large drop of olive oil is applied to the exposed surface of the protoplast the oil snaps on and starts to engulf it. Before the pro- cess is carried to completion the protoplast bursts. If the oil drop is very small you get the re- verse. The droplet is engulfed by the protoplast. Dr. Mudd: in both cases ? Dr. Chambers: was the same. Dr. Mudd: For the protoplast to engulf the oil, the tension at the oil-medium interface must be greater than the sum of the tensions at the oil- protoplast and the protoplast-medium interfaces. For the oil to engulf the protoplast, the tension at the protoplast-medium interface must be greater than the sum of the tensions at the oil-medium and oil-protoplast interfaces. Both conditions can not obtain for the same system. Dr. Chambers: For the small drop of oil, material dissolves in it, so that the surface forces change. In the large oil drop the surface forces remain of one order. Dr. Osterhoui: Would it be possible to apply the drops of oil to the inner surface of the proto- plasm which is in contact with the vacuole? When the outer surface is drawn away from the cell wall by plasmolysis it tends to secrete a new cell - wall and this may possibly begin as soon as plas- molysis takes place. No such formation of cell wall occurs on the surface in contact with the vacuole, so that here, if anywhere, we might ex- pect naked protoplasm. Is the oil of the same composition The oil applied in both cases A UGUST. 26, 1 933 ] THE COLLECTING NET 291 OSMOTIC BEHAVIOR OF RED CELLS. I. Eric PONDER When one succeeds, by using some well known physical principle, in reducing to order some com- plex biological phenomenon, one can sit down to write a lecture about it with all the élan which properly accompanies the production of a rabbit from a hat. But when, by closely examining a supposedly simple biological phenomenon, one finds that the physical principles which have al- ways been supposed to apply to it do not do so in fact, and that the more thorough one’s examination, the further do the results depart from expectation, one is not quite so comfortable. Under such circumstances there is nothing to be done except to give an account of one’s experi- ments, of why they were carried out, and of the results which emerge. This is what I propose to do in speaking about the osmotic behavior of red cells, and if this lecture seems to be too personal an account of matters which appear more specific than general, I must excuse myself by saying that the erythrocyte has not been disposed to be- have itself as accountably as might be wished. Some years ago, I and my collaborators, after having measured red cell diameter and thickness almost ad nauseam, decided to make some really good measurements of red cell volume. Quite a number of methods presented themselves, e. g., refractometric methods, methods employing vari- ous kinds of chemical analyses, viscosity methods, and so on, but all of these we rejected after ex- amination. Only two methods seemed promising, the haematocrite method, and a modification of an old colorimetric method of Stewart") '?)), This last I shall describe in some detail, and I shall tefer to the haematocrite method in its proper place. The principle of the colorimetric method con- sists in mixing a known volume of a solution of the animal’s own haemoglobin, dissolved in its own plasma, with a known volume of the animal's whole blood, determining the extent to which the pigment has been diluted by the plasma contained in the whole blood, and then calculating the vol- ume of such plasma present, and thence the vol- ume of the cells. Division of the latter figure by the number of cells present, as determined by a careful count, gives the mean volume of the single cell. If properly carried out, this method is very accurate indeed, and will give the mean volume of the red cells of such an animal as the rabbit (volume about 60 »*) to within about +13. Its use, however, is rather limited, as will he seen below, for while it is excellent for finding vol- umes in plasma, in isotonic solutions, and in cer- tain hypertonic and hypotonic solutions, technical difficulties prevent its use for finding volumes in very hypotonic solutions, and it is in these, of course, that we are most interested. The method, nevertheless, is a kind of “standard method”, to which others may be compared. Using this procedure, we first determined the mean cell volumes for a number of animals, and then went on to examine the alterations in cell volume in solutions of different tonicities, for such knowledge is clearly indispensable in con- nection with problems relating to red cell permea- bility in general), It is true that the relations between tonicity and red cell volume had been studied at least since the time of Hamburger’s investigations, but we soon appreciated the fact that the method almost invariably used for meas- uring cell volume, the haematocrite method, is subject to errors about as great as many of the changes which have to be detected. This point I shall return to directly; in the meantime it is sufficient to say that we began by trying to meas- ure the mean volume of rabbit red cells suspended in mixtures of plasma and NaCl of tonicities from about the equivalent of 1.6 p. c. NaCl (grams per 100 grams water) to about the equiv- alent of 0.8 p.c. “NaCl, and in mixtures of plasma and KCl and of plasma and glucose, covering about the same tonicity range. At this point I may remind you that rabbit’s plasma, which is presumably isotonic with the interior of the rabbit erythrocyte, has a tonicity corresponding to about 1.1 p. c. NaCl as determined by freezing point depression measurements, 1. e., a tonicity roughly in the middle of the selected range. I ought also to emphasize that the colorimetric method involves the addition of haemoglobin in plasma to whole blood, and that the systems therefore contain a very considerable amount of plasma, although the latter is rendered hypertonic by the addition of hypertonic NaCl, KCl, or glucose, or hypotonic, by the addition of hypotonic NaCl, KCI, or glu- cose, as the case may be. At first we adopted the plan of measuring the volume of the cells in all the systems of differing tonicity in one experiment, but the results turned out to be exceedingly irregular. For example, in one experiment we would obtain the following values : Tonicity 0.8 0.9 1.0 11 1.2 1.3 1.4 1.5 Plasma Vol., ps 60.7 58.6 57.1 55.9 54.2 53.1 51.951.6 53.8 the “normal volume” of the cells (i. e., their vol- ume when suspended in plasma) being maintained ina mixture of plasma and NaCl of an equivalent tonicity of about 1.2 p, ¢, NaCl, while in hypo- 292 THE COLLECTING NET [ Vor. VIII. No. 69 tonic solutions there was swelling and in hyper- tonic solutions shrinking. In another experiment, however, we would get: Tonicity 0.9 1.0 11 1.2 13 1.4 1.5 1.6 Plasma Vol. y3 77.1 75.5 73.5 71.9 70.4 69.9 65.1 63.8 63.7 all the solutions now bringing about swelling. Contradictory experiments of this kind soon showed us that some factor was being neglected, and this turned out, as so often happens, to be a time factor, the volume of the cells depending not only on the tonicity of the environment, but also on the length of time during which the cell had been exposed to the particular hypertonic or hypo- tonic medium surrounding it. Once this was taken into account, all the irregularities disap- peared, and we began to get consistent results of the kind shown in_ the following table, in which the volume of rabbit red cells, exposed to NaCl- plasma mixtures of various tonicities, were meas- ured colorimetrically after various intervals of time. All the volumes are expressed as percent- ages of the “normal volume” found for the cells suspended in plasma. Iinal tonicity Cell volume r. NaCl per 100 g. water Oo S)vobioly woLOinovbo Winhe Aone Shlohe 0.81 104 105, 05 ~~ 109 _ 0.91 OZ LOSS OSS LOST OS 1.02 102 100 104 #& 102! — Ll 99 100 101 100 ~—=—-100 1.22 103. 104 == IOS — Oe, 7 OOM LOZ LO: — 1.42 102 103 106 106 +#« 108 LS LO 109 S07 Or ee The most striking point about these results is the swelling, instead of the shrinking, which occurs in hypertonic solutions, although with considerable irregularity, even considering that the method has an error of at least 2 p. c. attached. This swell- ing, furthermore, often increases with time, as the figures for the tonicities 1.32 and 1.42 illustrate very well. There is, of course, one particular concentration of NaCl, in this case 1.12 p.c., in which the “normal cell volume” is maintained, but for concentrations both greater than this and less than this, the behavior of the cell is quite anomalous, for in the former there is often swell- ing instead of the expected shrinking, and in the latter, although the figures of this table do not show it unless calculations are made, the swelling is considerably less than one would expect it to be. One requires some explanation for these anomalies, especially as exactly the same sort of behavior is met with in plasma-KCl and plasma- glucose systems; clearly we are not dealing with a “simple” or “perfect’’ osmometer, which, being impermeable to cations and other osmotically ac- tive substances, loses or gains water only, and thus changes in volume as required by the Mar- riotte law in its simplest form. Now this conclusion, obvious though it is, is not far removed from physiological heresy, for if there is any one point which is generally accepted about the red cell, it is that it is impermeable to cations, the principal osmotically active substances which it contains. The validity of many of the calculations of Van Slyke!®) ‘®), L. J. Hender- son'"!, Peters, and many other investigators de- pend entirely on the existence of this impermea- bility, which is accepted by almost everybody, al- though Hamburger and one or two others have held that there is an Na-K exchange across the membrane, and have supplied evidence for it which, in my opinion, has been disregarded rather than disapproved in its entirety. One must bear in mind, nevertheless, that Henderson, Van Slyke, and others who work along the same lines, are concerned with the behavior of the erythrocyte in normal plasma, and not with the volume changes which it undergoes in grossly hypertonic or hypo- tonic media. Within the “physiological range” its behavior may quite well be much simpler than its anomalous behavior outside that range, or, if not actually different, at least not sufficiently anomal- ous to attract attention. The colorimetric meas- urements of volume show, indeed, that ina NaCl- plasma mixture of about the same freezing point as that of rabbit plasma (equivalent to about 1.1 p. c. NaCl), the rabbit red cell maintains its nor- mal volume more or less indefinitely, i. e., it be- haves as if it were impermeable to cations, al- though in hypertonic and hypotonic solutions, (as we shall see even more clearly below) it behaves quite otherwise. One can thus easily enough wriggle out of the apparent disagreement between the results of the experiments in hypertonic and hypotonic solu- tions and the accepted impermeability to osmoti- cally active substances, for the latter can be put down as being a property of the red cell mem- brane when in its normal environment of isotonic plasma, although not necessarily as a property of the cell when in a non-physiological medium. Un- fortunately, however, one cannot evade with equal facility the very definite claim that the erythro- cyte, when surrounded by hypertonic or hypotonic media, exchanges water according to the Boyle- Marriotte law, and, in doing so, still behaves as a “perfect osmometer” impermeable to cations and other osmotically active substances. This latter claim is not put forward as a postulate, as is the idea of impermeability in isotonic plasma (which one can scarcely, in the nature of things, prove or disproye), but as an experimental fact'*) ®, Aucust 26, 1933 ] THE COLLECTING NET. 293 and so I shall be required to describe briefly the kind of experiments on which it is based. These experiments consist almost entirely of determinations of red cell volume by the haema- tocrite method, in which a volume of red cells with the medium in which they are suspended is placed in a narrow capillary and spun at high speeds until the column of packed cells either shows no further decrease in length on further spinning, or shows a kind of translucence known as Koeppe’s criterion. If the same number of cells is suspended in solutions of different tonicity. (e. g., plasma, diluted plasma, etc.) the percentage swelling in each of the hypotonic solutions can be obtained by dividing the length of the column for each of the hy potonic solutions by the length of the column for the cells in plasma, the paliree of the latter in this way being regarded as 100 p.c., and all other volumes as percentages of it. And similarly for hypertonic solutions. Now let us imagine that we are dealing with cells which contain 67 p.c. of their volume (the usual figure) as water with osmotically active substances dissolved in it, the tonicity of the cell interior being the same as that of plasma, which we shall call 1.0. Then if the cells are placed in media containing plasma, plasma diluted with water so as to give a tonicity of 0.9, 0.8, 0.7, etc., and if the cells reach equilibrium with the hypo- tonic solutions by a water exchange alone, we must have the following volumes attained at equi- librium, the volume of the cells in plasma being denoted by 100, as before: Plasma Diluted plasma = 110) OS OlS Oe OlGmen Obs 100 107/129 SAA 167 Ultimately, of course, the cell attains a certain “critical volume” at which it can swell no further without haemolysing. This is the result which is consistent with the cell’s being a “perfect osmo- meter”, and swelling as a result of water exchange alone. Some investigations by the haematocrite method have shown that this kind ot result is obvained, but others have shown that it is not, and that the swelling is always considerably less than that de- manded by the Boyle-Marriotte law. Under any circumstances, the results are irregular and vari- able. The difficulty is one which is inherent in the haematocrite method itself, for the result one obtains depends very largely on the rate of spin- ning which one uses, and the “right rate’’ may be, and is usually, quite different for cells in plasma and for cells in a hypotonic medium. In a lecture of this sort any consideration of the fallacies of the method, or even on account of the divergent results which it has given in the hands of differ- ent investigators, rauld! he out of place, but we can summarize the situation, so far as haematoc- rite determinations go, by saying that the swell- ing and shrinkage ot red cells is usually, although not always reported to be less than it should be. ‘This is the same sort of result as emerges from my own colorimetric determinations. The latter, how- ever, are obtaimable only over a very limited range, and so we have to look for another method of determining red cell volume, and it is necessary that such a method shall be able to give values for volume even in very hypotonic solutions. Provided that a certain amount of care is used in applying it, the ideal method for measuring volume is a diffraction method in which the cells are converted into spheres without any change in their volume, and in which the mean volume is found from the diffractometric measurement of their mean radius"). To turn the normal dis- coidal cells into spheres, all that is necessary is to suspend them in a hypertonic, isotonic, or hypo: tonic saline, and then put them between a slide and a closely applied coverglass™’). lor some reason, at present obscure, they then become per- fectly spherical, and it can be shown in various ways that this change of shape does not involve a change in volume. lhe ditfratometric measure- ment of volume is very accurate, and, by making volume measurements for cells in plasma or in a saline isotonic with it, for cells in hypertonic so- lutions, and for cells in hypotonic solutions, one can show quite readily that the Boyle-Mariotte law roes not apply at all. In hypertonic solutions one gets the same rather irregular shrinkage as the colorimetric method shows, or even sometimes swelling. In isotonic solutions (about 1.1 p.c. NaCl) one gets a maintenance of the normal vol- ume, and in hypotonic solutions one gets much less swelling than the Mariotte law demands, al- though the results are perfectly regular, and the “swelling curves’ obtained by plotting relative cell volume against tonicity are perfectly smooth up to the point where lysis begins. On the basis of these experiments, together with the restricted results obtained colorimetrically and the rather unreliable results obtained by the use of high speed haematocrites, | have no hesitation in say- ing that the erythrocyte does not swell, or shrink, as if it were a “perfect osmometer’reaching equil- ibrium by water exchange alone. And at this stage I want to remark that this is a conclusion of fact, and that it does not necessarily involve the ac- ceptance of any particular theory which may be put forward to account for it, especially the theory which I shall put forward directly. If we admit, then, that the red cell does not behave as the Mariotte law requires, what ex- planation can we give for the divergence? Juite a number of observers have recognized, to a 204 THE COLLECTING NET { Vor. VIII. No. 69 greater or lesser extent, that the divergence ex- ists, and it will be simplest if I tabulate the ex- planations which have been offered. 1. It is conceivable that water should enter the cell from a hypotonic medium, that the cell should swell, but that its expected swelling should be prevented by its membrane being stretched and then exerting a “back pressure” equal to the difference between the osmotic pres- sure of the interior and the surrounding medium at equilibrium. In the case of the red cell, this explanation can scarcely be countenanced, for the membrane is less than lp thick, and could not stretch to the required extent within its elastic limits. 2. It is possible that either the cell contents or the medium which bathes the cell might under- go dissociation in a very anomalous manner, so that osmotic equilibria, as calculated in the usual way, would not be equilibria at all. Ege has con- sidered this possibility, and has rejected it. One must express one’s self guardedly nevertheless, for osmotic equilibria in systems containing large amounts of protein on one side of a membrane are sometimes very anomalous indeed, Consider- ing, however, that the electrolyte concentrations are relatively high, and that the range of tonicity over which we work is really exceedingly small, I doubt if anomalous dissociation could account for the relatively large divergences observed. 3. Ina similar way we have to consider the possibility that the red cell interior has initially an Osmotic pressure different from that of the surrounding plasma. This used to be thought to be the case, but recent investigations lead to the far more likely conclusion, viz., that the interior of the cell 1s initially in equilibrium with its sur- roundings. 4. When it was first noticed that the swell- ing which occurs in hypotonic solutions is less than that expected on the basis of Marriotte’s law, various observers introduced the idea of “bound water” rather than abandon the idea that the cell is a perfect osmometer. Thus, if the cell actually contains, as shown by analysis, 67 p.c. of water by volume, some of it was sup posed to be “osmotically inactive” or “bound,” and the’ rest “tree” If such a state of affairs were actually to exist, the cell would, of course, reach equilibrium with a hypotonic me- dium by taking in about half as much water as it would have to do if all its water were “free,” and so the small volumes met with in hypotonic solutions would be accounted for without aban- doning the idea of impermeability to cations and other osmotically active substances. This “bound water” idea has had quite a vogue, parallelled by the idea of “bound water” in muscle and in other cells which do not swell “as they ought to do” in hypotonic solutions. No one, of course, has ever demonstrated the existence of this bound water by independent means, and recently Hill has shown that, even if it exists at all, it makes up only about 5 p.c. of the total water present in the cell''*). There has nevertheless developed quite an extensive literature regarding the purely imagin- ary substance, because of the principle of sancta simplicitas, | imagine, rather than any other, 5. Although most physiologists have consid- ered the erythrocyte as being impermeable to ca- tions, etc., this assumption and its consequences have usually been confined to the erythrocyte when bathed in plasma, and when the cell is suspended in hypotonic media there is, as | have already re- marked, no reason to abide by it. In fact, there is plenty of evidence that under such circum- stances the cell actually loses cations (mainly Kk) into a hypotonic environment, and the existence of the cations lost has been demonstrated in the suspension medium by chemical analysis by Kerr (3) (4) (5) | Neubauer and! Bres- lin?®, and others. When the suspension me- dium is glucose, it is universally admitted that there is a loss of cations from the cell, and this can be shown conclusively by conductivity meas- urements. In fact, there is abundant evidence to show that while the cell may be a more or less per- fect osmometer when in plasma, in which it is not called upon to be much of an osmomter at all, it is a very imperfect one when the medium is NaCl, KCl, or glucose, especially if the solutions of these substances are hypotonic. I now propose to put together two facts: (1) If the red cell were a perfect osmometer, it would swell in hypotonic solutions according to a regular swelling curve, calculable from Marriotte’s law, and (2) the cell in fact swells in such a way as to give a regular swelling curve, but a different one from that calculable from Marriotte’s law. The fact that both the “theoretical” and the ob- served swelling curves are regular and of the same general form does not appear to have been commented upon, and so we shall examine the re- lation between the two curves more closely. At once we may observe that the difference between two curves of similar and regular form must it- self be describable in simple, if empirical, terms. To make things as simple as possible, I pro- pose to use the fiction of “bound water.” We shall think of the cell as a perfect osmometer. If all its water were “free” it would swell accord- ing to Marriotte’s law. In fact, it swells less, so we shall suppose that some of the water is “bound.” Now let us denote the tonicity of the medium by T, and let us put the tonicity of the cell interior as equivalent to a 1.1 p.c. NaCl. Let us also use v to mean the percentage increase in cell volume when the cell reaches equilibrium with a hypo- Aucust 26, 1933 ] tonic solution. Then, if everything is expressed in the proper units, we have as an equilibrium con- dition, 1.1 Os i = 3(Q)) O,+v 100—v where Ou. is the quantity of free water present in the cell. Let QO, be the total quantity present ; then let us use the fraction 1000 O2/O1 bans t2) as a measure of the fraction of the total water Rr = which is free, or, alternately, there being no bound water in fact, of the fallacious- ness of the idea that we are dealing with a simple osmometer. Then if R = 1.0, our osmometer is perfect; in fact, however, the values of v found experimentally are always sucn as to give a value of k of about 0.5, or even, in the case where glucose is the suspension medium, of 0.3, and so we require to find a reason, in quan- titative terms, why this should be so. There are many conceivable explanations, but the most likely is this. Let us imagine that the cell, when suspended in a hypotonic medium, is not wholly impermeable to osmotically active sub- stances, but that as it gains water it loses a quan- tity of osmotically active substance x, thereby reaching equilibrium. Then we must have Ota at — 53) Ore tay x being, in the units used, a quantity of osmotical- ly active substance in grams lost by a litre of cells, and expressed as equivalent to a NaCl solu- tion of the same freezing point. The units are troublesome, as they always are in equations in- volving tonicities, but if we express x as a func- tion of the original amount of osmotically active substance in the cell and call it X, we arrive at the curious relation dX/dT =const. « 1/R (4) Equal steps of tonicity thus result in the loss of equal amounts of osmotically active substances from the cell, the smaller the value of R, the greater being the loss per unit step. As an in- stance, take the case where R = 0.5, i. e., where the cells swell as if only half their water were “free,” and let us take the tonicity of the cell in- terior as 1.0. Then as we pass one tonicity to an- other, immersing the cells in 1.0 p.c. NaCl, 0.9 p.c. NaCl, 0.8 p.c. NaCl, and so on, the quantity of osmotically active substances lost increases in the following way: Tonicity, g.NaCl p.c..... 1.0 0.9 0.8 0.7 0.6 0.5 0.4 X, p.c. original NaCl lost.. 0 5 10 20 30 40 50 ‘THE COLLECTING NET 295 Equal steps in tonicity thus correspond to losses of equal amounts of osmotically active substances. The idea of the loss of cations, etc., from erythrocytes in hypotonic media is not in itself at all new, as I have already remarked; the interest- ing point, however, is the regular way in which the loss appears to occur, for the observed swell- ing curve differs from the theoretical one in just such a way as one would expect 1f some constant factor were operating to bring about the diver- gence. The presence of “bound water” would do as a factor, but this substance does not exist in appreciable quantities. The loss of cations, etc., is a sufficient explanation, especially as we have independent evidence that it occurs, but it is not the only possible explanation, nor it 1s necessarily completely adequate in a quantitative sense, for a cation loss, together with other modifying factors which operate as regular functions of tonicity might be necessary to account for the phenomen- on fully. The important point, to my mind, is that all these factors do in fact seem to operate as regular functions of tonicity, and, indeed, if ex- pression (+) is correct, as linear functions, at least when considered in the aggregate, and that the red cell, although it does not behave as a per- fect osmometer in the sense of Marriotte’s law, hehaves in a very orderly fashion according to a law of its own. So far I have been speaking of systems con- taining red cells in NaCl, KCl, or glucose of dif- ferent tonicities, plasma being absent in all those cases in which the measurements were made dif- fractometrically, for red cells will not become spherical in the presence of plasma. Plasma is cer- tainly present in those systems in which the vol- ume measurements were made colorimetrically, but, as I have said already, this method covers too small a range to provide us with much infor- mation regarding swelling in hypotonic solutions. The swelling curves and the expressions which apply to them (expressions 1, 2, 3 and 4) are thus based on systems in which the cells are sus- pended in plasma-free media, and we have now to take the next step and see what happens when the cells are suspended in media containing plasma which has been rendered hypotonic by the addition of water. This step is important, for it may be laid down as a principle that no red cell (and probably no other vertebrate cell) is “normal” unless it is suspended in the plasma of the animal from which it is derived. In the case of the erythrocyte, its form and metabolism are both dependent on its environment, and_ cells suspended in saline, however well buffered or balanced, are different in many respects from the saine cells in plasma. So far as their behaviour in hypotonic solutions is concerned, cells in NaCl, KCl, ete., might be very different from cells in 296 THE COLLECTING NET [- Vor. VIII. No. 69 plasma, for there are at least three factors which might bring about a difference. 1. The volumes attained in hypotonic NaCl, KCI, and glucose are found diffractometrically, the cells being converted into spheres by being placed between a slide and a coverglass. The measure- ments of volume are themselves reliable, but the objection can always be raised that the same kind of unknown forces which change the form of the cell may also alter its permeability, and so cause it to behave in an anomalous way. 2. \hen cells are suspended in NaCl, KCI, or glucose, changes in pH may occur, and these may be considerable. Particularly is this so in glucose, which is the very substance in which cation loss is most marked; the anomalous swelling of the cells in these media might therefore be related to undetected alterations in pH, bringing about changes in osmotic pressure. 3. Quite apart from these particular consid- erations, it is not only possible but probable that the red cell membrane, when surrounded by plasma, is a different membrane in a_ physical sense, from the membrane of the cell bathed in NaCl, KCI, or glucose. It would be very unsafe indeed to suppose that the properties of the mem- brane in these latter media would be any guide to its properties when in plasma. : Difficulties arise, however, when we try to measure red cell volume in hypotonic plasma. The colorimetric method cannot be used, haematocrite methods are worse than useless, and the diffrac- tion method is not applicable to the discoidal cells in plasma. We have therefore to employ a dif- ferent type of method in which we do not measure volumes in absolute units, but rather the percentage increase in volume, v, which results when the cell comes into equilibrium with the hypotonic plasma. These methods are three in number”, (a) If the cell takes in water from a hypo- tonic medium, its density will decrease, and from the extent of the decrease the amount of water taken in can be calculated. (b) Ina similar way the content of haemo- globin per unit volume of cells must diminish if the cells take in water, and from the extent of the diminution the amount of water which has en- tered can be computed. (c) If the cell takes in water from a hypo- tonic solution, the amount of water taken in can be calculated from the figures for the water con- tent of the cells in plasma and for the swollen cells in hypotonic plasma, both being obtained by drying a weighed quantity of cells to constant weight at 60°. Of the three methods, which all agree with each other sufficiently closely, the last is the most convenient. When applied to erythrocytes in hypotonic plasma, all three methods give the same result, viz., the cells swell as if only 50 to 70 p.c. of their contained water were “free,” a result very similar to that obtained in hypotonic NaCl and KCl, al- though perhaps a little nearer that which would be expected from a perfect osmometer. It is therefore clear that even in highly buffered plasma the cells do not follow Marriotte’s law, but rather the law expressed in expressions (1, 2, 3 and 4). I shall now summarise these results so far as the erythrocyte is concerned. 1. When the red cell is placed in an isotonic solution of NaCl, KCl, or glucose, it maintains its normal volume unchanged. The concentration of such an isotonic solution is one which is very nearly equivalent to the normal plasma of the ani- mal from which the cells are obtained. It is diffi- cult to be certain as to how nearly equivalent it is, and I have the impression that the isotonic sa- line solution is really a little more concentrated than the plasma; at all events, there is no great discrepancy. 2. When the cell is placed in a hypertonic solution of NaCl, KCl, or glucose, it shrinks less than it should if it obeyed simple osmotic laws, and its volume tends to increase with time. This is in accordance with the idea that cations pass through the membrane under such conditions. 3. When the cell is placed in hypertonic solu- tions of NaCl, KCl, or glucose, is swells much less than it ought to if simple osmotic laws are obeyed. We therefore suppose, with Kerr and others, that it loses cations or other osmoucaily active substances. 4. The cell nevertheless swells in a regular fashion as the tonicity decreases. This regular swelling could be accounted for by assuming that only part of the contained water is “free,” or (since the idea of free water is not admissible), by supposing that the loss of osmotically active substances is a simple function of the tonicity. 5. This applies also to cells in highly buffered hypotonic plasma, although in this medium the extent of leakage appears to be slightly less. The conclusion therefore is that the cell reaches equilibrium with a hypotonic environment in a way which cannot be accounted for by Marriotte’s law, although it does so in a way which is sus- ceptible of exact expression. I said a little time ago that it behaves in an orderly manner “accord- ing to a law of its own,” but this, in a sense, is a misstatement, for several other types of cell be- have similarly. Hill?), for example, has shown that that same sort of thing is true for the muscle cell, which also swells less in hypotonic solutions than it ought to do if it followed simple osmotic laws. It behaves, like the red cell, as if only part Aucusr 26, 1933 ] _THE COLLECTING NET 297 of its water were free, and Hill’s explanation is essentially the same as that which I have given for the erythrocyte, viz., that there is a loss of cations. Siebeck''*’ has reached the same conclu- sion, and given the same explanation, for the cells of kidney tissue, and Pantin") has used the same idea to account for the anomalous osmotic behaviour of Gunda. Even in the case of the cell which is supposed to be the typical perfect osmometer, the Arbacia egg, McCutcheon and Lucké'?") have found too small a degree of swell- ing 1f the egg is injured. If it makes the idea any more easy of acceptance, I am prepared to con- cede that a red cell, a muscle cell, or a kidney cell is “injured’’ when it is placed in a grossly hypotonic environment; again, however, I em- phasize the essential point, that the effects of this “injury” are expressible quantitatively, and pro- ceed in a regular fashion as the “injuriousness”’ of the environment changes. REFERENCES FOR PAPER 1 1. Stewart, G. N. 1899a. The behavior of the haemoglobin and electrolytes of the coloured cor- puscles when blood is laked. J. Physiol., 24, 211. 2. Stewart, G. N. 1899b. The relative volume or weight of corpuscles and plasma in blood. J. Physiol., 24, 356. 3. Ponder, E. and Saslow, G. 1930a. The meas- urement of red cell volume. J. Physiol., 70, 18. 4. Ponder, E. and Saslow, G. 1930b. The meas- urement of red cell volume. II. Alterations in cell volume in solutions of various tonicities. J. Physiol., 70, 169. 5. Van Slyke, D. D., Hastings, A. B., Murray, C. D. and Sendroy, J. Jr. 1925. Studies of gas and elec- trolyte equilibria in blood. VIII. The distribution of hydrogen, chloride and bicarbonate ions in oxygen- ated and reduced blood. J. Biol. Chem., 65, 701. 6. Van Slyke, D. D., Wu, H. and McLean, F. C. 1923. Studies of Gas and Electrolyte Equilibria in blood. V. Factors controlling the electrolyte and water distribution in the blood. J. Biol. Chem., 56, 765. 7. Henderson, L. J. 1928. Blood: A Study in Gen- eral Physiology. New Haven. Yale University Press. p. 199. 8. Ege, R. 1922a. Untersuchungen iiber die Vol- umveranderungen der Blutkorperchen in Lésungen von verschiedenem osmotischen Druck. III. Mitt. Studien tiber das osmotische Verhalten der Blut- korperchen. Bioch. Z., 130, 99. 9. Ege, R. 1922b. Untersuchungen iiber die Per- meabilitat des Blutkorperchenhautchens fiir Elek- trolyte. IV. Mitt. Studien iiber das osmotische Ver- halten der Blutkorperchen. Bioch. Z., 130, 116. 10. Ponder, E. and Saslow, G. 1931. The meas- urement of red cell volume. III. Alterations of cell volume in extremely hypotonic solutions. J. Physiol., 73, 267. 11. Ponder, EH. 1928-29a. On the spherical form of the mammalian erythrocyte. Brit. J. Exp. Biol., 6, 387. 12. Hill, A. V. 1930. The state of water in muscle and blood and the osmotic behavior of muscle. Proc. Roy. Soc. B., 106, 477, 13. Kerr, S. E. 1926a. Studies on the inorganic composition of blood. I. The effect of haemorrhage on the inorganic composition of serum and corpuscles. J. Biol. Chem., 69, 689. 14. Kerr, S. E. 1926b. Studies on the inorganic composition of blood. II. Changes in the potassium content of erythrocytes under certain experimental conditions. J. Biol. Chem., 67, 721. 15. Kerr, S. E. 1929. Studies on the inorganic composition of blood. III. The influence of serum on the permeability of erythrocytes to potassium and sodium. J. Biol. Chem., 85, 47. 16. Neubauer, B. S. and Breslin, J. E. 1923. Johns Hopkins Hosp. Bull. 34, 199. 17. Ponder, E. and MacLeod, J. 1933. J. of Physiol., 77, 181. 18. Siebeck, R. 1912. iiber die ‘osmotischen Eigenschaften” der Nieren. Pfliigers Arch. 148, 443. 19. Pantin, C. F. A. 1931. The adaptation of Gunda ulvae to salinity. III. The electrolyte ex- change. J. Exp. Biol., 8, 82. 20. McCutcheon, M. and Lucké, B. 1932. The liy- ing cell as an osmotic system and its permeability to water. Physiol. Rev., 12, 68. DISCUSSION Dr. Fricke: How long does it take to reach equilibrium in these hypotonic solutions? Could you follow, by any method, the volume changes which occur before equilibrium is reached ? Dr. Ponder: It takes about a minute to make a measurement of volume, diffractometrically, and by that time equilibrium has been reached in the systems which contain a large amount of NaCl and a small quantity of cells. I doubt if one could follow the volume changes; one would cer- tainly need to devise a new method for doing so. Dr. Cohen: Do you get intermediate values for cell volume in hypotonic solutions if you mix the NaCl and the glucose ? Dr, Ponder: Thus far I have not reached the stage of working with mixed solutions, but I imagine that one would get intermediate values of some kind. Dr. Briggs: Is the NaCl solution, in which the cells do not either shrink or swell, exactly isosmo- tic with the plasma? Dr, Ponder: That is a point upon which I would not like to commit myself, but it is cer- tainly very nearly isosmotic with the plasma. The difficulty is that we can measure cell volume only to within about +2 p.c., and that the three volumes, 99 p.c., 100 p.c., and 101 p.c. would be indistinguishable from each other. These three volumes would nevertheless correspond to three different tonicities, and so it is impossible to say just what the tonicity for the maintenance of normal volume is. We can, however, usually pick out one NaCl solution, within this tonicity range, which has the same depression of freezing point as plasma. 298 THE COLLECTING NET [ Vor. VIII. No. 69 Dr. Chen: Many preparations of NaCl con- tain silver, and it is known that this impurity has an effect on the haemolysis of fish cells. Dr. Ponder: Wahlbaum’s NaCl, I believe, is silver-free, and it was this preparation which | used. Dr. Abramson: ible ? Dr. Ponder: 1 do not know from my own ex- perience, but it is stated not to be. Dr. Abramson: In regard to the effect of the slide and coverslip on the red cells, are the cells, when they are spherical, very close to the glass surfaces ? Dr. Ponder: No. The diameter of the cell in its spherical form is about 5y and the distance be- tween the surfaces about 50u. The cells move about freely between the surfaces. Is the leakage effect revers- Dr, Abramson: What I had in mind was that the potential on the glass surface might be re- sponsible for the change in form. Dr. Ponder: That has been suggestea, but I do not think that it is so. Dr. Abramson: Would the cells become spherical if you put the glass surface into plasma and had on it an adsorbed protein layer? Dr, Ponder: The presence of plasma always prevents the assumption of the spherical form, but I do not think it is because it is adsorbed on the glass surfaces. It is rather an effect on the cell itself. Other proteins, such as haemoglobin and gelatin, are altogether without effect. Dr. Chambers: One of the first papers on microdissection described the same sort of thing. If you approach a red cell with a microdissection needle, the cell undergoes crenation not unlike that which occurs before the spherical form is produced. Dr, Abramson: Have you any explanation for the occurrence of the spherical forms ? Dr. Ponder: The only explanation I can of- fer is that it is a pressure effect. When the slide and coyverglass are as close together as they are, the pressure between them may be considerable, and I fancy that this may in some way affect the cells lying between the surfaces. Dr. Chambers: 1 have done rough measure- ments on Arbacia eggs in hypertonic, and hypo- tonic, NaCl solutions, and I have found that the shrinkage and swelling, in NaCl solutions, is less than it is in calcium chloride solutions. The changes in calcium chloride are reversible, but those in NaCl are not. Dr, Ponder: McCutcheon and Lucké say that the Arbacia egg is a perfect osmometer in hypo- tonic sea water but I do not know that it follows that it is one in hypotonic NaCl. Under those circumstances the eggs may be injured, and this might account for the small swelling which you observe, and for its irreversibility. In fact, | am quite prepared to admit that when you place a cell in anything except plasma, or sea water, as the case may be, you are doing it an injury. Dr. Cohen: Then one conclusion to derive from these results is that the integrity of the membrane, as an osmometer, depends on its ex- ternal environment, as well as upon its structure. Dr. Ponder: That is so, but it is not generally admitted, or, if admitted, is not sufficiently taken into account. The shape of the cell, and its metabolism, are influenced profoundly by the en- vironment, and I have no doubt that almost every other property is affected as well. Aucust 26, 1933 ] THE COLLECTING NET 299 THE EFFECT OF WING BUD EXTIRPATION AND TRANSPLANTATION IN CHICK EMBRYOS ON THE DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM Dr. VikTOR HAMBURGER Rockefeller Fellow in Zoology, University of Chicago These experiments deal with the problem of determination of the nervous system. This organ is, in the vertebrate embryo, determined as a whole in the early stages of development. But that does not mean that all the qualitative and quantitative details of its pattern are already fixed at that time. We know that long after the formation of the spinal cord and the separation of the spinal ganglia these parts are—at least in their quantitative development—submitted to the influence of different factors. We shall not deal with the group of itrinsic factors, working within the spinal cord itself. There exist growth correlations between the dif- ferent levels of the spinal cord, which have es- peciaily been revealed by Detwiler (1) and other authors. The same author and his collaborators have shown in extensive experimental work, that er- frinsic factors, especially the peripheral fields to be innervated, also play an important role in the determination of the growth of the nervous sys- tem. To prove this, one has to decrease the peri- pheral field, for instance by extirpating a limb bud, or to increase it by implanting an additional one. The effects of such experiments are striking. Extirpation is followed by hypoplasia, additional implantation is followed by hyperplasia of the spinal ganglia concerned in their nerve supply. But this effect is, according to Detwiler’s studies on Amblystoma, strictly limited to the spinal ganglia. The spinal cord does not react at all, either to decrease or increase of the peripheral held. Whereas all students of the problem agree in the reaction of the spinal ganglia, Detwiler’s statement concerning the spinal cord is nof in agreement with other results. I shall only refer to the experiments of Miss Shorey (2), per- formed 25 years ago in Dr. F. R. Lillie’s labora- tory at the University of Chicago. She extirpated one wing bud in 72-hour chick embryos and found a remarkable hypoplasia, not only in the spinal ganglia, but also in the spinal cord, especially in the motor centers of the anterior horn of the op- erated side. To settle these discrepancies, Dr. Lillie pro- posed to me to repeat these experiments. Miss Shorey had met with many difficulties in the tech- nical procedure by using the thermocauter. The extirpation can be done much more easily and successfully by using a glass needle and a hair loop. The wing buds in 72-hour chick embryos can be removed by a single cut of the needle, The chorion heals well. Before the operation, a rec- tangular window is sawed in the shell, which 1s sealed in again after the operation. The embryos were fixed 5-6 days after operation. They show in most cases complete absence of the wing. One can increase the loss of muscles by additional extirpa- tion of the anlagen of the shoulder girdle and its muscles. This variation proved to be valuable for further analysis. Histological study of four cases revealed the following facts: On the operated side, a variable portion of the shoulder girdle and its muscles is present. The spinal nerves of the wing level are much smaller on the operated side, but a normal brachial plexus is formed. The spinal ganglia, Nos. 13-16, show a remarkable hypoplasia. The spinal cord is strikingly affected too, Four groups of cells may be distinguished in it: (1) The most obvious hypoplasia can be seen in the lateral motor group, supplying the wing. The number of neurones is considerably reduced. (2) The median motor group is not affected. These neurones supply the axial trunk muscles whose anlagen were not injured in the operation. (3) The size of the posterior horn is reduced. (4) The medial part of the cord is not af- fected. These data completely confirm Miss Shorey’s results in all details. They also agree with the re- sults of R. May (3), who recently reported motor cell hypoplasia in frogs following limb bud ex- tirpation. The contradictory results of Detwiler concerning Amblystoma cannot be explained at this time. It seemed to be interesting to follow the hypo- plasia more in detail, especially that of the motor region, and to compare it with the actual muscle loss. To get exact quantitative data, counting of cell nuclei was begun in the spinal ganglia and the cord, and the muscle loss was calculated by drawing the muscles on cardboard, cutting them out and comparing the weight of the right and left brachial muscles. The hypoplasia in the lateral motor group, ranging from 62% to 30%, corresponds quanti- tatively to the loss of muscles, ranging from 92% to 43%; whereas the hypoplasia in the spinal ganglia does not vary, showing an average of 35%. This is to be expected, as the skin loss is nearly the same in all cases, independent of whether or not the shoulder muscles are present. These quantitative studies seem to indicate a correlation between each part of the peripheral field and its own nerye center, The muscles ap- 300 THE COLLECTING NET [ Vor. VIII. No. 69 parently control the growth of the motor centers, and the skin controls that of the sensory center, and both actions may be independent of each other. [| hope by continuing these quantitative studies to approach the main problem of this field, the problem of the mechanism by means of which the peripheral field manages to stimulate the growth of the nervous system. The argument would be as follows: If the different parts of the peripheral field influence their own nerve centers directly, then no other path of transmission for these stimuli need be imagined than the nerves themselves connecting the peripheral areas with their centers. Transplantations. It was to be expected that, as in amphibians, the overloading of the periphery in a given region, for instance by the implanta- tion of an additional limb, might result in hyper- plasia of the corresponding part of the nervous system. Therefore a series of hind limb and wing transplantations was performed. A limb bud was cut out of a 72-hour chick embryo and transplant- ed to another embryo of the same stage. Lateral- ly, between wing and hind limb bud, there is just rcom for a third bud (For details of technique see (4) ). The transplant is allowed to develop for 4-6 days and often grows normally. Only two cases have been sectioned up to the present time. Both show normal histological differ- entiation of muscles, cartilage, ete. One of them is completely nerveless, thus proving, as has been shown for frog development (5), that normal formation and histological differentiation are inde- pendent of nerve supply. The other transplant is innervated by two small nerves, emerging from the spinal cord at the ninetcenth and twentieth segment. The two spinal ganglia, numbers 19 and 20, show an increase in cell number of 28% and 23%. The spinal cord has not yet been studied. LITERATURE 1. A recent review of the experimental work of Detwiler and his collaborators may be found in: Detwiler, S. R., Biol. Rev. of the Cambridge Philos. Soc. 8, 1933. 2. Shorey, M. L. Jour. Exper. Zool. 7, 1909. 3. May, R. Bull. Biol. de la France et de la Bel- gique. 67, 1933. 4. Hamburger, V. Anat. Rec. 55, 1933. No. 4 Suppl. . 5. Hamburger, V. Roux Arch. 114, 1928. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 8). THE DIFFERENTIATION OF THE PROTOPLASM OF EGG CELLS DURING EARLY DEVELOPMENT (Continued from Page 265) the formation of the polar bodies. After the phenomena of maturation, some of the egg substances begin to migrate towards the pole Where the polar bodies have been formed, the others towards the opposite pole. This migration holds for both microscopical and ultramicroscopical particles. A series of ex- periments showed that this new arrangement can- not be caused by gravity, nor by differences of the surface tension. Its formation also is too com- plicated to be explained by diffusion phenomena. This conclusion alone makes it probable that the cause of this so-called “bipolar differentiation’’ must be some electrical phenomenon. In a study of the bipolar differentiation of the eggs of the neapolitan Nerets Dumerilii, it could be shown that the cell substances of these eggs were being distributed in such a way that sub- stances of a high pH migrated towards the ani- mal pole, substances of a low pH towards the other pole. In the egg cells of Nereis Dumerilii there is a homogeneously dissolved yellow pig- ment which is a natural indicator changing color at about pH 5.4 from yellow to violet from the alkaline towards the acid side. During the bipolar differentiation the pigment is accumulated in the vegetative half of the egg cell, while the animal haif becomes colorless. the first four blastomeres separated by meridional cleavage furrows show essentially the same organization, The bipolar ditferentiation of these blastomeres is still pro- ceeding. When the accumulation of the substances on the vegetative pole reaches a certain amount, the color of the natural pigment changes from yellow to violet, indicating an increase of acidity, ie., a pH of at least 5.4 in the vegetative part of each of the four blastomeres. This happens just before the third cleavage. Vital staining exper- iments with indicators also show an increased acid reaction of this part of the blastomeres from this time on. Moreover, these dyes also stain the other (animal) half of the blastomeres and here they indicate an alkaline color more pronounced than before. Each of the four blastomeres now consists of an alkaline part and an acid part. Be- tween them the indicators have an intermediate color. The preliminary explanation of these observa- tions is that the bipolar differentiation of the egg cells is a cataphoretic phenomenon, that somehow there is established in the living cell an electrical field and that this causes a migration of the mic- Aucust 26, 1933 ] roscopical and ultramicroscopical particles accord- ing to their electrical charge. The particles of cell substances which show an alkaline reaction mi- grate in one direction, those of the acid sub- stances in the other. The different cell substances apparently can have a very different pH, even if they are enclosed in one single cell. The establish- ment of the electrical field is somehow tied up with the presence or local concentration of cer- tain ions, since it is possible to start the bipolar differentiation of egg cells of many different ani- mals artificially by adding KCI to the culture me- dium. The succeeding cleavage furrows separate the two different parts of the Nereis egg. In this way there originate two different groups of blasto- meres, one, after vital staining with indicators, showing the color of the acid side and the other showing the color of the alkaline side. During the later development the alkaline cells form the ecto- derm, the acid cells the entoderm. It seems to be important that during segmentation bipolar dif- ferentiation phenomena still proceed in the single blastomeres in the same sense as in the undivided egg cell. these observations were first done on eggs of Nereis Dumerilii. In that first study only two vi- tal staining indicators were used, namely, neutral red and Nile-blue sulfate. In later investigations brilliant vital red, brilliant cresyl violet and cresylecht violet were also used as vital staining indicators with very good results. Brilliant cresyl violet and cresylecht violet have peculiar proper- ties which one must know if one wants to use these dyes as indicators (see Protoplasma, 1933, 18, 497). In some cases it is possible to obtain a beautiful vital stain with methyl red and brom cresol purple by adding the dyes to pure isotonic NaCl solution instead of to the normal culture medium. All the experiments described with ref- erence to the eggs of Nereis Dumerilii have been repeated with the whole series of indicators men- tioned above (with the exception of brom cresol purple) on the egg cells of Nereis limbata in Woods Hole with essentially the same results. Similar studies on the eggs of Chaetopterus per- gamentaceus showed that the principle of the bi- polar differentiation of these eggs is the same as in the Nereis eggs. __THE COLLECTING NET 301 Eges with a discoidal type of cleavage, as for example, those of the Teleosts and Cephalopods are exceptionally good material for studies on the bipolar differentiation. A great number of exper- iments were done on eggs of fresh water fishes in Munich and Heidelberg, and a series of vital staining experiments on the eggs of Loligo vul- garis at \WWoods Hole. The results obtained on fish eggs are important for the analysis of the phenomena, because on this material it is possible to control the vital staining with indicators by microinjection of the whole series of the pH in- dicators of Clark and Lubs, and to vary and ana- lyze the method from different physical view- points. Hydrogen ion determinations with both the vital staining indicators and also with the Clark indicators gave the same results. In both the fish eggs and the Loligo eggs, alkaline colloi- dal substances migrate after the tormation of the polar bodies towards the animal pole, and acid substances towards the vegetative pole. The pro- toplasm of the animal cap of the trout eggs has a pH of more than 7.6; the yolk substances have a pH of about 5.6 or less. In the egg cells which have a concentric organ- ization, the substances of the cortical layer and those of the central part of the cell body show also a great difference in pH, and are distributed in the egg cell according to their pH. Either the alkaline substances are accumulated below the surface and the acid colloids in the center of the cell body, or—in the eggs of other animals—the cortical layer shows the more acid reaction and the central part of the cell the alkaline one. In some types of cells of Teleosts (unripe ov- ary eggs) it was possible to show that even if the protoplasm is hyaline and microscopically not differentiated at all, it contains several substances or phases which have a very different pH. (Col- loid particles of different reaction must somehow be protected from each other in the living cell.) This fact seems to be of great importance for the whole analysis of differentiation phenomena and obliges us to review our methods and interpreta- tions of pH determinations in living cells. (This article is based upon a lecture presented at the Marine Biological Laboratory on August 11). GENERAL SCIENTIFIC MEETING THURSDAY, AUGUST 31, 1933 Part I. 9:00 A. M. 1. Dr. Paul Reznikoff and Mrs. Dorothy G. Rez- nikoff: Blood cell studies in dogfish. 2. Dr. W. H. F. Addison: Intracranial pigmen- tation in teleosts. 3. Dr. E. R. Clark and Mrs. Eleanor Linton Clark: The blood capillary in relation to contractil- ity. : 4. Mr. Herbert L. Eastlick: Striated muscles of the lamellibranch mollusc, Pecten gibbus. 5. Dr. Arthur W. Pollister: The centrioles of amphibian tissues. 6. Mr. Theodore G. Adams: The chromidium in Arcella vulgaris. (Continued on Page 302) THE COLLECTING WET { Vot. VIII. No. 69 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Jean M. Clark, Martin Bronfenbrenner, Margaret Mast and Anna- leida S. van't Hoff Cattell. Printed by the Darwin Press, New Bedford A NOTE OF APPRECIATION The lecture and motion picture on whaling lore was successful. The auditorium was comfortably full, and the value of the tickets sold amounted to $207.00. The expenses of the show, counting the fee for the lecturer, publicity and miscellan- eous expenses, was approximately $70.00. This enables us to deposit more than $135.00 to the ac- count of THe CoLLectinG Net Scholarship Fund in the Falmouth National Bank, and makes cer- tain again of the award of six one-hundred dollar scholarships for the students of the Marine Bio- logical Laboratory and of one for the Biological Laboratory at Cold Spring Harbor. We wish to express our deep appreciation to Dr. Conklin for his brief introductory talk and to thank sincerely all those who contributed to the success of the performance: especially Mr. How- land, the lecturer; Mr. Sherman, who brought down the exhibit of whaling equipment; Miss Emily Ann Lillie and Miss Ruth Burdett for their posters; and to Margaret and Kathleen Stokey, who sold many tickets for us. At this time we would like to acknowledge two gifts, each of ten dollars, to the Scholarship Fund from two Falmouth merchants, Dr. E. C. Cole and the Wood Lumber Company. Introducing DR. REID HUNT, professor of pharmacology at the Harvard Medical School, who is spending a few days here at Woods Hole, renewing old acquaintanceships and reading in the library of the Marine Biological Laboratory. Dr, Hunt is especially interested in drugs which act upon the parasympathetic nervous system, particularly in derivatives of choline. He was the first to discover the extraordinary activity of acetyl choline, which is perhaps the most active drug made. It is now being used to some extent in medicine. Dr. Hunt first discovered this com- pound many years ago in the adrenal glands; it is now classed with the hormones and is believed to be an important factor in connection with the circu- lation. He has also done much important research work on the thyroid gland and is in addition noted for his work on nitrites, quinine, arsphenamin, alco- hol, and the biological standardization of drugs. Dr. Hunt arrived in Woods Hole on August 17th. He plans to leave early in September. GENERAL SCIENTIFIC MEETING (Continued from Page 301) 7. Dr, Harold H. Plough. Selective fertilization in Styela. 8. Mr. M. Atlas: Relation of temperature and cleavage in frog’s eggs. 9. Dr. E. G. Conklin: Disorientations of devel- opment in Crepidula, caused by cold. 10. Dr. Ethel Browne Harvey: Changes in the Arbacia egg immediately following fertilization, as determined by centrifugal force. ss 11. Dr. P. S. Henshaw and Dr. D. S. Francis: Recovery from X-ray effects before fertilization in Arbacia eggs and its effect on development. 12. Miss Anna K. Keltch, Miss Lucille Wade and Dr. G. H. A. Clowes: Further observations on the contrasting sensitivity of eggs and sperm to various chemical agents. 13. Dr. G. H. A. Clowes, Miss Anna K. Keltch and Miss Lucille Wade: Variations in the sensitivity of eggs following fertilization. 14. Dr. E. Newton Harvey: Flattening of marine eggs under the influence of gravity. 15. Dr. L. V. Heilbrunn: The action of anaesthet- ics on the surface precipitation reaction. 16. Dr. Dorothy R. Stewart and Dr. M. H. Jacobs: The effect of certain salt solutions on the permeability of the Arbacia egg. 17. Dr. B. R. Speicher: The effective period in development of the mutant factor ‘eyeless’” in Habrobracon. 18. Dr. Anna R. Whiting: Variegated eye color in Habrobracon. 19. Dr. P. W. Whiting: Egg-trinuclearity in Habrobracon, Intermission. Part II. 2:00 P. M. 20. Mr. Heinz Specht: Relation between oxygen tension and respiration in Spirostomum ambiguum, with corrections for Ammonia. 21. Dr. C. S. Shoup: Respiration and lumin- escence of bacteria in carbon monoxide. 22. Dr. G. Wellford Taylor: The relation be- tween luminescence and respiration in bacteria with especial reference to the effects of narcotics. 23. Dr. Lyle V. Beck: Nature of the aerobic ap- parent reduction potential. 24. Dr. Eric G, Ball: The relative abundance of hydrogen isotopes in sea water. 25. Dr. Oscar W. Richards: Toxicity of some metals and Berkefeld filtered sea water to Mytilus edulis. 26. Dr. Herbert H. Jasper: Some new aspects of — the physiology of the nerve-muscle system in crus- tacea brought out by electrical excitation and re- sponse. 27. Dr. Margaret Sumwalt and Miss Kathryn McLane: The blood pressure of Limulus. 28. Mr. John C. Bridges and Dr. Margaret Sum- walt: The effect of pH upon potassium penetration into Fundulus eggs. 29. Dr. Arthur K. Parpart: A method for follow- ing volume changes of cells. 30. Dr. William R. Amberson, Mr. Frank Engel, Miss Dorothy Webster, and Dr. Edwin P. Laug: The influence of pH upon the passage of hemoglobin through the glomerulus of the perfused frog’s kid- ney. 31. Dr. M. H. Jacobs and Dr. Arthur K. Parpart: The influence of the escape of salts on the osmotic behavior of the erythrocyte. Vol. VIII. No. 10 THE COPEPOD PLANKTON OF THE LAST CRUISE OF THE NON-MAG.- NETIC SHIP “CARNEGIE” Dr. C. B. WiLson State Teachers College, Westfield, Mass. The last cruise of the non-magnetic ship Car- negie covered portions of the Atlantic Ocean north of the equator, and of the Pacific Ocean between latitudes 52° North and 40° South. During the entire cruise plankton was collected at the surface and at depths of 50 and 100 meters and at the same time data were obtained of the temperature, salinity, density, hydrogen ion, and phosphates at the three depths. As the towings were all made in the daytime by the same persons, with the same nets, using the same methods, and in as close succession as possible, they furnish the hest basis thus far ob- tained for a comparison of the plankton of the two cceans, and of the different. portions of each. An examination of the copepods of this plankton, just completed, yields the following general re- sults. 1. The Pacific plankton is 50 — 100% richer than that of the Atlantic, both in number of spe- cies and in number of individuals. On the other hand, there was not found in the Pacific any trace of such countless swarms of a single species as are often seen of Calanus finmarchicus at cer- tain seasons in the northern Atlantic. 2. The plankton of the southern Pacific is richer than that of the northern portion, and of course the tropical (Continued on Page 347) TABLE OF The Capepod Plankton of the Last Cruise of the) Carnegie”’,, Dr. C: Bi Wilson: 53.0. <<... 309 The Hormone of the Pituitary and the Thy- roid, Dr. Marie Krogh The Biological Laboratory: Reversible Two-Step Oxidation, L. Michaelis 313 Reversible Oxidation-Reduction Potentials in Dye Systems, Barnett Cohen......... 319 An Analysis of Determinations of Intracel- lular Reduction Potentials, R. Chambers 329 SATURDAY, SEPTEMBER 2, Annual Subscription, $2.00 Single Copies, 25 Cts. 1933 THE HORMONS OF THE PITUITARY AND THE THYROID’ Marre Kroea, M. D. Lecturer in Physiology; State School for Teach- ers; Examiner, University of Copenhagen Our knowledge about a thyroid stimulating substance from the hypophysis is of a fairly re- cent date. The first report concerning an effect of extracts of the anterior pituitary has been given by Leo Loeb and his associates, and shortly after, independent of the previous paper, ap- peared a paper by Aron on the histological changes in the thyroid caused by injection of an- terior pituitary extract. At this time, Dr. Harald Okkels and I had been working together on the thyroid problem, examining surgically removed human goiters, Dr. Okkels from a cyto-histological point of view and I studying the metabolic effect of the glands fed to guinea pigs in equi-iodine amounts. The results obtained were that im human exophthalmic goiters, contrary to colloid goiters, the Golgi apparatus in the thyroid cells was dis- tinctly hypertrophic, indicating a hyperactivity of the cells, and, furthermore, that the metabolic ef- fect of the dried gland fed in equi-iodine doses was less for the colloid-poor or nearly colloid- free exophthalmic goiters than for the normal thyroid and for the colloid goiters. So far, our results combined with earlier known histological changes in the thyroids and 1 These experiments have been made in collabora- tion with Dr. Harald Okkels. CONTENTS Eye Colors in the Parasitic Wasp Habrobra- con, Dr, Annas Wbitine ere riers cris aren 339 Anterior Pituitary-Like Hormone Effects, Bl Bie Chidestersere ia apatites cient temas atecel mates = 340 The Cardiac Paradoxes of Limulus Polyphe- mus, pine Chae sna. senate ee aie ste «ese « 341 A Response of Arbacia Eggs to X-rays, Dr. P. S. Henshaw and Dr. D. S. Francis...... 342 Striated Muscle of the Lamelli-Branch Mol- luse, Pectin Gibbus, Herbert L. Eastlick.. .343 310 AMEND, (CONLILIATEIMUNE: [ Vor. VIII, No. 70 Normal guinea pig anterior pituitary 200x. Anterior pituitary of guinea pig treat- ed with 1000R 200x. FIGuRE 3 with the clinical symptoms emphasized the differ- ence between the exophthalmic goiter and_ all other types of goiters. We therefore welcomed the possibility of pro- ducing thyroid hyperactivity by means of anterior pituitary extracts, and carried out a series of ex- periments according to the following scheme: (1) examining the influence on the metabolism; (2) the influence on the Golgi apparatus in the thy- roid cells; (3) the influence on the other morpho- logical conditions in the thyroid. As experimental animals in the experiments | am going to report this evening we used full grown guinea pigs which we have found to be suitable animals because of the constancy of their metabolic rate. The recording respiration appar- atus is connected with two or three respiration chambers, which can be used successively. After injection of anterior pituitary extracts we found: (1) Increase of the standard metabol- ism; (2) Enlargement of the Golgi apparatus in the thyroid cells; (3) Increased vascularisation, cell hypertrophy and diminution of the colloid content. In order to obtain an absolutely objective esti- mation of the histo-cytological conditions, my as- sistant, Miss Lindberg, and I remove the thyroids from the experimental and control animals and send them to Dr. Okkels, who is examining them under a number without knowing what has been done with the animals until he has made a writ- ten report on the microscopical findings. TABLE I ADULT MALE GUINEA PIGS g » § a ~~ aoe Gi sfiet | 38 Baas g a) » Vea g 2 bn w Sp 5 fash BE 2s He ee ea aie gs se § Fee Fa as 425s << qo85 Ov 'o injected | 7) 43/g) 539) ant. pit. fed Te AS ae 0 0 ant. pit. injected 6 180mu. 0 0) 0) prolan fed if Oufses stats 0 0 dried thyroid Table 1 shows the typical effect of injection of anterior pituitary, on the metabolism, on the Golgi apparatus, and on the other morphological conditions, colloid resportion, et cetera. Anterior pituitary extract by mouth has no effect; 180 mouse units of Prolan injected have no effect either. Feeding dried thyroid causes, as is known, a considerable metabolic rise, but no hyperactivity of the cells; on the contrary, we found that after feeding dried thyroid for eight months the guinea pig’s thyroid showed atrophic changes. SEPTEMBER 2, 1933 | THE COLLECTING NET 311 cc O pw "hos ise lea no D) fio ho io x Ee i we) 2) he , oa at Fig. 1. measured during the first 5 hours and 24 hours after intraperitoneal injection. ue Q injection of ant. pit. extract (average of 4 animals). 2. x injection of Ringer solution (average of 3 animals). Metabolism of adult male guinea pigs The effect of the thyro-stimulating hormone becomes visible very soon after injection; as early as twenty to thirty minutes after intraper- itoneal injection the cells have changed, and after one hour the Golgi apparatus is enlarged and the colloid resorption pronounced. A considerable metabolic rise compared with that of control ani- mals injected with Ringer’s solution indicates that an increased amount of thyroxin is given off into the blood stream. Having convinced ourselves about the fact that injection of anterior pituitary induces thyroid hyperactivity, we carried out some experiments in order to contribute to the exophthalmic problem. In preliminary chemical experiments we found that the thyroid stimulating substance in the pres- ence of protein can be precipitated by 70% alco- hol but dissolved in 48% alcohol. Combining the alcohol method with the trichloracetic acid and acetone method, indicated by Loeser, we were able to get a protein free preparation of which TABLE 2 Adult male guinea pigs daily treated with purified anterior pituitary preparations ry (3) . £ g 8 ve 3 Le oe = o By = ge b= 2 ga 3 5S a6 es q oA iS aa cs 8S 2 wSS m3 on 2 ® ef 49 3% L bo SG bo av > noe ae wo) q ° q 83 8382 $80 $8 as & ZaeSs 483 O89 ffi 70% alcohol, trichloracetic acid, acetone 3 4.2 1423 +++ ++ 48% alcohol, 70% alcohol, trichloracetic acid,acetone 3 66 +26 4++4+-+ 444+ about 4 mg. is enough to produce thyroid hyper- activity in guinea pigs. In contrast to Aron we had been unable to detect the thyroid active substance in fresh un- treated urine. We therefore made use of the in- formation gained about the chemical properties and tried to concentrate the substance in urine from patients with severe attacks of exophthalmic goiter and from guinea pigs injected with large doses of anterior pituitary extracts. As shown in Table 3, the urine concentrates did not cause changes at all in the thyroid gland. The explanation I can offer for the slight meta- bolic rise is that the fairly large amounts (about 15 cc. per day) of the unsterilized residue after the alcohol evaporation produced some tissue irri- tation, and the animals therefore did not keep as quiet during the metabolism determinations as re- quired for standard conditions. The control ex- periment with urine plus anterior pituitary ex- tract shows that the urine itself does not inac- tivate or destroy the thyroid stimulating sub- stance. We therefore find it legitimate to conclude that the thyroid stimulating hormone is not eliminated by the kidneys. Dr. Okkels previously found on surgically re- moved human thyroids that after the Plummer TABLE 3 Urine from patients with exophthalmic goiter and from guinea pigs with experimental hyper- thyroidism. (Urine +1% plasma, precipitated with 70% alcohol and extracted with 48% al- cohol.) FI » § a. # eM ec z 3 3 3 n BS 5 rf 3 g om hy -_ 3% 58 HSos ws oo 5 82 gHS8- Sa 2a i) 5252090 58 as . BS Z28 4683 Of qo Urine from Patient 1 2830 4 +3 0 0 ¢ 2 3170 4 +7 0 0 j 8 5150 4 +19 0 0 Urine from 4 guinea pigs injected 4x23cc. ant. pit. extract 510 4 +19 0 0 4 guinea pigs injected 4x16 ant. pit. extract 725 5 +13 0 0 Normal urine +3x8cc. ant. pit. extract 2365 3441 + t+ 312 THE COLLECTING NET [ Vor. VIII, No. 70 pre-operative iodine treatment of exophthalmic goiter, there is no diminishing of the enlarged Golgi apparatus in spite of colloid storage in the alveoles. This means that the cells are still hyper- active, but to a certain extent able to regulate the amount of thyroxin given off into the blood stream. With this in mind, we tried to imitate the Plummer treatment on guinea pigs with experi- mental hyperthyroidism. From our previous ex- periments we knew that for full grown guinea pigs injected daily with 2-3 cc. of the Evans al- kaline extract, the metabolism rises to an average of about +40%, and the thyroid is morpholog- ically changed often to such a degree that it can- not be distinguished from the picture of a human exophthalmic goiter. We therefore injected daily anterior pituitary extract into the animals and after 4-5 days, when the metabolism was about +40, we started iodine treatment, continuing the anterior pituitary injections. — Figure 2 shows that the metabolism of a guinea pig with experimental hyperthyroidism reacts to iodine like that of an exophthalm‘c goiter patient. But when the thyroitoxic condition is produced by feeding thyroid preparation, the iodine does not lower the metabolism—an effect similar to that in human toxic adenoma. The microscopical examinations of the iodine treated experimental hyperthyroidism show the same picture as the 10- dine treated exophthalmic goiter, namely, an ac- cumulation of colloid but persistence of the en- largement of the Golgi apparatus. I shall add that in addition to a number of experiments like the one shown on the figure, where the animals were cm? OL yf Yimin, to Fig. 2. killed as soon as the metabolism went down to normal, we carried out just before I left Copen- hagen an experiment lasting for 44 days, where the guinea pig was injected with anterior pitui- tary extract and fed iodine. It kept a normal metabolism and a good health, while a control animal injected for a fortnight with the same daily amount of anterior pituitary, but without having iodine, had an average metabolism of +42% and loss of weight and hair. We are, of course, going to repeat this experi- ment to make sure whether or not a suitable iodine dose is able to control or neutralize the thyroid stimulating hormone for a longer time. In collaboration with an X-ray expert, Dr. Arntzen, we have tried to eliminate the anterior pituitary effect on the thyroid by exposing the hypophysis to an elective X-ray treatment. The smaller doses have no or uncertain effect, but doses of about 1000R cause damage to the hypophysis and corresponding, to this effect we find a metabolic drop of 13-14% below normal, atrophic changes of the thyroid cells and of the Golgi apparatus. To confirm the results we are going to repeat these experiments and also for the reason that X-ray treatment may be useful in in- operable cases of exophthalmic goiter. In our opinion the results make it probable that exophthalmic goiter as distinct from other types of goiter has its origin in anomalies in the fune- tion of the anterior pituitary gland. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 15). zo dage Standard metabolism of guinea pigs, I., injected ant. pit. and fed iodine, I1., fed dried thyroid and iodine. SEPTEMBER 2, 1933 | THE COLLECTING NET THE BIOLOGICAL LABORATORY | 313 COLD SPRING HARBOR REVERSIBLE TWO-STEP OXIDATION Leonor MICHAELIS In general, an organic dye stuff of quinoid structure is reduced by accepting from the reduc- ing agent two hydrogen atoms or two electrons at once. Until recently no case had been known in which the reduction would occur in two sep- arate steps in succession, each involving the ac- ceptance of one electron (or hydrogen atom). This experience seemed to be quite natural and in agreement with the customary formulae, used in organic chemistry, for a quinoid or a benzoid compound. No intermediary form seemed to be imaginable, because one valence in this imaginary “semiquinoid” form would have to stand aloof and unsaturated : Benzenoid On Q@uinoid structure structure OH fo) (hydroquinone) (quinone) ° Hypothetical semiquinoid structure . with one 4) unsaturated OH bond. However, a substance at an oxidation level inter- mediary between the benzoid and the quinoid form had been known for a long time, to wit, quinhydrone. In order to avoid the seemingly un- acceptable semiquinoid formula Willstaetter and Piccard proposed the following structure for Quinhydrone as a “meriquinone” quinhydrone, One molecule of quinone and one of hydroquinone were supposed to be combined to form one molecule of the double size, held to- gether by some kind of residual valences. The term “meriquinone” implied that the oxidized half and the reduced half of this double-m.lecule may not be fixed but shifting continuously so as to dis- tribute the state of oxidation in the time average equally between both halves of the molecule, There was, however, no definite proof for this hypothesis because of the fact that quinhydrone exists only in the solid crystalline state in wh.ch no determination of molecular weight can he per- formed. In the dissolved state it is completely “dissociated” into hydroquinone and quinone. In the meantime other substances at the semi- quinoid level of oxidation have been discovered, which are much more favorable for the study of their structures. Some of them are constituents or products of living organisms. The number of cases of this kind is increasing at such a rate that there can be no doubt of a much wider occurrence in Lving organisms than has been known as yet. There can be little doubt, furthermore, that the occurrence of such substances in living cells has some biological significance. [I am thinking of quite a definite significance, of quite a definite réle played by this kind of substance in the process of oxidation metabolism. This, however, is still a vague idea and has to wait for further elabora- tion. Therefore, I shall, at the present time, re- frain from discussing the biological importance and present only the chemical side of the problem with the definite idea in mind that this matter will very soon be taken up successfully from the bio- logical standpoint. The particular substance in which the first ob- servation pertaining to this problem has been made is the blue pigment pyocyanine produced by Bacillus pyocyaneus. In a systematic search for reversible oxidation-reduction systems it was de- cided to study this pigment, which was known to he easily reducible to a leuco-compound and easily re-oxidizable. The chemical constitution of this dye has been found by Wrede and Strack to be N-methyl a-oxyphenazine. These authors in- deed attributed to it the double molecular size on the ground of an apparently inadequate determina- tion of its molecular weight. The method about to be presented shows that the simple structure of the following formula is correct: ° if cH; This is an orthoquinoid molecule and could be expected to behave like a regular quinoid dye stuff 314 THE COLLECTING NET [ Vor. VIII, No. 70 when subjected to a reductive or oxidative poten- tiometric titration such as inaugurated by W. Mansfield Clark and his associates, especially Bar- nett Cohen. As a matter of fact, this was true provided the titration was executed in an alkaline solution. In acid solution, an unexpected phe- nomenon occurred, which seemed to be quite unique at that time but has in the meantime turned out to be one instance among many others. The color of pyocyanine at pH < 4.9 is red. When it is being reduced, it first turns green and then colorless. In strongly acid solutions these two steps are distinctly separate, in less acid solu- tions they overlap and with increasing pH the overlapping becomes so complete that the green intermediary form does not appear at all during the course of the titration. It was very tempting to ascribe to this green intermediate form the meriquinoid structure of Willstaetter and Piccard. easily be tested by an analysis of the titration curve, the potentials being plotted against the per- centage of oxidation or reduction. Let us first consider which curve can be expected on the basis of such an assumption. The reversible chemical reaction in the oxidation of the completely re- This suggestion could E “100 200 Ce.0 i : Fig. 1. Electrometric titration curve obtained when reduced pyocyanine is titrated with K3Fe(CN )¢ at pH 1.82. duced form to the intermediary form would be: 2 Mol Reduced form=1 Mol Meriquinone + 1 H Hence, the potential during the first step of oxi- dation must depend on the ratio of the two forms, when pH is kept constant, in the following way: RT (Meriqn) E = Constant + In a F (Reduced form )? This formula shows that the potential depends not only on the ratio of the two forms, as is usual in other oxidation-reduction systems, but also on the absolute amounts. In the second place, it shows that the potential curve is not symmetrically ar- ranged around the mid-point of the titration. The experiment, however, was not in agreement with this formula, as shown in the following graph (Fig. 1). This curve shows the two steps of oxi- dation as they occurred in a very acid solution. The curve of the first step should behave as has just been demonstrated, but it does not. The second step should behave also as developed be- fore, but it does not either. Each of the two steps shows a course perfectly symmetrical around the individual mid-point, and the whole curve is the same no matter what the total concentration of the dye at the start of the experiment. The slope of each half of the curve is the one for an oxidation involving only one hydrogen atom or electron, and of necessity the above chemical equation must be discarded and replaced by the following much simpler one: Ist step: 1 Mol reduced form = 1 Mol inter- mediate form + 1H; 2nd step: 1 Mol intermediate form = 1 Mol oxidized form + 1H. This shows that the intermediate form has the same molecular size as either the reduced or the oxidized form, and that the intermediate form differs from either the reduced or the oxidized by nothing but one H-atom (or one electron). The intermediate form therefore must have the seem- ingly unacceptable formula with a semiquinoid structure and is therefore of the character of a chemical radical, which may be written as fol- lows: OH On ou a N t @ 4 : : s CHs CH I II Il Formula I shows the simplest possible way of writing. It contains what we may call a bivalent SEPTEMBER 2, 1933 ] THE COLLECTING NET 315 nitrogen atom. On considering that this semi- quinoid form appears only in acid ~ solution, we may add a proton to it in the same way we add a proton to ammonia to make it an ammonium ion (see Formula IL). In order to explain the unex- pected stability of such a radical we may add the following hypothesis: the positive charge is not definitely located at the one nitrogen atom but shifts periodically to and fro between the two ni- trogen atoms, as a result of a periodic oscillation of one electron between two N-atoms. Each ni- trogen atom may be said to possess an outer elec- tron shell of only seven electrons, which by the odd electron is alternately supplemented to a reg- ular octet. This odd electron, loosely held and oscillating with a rather slow frequency may be imagined to be responsible for the very intense color of all these semiquinoid compounds and for the very distinct band spectrum exhibited by most, though not all, of the semiquinones. Radi- cals of this kind are not entirely unknown in or- ganic chemistry. The best known instances are triphenyl-methyl, discovered by Gomberg, and di- phenyl-nitride, discovered by Wieland. These radicals are as a rule in part associated as double molecules of a saturated character. Such an association, however, does not take place in the semiquinoid radicals, obviously for electrosta- tic reasons. These radicals are monovalent ca- tions and it is unlikely that two monovalent ca- tions would combine to form a bivalent cation. Once this phenomenon had been observed in the particular case of pyocyanine, many other examples were found. In the first place, a great number of phenazine compounds turned out to behave in the same manner, among them also the simple phenazine itself. Practically all those phenazine compounds show this phenomenon, ex- cept those containing one amino group or several as a side chain. To this group there belongs also, according to Kogl, another bacterial p gment, chlororaphine. Of other groups of organic com- pounds which can be oxidized through the oxida- tion level of a semiquinone are the aromatic para- diamines. A certain difficulty is involved in the study of these compounds because of the fact that the fully oxidized forms, at the oxidation level of a quinone, are very labile substances under- going a rapid irreversible disintegration. The semiquinoid forms are what were called a long time ago Wurster’s dyes, which were considered by Willstaetter and Piccard as double molecular meriquinones as a result of a now well under- standable misinterpretation. When for each of the two H-atoms in the two amino groups, phenyl groups are substituted, all of the three possible forms are perfectly stable molecules, the reduced, the semiquinoid and the quinoid forms. Another especially interesting group of this kind are the derivatives of y-y’-dipyridyl, es- pecially the quaternary ammonium derived from it. In these cases, in contrast with all the others, it is the reduced form which exhibits the quinoid structure, and it is the oxidized form which has the benzenoid structure. The form intermediary between these two, the semiquinoid, is an intense blue-violet dye stuff, whereas both the fully ox- idized and the fully reduced forms are colorless. The fully reduced form happens to be a rather labile compound, whereas the two other forms are perfectly stable substances. The preparation prac- tically used is the oxidized form; on reduction it turns blue-violet reversibly. The normal potential of this first step of reduction is extremely neg- ative, amounting to about —.45 Volt, independent of pH within any practically available range. This potential is, at pH 7, virtually equal to the potential of a hydrogen electrode at one atmos- phere pressure. It is easy to see that under these circumstances the normal potentials of these sub- stances are in the range of hydrogen overvoltage in acid solution but, on the other hand, are more positive than the hydrogen potential in an alka- line solution. The very negative range of poten- tial makes these substances very desirable oxida- tion-reduction indicators for potential range not accessible to the well known ind.cator series es- tablished by W. M. Clark and associates. [or brevity’s sake they may be called viologens. 1 have prepared methyl-, ethyl-, benzyl-, and be- tain-viologens, of which the benzyl compound has a normal potential about ninety millivolts more positive than the three others, all of which are close to —.45 Volt. The substances are rela- tively very little poisonous and I hope they may become useful as indicators in biology. A mathematical analysis of the oxidation or re- duction titration curve has been developed for those cases in which all of the three forms are stable and we have to deal with true equilibria. The result of such an analysis is very striking and most instructive for various problems. It will especially find application in what is called the Cannizzaro reactions, in which two molecules of the same kind act upon each other in such a way that one is reduced and the other is oxidized, so that the original substance is on an oxidation level intermediary between the two others. The mathematical theory cannot be given in a lecture with any profit to the audience, and may be studied in the original papers by myself and by Elema; but I wish to demonstrate some of the results. Suppose we have a substance in its reduced form and we titrate it with an oxidant. Let this substance be capable of forming two successive steps of oxidation, each one by acceptance of one 316. THE COLLECTING NET [ Vor. VIII, No. 70 electron. There are two limiting cases possible. In one case, on oxidation the semiquinone alone is formed first and after the whole of the substance has been oxidized to the semiquinone the oxida- tion to the higher level begins. In this case the curve of the potential plotted against percent of oxidation distinctly shows two separate steps, each of which has the character of an ordinary titration curve in a one electron system, such as ferrocyanide-ferricyanide. In the other limiting case, upon addition of the oxidant, the second step of oxidation is formed already before the first step is completed. In this case, an over- lapping of the two steps takes place, and_ this overlapping may reach different degrees of com- pletion, until it is so strong that the intermediary form does not appear at all during the titration. This case is the one usually encountered in or- ganic dye stuffs. The problem is now: to what magnitude can the degree of overlapping be cor- related? This quantity is understood from con- sidering the following chemical process : 2 Mol Intermediate = 1 Mol Oxidized + 1 Mol Reduced. This formulation symbolizes the fact that there must be established a chemical equilibrium among the three forms. According to the mass action law this equilibrium is fixed by the equation: (Intermediate )” =a 1; (Oxidized) (Reduced) The constant K may be called the formation con- stant of the semiquinone ; its reciprocal value may be called the dismutation constant, because this reaction, resembling the Cannizzaro process, 1s often referred to as dismutation. It is the magni- tude of this formation constant which is corre- lated with the degree of overlapping. This is best shown in the following graph (Fig. 2). Whea K is very large, say 100,000, no overlapping takes place at all, the two steps are widely separated. As Kk becomes smaller, the two steps still remain separated but the jump in the middle of the curve is smaller. As K becomes still smaller, the over- lapping becomes more manifest, but still in such a way that a kind of jump is visible in the middle of the curve. This is true until K becomes equal to 4. Here the jump disappears, the curve is en- tirely smooth and has precisely the same form as in an ordinary one-step system such as ferro- ferri-cyanide. However, the two-step nature of the system can be detected during the titration by the fact that a twofold shift in color takes place. When K becomes smaller than 4, the character of the curve is the same, except for the fact that the steepness of the curve is diminished, and as K approaches 0 the steepness is reduced to such an extent that we have to deal now with an ordinary curve of organic dye stuff with no in- termediate step at all. In a two-step system, it is insufficient to speak of “a” normal potential of the system. There are three reversible oxidation-reduction systems with- in the solution in equilibrium with each other. The first system consists of the reduced and the intermediate form; the second, of the interme- diate and the oxidized form; and the third sys- tem of the reduced and oxidized form. When the titration has reached that point where the re- duced and intermediate forms are present in equal amounts, the potential may be termed the normal potential of the first step, E;. When the titration has come to the point where the intermediate and the oxidized forms have the same concentration, the potential may be designated as the normal po- tential of the second step, Ez. At the mid-point of the titration the reduced form will always be present in the same amount as the fully oxidized. The potential at the mid-point of the titration may be termed the middle normal potential, E,,. In any case, there will be: Ei + Es Ex LE 2 and E,, will always be the potential at the mid- point of titration. The problem is now: At which vA Fig. 2. Electrometric titration curves when the reduced form of a two-stage reversibly oxidizable substance is titrated with an oxidiz- ing agent. Abscissae : Quantity of added oxidizing agent. Ordinates: Potentials referred to the potential at half-oxidation (x= 100) as zero. Each curve applies for the indicated yalue of K. SEPTEMBER 2, 1933 ] THE COLLECTING NET 317 point of the titration will the potential = Ej, or E,? The answer is easy when no overlapping takes place. Then EF, is the potential at 25% oxi- dation, and E, that at 75% oxidation. When K becomes smaller, and the overlapping more man- ifest, the two points of the curve corresponding to E; and E» are shifted towards the middle and approach each other. When K equals 4, all three normal potentials coincide at 50% oxidation. ‘hen K becomes smaller than 4, E; shifts over to the right hand side and Ez to the left hand side; as K approaches 0, E; has shifted to the point of 100% oxidation, whereupon obviously FE, becomes c and in the same way E» lies at O% oxidation and equals —%. The meaning of this infiniteness is of course that one can no longer speak of a two-step system. Now, looking again at the above formula which defines K, we have to consider the fact that all of the three substances according to pH may be present in various states of acidic dissociation, and so the value of K will be dependent on pH. Accordingly, as we vary pH in a series of titra- +100, 3 4 GROEN 2: I ETT 4 Fig. 3. The three normal potentials of pyocyanine : E, = normal potential of the first (more neg- ative) step. E, = normal potential of the second (more positive) step. E,, = middle potential. K,, K, and K, are dis- sociation constants of the oxidized, interme- diate and reduced forms respectively. tion experiments, the degree of overlapping in the two steps will vary. For each individual titra- tion curve we can compute the value of Ej, Es, and E,, by a mathematical analysis of the experi- mental titration curve. The method of computing these data is too complicated to explain in a lec- ture, and I shall show only the result for the in- stance of pyocyanine. In Fig. 3 the three normal potentials E,, E», and E,, are plotted against pH for pyocyanine. In order to interpret the slope and the bendings in such curves we have to recall some rules de- veloped by W. M. Clark and Barnett Cohen: In general such a curve will consist of rectili- near parts connected by smoothly curved bend- ings. The slope of each rectilinear part is either Q (the line is horizontal), or, according to cir- cumstances, amounts to .03 Volt or an integral multiple of it, per pH unit. The point of inter- section of two adjacent slopes projected on the abscissa indicates a dissociation constant. When the curve becomes steeper with increasing pH, the dissociation constant is of the oxidized form; when it becomes flatter it is the dissociation con- stant of the reduced form. When the system is a one electron system, each dissociation constant causes a shift of the slope by .06 Volt per pH unit. When the system is a two electron system, the shift amounts to .03 Volt per pH unit. The normal potential termed E,, belongs to a two electron system, whereas both E, and Ey be- long to a one electron system. To give an ex- ample: The oxidized form of pyocyanine has a dissociation constant p* = 4.9, at which the color shifts from blue to red. In fact, at pH4.9 there is a bend in the Es and in the E,, curve but there is no bend in the EF, curve because the oxidized form does not belong to the E; — system. The shift of this slope amounts to .06 Volt per pH unit in the Ey system, as it is a one electron sys- tem. The corresponding shift in the E,, curve amounts to but .03 Volt, as this is a two electron system. As a result of the various dissociation constants of the three forms it happens that the three curves not only are not parallel, but even cross each other. The crossing point is the one at which the three potentials equal each other, the forma- tion constant of the semiquinone K being unity. To the right hand side, E; is more positive than E». This causes a distinct overlapping of the two halves of the individual titration curve. At very high pH values the divergence of E; and Ey» be- comes very great. The significance of the latter phenomenon is this: when even a small amount of oxidant is added to the reduced form of the dye, the second step of oxidation arises rather than the first, in the same way as in the oxidation of copper the cupric state arises rather than the 318 THE COLLECTING NET { Vor. VIII, No. 70 cuprous, This is what usually happens on oxidiz- ing the reduced form of a quinoid dye of the reg- ular type. It is remarkable that for all reversible two- step dyes which as yet are known as occurring in living organisms, the behaviour within the physiological pH range (say from 6 to 8) is such that the two steps are not very widely separated indeed, yet the overlapping is not very great, the formation constant K being, say, between 4 and 1. Therefore, these dyes will in general be pres- ent, either within the cells or in the surrounding liquid, to a small but finite and measurable per- centage in the form of the semiquinoid. It is im- possible to imagine that such an occurrence is a pure chance. Once more, however, I wish to em- phasize that the time is not yet mature to utter those speculations on the biological significance which one may have in mind. LITERATURE W. M. Clark and B. Cohen, Pub. Health Report 38, 666 (1923). B. Elema, Rec. Tray. chim. des Pays Bas, 50, 807 (1931). B. Elema, J. Biol. Chem. 100, 149 (1933). E. Friedheim, Biochem. Z. 259, 257 (1933). E. Friedheim and L. Michaelis, J. Biol. Chem. 91, 355 (1931). L. Michaelis, Biochem. Z. 250, 564, (1931). L. Michaelis, J. Biol. Chem. 92, 211 (1931). L. Michaelis, J. Am. Chem. Soc. 53, 2953 (1931). L. Michaelis, E. S. Hill and M. P. Schubert, Biochem. Z. 255, 66 (1932). L. Michaelis and E. S. Hill, J. Am. Chem. Soc. 55, 1481 (1933). L. Michaelis, ‘““Oxidations-Reductions Potentiale,” 2nd. Ed. Berlin 1933. Discussion Dr. Cohen: What evidence is there as to the molecular weights of these semiquinones ? Dr. Michaelis: \Veitz determined molecular weights of some of the Wurster dyes by the boil- ing-point method. He arrived at the same conclu- sion about a year prior to me and pointed out that Willstaetter’s interpretation of a bimolecular compound was incorrect. Ten years previously, Hantzsch suggested the same idea but had no way of proving it. Dr. Stiehler: \Nere the molecular weight de- terminations made in water? Dr. Michaelis : Dr. Cohen: The existence of free radicals in organic solvents is well-established ; but their oc- currence in benzene, for instance, is no proof that they can exist in water. The molecular weight of such a compound in benzene may be the same or different in water. The organic free radicals postulated by Dr. Michaelis are apparently stable in water under acid conditions, whereas the known free radicals are highly unstable in this environment. There has been presented a plaus- ible extension of the concept of radicals, and what may turn out to be a useful generalization of oxidation-reduction theory. The idea of this new type of radical should stimulate the organic chemist to search such compounds out and char- acterize them adequately, Dr. Michaelis: There are other instances of this in the field of organic chemistry. Similar phenomena have been encountered with tri- phenylmethyl. It is true that these exist only in organic solvents. No, in some organic solvent. Dr. Barron: In biological systems, we think that the oxidizing catalyst is an electromotively active system with a one-electron transfer, e.g., hemin and its derivatives. These one-step changes are true of a number of compounds found in biological systems. SEPTEMBER 2, 1933 | THE COLLECTING NET 319 REVERSIBLE OXIDATION-REDUCTION POTENTIALS IN DYE SYSTEMS BARNETT COHEN Department of Physiological Chemistry, The Johns Hopkins School of Medicine When a noble metal electrode (such as _plat- inum or gold) is placed in an acid solution of a mixture of ferrous and ferric chlorides it will very quickly assume a stable potential which is determined by the ratio of ferrous to ferric ions. This relation may be written. RT iDy, = 1B In F (Petts ) (Fe++) a@l3) FE, is the observed difference in electromotive force between the electrode and the normal hy- drogen electrode; E, is a constant characteristic for the ferrous-ferric system (the so-called nor- mal potential) ; R, T, and F have their customary significances; the parentheses represent concen- trations of the corresponding components. There are a large number of systems organic as well as inorganic which behave in a similar way. Each of them is able to induce on the electrode a reversible potential which is thermodynamically definable. Such systems may be described as elec- tromotively active and easily reversible. There is another, larger group of oxidation-re- duction systems which apparently are electromo- tively inactive. These fail to impose definable po- tentials on the electrode. The reasons for these failures are often obscure. In some cases they act as if the proper catalyst were absent; in others, there is evidence that one of the active compon- ents may be rapidly destroyed thereby upsetting the equilibrium conditions which are essential to the establishment of significant potentials in an oxidation-reduction cell. The present discussion will be limited to a con- sideration of certain simple reversible systems, es- pecially certain groups of organic dyes which can be employed as indicators of oxidation-reduction. Let us return to the iron system for the elab- oration of certain important concepts. (cf. Clark, 1923; Clark and Cohen, 1923). The oxidation or reduction may be imagined to occur in several different ways. One may consider the oxidation of the ferrous iron to be due to oxygen, or to chlorine or to its electrochemical equivalent, i.e., the removal of an electron. Re- versely, the reduction may be imagined as due to hydrogen or its equivalent: the addition of an electron to the ferric ion. These processes may be represented schematically as follows: —— reduction oxidation — —> 2 FeO + O = FeO; + - 4 HCl 6 HCl 4 U 2 FeCl, + 2 C1=2 FeCl 2 FeCl, + 2 HCl = Hz + 2 FeCls 2Rett 3; Ze 2 Revit (2) It seems quite evident that one of the fund- amental parts of an oxidation-reduction process is a transfer of electrons. Whether or not free elec- trons really exist in aqueous solution, it is con- venient from the standpoint of formal treatment and discussion of interrelations to assume that all “electromotively active” oxidation-reduction sys- tems produce a virtual free-electron tension which can be picked up by a suitable electrode. Then for the reaction (equation 2) involving the electron transfer in the iron system, the equilibrium state may be defined by (kes) (Gx) Se IN .(3) ( Fett ) which gives for the electron transfer tendency in the solution the relation (Bess) (4) (e,) = Kye (Fe+++) The electron activity (e,) in the noble metal electrode is experimentally a constant and we shall so consider it. Then, for the process oc- curring at the electrode, the work W required to transfer isothermally one faraday of electrons from activity (e,,) to activity (e,) is (en) W = EF —=RT in = go hl) ; (€s) RT RT that is, E = —— In (e,) — —— /n (e,) F RT = Constant — —— In (e,) . (6) 320 THE COLLECTING NET [ Vor. VIII, No. 70 Substituting in (6) the value for (e,) from equation (+), we obtain RT EjG== (Her ) In kk F (Fe+++) or, when the potential is referred to the normal hydrogen electrode as a standard, RT (Fett) In Peat) F Gers) Ey = E; For the oxidation-reduction in the hydrogen system the reaction may be described as 2H++2e=Hp and by similar reasoning there is obtained the equation RT Vv (Hz) En = Eq In 15 (H+) or more familiarly, RT V Paz Ey — Eu In F (8) F (Galas) This is the general equation for a hydrogen elec- trode. When P = 1 and (Ht) = 1, Eg 1s zero by definition. For the oxygen system, O. +2H+ +4e=20H- RT Ey, = Eos In F 4\/Po2 For an oxidation-reduction system represented by the dye compounds to be discussed presently, the type reaction is the following (in one or an- other of its variations) : @Ox-= Ze — Redes RT GRedm a) ite, == 16. In 2F (Ox) where Ox represents the oxidant, and Red, the reductant. If we plot E, against percentage re- duction, we obtain an S-shaped curve the position of which on the E, axis depends on the value of E, which fixes the middle.point of the curve, i. e., when the ratio of reductant to oxidant is unity, E), = E,. The slope of the curve is determined by the value of n, the number of electrons involved in the reaction: it is flatter when n = 2 than when n=1. Within any particular system, E, depends on the ratio of reductant to oxidant. (OH) . (9) 5 6 C0) and, It is thus possible to express relative oxidation- reduction intensities in terms of electrode poten- tial. One of the problems that has occupied our attention for a number of years is the develop- ment of a series of indicators to register differ- ences in oxidation-reduction intensity analogous to those employed in the differentiation of hydro- gen ion intensities or activities. The Participation of Hydrions Since, as we have seen, the oxidation-reduction reaction involves the virtual transfer of one or more electrons, it follows that the electrical charge on each reactant is susceptible to change, with alteration in hydrion concentration. It fol- lows also that one or both of the reactants can be a cation, an anion or, one of them, a neutral molecule as can be seen from the following type reactions : Ox + 2e = Red Ox +2e= Red " Oxt+ +2e= Red Oxttt + 2e = Redt It is evident, therefore, that the hydrion con- centration must play an important role in deter- mining the particular ionic species present. More- over, the electrode potential which is a measure of the free energy change will measure, pari passu, the energy of ionization of the ionized reactants. The simple electrode equation, to be useful exper- imentally, must therefore be amplified to take into account possible ionizations. That is, it must include the ionization equilibrium constants that may be encountered experimentally. For example, in the case of the reaction: Ox* -+- 2e= Red, the simple electrode equa- tion for which is RT lop, == 12 Tn 2F (Ox* ) (Red-) eA (iltl ) the total reductant may be defined as the summa- tion: (Sr) = (Red-) + (HRed) .. . 7(12) and total oxidant as (Sw) =" (Oxat ) =] (OxOR) ye Gaia The ionization equilibrium for the reductant is (H+) (Red-) ee es (HRed) *—For the sake of simplification, concentrations and activities are considered as equivalent in the present discussion, SEPTEMBER 2, 1933 ] THE COLLECTI NG_ NET 321 and for the oxidant, (Ox+) (OH) (Oxt+) Ky = = I, (ld) (OxOH ) (OxOH) (H+) When (14) and (15) are substituted in (12) and (13) we obtain expressions for (Red~) and (Ox?) respectively which can be substituted in equation (11) to give RT (Sr) 3p, Slee In +- In 2F (So) 2F Ben Gilt ict Clea) ae exe l'6)) K, (H*) + Ky When (H*) is kept constant by buffering strongly, the last item in (16) is constant and (16) may be written RT E, = E’, — —— ln 2F (So) (Sr) 5 (GIZA) This equation contains the quantities that are ex- perimentally determinable, and with it can be con- structed the S-shaped titration curves already mentioned. When ratio of (Sr) to (So) is equal to unity, then the last term in equation (16) is the variable which determines the manner in which E’, varies with change in pH. Each class of oxidation-re- duction reaction has its own peculiar variable term which determines the slopes of the E’,:pH curve. Within the experimentally determinable pH limits of this curve, its analytical geometry shows two important characteristics. (1) When the dis- sociation constant of an ionic species is altered in the process of oxidation-reduction, the two disso- ciations (the new and the old) are characterized by bends in the E’,:pH curve which are centered at the points, pH = pK. (2) Not only are detect- able dissociations in the oxidant or reductant determined by the bends in the curve, but such dissociations may be identified as belonging to the oxidant or to the reductant from the change in slope that occurs. Thus, when the change in slope —-dE (defining slope as ) is negative, the disso- dpH ciation causing this change is due to an ionic species in the reductant ; when the change is pos- itive, the corresponding dissociation belongs to the oxidant. Such dissociations are, of course, also determinable experimentally by acid-base ti- trations. (Hall, Preisler and Cohen, 1928). One may summarize the important elements of examination and formulation of a simple oxida- tion-reduction system by an illustration. Consider the transformation of simple phenol indophenol to its leuco-product o=-C_>=n-<_»-on: «=F 2e + He = Ko Ky Os Its complete electrode equation in the exper- imentally determinable region of pH is at 30° C.; (Sr) E, = E, — .03 log ——— + .03 log (So) Kr Ke (H+) + Ky (H+)? + (Ht)? Kr (Ky negligibly small) Ko + (H*) First Step. Make (H+) constant. Then the last term of the equation is a constant which, when combined with E,, is called E’,. Then (Sr) (So) Changing the ratio of total reductant (Spr) to total oxidant (S,) gives the typical sigmoid titra- tion curve. EF, = EF’, — .03 log (Sr) (So) term is zero and E, = E’,. Now vary (H+). At each value of (H*) where it equals one of the dissociation constants, K,, Kp or Ks, there will be a center of inflexion of the curve relating 1, uo) (lala), Inasmuch as the electrode potential is prim- arily determined by the percentage reduction (or oxidation) and by the pH, these systems should be visualized as three dimensional surfaces, the coordinates being Ey, pH and percent reduction. = 1. Then the second Second Step. Make Oxidation-Reduction Indicators The literature contains numerous references to the use of various substances as indicators of ox- idation-reduction change in the solutions contain- ing them. The reduction of litmus in bacterial cul- tures is a well-known phenomenon which dates back at least as far as Helmholtz (1843). Methylene blue has considerable vogue as an indi- cator of reduction in a variety of applications which we shall not pause to discuss. It must be pointed out, however, that such applications of 322 THE COLLEGRING NED [ Vor. VIII, No. 70 this and other dyes have been based on empirical observations. For the development of systematic indicator theory in the field of oxidation-reduc- tion there is required quantitative information on equilibrium potentials such as we shall now pre- sent in brief summary. Most of the organic compounds which have thus far lent themselves readily to potentiometric measurement of their oxidation-reduction equil- ibria have the quinonoid structure as a common denominator, Quite a number of such quinonoid compounds can be reversibly reduced to their cor- responding benzenoid products; and each oxidant with its reductant sets up reproducible electrode potentials that are thermodynamically definable. It is rather curious, as well as a challenge to investigation, that the ordinary ethylenic linkage which can often be so readily reduced chemically is not amenable to simple electrometric investiga- tion. In the best known example, the succinate- fumarate system, an enzyme is required to cata- lyze the equilibrium process ; but this is apparent- ly not enough, for a go-between like methylene blue (a quinonoid compound) seems necessary to help establish significant potentials. Another type of compound that seems to yield significant oxidation-reduction potentials is that included in the class of semiquinones and certain types of “free radicals.” These were first studied by Conant, Small and Taylor (1925) and are now being actively investigated by Michaelis; and the elucidation of their peculiar phenomena promises to extend our knowledge of the theory of reversible oxidation-reduction, We shall now present the essential data for those indicator systems which have been tematically studied and adequately characterized, proceeding along the scale of electrode potential from the the tronegative. visualization ot sys- electropositive zone to elec- An aid relations is to imagine a diagram having as abscissae the electrode ordinates the pH. of the hydrogen electrode (at the left) would slope away, decreasing .06 volt (for 30°C.) with in the potentials and as On this diagram the line each tenfold decrease in hydrion concentration. With this as a baseline the relations of other sys- tems may be gauged. Another fixed base of ref- erence is the line of the theoretical oxygen elec- trode which runs parallel to that of the hydrogen electrode at a distance of 1.23 volts. The systems which we shall consider lie within these limits. Unfortunately, the indicator systems now known are not evenly spaced between these limits ; most of them are crowded more or less near the mid- dle zone and toward the hydrogen electrode. Indophenol Systems—This group of com- pounds was studied by Clark, Cohen and co- workers (1923-1928) The type structure of the oxidant is = €-N- C )-o The type oxidation-reduction reaction is Ox + 2e+ H+ = HRed The electrode equation (for 30°) is (Sr) E),, = E, — 0.03 log (So) Kr Kz (H+) + Kp (H*)?+ (Ht )8 + 0.03 log K, + (Ht) K, = dissociation constant of phenolic group in the oxidant Kr = dissociation constant of same group in the reductant K. = dissociation constant created by reduction of phenolic group Some thirty different derivatives were studied. The characteristics of a few which seem most suitable for indicator purposes are given in the following table. Characteristics of Certain Indophenols Useful as Indicators E’, at Name pH7.0 Eo pK. pikepks Phenol-m-sulfonate indo- 0.273 0.6906 7.40 7.12 8.93 2, 6-dibromophenol m-Chlorophenol indo- 0.254 0.6919 616 6.89 9.21 2, 6-dichlorophenol Phenol-o-sulfonate indo- 0.242 0.6834 6.07 7.01 10.22 2, 6-dibromophenol o-Chlorophenol indophenol 0.233 0.6627 7.00 8.44 10.30 2, 6-Dichlorophenol- 0.217 0.6684 5.70 7.00 10.13 indophenol 2, 6-Dichlorophenol- 0.181 0.6394 5.50 7.10 10.43 indo-o-cresol 1-Naphthol-2-sulfonate indo- 0.119 0.5630 6.14 745 9.32 2, 6-dichlorophenol Note: In this and the following tables, E’, rep- resents the potential at any given pH of a sys- tem in which the ratio of oxidant to reductant is unity, E, is the ‘normal’ potential, i.e., at pH = 0. SEPTEMBER 2, 1933 ] THE COLLECTING NET Amino Indophenols—Several members of this group were studied by Cohen and Phillips (1929). These are amphoteric compounds which may be of advantage under certain conditions of indi- cator application. The type structure of the oxi- dant is The type oxidation-reduction reaction may be written ie ve Ox + 2e= Red The electrode equation is (Sr) E, = E, — .03 log —-— — 0.06 pH —.03 log ( Sin) K Ky —— a IK, (EI) 4b re on sie Kon 03 log [ Kr Koks -f- Kak (ilar) +- Kg (H+ We S(delay | K, = dissociation constant of oxidant’s phenolic hydrion Kom = dissociation constant of oxidant’s polar amino group Kr = dissociation constant of reductant’s phen- olic hydrion K. = dissociation constant of reductant’s Ist amino group Ky = dissociation constant of reductant’s 2nd amino group Ky = dissociation product of water Two compounds in this group may find utility as indicators. These are m-toluylene diamine indo- phenol and phenol blue. The characteristics of these systems at 30° are given below 1D at 1B pK, pKon pKr pKe pH 7.0 0.225 0.677 4.85 high 9.88 5.96 0.125 0.567 8.07 2.31 10.32 4.96 System Phenol blue m-Toluylene diamine- indophenol pK, — 2.72; pK, — 13.73 Indamines—Bindschedler’s green and toluylene blue were examined by Phillips, Clark and Cohen (USZ7Ne The type structure is n-ne The type reaction is + — Ox + 2e= Red The electrode equation for 30° is (Sr) yy ig = OSnlog: (So) KoKoo( Ht ) + Kooky + Kor (Ht)? — 06 pH — .03 log KoK3Ky + KsKy(H+) + Ky(H*)? + (Ht)§ Ki. = dissociation of oxidant’s polar group K,» = dissociation of oxidant’s first non-polar group Ky = dissociation of reductant’s first basic group Ks = dissociation of reductant’s second basic group ‘ Ky, = dissociation of reductant’s third basic group The characteristic constants of these indamines at 30°C. are given below Sys- By’, at tem pH7.0 E, pKa pKoz pK» pKs pK Bindschedler’s green 0.224 0.680 11. 3.27 6.46 5.10 —— Toluylene i blue 0.115 0.601 10.48 3.80 656 440 2.14 It will be noticed that these systems lie in or near the zone of the indophenols. Thiazines. Two well-known representatives of this group of dyes, methylene blue and Lauth’s violet, were examined by Clark, Cohen and Gibbs (1925). The type structure is —N= rnd J-s-\ Janey The type reaction and electrode equation are the same as for the indamines. The characteristic con- stants of these thiazines at 30° are given below 18; ane System pH7.0 E, pKa pKoo pKe pKs Lauth’s violet 0.062 0.563 11.0 low 5.30 5.85 low 4.38 4.52 Rapkine, Struyk and Wurmser (1929) have examined the thiazines toluidine blue and azure I. Vellinger (1929) also examined tolui- dine blue. Although these authors do not report the constants that define these systems, they pre- sent curves of the potentials between pH 4 and 9. These show the two systems to lie close to that of methylene blue. It will be observed that the thiazine systems lie in a zone slightly electronegative to that of toluy- lene blue. Methylene blue 0.011 0.532 high Oxazines—The oxazine nucleus differs from the thiazine by the substitution of an atom of oxygen for the sulfur of the latter, Four mem- 324 THE COLLECTING NET { Vor. VIII, No. 70 bers of this group were studied by Cohen and Preisler (1931). The type structure —N= RNA /-9- =NR, The type reaction and electrode equation are the same as those for the methylene blue system. The characteristic constants determined for 30° are as follows Eyarit System pH7.0 E, pKa pKos pKs pKs Cresyl blue +.047 0.583 10.7 low 6.3 4.6 Methyl Capri blue -.061 0.477 high low 6.10 4.85 Ethyl Capri blue —.072 0.540 high low 7.14 6.70 Nile blue - HSO, —122 0.406 9.7 low 6.90 3.92 Methyl Capri blue, the analog of methylene blue, is more electronegative than the latter. The oxazine dyes as a group occupy a zone of elec- trode potential which overlaps the thiazine range and extends a short distance to the electroneg- ative side. Nile blue exhibits very interesting phenomena of reversible molecular aggregation which exert peculiar effects on the equilibrium potentials. Vellinger (1929) has reported studies on cresyl blue and Nile blue (temperature not given). Rapkine, Struyk and Wurmser (1929) have reported on cresyl blue, Nile blue and cresyl violet at 18°C. Letort (1932) has presented data for 20°C. on the following oxazines: Nile blue 2B, Capri blue. new methylene blue sold cotton blue (Rowe, 910) and muscarine DH. Michaelis (quoting Michaelis and Eagle) reports on the ox- azines: brilliant alizarin blue, gallophenin and gallocyanin at 25°. In all the above references the authors present data on potentials in the form of charts or tables which serve as a basis for empir- ical use as indicators of the dyes named. It is to be noted, however, that more or less serious discrepancies appear in the data on some dyes studied in different laboratories. While small differences may be accounted for by differ- ences in temperature and technique, a far greater element of error lies in the inadequate identity of the dyes studied. This has two aspects. One is the purity of the material, which is more or less un- der the control of the experimenter. Another, and more serious danger to the unwary is the accep- tance of the manufacturer’s name of a dye as being identical in chemical constitution with that having the same name and listed in Schultz’s “Farbstofitabellen” or Rowe’s “Colour Index.” There is no such standardization in the d: e indus- try and we have encountered several instances where unsuspected substituents were present in the compounds obtained on the market. Indigo sulfonates—This group of acid dyes differs in structure from the basic compounds which we have been discussing. They were studied by Sullivan, Cohen and Clark (1923). The type structure is The type reaction is Ox) Ze Radt The electrode equation at 30° is (Sr) E, = E, — 0.03 log + .03 log ESCH sts (Get eal KX, = first dissociation constant of the group cre- ated by reduction. The characteristic constants at 30°C. for the four indigo sulfonates are given below. E’, at System PH 7.0) Es pki Indigo tetrasulfonate —.046 0.365 6.9 Indigo trisulfonate =081 0332) 471 Indigo disulfonate =125 0:29) [73 Indigo monosulfonate* —.159 0.262 78 * The monosulfonate is rather poorly soluble when salts are present. The indigo sulfonates occupy a zone of poten- tial covered by the oxazines and extending some- what toward the hydrogen electrode. Safranines—Five of these compounds were ex- amined by Stiehler, Chen and Clark (1933). The type structure is —N= RNA) -N- =NR, The type reaction and the electrode equation are the same as for methylene blue. The characteristic constants at 30° are as follows. Bat - System pH7.0 E, pKe pK, Phenosafranine (Rowe 840) —.252 0.280 4.95 5.8 (Rowe 847) —.254 (Rowe 842) —.260 —.273 (Rowe 841) —289 0.355 6.4 7.7 0.286 4.9 6.3 0.288 5.3 6.5 0.235 4.7 5.7 Tetraethyl " Dimethyl ” Tetramethyl ” Safranine T SEPTEMBER 2, 1933 ] THE COLLECTING NET 325 The dye concentration affects the constants to some extent. The above values apply specifically to 0.0001 M solutions. These systems are all primarily reversible, but the reductants are subject to progressive altera- tions that must be considered as limiting the re- liability of these dyes as oxidation-reduct.on ind- cators. Another unfortunate characteristic is that the alterability of the reductants seems to be most marked in the pH range 3 to 7.5. Neutral Red—This azine is mentioned in pass- ing because it is available in many laboratories and has been employed occasionally as an indica- tor of oxidation-reduction. However its pecul- iar irreversibility in the physiological range of pH was reported by Cohen, Chambers and Reznikoff in 1928. This compound was recently examined by Clark and Perkins (1932). The system was found to be primarily reversible and to lie in the region of potentials covered by the safranines. However, it undergoes rather rapid transforma- tion in neutral pH regions to a stable form which does not easily reoxidize. This change is much more pronounced than that occurring in the sa- franines and rosindulines. We shall therefore omit further consideration of neutral rcd. Rosindulines and Rosindones—Six of these were examined by Stiehler (1933). Poor solu- bility makes four of them unsuitable as indicators. The remaining two, Induline scarlet (Rowe 827) and sulfonated rosindone, seem to be satisfactory. The structures of these are given below Induline scarlet H,cC— -N= (Rowe 827) rah as =Nh,Cl Cos Sulfonated rosindone S03 (2) (position of sulfonic acid group uncertain ) re (C,H,)N \/-N-\/=0 CHs The type reaction and electrode equation are the same as for the methylene blue system. The characteristic constants at 30° are given below. iY, Bie System jolsl FQ "18s, pK. pKs Rosinduline scarlet -0.296 0.047 4.5 ? (Rowe 827) Sulfonated rosindone —0.380 0.25 7.5 9.5 Of particular interest is the fact that these two systems lie close to the hydrogen electrode. In- deed, the potential of the rosindone when 90% reduced at pH 7 equals that of the hydrogen electrode. Mention should be made at this point of the work of Michaelis (1931) on Rosinduline 2 G (Rowe 830). This compound is a simple unsub- stituted monosultonated rosindone, the [’, or which at pH 7 was found by Michaelis to be —0.281. It will be noted that this is about 100 my. more electiopositive than the compound Stiehler examined. Data on some of the azines have been reported by other workers, but unfor- tunately important details are absent in the pub- lished papers. Vellinger (l.c.) gives curves for phenosatranine, safianine (?) and neutral red. Letort (l.c.) gives a curve for phenosatfranine wich approximately superposes that of Vellinger. They show general agreement with the more de- tailed data presented by Stiehler, Chen and Clark. Rapkine, Struyk and Wuimser (l.c.) give curves for neutral red, neutral violet and Janus green. ‘the latter is an azo-c.mpound of safranine. On reduction the hydrazo system formed is appar- ently unstable because the end-product obtained is safranine. The curve given by these authors for Janus green seems to be in fact that representing the system safranine-leuco safranine. SUMMARY The foregoing account of oxidation-reduction indicators includes only those well defined s) stems which are relatively unccmplicated and involve a two-electron transter in the process of oxidation- reduction. It includes also only those structures in which the oxidant has high tinctorial power, which is important for indicator applications. Con- sequeatiy no menton has been made of the signi- ficant contributions by Conant, Fieser, LaMer, Bulmann and others on various quinones and other compounds of low tinctorial power which give well defined oxidation-reduction potentials. ‘ Certain “meriquinones’, “semiquinones” and “free radicals” are known to yield highly colored oxidation products and to give definite equilibri- um potentials. The formulation of these systems is now being actively pursued by Michaelis. Rel- atively few such systcms are at present worked out sufhciently to serve as a basis for general ap- plication; but more and more of them will no doubt become available as their properties become established.’ It will be noted that the adequately defined dye systems at present available do not cover all the possible ranges of potential. There are certain gaps that need to be filled in. Although recent work on the rosindulines and safranines has helped somewhat to fill the gap between the indigo sulfonate zone and the hydrogen electrode, there 326 THE COLLECTING NET [ Vor. VIII, No. 70 is still a need for good stable systems in this region. Another notable lack is for indicator systems on the positive side of the indophenols toward the oxygen electrode. A number of the simple para- and ortho-quinones lie in this region, but these are weak tinctorially. It should be pointed out that many of the oxi- dation-reduction indicators are also acid-base indi- cators. These two distinct properties should therefore be kept in mind. It is a safe attitude to assume that none of these dyes will remain in aqueous solution indefinitely without decomposi- tion. Indeed, scme of them, notably the oxazines, decompose rather rapidly when exposed to water. Consequently difficulties will be avoided if such indicator solutions are made immediately before use. There appear to be three fields for the applica- tion of oxidation-reduction indicators: (1) in the field of inorganic and organic chemistry as an aid in separations, assays and energy studies; (2) in biochemistry, as go-betweens in certain enzymic transformations; (3) in general, as indices of oxidation or reduction intensity in solutions to confirm or substitute for potential measurements, and in solutions where the presence of metallic electrodes is impossible or contraindicated. LITERATURE Clark, W. M., 1923, Public Health Repts., 38, 443. Clark, W. M. and Cohen, B., 1923, Public Health Repts., 38, 666. Clark, W. M. and Cohen, B., Studies on Oxidation- Reduction, III. Publ. Health Repts., 1923, 38, 933. Clark. W. M., Cohen, B. and Gibbs, H. D., 1925. Publ. Health Repts., 40, 1131. Clark, W. M. and Perkins, M., 1932. J. Am. Chem. Soc., 54, 1228. Cohen, B., Chambers, R. and Reznikoff, P., 1928. J. Gen. Physiol., 11, 585. Cohen, B., Gibbs, H. D. and Clark, W. M., Papers V and VI. Publ. Health Repts., 1924, 39, 381, 804. Cohen, B. and Phillips, M., 1929. Publ. Health Repts., Supplement No. 74. Cohen, B. and Preisler, P. W., 1931. Publ. Health Repts. Supplement No. 92. Conant, J. B., Small, L. F., and Taylor, B. S., 1925. J. Am. Chem. Soc., 47, 1959. Gibbs, H. D., Cohen, B. and Cannan, R. K., Paper VII, Publ. Health Repts., 1925, 40, 649. Hall, W. L., Preisler, P. W. and Cohen, B., Paper XIV, Publ. Health Repts., Supplement No. 71, 1928. von Helmholtz, H., 1843. Arch. Anat. Physiol., 453. Letort, M., 1932. C. R. Acad. Sci., 194, 711. Michaelis, L., 1931. J. Biol. Chem., 91, 369. Michaelis, L., 1933. Oxydations-Reduktions-Poten- tiale. 2nd Ed. Berlin. Phillips, M., Clark, W. M. and Cohen, B., 1927. Publ. Health Repts., Supplement No. 61. Rapkine, L. Struyk, A. P. and Wurmser, R., 1929. Jour, Chim. physique, 26, 340, Stiehler, R. D., 1933. Dissertation. Johns Hopkins University. Stiehler, R. D., Chen, T. T. and Clark, W. M., 1933. J. Am. Chem. Soc., 55, 891. Sullivan, M. X., Cohen, B. and Clark, W. M., 1923. Publ. Health Repts., 38, 1669. Vellinger, E., 1929. Arch. physique biol., 7, 113. DIscUSSION Dr. Fricke: Is it possible for you to say more about the actual mechanism whereby the electrons are transferred to the electrode? Dr. Cohen : This question emphasizes the point that there are two distinct aspects to the theory of oxidation-reduction electrodes. My paper dis- cusses their application to the measurement of free energy change in the transfer of electrons. The matter of mechanism at the electrode is of fundamental importance but the problem is un- solved. One view has it that the noble metal ad- sorbs atomic hydrogen so that it is in effect a gas electrode. This is conceivable for systems lying close to the hydrogen potential. On the other hand, it is difficult to reconcile this view with the fact that the theoretical hydrogen pressure in equilibrium with a positive indophenol system is of the order of 10° atmosphere. With certain types of compounds the equilibri- um is reached with remarkable rapidity and po- tentiometric balance stays constant. In other cases there is a lag, sometimes appreciable—a matter of minutes, hours and even days. If there is decomposition of one of the reactants the time curve of potential may be difficult to interpret. In general, freshly prepared electrode surfaces (e. g. gold plate) reach equilibrium more rapidly than old ones. Dr. Fricke: Did you in your experience meet with catalytic agencies which are effective in pro- moting rapid establishment of equilibrium ? Dr, Cohen: The platinized electrode for the H:H~ system is a classical example. The enzyme for the succinate-fumarate system is another, Dr. Michaelis: The reduction of ferricyanide with hydrogen reaches equilibrium ordinarily in a day, but the addition of a small amount of dye causes it to take place immediately. Dr. MacInnes: What electrodes did you use for reference in general ? Dr. Cohen: The saturated calomel cell which had been compared with the hydrogen electrode in M/20 potassium acid phthalate. No provision was made for liquid junction potentials. Dr. Mudd: Were your oxidation-reduction potentials measured by a null-point method ? Dr. Cohen: Dr. Mudd: If current were allowed to flow through the detecting circuit, how would that Yes. SEPTEMBER 2, 1933 ] THE COLLECTING NET 327 affect the equilibrium of the oxidation-reduction system ? Dr. Cohen: The experimental concentration cell consists of a calomel half-cell coupled to the oxidation-reduction half-cell. On closed circuit, the whole unit would run down. Thus, if the Hg:Hgt system is the more positive, it will tend to become reduced while the reductant in the other half-cell will be oxidized an equivalent amount. If the Hg:Hg* system is the more negative, it will tend to become oxidized at the expense of the oxidant in the other half-cell. The process will continue until both half-cells reach an equipotential value. Dr. Mudd: Should not an oxidation-reduction system in the body constitute a source of bio- electric currents ? Dr. Cohen: In a spontaneous oxidation-re- duction reaction there is of course a loss in free energy which becomes available for work on the environment. The total amount of it may be ex- pressed in any convenient units, e. g. volt-cou- lombs, gram-calories, B.T.U., ete. The work may be expended as electricity, heat, light, etc., de- pending on the particular conditions of the envir- onment. It follows therefore that oxidation-re- ductions in the body can be a source of bioelectric just as they can be of biothermic or biolumin- escent phenomena depending on the path or paths provided by the body for the manifestation of these phenomena. Dr. Blinks: Can oxidation-reduction poten- tials exist in liquid junctions or must they be at an electrode? Dr. Cohen: If a liquid junction were made between two oxidation-reduction systems, a re- action would start at the junction and the free energy liberated would be dissipated by any avail- able path. If a metallic conductor be present, it will filter off the electrons and carry them (pro- ducing a negative current of electricity) to the other end of the metallic circuit, provided that (1) there is also available a path whereby neg- ative ions can travel in the opposite direction to maintain ionic electroneutrality in the solution, otherwise no current will flow, and (2) the solu- tion at the other end of the conductor has a lower ‘electron pressure,’ otherwise the current would flow in the opposite direction. It is by this means that electrochemical potential differences are es- tablished. Dr. Blinks: Can one get an effective electron transfer that can give rise to the currents you speak of except by means of a metallic surface ? Dr. Cohen: Reversible electron transfer from the reducing to the oxidizing system occurs spon- taneously or may be speeded up by a catalyst. In the case of electromotively active systems, a metallic electrode can pick up the ‘electron-pres sure’ and conduct the electron current to a region of lower pressure. Whether effective electron con- duction such as this can occur along non-metallic films or membranes remains a question that is un- answered. Dr. MacInnes: In any purely electrolytic solution the potentials will be due to differences in mobilities and concentrations, and unless there is something analogous to a metal electrode one 1s not dealing with oxidation-reduction potentials. Dr. Michaelis : reaction. Dr. MacInnes: Just what a potential means if there is no electrode present is worth following up. It would appear to be something like a prin- ciple of uncertainty; because anything, (an elec- trode or a chemical reagent) used as a detector of the potential, changes the latter to some degree. Dr. Michaelis: Without an electrode there would still be present a level of free energy. There must be some chemical Dr. MacInnes: In the body of the electrolyte one could change the ratio of oxidant to reductant from one region to another and follow the poten- tial change on a probe electrode, but I cannot see that the oxidation-reduction potential at that point would have much meaning except as the electrode is inserted. Dr. Michaelis: It might be stated in this way: the oxidant-reductant mixtures at each point would be in an equilibrium that determines the potential. The potential manifests itself as an index of the electron pressure of the particular equilibrium present in the vicinity of the elec- trode. Dr. MacInnes: thetical. Dr. Cohen: That seems to be quite true for aqueous solutions; but it is also true that when an oxidant is reduced its electron number is in- creased, therefore an electron transfer must have occurred somehow. Dr. Miiller: Is it possible to have free elec- trons ina solution? Dr. Cohen: They are stated to exist in liquid ammonia systems ; but in water they could not ex- ist except when attached to something. Dr. Miiller: Is there any reason why elec- trons cannot exist in water? Dr. Michaehs: There would be a great ten- dency for the electrons to reduce hydrogen ions to hydrogen. Dr. Cohen: The matter of the meaning of oxidation-reduction potentials may be put in an- other way. They are the index of the energy The electrons are quite hypo- 328 THE COLLECTING NET [ Vor. VIII, No. 70 state of a system which corresponds to the chem- ical potential of Gibbs. Whether an electrode is present or not, two reversible oxidation-reduction systems will interact if mixed, and the energy change is measurable in a calorimeter as_ heat. From this and other thermal data, the oxidation reduction potential may be calculated. The elec- trode is merely an elegant means for determin- ing the free energy change directly. The phenomenon of ‘oxidation at a distance’ should be cited. It is well known that if chlorine be added to iodide solution, the latter will be ox- idized and free liberated. Now, put chlorine solution in one vessel and iodide solution in another. Connect the two by a salt bridge plugged with agar to prevent convection and re- tard diffusion. In the solutions place electrodes and connect them with a metallic conductor (our electron filter). Very soon it will be noted that the region about the electrode in the iodide solu- tion is colored brown from the free iodine lib- erated. It is as if the iodide ions gave tp their iodine negative charges to the electrode which carried the electrons to the region of lower “electron pressure.” The discharged iodide ions have be- come free iodine atoms, while at the other end of the metallic circuit chlorine atoms have been charged (reduced) to become chloride ions. This appears to be a convincing illustration of the phenomenon of electron transfer and of chemical potential as the determinant of oxidation-reduc- tion potential. Thus it is that the potential of a reversible sys- tem is detectable not only by its action on a suit- able metallic electrode but also by its action on suitable chemical systems. If the latter are irre- versible, interpretation of the result may be rather involved. If they are smoothly reversible and reach quick equilibrium, their properties connect- ed with the equilibrium state may be used as an index of that state; that is, they may serve as in- dicators. The compounds discussed in my paper are such indicators in which the differences in visible color between oxidant and reductant are utilized. SEPTEMBER 2, 1933 ] THE COLLECTING NET _ 329 AN ANALYSIS OF DETERMINATIONS OF INTRACELLULAR REDUCTION POTENTIALS BY MEANS OF INDICATORS! ROBERT CHAMBERS We are safe in assuming that the protoplasm of a living cell contains three types of systems which are oxidizable or reducible. They are (a) elec- tromotively active and truly reversible oxidation- reduction systems, (b) sluggishly reversible sys- tems which need to be activated in order to behave reversibly, and (c) systems which are irreversibly affected. A determination of the reduction potential of such a heterogeneous mixture should be affected primarily by the system present having the most negative potential. It is also conceivable that the presence of irreversible systems which are con- tinually being oxidized during the period of the determination may be affecting the apparent values obtained. At present we do not know what significance can be ascribed to the values of reduction poten- tial deduced from the behavior of indicators brought into contact with the protoplasm of dit- ferent cells. The reactions within a living cell presumably never are in a state of equilibrium, and the reducing intensity of one part of a cell may differ widely from that of another. The faci remains that certain definite results have been ob- tained both under aerobic, and under anaerobic, conditions. These results are fairly constant ana reproducible, provided that proper precautions are taken to be sure that the indicator is non-toxic and enters the cell in minimum amounts and does not become too much localized in certain regions. Moreover, the values obtained can be made tu shift in the expected direction with varying pH (6) and other conditions, and the rate of reduction of the indicators employed can be. changed by agents which presumably affect activating agen- cies within the cell. It will suffice to describe the experimental re- sults obtained, the difficulties involved in the technical procedure, and to analyze possible dis- crepancies in the results obtained by different m- vestigators. , Up to the present time electrometric methods of measuring the potential of protoplasm have been fruitless. It has not been possible to make tips of metal electrodes sufficiently stiff and yet fine enough to penetrate a living cell without oc- casioning serious injury. It has been possible to make glass microneedles of the proper order of fineness and to convert them into metal electrodes by coating with platinum, or with silver and silve: chloride, but they have been unsatisfactory be- 1 From the Eli Lilly Research Division, Marine Bio- logical Laboratory, Woods Hole, Mass, cause of their high resistance. Even with ideal electrodes there is still the difficulty of keeping their tips within the protoplasm of a living cell long enough to arrive at some kind of a condition of equilibrium between the electrode and the pro toplasm. A microneedle, after being thrust into a starfish egg or an ameba and held stationary for longer than a few seconds, is usually no longe: within the protoplasm but is walled off from it by a newly formed plasma-membrane. The colorimetric method, with the use of oxida- tion-reduction indicators, has proved much more satisfactory. These indicators, largely prepared and studied by Clark, Cohen and their co-workers, (see Figure), are easily reversible and, if present in sufficiently dilute quantities, readily adjust themselves to the potential of the system into which they are introduced. In general this meth- od of determination parallels the analogous one of determining acid base conditions by means of pH indicators. Owing to the fact that the potential can be de- termined only by the maintenance of truly re- versible systems, it is imperative that the cell in- terior be brought into contact not only with the oxidant but also with the reductant of the indi- cator. Otherwise there would be no way of indi- cating whether the indicator, which should shift to the potential of the electromotive system pres- ent, is behaving in a truly reversible manner. The main advantage of the microinjection method is that it permits the use of indicators to which the living cell is impermeable. Also, owing to the rapidity of the method and to the minute amounts of indicator necessary, there is less dan- ger of upsetting the original equilibria of the cell than by the immersion method. Its disadvantages are that it cannot be used on many types of cells, either because of their ex- treme susceptibility to mechanical injury or be- cause of their minute size. Also a quantitative study of capacity and rate factors is not possible because of the inability to inject the same amount into several successive cells, a procedure which is necessary in order to obtain more than a very ap- proximate value. The immersion method is far better suited for the quantitative studies neces- sary for the determination of capacity and rate factors. However, it is satisfactory only with pen- etrating indicators. The micromanipulative technique permits the use of micropipettes having tapering tips as small as 4 a micron in diameter through which aque- ous fluids exude easily into such cells as marine 330 THE COLLECTING NET { Vor. VIII, No. 70 13 2 1012 1¢ 8 9b 3b 9a 9c Anaerobic Along the ordinate are plotted the potentials (E’o) in volts, with reference to the hydrogen elec- trode at pH 0.0, exhibited by mixtures, in equal parts, of the oxidized and reduced forms of the re- versible indicators. Along the abscissa are plotted the pH values from 6.0 to 7.5 covering the range of the physiological systems investigated. The curves (lettered according to the scheme in previously published investigations, see Table 1) represent the (E’o values of the indicators used (H to U,,) with variations in pH. Values for the in- dicators H to U,, are taken from Clark and co- workers; as follows: U,, (35), I (36), Q) (37), H, L, O, P, Q, R,sS and U (38), Q & S (389). Those for in- dicator U,, are taken from Wurmser(40). The crosses on the curves represent the Eo values of the indi- cators on or next to the borderline of reducibility in the cells. The horizontal lines extend these levels to the right where vertical lines join the levels of the pairs of indicators which delimit the potential value of the particular cell or cells. For example, the figures, lc and 3b, representing Amoeba proteus and Paracentrotus, (see Table 2) are placed over a ver- ticle line joining levels at pH 7.0 of R and §, the former being reduced and the latter being oxidized when injected. The two Ts on the vertical line show the probable outer limits for the aerobic potential on the as- sumption that R is about 90% reduced and that S is about 75% oxidized. In a similar manner the anaerobic value for 1c and 3b lies somewhere below the T on the vertical line starting from the indicator U at pH 7.0. The level at which this T is placed is based on the as- sumption that U is at least 95% reduced, such a condition being assumed since a much larger quan- tity can be reduced in anaerobiosis than in aero- biosis. In those cases in which partial reduction is re- ported only one cross is given. For example, in the case of 8, 9a, 9b and 9c, the reported result is given as a cross on S, at pH 7.0. The two Ts on the ver- tical line at the end of the horizontal extension from this cross mark the probable limit of the potential on the assumption that an indicator reported as par- ially reduced is in all likelihood not more than 75% oxidized nor more than 90% reduced. In the case of 16, Valonia (see Table 2), the ver- tical line joining the extensions from I and Q, at PH 6.0 represents the range between the two indi- cators used. The two Ts show the limits of potential calculated by Brooks who claims that I is at least 99.9% reduced and that Q, is at least 99.9% oxi- dized. ova and protozoa. The most reliable indicators for injection are those which do not penetrate living cells from outside, but, when injected, spread im- mediately through the cytoplasm. When injecting a solution of a readily pene- trating indicator the experiment may be vitiated if some of the solution is spilled around the cell during the process. The spilled dye may then dif- fuse into the cell and accumulate there until the possible effects of reduction of a minimum amount are swamped out. Methylene blue, whose reduction potential lies close to the physiological range, is difficult to inject. It tends to coagulate the extraneous coatings of cells so that the pi- pette clogs before an appreciable amount can be injected into the cytoplasm, where a_ localized coagulum may also be produced. It tends to cause precipitations and has a strong affinity for certain cell inclusions in which it seems to be more re- sistant to reduction than when free in solution. Successful injections of indicators of this sort have been made by introducing very dilute solu- tions in successive small doses so as to permit the cell to exert its reducing capacity on a little at a time. The indophenols, and especially the sulphonated indicators, have proved to be the best for injec- tion. No coagulation occurs, spilled sulphonated indicators do not penetrate, and when introduced into the cell they diffuse readily and are either re- duced or impart to the cytoplasm the homogen- eous blue color of the oxident. In the experiments conducted in the series | to 7) precautions were always taken to test the presence of reduced indicators within the cells by subsequently introducing an oxidizing agent, e.g., potassium ferricyanide. It has been known for some time'®) that dye stuffs which contain sulfonate radicals, do not in general penetrate living cells: Brooks'®) showed this to be true for Valonia in regard to (1)naph- SEPTEMBER 2, 1933 ] THE COLLECTING NET thol(2)sulfonate indolphenol. More recently‘) in studies on the permeability of echinoderm ova to the Clark’s series of reversible indicators, it was shown that all those indicators possessing a sul- fonate radical (irrespective of their position on the E’o scale) do not penetrate the ova and con- sequently are not reduced either under aerobic or anaerobic conditions. This has also been found true for starfish spermatozoa’) and for yeast cells‘. Another feature to be taken into consideration is the difference in affinity of living cells for various penetrating indicators. Moreover, the ac- cumulation of some of these indicators in a cell is often largely a matter of the difference in the pH of the medium and that of the cell interior. In short, the several indicators which are at our disposal for a colorimetric study of oxidation-re- duction potentials vary so greatly in their inter- actions with biological systems that the greatest precautions must be taken in order to secure ade- quate interpretations. Many of the observed dis- crepancies of various investigators may largely be eliminated by a proper consideration of the in- dividual differences among the indicators used. Special precautions are also necessary if buf- Table 1 H Phenol indo-2, 6-dichlorophenol (pene- ieates)) =. I Phenol indo-2, 6-dibromophenol (pene- trates ) L o-Cresol indo-2, 6-dichlorophenol (pene- trates ) © 1-Naphthol-2-sulfonate indophenol (does not penetrate ) P 1-Naphthol-2-sulfonate indo-2, 6-dichloro- phenol (does not penetrate ) © Toluylene blue chloride (penetrates) ©, Thionine (Lauth’s violet) (penetrates ) Q, Brilliant cresyl blue chloride (penetrates ) R_ Methylene blue chloride (penetrates ) S_ Ky, indigo tetrasulfonate (does not pene- trate ) S: Ethyl Capri blue nitrate (penetrates) U_ Kz indigo disulfonate (does not penetrate ) Ui. Ky indigo monosulfonate (does not pene- trate ) Uz Cresyl violet (penetrates ) Table 1. Indicators used which, by their oxidized or reduced state, show the limits of potential of various cells, fered solutions are to be used in which to sus- pend cells. When a buffer is used the mainten- ance of a definite pH in the environment does not necessarily mean that corresponding pH con- ditions are being maintained within the cell. The buffering capacity of the protoplasm of a cell may be temporarily swamped out, and it has beea shown that the vacuoles within the cell very read- ily shift their pH in the presence of penetrating acids‘), Of the the acids usually used in buf- fers acetic acid penetrates more readily than phthallic acid which also is able to enter, while citric acid, which is highly polar, and phosphoric acid penetrate much more slowly. Aside from the special precautions to be kept in mind in detecting the oxidized or reduced state of indicators in the cells, there is still the prob- lem of interpreting results in determining the pH of the cell which must be known before the re- sults from the reactions of the oxidation-reduc- tion indicators can be stated in terms of potential. Investigators do not agree on the pH value of even the same type of cell™?). However, most of the publications on such diverse cells as certain protozoa'!*) 1), marine ova, both invertebrate (15, 16, 17, 18) and vertebrate™®) | several somatic cells (4 1% 20.21) and the protoplast of plant cells (°2) agree that the pH of the protaplasm of all the cells investigated is within two or three decimal points of neutrality. The pH values given by Vles, Reiss and co- workers" 74)) as 5.4 — 5.8 have been objected to 4) (4) on the following grounds. In the ex- periments in which they crushed the eggs'?*) the low values were undoubtedly due to the de- velopment of an acid of injury, and their spec- troscopic experiments’**) deal with pigment changes within granules the pH of which does not necessarily have anything to do with pH of the cytoplasmic mating''®: 1), Another reported value which seems to be too low is the pH of 6.0 which Rapkine and Wurnser claim for plant cells'°). This is identical with that obtained by Brceoks for the sap of Valonia cells'**), while in a recent pa- per'**) in which the pH of the protoplast and that of the sap have been separately determined, the pH of the protoplast was found to be 6.9 0.2. All the determinations of the protoplasmic pH which give values close to neutrality have been made principally by injecting indicators of Clark’s series. These indicators diffuse readily through the cytoplasm and give uniform degrees of coloration. They are similar in chemical con- stitution, all being weal sulfonic acids, and, therefore, are not open to the serious objection of being indiscriminately acidic or basic as is the case with the oxidation-reduction indicators. = ‘aporjzoela SSeLs ay} JO asn ‘soloeds sures Jo eAo Aq _| Aq peurusejep des jo Yd , pezeunxoidde enjea jo siseq uo pauinsse on[teA % “‘s1OyedIp iS) ‘peuinsse on[eA , OD -UI JO SUCT]NIOS UI UOISIaUIUTT Aq pauTe}qgo en[eA «+ a ‘pewinsse onteA 2 Hd jo aayynq azeydsoyd eynTIp ur paezAjo}A0 s8BqQ “S10}BOIpUl JO UOTJDefUIOIDIUI Aq peuTe}qo enTeA «x = S|} fez) of. A 40) I “WUT 09% > 4-7 Ra (deg) emo, 91 | & Ge) 82. "MU u O a =0'9 , wixBouds Cy f's 3 (eroydiyje9 ‘snuouosy)) = (eT) 82. “MU O I ‘fuy Sa) ‘Tuy S]Jeo purys ArBales fF] — s I ae (quadseurtuny ) (Ze) 60a sia) O Veni a s OZ oe Bape CT (OL) Ud “a 'O O Hf OZ 2 48a sea ZI Wea) Tae Gillan ni O I i sanssi} ueleUURy [| (Z) XD) WN Jal v6) at a OZ Wy vozoyeulieds svueisy QT (¢) Soo) ao Jal 1S PSI é Oz 2 SH pazAJOIAD SBLIaISY 26 ) ra) (S) (ei, ee) tel ID) i) ES YW eg UUWUT =99 ” (rwy) seuaIsy 46 . 70 : 7 (Z) 6c. 0 d YO ial S 1S MW 7 #39 ) (raul) SeLiasy %6 A ra) o (Z) (4; 2) ral “\Y{9) fal S ES Ps =3'9 (‘reury) sniuypeieulysy PANG) 192. N N al =9'9 Mi (ang) Bppsy Z| 2 PaCS) 192. ‘N “N Al I =99 ys (anq) waniydgQ 9 | & ca . z0 ( = a (pr). AS; Tal 30)! 0) AI - =9'9 E (cinsz) BLIeTPaqeS qg S | (ST) 192. N N “ I =9'9 Bs (cany]) BlaeTaqes eg | 3 O Z0 = (+) (AS; el 3) MO) MI Me 99 a (ang) wmipsesouryoy (lp = (ST) 192. N N O al , =99 P (sang?) wunipsesoulyay ep | is rae) (Z) A, 2) al UO) fl S Psi an +=3'9 4 (camzz) snjosjuesevieg q¢ svliajsy pure (CT) 192. N N O al es +-99 Pe (sang) snjorjue RIE ee J (¢T) 29d. NN lal O all ne SEY ue (canq_) snasay{OWAN Z bo (+) ASX, “el YD) {ol S ua ve ti : (cany]) snajoid eqaouny oT 2 (CD) 8, “eat MO) “D) n eon a +=0Z > (-reury) BIqnp eqsowy qT | 9 | (Sl 82)! 29% Sa Ni (NI O O “fuy FOL fu] (-inq_) snajoid eqsouy ey E ‘per ‘per ‘ ; ‘pIxo “ANd ‘peu 'PIXO “ANd 'P8U_- Surureqqo Surure}qo seoualejoy _ SISOIHOYAVNV SISOISOUaV Jo jo yeezew :Jepun pou an[eA poyien | peZziIprxo a1e Yorum eatqisod soul pue Hd ‘pesonpel 318 YOIGM seuo dA} eSou SOW | :s10}BOIpUy SUIqIWIT a : Oo *s]]90 Ule}Ie0 UT SeyISUaJUT Sujonpas ayy ur Suypuy poyioder ayy Jo AreuruMg -Z eIqGeL SEPTEMBER 2, 1933 ] THE COLLECTING NET 333 Extracellular Reduction In all the cells studied by means of microin- jection there has been no evidence for the exis- tence of extracellular reduction. In immersion ex- periments in which indicators known to be non- penetrating have been reported as reduced?" 7"), it is possible that the results are due to the pres- ence of cellular disintegra which possess a pro- nounced reducing intensity. However, Brooks'=”’ describes having found that the sap within the vacuole of the Valonia cell possesses a definite re- ducing intensity and Dr. Armstrong (personal communication) reports that the fluid within the brain ventricle of the pipe fish embryo (Syngna- thus fuscus) reduces the several indolphenols supplied by La Motte. In both cases the extract- ed fluid shows no reducing intensity. A similar case appears to be that described by Voegtlin'?”) who states that plasma and lymph, in the absence of cellular constituents, possesses no reducing power. The Intensity Factor of Oxidation-Reduction in the Cell Needham and Needham were the first to con- sider the idea of making determinations by in- jecting indicator solutions into living cells. They obtained varying values of potential for different marine ova (See Table 2—3a, 4a, 5a, 6, 7) the internal pH of which they stated!) as being uni- formly in the neighborhood of 66. For the fresh water ameha (Table 2-la,) they obtained'**) a more negative potential, and this they related to their claim that the ameba protoplasm possesses a pH of the more alkaline value of 7.6. Most of their work was carried on under aerobic condi- tions; they made anaerobic experiments‘! only with lmoeba proteus and Nyctotherus, (Table 1, 2), the latter a facultative anaerobe. In the ameba they found no difference between the aerobic and anaerobic reduction potential. This led them to be- lieve that the reduction potential of protoplasm of the ameba is poised at a definite level indepen- dent of the oxygen tension. With Nycotherus, on the other hand, they found an anaerobic value which was definitely more negative than the aerobic. In later investigations''*) special precautions were taken to obtain more strictly anaerobic con- ditions, and any evidence of intracellular reduc- tion was carefully checked by subsequently inject- ing an oxidizing agent. It was found that, under aerobic conditions, such diverse organisms as the fresh water ameba and the several marine ova, both European and American, if their. protoplasmic pH be reckoned at 7.0, possess an apparent aerobic potential more negative than -+ 0.011 and probably about —0.072V, "2; 3), See Figure. With two excep- tions all the indicators, which at present number over 30, are consistent in their reactions to indi- cate the same value. The two exceptions are Ky indigo tetrasulfonate and ethyl Capri blue. The former, which possesses a less negative E’o po- tential at pH 7.0 of —0.046V, shows no sign of reduction within the cell, while the latter with a more negative :’o potential of —0.072V appears to be at least partly reduced. Both are relatively non-toxic. A possible explanation is that the tetrasulfonate, being the salt of an acid dye, col- ors the cell-interior diffusely, while the Capri blue, as a basic salt, tends preferentially to stain certain cytoplasmic granules. If these structures are the seat of more intense reduction it might be expected that ethyl Capri blue would be more readily reduced than would the sulfonate. Under anaerobic conditions all cells investigat- ed by means of microinjection gave uniform indi- cations of a potential definitely more negative than —0.125V" *; #), a value which Needham and Needham''*) considered to be the lower limit for Nyctotherus. Recently the indicator, cresyl violet, with a still more negative potential of —0.167 at pH 7.0, has been found to be reversibly complete- ly reduced by starfish eggs'®), by starfish sperm- atozoa'") and by yeast cells). These values were obtained by observing the decoloration of stained masses of cells in exhausted Thunberg tubes and noting the return of color on the admission of air. It is of interest to note that a suspension of osmotically cytolyzed starfish eggs gives indica- tions of the same. reduction potential as intact eggs, both under aerobic and under anaerobic con- ditions". In regard to plant cells with large vacuoles filled with sap, Brooks'?® 75) found, by immer- sion experiments, that the sap, within the intact Valoma plant cell, can maintain certain dyes in the reduced condition. If the sap is removed, the reduced dye quickly oxidizes. The sap of Valonia macrophysa, according to Brooks, consists of a nearly pure solution of KCl, NaCl and CaCly and can have no reducing ability of itself. She main- tains that it accepts the oxidized and reduced form of the dye as it passes through the proto- plasm. It is surprising that sufficient anaerobic conditions to prevent oxidation of the indicators can be maintained in the voluminous vacuole of the size of a hen’s egg and surrounded by a layer of protoplasm not more than 3-10 micra in depth. In her later paper °°) Brooks made a revision of her earlier data ‘°®) on the value of the aerobic potential by using thionine as an indicator. She considered the reduction potential of the sap to be an index of that of the protoplasm and as- sumed them to be the same, see Chart 334. THE COLLECTING NET [ Vor. VIII, No. 70 and Table 1. Rapkine and Wurmser'‘!® found values for the aerobic potential of Spirogyra which are somewhat more neg- ative than those of Brooks for Valonia and pro- posed the generalization that the reduction poten- tial of plant cells is more negative than that of animal tissues. However, they were dealing only with the apparent reduction potential in aerobiosis and by methods in which there is considerable doubt as to whether or not the values obtained were that of the sap alone and not of the proto- plasm. Moreover, later publications have shown that the reducing intensity of animal cells is even more negative than the values reported by Brooks and by Rapkine. One of the earlier papers in which a determi- nation of the reduction intensity of cellu- lar tissues was attempted with the use of indicators of known potentials is that of Voegtlin, Johnson and Dyer? on mam- malian tissues. They made anaerobic experiments on thin sections of tissues in solutions of various indicators down to and including indigo disulfo- nate (U in Figure), all of which were reduced. An important feature of the work was that the reduction time of the various indicators was found to decrease with an increase in the elec- trode potential of the indicators. As can be seen from the figure, the reducibility of the indicators reported by Voegtlin, Johnson and Dyer corresponds to the anaerobic values ob- tained for other forms. They also injected the in- dicators into living animals and obtained, in the majority of cases, an aerobic potential which was considerably more positive, since indicator O was not reduced. It is significant that this indicator is a sulfonated compound and might not have been reduced because of its inability to penetrate the cellular tissues. It is also possible that the reason why they secured reduction of all the indicators, including the sulfonated ones with tissue sections under anaerobic conditions, might have been be- cause of the presence of cytolyzed material. By eliminating the values (see Figure and Table 2) shown by subsequent investigations to be faulty, we are brought to certain conclusions regarding the reducing intensity of the cytoplasm of at least such cells as the ameba and the var- ious marine ova. All tend to possess an apparent aerobic potential which approaches the E’o value of —0.072V and an anaerobic potential which is definitely more negative than —0.125V and, in some cases at least, more negative than —0.167V. Machlis and Green, working in my labora- tory, have found by the immersion method that starfish spermatozoa do not possess the same re- ducing intensity as starfish eggs, the indicators which are reduced by the former indicating an apparent aerobic potential more positive than + 0.047V. This may possibly be related to the fact that the spermatozoa contain extremely little cytoplasm, their heads consisting almost entirely of nuclear material. Fragmentary results'?) sug- gest that the nucleus at least of the immature starfish egg, possesses no observable reduction po- tential. With the very scanty cytoplasm of the spermatozoa it is possible that a capacity factor may influence the observed results on reducing intensity. At any rate no significant conclusions should be drawn from observations which entail only aerobic determinations since, under anaero- biosis, the reducing intensity of the starfish sper- matozoa gives indication of being of the same order (viz. <—0.167V) as the reduction potential of starfish eggs. No support is given by these ex- periments to the claim of Joyet Lavergne'*”) that male cells possess a more positive potential than female cells. Conditions Affecting the Reduction Potential 1. Effect of Cytolysis. With properly con- trolled experiments there is no evident for an in- creased reduction intensity at the moment of cy- tolysis. The observations of Needham and Need- ham) indicating that such a phenomenon exists were based on the disappearance of color in an in- jected egg with the onset of cytolysis. Later ex- perimental evidence’: *) offers an alternative ex- planation that the dye diffuses out of the cyto- lyzed cell, a view supported by the fact that a subsequent injection of an oxidizing agent shows no return of color which should be the case if the dye were present in its reduced state. Moreover, it has been recently shown ‘*) that the reduction potential of a mass of osmotically cytolyzed star- fish eggs exhibits no difference in its reducing in- tensity (aerobic or anaerobic) from that of intact eggs. 2. Effect of variations in intracellular acidity. It is well known that the potential value of a truly reversible oxidation-reduction dye system is de- termined by the pH of the system, being more negative with increasing alkalinity, and more positive with increasing acidity. Beck'®) has investigated this point with respect to the effect of variations in intracellular acidity. He found that the aerobic apparent reduction potential of intact living starfish eggs, determined colorimet- rically, becomes markedly more negative than normal in the presence of the penetrating base, ammonia, and it becomes markedly more positive than normal in the presence of the penetrating acid, carbon dioxide. For cytolyzed eggs the anaerobic potential shifted in acid solutions to a markedly more positive value. Anaerobic exper- iments with intact eggs internally acidified were inconclusive. The cytolyzed material reacted equally well to the organic and inorganic bases -SEPTEMBER 2, 1933 ] THE COLLECTING NET 335 and acids used, while intact eggs reacted only to those acids and bases which are known to pene- trate living cells. 3. Effects of Various Reagents on the Inten- sity Factor. In the experiments of Machlis and Green'?’ on suspensions of starfish spermatozoa it has been possible to show that ethyl urethane renders more positive the apparent aerobic poten- tial of the suspension while cyanide and H.S render it more negative. On the other hand, the anaerobic potential is unaffected being the same as that of untreated sperm suspensions. For the lower range of oxidation-reduction po- tentials there are at present no available indica- tors which are truly reversible. Hence, there is no way of dealing with possible effects of many reagents, such as narcotics and oxidation inhibi- tors, on the anaerobic potential. Machlis and Green found that the anaerobic potential was destroyed on heating the sperm sus- pension to 100°C., the anaerobic value obtained being of the same order as that of both heat killed and living spermatozoa under aerobic con- ditions. This finding indicates that the systems re- sponsible for the anaerobic high reducing inten- sity of normal cells are largely thermolabile. Rate Factor All experimental evidence ‘?7 4 #15) has indi- cated clearly that the speed of reduction is great- est for the most electropositive indicators and be- comes progressively slower as the potential of the indicator approaches that of the cell. It is of in- terest to note that there are reactions in biological systems which are affected by the E’o value of the indicators. This is shown by Barron and Hoffman‘*?) who found that the catalytic effect of the oxidation-reduction indicators on oxygen con- sumption depends on the position of the indicator on the oxidation-reduction potential scale. Of considerable significance is the fact that cytolysis has a marked retarding effect on the rate of reduction. This has been shown for star- fish eggs ‘) osmotically cytolyzed by treatment with distilled water and with hypotonic solutions of sodium phosphate (M/15) at pH 7.0. The ap- parent potential of the system, however, as we have already noted, remains the same as that of intact and living eggs. Of the narcotics, phenyl urethane saturated in sea water was not found to have any retarding effect, but this may be due to the extreme dilu- tion of the narcotic since we have been able to obtain a definite retardation in the speed of re- duction by using the more soluble ethyl urethane in concentration of 3 per cent. It is of interest to note that both phenyl urethane (saturated ethyl urethane (3%), ether (1/100 saturated) and ethyl alcohol ,0.017) completely destroyed the ability of cytolyzed eggs to reduce cresyl blue and methylene blue within the period of observation of three hours. Potassium cyanide in concentration of N/10, N/20 and N/100 in sea water was not found to have any effect on the rate of anaerobic reduc- tion of cresyl blue or of methylene blue either by intact or by cytolyzed eggs. For further information on the effects of var- ious reagents and experimental conditions on the rate factor, a review by Ahlgren'**) may be con- sulted. The meaning of the experimentally found re- duction potential in cells is still very uncertain and it should be realized that the results of the colorimetric method are based on an indiscrimin- ate average of the cytoplasmic mass of the cells investigated. The findings that the reducing intensity of spermatozoa'’) and of yeast'!") is shifted by nar- cotics and oxidation inhibitors agrees with the findings of Keilin'*’ on the reaction of cytoch- nine and suggests that the value of the apparent aerobic potential is determined by the relative rates of activity of intracellular respiratory en- zymes. Regarding the anaerobic reduction potential nothing can be stated at present since the exper- iments in which due precautions have been taken to obtain as strict anaerobic conditions as possible indicate that the most negative value of the an- aerobic potential has not yet been determined. I take this opportunity of expressing: my ap- preciation’to Dr. Lyle V. Beck for his assistance and for preparing the figure illustrating the plot- ted electrode potentials with reference to the cells investigated. LITERATURE Papers in the series Intracellular Oxidation-Re- duction Studies. 1. B. Cohen, R. Chambers and P. Reznikoff. 1928. I. Reduction potentials of Amoeba dubia by microinjection of indicators. J. Gen. Physiol., 11, 585. 2. R, Chambers, H. Pollack and B. Cohen. 1929. II. Reduction potentials of marine ova as shown by indicators. Brit. J. Exp. Biol., 6, 229. 3. R. Chambers, B. Cohen, H. Pollack. 1931. Ill. Permeability of Echinoderm ova to indicators. J. Exp. Biol., 8, 1. 4. R. Chambers, B. Cohen and H. Pollack. 1932. IV. Reduction potentials of European marine ova and Amoeba proteus as shown by indicators. Pro- toplasma, 17, 376. 5. R. Chambers, L. V. Beck and D. E. Green. 1933. V. A comparison of intact and cytolyzed star- fish eggs by the immersion method. J. Exp. Biol., 10, 142. 6. L. V. Beck. 1933. VI. The effects of penetrat- ing and non-penetrating acids and bases on the oxi- dation-reduction phenomena of starfish eggs. J. Cell. and Comp. Physiol., 3, 261, 336 THE COLLECTING NET [ Vor. VIII, No. 70 7. S. Machlis and D. E. Green. 1933. VII. Mech- anism of reduction potentials in starfish sperm. J. Cell. and Comp. Physiol. (In press). 8. R. Hoeber und O. Nast. 1913. Weitere Beit- rage zur Theorie die Vitalfarbung. Biochem. Z., 50, 418. 9. M. M. Brooks. 1931. The penetration of 1- naphthol-2-sulphonate indolphenol, o-chlorophenol indophenol and o-cresol indophenol into Valonia. Proc. Nat. Acad. Sciences, 17, 1. 10. L. V. Beck and J. P. Robin. 1933 The ap- parent reduction potential in yeast. (In press). 11. R. Chambers. 1928. Intracellular hydrion con- centration studies. I. The relation of the environ- ment to the pH of protoplasm and of its inclusion bodies. Biol. Bull., 55, 369. 12. R. Chambers. 192$. Hydrogen ion concentra- tion of protoplasm. Bull. Nat. Research Council, No. 69, 37. 13. J. and D. M. Needham. 1926. Further micro- injection studies on the o/r potential of the cell interior. Proc. Roy. Soc. B., 99, 383. 14. R. Chambers, H. Pollack and S. Hiller. 1927. The protoplasmic pH of living cells. Proc. Exp. Biol. and Med., 24, 760. 15. J. and D. M. Needham. 1926. The hydrogen ion concentration and o/r potential of the cell in- terior: a microninjection study. Proc. Roy. Soc., B., 99, 173. 16. L. Rapkine and R. Wurmser. 1928. On in- tracellular o/r potential. Proc. Roy. Soc., B. 102, 128. 17. R. Chambers and H. Pollack. 1927. Micrur- gical studies in cell physiology. IV. Colorimetric de- termination of the nuclear and cytoplasmic pH in the starfish egg. J. Gen. Physiol. 10, 739. 18. C. G. Pandit and R. Chambers. 1932. Intra- cellular hydrion concentration studies. IX. The pH of the egg of the sea-urchin, Arbacia punctulata. J. Cell. and Comp. Physiol., 2, 243. 19. R. Chambers. 1932. Intracellular hydrion concentration studies. V. The pH of the protoplasm of the Fundulus egg. J. Cell. and Comp. Physiol., 1, 65. 20. R. Chambers and R. J. Ludford. 1932. Micro- dissection studies in malignant and non-malignant cells. Arch. f. exp. Zellf., 12, 555,. 21. R. Chambers and G. Cameron. 1932. Intra- cellular hydrion concentration studies. VI. Colori- metric pH of malignant cells in tissue culture. Proc. Roy. Soc., B, 110, 120. 22. R. Chambers and T. Kerr, 1932. Intracellular hydrion concentration studies. VIII. Cytoplasm and vacuole of Limnobium root-hair cells. J. Cell. and Comp. Physiol., 2, 105. 23. P. Reiss. 1926. Le pH interieur cellulaire. Les presses Universitaires de France, Paris. 24. KF. Viles. 1924. Recherches sur le pH interieur cellulaire. Arch. Phys. Biol., 4, 1. . 25. M. M. Brooks. 1930. The pH and the rH of the sap of Valonia and the rH of its protoplasm. Protoplasm, 10, 505. - 26. E. N. Harvey. 1929. A preliminary study of the reducing intensity of luminous bacteria. J. Gen. Physiol., 13, 13. -27. C. Voegtlin, J. M. Johnson and H. A. Dyer. 1924. Quantitative estimation of the reducing power of normal and cancer tissue. J. Pharm. and Exp. Therap., 24, 305. 28. J. and D. M. Needham. 1925. The hydrogen- ion concentration and o/r potential of the cell in- terior: a microinjection study. Proc. Roy. Soc., B. 98, 259. 29. M. M. Brooks. 1926. Studies on the permea- bility of living cells. VI. The penetration of certain o/r indicators as influenced by pH; estimation of the rH of Valonia. Amer. J. Physiol., 76, 360. 30. Ph. Joyet-Lavergne. 1931. La physico-chimie de la sexualité. Protoplasma. Monographien, 5. 31. E. Aubel and R. Levy. 1931. Etude du poten- tial d’oxydo-réduction dans des organismes vivants, Ann. Physiol. physicochim. biol., 7, 477. 32. E. S. G. Barron and L. A. Hoffman. 1930. The catalytic effect of dyes on the oxygen consump- tion of living cells. J. Gen. Physiol., 13, 483. 33. G. Ahlgren. 1925. Zur Kenntnis der tierischen Gewebes oxydation Skand. Arch. fur Physiol., Suppl. to 47. 34. D. Keilin. 1928. Cytochrome and Respiratory Enzymes. Proc. Roy. Soc., B, 104, 206. Papers in the series—Studies on Oxidation-Re- duction. 35. M. X. Sullivan, B. Cohen and W. M. Clark. 1928. IV. Electrode potentials of indigo sulphonates, each in equilibrium with its reduction product. Hy- gienic Laboratory Bull. No. 151, U. S. P. H. S., Washington, D. C., 1928. 36. B. Cohen, H. D. Gibbs and W. M. Clark. 1928, VI. A preliminary study of indophenols. Ibid. 37. W.M. Clark, B. Cohen and H. D. Gibbs. 1928. VIII. Methylene blue. Ibid. 38. M. Phillips, W. M. Clark and B. Cohen. 1927. XI. Potentiometric and spectrophotometric studies of Bindschedler’s Green and Toluylene Blue. Suppl. No. 61 to Public Health Reports. 39. B. Cohen and P. W. Preisler. 1931. XVI. The oxazines: Niie blue, brilliant cresyl blue, metnyl capri blue and ethyl capri blue. Suppl. No. 92 to the Public Health Reports. 40. R. Wurmser, 1930. tions. Les Presses Universitaires de Boulevard St., Michel, Paris. Oxydations et Réduc- France, 49 DISCUSSION Discussion is omitted because the author includes in his manuscript as presented for publication sali- ent points of the discussion. Those who took part in the discussion of Dr. Chambers’ paper are Dr. Harold Abramson, Barnett Cohen, Hugo Fricke, Stuart Mudd, and Oscar Riddle. SEPTEMBER 2, 1933 | THE COLLECTING NET 337 DIRECTORY FOR 1933 INVESTIGATORS, ASSISTANTS AND TECHNICIANS Abramson, H. A. Research, dept. of biochem., Col- lege of P. and S., Columbia. Bacon, Annette L. Research, ass’t. Temple. Baker, R. F. Research, grad. stud. Penn. State. Bernstein, Felix, Research, formerly director Insti- tution for Mathematical Statistics, Univ. of Goettingen, Germany. Birnie, James H. Research, grad. ass’t. Brown. Briggs, David R. Research, investigator in chemis- try, The Otho S. A. Sprague Memorial Institute, Univ. of Chicago. Brown, Catherine R. Secretary, Biol. Lab. Brownscombe, E. R. Research, chemist, Biol. Lab. Chouteau, Ellen M. Librarian, Biol. Lab. Chen, T. T. Research, grad. stud., physiol. chem., Johns Hopkins Univ. School of Med. Cohen, Barnett, Research, assoc. prof. physiol. chem., Johns Hopkins Univ. School of Med. Cole, K. S. Research, ass’t. prof. physiol., College of P. and S., Columbia. Conard, H. S. Inst. and research, prof. botany, Grin- nell. Cover, Nelson, Inst., grad. Princeton. Crescitelli, Frederick, Inst., grad. ass’t., Brown. Cunningham, Bert, Inst. and research, prof. biol., Duke. Curtis, Howard J. Research, physicist, Biol. Lab. Deery, Edward, Glassblower, Bell Telephone Labs. ING ers, Elliott, H. C. Research, ass’t. anat., Univ. of Tor- onto. French, Helen, Research, grad. Grinnell. Fricke, Hugo, Research, in charge biophysics, Biol. Lab. Gallagher, D. M. Research, radio engineer, Biol. Lab. Gallagher, Anne H. Research, ass’t. Brooklyn Inst. Arts and Sciences. Gaunt, Robert, Research, Charleston. Gold, Dorothy, Secretary, Biol. Lab. Grout, A. J. Bryologist, Biol. Lab. Harris, R. G. Director, Biol. Lab. Haterius, H. O. Research, ass't. prof. biol., Wash- ington Square, New York Univ. Hays, H. W. Inst., grad., Franklin and Marshall. Henderson, C. Instrument maker, Biol. Lab. Hodge, Charles, Inst., inst. biol., Temple. Hogg, Bruce, Research, med. stud., College of P. and S., Columbia. Huggins, John R. Research, grad. stud., Univ. of Penn. Kornhauser, S. I. Inst. and research, prof. embry., Univ. of Louisville Med. School. Kutchka, G. M. Research, Carnegie Museum, Pitts- burgh. Morse, Miriam, Research, inst. zool., Mass. State. Mudd, Stuart, Research, prof. bacteriology, Univ. Penn. School of Med. Miiller, Hans, Research, prof. physics, Mass. Inst. Tech. prof. biol. College of Outerbridge, Marion, Research, grad. Smith. Parkins, W. M. Inst. and research, grad. Princeton. Ponder, Eric, Research, prof. physiol., Washington Square, New York Univ. Rybachok, O. G. Research, grad. stud., Temple. Sargent, Louisa M. Inst., ass’t. prof. bot., Grinnell. Schaeffer, A. A. Research, prof. biol., Temple. Schaeffer, Olive, Research, Biol. Lab. Smith, Marion, Ass’t., Biol. Lab. Swingle, W. W. Inst., prof. biol., Princeton. Taylor, I. R. Inst. and research, ass’t. prof. physiol., Brown. Viitoreen, E. Research, technical ass’t. Biol. Lab. Walzl, Edward, Research, grad. stud. Johns Hop- kins. Williamson, Cornelia, Research, undergr. Smith. stud. STUDENTS Atsatt, Sarah B. S. M., assoc. prof. biol. Scripps. Bittman, Myrtle F. Z., ass’t. Adelphi. Boettiger, Edward F. Z., grad. stud. Brown. Bookman, John G. P., undergr. Brown. Clark, C. C. F. Z., inst. biol. Washington Square, New York University. Cornwell, Alfred G. P., undergr. Dickinson. Day, Dorothy P. S., ass’t. prof. bot. Smith. Dean, Wynant F. Z., undergr. Hampden-Sydney. DeLamater, E. E. F. Z. and research, grad. stud. Johns Hopkins. Dreier, Dorothea F. Z., undergr. Univ. of Wisconsin. Earle, D. P., Jr. S. M., grad. Princeton. Ferrer, J. M. S. M., undergr. Princeton. Fraad, D. J., Jr. G. P., undergr. Brown. Freeman, J. W., Jr. F. Z., inst. biol. Moses Brown. Goodman, 8S. K. S. M., undergr. Princeton. Harrison, J. S. G. P., undergr. Brown. Horner, Helen E. P. S., undergr. Grinnell. Kuchler, Frances W. S. M., grad. stud. Yale. Laity, Elsie P. S., undergr. Grinnell. Lockhart, J. H., Jr. S. M., undergr. Princeton. Meneely, G. R. S. M., grad. Princeton. Pomerat, C. M. S. M., inst. biol. Clark; grad. stud. Harvard. Reese, W. E. F. Z., undergr. St. John’s. Rizer, D. K. S. M., undergr. Princeton. Rizer, R. I. S. M., undergr. Univ. of Minnesota. Rouse, Sylvia G. P., and research; grad. ass’t. Brown. 5 Solinger, J. L. S. M., grad. stud. Brown. Stix, Helen D. G. P., undergr. Wellesley. Tack, Frank’ M. S. M., undergr. Princeton. Thompson, Ruth E. F. Z., inst. zool. Univ. of New Hampshire. Vandam, L. D. G. P., undergr. Brown. Wahlers, Alice F. Z., undergr. Adelphi. Walker, Philip P. S., undergr. Univ. of Pittsburgh. Warner, Douglas S. M., grad. stud. Claremont. Whipple, Ralph V. S. M., undergr. Princeton. Woodbridge, Anna F. Z., undergr. Mt. Holyoke. THE COLLECTING NET [ Vor. VII, No. 70 LTE MisstOrr DINE Eee Sk Recent visitors to the Laboratory include, Prof. W. J. Crozier of Harvard University, Dr. Rob- ert Hegner of Johns Hopkins University, Prof. and Mrs. Charles J. Lyon of Dartmouth College, Dr. L. Reiner of New York University, Dr. Jules Ireund of Cornell Medical College, Prof. Harry A. Charipper of New York University, Dr. Hen- ry G. Barbour of Yale University School of Med- icine, and Dr. andMrs. Leslie E. Sutton of Dr. N. V. Sidgwick’s laboratory, Oxford. Dr, Sut- ton is on his way to Pasadena where he will work with Dr. Linus Pauling on a Rockefeller Founda- tion Fellowship. He plans to study electron dif- fraction. Prof. John T. Buchholz of the University of Il- linois has recently arrived with his family. He will spend the remainder of the summer at Car- negie Institution here, as guest investigator. The annual award of John D. Jones Scholar- ship, formerly made at Columbia University for use at the Biological Laboratory here, has been turned over to the Scholarship Committee of the Laboratory. The stipend of $250 will be given to one applicant, or divided between two or more applicants, as the Committee decides in any given year. Mr. Jones was a founder of the Biological Laboratory. The Scholarship bearing his name was established some years ago by the Wawepex Society. Professor Harry A. Charriper, Chairman of the Department of Biology at New York Univer- sity, has completed arrangements whereby the University will have a research table at the La- boratory each year. At present that table is being used by Prof. H. O. Haterius. Evening lectures, given at the Laboratory this summer, in addition to those already published in THe CoLttectine Net, include: Dr. Oscar Riddle, Department of Genetics, Carnegie Institution: “Prolactin and Other An- terior Lobe Hormones.” Dr. Stuart Mudd, University of Pennsylvania Medical School: “Infection and Resistance.” Dr. M. Demerec, Carnegie Institution of Wash- ington: “Genes—An Element Essential for the Life of the Cell.” Dr. S. I. Kornhauser, School of Medicine, Uni- versity of Louisville: “Studies on Anisolabis.” Dr. A. A. Schaeffer, Temple University : “Pro- toplasmic Organization of Movement.” Dr. H. S. Conard, Grinnell College: “A Criti- cism of Succession.” Prof. A. M. Banta, Brown University and Car- negie Institution of Washington: “Some Newer Data on Control of Sex in Cladocera.” Carnegie Corporation has just made a contribu- tion of $2500 to the Laboratory in addition to one of equal amount reported in THE COLLECTING Net of July 15th. This completes payment of a special grant of five thousand dollars from the Corporation this year for the purchase of equip- ment. Dr. H. O. Haterius of New York University has recently arrived at the Laboratory to spend the rest of the summer. Dr, John R. Huggins of the University of Pennsylvania has just come to the Laboratory for a few weeks’ residence. His family is with him. END OF COLD SPRING HARBOR SECTION SEPTEMBER 2, 1933 ] THE COLLECTING NET 339 EYE COLORS IN THE PARASITIC WASP HABROBRACON AND THEIR BEHAVIOR IN MOSAICS AND IN MULTIPLE RECESSIVES Dr. ANNA R. WHITING Professor in Biology, Pennsylvania College for Women The eyes of wild-type Habrobracon are black. All mutations are therefore necessarily lighter than wild-type and all are recessive to it. lour independently segregating loci have been in- volved in eye color mutations. In the orange locus there are four mutant types. These are light-ocelli (o'), dahlia (o0"), orange (0), and ivory (0'), forming with type (O), a multiple allelomorphic series of five factors. Dominance is in the order of intensity of pigmentation, type being dominant to all the others and ivory recessive to them. Two different mutations have occurred in the white locus, white (wh) and carrot (wh*), which with type (Wh) form a triple allelomorphic series. White is partially dominant to carrot since the heterozygote is cream. The two remaining loci have been identified by one mutation each. They are cantaloup (c) and maroon (ma), each re- cessive to its type allelomorph (C and Ma). In Habrobracon the second polar nucleus oc- casionally undergoes division as well as the egg nucleus. If such a binucleate egg is unfertilized and from a mother heterozygous for eye color it may produce a male with mosaic eyes. The be- havior of eye colors in relation to each other in mosaics is of considerable interest. Whenever any color in the orange locus is associated in a mosiac with an allelomorph there is no sharp line separ- ating the genetically different regions. Instead, one color shades gradually into the other. An eye, mosaic for black and ivory, shows black pigment at one side, then orange, and finally, at the oppo- site side of the eye, ivory. An eye mosaic foi orange and ivory likewise shows grading. There appears to be diffusion of a chemical substance from the dominant to the recessive region so that some facets genetically recessive show the dom- inant color. In striking contrast to this, eye mosaics involy- ing the white locus show a clear cut line between the genetically different tissues. An eye mosaic for black and white or for carrot and white shows each region clearly marked off from the other and obviously autonomous. The cantaloup locus resembles the white in its behavior in mosaics. No mosaics for maroon have yet been found. A particularly striking type of interaction is illustrated by eyes mosaic for cantaloup and ivory. Mothers were heterozygous for both (Cc.Oo') and had black wild-type eyes. There can be no genetically black (OC) tissue in the eyes of the mosaic sons in question since they show cantaloup and ivory regions and would have to have arisen from three ditferent kinds of nuclei if genetically black tissue were present. The cantaloup region is clear cut and marked off sharply froma black band which grades into orange. In the abseace of gene- tically black tissue this black band must be due to the diffusion of a substance from the cantaloup region into the ivory and a physiological recon- stitution of the double dominant color. A black-eyed mother, heterozygous for ivory (O.0') occasionally produces mosaic sons in he the line of division between black and i ivory nuclei does not fall in the eye but in the thorax or abdomen. Gonads of these males are black as shown by breeding tests but the light eyes are not ivory as expected. Instead they are, in most cases, a uniform orange. This likewise appears to be the result of diffusion from the wild-type tissue, pos~ sibly the gonads, into the ivory tissue of the eyes. The complete change in color of these eyes, in contrast to the grading of the mosaic eyes, may be explained possibly by supposing that testes or other abdominal tissues develop more rapidly than eyes and produce the darkening chemical sub- stance so early that there is opportunity for com- plete distribution before the eyes begin to form. The interactions here described are of interest in view of the opinion held by many that inver- tebrates, and perhaps more especially insects, dif- fer from the vertebrates in having their tissues autonomous with little chemical or hormonal ef- fect in growth and development. If light-ocelli (o') which was lost several years ago be omitted the mutant eye colors can be com- bined in forty-eight different ways in the haploid males. Twenty-six of these combinations have been made. Of these nine show pigmentation while seventeen are colorless. The indications are that the remaining twenty-two will likewise be colorless although no predictions can be made with certainty. The intensity of a pigment in a single mutant type has little or no relation to its effect when combined with another. In summariz- ing these combinations it is clear that every mul- tiple recessive is lighter than the lightest mutant entering into the combination, and the majority of them are colorless. The white resulting from combinations of other mutants is usually a trans- lucent white so that dark underlying tissues show through whereas this is not the case in the single mutant forms white and ivory which are opaque. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on Au- gust 29.) 340 THE COLLECTING NET [ Vor. VIII, No. 70 ANTERIOR PITUITARY-LIKE HORMONE EFFECTS F. E. CHIDESTER From the Zoological Laboratory, West Virgina University, and the Marine Biological Laboratory, Woods Hole, Mass. For a number of years, our experimental work has led us to an appreciation of the close relation- ship between the endocrine glands and problems of nutrition. Quite recently it has seemed advis- able that we bring to the attention of investigators certain correlations that have been emphasized by our converging lines of work, but have apparently been overlooked by others. The specific points that we wish to stress in this report are (1) the role of iodin in the endocrine chain, and in nutritional conditions favoring re- production; (2) the role of highly unsaturated hydrocarbons and fatty acids as modifying fac- tors in the action of iodin, calcium and other ele- ments. Our endocrine studies began in 1912, when, during a summer appointment at the Carnegie Station for Experimental Evolution, Cold Spring Harbor, L. I., we learned that pituitary, thyroid and adrenal extracts apparently inhibited egg lay- ing and somewhat affected body weight of fowls, but possibly exerted a stimulating effect on the reproductive capacity of rabbits. Heavy doses were resisted by pregnant rabbits, but the young were affected in various ways, some exhibiting deformity. During lactation the young were very susceptible to heavy dosage of the mothers. Later, in 1916 and 1917, we noted the depleting effects in rabbits of nursing large litters, and also receiving thyroid extract, as possibly contributory to paralysis. In 1918, at the Wistar Institute, we found that heavy doses of thyroid extract or thyroxin (Ken- dall) apparently induced sterility or resorption of the young in rats. This fact, had been recorded, however, earlier by Gudernatsch (1915) and was reported in 1925 by Pighini. It has been noted by Weichert (1930). We found that with basic diets (Chidester et al, 1928) rats and rabbits required increased fats and carbohydrates when receiving KI or thyroid ex- tract. Similarly (Chidester et al, 1929) with acid diets, glucose and thyroid together, induced in- creased weight, and glucose alone apparently has- tened maturity in rats. Two grains of thyroid ex- tract daily did not inhibit pregnancies or cause resorption of the young in rats. In chicks and rats, low iodin favored growth including elonga- tion of the bones. This we immediately correlated with the height and body size of mountaineers, living in a low-iodin, goitrous region. We found that orally administered adrenal cor- tex, induced prematurity in chicks (Eaton et al, 1929) and in rats, (Chidester et al, 1929). Brit- ton and associates reported similarly in 1931 for mammals. Nice and Schiffer (1931) found that implants of adrenal cortex tissue produced pre- maturity in young rats. We had been much impressed by the studies of Crew (1925) who found that thyroid extract re- juvenated aged fowls and caused them to lay eggs that were fertilized by aged cockerels. Experimental studies since 1927 by our students, Addair, Eaton, Thompson, and Wiles, working with tadpoles, blow flies, mosquitoes and fruit flies, have furnished important information regarding the action of endocrine extracts. Ad- dair learned (1928) that pineal extract apparently caused acceleration of metamorphosis in tadpoles. Other unpublished experiments suggested that ad- renal cortex extracts might act similarly. And Eskin (1932) reported that adrenal cortex in- duced metamorphosis in Axolotl. It was also shown (Thompson et al., 1928) that orally administered whole pituitary caused increased growth in young rats which was more marked in males. Wiles (1931), in the most important studies made by us with insects, has shown that adrenal cortex, anterior pituitary and thyroid extract, in- duced marked acceleration in gonadal develop- ment in female Drosophila, but that males were retarded. Similar results to be reported else- where (Wiles, Tomblyn, Zucchero, and Chides- ter) show that iodin induces the same prematur- ity, thus ruling out possible objection to the na- ture of the extracts. Males have less of the pro- tective unsaturated fatty acids, but we believe that reduced doses of iodin will act similarly with them. Since 1930 we have studied the influence of extracts of the anterior pituitary hormone on chicks. We found (Chidester and Wiles, 1931), that intravenous injections did not inhibit, but subcutaneous injections stopped ovulations in young laying pullets. Using the pregnancy urine extract, Antuitrin S, furnished through the courtesy of Dr. Kamm of Parke Davis & Co., we found (Chidester, Ashworth, Ashworth, and Wiles, 1932 a and b) that when it was intravenously injected into young fowls, 7 cases of leg weakness developed in a lot of 14 treated birds. Thyroid extract, fur- nished in capsules with balancing fatty acids of a wheat preparation, failed to induce such a condi- tion. Our work was checked by a poultryman, who knew of our earlier iodin studies and who SEPTEMBER 2, 1933 ] THE COLLECTING NET sal cured leg weakness and protected his flocks with iodized buttermilk. We believe that in our cases the hormone effect was exerted in a similar way to that shown by Loeb, Schockaert, Loeser, Adams, and _ associates, through the thyroids, when they induced Graves’ disease. Notthaft’s pa- tient, who took many thyroid tablets for obesity and developed Graves’ disease, naturally recurred to our mind. In hyperthyroidism, one finds that osteoporosis may result, since iodin will disperse calcium, Clinical reports support this statement. THE CARDIAC PARADOXES Mr. Brinton, the poultryman, caused leg weak- ness in his flock by feeding a diet rich in wheat, but apparently adequate in cod liver oil and fish meal. We believe Mellanby’s “toxic factor” of wheat and oats to be the same excess of unsat- urated fatty acids that he had noted in 1921 as a cause of goiter. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 15. It will be completed in the reprints.) OF LIMULUS POLYPHEMUS IpING CHAO Department of Physiology, University of Chicago It was shown by Libbrecht, (Arch. internat. de phystol, 1920, 15, 446) that when a frog’s heart is perfused for some minutes with a potassium-free Ringer’s solution and is then again perfused with the normal Ringer, the ventricular contraction is completely inhibited for a short period; by de- grees the normal rhythm is resumed. This prim- ary inhibition of the cardiac activity on change from the K-free to the normal Ringer’s solution was called by Libbrecht the potassium-paradox (K-P). Libbrecht, (Arch. internat. de physiol., 1921, 16, 448) also reported a similar primary in- hibition on change from a warm to a cold Ring- er’s solution—the thermo-paradox. More recently Kisch (Arch. f. exp. Pathol. u. Pharmakol., 1930, 148, 140) found two similar paradoxes in the frog’s heart; namely, the calcium-paradox and the strontium-paradox. Potassium-paradox was recently observed in the Limulus heart by the author (Chao, Biol. Bull., 1933, 64, 358). This summer a more tho- rough study of the conditions for its production was carried out, and observations on the calcium- paradox and thermo-paradox were also made; so far the experiments have been performed on the ganglion of the heart only, The results may be summarized as follows: The K-P is more readily obtained on a second or third repetition than on the first immersion of the ganglion in the K-free solution. When a fresh ganglion is immersed for the first time in a K- free Ringer’s solution for ten minutes and is then returned to a normal Ringer’s solution the K-P is usually slight or may not appear at all; but on a second trial under similar conditions it invariably appears; and a third trial gives a still more marked result. The effects designated as paradoxical (the primary and temporary decrease in the rate and amplitude of the heart beat) become more pro- nounced with increasing duration of the prelimin- ary immersion in the K-free solution. Thus the effect obtained after an immersion in a K-free Ringer’s solution for twenty minutes is always greater than that obtained after an immersion of ten minutes. The K-P is obtained not only on transfer from a K-free to a normal Ringer's solution but also on transfer to a Ringer’s solution containing an ex- cess of KCI. It is also obtained on change from a K-poor to a normal Ringer’s solution and even on change from a normal Ringer’s solution to a Ringer’s solution containing a higher concen- tration of KCl. In other words, the factor determining the K-P is the sudden change in the K-concentration, not the absolute con- centration of K; and this change must be in the direction of increasing the K-concentration in the second solution. The presence of NaCl in the K-free solution is absolutely necessary for the production of the K-P; the K-P can be obtained on change from a pure isotonic NaCl solution, but not from a pure isotonic sucrose solution, to an isotonic sucrose solution containing KCl in the concentration normally present in the Ringer’s solution. The presence of CaCl» in the first solution or in the second solution is of only secondary importance— e.g. because of its antagonistic action. Ca-paradox has been observed in the gangliou of the Limulus heart on changing from a Ca-free to a normal Ringer’s solution. The Ca-paradox differs, however, from the K-paradox in that the primary decrease affects only the amplitude and not the rate of the contractions ; while in the K-P both rate and amplitude are decreased before the final recovery. In the Ca-paradox also the pri- mary decrease in amplitude is greater, the longer the duration of immersion in the Ca-free Ring- er’s solution. The effect is obtained only on change from a Ca-free to a normal Ringer’s so- lution (not from a normal to a Ca-free Ringer’s solution). Thermo-paradox can also be obtained in the ganglion of the Limulus heart. Within a certain 342 THE COLLECTING NET [ Vot. VIII, No. 70 range of temperature (up to about 50°C.) the cardiac rhythm increases in rate as the tempera- ture rises. If the temperature is suddenly de- creased (e.g. from 40° to 24°C.), both the rate and the amplitude of the beat are temporarily de- creased, and in some cases the beat may even be completely arrested for a short period; the beat then returns to the normal rate for the particular temperature. The K-P and Ca-paradox are ob- tained only on change from lower to higher con- centrations of KCl or CaCls; in an analogous manner the thermo-paradox is obtained only on change from a high to a low, not from a low to a high, temperature. The fact that the same kind of paradoxical ef- fect (a primary and purely temporary de- crease in rate or amplitude, or in both) can be obtained under such different changes of ex- ternal condition suggests that some general or unitary physiological condition underlies the ef- fect. The nature of that condition is not clearly understood at present. (The work was partly aided by a scholarship from THE COLLECTING NET, for which the author wishes to express his gratitude). A RESPONSE OF ARBACIA EGGS TO X-RAYS Dr, P. S. HENSHAW AND Dr. D. S. FRANCIS Biophysical Laboratory, Memorial Hospital, New York, N. Y. The mode of action of radiation on organisms may be represented as taking place in four steps: 1) irradiation; 2) ionization; 3) chemical change; and 4) biological response. Radiant en- ergy as it moves from its source impinges on atoms and molecules which lie in its path. The result of the interaction of such energy and mat- ter, living or non-living, is that the atoms and molecules lose temporarily some electrons and thus become positive ions. The electrons liberated become attached to other atoms or groups of atoms which thereby beccme negative ions. The presence of such oppositely charged ions in a compound facilitates a regrouping of the atoms to form new compounds. All of this, (irradiation, ionization, and chemical change) takes place dur- ing treatment and is probably entirely compl-ted within a small fraction of a second after the end of irradiation. The biological responses observed are due either directly or indirectly to the chem- ical changes produced. From this it is apparent that one of the more important phases of an investigation of the ac- tion of radiation on organisms is a consideration of the nature of the chemical changes produced. This, however, is difficult for a number of rea- sons. Irradiation is the factor controlled and the biological response is the effect observed. But, such a procedure does not indicate how the chem- ical changes vary with the amount of treatment, nor does it indicate how many types of chemical changes combine to bring about the particular re- sponse under consideration. In order to investi- gate such changes it is necessary to find a re- sponse, preferably one which can be measured, which is closely connected with one of the initial changes produced by the radiation. Such a re- sponse has been found in the egg of 0.5. Similar situations are found for metallic elec- trodes in electrolytes where this type of reactance has been interpreted as due to a condenser-like element having a capacity which depends upon frequency. These polarization capacities usually have associated with them a polarization resis- tance which also changes with the frequency. In SEPTEMBER 9, 1933 ] THE COLLECTING NET 369 many cases, however, the ratio V,/R, remains constant over quite a frequency range, and since tan 6, = X,/R, the phase angle. ®, is constant (q. v. Fricke, 1932). When the condenser C of Fig. 3a. is replaced by a polarization element /, Fig. 3b, having the polarization resistance R, and reactance X, in series for instance, the formula- tion of Philippson has not been shown to be ap- plicable. Unless « = 1, it is probably safe to as- sume that polarization phenomena are present and it is to be expected that any value of « = 1 is not correct. Resistance and Reactance Measurements As has been stated, a two terminal electric net- work is completely characterized at any one fre- quency either by the impedence and the phase an- gle or equally well by a resistance and a react- ance. In general, the measured resistance and re- actance are functions of the frequency, and these two functions / (w) and X (w) contain all of the electrical information to be obtained from alter- nating current bridge measurements. The inter- pretations of these functions must be based large- ly upon assumptions as to the electric and geo- metric structure of material between the two elec- trodes. It seems reasonable to assume that the re- sistance of the internal and external electrolytes is low compared to the thin but highly resistant cell membrane, and, as a first approximation, that all of the capacity is located in the membrane. Single Cells The most direct method of attack is to measure the resistance and reactance between the interior and exterior of a single cell, or between two points on the exterior such that most of the cur- rent traverses the cell membrane twice. This has been done with the large plant cells, Valoma and Nitella by Blinks with alternating current (1926) and direct current (1929 a, b). The alternating current data will be mentioned later. The direct current measurements lead to values of capacity between 0.1 and 1.0 » F per cm.?, Suspensions On the other hand, quite a bit is known of the current and potential distribution in and around the cells of a suspension which is not too concen- trated. Considering the red blood cell as a spher- oid, Fricke (1924) has derived a generalization of the Maxwell formula for spheres which predicts the resistance of a suspension of red cells ex- tremely well. The membranes give a reactance component, and it is possible to calculate the ca- pacity to, be 0.8 » F per cm? of the cell membrane (Fricke, 1925a,b). Fricke and Morse (1925) then showed that a blood suspension could be rep- resented over a wide frequency range by the cir- cuit of Fig. 3c and found the corpuscle interior to have a resistance 3.5 times that of serum. On the other hand McClendon (1925) represented blood by the circuit of Fig, 3a. It should be em- phasized that these circuits can be made identical in both impedance and phase angle for all fre- quencies, so the choice between the two is to be made on the basis of convenience of computation and interpretation. Intuitive interpretation may be misleading as can be shown by an extension of the Maxwell equation for spheres to include the reactance of a high resistance surface layer (Cole 1928a). It is then found that the specific resis- tances of the medium and the cell interior enter into the expressions for both Rk, and Fk, (Fig. 3a). In the other circuit (Fig. 3c) both specific resistances enter into r;, but r2 involves only that of the medium. Calculations of the specific capacity of the surface from the values of C (Fig. 3a) and c (Fig. 3c) are quite different and partially account for the discrepancy between values given by McClendon and Fricke for the red blood cell. At present, the resistance and re- actance of suspensions of cells is the most power- ful method available for the study of the electric characteristics of living cells, and it is unfortunate it has been applied to only the red blood cell in a satisfactory manner. Tissues In most tissues, the individual cells are so ir- regular in shape and the spaces between cells are so small that it has not been possible to formulate the gross tissue resistance and reactance in terms of the electric and geometric characteristics of the cells. Since the underlying assumptions become invalid for high cell concentrations, the analysis of suspensions should not be applicable to tissues, but may, nevertheless, be taken as a guide. The work on suspensions seems to justify the assump- tion that the cell membranes are the only elements having electric characteristics which depend upon frequency. When all the cells of a suspension are identical, it is found that the total effect of all the cell membranes is equivalent to a single element such as p of Fig. 3b having an impedance which is the same function of the frequency as the im- pedance of the individual cell membranes.* It is then to be expected that in tissues, the variable impedance element is situated at the cell mem- brane, and that for tissues composed of a single cell type, these membrane impedances are equiv- alent to a single variable impedance element. The circuit of Fig. 3b may then represent the tissue, since any resistance network containing a single *—For the red blood cell this impedance is very nearly a pure reactance such as C of Fig. 3a but this is not generally true of tissues. THE COLLECTING NET [ Vor. VIII, No. 71 a7 variable impedance element may be made equiv- alent to this circuit. Interpretations of the resistances F;, Fk, should be made with caution, but it is still possible to draw some conclusions as to the membrane char- acteristics from a consideration of this circuit and the measurements of the series resistance and re- actance of the tissue R (m) and X (w) as func- tions of the frequency. Since the magnitude of the impedance Z = \/ k* + X? and its direction ®& = tan 'X/R, R and X may be considered as rectangular components of the impedance vector, so any pair of Rk and X at the same frequency de- termine the terminal point of the impedance vec- -tor as in Fig. 4. As the frequency is varied, this vector will change both in magnitude and direc- tion, and its terminal will trace some form of curve. If the element p behaves like a condenser in that its impedance is very high at low frequen- cies, then no current will pass through p, X (O) will be zero and the impedance will be a pure re- Fig. 4. Schematic circular are locus of the impedance vector Z of an entire tissue having a single variable impedance element of constant phase angle. sistance, kK, = R,; + R,. At high frequencies, the impedance of p will be so low that there will be no potential drop across it, X (0) will again be zero and the impedance will be R,, = Rz. The impedance locus will then have its end points at KR, and R,. In case p is a pure capac- ity, it is well known that the locus is a semi-circle of diameter , — R,. Now let p be a polariza- tion element consisting of a resistance R, and a reactance \’, both of which change with frequency in such a way that their phase angle ®, remains constant or tan 6, = X,/R,. For this assumption it has been shown that the impedance locus is a circular arc—less than a semi-circle since the center is below the R axis—and that ®, is the half angle between the radii to R, and R, (Cole, 1928 a, 1932). There are several ways in which the value of the phase angle may be computed analytically, such as those used by Lullies (1930) and Fricke (1932). These may be preferable in cases where a good value of R, can be obtained. The experiments of Gildemeister on human skin and frog skin have already been mentioned Fig. 5. tance 7 and reactance + in ohms. Circular are locus for potato. Resis- to indicate their historical importance. With hu- man skin Fk, is approached at a relatively low frequency, and measurements have never been made at a low enough frequency to go beyond the maximum of the arc. The data of Gildemeister (1919) Einthoven and Bijtel (1923) and Hoza- wa (1925) give ®, in the neighborhood of 55° for human* and frog skin on the high frequency portion of the curve. Blinks (1926) found a sim- ilar frequency situation for Valoma with ®, about 55°. There are measurements on other tissues which fall in a convenient frequency range and may be used to test the hypothesis of a single constant phase angle element (Cole, 1932). It may be stated in general that, in the low and in- termediate frequency ranges, the data are well represented by a circular arc, but that there may be deviations at the high frequency end. Fig. 5 shows data: for potato which fit quite well, while the data of Lullies (1930) in Fig. 6 show the greatest divergence of any so far. The effect at high frequency is found in several tissues and may be due either to a failure of the polarization phase angle to remain constant at high frequency or to the presence of another impedance element which is masked by the high polarization imped- ance at low and intermediate frequencies. We may conclude that for at least the major portion *—The recent data of Hozawa (1932a) give a value of 6, = 89° for human skin. Fig. 6. Circular are locus for nerve (Lul- lies). Resistance r and reactance + in ohms. SEPTEMBER 9, 1933 ] THE COLLECTING NET 371 of the frequency range, the variable impedance element of living tissues—which is probably the cell membrane—has a constant phase angle. The constant value of ®, are given below for several tissues. Tissue ®, Biopanerves (ullies)) 2. ctiaemier cerns 64° Rabbit muscle (Fricke).............. 65° BROGUSKIN 20+... aVeeNea yi Stic SE)e iumaneskin: (above))...22.+..sn.<.-.- 55° Gaerdiaphrasmigscc) sca, at eet are: ZN [OWE OT" Seat ie ca easier Ta eatery ictinetr een 64° LL OVPQOT LEO aes See, mere A chee Me Berets 78° aaronign (Blinks))\ierr.y. eile cee ieee 55)2 Polarization The classical polarization theories are those of Warburg (1899) and Nernst and Riesenfeld (1902) for reversible electrodes and diphasic sys- tems. The former gives a phase angle of 45°. The latter gives a counter e.m.f. proportional to the square root of the time of constant current flow. This demands a 45° phase angle for alter- nating current. The reversible electrode may be considered the equivalent of a membrane which is perfectly permeable to one ion of a binary elec- trolyte. The alternating current extension of the diphasic system has been worked out in some de- tail by Hozawa (1931, 1932, b). From the data which are available, it seems that insofar as the phase angle of the polarization elements are con- stant, they lie between 45° and 90° for biological systems. The frequency variations of capacity and resistance found for irreversible electrode-elec- trolyte systems and dielectrics are examples of polarization in non-living systems. Usually when it is possible to make any generalization, the phase angle is approximately constant indepen- dent of frequency. There are, however, no satis- factory theoretical explanations. Hozawa (1932 a) has interpreted his recent data on human skin in terms of a polarization of 45° phase angle and a static capacity in series. This accounts for the observed capacity very well, but does not give as satisfactory an explanation for the constant phase angle of nearly 90° ob- served above 1000 cycles. Transient Conductance of Skin. Hozawa The direct current flow in human skin at very short times after the application of a constant po- tential was measured accurately with a Helmholtz pendulum by Hozawa (1928a). The assumption that there was no polarization—except that of a static capacity—explained the data very well as an exponential curve up to 50 micro-seconds*, and the progressive divergence after that time was at- tributed to diffusion polarization. These data then gave values of the capacity C and resistances R, AR, of circuit Fig. 3a. C = 0.02 p F, R, = 15,000 to 116,000 ohms per cm?. of skin area, and R, = 400 ohms. In the succeeding paper, Hozawa (1928 b) gave measurements of the damped oscillatory cur- rent when an inductance was included in the cir- cuit and a constant potential applied. From the inductance and the observed frequency, the value of the capacity C could be computed. It agreed with the non-oscillatory value and was indepen- dent of the frequency in the range measured. This was extremely difficult to understand when compared with earlier alternating current bridge measurements where the capacity decreased with frequency and the phase angle was between 50° and 60°. The Fourier Integral analysis shows that an element which gives an exponential time relation for the current in a dissipative circuit with a constant potential applied, must give a capacity which is independent of the measuring frequency. Furthermore, an element having a constant phase angle, different from 90°, can not give an exponential direct current transient. The recent alternating current bridge data of Hozawa (1932a) give a phase angle of almost 90° in agreement with the transient observations. This suggests that the earlier bridge measurements may have had some unrecognized source of er- ror, and one is inclined to suspect that the meas- uring current may have been too large. Conclusion It was suggested at the beginning of this paper that electric conductance might be expected to furnish a clue to the mechanism of cell membrane phenomena. This it has so far failed to do. The alternating current conductance at low frequen- cies and the steady direct current conductance are found to be indices of the physiological condition of the cells. The high frequency alternating cur- rent conductance and the initial direct current conductance are comparatively independent of the physiological condition, and show that the cell in- terior has a comparatively high conductance. It may then be said that the cell membrane has a very low conductance which changes as the permeability of the membrane is altered. Although electric conductance is a_ useful empirical tool, it has not yet been possible to formulate it adequately in terms of membrane structure and mechanism. The experimental ev- idence at present indicates that the electrical be- *—When one postulates a constant but unknown polarization in addition to a static capacity, a time interval of 100 micro-seconds may be accounted for. 372 THE COLLECTING NET [ Vou. VIII, No. 71 haviors of cell membranes are very similar to those of the polarizations in solid dielectrics and at irreversible electrcde surfaces—which are also unexplained. BIBLIOGRAPHY Bernstein, J., 1902, Arch. ges. Physiol., 92, 521. 1912,Elektrobiologie. Braunschweig. 1926, Harvard Thesis, Cambridge. Blinks, L. R., 1929 a, J. Gen. Physiol., 13, 361. b, ibid. 13, 495. Bronk, D. W. and Gesell, R., 1926, Am. J. Physioh, 76, 179. Cole, K. S., 1928 a, J. Gen. Physiol., 12, 29. b, ibid. 12, 37. 1932 ibid. 15, 641. Crile, G. W., Hosmer, H. R. and Rowland, A. F., 1922, Am. J. Physiol., 59, 458. Dixon, H. H., 1924, Proc. Roy. Dublin Soc., 17, 3494 Ebbecke, U. and Hecht, G., 1923, Arch. ges. Physiol., 199, 88. Einthoven, W. and Bijtel, J., Physiol., 198, 464. Fricke, H., 1924 Phys. Rev., 24, 575. 1925 a, ibid. 26, 678. 1925, b, J. Gen. Physiol., 9, 137. 1931, Physics, 1, 106. 1932, Phil. Mag., (7) 14, 310. Fricke, H. and Morse, S., 1925, J. Gen. Physiol., 9, 153. Gildemeister, M., 1919, Arch. ges. Physiol., 176, 84. Gray, J., 1913, J. Marine Biol. Assoc. United King- dom (N. 8S.) 10, 50. 1916, Trans, Roy. Soc. London, B 207, 481. Green, R. G. and Larson, W. P., 1922, J. Infect. Dis., 1923, Arch. ges. 30, 550. Hober, R., 1910, Arch. ges. Physiol., 133, 237. 1912, ibid. 148, 189. 1913, ibid. 150, 15. Hozawa, S., 1925, J. Biophysics, 1, 185. 1918, a Arch. ges. Physiol., 219, 111. ; ibid. 219, 141. 1931, Z. Biol. 91, 297. 1932, a, ibid. 92, 209. b, ibid. 92, 373. Luyet, B. J., 1932, J. Gen. Physiol. 15, 283. Lullies, H., 1928, Arch. ges. Physiol., 221, 296. 1930, ibid. 225, 69. McClendon, J. F., 1910, Am. J. Physiol., 27, 250. 1912, ibid. 29, 302. 1927, Protoplasma, 3, 71. W. and Reisenfeld, E. H., 1902, Ann. d. Physik., (4) 8, 600. Osterhout, W. J. V., 1922. Injury, Recovery and Death in Relation to Conductivity and Permeability, Philadelphia. Peserico, E., 1926, Atti. Accad. Lincei, 3, 346 and Nernst, 615. Philippson, M., 1920, Compt. rend. Soc. biol., 83, 1399. 1921, Bull. Acad. roy. Belgique, Cl. Sc., 7, 387. Sapegno, E., 1930, Arch. ges. Physiol., 224, 187. Sen, B., 1923, Proc. Roy. Soc., B. 94, 216. Shearer, C., 1919, J. Hygiene, 18, 337. Warburg, E., 1899, Ann. d. Physik., 67, 493. Waterman, N., 1922, Biochem. Z., 133, 535. 1923, Z. f. Krebsforsch., 20, 375. Zoond, A., 1927, J. Bact., 14, 279. GENERAL REFERENCES Gellhorn, E., 1929, Das Permeabilitatsproblem, Ber- lin. Gildemeister, M., 1928, Die passiv-elektrischen Ers- cheinungen, Bethes Handb. d. Physiol., VIII, 12, p. 657. HO6ber, R., 1926, Physik. Chemie der Zelle und der Gewebe, 6. Aufl. Kap. 8, 9, 10 and 12 Leipzig. Osterhout, W. J. V., 1922, Injury, Recovery and Death in relation to Conductivity and Permeabil- ity, Philadelphia. Strohl, A., 1925, La conductibilité electrique du corps humain, Paris. Discussion Dr. Blinks: 1 might add to what Dr. Cole has reviewed of the work on tissues and cell sus- pensions, a few words on the same type of meas- urements with the large plant cells Valonia and Nitella. It was early recognized that such meas- urements should be made on these cells, and they proved to have certain advantages as well as dis- advantages. The advantage lies chiefly in the large area with only two surfaces in series, so that the actual capacity is extremely large—up to 0.1 or 1.0 microfarad. ‘his causes the complete change of impedance described by Dr. Cole to fall in a fairly low frequency range, usually below 20,000 cycles, which has the technical advantage of keep- ing the measurements largely in the audio range. In general the same results have been found as Dr. Cole indicated for most plant and animal tis- sues except erythrocytes, namely, a decrease of the capacity itself with frequency, again less rap- idly than the square root relation given by revers- ible electrodes, and a similar, though more rapid fall, of the associated series resistance, with in- creased frequency. This so resembles the phenom- ena at an indifferent electrode such as platinum in KCl, that we call it a polarization capacity and resistance. But when we attempt to describe these time or frequency relations more accurately than by this rough characterization, we find a variety of diffi- culties, some peculiar to the organisms employed, others perhaps more widespread than is generally realized. One of these latter difficulties is implicit in the electrical circuit itself. The Maxwell form- ula for the relations of cell volume and surface to the outside solution seems to take care of this for suspensions: its best justification is the fact that Dr. Fricke finds a nearly constant capacity for the surface of erythrocytes. But for the close- ly packed, non-spherical cells of tissues, as well as for Nitella and Valonia where electrical con- tacts are made at the ends_ or other localized re- gions, we must consider also the capacity between the exterior and cell interior across the proto- plasm along the cells, not perpendicular but par- SEPTEMBER 9, 1933 iL THE COLLECTING NET 373 allel to the general axis of current flow. This may occur not only between the electrode-contact re- gions, but in some cases, as in nerve, and Nitella, under the contacts themselves. This is in essence the problem of “distributed” capacity, met with in telephone and other cables, where it has been mathematically solved by Kennelly. Both theoret- ically and experimentally such distributed capac- ity can give rise to frequency changes in the equivalent “lumped” capacity usually assumed for the circuit, in a manner so like those at a polar- izing electrode that we must know pretty accur- ately the constants of the circuit before deciding which factor, polarization, or distribution, or both, are involved, and to what extent. I think this can be solved when we can keep some of the other things involved in the living protoplasm constant. Here we run into those “active” effects of the electrical current, as distinguished from the “‘pas- sive” ones we always hope to maintain during the measurement. In other words the current “does something”’ to the protoplasm when it pass- es across it. Valoma and Nitella, as normally em- ployed in experiments, are in such condition that two opposite effects of current flow are beauti- fully exemplified in them. An impaled cell of Valoma has a small negative potential and polar- izes only slightly to small currents, or even to very large currents passing outward across the protoplasm. But to increased currents passing in- ward, there is a striking response, the potential going through an S-shaped reversal curve, and reaching values of 200 my. positive. It then pol- arizes quickly and regularly to increments or de- crements of this current, and remains polarizable for a short, variable period after the current has ceased to flow. This seems to be a ‘restorative’ effect of current flow, if we consider the cell in a normally “stimulated” state. On the contrary, Nitella has normally a very high positive potential (up to 200 my.) and pol- arizes promptly and regularly to small currents in either direction, or even to very large ones pass- ing inward. But at the break of large inward cur- rents, or at the make of rather small outward cur- rents, the cell becomes stimulated, the potential drops to nearly zero, and the polarizability temp- orarily disappears. Alternating currents of suff- cient intensity can also cause this stimulation, so we must work with very low currents. During the past year methods have been found for “restoring” Valonia to polarizability, and maintaining it in that state, so that capacity measurements of impaled cells could be made, thereby avoiding the difficulties due to distributed capacity. Other difficulties due to the high resis- tance of the inserted capillary still exist, so that the problem is not solved. Osterhout and Hill have also found means for greatly decreasing the sensitivity of Nitella to electrical stimulation; I hope to employ such insensitive cells for capacity measurements. One other disturbing point brought out by the direct current study of capacity is that the charg- ing curve of the protoplasm has in many cases form, as well as speed, very different from the discharge. This would necessarily be also reflected in the A. C. measurements to some extent, and might give very strange frequency relations. Dr. Cole: The possibility of distributed ca- pacity and resistance must certainly be considered in all cases. These effects are, of course, present in the case of a suspended spher ical cell, ‘but they do not put in theoretical appearance in the un- pleasant ways found for even the simplest cable problem, The impedance of suspended Arbacia eggs is then at least partial support for the belief that the membrane of itself has polarization characteristics. Fricke has found that the nearly constant capacity of red cells as found in suspen- sions is practically unchanged when the cells are centrifuged down to a cencentration quite com- parable to that of cells in tissues. This may be taken as a further indication that the frequency characteristics of tissues are due more to polariza- tion membranes than to distributed effects. Nitella and nerve ,on the other hand, should be expected to show more of the characteristics of cables. It may be possible that these difficulties are somewhat avoided by suspending in air and making the electrode regions of short length. The Nitella cell wall is imbibed with tap water and not an especially good conductor so that most of the current will cross the membrane and travel through the cell interior. With short electrode re- gions the potential drop along the interior under the electrode would then be small compared to the potential drop across the protoplasm and distrib- uted effects should be small. Nerve is more com- plicated because of the presence of intercellular electrolytes of high conductivity. Lullies data give a series of complex plane circular arcs for differ- ent lengths of nerve which are practically identi- cal for lengths greater than 8 mm. except for a displacement along the resistance axis. This indi- cates that there is no current flow across mem- branes more than 4 mm. from an electrode re- gion, An analysis similar to that of Rushton should give the current paths in the electrode re- gion when the latter is long, and show what might be gained by the use of a short electrode region. It should be pointed out that distributed capac- ity and resistance must of necessity enter in ex- actly the same manner whether alternating or di- rect current is used. The difference of charge and discharge curves uggests a rather complicated mechanism, for, as 374 THE COLLECTING NET [ Vou. VIII, No. 71 Kelvin said, “The charges come out of the glass in the inverse order in which they go in.” Dr. Fricke: 1 may mention some measure- ments on collodion membranes which I think may prove to be quite significant. A collodion mem- brane behaves very much like a cell membrane in that the current passing through it is strongly polarized. The polarization appears to be derived from the aqueous phase of the membrane, as ev- idenced by the changes produced when the mem- brane is placed in different salt solutions. In one case there may be a polarization of the type ob- served for the red corpuscle membrane with a very small frequency dependence of the capac- itance. In another case, the polarization may be of the type found in tissues or at metal electrodes. At very high frequencies, the capacitance is in- dependent of the salt solution and considered as the ordinary static capacitance of the membrane. This gives a dielectric constant of about 5, which is within the range of values found for the dielec- tric constant of cellulose. Dr. Cole: Experiments with artificial mem- branes are extremely important. An attempt was made several years ago to find changes in conduc- tance of frog skin as the selective permeability was reversed by altering the pH of the salt solu- tion, but the measurements were not reproducible. More recently, a start was made on protein coat- ed glass and collodion membranes for the same purpose, but the work has been delayed. The wax cuticle of the onion membrane has been found to give a constant phase angle, and is known to show a selective permeability. The non-aqueous liquid artificial membranes also need investiga- tion. Dr. Kornhauser: Do skeletal and smooth muscle show the same conductance phenomena ? There has been so much disagreement on wheth- er or not striated muscle really has true discs of Krause through the fibrils and sarcoplasm. Dr. Cole: Ido not know of any conductance data taken parallel and transverse to smooth muscle fibers. Sapegno’s impedance data parallel to the fibers show polarization elements effective- ly normal to the current flow, but the frequency characteristics are the same as for the transverse measurements. This suggests that the fiber in- terior may be electrically isotropic and that the polarization observed is due to membranes which are not effectively parallel to the current flow. Dr. Kornhauser: Then that would seem to in- dicate that there are no membranes across each minute distance between the middle of the iso- tropic bounds where Krause’s membranes are lo- cated. Half way in the singly refractive band of each fibril there is a distinct disc shown in stained preparations. Dr. Cole: I would think it probable that the discs are at least not electrically differentiated. SEPTEMBER 9, 1933 | THE GCOLEECTING, NET 375 PHAGOCYTOSIS Stuart Mupp Phagocytosis* may be defined as the ingestion of a particle by a living cell. The particle may be non-living, or may be a bacterium or other cell, to whose death and digestion phagocytosis may or may not lead. In the present discussion, we shall be concerned merely with the act of ingestion. For historical treatment of phagocytosis and for discussion of many aspects not here dealt with reference is made to the classical monographs of Metchnikoff" *) and to more recent reviews by Ernst’), Marchand‘), Hamburger, Neu- feld™, Neufeld and Loewenthal'*’, Leding- ham®), Muir?®, Gray@), Fleishmann“?) and Hirschfeld), The phenomenon of phagocytosis has been traced by Metchnikoff through different groups of animals from protozoa to man. He pointed out that in amoeba and similar protozoa phagocyto- sis of bacteria and other organic material is the usual way of obtaining food. After ingestion the engulfed particles are either digested by cytoplas- mic enzymes or extruded. In the lower metazoa, phagocytosis and intracellular digestion still play the principal role in the nutrition of the animal. Here cells, living in the body cavity, phagocytize and digest the food material. With greater spe- cialization this primitive method of digestion gives way to the more complicated extracellular digestion in specialized hollow organs, such as the gastrointestinal tract. There remain in all metazoa, however, motile cells which have retained the primitive power of phagocytosis and intracel- lular digestion. In the higher vertebrates two principal categories of phagocytic cells may be distinguished, the polymorphonuclear leucocytes or microphages, and the macrophages or cells of the so-called reticulo-endothelial system™*’. But Lubarsch") and others‘! have stated that even in the higher vertebrates nearly all types of cells may, under some conditions, act as phagocytes. Phagocytosis by the microphages and macro- phages of vertebrates has been extensively studied both in vivo and in vitro. Various kinds of par- ticles such as carmine, carbon, erythrocytes and other animal cells, and living or dead bacteria have been introduced by various routes (intra- venously, subcutaneously, by injections into the serous cavities) into the intact animal. The fate of the injected particles has then been studied by making direct smears from exudates of organs, or by histological methods. * In preparing this lecture the writer has drawn upon an article for “Physiological Reviews” in pre- paration by Stuart Mudd, Morton McCutcheon and Balduin Lucké. In vitro methods lend themselves to more quan- titative treatment. Phagocytic cells, usually ob- tained from the blood or from exudates, are brought into contact with the test particles under various controlled experimental conditions. Smears are then made and stained, and the amount of phagocytosis determined either as the mean num- ber of particles ingested per leucocyte, or as the percentage of leucocytes which have ingested par- ticles under the given experimental conditions. Still another procedure was elaborated by Fenn (7) In a phagocytic system with known numbers of cells and test-particles, Fenn determined the number of uningested particles as a function of time. In this lecture attention will be confined to the two principal types of mammalian phagocytes, the polymorphonuclear leucocytes, and the ma- crophages or large mononuclear phagocytes. These cells are a principal factor in the interception and removal of any parasites or other foreign material which may reach the tissues or circulating fluids, and, also, in the removal of any débris resulting from tissue injury or tissue replacement. In attempting to analyse the mechanism of pha- gocytosis, it will be convenient also to restrict at- tention to phagocytosis in vitro. Consider the process in two stages: first, the cell and particle come into contact ; second, the cell ingests the par- ticle. The Probability of Contact Between Phagocyte and Particle The first stage is best studied by simplifying the system as much as possible; this is done by suspending cells and particles in some liquid con- tained in tubes, which are slowly rotated. Under these circumstances it is evident that contact be- tween cell and particle occurs purely by chance. Chemotropism is not involved since leucocytes are capable of locomotion-only when attached to a solid surface ; in other words they can crawl, but not swim.* Under these simplified conditions it might be expected that the amount of phagocytosis would be proportional to the chances of collision be- tween cell and particle, such was found by Fenn to be the case'7 19, 20), With a system consisting of rat leucocytes, quartz or carbon particles of known size and a suspending medium of isotonic NaCl solution and serum, he found that the rate of phagocytosis was * Philipsborn finds some evidence to the contrary; see page 157 of his review on locomotion of leuco- cytes (18), 376 THE COLLECTING NET [ Vor. VIII, No. 71 proportional to the number of uningested parti- cles. This relation is what one would expect at the beginning of an experiment, but that it would still hold after leucocytes had already ingested particles is interesting; it depends on a type of behavior previously found experimentally by Mc- Kendrick'*"), i. e., that there is no decrease in the ease with which a cell engulfs one small particle after another. Observations of our own indicate that this principle is only true within limits; we have observed phagocytes so full of erythrocytes or Monilia cells that further phagocytosis was in- hibited ‘?). Fenn showed in two additional ways that pha- gocytosis is ordinarily proportional to the chances of collision. It is evident that the chances of col- lision depend on the size of particle and of cell— that is, according to Fenn’s formulation, on the square of the sum of the diameters of cell and particle. The size of both cells and particles being known, it was possible to test this hypothesis by using particles of different size, and the relation was found to hold 1%, Secondly, Fenn showed that the chances of col- lision depend on the relative velocities of cell and particle in the rotating tubes. These velocities can be increased or decreased at will by varying the speed of rotation; for when the tube is slowly rotated there is a relatively long time for the ob- ject to fall under the influence of gravity, before completion of rotation brings it back to its origi- nal position; whereas rapid rotation allows it less time to fall, so that it moves fewer millimeters per minute, until under very rapid rotation its rate of motion becomes practically zero. Evi- dently when both cell and particle have zero ve- locity there is the least chance for collision be- tween them—theoretically no chance, while there is the greatest chance for collision during slow rotation. These predictions were confirmed ex- perimentally"”, Thus it was shown that the rate of phagocyto- sis is proportional to the chances of collision, But this is true, of course, only under constant condi- tions. Thus, change in the kind of test object greatly changes the rate of phagocytosis. Such a difference was shown by Fenn to exist between quartz and carbon: under certain conditions car- bon was phagocytized 4 times as readily as quartz (20) And this occurs even though the numbers of collisions be equal, as was demonstrated by Fenn with another method. In this, the suspension was run under a coverslip onto a slide, and observed under the microscope. In one such experiment in a certain microscopic field in 24 minutes, 36 con- tacts between leucocytes and quartz particles were observed and 37 between leucocytes and carbon particles; yet at the end of the period only 1 quartz particle had been ingested as compared with 12 carbon particles'**). Serum was present in Fenn’s phagocytic mixtures, and Fenn states that little phagocytosis of either carbon or quartz occurred without serum. The differences between carbon and quartz are probably referable, there- fore, chiefly to differences in the ability of carbon and quartz to adsorb phagocytosis-promoting sub- stances from the serum under the given experi- mental conditions. With this method, called by Fenn the “film” method, contact between cell and particle results not from motion induced by gravity, as in the preceding experiments, but from amoeboid mo- tion, the cells crawling about, coming in contact with particles which they then may or may not ingest. Under these conditions a complicating factor may be introduced, chemical attraction of particle for cell. This was not the case with par- ticles of quartz and carbon; with these, contacts appeared to be purely by chance. When, how- ever, particles of MnOs. and of MnSiOs3 were used, it was evident by direct observation that the collisions were no longer fortuitous. Though equal numbers of the two kinds of particles were present, 2.4 times as many encounters occurred with MnO. as with MnSiOg; this resulted in 20 times as many MnO, particles being ingested as MnSiO3. There was evidently definite chemical attraction exerted by the MnOz particles, a con- clusion which Fenn confirmed by observing that leucocytes frequently advanced directly toward these particles instead of exhibiting the usual ran- dom movements. The chemical attraction he sup- posed to depend on the fact that MnO, is soluble in water, though only slightly so). Similarly Commandon'**) was able to demonstrate by moy- ing pictures that leucocytes are attracted toward starch grains which are subsequently ingested. Electrokinetic Potential and Surface Charge An obvious question in considering the ap- proach and contact of leucocyte and particle in vitro is that of the effect of their electrokinetic potentials.* It is not to be anticipated that ¢-po- tentials would have the same critical importance in phagocytosis as, for instance, in phenomena of aggregation of hydrophobic colloids. One reason for this is that the opportunities for collision of colloidal particles are ordinarily afforded by forces of a low order, namely Brownian motion, whereas the contact of leucocyte and particle in experiments in vitro are afforded either by me- chanical agitation, or by the locomotion of the leucocytes. Another reason is that the ¢-poten- tials of leucocytes are relatively low; Abramson * Discussion of these factors in vivo has been given by Abramson in a previous lecture of this series, SEPTEMBER 9, 1933 ] THE COLLECTING NET 377 gives for horse polymorphonuclear leucocytes in serum the value of approximately 12.5 millivolts (25) The attempt to determine from experimental data the effect of ¢-potential on phagocytosis is rendered difficult by the fact that the conditions which have modified the ¢-potentials have often simultaneously modified also the interfacial free energies, upon which phagocytosis is more direct- ly dependent. Cases in point are the promotion of phagocytosis by treatment of bacteria with tan- nin'®), or with serum'*"), Moreover, influences which have been attributed to the direct effects of ions on phagocyte or particle have often not taken into account the effects of such ions on the ad- sorption of serum proteins on the bacteria. One interesting set of experiments in which such disturbing effects seem to have been reduced to a minimum, is that of Neufeld and Etinger- Tulezynska‘**). In these, leucocytes washed and resuspended in isotonic NaCl solution were used. The bacteria were grown on solid media, and were washed and resuspended in distilled water. To these suspensions dilute electrolyte solutions were added. Solutions of salts of polyvalent ca- tions in certain ranges of concentrations both ag- glutinated and caused phagocytosis of a strain of virulent pneumococci and of a typhoid bacillus. The minimal concentrations able to bring about agglutination and phagocytosis were approximate- ly the following: Th(SO,4)s, N/2000; “aluminum alum,” 1:5000; Alo(SO,4)3.18H20, N/2000; AICls. 6H.O, N7/1000; Als( NO3)¢.18Hs0, N/2000; Fes ( SO, )3. 9 H.0O, N/500 ; Cre ( SO, )g, N/500; CeCls, N/1000; Pb( NO3)2, N/2000. Di-divalent salts were not found to be effective. Although ¢-potentials were not determined, these experiments were interpreted as being due to re- duction of electrokinetic potentials by these salts. Numerous parallel determinations of ¢-potential and phagocytosis have been made by Mudd, Lucké, McCutcheon and Strumia. A number of acid-fast bacteria'*® °°), and collodion particles with proteins adsorbed on them‘), were exam- ined in 0.85% NaCl solution, and also after treat- ment with serial dilutions of specific immune sera. It was found that in general the highest ¢-poten- tial and the least phagocytosis occurred in the pure NaCl solution, and conversely the lowest ¢-potential and greatest phagocytosis occurred after treatment with the highest concentrations of serum. The opposite relation between ¢-potential and phagocytosis is illustrated in experiments with ty- phoid bacilli'**). These bacteria (in their “smooth” form) have only a minimal ¢-potential?) ; in the presence of homologous immune serum they ac- quire a negative ¢-potential. Yet sensitization greatly promotes phagocytosis. These correla- tions, both direct and inverse, between -potential and phagocytosis we believe to be accidental and dependent upon the fact that sensitization involves the deposition of a film of antibody-globulin on the bacterial surface. That €potential is not the decisive factor in determining phagocytosis appears also when we consider different acid-fast bacteria in NaCl solu- tion, and attempt to correlate their ¢-potential and ingestion. No correlation is found. Thus in absence of serum, virulent human mammalian tubercle baccilli were freely phagocytized although they have a relatively high ¢-potential, while, con- versely, M. avium (Prague strain) with a rela- tively low ¢-potential was not spontaneously pha- gocytized'*7 2%), Other bacteria might be selected to show the opposite relation. The subject of the influence of surface charge (not to be confused with ¢-potential of which it is a complex function) on the phagocytosis of a particle has been treated by Ponder'**), who has elaborated Gyemandt’s equations, for the effect of surface tension and charge on the contact of two like particles, to cover the case of the in- gestion of a particle by a cell. The results are exceedingly complicated, and the equations are experimentally unverifiable, but three interesting conclusions are arrived at: (1) Only large surface charges at the cell-fluid and particle-fluid interfaces can in- fluence the ingestion. Very large charges at these interfaces will prevent ingestion. (2) Ifa very large charge occurs at the cell-particle interface ingestion may be pre- vented. (3) The effects on ingestion of the charges at these three interfaces vary with the radius of the cell, the radius of the parti- cle, and the surface tension at the interfaces, in such a complex way that the effects of surface charge on phagocytosis is probably comparatively small. Interfacial Tension Relations It was first shown by Fenn‘**) that the same formulation of the interplay of surface forces in phagocytosis may be reached from considerations of surface tension or of free surface energy. We shall use the. former as simpler. Let text-figure 1 be a section through suspending medium, phago- cyte and partially ingested particle. Let O be a representative point in the line of contact between the three phases; let the vectors S;, Se and Sie be the interfacial tensions, respectively, in the particle-fluid, phagocyte-fluid, and phagocyte- particle interfaces. THE COLLECTING NET [ Vor. VIII, No. 71 Text-Fig. 1 If Si > Sie + Se the surface of the phagocyte would be drawn completely around the particle and ingestion would occur, provided viscosity, or other forces, did not interfere with the action of the surface forces. If Si2 > S; + So neither ingestion nor adhesion of particle and phagocyte would occur under the action of surface torces. When S; < Sy + So and Sie < S; + So, the surface forces are in equilibrium with the particle in a position of partial ingestion, as shown in the figure; the position taken by the particle at equili- brium is such that S; = Siz + S»e.cos 6, or Si— Sie cos 96 = ————_. Se The three main possibilities have been ex- pressed by Ponder in terms of the value of (Si — Sie)/Se as follows: (Si — Si2)/Se is less than or equal to (—1). The cells will not ingest the particle under these circumstances, for the particle will either stay in equilibrium at the cell surface, or, if its physical nature permits, will flow over the cell. (Si — Sie)/Se is greater than (—1) and less than (+1). In this case there will be a real value of @, and there will be equilibrium at in- complete ingestion, i. €., when the particle is only partly inside the cell. (Si — Sie)/S2 is equal to or greater than (+1). In this case, given sufficient time, the cell will completely ingest the particle. It follows immediately from these considera- tions that the probability of phagocytosis is fav- ored by increase in the value of the particle-sus- pending medium interfacial tension (S;), and by decrease in the particle-phagocyte interfacial tension (Si2). Phagocytosis in the body is pro- moted by the deposition on the particle surface of specific and non-specific serum proteins, and such serum sensitization is also the most reliable and powerful means yet discovered of promoting phagocytosis in vitro. The action of sensitizing serum in forming on bacteria a surface deposit of serum-globulin is to give them surfaces presum- ably with high interfacial tension against the me- dium (as evidenced by the hydrophobic aggrega- tion behavior of sensitized bacteria) ,‘**) and pre- sumably with low interfacial tension against the leucocyte. Unfortunately none of the three interfacial tensions in the phagocyte-particle-liquid system can be directly measured. We are forced to make such inferences as we can from analogy, and from indirect evidence. In the case of the eggs of certain marine in- vertebrates K. S. Cole’) and Harvey‘®) have been able by independent methods to assign an upper limit to the protoplasm-suspending medium interfacial tension. The maximum values for the tensions at the surface of eggs studied by Har- vey by the microscope-centrifuge method were: Unfertilized egg of Arbacia punctulata, 0.2 dynes per cm. Unfertilized egg of the annelid Chaeptopterus, 0.33 dynes per cm. Egg of the mollusk Cumingia, 0.54 dynes per cm. Fertilized egg of the mollusk //lyanassa, 1.1 dynes per cm. Dr. Cole by computation from observations of unfertilized Arbacia eggs under compression, reached a value of 0.08 dynes per cm.—a _ re- markable agreement with the result of Harvey. He concluded: “It thus seems reasonable to sup- pose that there are no capillary forces acting, and that the initial pressure and tension are due to an initial stretch of this elastic membrane.” To regard such results as affording more than a suggestive analogy with the phagocyte-plasma interface would, of course, be unwarranted. There are however, independent reasons for be- lieving that the tension at the phagocyte-liquid in- terface is low. In the blood stream leucocytes are typically spheroidal. However direct observation of these cells, when spread out on a surface, shows that their surfaces are highly mobile; the mononuclear phagocytes often project and retract into the medium in response to internal stimuli, delicate veil-like pseudopods; the polymorpho- nuclear leucocytes in their locomotion often leave long thread-like processes attached to the surface on which they are crawling‘); these may pull loose and be retracted into the cell. The even more elaborate development of these “pseu- dopodes pétaloids”’ in the amoebocytes of inverte- brates has been described by Fauré-Fremiet®). It is interesting to compute the amount of work involved in the extension and retraction of such processes. The phagocytes of mammals and the amoebocytes of invertebrates can both pass re- IBER 9, 1933 } 8 +++ y + iM & s Q N +H mae q ae Q 2 Ree +++ « N Ww + S$ « 0 8 (2) YW. 0 Sg 80 cy Ne At) FHAECEY TOSS g Text-Vig. 2. washed. fractionation. THEE JLLECTING 379 PSEUDOGLOBULIN ALBUMIN & * S< fc/| s 2 JS Nay NET Parallelism between surface changes and phagocytosis after sensitization with globu- lin fractions of immune serum. M. avium (Arloing strain) sensitized with serum fractions and then Open circles: fractions from Anti-Arloing serum heated for 30 minutes at 56°C. before Black circles: fractions from same Anti-Arloing serum unheated. Ordinates are the intensities of the several reactions. Abscissae are successive dilutions of antiserum fractions expressed in powers of 4. versibly from the spheroidal form to the form in which surface processes are extended. According to the computations of Fauré-Fremiet, the amoe- bocytes of Asterias equivalent to 1 gram dry weight, in the condition of maximum extension have an aggregate surface area of 21.41 sq. meters; their surface in the form of minimal area has been computed as 3.03 sq. meters. Let us assume that some metabolic process provides to these extended processes sufficient surface energy to cause their retraction into the cell. The loss of surface area is then 21.41 — 3.03 = 18.38 sq. meters = 1.838 x 10° sq. cm. The work (W) required to increase the energy at the protoplasm-liquid interface by 1 erg per em.” is thus 1.838 x 10° ergs. 1 1 erg = —— x 10” gram-calories. 4.18 esetet OY We oe SS Se 108 418 10° (Thus 3 is a dilution of 1:43 or 1:64.) calories per erg/em?. per gram dry weight of amoebocytes. Warburg'*! gives the energy liberated by sur- viving carcinoma tissue as 0.04 calories per milli- gram dry weight per hour, Fleishmann and Ku- bowitz, using Warburg's technique''”) obtained for rabbit exudative leucocytes 0.021 calories per milligram dry weight per hour, The latter figure is equivalent to 21/60 = .35 calories per gram per minute. Were this energy transformed wholly into free surface energy it would thus be sufficient to in- crease in one minute the surface energy over an area equivalent to that of the maximally extend- . 8 ed amoebocyte cells by —— x 10% = 80 ergs per 4.4 sq. centimeter. It is thus clear that the energy made available in metabolism is far in excess of that required in the reversible extension of protoplasmic surface pre CESSES, » DHE COLLECHING NEG Ss FVoL. Vliet: Reprinted from J. Gen. Physiol. 1933, 16, 635 PLATE 1 SEPTEMBER 9, 1933 ] Experimental Analysis and Direct Observation of Phagocytosis It remains to consider how far the formulation of phagocytosis as primarily determined by inter- facial tension relations at the particle-cell-liquid interfaces has heen verified by experimental an- alysis and by direct observation. Three deduc- tions may be examined. These are (1) that a quantitative correlation should exist between phagocytosis and the surface properties of the particles ingested; (2) that phagocytosis is es- sentially a phenomenon of spreading of the phagocyte surface over the surface ingested ; and (3) that partial ingestion should occur under cer- tain circumstances. Correlation Between Phagocytosis and Surface Properties. The first obvious deduction from a theory which assigns to surface forces a principal part in phagocytosis is that phagocytosis should be related, in some orderly way, with the surface properties of the particles phagocytized. This re- lation has been verified over a very considerable range of experimental conditions, (7% 7% 0 31), Various bacteria, erythreytes, and protein adsorbed on collodion particles have been treated with graded concentrations of the phagocytosis-pro- moting substances of sera. The é-potentials, iso- electric points, wetting properties, and cohesive- ness of such series of sensitized particles have heen estimated in independent tests, and the phagocytosis of the particles has been quantitative- ly determined, using mammalian phagocytes both of the polymorphonuclear and large mononucl) of the leucocyte which contains one partially ingested erythrocyte continues to migrate toward the lower right hand corner of the field until the prot-plasm of the leucocytes is stretched into a thin filament. While under direct obserya- tion the adherent er threcytes in the upper left hand corner were pulled loose frem the glass and the protoplasmic filament contracted. Successive stages in ingestion sly sensitized sheep erythro- e; the macrophage contains [ Vor. VIII, No. 71 16, 635 oo ise) a ri gs n > i A a o a) 5 2 3 mu HoH s Ey YLLECTING Reprint (CX THE SEPTEMBER 9, 1933 ] THE COLLECTING NET 383 films some leucocytes were- freely suspended, some were spread out on the slide, and some on the cover-slip; only rarely was a single cell in contact both with slide and cover-slip. The prep- arations were put immediately under microscopic observation in a warm-box kept near 37°C. Particles Phagocytized. Suspensions of washed sheep and washed chicken erythrocytes, Bacterium typhosum, Bacillus subtilis, and Monilia albicans were used. Specific rabbit antisera were prepared for the sensitization of each type of cell. For ob- servation and photography of the bacteria with the brightfield, they were first stained with car- bolfuchsin and then washed four to five times. Erythrocytes and monilia were not stained. With the dark-field no staining was necessary. Optical Apparatus. For transmitted light, Zeiss aplanatic N. A. 1.4 condenser. For dark- field, Zeiss cardioid condenser. Zeiss apochro- matic 60 x objective with iris diaphragm. Zeiss 20 x compensating ocular. Zeiss microscope in- candescent lamp No. 1 with 165 watt Mazda pro- jection bulb. Zeiss Phoku camera. Hypersensitive panchromatic plates. For transmitted light, yellow G filter No. 15. Exposure time with transmitted light, 3 seconds; with dark-field, 30-60 seconds. Developer D 11 contrast (Eastman). Developed 5 minutes, room temperature. The superficial protoplasm of the phagocyte and the multiform processes and membranes to which it gives rise can be seen more clearly with the cardioid con- denser than with any other optical arrangement with which we are familiar. Phagocytosis a Phenomenon of Spreading. It follows both from the mathematical formulation of Fenn ‘°) and Ponder “®, and from the ex- perimental analysis, that the capacity of the phagocyte to spread over the surface of the par- ticle undergoing ingestion is a principal factor in determining phagocytosis. Is this deduction in agreement with the process of phagocytosis as di- rectly observed? This question we have examined with especial care. Prediction and observation have been found to be uniformly in agreement. PLATE 2 Figs. 15-19. Successive stages in ingestion of clumps of strong sensitized typhoid bacilli by a polymorphonuclear leucocyte. In Figs. 15, 16, and 17 a process of the leucocyte spread over a clump of sensitized bacteria shown above the leucocyte. In Figs. 18 and 19 this process contracted, draw- ing the ingested bacteria toward the center of the cell. In Fig. 19 a second process began to spread over a clump of bacteria to the right of the leu- cocyte. Figs. 20-24. Successive stages in contraction of the process of a polymorphonuclear leucocyte which has spread over a clump of sensitive ty- phoid bacilli. In Fig. 24 the process has contract- ed and the bacteria have moved in toward the center of the cell. Figs. 25 and 26. A polymorphonuclear leu- cocyte spread over agglutinated B. subtilis. In Fig. 26 the cell is tending to round up and some bacteria have moved toward the center. Fig. 27. A polymorphonuclear leucoctye spread over a A-shaped chain of subtilis bacilli. Fig. 28. Two polymorphonuclear leucocytes each filled with monilia cells. A process (ps) of the upper leucocyte has just spread around a monilia cell, (mo), and the lower leucoctye is spreading over another half-ingested monilia cell. Fig. 29. A macrophage ingesting subtilis ba- cilli. Two chains of bacilli are adherent to the macrophage surface; the right hand arm of the upper Y-shaped chain has been drawn into the macrophage. Fig. 30. Subtilis bacilli being drawn into a macrophage. Fig. 31. Macrophages ingesting or spreading on the droplets of an emulsion of mineral oil. Fig. 32. Macrophages spread out on a penin- sula of mineral oil. Figs. 33-37. Are dark-field photographs. Fig. 33. Macrophage spread out on glass. In order to bring out the detail of the peripheral hy- aline protoplasm, the detail of the central granu- lar protoplasm has been lost by overexposure. Fig. 34. Macrophage extended to a_pear- shape by spreading over a subtilis chain. The subtilis chain (s) is the stem of the pear and the vague white around it (ps) a process of the mac- rophage. Fig. 35. The same macrophage a few minutes later. The cell has thrown out thin “veil-like processes” toward the top of the picture. Fig. 36. Macrophage with hyaline protoplas- mic process (ps) spreading over a subtilis chain (s). The latter becomes out of focus in the upper right hand corner of the picture. Fig. 37. Two macrophages with subtilis chain (s) between them. Each cell has ingested one end of the bacterial chain and has extended a hyaline process (ps) on that portion which lies between the cells. The subtilis appears as a white chain and the two processes as delicate sheaths with dim outlines separated from the bacteria by dark spaces. The processes from the two cells met and remained approximated for some minutes; both were then withdrawn into their respective cells. One process was observed to extend again out over the subtilis chain before the field was lost to view. 384 THE COLLECTING NET [ Vou. VIII, No. 71 In mixing the phagocytes, erythrocytes or bac- teria and serum as described many collisions be- tween phagocytes and test particles are brought about. Additional contacts between particle and phagocytes may later be made by the locomotion of the latter. In the absence of sensitizing serum the test particles typically neither stick to one another nor to the phagocytes (Figs. 1 and 2), and the particles are not ingested. In the presence of dilute sensitizing serum, agglutination of the particles and adhesion to the phagocytes may be much in evidence with little complete ingestion occurring (Figs. 5, 6 and 11-14). In the presence of more concentrated sensitizing serum, the test particles adhere to the phagocytes and are drawn into their cytoplasm in great numbers (Figs. 7-10, 29 and 30). Text-Fig. 3 The test particles may be drawn into the phagocytes with comparatively little distortion of the latter (Figs. 29 and 30). Or a process com- posed of the hyaline superficial protoplasm may flow out over the surface of the particle under- going phagocytosis (Figs. 3,4, 15-19, 28 and 34- 37). A semidiagrammatic tracing of Figs. 15-19 is shown in Text-fig. 3. Or the spreading of the leucocytes over the sensitized particles may cause marked deformation of the leucocytes (Figs. 20- 24 and 25-27). A semidiagrammatic tracing of Figs. 25-27 is shown in Text-fig. 4. Text-Fig. 4 The types of ingestion described of course merge into one another. For instance four chains of sensitized subtilis bacilli were seen arranged in, a diamond-shaped figure with a suspended spherical macrophage in their center. When first observed the chains were merely tangent aad ad- herent to the macrophage surface. Gradually the areas of contact between subtilis chains and phagocyte surface increased, the adherent tan- gents becoming arcs of circles, which were slowly drawn into the macrophage protoplasm. The ends of the chains projected for a time beyond the macrophage surface, but these eventually were drawn in also; in several instances hyaline pro- cesses were observed to flow out over the pro- jecting end of the subtilis chain as the last step in its ingestion. For purposes of comparison between observa- tion and deduction from theory the essential point is that in all instances observed the particles were not taken up in vacuoles of the suspending me- dium; on the contrary the protoplasm of the phagocytes was in immediate contact with the surface undergoing phagocytosis. Phagocytosis as observed, then, is primarily a phenomenon of surface spreading—the spreading of the phag- ocyte surface over the surface of the object un- dergoing ingestion. Prediction from theory and from experimental analysis is thus in agreement with observation on this second’ essential point. 3efore leaving this point, however, two pos- sible sources of confusion should be mentioned. Phagocytes of the large mononuclear type, al- ready mentioned, are able to form delicate petal- like extensions of their peripheral hyaline proto- plasm—the “‘sheet-like pseudopods” of Smith, Willis, and Lewis “*), the “undulating mem- branes” of Carrel and Ebeling 4), the “pseudo- podes pétaloides” of Fauré-Fremiet “4. Figs. 33 and 35). W. H. Lewis'*) has described under the term “pinocytosis,” and shown in moving pic- tures, the engulfing of tiny vacuoles of the fluid medium by these processes. Should such a vacu- ole contain a minute particle it would of course be engulfed also. However, although we have seen the phagocytosis of a large number of bac- teria and erythrocytes by direct extension of the phagocyte surface over the surface of the particle ingested, we have never observed ingestion in a vacuole. Phagocytosis and pinocytosis we believe to be quite different phenomena. Another possible source of confusion is the fact that in stained films showing phagocytosis, bacteria can often be seen to lie in little vacuoles in the cytoplasm ‘4: 46), These digestive vacuoles are seen especially about the bacteria which have been ingested for some minutes and have been moved in toward the center of the cell. These vacuoles are a phenomenon not of ingestion but of intracellular digestion. Partial Ingestion. Fenn’s formulation of sur- face forces in phagocytosis predicts that under certain conditions partial ingestion should occur; this is an important point of departure from the earlier formulations of Rhumbler“@” and Tait"). Fenn recognizes two conditions : SEPTEMBER 9, 1933 ] THE COLLECTING NET 385 Text-Fig. 5. Tracing of Figs. 31 and 32. Macro- phages white with black outlines; mineral oil stippled. (a) The free surface energy is at a minimum when the particle is partially ingested; surface forces are therefore in equilibrium and satisfy the equation S; = Sy + Sy» .cos 6. (b) Surface forces would tend to bring about complete ingestion, but this is prevented by the resistance to deformation of the phagocyte; sur- face forces are therefore not in equilibrium but are held in check by viscosity. The second condition has certainly been realized in our experiments, and to the best of our belief also the first. Figs. 31 and 32 show fields in which macrophages were mixed with an emulsion of light California mineral oil. A tracing of Figs. 31 and 32 is shown in Text-fig. 5. Emulsion droplets of small size are readily and completely ingested by the macrophages. On the larger drops the macrophages spread (Figs. 31 and 32) to positions determined by the balance between surface forces and their own resistance to deformation. Incidentally it may be mentioned that such small emulsion droplets are very readily ingested by macrophages, but ordinarily not by polymor- phonuclear leucocytes. Such a difference cannot be explained by differences in resistance to de- formation, since the polymorphonuclears are on the average more fluid cells than the macrophages. This is evidently an instance in which a difference in the surfaces of the two types of phagocyte is a critical factor in determining phagocytosis. An- other such instance was found in the quantitative phagocytosis study ;—collodion particles are read- ily ingested by macrophages but not by polymor- phonuclear leucocytes ‘”). Partial ingestion with surface forces in equil- ibrium is more difficult to demonstrate conclu- sively. When weakly sensitized erythrocytes are mixed with phagocytes partial ingestion often oc- curs (Figs. 5, 6 and 11-14). Such partially in- gested cells are not completely ingested during the time they are kept under observation, even though this may be far longer than is required for complete ingestion of more strongly sensi- tized erythrocytes. It is difficult to believe that the partial ingestion by sucha fluid cell as is shown in Figs. 11-14, could represent anything other than equilibrium under surface forces. Moreover in stained preparations ‘®) it has very frequently been observed that strongly sensitized bacteria were completely ingested, whereas weakly sensi- tized bacteria under otherwise similar conditions were merely adherent to the surfaces of the phagocytes. Although we realize that such obser- vations fall somewhat short of rigorous proof that the surface forces are in equilibrium, we be- lieve that this is by far the most probable inter- pretation. The third deduction from theory, name- ly the occurrence of partial ingestion, is thus like- wise in agreement with observation. Viscosity. L. Loeb\®) has related the amoeboid motion of the amoebocytes of Limulus to, “1) changes in consistency in the ectoplasmic layér as well as in the granuloplasm, 2) phenomena of contraction and 3) surface tension changes.” Loeb in 1927 sought to carry over these concep- tions to the explanation of phagocytosis by mam- malian cells, assigning a primary importance ‘to softening of certain parts of the surface layer of the cell in contact with a foreign body. Whether or not such local softening occurs on contact of phagocytes with foreign particles, it is evident that the quantitative correlation which has since been demonstrated between phagocytosis and the surface properties of the particles phagocytized is not explained by viscosity changes, and is ex- plainable in terms of interfacial tension relations: The resistance of the protoplasm to deforma- tion is, on the other hand, a modifying factor in phagocytosis which, under certain conditions, may reach critical importance. Fenn'*®), for instance, found very high temperature coefficients for phagocytosis below 30°C. as compared with those above 30°. He interpreted his data as indicating that below 30° the viscosity of the phagocytes was so high as to become the limiting factor for phagocytosis. Ponder'**’has also considered the effect of cytoplasmic viscosity as influencing the rate of ingestion. He has also dealt with the case in which movements in the surrounding fluid tend to dislodge particles which might otherwise be ingested, and has shown that either great cy- toplasmic viscosity, or great turbulence of flow in the fluid, tends to prevent phagocytosis. This has since heen vérified experimentally ‘°). An average difference in viscosity between phagocytes of the large mononuclear and the polymorphonuclear types has been observed by E. R. and E. L. Clark “?), by Goss 8), and by our- selves ‘°4). The macrophages offer, on the aver- age, more resistance to deformation than the poly- morphonuclears. This difference has been evi- denced in our own study in two ways. In the first 386 THE COLLECTING NET [ Vor. VIII, No. 71 place the act of ingestion is, on the average, more quickly accomplished by the polymorphonuclears, and in the second the polymorphonuclears are more readily distorted to all manner of bizarre shapes in spreading over the larger bodies phago- cytized (Figs. 25 and 27). Unformulated Factors. It seems clear then that surface forces are a principal factor in deter- mining ingestion, and that viscosity is an impor- tant factor in controlling its rate. It is perhaps worth emphasizing, however, that a complete ex- planation of the behavior of phagocytes is not af- forded by these factors alone. A particle phago- cytized under the action of surface forces does not enter a homogeneous liquid, but a system pos- sessing internal organization in high degree. The process which has spread over the particles under- going phagocytosis is frequently retracted (Figs. 15-26). The protoplasm of the phagocytes pos- sesses elastic properties (Figs. 11-14).* The in- gested particles are commonly moved in toward the center of the cell. They frequently undergo rapid intracellular digestion. The formation and retraction of pseudopods appears to be the con- sequence of internal changes within the cell, as well as of the tendency of the cell surface to spread upon external surfaces. Reversible changes in viscosity, as evidenced by the appearance and disappearance of Brownian movement, may be seen to occur in local areas within the cell. The phagocyte, then, is a complex system deli- cately responsive to internal and external influ- ences. Interfacial tensions, and under certain con- ditions viscosity, are critical factors in determin- ing the ingestion of particles with which the phagocyte has come into contact. Deductions from the formulation of these factors by Fenn and Ponder are in agreement with observation and with experimental analysis. However, other and still unformulated forces also enter into the be- havior of these remarkable cells. BIBLIOGRAPHY 1. Metchnikoff, E. Lectures on the comparative pathology of inflammation. English translation by F. A. and E. H. Starling, London, 1893. 2. Metchnikoff, E. Immunity in infective dis- eases. English translation by F. G. Binnie. Cam- bridge, 1905. 3. Ernst, P.; in Handb. d. allg. Pathologie. Ed. by L. Krehl u. F. Marchand, III, 1 pt., 152-175, Leip- zig, 1915. 4. Marchand, F., in Handb. d. allg. Pathologie. Ed. by L. Krehl u. F. Marchand IV, 1 pt., 607-643. 5. Hamburger, H. J. Physikalisch - chemische Untersuchungen iiber Phagozyten. Wiesbaden, 1912. 6. Hamburger, H. J., in Handb. d. biol. Arbeits- meth., Ed. by E. Abderhalden, 1927, IV. pt. 4, 953- 992. 7. Neufeld, F. in Handb. d. path. Mikroérgan- ismen, by W. Kolle, R. Kraus u. P. Uhlenhuth, 1929, Il, pt. 2, chapt. XT1. 8. Neufeld, F. and Loewenthal, H. in Handb. d. norm, u. path. Physiol. 1929, 13, 813-831. 9. Ledingham, J. C.G.; In “System of Bacteriol- ogy,” VI, chapt. II, London, 1931. 10. Muir, R., In “System of Bacteriology,” VI, chapt. XI, London, 1931. 11. Gray, J., Textbook of Experimental Cytol- ogy, New.York, 1931. 12. Fleishmann, W., Ergb. der Physiol. 1928, 27, 1-46. 13. Hirschfeld, H., in Lehrbuch der Allg. Haema- tologie I, part 1 by Hirschfeld, H. u. Hittmair, A., Berlin u. Wien, 1932. 14. Gay, F. P., J. Am. Med. Asso., 1931, 97, 1193. 15. Lubarsch, O., Klin. Wochenschr., 1925, 4, 16. Hogue, M. J., Anat. Rec., 1932, 54, 307. 17. Fenn, W. O., J. Gen. Physiol., 1921, 3, 439. 18. Philipsborn, E., von, Fol. Haem., 1930, 43, 19. Fenn, W. O., J. Gen. Physiol., 1921, 3, 465. 20. Fenn, W. O., J. Gen. Physiol., 1921, 3, 575. 21. McKendrick, A. G., reviewed by Ponder E.; Protoplasma, 1927-8, 3, 611. 22. Mudd, E. B. H., and Mudd, S., J. Gen. Physiol., 1933, 16, 625. 23. Fenn, W. O., J. Gen. Physiol., 1923, 5, 311. 24. Commandon, J., C. R. Soc. Biol., 1919, 82, 1171. 25. Abramson, H. A., in Alexander, J., Colloid Chem., New York, 1928, 2, 708. 26. Reiner, L., and Kopp, H., Z. f. Immunitats- forsch,. 1929, 61, 397. 27. Mudd, S., Lucké, B., McCutcheon, M., and Strumia, M., J. Exper. Med., 1929, 49, 779. 28. Neufeld, F., and Etinger-Tulczynska, tralbl. f. Bakt. Orig., 1929, 114, 252. 29. Lucké, B., McCutcheon, M., Strumia, M., and Mudd, S., J. Exper. Med., 1929, 49, 797. 30. Strumia, M., Mudd, S., Mudd, E. B. H., Lucké, B., and McCutcheon, M., J. Exper. Med. 1930, 52, 299. 31. Mudd, S., Lucké, B., McCutcheon, M., Strumia, M., J. Exper. Med., 1930, 52, 313. 32. Mudd, S., Lucké, B., and Strumia, M., J. Im- munol., 1933, 24, 493. 33. Mudd, S., see Lecture this volume. 34. Ponder, E., J. Gen. Physiol., 1927-8, 11, 757. 35. Fenn, W. O., J. Gen. Physiol., 1921-2, 4, 373. 36. Ponder, E., Protoplasma, 1927-8, 3, 611. 37. Cole, K. S., J. Cell. and Comp. Physiol., 1932, Cen- and “Agelutination,” ip 38. Harvey, E. N., Biol. Bull., 1931, 61, 278. 39. Bond, C. J., ‘The Leucocyte in Health and Disease,”’ London, 1924. 40. Fauré-Fremiet, E., Protoplasma, 1929, 6, 522. 41. Warburg, O., Uber den Stoffwechsel der Tu- moren, 1926, Berlin, p. 101. 42. Lucké, B., Strumia, M., Mudd, S., McCut- cheon, M., and Mudd, E. B. H., J. Immunol., 1933, 24, 455. 43. Smith, D. T., Willis, H. S., and Lewis, M. R., Am. Rev. Tuber. 1922, 6, 21. 44. Carrel, A., and Ebeling, A. H., J. Exper. Med., 1926, 44, 285. 45. Lewis, W. H., Bull. 1931, 49, 17. 46. Lucké, B., personal communication. 47. Rhumbler, L., Arch. Entwicklngsmechn. Or- gan., 1898, 7, 199. Ergebn. Physiol., 1914. 14, 477. 48. Tait, J., Quart. J. Exp. Physiol., 1918-20, 12, 1. 49. Loeb, L., Protoplasma, 1927, 2, 512. Johns Hopkins Hosp., SEPTEMBER 9, 1933 ] THE COLLECTING NET 387 50. Fenn, W. O., J. Gen. Physiol. 1922, 4, 331. 51. Ponder, E., personal communication. 52. Clark, E. R., and Clark, E. L., Am. J. Anat., 1930, 46, 111. 53. Goss, C. M., Arch. Zellforsch., 1930, 10, 213. 54. Mudd, S., and Mudd, E. B. H., J. Gen. Physiol., 1931, 14, 733. Discussion Dr. Abramson: In these observations of Fenn’s does the specific effect of MnOz occur with each particle and every leucocyte, or does it happen once in a hundred? Is this a small per- centage observation or is it a regular thing? Dr. Mudd: I do not know the percentage of instances in which attraction of particle for leu- cocyte was demonstrable. But Fenn analysed his data carefully, and was convinced that in the case of manganese dioxide particles the contacts were more than could be accounted for by chance col- lisions. Dr. Fricke: Were the suspending solutions in Fenn’s experiments saturated with manganese dioxide ? Dr. Mudd: No. Dr. Fricke: Would not saturation of the me- dium with manganese dioxide afford a method of testing the theory of chemical attraction? Dr. Mudd: I should think it would. Dr. Abramson: Did you say that serum al- bumin is not much adsorbed ? Dr. Mudd: If bacteria are suspended in the euglobulin, pseudoglobulin, or albumin fractions of unheated normal serum, phagocytosis is usual- ly somewhat promoted in each case. But if the bacteria so treated are washed, the surface altera- tions, and phagocytosis-promoting effect, persist, at least partially, after the globulin treatment, but not after treatment with albumin. It would seem, then, that the albumin is less firmly adsorbed than the globulins. Dr. Abramson: You mentioned that the leu- cocytes were able to phagocytize bacteria, even in the presence of such relatively high concentrations of aluminum ion as N/2000, and thorium, also, I believe. Do you think that the thorium was se- lectively adsorbed by the bacteria and did not poi- son the leucocytes ? Dr. Mudd: At least it did not poison the leu- cocytes sufficiently to prevent ingestion. Dr. Abramson: J am thinking of it from this point of view: if you had the bacteria positively charged by thorium ions, and the leucocytes still remaining negative, you could account for that rather exceptional case of phagocytosis by the ad- hesion resulting from the difference in charge. Do you not think that is possible, even probable? Dr. Mudd: Yes. Dr. Abramson: In all your cases of phago- cytosis, the bacteria and the leucocytes have the same charge. Dr. Mudd: Yes, but wouldn’t you, taking your case, expect the leucocytes to be reversed, too ? Dr. Abramson: Not necessarily. I would ex- pect that if you have two types of surfaces, “a” and “b,” there would be a difference in adsorp- tion of polyvalent ions by “a” and “b.” That might account, then, for the phagocytosis in the presence of the polyvalent cations. Dr. Ponder: That would do it. Dr. Abramson: It is a sort of cross-aggluti- nation between the bacteria and the leucocytes. This case, of course, is analogous to the precipita- tion of one colloid by another, opposite in sign of charge. Dr. Chambers: Your proposition presents the idea that protoplasm is of such a nature that when a particle on the outside is wetted by the proto- plasmic surface, it will be drawn into the sub- stance of the protoplasm. This may happen if the protoplasmic surface possesses a monomolecular oriented palisade structure of matter which is of the same nature as that of the interior, except for its oriented disposition. However, there is another view, viz., that the protoplasmic body possesses a differentiated plas- ma membrane which is qualitatively different from the internal protoplasm. In other words, the plasma membrane is a layer of material with two interfaces, an external one, in contact with the aqueous environment, and an internal, in contact with the internal protoplasm. Evidence for this is suggested by (1) the high electrical resistance of cells, and (2) the impermeability of the surface, both from without and from within, to non-pene- trating dye solutions, e.g., phenol red. I would postulate that surface tension forces, existing at the interfaces of the plasma mem- brane, are of different orders, the interface in contact with the aqueous environment possessing a higher tension than the interface in contact with the heterogeneous, but freely water-miscible, internal protoplasm. The particle being ingested might then be wet- ted by the plasma membrane at its external inter- face and be pulled in, in the manner you suggest. Owing to the relatively high tension of the ex- ternal interface, this interface would tend to de- velop an even contour, with the consequence that the particle would form a protrusion of the plas- ma membrane along its inner interface. The very low forces at play along this inner interface may result in the continued formation of myelin-like protrusions, into the internal protoplasm, which 388 THE COLLECTING NET [ Vou. VIII, No. 71 pinch off periodically, one of them carrying along the particle with it. This is not a far cry from the formation of food vacuoles by certain protozoa. In short, the object being ingested is wrapped or infolded within plasma membrane material, and in this condition is pinched off into the interior of the cell. Dr. Mudd: In the formulation of surface forces in phagocytosis no assumption is made as to whether the cell surface is a monomolecular film or a thicker layer, except that it is implicitly assumed that the cell surface is not significantly altered in spreading over the particle phagocy- tized. Kite described the mammalian leucocyte as naked protoplasm. Our observations agree with this, in that no microscopically visible membrane, such as can be seen on the erythrocyte, can be made out even with cardioid condenser and oil immersion objective. Of course, however, I be- lieve that we must postulate a differentiated sur- face film to account for impermeability and other surface effects. We have never seen with dark or bright field illumination, nor received an account oi such myelin-like protrusions into the interior of the phagocyte, although they may often be seen on the external surface. Moreover, the pinching off of protrusions carrying particles with them suggests, to me, particles small in relation to the dimen- sions of the phagocyte. We have often watched leucocytes spreading over and _ phagocytizing chains of bacteria considerably longer than the diameter of the resting leucocyte. Fee The idea of negative surface tension has re- cently appeared in the literature on amoeboid cells and phagocytosis; discussion of this idea by this group would be most desirable. The idea was introduced by Leathes in treating the myelin forms of lecithine in the following passage: “Lecithine is a fat containing two fatty acid radicals instead of three, one of them certainly unsaturated, the other probably saturated; in place of the third there is phosphoric acid con- densed by one of its hydroxyl groups with the glycerol and by a second with a basic alcohol, the third hydroxyl group being free... . “But though lecithine is a fat and in a film behaves as one, lecithine in bulk behaves very differently from a fat or oil in contact with water. The ‘surface tension” of oil against water is such that the number of molecules of either which are allowed by the attractions of other molecules in their neighborhood to remain in the surface is kept as small as possible, that is to say, the sur- face area is kept as small as possible. A drop of oil suspended in water remains spherical. With lecithine it is just the other way. The number of molecules of lecithine in contact with water tends to increase and to become as large as possible; that is why the surface is protruded into the water and the myelin forms appear. The attrac- tion exerted by water on that part of the mole- cule of lecithine by which it differs from a simple fat is so great that the molecule is drawn to and held by the water more strongly than it is by the other lecithine molecules in its neighborhood. This attraction prevails over the attractive forces between the paraffin chains, partly because these can not be packed so close as in a triglyceride. The cohesion between the lecithine molecules is consequently weakened at a water surface, and the surface tends to grow and not to be kept small; if the term surtace tension must be used, the surface tension is negative.” (The Lancet, 1925, 208, 960). Fauré-Fremiet, in his beautiful studies of the surface hyaloplasmic processes of invertebrate amoebocytes, brought out many points of resem- blance of these processes to the myelin forms of lecithine, and adduced evidence to indicate that the formation of these processes was probably de- pendent on the presence of lecithine in the sur- face protoplasm. He adopted Leathes’ conception of negative surface tension. “Concerning the hyaloplasmic processes (peta- loid pseudopods) we can now imagine that fol- lowing the schema suggested above, their forma- tion results from a local swelling, by imbibition, of the hyaloplasmic zone ; we shall make no hypo- thesis as to the cause of such localized variations, but we shall recall that our measurements of re- fractive index and volumetric calculations are favorable to this idea. In this case all the proper- ties of the hyaloplasmic processes, their negative surface tension, their lamellar expansion, indicate an anisotropy in the distribution of capillary ten- sion, make us think of a swelling of lecithine in the presence of aqueous solutions and of the for- mation of myelin figures of a special type.” ( Pro- toplasma, 1929, 6, 603). Most recently Volkonsky has taken up the idea of negative surface tension, and has tried to re- write Dr. Ponder’s equations for the conditions of phagocytosis, giving the cell-plasma interfacial tension a negative value. The predictions drawn from Volkonsky’s treatment are the opposites of the correct ones. He then justifies his predictions by making interpretations of the experiments of Mrs. Mudd and myself which are quite unaccept- able to us. (Bulletin Biologique de la France et de la Belgique, 1933, 67, 168). Dr. Ponder: One of the most interesting points for discussion concerns Volkonsky’s idea that the cell-plasma interface may have a neg- ative surface tension. The conception of negative SEPTEMBER 9, 1933 ] THE COLLECTING NET 389 surface tension is becoming dangerously preva- lent among biologists, and so I want to make four points about it, and then leave the matter to dis- cussion. The first point is that the lowness of the cell- plasma interfacial tension, even if we admit that it is very low, is not an essential point, for the equations which deal with the conditions for in- gestion are not concerned with the cell-plasma in- terfacial tension in absolute units, but with the relative values of three interfacial tensions. If one allows that the cell-plasma tension is low in absolute units, I do not see why one should not imagine the particle-plasma and cell-particle ten- sions to be of an equal order of lowness, It is a logical fallacy to think of a tension of 0.5 dynes/cm. as “‘virtually zero’’; compared with the other interfacial tensions existing in the system, 0.5 dynes/em. may be a very considerable value. In the second place, Volkonsky is not content with a small tension at the cell-plasma interface, but makes the tension negative. This, of course, turns everything upside down, and the condition for ingestion becomes the condition for no inges- tion, or vice versa, for he changes the sign of S» from positive to negative. His idea seems to be that one can properly make a very small value equal to zero, and then make zero equal to a neg- ative quantity. Thirdly, even if the cell-plasma tension were negative, | am sure that the equilibrium condition would not be given by cos 0 = (S;—Si2)/—S», as Volkonsky supposes. The equilibrium condi- tion, if any, would have to be worked out from first principles, and not merely by changing a sign. Lastly, I simply do not know what a “negative surface tension” is. In Leathes’ idea of negative surface tension it is not contended, I suppose, that the volume of the myelin forms remains con- stant as their surface expands, and I know of no evidence against the expansion of surface being really due to an imbibition of water by the leci- thine. It seems to me that if one has a surface which is going to expand continually because of a “surface tension,’ one will have a surface which will disintegrate and undergo spontaneous emulsi- fication. Dr. Abramson: I can conceive of a negative surface tension exerted under these conditions. Take a stable particle in a liquid and let the in- terfacial tension be very small. Charge the sur- face of the particle so that the opposing force ex- erted by the charge will be greater than the torce of surface tension. Dr. Ponder: That is essentially Ostwald’s old model of a charged and liquid surface which would break into a fountain. Dr. Cole: It may be of interest to point out that under certain conditions, instability occurs before a net or effective surface tension is reduced to zero. When a spherical droplet is electrically charged, the mutual repulsions of the charges ex- ert a force which acts against the component of surface tension directed inward. The electric po- tential necessary to reduce the force normal to the surface to zero may be computed easily. A complete analysis shows, however, that the drop- let will become. unstable and separate into two at one-half the value of potential necessary to “‘re- duce the surface tension” to zero. Dr. Ponder: I have been interested to see the pictures of the rod-shaped bacteria being ingested in an end-on position. You will notice that this is the condition for @ being single-valued, and it seems to be the position in which the bacillus ought to go in. Does a rod-shaped particle al- ways go in end-on? Mrs. Mudd: Bacterial rods after contact with the leucocyte are often seen to be oriented so as to be ingested end-on. This has been the more usual method of ingestion in our observations, although occasionally the bacterium may be taken into the leucocyte with its long axis tangent to the leu- cocyte surface. Dr. Cole: If it has not already been tried, it should be interesting to investigate the phagocy- tosis of air bubbles where the surface tension is high. 390 THE COLLECTING NET [ Vor. VIII, No. 71 OSMOTIC BEHAVIOR OF RED CELLS. II. Eric PoNDER In my last lecture I dealt with the applicability of the Boyle-Marriotte law to the volume changes observed when red cells, and probably most other types of vertebrate cell, are immersed in hyper- tonic or hypotonic solutions, and concluded that the law does not apply in its simplest form, but that the cells shrink in hypertonic solutions, and swell in hypotonic solutions, as if part of their contained water were “bound,” or, al- ternatively, as if osmotically active substances were exchanged between cell and environment. Now if the medium is sufficiently hypotonic, the swelling of the red cell is so great that it bursts and liberates its haemoglobin, and so some light can be thrown on the foregoing conclusion by considering the relation between the volume of the cell and that extreme hypotonicity of the me- dium in which haemolysis occurs. The matter can best be approached by seeking the solution to two classical problems: 1. Why do the erythrocytes of the same ani- mal (e. g., the rabbit) haemolyse in quite differ- ent equivalent concentrations of different sub- stances, e. g. in 0.52 p.c. NaCl, but in a glucose solution whose osmotic pressure is equivalent, not to a 0.52 p.c. NaCl, but to a 0.38 p.c. NaCl? 2. Why do the red cells of one animal (e.g., man) haemolyse in a 0.40 p.c. NaCl, while those of another animal (e. g., the sheep) haemolyse in a solution of a different concentration, e. g., 0.56 p.c. NaCl? I may remark at the outset that there is no doubt about the experimental facts, and that both problems have well known practical applications associated with them. The comparison of the con- centrations of different solutions in which ery- throcytes just begin to haemolyse is one method of finding the so-called “isotonic coefficients,” and the observation of the concentration of the solution in which different types of cell, or cells from the same kind of animal under different conditions, just begin to haemolyse is the method used for determining what is commonly known as “red cell fragility,” a property of some clinical and general biological interest. The generally accepted explanation for hypo- tonic haemolysis, introduced by Hamburger and supported by the investigations of Koeppe, Ege, Warburg and Winge, Jacobs, and many others, is that the cells take in water, swell, and _ finally burst or leak pigment because their membranes become so stretched. Some observers have pos- tulated the existence of “bound water,” while others have treated all the water as “free”; such postulates influence the computation, but the general idea is the same for all. It is always as- sumed, however, except by those few observers who look upon the lysis as a result of imbibition, etc., that the volume of the cell is calculable from simple osmotic laws, “bound water,” perhaps, having to be taken into account. Now if this is so, it is very difficult to account for the fact that the red cells of a given animal undergo lysis in such concentrations of different substances as are quite different from an osmotic standpoint. If simple osmotic laws are sufficient, even if we allow a constant quantity of “bound water” to be present, it is hard to see why rabbit red cells, for instance, should haemolyse in 0.52 p.c. NaCl but in glucose equivalent to 0.38 p.c. NaCl, for the two concentrations should be the same and not different. Moore and Roaf have rejected the entire osmotic theory because of this difficulty, and have fallen back on a rather indefi- nite hypothesis which accounts for haemolysis as the result of the imbibition of water by a dense in- tracellular stroma. The difficulty is usually ex- plained, however, by introducing subsidiary as- sumptions, some of which are the following. 1. Ege has considered the possibility of the dissociation of osmotically active substances with- in the cell changing when the erythrocyte is im- mersed in solutions of different substances, but he dismisses the explanation on the ground that the possible changes are too small to account for the observed phenomena. I have no doubt that we could account for small differences in this way, and also by taking account of possible pH changes, but the observed differences are far too large. 2. One might suppose that the immersion of the cells in solutions of different substances, e.g., NaCl, glucose, or sucrose, brings about an al- teration in the quantity of “free water.” The amount of this substance would have to be differ- ent for almost every different suspension medium, so the explanation would not be a very good one even if “bound water” existed in appreciable quantities; as it does not, we need not consider this explanation further. 3. _The next explanation is that the erythrocyte has a membrane which is more resistant to stretching when in glucose (say) than when in NaCl, and not unlike this hypothesis is one which depends on Brinkman’s observations ") ©), (now disproved'*) that the fragility of the cell depends on the lecithin/cholesterol ratio in the membrane ; different substances, affecting the ratio in differ- ent ways, might conceivably cause such differ- ences in the membrane as would allow it to stand SEPTEMBER 9, 1933 ] THE COLLECTING NET 391 more stretching in a solution of one substance than in a solution of another. Now this idea is one which can be tested ex- perimently, for we can measure the volume at which the erythrocyte begins to haemolyse in so- lutions of different substances, and we can then tell whether the surface is really more stretched in a solution of a substance such as glucose than it is in one of NaCl. The cells are placed in solu- tions of NaCl of descending degrees of tonicity, and similarly in a series of solutions of glucose. In a certain concentration of NaCl, and in a cer- tain solution of glucose, commencing lysis is ob- served, and the volume of the cells in these two so- lutions is then determined diffractometrically. The experiment is easy to do, and the result is always the same, allowing, of course, for the small errors inherent in the method: the cells begin to haemolyse at the same “critical volume,” irrespec- tive of the nature of the substance in the sur- rounding medium. There is, of course, a little variation from animal to animal, and more in some animals than in others, but, as an instance, the rabbit erythrocyte, the normal volume of which is about 583, shows commencing lysis when its volume has increased to about 783, irre- spective of whether the surrounding medium is hypotonic NaCl, KCl, glucose, sucrose, or any one of the monovalent chlorides. The existence of this “critical volume” was first recognized by Jacobs"), and is a very important point. 4. This being so, we can proceed in the fol- lowing way. Let us regard the cell as an imper- fect, instead of as a perfect, osmometer, and let us again measure the degree of imperfection, or the amount of leakage of osmotically active sub- stances, in terms of the fiction of “bound water.” Then we can write as a general equilibrium con- dition ’ 11.0 (Q,) , (R/100) a = (1) (Q1) . (R/100) + v 100—v in which all the symbols have the same meaning defined in the last lecture, i.e., T the tonicity in which a percentage increase in volume v occurs, QO, the total water in the cell, and R the ratio Ov/Q1, where Q» is the quantity of water which would have to be assumed to be contained in the cell if it could be treated as a perfect osmometer. Now this expression is true for all sorts of values of T and v, and so we can immerse the cells in so- lutions of different tonicities and measure v for each; then remembering that Q, is constant, we can use the results to give a series of equations from which we can get a “best value” for R. Having obtained this for a series of solutions of NaCl, (say), we can insert in the expression the proper value of R and of Q,; we then have a numerically soluble equation in T and vy, applic- able to systems containing red cells suspended in NaCl of various degrees of tonicity. Next we can do the same thing for the cells suspended in a series of hypotonic solutions of an- other substance, say glucose, and obtain in a sim- ilar way a numerically soluble equation in T and v for this system. If this is done, the first thing which strikes one is that the “best values” for kK for the system of cells in hypotonic NaCl is much higher than that for the system of red cells in glucose, the former being usually about 0.5, and the latter being more nearly 0.25. This, of course, is not surprising, for all that it means is that the leakage of osmotically active substances 1s greater in NaCl than in glucose, which is generally admitted. But we can go a_ step further, and use the equation in the following way. Let us suppose that we find by experiment that in a solution of NaCl of tonicity 0.52 we get commencing lysis, and that in such a solution the volume of the cells is 135 p.c. of their normal volume. Then remembering that this is the “crit- ical volume,” and that it is the same when the cells are in glucose as when they are in NaCl, let us insert the proper value for Qy, the value which we have just found for R for the systems of cells in glucose, and the same value of v as found for the NaCl systems, in expression (1), and let us solve for T, i.e., from the value of R for the glucose systems and from the value for the “‘crit- ical volume” found for the NaCl systems, let us predict the tonicity of glucose in which the cells will just undergo lysis. The result will only be correct if the assumptions and measurements are correct, and it is surprising how closely the pre- diction can be made, not only for systems of cells in glucose, but for systems of cells in quite a variety of hypotonic solutions. The following table shows one series of results: ‘? Predicted Observed Substance Tonicities used tonicity tonicity for finding R. for lysis for lysis NaCl 0.7, 0.6, 0.54 0.52 0.57 KCl 0.78, 0.76, 0.7 0.62 0.66 LiCl OOF 07 0.67 0.63 Glucose 0.66, 0.5, 0.46 0.38 0.36 Sucrose 0.08, 0.06 0.52 0.50 KNO3 0.8, 0.7, 0.6 0.59 0.59 Considering the errors inherent in the methods, these results are quite striking, and _ the puzzling fact that red cells haemolyse in solu- tions which are far from osmotically equivalent can be quite well explained by assuming that the cells undergo lysis when they reach a_ certain critical volume, constant for all these substances, but that the leakage of osmotically active sub- stances, as shown by the value of R, is different 392 THE COLLECTING NET [ Vor. VIII, No. 71 in the case of each. That this hypothesis is ade- quate is shown not only by the fact that direct experiment reveals the same critical volume and the different values of R, but by the fact that by taking account of these two factors alone we can predict with considerable accuracy the tonicity ot each substance in which lysis will begin to occur, even although there are wide differences in the concentrations which bring about haemolysis. 3efore passing to the next problem, I may re- mark upon the rather curious fact that the cell is “most fragile” or “least resistant’? in hypotonic solutions of the very substances in which it loses osmotically active substances least, and “most re- sistant” in solutions of substances in which the leakage is great. The most resistant cells are thus the least “perfect”’ from the osmotic standpoint, and, far from their membranes being peculiarly “strong” or “extensible,” as seems to be the gen- eral idea among clinicians and biologists, they are peculiarly liable to leak and to lose cations into the surrounding fluid. We have now to consider the second classical difficulty, that of explaining why the cells of one animal are “fragile” and easily haemolysed by hypotonic solutions of a particular substance, e.g., NaCl, whereas those of other animals are “re- sistant’’ and can be haemolysed only by much more hypotonic solutions. There are considerable differences among the cells of various animals in this respect; those of the sheep and ox, for in- stance, haemolyse in NaCl solutions of about 0.72 to 0.66 p.c., whereas those of the rabbit and man are much more resistant and haemolyse only when the tonicity of the medium falls to about 0.40 p.c. There are, of course, considerable differences also between the cells from different animals of the same species, but, taking average values, the types of cell can be arranged in a series known as the Ryvosch Series®), which runs: man, guinea-pig, rat, rabbit, dog, pig, cat, ox, goat, sheep,—the most resistant kind of cell occurring first. The subject of this different fragility has been widely studied, but the only two suggestions of any consequence which have been advanced are that of Brinkman and van Dam, who regard the fragility as dependent on the lecithin/choles- terol ratio in the membranes of the different types of cell, and a suggestion that the fragility is a function of cell diameter, the larger cells being stated to be, in general, the more resistant. 3rinkman’s hypothesis has been contradicted, and the theory that the fragility is determined by diameter will not stand very close examination, although, as we shall see, it has an element of truth in it. The problem of accounting for the differences in fragility is a very complicated one, for there are at least four factors upon which the fragility must necessarily depend. (1) Even if the red cell were a perfect osmometer, the concentration of NaCl in which it would haemolyse would de- pend on the critical volume which it could at- tain, for the greater this volume, the less would be the concentration of NaCl which would bring about lysis, other things being equal. The critical volume, of course, might vary for the cells of dif- ferent animals. (2) Even if the cell were a per- fect osmometer, the swelling in any hypotonic so- lution of NaCl would depend on the quantity of water which the cell contained, and the smaller the amount of water the greater would have to be the degree of hypotonicity of NaCl which would bring about swelling to the same critical volume. (3) Still regarding the cell as a perfect osmometer, the swelling which it would undergo in any given tonicity of NaCl would depend on the osmotic pressure of the cell interior, and this is not the same for the cells of all mammals. (4) And lastly, if the cell is an imperfect instead of a perfect osmometer, the extent to which it would swell, and therefore the tonicity of hypo- tonic NaCl in which it would reach its critical volume, would depend on the extent to which it could lose osmotically active substances, i.e., on the value of R. Our problem is to decide which one of these factors is responsible for the different fragilities of the cells of different mammals, and a very dif- ficult problem it is, particularly when one con- siders that not one factor alone, but several act- ing in conjunction, might be responsible for the result. But even a roughly quantitative explana- tion is better than none, and so we shall see how far our experimental methods will take us. The quantity of water present in the cell is easily determined, so factor (2) offers no diffi- culty, but the osmotic pressure of the interior is much more difficult to find. Assuming that it is the same as that of the surrounding plasma, we might determine it by finding the depression of freezing point of the latter, but a far more con- venient way is to call the tonicity of the plasma unity, and that of the cell interior unity too. This gets rid of factor (3) immediately, but deter- mines that all the experiments are to be carried out in the hypotonic plasma of the animal whose cells are concerned. This, perhaps, is more advan- tageous than otherwise, for pH changes are much less liable to affect the results when the cells are suspended in plasma than when they are sus- pended in saline. As soon as we decide to use hypotonic plasma as the suspension medium our choice of methods for measuring red cell volume becomes severely limited. The colorimetric method is use- less for determination of cell volume in solutions in which lysis is likely to occur, the haematocrite SEPTEMBER 9, 1933 ] THE COLLECTING NET 393 method is out of the question, and diffractometric measurements cannot be made, for the cells are discoidal. We are therefore left with the methods which determine percentage increases in cell vol- ume from changes in cell density, changes in haemoglobin content, or changes in the water con- tent of the cells after swelling’), and, as the last mentioned is the simplest, we can select it for the determination of the relative increases in volume which the cell shows when immersed in hypotonic plasma of various degrees of tonicity, and for the evaluation of R. This disposes of factor (4). Lastly, we have to find the critical volume at which the cells haemolyse (factor 1), and here we encounter a difficulty which can only be over- come by the use of a special method. The methods which measure swelling from changes in water content, density, and haemoglobin concen- tration are useless, for they do not measure vol- ume in absolute units, but only the swelling which would occur if the cell were a perfect osmometer (which it is not) containing a quantity-of “free water,” O:/O; = R, (which it does not). The fiction of “bound water” is thus used as a meas- ure of the fictitiousness of the idea that the cell is impermeable to cations, and volume is not meas- ured directly at all; consequently these methods are useless for the determination of the critical volume, which must be found in absolute units. The colorimetric method is excluded, for the same reason as mentioned above, and haemato- crite methods are too unreliable; we have there- fore to determine the critical volume in hypotonic NaCl solutions diffractometrically, and to be con- tent with the result, although the effect of factor (4) is measured in hypotonic plasma. But this does not end the difficulty, for what we are interested in is not the critical volume per se, but the percentage of the normal volume to which the cell will swell, attaining its critical vol- ume, before it begins to lose pigment. We have therefore to measure the normal volume in addi- tion. Now we might do this colorimetrically, but the technical difficulties of making colorimetric determinations of volume side by side with all the other determinations is too formidable a task to be attempted. We can, however, avail ourselves of a very neat method of measuring cell volume in undiluted plasma. If a small amount of lecithin is added to the plasma, and cells added thereafter, the cells become perfectly spherical without change in volume, and diffractometric measure- ments of volume can be made in a few minutes. To find the percentage of the normal volume to which the cell will swell without haemolysing, we accordingly find the normal volume diffractomet- rically in lecithin-treated plasma, and the critical volume in a hypotonic saline: divison of the latter figure by the former then gives us what we re- quire, the maximum increase in volume which the cell can undergo while still remaining intact. I can pass over the details of the methods em- ployed, and go at once to the results. There is little point in making determinations of the effects of all the factors for all the types of cell in the Ryvosch Series, for the differences of re- sistance between some of them is very small; I shall therefore refer to the results for the cells of man, the rabbit, the sheep, and the ox, ignor- ing the remainder in the meantime. The determinations of the value of R for these various types of cell, each immersed in the hypo- tonic plasma of the same animal from which it was obtained, leave little doubt that R is essen- tially the same for all. It varies between 0.5 and 0.7, but the variation between the values for the cells of individual sheep, for example, is as great as is the variation between the values for sheep and rabbit cells. If a very large number of ex- periments were carried out, some differences, in a statistical sense, might emerge; after carrying out some twenty experiments or so, I am left, how- ever, with the very strong impression that what- ever factor may account for the different fragil- ities, it is not differences in the extent to which the different types of cell lose osmotically active substances. The cell of one type of animal seems to be just about as imperfect an osmometer as is the cell of another. Similarly there is nothing striking about the quantity of water present in the cells; there are differences, but they are as great between cells of individuals of the same species as between cells of different types. Nor does the figure obtained for the total amount of water contained in the cell seem to have much to do with the figure obtained for the tonicity of plasma in which the cells haemolyse; again, large numbers of experiments might indicate some effect, but the effect is cer- tainly not at all prominent. It is a very different matter when we come to consider the critical volume at which the cells of the different types undergo haemolysis, for one immediately observes that the more resistant types of cell, e.g., those of man and the rabbit, assume far greater critical volumes than do the cells of the ox and the sheep, which are much more fragile. Calling the normal volume in plasma 100, and taking average figures, the cells of the sheep haemolyse at the critical volume of 126, those of the ox at 130, those of the rabbit at 137, and those of man at 146. There are, of course, individual variations, for the critical volume for rabbit cells may vary between 134 and 138, and that for sheep cells between 124 and 128: the differences, nevertheless, are unmistakable. We therefore arrive at the conclusion that the factor mainly responsible for the different fragil- 394 THE COLLECTING NET [ Vor. VIII, No. 71 ities of the cells of different animals is that different kinds of red cell are able to assume different critical volumes, the more resistant cells, such as those of man and the rabbit, being able to withstand much more distention and stretching of their membranes than are the less resistant cells of the ox or sheep. Other factors, such as the water content, the initial osmotic pressure of the cell interior, or the extent to which the cells can lose osmotically active substances, may contribute to the final result, but their effect is of secondary importance. The fragility of the cells of the various kinds of mammals is thus determined by a factor quite different from that which deter- mines the resistance of any one kind of cell to hypotonic solutions of various substances; in the first case the most resistant cells are actually those which can withstand stretching of their mem- branes most, while in the second case it is the ex- tent to which the cell can adapt itself to its en- vironment by losing osmotically active substances, and not the extent to which the membrane can be stretched, which determines the result. In conclusion, it is interesting to work out the extent to which the membranes of the different kinds of cell can be stretched before they either rupture or become permeable to haemoglobin. Taking average figures, we have: Normal Stretched Extension Animal area area ratio Sheep 462 542 0.18 Ox 60p2 72y2 0.19 Rabbit 72,7 89, 0.24 Man 942 1212 0.28 All these figures for area, of course, refer to the cells in their spherical form. The table shows that the membranes of the cells which have the great- est resistance can be extended to a greater extent without rupturing than can those of the less re- sistant cells, for the extention ratio rises from 0.18 to 0.28, a very considerable increase. At first sight it is difficult to find an explana- tion for this fact, unless we fall back on the very unsatisfactory hypothesis that the structure of the membrane of the human cell, for instance, is dif- ferent from that of the membrane of the sheep cell, and that this difference, whatever it may be, results in the greater extensibility of the former. The whole matter becomes more comprehensible, however, if we put down, side by side, the figures for the volumes of the cell and for the absolute increase in area which can occur before rupture: Animal Volume Increase in area dA/V Sheep 30.3 8.47 0.28 Ox 448 11.4p7 0.26 Rabbit 583 17.32 0.29 Man 86.8 26.7 7 0.31 We thus have the relation dA/V or dR/R* = const. to within a very small degree of error. It is interesting to speculate on the meaning of this relation, and to do so we shall think of the cell membrane as a fluid or semi-fluid film, the permeability of which is governed by the proper- ties of a few layers of molecules, the thickness of this layer being of the order of 0.01, and much thinner than the “morphological membrane” as a whole. This conception is the one which we have to arrive at if we consider Fricke’s results for the capacity of the layer which prevents the mi- gration of ions, and is quite in keeping with all that is known about the membrane. Now let us stretch the membrane as a whole, and with it the thin layer just referred to, and let us take ac- count of the hypothesis advanced by Osterhout, that substances can be drawn from the interior of the cell (or from other parts of the membrane, whose volume is probably proportional to the cell volume) in order to augment, or even repair, the cell surface. Then we shall have what we may call a ‘‘reserve” of material available to be drawn into the thin layer when the latter is stretched, and the quantity of this will be proportional to the cell volume, or, more strictly, to the volume of the membrane as a whole. As the thin layer is stretched, it will be thinned even further, but the “reserves” will be able to make good the deficiency until they are exhausted. The greater cell volume, and the greater the quantity of these reserves, the more will we be able to stretch the thin layer without rupturing it, and this will give us the relation between increase in area and cell volume just referred to. Further, during the stretching of the thin layer and its continuous repair by a redistribution of the “re- serves,” it is not unlikely that the layer should be- come partially permeable to small ions, and this would explain the continuous loss of cations which has been the subject of these lectures. This theory, of course, is highly speculative, but it at least explains the observed facts in a way consistent with what we know about the properties of the membrane and the possibilities of its repair. REFERENCES FOR PAPER 2 1. Brinkman, R. and van Dam, E. 1920a. Studien zur Biochemie der Phosphatide und Sterine I. Bioch. Z., 108, 35. 2. Brinkman, R. and van Dam, E. 1920b. Studien zur Biochemie der Phosphatide und Sterine II. Die Bedeutung des Cholesterins fiir die physikalisch- chemischen Higenschaften der Zelloberflache. Bioch. Z., 108, 52. 3. Saslow, G. 1932. The effect of washing upon the resistance of erythrocytes to hypotonic solu- tions. J. Physiol., 74, 262. SEPTEMBER 9, 1933 ] THE COLLECTING NET 395 4. Jacobs, M. H. 1932. Osmotic properties of the erythrocyte. III. The applicability of osmotic laws to the rate of haemolysis in hypotonic solutions of non-electrolytes. Biol. Bull., 62, 178. 5. Ponder, E. and Saslow, G. 1931. The measure- ment of red cell volume. III. Alterations of cell volume in extremely hypotonic solutions. J. Physiol., 73, 267. 6. Ryvosch, D. 1907. Vergleichende Untersuchun- gen uber die Resistenz der Erythrocyten einiger Saugetiere gegen hamolytische Agentien. Pfliigers Arch., 116, 229. 7. Ponder, E. and MacLeod, J. 1933. J. Physiol., 77, 181. DIscuSSION Dr. Cohen: If cations are lost from the cells, would they not be lost in sufficient quantity to enable them to be determined quantitatively in the external medium? Dr. Ponder: That has been done for muscle, and an excellent correspondence found. In the case of the red cell, it is not quite so easy to do. But it is easy to show that cations are lost intu such a medium as hypotonic glucose, either by analysis, or by conductivity measurements. Dr. Blinks: Wave you, or Dr. Fricke, meas ured the capacity of cells which are swollen? Dr. Ponder: ‘Ve have done so, and it is the same as in the normal cell, per unit area. Dr. Blinks: That would show that the mem- brane does not undergo thinning. Dr. Ponder: Exactly. When its area is in- creased it is not increased by thinning, but by ad- dition of the substances which I call “reserves’ . Dr. Blinks: How long may a definite amount of swelling be maintained ? Dr, Ponder: Once a particular volume is at- tained it is maintained for hours. Dr. Harris: There being differences in the haemolysis curves for different animals, data in terms of the volumes attained at 50 p.c. haemol- ysis, instead of at the beginning of lysis, might give different results ? Dr. Ponder: Yes, but not substantially differ- ent, for, although the percentage haemolysis curves, for the different types of cell, show slightly different scatter, the differences in scat- ter are not very great. Indeed, there are as great differences between ox and ox, say, as there are between ox and rabbbit. END OF COLD SPRING HARBOR SECTION 396 THE COLLECTING NET [ Vor. VIII, No. 71 HETEROCHROMATIC RADIATIONS AND EARLY AMPHIBIAN DEVELOPMENT Roserts RuGu Instructor, Department In dealing with radiations of the Amphibian egg within the limits of the solar spectrum we are immediately concerned with the three prin- ciple light factors, namely: luminous intensity, quality, and radiant energy. This is particularly true of the Frog’s egg which has an abundance of black pigment and therefore represents the theo- retically perfect, non-selective absorption me- dium. The first factor, luminous intensity, refers only to light of the visible spectrum and is the density of luminous flux per unit area known as the In- ternational Candle. Foot-candles by definition re- fer to composite candle-light, never monochro- matic light, and may be measured by photronic photoelectric cells which are sensitive to total luminous intensity of a specific light source. Ac- cording to Cady: “Luminous flux is the rate of flow of radiant energy evaluated with reference to visual sensation” and according to Trotter: “Photometry” (which is the method of measur- ing illumination) “is not measurement of an ex- ternal or objective dimension or force but of a sensation.” Here, then, we are dealing with a sub- jective factor which has interest from the stand- point of special sense organ physiology. The il- lumination factor need not be considered apart from the sense organs adapted to respond to lum- inous intensities. Experimental evidence for this of Zoology, Hunter College is here presented, The second factor, quality or wave-length, is measured by the spectrophotometer. Colored or- ganisms cannot absorb light of the same color and yet the literature is abundant with references to the specifically lethal effect of monochromatic green on green plants, and the beneficial effect of violet, blue, and red on the same plants. Yung has reported similar results with the Frog tad- poles which evidence we are to examine here. The third factor, radiant energy, is measured either with a thermopile or by the paired ther- mometer method. In either case the blackened radiometer is non-selective, being sensitive to all wave lengths of the radiant energy spectrum. In this work such relatively great amounts of energy were involved that the paired thermometer method was used. By this method the direct sun- light at zenith in April (the normal breeding sea- son of Rana pipiens) registers about 25°C. In absolute units 1°C. by this method represents ap- proximately 0.2 gm. cal. per sq. cm. per minute. As a specific example of the distinction be- tween various light factors, we can by direct ex- periment show that 3 cm. of distilled water will not reduce the luminous transmission from a G. FE. —CX lamp in the slightest but will reduce the transmission of radiant energy by 48%. Radiant energy, then, is a physical factor characteristic of CORNING GLASS FILTERS Number’ Spectral Range Color 241 6560-7060 HR. Pyrs red #243 6200-7000 Red #254 lu-3u Infra red #396 4000-6600,2.5u- Light Alko *586 3300-3900 Violet Ultra 774 3650-3u Pyrex Rel. Luminous Trans. Rel. Energy Trans. 14% ft. candles 62.10% C®. 25% “ 66.94% C°. 00% ss SION 38% z 9.86% C°. 00% « 9.80% C®. 95% H 91.65% C°. (Data on other Corning Filters may be secured through the author). E. YUNG’S LIQUID FILTERS Spectral Range Color Aq. gent. viol. 4200-5000 Violet 6600 plus Alc. lyons»bl. 4200-5800, Blue 7000. plus Aq. Nic. Nitr. 5000-6000 Green Aq. K-chrom. 5200-7600 Yellow lus Alc. fuchsine 6400-7600 Red (basic ) plus (In Yung’s experiments the filters included two mission but not luminosity. The absorption of 8 Rel. Luminous Trans. Rel. Energy Trans. 31% ft. candles 45:59 1G 14% zt 43.0% C°. 10% 3 44% C°. 78% 2 39.4% C°. 30% sf 41.0% C°. thicknesses of glass which reduced energy trans- mm. of clear glass was 14% of the radiant energy). SEPTEMBER 9, 1933 ] THE COLLECTING NET 397 radiations from the shortest gamma rays to the longest Hertzian waves while luminous intensity refers only to the narrow limits of human vis- ibility. The following table gives the transmissions of some of the filters used, distinguishing between luminous intensity, quality, and radiant energy. It will readily be seen that there is no correlation whatever. Using the Bunson-Roscoe Law where K = IT and referring to I as intensity in terms of centi- grade degrees on the thermometer and T as time in minutes, the value for K was kept uniform ex- cept where otherwise stated. This meant, for in- stance, that radiations under monochromatic green were twice as long as exposure under the red because of the particular energy transmis- sions of these two filters. From the standpoint of the egg or tadpole there are other factors to be considered. Water alone absorbs radiant energy and because of its specific absorption tendency toward the red and heat rays, radiation of the Frog’s egg must be without intervening water. Further, since Frog jelly is 78% water, we would expect that the jelly would be a protective layer against the heat rays. By direct experiment this proves to be the case. Also, the efficiency of the black pigment to- ward the various light factors should be consid- ered. The following data indicates that as long as K (radiant energy x time) is constant the growth rate of tadpoles will be the same regardless of the wave lengths or luminous intensities used. The eggs and tadpoles were placed in blackened finger bowls immersed in running constant temperature bath and water removed until the upper surfaces of eggs or tadpoles were exposed during radia- tions. Between radiations, eggs or tadpoles were placed in large containers with compartments separated by wire, so that there was diffusion con- tinuity to equalize any food or metabolic condi- tions. RANA PIPIENS Filter Spectral Trans. Lum.Tr. Energy Tr. Dark 00.00 00.00 00.00 1 #774 3650-3u 95.0% 91.65% #554 bl. 3900-4900. 4.0% 24.05% #401 gr. 4800-5800 3.0% 20.04% #349 y-r 5500-7000 52.0% 61.92% #243 red 6200-7000 25.0% 66.94% #254 ir. lu-3u 00.0% 59.19% 15 days 30 days Exp. Min. K Length (mm) Length (mm) 00.00 00.00 15.50 0.00 22.00 0.00 974 125.0 15.58 +0.08 © 22:10 +0.10 24.75 125.0 15.52 +0.02 22.00 0.00 29.69 125.0 15.50 0.00 22.14 +0.14 14.41 125.0 15.52.+0.02 22.10 +0.10 13.34 125.0 15.54 +0.04 22.00 0.00 15.08 125.0 22.12 +0.12° 15.60 +0.10 K = IT where I is intensity in terms of centi- grade degrees and T is time in minutes. (Bunson- Roscoe Law). Experimental data presented led to the follow- ing conclusions : 1. Frog’s eggs, sperm, and zygotes without jelly show essentially the same limits of tolerance of total direct sunlight. 2. While the Frog jelly is permeable to lum- inous intensities of various wave-lengths it is rel- atively impermeable to radiant energy, thereby protecting the egg against heat injury. This is contrary to the traditional function ascribed to the jelly as a device to focus the rays of the sun and increase the temperature of the egg. 3. There is marked sensitivity to radiations at gastrulation expressed in high percentage of cases of spina bifida and failure of the blastopore to close. Many cases of spina bifida reported in the literature as due to Ultra-violet rays may be due to specific heat injury of the dorsal lip by the Infra-red or the entire spectrum of the mercury K = centigrade minutes as measured by the paired thermometer method. Direct zenith midsum- mer sun registers about 32°C. and corresponding midwinter sun about 19°C. are used. This suggestion is supported by the fact that these abnormalities can be induced by mono- chromatic radiations of any wave-length provid- ing the radiant energy is sufficient. 4. The black pigment of the Frog’s egg or embryo is not sensitive to various luminous in- tensities or wave-lengths if the total energy is constant. The absorption efficiency of this pig- ment toward radiant energy will shortly be meas- ured. 5. From the embryological point of view the radiant energy factor is the most important. This probably applies also to forms which are not so efficiently pigmented. 6. Yung’s results, showing the specifically de- leterious effect of monochromatic green and the beneficial effects of violet, blue and red in respect to tadpole growth rate, can be attributed to the technical difficulties which were encountered in 398 THE COLLECTING NET [ Vor. VIII, No. 71 1881 as well as to the fact that he did not realize that the plant food of the tadpoles had a sur- vival and growth curve under colored lights sim- ilar to that of his tadpoles. Also, Yung’s filters had a minimum transmission in the green in re- spect to all three radiation factors, namely: lum- inous intensity, quality, and radiant energy. This would partially explain the deleterious effect of green on the tadpoles as secondary to the effect on tadpole food. This statement would also apply VARIEGATED EYE COLOR IN THE to the beneficial effects of violet, blue and red. 7. The various radiation factors must be more rigidly calibrated and controlled in biological ex- periments so that no longer will luminous inten- sity and radiant energy be confused, or Ultra- violet mean the total radiation from a mercury are. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 22). PARASITIC WASP HABROBRACON Dr. ANNA R. WHITING Professor of Biology, Pennsylvania College for Women The semi-dominant factor shot-veins (sv) arose simultaneously in three different lines after extreme heat treatment of larvae. It causes veins of the wings to be broken up and distorted. Shot- veined stock has proved to be fully fertile and stable. In connection with studies of linkage made in 1931, shot-veins was crossed to white eyes and the F, daughters (Whwh.Svsv) bred as vir- gin. Wild-type (Wh.Sv), white (wh.Sv), and shot-veined (Wh.sv) males appeared in expected ratios and with them the double mutant type (wh.sv) with shot-veins and white eyes, the latter all showing a mottling of red spots in the poster- ior ventral region. These spots always appear in both eyes. Their distribution and intensity show some variation but they can never be seen from the dorsal side. The homozygous white shot-veins stock is called variegated. It has been followed for almost two years and has been found to breed true and to be perfectly viable, in haploid males as well as in their diploid sisters. When white- eyed females are heterozygous for shot-veins va- riegation is present but less extensive. Combinations have been made of white and shot-veins with the other eye color mutants but in no cases does the variegated condition occur ex- cept with three of the four allelomorphs in the orange locus, type (QO), dahlia (o*), and orange (o), and with the carrot allelomorph to white. Amount of spotting and intensity of color de- crease in the orange series from type to orange. Shot-veined females having the factor for white eyes and for its incompletely recessive alle- lomorph carrot: (whwh*.svsv) show the variega- tion on a cream background. Such females heter- ozygous for shot-veins (whwht*.Svsv) show slight variegation on cream background. Several investigators have reported mottling ef- fects in eyes of Drosophila but none so far re- ported resembles the type of variegation here de- scribed which always shows in the same region, is perfectly stable and fully viable in homozygous and azygous (haploid) condition. The dominance of variegation over non-varie- gation suggests that shot-veins is the result of a stable translocation. The possibility of its being a dominant gene mutation is not ruled out, how- ever. The reason for the appearance of red spots in the double mutant type, white shot-veins, is not easy to give with any degree of assurance. It is suggested that the shot-veins factor or condi- tion is associated with the constant habit of so- matic mutation occurring at a definite stage in the development of the compound eye, such that only facets in the posterior ventral region are affected. This would make every eye a mosaic. Mosaicism involving always the same small area of somatic tissue and never the gonads would be required to explain it. It is likewise possible, and to the au- thor probable, that the shot-veins condition or gene has a spotting effect on the eyes when the residual heredity is such as to allow its expres- sion and that the cells of the red region are of the same genetic constitution as those of the white. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 29). SEPTEMBER 9, 1933 ] THE COLLECTING NET 399 EGG-TRINUCLEARITY IN HABROBRACON Dr. P. W. WuITING Professor of Zoology, University of Pittsburgh The majority of mosaics in Drosophila have been shown to result from chromosome elimina- tion in early development. A few examples have appeared however which must be explained by egg-binuclearity. According to the various theories of egg-binu- clearity, the two nuclei from which the embryu develops differ from each other in hereditary con stitution. Thus the parts of the body descended from one cell differ from the parts tracing back to the other. Morgan (1905) suggested that sex-mosaics (gynandromorphs) in bees might arise if the re- duced egg nucleus were fertilized by one sperm nucleus, while a second sperm nucleus segmented independently. Boveri (1915) held that one of the first two blastomeres of a parthenogenetically dividing egg might be fertilized while the other continued in parthenogenetic cleavage. Doncaster (1915) obtained cytological evidence for fusion of two separate eggs of Abraxas. The nuclei of each underwent normal reduction, resulting in a binu- cleate egg with nuclei of different composition. Whiting (1924) assumed that in the origin of male mosaics of Habrobracon, the second polar “nucleus as well as the mature egg nucleus took part in parthenogenetic cleavage. Goldschmidt (1931) presented both cytological and genetic proof of this hypothesis for Bombyx except that both nuclei were fertilized, there being no parthe- nogenesis. Over 300 mosaics have thus far been obtained in Habrobracon and it has hitherto been assumed that these have been developed from binucleate eggs according to the hypothesis proposed in 1924, Evidence has now accumulated that in certain cases at least mosaics arise from trinu- cleate eggs. It may be supposed that three of the four nuclei from one unreduced egg nucleus (oocyte) function in the development of the mo-~ saic embryo. As an example of a mosaic arising from a trinucleate egg the following may be cited : A virgin female heterozygous for honey body color and for stumpy legs produced in addition to the expected wild-type, honey, stumpy, and honey stumpy males, a mosaic male with three legs wild- type, one leg stumpy and two legs honey stumpy. Raymond J. Greb has now obtained several cases of similar trinucleate mosaics. Kathryn A, Gil- more was the first to find an example which must be explained by egg-trinuclearity. In 1931 she ob- tained a mosaic male among the progeny of a fe- (cl/n.d) for the linked genes cantaloup, long, narrow and defective. The left eye was wild-type (black) while the right was cantaloup. Left wings were narrow, right were long defective. Breeding test showed that some of the sperm were cantaloup long defective while some were wild-type in all respects. Thus at least three combinations of the maternal genes male heterozygous were present in the mosaic; +, n, and c.l.d. It is altogether possible that four different com- binations of the maternal factors may occur in a male mosaic, but such a condition has not yet been found. If more than four combinations should oc- cur or if one member of an allelomorphic pair should be found in association with three differ- ent combinations while the other member should likewise be present, it would prove that the mosaic developed from more than one unreduced egg nucleus, thus favoring the hypothesis of Don- caster. It is highly probable that many of the mosaics previously explained by egg-binuclearity may have come from trinucleate or quadrinucleate eggs, but evidence for this would be obtainable only if the mother were heterozygous for more than one gene affecting the same structure. Thus far breeding tests of mosaic males have shown gonads to consist of not more than two of the possible combinations of factors, but embryolog- ical studies of the origin of the gonads indicate that more than two combinations are possible. REFERENCES Boveri, Th. 1915. Uber die Entstehung der Eug- sterschen Zwitterbienen. Arch. Entw. mechan. 41. Doncaster, L. 1915. On the relations between chro- mosomes, sex-limited transmission and _ sex-deter- mination in Abraxas grossulariata. J. Genet. 4. Goldschmidt, Richard. 1931. Die Sexuellen Zwis- chenstufen, Julius Springer, Berlin. Morgan, T. H. 1905. An alternative interpretation of gynandromorphous insects. Science (N. S.) 21. Whiting, P. W. 1924. Some anomalies in Habro- bracon and their bearing on maturation, fertilization and cleavage. Anat..Rec. 29, p. 146. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on Au- gust 31.) 400 THE COLLECTING NET [ Vor. VIII, No. 71 The Collecting Net An independent publication devoted to the scientific work at Woods Hole and Cold Spring Harbor Edited by Ware Cattell with the assistance of Mary L. Goodson, Rita Guttman, Jean M. Clark, Martin Bronfenbrenner, Margaret Mast and Anna- leida S. van’t Hoff Cattell. Printed by the Darwin Press, New Bedford IMPRESSIONS OF TKE THIRD INTERNA- TIONAL CONGRESS FOR EXPERI- MENTAL CYTOLOGY AS OBTAINED FROM PROFESSOR CHAMBERS! In spite of the fact that the General Secretary of the Third International Congress of Cytology, Dr. Rhoda Erdmann, who is editor of Archiv Fiir Experimentelle Zellforschung, was incar- cerated for three weeks in one of Hitler’s con- centration camps near Berlin, the meetings of the Congress were a great success. One of the unex- pected results of the Congress was her release so that she need not function by proxy. The Congress was one of the most successful of its kind largely due to the homogenity of in- terest of a not too large a group of persons in- terested in the functional activities of the cell. There were about 130 attending the meetings, and the excellent planing out of the social activities during the week made all of its members come to know each other well by the end of the week. The evening functions were (1) a reception at Kings College by Dr. Gray, local President of the Congress, and Mrs. Gray. (2) A motion picture presentation of scientific films in the auditorium of the newly erected physiology laboratory of Professor Barcroft. (3) A dinner in the Hall, replete with traditions, of Trinity College. (4) Last, but by no means least, a delightful dancing party at the famous Dorothy Cafe, at which the ladies of Cambridge served as hostesses. The Congress was opened by the Mayor of Cambridge, Mrs. Keynes, mother of J. Maynard Keynes, the distinguished English economist, and mother-in-law of Professor A. V. Hill. Mayor Keynes also graced the Congress with her pres- ence at the dinner in Trinity and at the Presen- tation of the films. Among the Americans present were: Drs. Har- rison, Streeter, Brooks, Lund, Hope Hibbard, Mary J. Hogue, Chambers, Speidel, Beutner, Raymond Parker, and H. Pinkerton. All Europ- ean nationalities were well represented, including Soviet Russia. 1 Dr. Chambers and Dr. Carrell served on the or- ganizing committee appointed by the previous Con- gress which convened in Amsterdam in 1930. The former presented a paper at Cambridge on “Some features of cell permeability in relation to kidney function.” Selected topics for the meetings gave a well rounded picture of cellular physiology. The first day was devoted to cell respiration and metabolism which included metabolism in plant tissues, bacteria and amphian ova. The second day dealt with the relation of cel- lular structure to function. Electro-physiology, the topic of the third day, was started by Professor Adrian who made a unique demonstration which enabled the audience to hear, in the form of varying booming sounds, the response of successive muscle fibres through a needle electrode inserted into the fleshy part of his own arm. The fourth day dealt principally with develop- mental mechanics in which one of the interesting papers was that of Dr. and Mrs. Needham and C. H. Waddington who presented evidence of having obtained cell-free aqueous extracts, pos- sessing organizer-activity in early embryonic de- velopment. The Golgi apparatus furnished a topic for considerable discussion in which Irish eloquence played a prominent role. The fifth day dealt with animal and plant vir- uses, a topic which stimulated interest among the cytologists because of observable effects in the nuclear and cytoplasm of living cells caused by the activity of viruses. One of the many interesting motion pictures was that of Dr. F. M. L. Sheffield of Herpendon in which the progressive increase of coagulum- like lumps could be observed forming in the pro- toplasmic strands within hair cells of a plant which had been infected with a virus disease. Each afternoon tea was served in the Depart- ment of Pathology. A large series of very inter- esting demonstrations were open to view in an adjoining room. Of especial interest were slides of Dr. Holtfreter from Spemann’s laboratory which showed the organizing activity of pieces of a variety of foreign tissues planted under the skin of larval frogs. A yery significant demonstration of the physi- cal properties of protein films was that of E. K. Rideal, professor of physical chemistry at Com- bridge, in which he showed the effects of enzyme action in changing their properties. A new and simple form of a micro-manipula- tor to be used with the ultrapak nucroscope was demonstrated by Dr. Himmelweit, who has been given an asylum in St. Mary’s Hospital because of his sudden and forcible expulsion from the Pathological Institute in Berlin. There were several conducted parties to points of historical interest such as the colieges, Ely Cathedral, etc., the attraction of which proved to be a sore temptation for some members to absent themselves from meetings. SEPTEMBER 9, 1933 | THE COLLECTING NET 401 Among those who took active part in the ar- rangements of the Congress were Dr. and Mrs. Shearer. Dr. Shearer, who is well known for his contributions to experimental embryology, knows the Marine Biological Laboratory from the days of its inception tor he was a student of the late Professor Whitman and took part in many of the pranks instigated by the youngsters of those days such as E. B. Wilson, Jacques Loeb, T. H. Morgan, ete. He was present when Professor Whitman rescued the Laboratory from its financial difficulties at the time when influen- tial Bostonians sought to destroy it. For the extraordinary success of the running of the Congress credit is due to the members of the Strangeway’s Research Laboratory, especially to Dr. Honor B. Fell and Dr. F. G. Spear. Dr. kell visited Woods Hole last summer. Mention must also be made of the services of Professor D. Keilin, professor of cellular biology at Cam- bridge, Dr. and Mrs. Needham of Sir Gowland Hopkins’ Laboratory, and Dr. R. A. Webb of the Department of Pathology at Cambridge. Dr. and Mrs. Gray, well known to those of us at Woods Hole, together with their enthusiastic group of colleagues, are to be congratulated in making the Third International Congress for Ex- perimental Cytology one of the most successful of its kind. ‘heir delightful hospitality will long be remembered. The perfect weather, the beauti- ful gardens, and the uniqueness of the old univer- sity town of Cambridge, combined with the other features, made the visit there an unforgettable occasion. A full report of the papers presented together with discussion of the papers will be printed in Erdmann’s journal. Dr. Chambers had as his companion on the train from London to Cambridge a prominent German biologist who was thrown out of his position as Privat-dozent at a German University. During their conversation on the train Dr. Cham- bers learned that he was in the tropics during the Great War, but that he rushed back to his Father- land to join the conflict. He went through the entire period of the war in which he distinguished himself and was awarded the Iron Cross. He carried with him his honorable discharge from the Army. He was a member of one of the time- honored student corps during his university ca- reer and had prominent scars on his forehead and cheeks as testimony of numerous sabre contests. He is a typical Prussian in appearance, but be- cause one of his grandmothers had Jewish blood he was thrown out of his position in one of the German universities. . His only recompense for his war service is permission, to start up medical practice where and when he can. ITEMS OF INTEREST Among the investigators still in Woods Hole on Monday, September 18 were: W. R. Amber- son, Louise S. Armstrong, P. B. Armstrong, G. A. Baitsell, L. G. Barth, L. V. Beck, Louise E. 3oydon, G. N. Calkins, W. Cattell, R. Chambers, T. T. Chen, F. E. Chidester, Eleanor Clark, E. R. Clark, Frances Clark, G. H. A. Clowes, E. G. Conklin, S. A. Corson, Margaret Crane-Lillie, K. Dan, W. L. Doyle, Dorothy Francis, H. J. Fry, W. E. Garrey, Ethel B. Harvey, E. N. Harvey, L. V. Heilbrunn, Ella N. Hoppe, H. E. Howe M. H. Jacobs, J. M. Johlin, Takeo Kamade, J. B. Katz, Anna K. Kelch, F. R. Lillie, R. S. Lillie, Ruth S. Lynch, S. O. Mast, A. P. Mathews, J. A. Miller, F. B. Moreland, Lilian V. Morgan, ‘I. H. Morgan, Helen kK. Newton, J. F. Nonidez, C. Packard, G. F. Papenfuss, S. E. Pond, G. S. de Renyi, Florence M. Scott, F. M. J. Sichel, H. Specht, H. B. Steinbach, C. R. Stockard, O. S. Strong, Miss E. M. Vicari, Lucille W. Wade, and Edith M. Wallace. Dr. R. F. Pitts, who took the course in physi- ology here in 1930, has recently been appointed instructor at the New York University Medical School. Dr. Kenneth S. Rice was recently appointed as the head of the department of zoology at the University of Maine, where he has worked for the last six years, succeeding Dr. D. B. Young who has accepted a position at Washington Uni- versity. Dr. Ancel B. Keyes has been appointed instruc- tor in the biochemical sciences in the Fatigue La- boratory of Harvard University. Dr. Keyes has held a National Research Council Fellowship for the past two years, working in 1930-31 in Copen- hagen at the Laboratory of Zoophysics under Dr. Krogh, and in the following year under Dr. Bareroft at Cambridge. The Imperial University of Tokyo has dele- gated Mr. Takeo Kamade to visit the marine laboratories of the United States in order to in- spect their equipment with a view to aiding the planning of similar laboratories in Japan. Mr. Kamade is now visiting Woods Hole. Dr. Ralph A. Kekwick, who has been studying for the past two years at New York University and Princeton, occupied the last few months of his tenure of a Commonwealth Fund Fellowship at Woods Hole in the investigation of the perme- ability of drbacia eggs under anaerobic condi- tions. He has returned with Mrs. Kekwick to the University of London, where he will be en- gaged in teaching biochemistry. 402 THE COLLECTING NET { Vor. VIII, No. 71 THE EFFECT OF X-RAYS UPON CELL OXIDATIONS Dr. Leon C. CHESLEY Biophysical Laboratory, Memorial Hospital, New York, N. Y. Exposure of young organisms to sufficiently large doses of X-rays inhibits subsequent devel- opment. In considering the primary seat of at- tack of radiation in the cell, it is only natural that attention should be directed to the energy releas- ing mechanisms; of these, respiration is predom- inant, The view is commonly held that the flux of ac- tivity—so characteristic of living protoplasm—re- quires free energy. In the cell, free energy is ob- tained chiefly by the processes of oxidation. Hence to study fundamental life processes, study respiration and factors controling it, as respira- tion is one of the ultimate processes governing vital activity. Loeb proposed that the essential feature of fer- tilization is the increasing rate of oxygen con- sumption which causes or permits development to proceed. This hypothesis is based upon certain of his findings in the sea urchin egg. The litera- ture is reviewed and discussed at length by Whit- aker (J. Gen. Physiol. 16:497). It seemed profitable to investigate the effect of radiation upon respiration, particularly as iron is so important in the respiratory process. Presum- ably iron exerts its catalytic effect by alternate oxidation and reduction, i.e. by the gain and loss of electrons. The primary action of X-rays is to ionize the atoms and molecules in the permeated medium by this very process of electronic change ; the action is most marked on the heavy elements, such as iron. It was thought that some light might be shed upon the problem of development by a study of X-ray effects upon respiration and development concomitantly. There is but little in the literature which. bears directly upon this question. Most of the work dealing with radiation effects upon res- piration has been done on mature differentiated cells. Recorded results are discordant. Hubert (Pfliiger’s Arch. 223 :333) found that exposure of chick embryos to X-rays affected growth before glycolysis. Adler (Strahlentherapie 36:1) and Crabtree (Report Imp. Cancer Res, Fund 10:33) aemon- strated that while respiration is finally influenced by irradiation, there is a latent period of eight to twelve hours before any effect is noticed. Redfield and Bright (J. Gen. Physiol. 3 :297) studied the effects of radium emanation upon COz production and growth in the radish seed. They found that “changes in the rates of COs production and cell division do not always go hand in hand. One is increased by exposures which retard the other.” In the present study, the oxygen consumption was determined at different times after irradia- tion in wheat seedlings and the eggs of Arbacia and Chaetopterus. Vhe anaerobic metabolism of the eggs was studied as well. All measurements were made manometrically, using the Barcroft- Warburg apparatus. In the wheat seedling experiments, the dose of X-rays used (2260 r) was sufficient to inhibit growth by forty per cent., as measured twenty- four hours after irradiation. The routine em- ployed in preparing the seedlings for irradiation has been described by Failla and Henshaw (Radiology 17:1). Wheat seeds were soaked for an hour and placed into moist chambers at 26°C for eighteen hours in the dark. The seedlings were then selected for uniformity, irradiated, and put into moist chambers which were kept at 26°C or 6° C. Respiration was determined 4, 24, 48, and 72 hours after irradiation. Fresh weight was determined by stripping the sprout from the grain and weighing at once. Arbacia eggs were obtained by cutting the ani- mal in two equatorially and inverting the upper half in a syracuse watch glass; mature gametes are then shed. The eggs were washed and concen- trated to one volume in forty of sea water, and irradiated (22,000 r). Chaetopterus eggs were collected in the usual way and treated in the same manner. Anaerobic metabolism was studied by adding methylene blue (final concentration 0.005 per cent) to the egg suspensions. Barron (J. Biol. Chem. 81:445) states that the resulting increase in Oy, consumption is proportional to the anaero- bic metabolism. Under all conditions investigated, the results were the same. Irradiation sufficient to impede or stop growth has no effect upon cell oxidations. While growth in the wheat seedling was inhib- ited forty per cent., the oxygen consumption per gram fresh weight was the same in control and irradiated samples. This result was corroborated in another way. The seedlings kept at 6°C did not grow perceptibly. When the oxygen consump- tion for this series was determined, it was found to be the same in control and irradiated seedlings whether calculated on the basis of fresh weight or per seedling. In the cases of the marine eggs, the doses given were sufficient to kill the embryos after a SEPTEMBER 9, 1933 ] THE COLLECTING NET 403 period of a day or two. Early development was seriously hampered. In the Arbacia eggs, cleavage was considerably delayed and abnormal cleavages were frequent. The largest dose used (65,000 r) was almost immediately lethal to Abracia eggs. No effect upon respiration was found. All measurements of respiration of eggs were made within six hours of irradiation. This should detect any direct influence that irradiation might have on respiration. Later changes in metabolism are almost certainly secondary, if, indeed, they occur at all. As for anaerobic metabolism, no constant re- sults have been obtained. However, there is ap- parently no influence of radiation upon this phase of cell activity. These experiments do not prove that growth and development are independent of respiration. Development may depend upon several factors, concomitantly or concatenately, of which respira- tion is but one. One or more of these factors is radiosensitive to such a degree that doses of X-rays may stop development without affecting respiration. We can conclude only that respiration certain- ly, and all of the energy releasing mechanisms probably, are not the seat of radiation attack in the cell. If, and when, respiration is affected by irradiation, it is a secondary reaction. (This article is based upon a seminar report pre- sented at the Marine Biological Laboratory on August 22). BOOK REVIEW Science and Sanity. An Introduction to Non- Aristotelian Systems and General Semantics. A\- fred Korzybski. Ca. 800 pages. $7.00. The Science Press, 1933. This interesting and original book deserves careful study by all who are concerned with science and its applications. It is unusually com- prehensive in its scope, and any adequate survey of its contents would require a much fuller review than is here possible. Broadly speaking, two main fields of general scientific interest are cov- ered: first, the, methods, aims and presuppositions of scientific method in general; and, second, the applications, actual and possible, of science in the individual and social life of human beings. Al- though not primarily biological, the book is per- vaded by the biological conception of life as con- sisting primarily in the adjustment of organism to surroundings. The failures and difficulties of modern society are largely the sign of deep- seated biological maladjustments which it would be quite possible to correct by the intelligent and systematic application of existing scientific knowl- edge. The methodological part of the book is re- markable chiefly for the author’s vigorous advo- cacy of what he calls “non-Aristotelian” methods of logical procedure. He exposes clearly the fallacies which arise from a too exclusive de- pendence on reasoning of the classificatory or syl- logistic type derived from Aristotle, with its tacit assumption of the equivalence or essential identity of all units belonging to a given class. Individuals of the same class usually receive the same verbal designation or name; and being thus identical in name it is automatically taken for granted that they are identical in all other res- pects. False identifications and failures of dis- crimination inevitably follow. The author wages a vigorous polemic against the habits of non-dis- crimination based on the unreflecting acceptance He shows that when- ever there is an unconscious or automatic identi- of purely verbal reasoning. fication of the symbol (word, image, diagram, formula) with the thing symbolized, fallacies of this type are likely to arise. Verbalism, with its many pitfalls and confusions, is clearly analyzed. Evidently the affixing of the same label to two different objects does not render them identical in their actual properties and activities. The only permanently valid, i. e., scientific, procedure is to face the reality in an objective and disinterested spirit and not be deflected from a realization of its true character by the representative signs em- ployed. “Words are simpler and take less effort to handle than objects.” (p. 480) But real things “are what they are”; their existence is prior to their designation; real existence is always indef- initely complex; hence all mental representation of real objects, including scientific representation, necessarily involves abstraction, 7. e., a singling out of partial features or aspects for special or exclusive attention and a neglect of other aspects. On the objective level things have their own actuality which is only partially representable. In other words, in all mental representation of real- ity—t. e., in all knowledge—certain characters of the reality are-“left out” of consideration. The truly educated person is conscious of this; the verbalist is not; he attaches more importance to the sign or word than to the actuality. Thus being a liar or thief seems to the unregenerate man a matter of less importance than being called one; he is hugely indignant at having the label attached, while having little or no objection to ex- emplifying the deplorable actuality in his own 404 THE COLLECTING NET [ Vor. VIII, No. 71 person. This is one illustration of the manner in which verbalism deflects attention from reality and so makes for confusion and falsification, with often disastrous consequences. As Count Korzyb- ski puts it (p. 486), “the ignorant or pathological use of language is a public danger.” As a counter- measure he would have children early trained in habits of discrimination, based on close observa- tion of actual things rather than on the study of language alone. They should be made to realize that things exist on their own objective or “un- speakable” level and have their own obstinate reality to which we must learn to adjust ourselves. In his dealings with things, the educated person should be “conscious of abstraction”; naming, classifying and reasoning necessarily involve ab- straction and hence incompleteness, and he is al- ways aware of this. Such a person is immune to the virus of verbalism. The basis of true knowledge is discrimination—not the equalization or identification of differents. The importance of this “principle of non-identity” is insisted on throughout the book. The legitimate use of abstraction, for the pur- pose of gaining real insight into and control over actuality, is the special province of science; and the nature of scientific abstraction is considered at length, with a wide range of illustrations from mathematics and the natural sciences. The dis- cussion of mathematical procedure and its appli- cation in the sciences of nature is very full and clear. Mathematics gives the most exact and complete account of the purely formal or struc- tural side of nature, i. ¢., of the orderly and permanent conditions exemplified by all pheno- mena. Permanently valid or scientific knowledge is always knowledge of structure and relations rather than of complete reality. Such knowledge consists essentially in a formal correspondence, best expressed mathematically whenever possible, between the representation and the reality repre- sented. Since knowledge is correspondence or ad- justment, we should expect to find certain definite parallels between the structure of the nervous system (the chief physiological instrument of ad- justment) and the general structure of the ex- ternal world; and in an interesting section of the book these parallels are described and discussed. The fundamental problems of physical science have reference to the general or foundational characteristics of world-structure ; and in the con- cluding section the author gives a concise and lucid account of the elements of quantum theory and relativity. The applications of science to the general prob- lems of human society meet with the obstacle that many classifications and social procedures, which to the objective and disinterested view of science appear fundamentally fallacious, are based on conceptions of long standing which are sanctioned or stabilized by language and traditional usage. Hence they become the objects of misplaced and often passionate loyalty. There is also the general human tendency to undue simplification, and to action based on over-simple conceptions rein- forced by verbalisms. Men are actuated by language and tend to identify and to treat alike all things that are similarly labeled. Other im- portant fallacies are based on identity of money value; men regard as equally valuable and desir- able all things which have the same price, or all activities which have the same remuneration. For permanent social progress a far-reaching revision of concepts is required; this revision requires in its turn an extensive program of reform in edu- cation, economics and government. It is largely for lack of sound scientific control that political, social and economic affairs meet with such fre- quent collapse or failure. As the author remarks toward the end of his book (p. 538): “world affairs have seemingly come to an impasse, and probably without the help of scientists, mathema- ticians and psychiatrists included, we shall not be able to solve our urgent problems soon enough to prevent a complete collapse.” Ravpu S, LILvir. Department of Physiology, University of Chicago. SCIENCE AND PROGRESS (Continued from Page 356) nesses, the duel, and a thousand other customs of savagery and barbarism, is due not to eugenics, but to education—and war can be banished by the saine means. Fortunately the opportunities for world wide education were never so good as they are today. The printing press, the telegraph, the telephone, radio, moving pictures, rapid transit on land and sea or in the air, have put information concern- ing the whole world within the reach of everyone. World opinion can now be formed and expressed, not years and centuries after an event, but while it is happening. No nation can long stand against the sober judgment of the majority of mankind. Japan and Germany are showing that they are sensitive to world opinion. There is great force in what our Declaration of Inde- pendence has so well expnessed in the phrase, “A decent respect to the opinions of mankind.” This is no easy or rapid cure for the ills of the world, but it is the only rational one. Science and education, knowledge and ethical character, are the chief hopes of human progress. , al ES PELE : Ag - aH a ia