LABORATORY TECHNIQUE IN BIOLOGY AND MEDICINE LABORATORY TECHNIQUE IN BIOLOGY AND MEDICINE BY E. V. COWDRY Research Professor of Anatomy and Director Wemae Cancer Research Laboratoru, Washington University, St. Louis THIRD EDITION BALTIMORE THE WILLIAMS & WILKINS COMPANY 1952 Copyright, 1952 The Williams & Wilkins Company Made in the United States of America Published 1943 Second Edition 1948 Third Edition 1952 First Edition known as Microscopic Technique in Biology and Medicine Composed and Printed at the WAVERLY PRESS. INC. FOR The Williams & Wilkins Compant Baltimore, Md., U. S. A. PREFACE TO THE THIRD EDITION The request by The Wilhams & Wilkins Company to prepare a Thh-d Edition so soon after the Second Edition was pubhshed (1948) was unexpected. It would have been a simple matter for them simply to reprint the Second Edition in whatever number they thought that they could sell within a reasonable period. This, however, would have been a short-sighted policy for there have been great advances in laboratory technique since the material for the Second Edition was assembled in 1947 prior to publication in 1948, which advances should be in- cluded. In fact the advances made, or reported, in the four years 1948, 1949, 1950 and 1951 are probably greater than those accomplished in any previous 10 year period. Though some of the modifications of standard techniques that proved useful during the war as well as altogether new techniques were published before 1948, many, while known to specialists in various fields, had not been gathered together in convenient form from which laboratory workers as a whole could select those most hkely to be helpful in their particular problems. It is hardly necessary to note that the vast amount of new work in the four years mentioned, reflecting the appreciation of the value of research in medicine and biology gained in the war, and implemented by an unprecedented outpour- ing of funds for research, has resulted in the discovery of new and better means of revealing the structure of organisms from the highest to the lowest in health and disease. The electron microscope has found its way into about 300 labora- tories. Satisfactory methods for cutting the extremely thin sections required have been devised. There is a rapprochement between what one can see at very high magnification and increasing knowledge of molecular structure and orientation determined by several methods. The phase microscope likewise has been produced in quantity in this country. The quality of moving pictures of living cells has thereby been greatly unproved. Thanks to the Atomic Energy Commission radioactive isotopes have been made available. Plastics have been introduced in great variety. Microscopic localization of enzyme activities has been advanced. Microchemistry has leaped ahead. Quantitative analyses of extremely small amounts of material reduce the gap between chem- istry and microscopy. The separation and collection en masse in a condition suitable for analysis of certain cellular components has been most helpful. And so on ahnost without end. Obviously no single individual can authoritatively present these new tech- niques, as well as mjTiads of others of great value, because he cannot have personal experience with all of them. Partly to compensate for such limitations I have included descriptions of some of the key techniques written by the in- vestigators who introduced them, or by others who have had extensive experience in their use. Most of these descriptions are new while others are revisions of accounts given in previous editions of this book. The name of each contributor Vl PREFACE TO THE THIRD EDITION is given followed by the address (so that questions can be asked if desired) and the date (so that it can be seen at a glance when it was last checked for improve- ments). To all of these friends I am very grateful. Proofs of their contributions have not been submitted to them for corrections, because there are so many such accounts — ^most of them very brief — and the material presented by them was clear and concise. For typographical errors the responsibility is to be shared by me and the publisher. Most of the text I have written or revised and in doing so I have relied heavily on the reference books listed on page xxxi. It is hardly necessary to explain that the policy is to provide brief accounts of techniques, and leads to others, and to make this information quickly available by alphabetical arrangement. It is better to give some data not required by experts than it is to write mainly for well-informed but limited groups. Ob- viously the said experts must approach fields other than their own as beginners handicapped by unfamiliarity with speciahzed technique and equipment. Be- cause the several biological and medical sciences have so much to offer each other in the way of laboratory technique this exposure of opportunities may facilitate inter-specialty cooperation. My thanks are due to Mrs. Theresa Bresnahan for help in preparing the manuscript. E. V. COWDRY St. Louis COOPERATION My friends have generously contributed techniques written by them as follows : G. Adolph Ackerman, Hinsman Hall, Ohio State University, Columbia 10, Ohio, May 24, 1951. Auer Bodies. Paul M. Aggeler, University of California Medical Center, San Francisco 22, California, November 15, 1951. Blood Platelets. Glenn H. Algire, U. S. Public Health Service, Bethesda Maryland, June 15, 1950. Transparent Chamber Techniques. James W. Bartholomew, Department of Bacteriology, University of Southern California, Los Angeles 7, California, July 9, 1951. Gram Stains Mechanism. H. W. Beams, Department of Zoology, University of Iowa, Iowa City, Iowa, September 27 1951. Ultracentrifuges. R. Bogoroch, Department of Anatomy, McGill University, Montreal, Canada, September 12, 1951. Radioautographic Technique (With C. P. Leblond). L. R. Boling, Department of Anatomy, Washington University School of Dentistry, St. Louis 10, Missouri, December 16, 1950. Teeth Blood Vessels. Teeth Decalcification. Geoffrey Bourne, Department of Histology, London Hospital Medical School, London, England, November 5, 1951. Golgi Apparatus. Mitochondria. Marian Pfingsten Bryan (Mrs. WilhamT. K. Bryan), Department of Otolaryn- gology, Washington University School of Medicine, St. Louis 10, Missouri, September 20, 1951. Ear Cell Smears. Nasal Cell Smears. E. J. Carey, Department of Anatomy, Marquette University School of Medi- cine, Milwaukee, Wisconsin, 1942 (now deceased). Carey's Method for Motor End Plates. Christopher Carruthers, Division of Cancer Research, Washington Uni- versity, St. Louis 10, Missouri, May 12, 1950. Oxidation-Reduction Potential. Vitamins. Polarographic Technique. vii 67B79 VUl COOPERATION Jane E. Cason, Department of Pathology, Medical College of Alabama, Bir- mingham, Alabama, January 27, 1951. Mallory-Heidenhain Rapid One-step Stain for Connective Tissue. Robert Chambers, Department of Biology, Washington Square College of New- York University, New York, New York, May 15, 1950. Micromanipulation. E. R. Clark, Department of Anatomy, University of Pennsylvania, and the Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania, No- vember 28, 1951. Transparent Chamber Technique. Barry Commoner, The Henry Shaw School of Botany, Washington University, St. Louis 5, Missouri, November 27, 1951. Microspectrophotometry. A. H. Coons, Department of Bacteriology and Immunology, Harvard Medical School, Boston, Massachusetts, August 31, 1951. Antigen Localization. W.P. CovELL, Departments of Anatomy and Otolaryngology, Washington Uni- versity School of Medicine, St. Louis 10, Missouri, October 26, 1951. Ear. E. W. Dempsey, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, February 26, 1951. Cholinesterase. Dehydrogenase. Enzymes. Esterases. Nucleases. Phosphatases. W. T. Dempster, Department of Anatomy, University of Michigan, Ann Arbor, Michigan and R. C. W^illiams, Department of Biochemistry, University of California, Berkeley, California, June 9, 1950. Shadow Casting. 0. H. Duggins, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, May 18, 1950. Hairs (With Mildred Trotter). F. Duran-Reynals, Department of Microbiology, Yale University School of Medicine, New Haven, Connecticut, October 8, 1951. Spreading Factors. W. R. Earle, National Cancer Institute, Bethesda, Maryland, July 10, 1951. Tissue Culture. LXrus Einarson, Normal-Anatomisk Institut, Aarhus Universitet, Aarhus, Denmark, February 27, 1951. Gallocyanin-Chromalum Staining of Basophilic Cell Structures. COOPERATION IX Joseph A. Falzone, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, October 26, 1951. Desoxyribose Nucleic Acid, Method for Determination In Isolated Hepatic Nuclei. Differential Centrifugation of Cell Particulates. Honor B. Fell, Strangeways Research Laboratory, Cambridge, England, June 8, 1951. Organ Culture in Vitro. F. H. J. FiGGE, Department of Anatomy, University of Maryland Medical School, Baltimore, Maryland, October 10, 1951. Porphyrins. Hematoporphyrin. H. L FiRMiNGER, Pathology Section, National Cancer Institute, Bethesda, Maryland, February 9, 1951. Carbowax Embedding. E. D. Gardner, Department of Anatomy, Wayne University School of Medi- cine, Detroit, Michigan, June 15, 1950. Articular Nerve Terminals. David Glick, Department of Physiological Chemistry, University of Minne- sota Medical School, Minneapolis 14, Minnesota, October 17, 1951. Linderstr0m-Lang, Kaj. U., and Holter Heinz, Histochemical Advances. Morris Goldman, Department of Parasitology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland, January 29, 1951. Iron Hematoxylin Single Stain. G. Gomori, Department of Medicine, University of Chicago, Chicago, lUinois, May 7, 1950. Gomori's Method for Reticulum and Acid Phosphatase. A. R. Gopal-Ayengar, Barnard Free Skin and Cancer Hospital, St. Louis, Missouri, September 10, 1946 (now Tata Memorial Hospital, Bombay). Chromosomes. Hyaluronic Acid. H. S. N. Greene, Laboratory of Pathology, Yale University School of Medi- cine, New Haven, Connecticut, September 11, 1951. Anterior Chamber Transplantation. Joan Haberman, Parkland, Washington, March 10, 1951. Anethole Clearing Agent. J. D. Hamilton, Department of Medical Research, University of Western On- tario, London, Canada, February 13, 1951. Cell Measurement, The EUiptometer. N. L. Hoerr, Department of Anatomy, School of Medicine, Western Reserve University, Cleveland 6, Ohio, November 28, 1951. Frozen -Dehydration Method. Isolation of Mitochondria. X COOPERATION R. D. HoTCHKiss, The Rockefeller Institute for Medical Research, New York 21, New York, November 14, 1951. Polysaccharides. M. H. Knisely, Department of Anatomy, University of South Carolina, Charles- ton, South Carolina, June 27, 1950. Quartz Rod Technique for Illuminating Living Organs. N. B. KuRNiCK, Department of Medicine, Tulane University, New Orleans 12, Louisiana, January 31, 1951. Aceto-Orcein-fast Green. Edward L. Kuff, Department of Anatomy, Washington University School of Medicine, St. Louis, Missouri, December 18, 1951. Nucleic Acid-Dye Interactions. A. I. Lansing, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, October 5, 1951. Collagenic Fibers. Elastic Fibers. A. Lazarow, Department of Anatomy, Western Reserve University School of Medicine, Cleveland, Ohio, November 28, 1951. Separation of Cell Components by DiflFerential Centrifugation. C. P. Leblond and R. Bogoroch, Department of Anatomy, McGill University, Montreal, Canada, September 12, 1951. Radioautographic Technique. R. D. LiLLiE, Division of Pathology, National Institute of Health, Bethesda, Maryland, May 8, 1950. Azure or Toluidin Blue Eosin. A. M. Lucas, U. S. Regional Poultry Research Laboratory, East Lansing, Michi- gan, August 10, 1951. Cilia (with M. S. Lucas). M. S. Lucas, Department of Biological Science, Michigan State College, East Lansing, Michigan, August 10, 1951. Cilia (with A. M. Lucas). Plastics. B. J. LuYET, Department of Biology, St. Louis University, St. Louis, Missouri, January 15, 1951. Revival after Ultra Rapid Cooling. C. C. Macklin, Department of Anatomy, University of Western Ontario, Lon- don, Ontario, Canada, November 28, 1951. Alveolar Epithelium. Gash Irrigation Recovery Method Alveolar Fluid. for Lung CeUs (GO- Alveolar Foam Cells. Heart Failure Cells. Alveolar Pores. Lungs, Uncollapsed, Fixation. Ammoniacal Silver. Pneumonocytes. Aquax. Silver Lineation on Pulmonary Al- Bronchiolar Epithelium. veolar Walls. COOPERATION XI Carmine Dusting. Tissue Phagocytes. Dust Cells. Vacuoloids. Wash-out Recovery Method. Paul Masson, Department of Pathology, University of Montreal, Montreal, Canada, October 24, 1951. Masson's Trichrome Stain. Morris Moore, Barnard Free Skin and Cancer Hospital, St. Louis, Missouri, December 10, 1951. Fungi. Norman Moskowitz, Department of Zoology, University of Pennsylvania, Philadelphia, Pennsylvania, January 24, 1951. Protein Silver for Staining Protozoa. J. L. O'Leary, Department of Neuropsychiatry, Washington University School of Medicine, St. Louis 10, Missouri, May 8, 1950. Golgi-Cox Method. Golgi Method, Quick. O'Leary's Brazilin Method. Jean Oliver, Department of Pathology, State University of New York, Brook- lyn 2, New York, September 4, 1951. Kidney. Donald L. Opdyke, Department of Anatomy, Washington University School Medicine, St. Louis 10, Missouri, November 8, 1951. Keratohyalin Granules, Separation and Analysis. Robertson Pratt, University of California, College of Pharmacy, San Fran- cisco, January 29, 1951. Triphenyltetrazolium Chloride. Eugene Roberts, Division of Cancer Research, Washington University, St. Louis 10, Missouri, July 15, 1951. Paper Chromatography. T. B. Rosenthal, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, June 6, 1951. Fluorescence Microscopy. Radioactive Isotopes. Electron Microscopy. C. H. Sawyer, Duke Hospital, Durham, North Carolina, December 16, 1950. Safranin-Light Green. Francis 0. Schmitt, Department of Biology, Massachusetts Institute of Tech- nology, Cambridge, Massachusetts, May 19, 1950. Polarization Optical Method. Gordon H. Scott, Department of Anatomy, Wayne University School^of M«di-^ cine, Detroit, Michigan, January 16, 1951. yWo^'^^' '"^ '-- /^ Altmann-Gersh Frozen Dehydration Method. /^, ^s Hq "f^py Microincineration. !>• ^ ^ \ '^ Cryostat. / ^^' / i r* -if \ ■ ;■ ■'• / ^~: Xll COOPERATION W. M. Shanklin, American University of Beirut, Beirut, Lebanon, March 30, 1951. Pineal. Silver Diaminohydroxide after Sensitizing with Sodium Sulfite for Neu- roglia. R. E. Stowell, Department of Oncology, University of Kansas Medical Center, Kansas City 2, Kansas, January 19, 1951. Photoelectric Microphotometer. Charlotte M. Street, Department of Anatomy, Cornell University Medical College, New York, New York, May 21, 1951. Papanicolaou Techniques in Exfoliative Cytology. Lloyd E. Thomas, Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri, July 8, 1951. Arginine Reaction. Mildred Trotter and O. H. Duggins, Department of Anatomy, Washington University School of Medicine, St. Louis 10, Missouri, May 8, 1951. Hairs. L. F. Wicks, Veterans Administration Hospital, Jefferson Barracks, Missouri, February 1, 1951. Hydrogen Ion Indicators. Piccolyte Resins. G. B. WiSLOCKi, Department o Anatomy Harvard Medical School, Boston 15, Massachusetts, March 8, 1951. Placenta. R. C. Williams, Department of Biochemistry, University of Cahfornia, Berkeley, California, June 9, 1950. Shadow- Casting (with W. T. Dempster) PREFACE TO THE FIRST EDITION What appeared altogether impossible twenty-five years ago has in several cases been attained by improvements in technique. Who would have believed at that time that ultramicroscopes would now be manufactured in quantity, built without any optical lenses, and capable of revealing a world of structures quite beyond their ken? Who would have thought that a whole series of dif- ferent atoms could be tagged and their distribution to the several tissues, when introduced into the body, accurately measured? Who would have anticipated the significant and unexpected new developments which have been made in polarization optical methods? Had we been told twenty-five years ago that the cell itself can be broken up into parts several of which can be collected in quantity and chemically analyzed, we would have been incredulous. All this and more has been achieved as a result of team work between the biological and physical sciences. And we may believe that more surprises are in store. Yet some of us individually are still extraordinarily conservative in the methods we use. The possibilities of improving old techniques, of replacing some of them by new ones and of reljdng more upon microchemical and physical procedures are not explored as they should be. The purpose of this book is to extend the horizon by exposing in an introductory way a few of the many oppor- tunities awaiting workers in biology and medicine interested in the minute struc- ture of living things. Success will depend upon ability to anticipate and meet the needs of those likely to consult it. Definite information about specific matters is likely to be more in demand than general statements. The latter are limited to a few pages and deal with "choice of methods" and "organization of laboratory." Some may turn to the names of the structures in which they happen to be most interested at the moment — Nissl Bodies, Nerve Fibers, Capillaries and so forth — on the off chance of finding some useful hints as to methods better adapted for their microscopic study, the most likely experimental errors and so on. Be- cause the range of cells, parts of cells, tissues, organs and systems is obviously so immense, mention is only possible of a small proportion of them so that much depends on the selection made. Others may seek information under the headings of elements such as Iron, Potassium and Calcium, of enzymes Hke Pepsin and Phosphatase and of many other components of living material. It is difficult to draw the line but most of those that can be localized microscopically are mentioned, likewise techniques for the determination of permeability, viscosity, pH and other properties of tissues. It often happens, however, that data are required about a particular technique, which the workers are using or expect to use, and which is known to them by the names of those who discovered it, as for example the methods of Giemsa and xiii XIV PREFACE TO THE FIRST EDITION Mallory. Consequently information also must be supplied under various names though this is usually less satisfactory than under subjects. A very annoying handicap is the host of synonyms for dyes. Being ignorant of chemistry, I have with confidence listed those given by Dr. H. J. Conn. Many more will be found in The Colour Index of the Society of Dyers and Colourists. Since all are busy people, time is a factor and they will wish to dig out what they want as directly and quickly as possible. It is for this reason that every- thing is listed alphabetically. Obviously this book can be nothing more than a brief entr6 to microscopic technique. Therefore, numerous references to the literature are supplied for follow up. Again to save time, these are given each in its appropriate place, thus avoiding the necessity of turning the pages and locating them in a large bibliography. But no attempt is made to trace the techniques to their original exponents and to apportion credit for numerous modifications. Often the most recent and accessible reference is provided re- lying on the author to state history fairly. Evidently, in order to keep up to date as to methods, the reader must repeatedly consult the latest issues of many journals. Stain Techn.; J. Lab. & Clin. Med.; J. Tech. Meth.; Bull. d'Hist. Appl.; and Zeit. f. mikr. Tech. are particularly valuable. Finally I wish to thank my colleagues for their help, particularly Drs. L. R. Boling, C. Carruthers, William Cramer, Morris Moore, J. L. O'Leary, W. L. Simpson, R. E. Stowell, Lester Wicks and Dr. H. J. Conn, Chairman of the Biological Stain Commission, who very kindly read the manuscript and made several useful suggestions. CHOICE OF METHODS The selections will depend on several considerations. The first is what one wants to discover. Many investigators in biology and medicine wish to obtain more information about structural components of the body whether gross, micro- scopic or submicroscopic in size. They desire to obtain further data on the physical and chemical properties of these components whether solid or fluid in different physiological stages of activity and in disease in both man and in animals. They are interested in subjects, not personalities, so that in this alphabetically arranged presentation the names of individuals are seldom listed. Emphasis is given to subjects. After all the problems continue while the names of generation after generation of individuals fade out. Those who perchance may consult this book will need help at two levels. It may be a simple question of the nature of some dye, or the composition of some solution, or the making of a well known test, or it may be the selection of a technique to be employed in a comprehensive series of experiments. In the latter case it involves a major decision. It is necessary not only to choose the technique most likely to lead to the answer but also give some thought to the equipment required and the training demanded for its proper use. A few leading references to the most recent utilization of the technique in question may be helpful. But to discuss the history of its development and to assess priority is not attempted. A complete discussion of the literature may in- deed constitute a handicap by providing an excuse for doing nothing. To be stuffed with information may give a feeling of frustration. Some chemical and physical procedures are well within the reach of people who are neither chemists nor physicists while others will simply lead them beyond their depth into futility. It is equally true that well trained chemists and physicists are Ukely to fail to reahze the complexity of vital processes and to make little progress through ignorance of physiology and pathology. Conse- quently one should never hesitate to seek advice from friends in other depart- ments. 1. To Examine Directly in Vivo The ideal arrangement is to look into the body and to study its parts as they function without causing any disturbance. With protozoa and certain small transparent invertebrates this is relatively simple. The web of a frog's foot is thin and can easily be looked through without seriously interfering with the frog. Some other parts of the bodies of various aquatic lower forms lend them- selves to direct examination in vivo ; but there are definite limitations in such a study of what is going on in the human body. It is possible to peer into the various apertures but to get close enough to the living tissues to use high mag- nifications is not feasible. The cornea and lens of the eye are transparent and XV XVI CHOICE OF METHODS much valuable information can be secured by direct examination of the retinal blood vessels. Even here their distance from the surface is considerable and magnification is therefore limited. As far as we know at present the best that can be done is to take advantage of a discovery, made by Lombard (W. P., Am. J. Physiol., 1911-12, 29, 335-362) that the epidermis can be rendered transparent by the addition of a little highly refractile oil without noticeably injuring it or disturbing the underlying tissues. By this means the blood vessels of the dermal papillae in the fold of skin over the nail bed, which are very near to the surface, can be studied directly at fairly high magnification and over long periods of time thus permitting the making of excellent pictures. See review of literature by Wright, I. S. and Duryee, A. W., Arch. Int. Med., 1933, 52, 545-575. That the lymphatics in the human skin can be made visible in vivo by the injection of small amounts of Patent Blue V has been demonstrated by Hudack, S. S. and McMaster, P. D., J. Exp. Med., 1933, 57, 751-774. The vessels in the ears of living mice can readily be seen without any surgical procedure. It is even possible to directly watch the dye, Chicago blue, after intravenous injection elsewhere in the body, leak out into the tissues especially through the walls of the venules (Smith, F. and Rous, P., J. Exp. Med., 1931, 54, 499-514). Ideas as to the relative hydrogen ion concentrations of some of the tissues visible from without can be secured by the injection of Hydrogen Ion Indicators (Rous, P., J. Exp. Med., 1925, 41, 739-759). The opportunities are many especially in animal experimentation. Another way to examine structure in vivo is to record the structure by x-ray photographs and to magnify the photographs, see Microradiographic examina- tion. 2. To Examine through Windows in Vivo The construction of windows in the skin or body wall through which the tissues can be examined in vivo is a less ideal technique because it involves surgical interference with the body. In the most used of these techniques a hole is made through a rabbit's ear from one surface to the other. A glass chamber is then sewed into the hole in such a way that a blood vessel is included between a thin layer of glass (serving as a cover glass) and a thicker one serving as a slide. After a time the epidermis adheres to the edges of the chamber and blood vessels, nerves and other tissues grow into it where they can be studied under oil immer- sion objectives. This technique was first reported by Sandison (J. C, Anat. Rec, 1924, 28, 281) working under Dr. E. R. Clark at the University of Penn- sylvania. It has since been very greatly improved (Clark, E. R., et al., Anat. Rec, 1930, 47, 187-211 and Abell, R. G., and Clark, E. R., Anat. Rec, 1932, 53, 121-140) by the introduction of "round table" and "moat" chambers. To place a window in the wall of the skull and to observe what is going on within has been done with more or less success on several occasions. The tech- nique devised by Forbes (H. S., Arch. Neurol, and Psych., 1928, 19, 75) permits direct observation at low magnification of the blood vessels over the cerebral CHOICE OF METHODS XVU convolutions with so little injury that their behaviour in various experimental conditions can be investigated (see also Clark, E. R., and Wentsler, N. E., Proc. Assoc. Res. Nerv. and Ment. Dis., 1937, 18, 218-228). Through a window in the thoracic wall Wearn and his associates (Weam, J. T. et al.. Am. J. Physiol., 1934, 109, 236-256) have similarly studied the pulmonary arterioles and capil- laries. They employed a fused quartz cone to conduct light to the tissue. Other investigators have availed themselves of the natural window, the cornea, through which what goes on immediately within it in the anterior cham- ber of the eye can be observed. Several tissues have been successfully trans- planted into this chamber. Perhaps the most dramatic is the behavior of trans- planted uterine mucosa in the rhesus monkey. In it the menstrual changes can be seen in detail and the influence of hormones noted (Markee, J. E., Con- trib. to Embryol., Carnegie Inst, of Washington, 1940, 28, 219-308). For some kinds of work the fact that the tissue fluid (aqueous humor) in this chamber differs from others in the same animal by the absence of certain species specific growth inhibiting factors is a priceless asset. Thus Greene (H. S. N., Science, 1938, 88, 357-358) was able to grow pieces of human cancers, which ordinarily quickly die in other species, in the anterior chambers of the eyes of some mam- mals. The existence of a barrier protecting this fluid against the entry of anti- bodies from blood plasma and thus making possible the growth of tumor trans- plants, while all other tissues are resistant to their growth, has recently been emphasized (Saphir, O., Appel, M. and Strauss, H. A., Cancer Res., 1941, 1, 545-547). In order to view the less accessible living tissues, techniques have been devised that include opening the body and partly withdrawing the organ so that it can be placed on the stage of a microscope but with circulation and nerve supply intact and adequate regulation of temperature and humidity. Particularly fruitful has been the direct observation through oil immersion objectives of secretion by acinous cells of the Pancreas by Co veil (W. P., Anat. Rec, 1928, 40, 213-223) and of islet cells by O'Leary, (J. L., Anat. Rec, 1930, 45, 27-58). Thus the influence of drugs on the secretory process can now be followed in minute detail. Knisely (M. H., Anat. Rec, 1936, 64, 499-523; 65, 23-50) has perfected a technique for the study of the living Spleen at somewhat lower magnification. The essential features are slight displacement of the spleen so that it can be transilluminated by light delivered through a quartz rod. This allows for the first time direct examination of the behavior of the venous sinuses. Undoubt- edly the Quartz Rod technique will be of great service in providing light for similar examination of other organs. 3. To Study the Arrangement of Parts in the Body Since the body is structurally so very complex it is often illuminating to view parts of it in their normal shape and size but unobscured by all the neighboring components. There are several ways by which this can be accomplished. XVlll CHOICE OF METHODS The first method of Reconstruction from serial sections is well known. Briefly stated the particular tissue, organ or system is outlined, as it appears in section after section, at the desired magnification on sheets of material of uniform and carefully selected thickness. The outlined areas are then cut out and when superimposed they constitute a reconstruction of the original structure. This technique is tedious but it may reveal topographical relations that can be dis- covered by no other means. The second kind of technique is to make casts of vascular, respiratory and other lumina. Woods' metal, formerly used for this purpose, has now been almost displaced by Celloidin and other substances. The surrounding tissue is freed from the cast by digestion in concentrated hydrochloric acid and gentle brushing away in a stream of water. Very beautiful Corrosion preparations of the lungs and kidneys have been obtained by this method which should be more widely employed. The third is by Maceration to soak the organs, without previous preparation, in fluids that either digest away the tissues which it is desired to eliminate or loosen their connections with those under investigation, which, latter, can then be individually examined. Techniques of this sort are the only available means for the isolation of individual seminiferous and renal tubules. Oliver's researches on the kidney illustrate the value of reconstruction and maceration in pathology. Only three other examples will be submitted. Thyroid follicles can be isolated by maceration (Jackson, J. L., Anat. Rec, 1931, 48, 219-239). Their study as individuals provides data as to size and shape only obtainable otherwise by the tedious examination of serial sections. The Epidermis is so tightly bound to the underlying dermis that separation is extremely difficult; but, after treatment of skin with dilute acetic acid, the attachment is loosened and the epidermis can readily be removed as a complete sheet of tissue which can be stained, made transparent and examined as a whole mount. Opportunities are thus afforded for the detection of regional differences which might not be located even by pains- taking study of sections and the making of mitotic counts is greatly facilitated. By macerating in the same fashion the nasal mucous membrane covering the septum can also be removed for study. Perhaps still other epithehal sheets can be similarly isolated. However such sheets are of Httle value for chemical analysis because of the action of the acetic acid. Fortunately it has been found that the epidermis may also be quickly loosened by simply heating the skin to 50°C. when it can be peeled off like the covering of a scalded tomato (Baum- berger, J. P., Suntzeff, V. and Cowdry, E. V., J. Nat. Cancer Inst., 1942, 2, 413-423). There is still another alternative. Instead of simply omitting the unwanted material by reconstructing only the structures chosen for demonstration, or of removing the material by corrosion or maceration, it can be left in and rendered transparent so that it does not obstruct the view. After marking the particular structures by vital dyes or other means the whole tissue is cleared by the method of Spalteholz or Schultze. These techniques give admirable results in the study CHOICE OF METHODS XIX of Cartilaginous Skeletons, Ossification centers, Blood Vessels and so on almost without end. 4. To Employ the More Routine Method of Fixation and Staining Here there is wide latitude of choice. For some purposes thin Smears are just fixed and stained without resort to sectioning. In the case of the denser tissues which must be cut in sections one first has to decide which of many Fixatives is likely to give the best results. Then, whether fixation is to be by immersion or injection has to be determined. The purpose of fixation by vascular injection is to bring the fixative into close contact with the tissues as they exist in the freshly killed animal without sub- jecting them to mechanical trauma or disturbing their topographic relations one to another. In choosing this procedure it is well to remember: (1) That it is usually necessary first to wash out most of the blood by perfusion with physio- logical salt solution for otherwise the fixative often clogs the vessels. This wash- ing unfortunately also facilitates chemical change. (2) That, even when it is not done, the concentration of the fixative about the cells is gradually increased and at different rates, rapidly in highly vascularized tissues (kidney, liver, etc.) and very slowly in avascular ones (epidermis, cornea and cartilage). The time for chemical change before fixation is therefore variable depending upon the tissue. (3) That the pressure may bring about an unnatural swelling of the tissues so located that they can enlarge, especially the abdominal organs as compared with brain and bone marrow which are confined within rigid walls. Fixation by immersion is the usual and easiest method. If small pieces or thin slices are used the preservation is quicker and more uniform than by vascu- lar injection. The cells are suddenly killed while active. The factor of slow death at uneven rates, present in supravital examinations, does not have to be reckoned with; but many precautions are required. Under Fixation is given a general account of the procedure. Under the several organs, Lungs, Small Intestine, Skin, etc., some special suggestions are provided. There are many fixatives to choose from. For routine purposes Zenker's Fluid as originally described or in one of its numerous modifications is suggested. Bouin's is also a very popular fixative especially among dermatologists. Formalin is an ex- cellent one. It is good practice to set aside some tissue in formalin for examina- tion as may be needed later. Both formalin and alcohol are the most useful fixatives preliminary to microchemical determinations. When preparations must be made very quickly, Alcohol Formalin and Caraoy's Fluid are suggested (see also Frozen Sections). For microincineration, formalin-alcohol is ordinarily employed; but the Altmann-Gersh method of freezing and drying, by which contact with fixatives is altogether dispensed with, is much less open to criticism. Osmic acid containing fixatives penetrate poorly and are therefore only useful for very small pieces of tissue. Regaud's fluid with subsequent mordanting in bichromate is the best for mitochondria. Heat fixation is useful for blood cells. Fixation in various vapors is called for in special cases. See Fixatives. XX CHOICE OF METHODS After fixation some Washing of the tissue in water is necessary unless it has been fixed in alcohol, Carnoy or similar mixtures. The next step is Dehydration and a choice must be made between slow and rapid methods. Sometimes a substitute for alcohol is indicated. If Imbedding is to be in celloidin Clearing in a xylol-like fluid is omitted and heating is unnecessary. There are many ways of clearing preliminary to paraffin imbedding. In Sectioning the thickness depends upon the purpose in view. Thick sections may be as necessary as thin ones and serial sections are often required. In the Mounting of sections on slides the use of water must occasionally be avoided. Numerous techniques are applicable to the sections and are given individually later either under the head- ing of the substance or structure to be demonstrated or under the name of the technique or its introducer. For choice see Staining. Many beautifully stained sections of well fixed tissue are of but little value, because the investigator failed to note the exact location in the organ or tissue whence they were excised and omitted to have the sections cut in the most favorable plane. 5. To Mark Selected Individual Cells or Tissues in Vivo FOR Later Examination In this connection we at once think of the vital stains, trypan blue, carmine, India ink (carbon) and hundreds of others, which, when injected into the body, are phagocytosed by the reticulo-endothelial cells (or macrophages). Pieces of tissue can then be excised and the accumulations of stains can be studied within the still living cells, that is supravitally, for unless cultured the cells are slowly dying. But, if desired, the tissues can be fixed and the results observed at leisure in sections. It has long been known that bone laid down in the presence of Madder fed to the animals is marked by the madder and can thus be distinguished from bone deposited beforehand and afterwards. In the same way dentine can be marked in vivo with Alizarin Red S. Another example of in vivo marking is the deposition of Prussian Blue. Thus a slightly hypertonic solution (potassium ferrocyanide 0.5 gm., iron ammonium citrate, 0.5 gm. and aq. dest. 50 cc.) injected into the subarachnoid space of the spinal cord is useful in the localization of the pathways of drainage of cerebro- spinal fluid, because of the marking secured when the tissues are fixed in 40% formalin plus 1% concentrated hydrochloric acid by the deposition of Prussian blue (Weed, L. H., J. Med. Res., 1914, 26, 21-117). The tissues of animals recently killed or under anesthesia can be selectively marked with various dyes by Perfusion of the blood vessels with dilute solutions of dyes. The outstanding methods in this group have been devised by Bensley (R. R., Am. J. Anat., 1911, 12, 297-388) for histological analysis of the epithelial components of the pancreas and stomach. Dilute solutions of the dyes in physio- logical saline are injected into the thoracic aorta of an animal killed by bleeding. Pieces of pancreas and gastric mucous membrane are then removed and examined CHOICE OF METHODS XXI fresh. Neutral red picks out the Islets of Langerhans of the pancreas, pyronin the duct system of the pancreas, naphthol blue the parietal cells of the Stomach and so on. In the same way Nerve Fibers can be marked for subsequent study by vascular perfusion with methylene blue and degenerating nerve fibers in poUomyelitis (and presumably in other conditions) can be sharply differentiated from uninjured ones by the fact that they take up neutral red (Covell, W. P. and O'Leary, J. L., Arch. Neurol. & Psych., 1932, 27, 518-524). It has long been known that the best way to mark renal glomeruli is to perfuse in the same fashion with a dilute solution of janus blue. The glomeruli stand out clearly in the fresh kidney by their deep blue color in a red background (Cowdry, E. V., Contrib. to EmbryoL, Carnegie Institution of Washington, 1918, No. 25, 39- 160). A similar selective staining in less brilhant colors is obtainable with janus green. Relatively permanent preparations can be made of some of these specimens. The same dyes, and many others, can also be applied in dilute solutions to cells freshly removed from the body and which are still living. Such methods have become very popular in hematology. However, the cells thus colored live only for a limited time and it is important to cut short the observations before they are vitiated by approaching death. It is feasible to employ a wide variety of Tracer Techniques, that is substances can be traced through the body by the markings given to them. The largest group is made up of Radioactive Isotopes. Because of their radioactivity they, and substances in which they are chemically combined, can be quantitatively measured by a Geiger Counter. Wherever they go in the body, they are ap- parently accepted by the tissues and play their roles in metabolism in the same way as if they were not radioactive. Thus Radiocalcium is found to be stored almost entirely in bone and the amount taken in in a given time is an indication of the amount of nonradioactive calcium given out in conditions in which the total amount of calcium is not changed. The turnover of calcium can therefore be estimated. Radioiodine tends to be stored in the thyroid, and, again, when the total amount of iodine does not change, the amount stored in a given time balances the amount lost and is a measure of the iodine replacement. By the technique of Autoradiography the exact location of the radioelements can be determined by holding a section of the tissue in contact with a photo- graphic film. The images on fine grained films can then be magnified. Con- sequently, by selection of radioelements based on information as to where they are stored in largest amounts and by their use, heavy radiation can be brought to bear upon several kinds of tissues leaving others influenced but little or not at all. An excellent account of Isotopes in Nutrition Research is given in Borden's Review of Nutrition Research, 1945, 6, Nos. 8 and 9. 6. To Employ Culture Methods The common feature in these techniques is to plant cells, tissues or organisms in new and different fluid environments and to observe their behavior therein. XXll CHOICE OF METHODS Thus cells can be grown in Tissue Cultures of chemical composition suited to their requirements. Mixed cultures are those containing several types of cells and pure cultures those containing but one sort. This technique affords un- rivalled opportunities for experimentally changing the fluid environments of cells, for the study of nutritional factors, growth stimulating and growth in- hibiting factors, and the influence of cells on one another. Individual cells can be observed at high magnification and the phenomena of motility, phagocytosis, mitosis, cell death, etc. can be recorded by moving pictures so that the analysis of form and function is possible with a high degree of accuracy. The limitation of the method is the obvious one that the fluid environments are artificial and must be changed at intervals to keep the strains of cells alive. Consequently tissue cultures are unsatisfactory for the investigation of inter- cellular materials, like fibers, hyaline deposits and so on. Moreover the cells cannot properly organize to form tissues and organs as they do in vivo since they are isolated from normal influences by other tissues of the body. But they make the effort. Methods have recently been advocated for the culture of organized tissues, bones, teeth, etc. (Fell, H. B., J. Roy. Micr. Soc, 1940, 60, 95-112). In selecting the technique of tissue culture for the solution of any problem it is well to remember that considerable equipment and several years training are required to realize its full usefulness. For this reason valuable time will be saved by learning the technique from an expert. The new and highly productive technique of analysing cellular responses by their observation in Motion Pictures offers more attractive leads when applied to living cells in tissue cultures than to cells viewed in other situations. In tissue cultures they can be photographed at high magnification, both by direct illumination and in ths dark field, because they occur as individuals or as thin clumps in the fluid. Moreover, their behavior can be followed in successive photographs over long periods of time and it is possible directly to observe how this is modified by a host of different influences experimentally brought to bear on them. For teaching Motion Pictures are helpful, but can be used too much. Easy come, easy go is true of instruction. Unless learning is combined with some sort of effort it will be of very transitory value. Transplantation of tissue from its original location to a new and different position, such as the Anterior Chamber of the Eye, is also a culture method of value in the solution of certain problems. The factors that condition the growth and the behavior of the transplant are of importance. Some organisms can best be grown, and viruses increased in amount, by implanting them into the Chorioallantoic Membrane of chick embryos. This technique has abundantly proved its worth. The feasibility of culture in this membrane depends essentially on the lesser development of growth inhibiting factors in young tissues than in older ones. Viruses will "take" and increase in amount in some locations better than in CHOICE OF METHODS Xxiii others. Intracerebral and intratesticular inoculations are often made and, again, young animals are in general most susceptible. The culture of Bacteria and Protozoa has for generations been a fine art based on meticulous study of their needs. These relatively simple organisms provide wonderful material for the investigation of the most basic of vital phenomena. 7. To Investigate Composition by Chemical Means This cannot be done blindly — by just taking a chunk of tissue and analysing it. The investigations must be guided by knowledge of the structure and func- tion of the materials analysed. Blood can, for example, be collected in suf- ficient volume for routine chemical analysis; but the results will differ depending upon whether it is arterial blood, portal venous blood from the intestines, or venous blood from the extremities. Analyses of whole skin are practically worthless because the skin is a structure made up of two parts: avascular epi- dermis of ectodermal origin and underlying dermis made up of connective tissue differing in vascularity, fiber, fat, tissue fluid and gland contents in various regions of the body. Only since a technique has been devised whereby whole Epidermis freed from dermis can be obtained in a condition suitable for analysis, not having been exposed to any fluids, has progress been possible. Results of direct chemical analysis of any tissue may be misleading unless interpreted in terms of its structural make up and of what has happened to it since it existed in vivo. Among the experimental errors to be guarded against are variability in sacrificing the animal, or the manner of death of the patient, in excision of tissue allowing more or less blood and other fluids to drain out or evaporate, in time and in temperature, in age, sex, and in conditions before death. The extracellular and intracellular fluids or phases, are large in volume, w^hen all are taken together, but difficult to get at directly. To obtain data "the deducive histochemical method" is suggested. This is described by Lowry, 0. H. and Hastings, A. B. in Cowdry's Problems of Ageing, 1942, 728-755. Those wishing to analyse extremely small volumes of fluid which by contrast can be collected for direct determinations cannot do better than to familarize themselves with the techniques elaborated by A. N. Richards and his associates at the University of Pennsylvania for the study of glomerular urine. By the useful technique of Microincineration minerals which are not volatilized at high temperature can be directly studied in the tissues in the positions which they previously occupied in living organisms. They appear as shining particles when viewed by the Dark Field Microscope. Microincineration is truly a microchemical method for the localization of structure which is microscopic in its fineness. Quite a number of Microchemical Reactions capable of demonstrating the precise location in the cells of minerals, fats, carbohydrates and proteins are available. XXIV CHOICE OF METHODS By a Photoelectric Microphotometer it is possible to estimate quantitatively reactions like that of Feulgen for Thymonucleic Acid which give distinctive colors and numerous stains which are specific for tissue components and can be standardized in their action. But the data obtained are relative, that is it can be said that the reaction is say 60 per cent greater in one specimen than in another. The absolute amount of the component demonstrated per gram of tissue cannot yet be arrived at. Several Enzymes (phosphatase, dopa-oxidase, arginase) can now be micro- scopically identified and their position within cells determined. By close com- parison of enz5Tnatic properties with the cellular composition of tissues, the localization of many others can be inferred. In the case of these and other microchemical methods the treatment of the tissue after excision and before the special procedures are commenced is of con- sequence. Even in the preparation of routine frozen sections, and much more so when the specimens are fixed, dehydrated, cleared, imbedded and sectioned, there are many opportunities for the loss of chemical substances and of change in their position in the tissue and within cells. The best way to hold the com- ponents in the positions they occupy in the living state is to instantaneously freeze the tissue and dehydrate in vacuum while still frozen, thus avoiding all fixatives, by the Altmann-Gersh technique. Moreover, the reagents used in testing must contact all the tissue equally for unequal contact may well be followed by stronger reactions in some areas than in others. Quite recently chemical analysis has been accurately focussed, not merely on cells, but on parts of cells. Nuclei, Mitochondria and many other cellular components including even Chromatin Threads can now be collected en masse by Centrifugation of broken up cells and analysed. This is a departure of con- sequence. Finally standard qualitative chemical methods are often appUcable on a microscopic basis. The reader wishing to do so may well consult Chamot, E. E., and Mason, C. W., Handbook of Chemical Microscopy. New York: John Wiley & Sons, 1940, vol. 2, 439 pp. Another book that will be found of service, especially for analysis on microscopic slides, is Benedetti-Pichler, A. A., In- troduction to the Microtechnique of Inorganic Analysis. New York: John Wiley & Sons, 1942, 302 pp. Sometimes one is held up by having to deal with some unfamiliar chemical substance in which case aid may be secured from the large and comprehensive "Dictionary of Organic Compounds" edited by Heil- bron and published in 3 volumes, 1934, 1936 and 1938, by Oxford University Press, New York. No attempt is made in this dictionary to include dyes but thousands of other organic compounds are conveniently arranged in alphabetical order. If the wanted material is some sort of medical preparation seek informa- tion in the following reference books. (1) New and Nonofficial Remedies, 1946. Chicago: Am. Med. Assoc, 770 pp.; (2) The National Formulary. CHOICE OF METHODS XXV VII. Washington: Am. Pharmaceutical Assoc, 1942, 690 pp.; (3) The Phar- macopoeia of the U. S. XII. Easton: Mack Printing Co., 1942, 880 pp. 8. To Employ Physical Techniques in the Investigation OF Composition Chemistry is, at rock bottom physics so that the distinction here made is convenient but without vaUdity. Hydrogen Ion Indicators and Oxidation- Reduction Potential could come under either heading. Histospectrography is a quick and reliable method to gain information on the presence or absence of many minerals. It is a kind of survey technique, for the absorption lines of many elements can be obtained in a single spectrogram. The density of the lines can be determined photometrically but data obtained on concentration of a particular element are relative (more in one tissue than in another) but not absolute (in mgm. per gm. of tissue). Ultraviolet Absorption Spectra have been employed to advantage by Caspersson and others in the intracellular determination of certain components but the technique requires elaborate and costly instrumentation. It gives promise, however, of being of great value in the solution of fundamental problems. Utilization of physical techniques in biology and medicine is now the order of the day and the limitations thereof cannot be envisaged. In this elementary survey only a few others can be mentioned briefly in passing as examples. By Electrophoresis measurements the electric charge on particles can be determined. The Polarization Optical Method is of surpassing value and Fluorescence Micros- copy, supplemented by fluorescence spectrography, is coming into its own. Surface Tension measurements can be made in numerous ways. Particle size can be measured by a flock of different techniques from which the one must be chosen that best suits the material. The simplest way is to compare the objects with rulings of a micrometer slide. DiflTraction methods are labor saving and often preferable. Filters of d^erent porosity are available so that the sizes of particles passing through can be roughly gaged. To employ Ultra- centrifugation techniques are among several other possibilities. There are now Microscopes of many varieties to choose from. The Electron Microscope is a physical tool which can be used only by a spe- cially trained individual, and it has the limitation that the cells and other ma- terials must be very thin, sections not more than about j of a micron. See Burton, E. F. and Kohl, W. H., The Electron Microscope. New York: Rein- hold Publishing Corporation, 1946, 325 pp. In biology and medicine it is clearly evident that the techniques of physics and chemistry are so revealing that some knowledge of these basic sciences is necessary. A little knowledge can however be a dangerous thing often leading to half baked conclusions. Cooperation with real physicists and chemists is essential and team work must take the place of isolated individual endeavor, moreover a laboratory of whatever kind must be well organized to be effective. XXVI CHOICE OF METHODS An untidy laboratory is not a sign of industry but an indicator of carelessness, and sometimes a source of actual danger to the occupants. 9. To Detect Deviations from Normal The Normality of a tissue or organ is often in doubt. There is no single technique capable of yielding an unqualified answer. Since some properties may be normal while others are abnormal (pathological) we need first to be told the property under consideration. If it is, for instance, the amount of contained pigment, this can be said to be normal when it is the amount usually present in a particular tissue under the same conditions. By the word "usually" is in- tended in the majority of cases, that is in 51 per cent or in any higher percentage. The phrase "same conditions" means that the conditions likely to influence the amount of pigment are so nearly alike as to be not responsible for any difference observed between the property of the tissue where normality is in question and that of others of the same kind. Thus, we could say with reasonable assurance that the amount of pigment is normal if it is that usually demonstrated by the same technique in tissues of the same kind of animals of the same species, sex and age living under the same conditions. Judgment is necessary in specifica- tion of possibly modifying conditions which will depend to some extent on the property under consideration and on the number of observations necessary to establish the percentage within the limits of probability. It would not do to compare the amount of pigment in the specimen, the normality of which is in question, with that in too few others. This is the statistical definition of nor- mality which is not universally accepted but which is useful and easily under- stood. Only a few samples of the various kinds of technique have been mentioned in this survey as a kind of menu from which to make a selection or to obtain clues to other methods that may fit the case. Many of them are very ingenious and were only discovered after wisely conceived attempts to overcome practical difficulties. This overcoming of obstacles is a pleasant experience. It calls for actual work and experiment and appeals to many of our best minds. The techniques may be regarded as keys by which scientific treasure can be unlocked. Unused they are worthless. STANDARDIZATION OF STAINS* In the use of stains one encounters a multitude of names, many of which are synonyms, and it is difficult to be sure of their meaning. Two comprehensive dye indexes have been pubhshed. One, "Schultz' Farbstofftabellen", is now in its 7th edition (1928 to 1939) but confusion is created by the fact that the index numbers of the dyes given in it do not correspond to those in the earlier editions. The other, the "Colour Index of the Society of Dyers and Colourists", was edited by F. M. Rowe and published in 1924. It was followed in 1928 by a supplement, but there has been no second edition. This Colour Index gives (1) the com- mercial name, or much more frequently names for there are so many synonyms; (2) the formula, (3) the preparation, (4) the discoverer and (5) the properties of a vast assemblage of dyes. It is the standard of reference in the United States and other English-speaking countries. When one wishes to be specific it is customary to list after the dye used its colour index number, for example vital red, C. I. No. 456. The most recent Year Book of the American Association of Textile Chemists and Colorists, New York: Hawes Publishing Co. 1945, 743 pp. is often of as- sistance. It provides an alphabetical list of over 6,000 American made dyes with classification, manufacturer and Colour Index Number if any. A listing of American made Dyes arranged by Colour Index Numbers is also useful. For example, if one is interested in Orange II, CI. 151, it will be seen that this is avail- able under 26 names from 12 different makers. In another place the foreign prototype names of dyes without Colour Index numbers are listed alphabetically with the corresponding American dyes and their manufacturers so that the available American substitutes for foreign dyes can be found. This Year Book is unfortunately often lacking in medical school libraries but it is usually on hand in the better Public Libraries like that of St. Louis. Much aid is given to investigators by the Biological Stain Commission and particularly by its distinguished Founder, Dr. H. J. Conn. This commission is concerned with the inspection and standardization of stains, not with their manufacture as is sometimes supposed. It was found in 1920, while the post- war embargo on dyes was still in effect, that American scientists were being sup- plied with dyes from three or four different stain companies and that their products were not sufficiently uniform to be reliable. Accordingly, through the cooperation of the National Research Council and of several national scientific societies, the Commission on Standardization of Biological Stains (now the Biological Stain Commission) was established. The Commission is now an independent organization but includes in its membership representatives of eight societies with which it cooperates. The work of the Commission is two- fold. First, by cooperation of biologists and chemists it gathers information * Kindly revised by Dr. H. J. Conn. xxvii XXVlll STANDARDIZATION OF STAINS concerning the nature of dyes as related to their use in microscopic technique ; secondly, by working with the manufacturers and dealers it endeavors to see that the supply of available stains in America is of the highest possible quality as judged by their performance in actual laboratory use. The first of these purposes has inspired a useful book on "Biological Stains" by Conn, now in its fifth (1946) edition, and at the same time has led to the pubhcation by the Commission of a quarterly, "Stain Technology." The second object is being brought about by the certifying of stains. The certification plan has been adopted because of the great difficulty of drawing up any chemical or physical standards to determine which stains are satisfactory'- and which are not. If such standards were formulated, it would be possible to prepare specifications with which manufacturers of stains ^^■ould be expected to comply. In the early work of the Stain Commission an attempt was made to draw up such specifications and they were published, in provisional form, for a few stains in the first edition of "Biological Stains." Full specifica- tions are given in the current edition and in the National Formulary. Instead of drawing up specifications, therefore, the Stain Commission instructs the manufacturers of stains to submit samples to it of every batch manufactured of any of the stains that are on the certification basis. The Commission submits these samples to certain definite tests which have now been formulated and published (see Conn, pp. 246-276). The methods in question include chemical, spectrophotometric, and biological tests, and only those dyes are certified which are satisfactory in all these tests. Such dyes the manufacturers are allowed to sell with a special label on the package indicating approval by the Stain Com- mission. The certification label on any bottle of stain means, therefore, that: (1) a sample of the batch bearing the label has been submitted to the Commission for testing and a portion of the sample is permanently on file in the chairman's oflBce; (2) the sample proves true to type, as judged by spectrophotometric tests; (3) its dye content is up to specification and is correctly indicated on the label; (4) it has been tested by experts in the procedures named on the label and has been found satisfactory by them ; and lastly, (5) no other batch can be sold under the same certification number except by such a flagrant breach of confidence on the part of the company as to risk losing the good will of the Commission. At present (1950) the following stains have been placed on the certified list. In descriptions of their use the names should be followed by C.C., indicating that the products were Commission Certified, for instance, alizarin red S (C.C.). Eight companies in the United States are now submitting their stains to the Commission for certification before putting them on the market. It must be realized, however, that no one of these concerns necessarily manufactures all the stains which it thus submits ; but in the case of any stain which is manufac- tured elsewhere, the company takes responsibility for its performance as a bio- logical stain, on the basis of tests made to show its adequacy, and in many in- stances carries out a certain degree of purification or other processing before STANDARDIZATION OF STAINS XXIX Alizarin red S Anilin blue, water soluble Auramine O Azocarmine G Azure A Azure B Bismarck brown Y Brilliant cresyl blue Brilliant green Carmine Chlorazol black E Congo red Cresyl violet Crystal violet Eosin, bluish Eosin, yellowish Erythrosin B Ethyl eosin Fast green FCF Fuchsin, acid Fuchsin, basic Giemsa stain Hematoxylin Indigo carmine Janus green B Jenner's stain Light green, S.F., yellowish Malachite green Martius yellow Methyl green Methyl orange Methyl violet 2B Methylene blue chloride Methylene blue thiocyanate Methylene violet Neutral red Nigrosin Nile blue A Orange G Orange II Orcein Phloxine Pyronin P Resazurin Rose bengal Safranin O Sudan III Sudan IV Sudan black B Tetrachrome stain (MacNeal) Thionin Toluidine blue O Wright's stain putting the stain on the market. One of these companies puts on the market every stain now on the certification list. Two other companies submit samples of over half the stains thus listed, while the other companies merely request certification of one or two dyes in which they speciaUze. No dyes have yet been certified by the Stain Commission submitted by any foreign concern except for one located in Montreal. Cooperation among the Americas is increasing (Conn, H. J., Stain Techn., 1942, 17, 5-6). In several recent editions of the National Formulary, published by the Ameri- can Pharmaceutical Association, a section has been included in which formulae of staining solutions are given. Originally there was no agreement between these formulae and the ones recommended by the Stain Commission. Begin- ning in 1937, however, it was decided that the National Formulary Committee and the Biological Stain Commission should cooperate in this matter. Accord- ingly, the chairman of the latter was made a member of the former and a member of the National Formulary was put on the Board of Trustees of the Commission. This interlocking membership is assurance that the work of preparing staining formulae for each edition of the National Formulary is carried on in close coopera- tion with the Stain Commission. This cooperation has resulted in two important steps : 1. Specifications of the most important stains now on the certification basis have been published in the National Formulary (1942, 1946). These specifica- XXX STANDARDIZATION OF STAINS tions are partly chemical and spectrophotometric, but also contain detailed state- ments as to how the stains should be tested as to their behavior for biological purposes and state the results to be expected from these tests. In every case these specifications have been made to harmonize with the tests as actually per- formed by the Stain Commission. 2. The formulae given in the National Formulary, in "Biological Stains" and in the "Manual of Methods for the Pure Culture of Bacteria," published by the Society of American Bacteriologists, have been compared and critically studied with the object of making them identical in all three. The years since the second World War have seen more progress in stain standardization than during any preceding similar period. Dr. E. H. Stotz, Biochemist, of University of Rochester Medical School, has been made an officer of the Stain Commission and is now in charge of its research and assay labora- tory at Rochester, N. Y. This laboratory is making a comprehensive survey of nearly every stain sample that has ever been submitted for certification, making a systematic comparison between their physical and chemical characteristics (notably spectrophotometric) and behavior in staining. If it is ever going to be possible to draw up specifications that correlate with staining properties, such a survey should furnish the necessary data. ABBREVIATIONS XXXI ABBREVIATIONS 1 fi (Greek letter for micron) = l/lOOOth part of a millimeter (mm.) = 0.001 mm. = 10~' mm. = 10,000 A = approximately l/25,000th of an inch. 1 m^ (millimicron) = 1/lOOOth part of a micron = l/l,000,000th part of a mm. = 10~* mm. = 0.001 M = 10 A. 1 A (Angstrom unit) = 0.1 m/ii = 0.0001 n = 10"^ mm. 1 MM (micromicron) = 1/1, 000,000th part of a micron = 1/1, 000, 000, 000th part of a mm. = 10-» mm. = 0.000,001 a« = lO"" A. 1 Kg. = approximately 2.2 lbs. 1 gm. = 10-' Kg., 0.001 K., 1000 mgm., 1,000,000 ng. 1 mgm. = 10-« Kg., 10-» gm., 1000 Mg- 1 /ig. = I7 = 10^ Kg., 10-8 gm., 10-' mgm. m^g = 1/1,000,000 mgm. A^ NaCl is normal solution of sodium chloride, see Normal Solution. M HCl is molecular solution of hydrochloric acid, see Molecular Solution. M = mole. mM => millimole. ME = milligram equivalent. 1 ml (milliliter = l/l,000th part of a liter = 1 cc. (approx.) that is 1 cc. + 0.000027 cc. at 40°C. M» = 1/1,000 ml 1 ml (milliliter) = l/l,000th part of a liter = 1 cc. (approx.). CI 76 means that the number of a dye is 76 in the Colour Index of the Society of Dyers and Colourists. CC. given after a dye signifies that it has been certified by the Biological Stain Commission The following publications are simply referred to by author, or senior author, or editor's name and page number (cf. Conn, p. 26). Bensley, R. R. and S. H., Handbook of Histological and Cytological Technique, Univ. Chicago Press, 1938, 167 pp. BouBNE, G., Cytology and Cellular Physiology, Oxford: Clarendon Press, 1942, 296 pp. (Second Edition, 1951, 524 pp.) Conn, H. J., Biological Stains, Geneva, N. Y.: Biotech Publications, 1940, 308 pp. CowDRY, E. v., Textbook of Histology, Philadelphia: Lea & Febiger, 1938, 600 pp., 3rd Edition, 1950, 640 pp. Craig, C. F., Laboratory Diagnosis of Protozoan Diseases, Philadelphia: Lea & Febiger 1942, 349 pp. Downey, H., Handbook of Hematology, New York: Hoeber, 1938, 3136 pp. Emig, W. H., Stain Technique, Lancaster: Science Press Printing Co., 1941, 75 pp. Glasser, O. (Editor), Medical Physics, Chicago: Year Book Publishers, 1944, 1744 pp. Glick, David, Techniques of Histo- and Cytochemistry. New York: Interscience Pub- lishers, Inc., 1949, 531 pp. Lee, Bolles, The Microtomists' vade-mecum. Philadelphia: P. Blakiston's Son & Co. (Tenth Edition, Edited by J. B. Gatenby and T. S. Painter, 1937, 784 pp. Eleventh Edition edited by J. B. Gatenby and H. W. Beams, 1950, 753 pp.) LiLLiE, R. D., Histopathologic Technic. Philadelphia: Blakiston, 1948, 300 pp. LisoN, L., Histochemie Animale, Paris: Gauthier-Villars, 1936, 320 pp. Mallory, F. B., Pathological Technique, Philadelphia: Saunders, 1938, 434 pp. McClung, C. a., Microscopical Technique, New York: Hoeber, 1938, 698 pp., 2nd Edition by Ruth McClung Jones, 1950, 790 pp. TECHNIQUES A-V Bundle, see Todd, T. W., Cowdry's Special Cytology, 1932, 2, 1173-1210. Abopon. For mounting amyloid stains (Leib, Am. J. Clin. Path., 1947, 17, 413). Absorption. Every solid surface attracts other substances more or less. This holding is referred to as absorption. The finer the structure of the solid the greater the combined surface area of the constituent particles and conse- quently the greater the degree of ab- sorption. An interferometer is an in- strument employed to measure change in concentration by absorption. There are many other ways of obtaining this information . See Water Absorption and fat absorption after previous coloration of fat with Sudan III or Sudan black (see Vital Staining). See X-ray Ab- sorption. Absorption Spectra. Methods are avail- able for the determination of absorption spectra of cell structures. Caspersson (T., J. Roy. Micr. Soc, 1940, 60, 8-25) has described apparatus for absorption from intracellular objects larger than 1 micron such as Nissl bodies. This line of investigation is just developing and is likely to be productive of im- portant results. See Histospectroscopy. Acacia, properties as a macromolecule (Hueper, W. C, Arch. Path., 1942, 33, 267-290). See V. Apathy's Syrup. Acanthocephala, see Parasites. Acarina, see Parasites, Ticks. Acetic Acid (L. acetum, vinegar). Widely used as a component of fixatives. The undiluted solution is often termed "glacial acetic acid." This contains 99.5%CH3COOH. Causes a distinctive swelling of fresh collagenic fibers. Employed in dilute solution to destroy red blood cells so that whites can be examined. In 1% solution separates epidermis from dermis. See Epidermis. Acetic-Osmic-Bichromate fixative of Bens- ley. 2% osmic acid, 2 cc; 2.5% aq. potassium bichromate, 8 cc; glacial acetic acid, 1 drop. Excellent for mitochondria but very small pieces of tissue must be used because the fluid penetrates poorly. The best stain is Anilin-Fuchsin Methyl Green, see also Copper Chrome Hematoxylin. Acetin Blue R (CI, 560)— Induline Alcohol Soluble — a basic dye of light fastness 4. Paraffin sections of plant tissues color dull light blue (Emig, p. 58). Acetic-Carbol— Sudan III, see Sudan III. Aceto-Carmine (Schneider's). Add 10 gms. carmine to 100 cc. 45% aq. glacial acetic acid. Dissolve with heat and bring up to boiling. Cool, filter, and store as stock solution. Used for smears this combines fixation with staining; but it causes a swelling of some cellular elements and is not recommended. Aceto-Orcein-Fast-Green. — Written by Dr. N. B. Kurnick, Dept. of Medicine, Tulane University, New Orleans 12. January 31, 1951 — This modification of La Cour's aceto-orcein stain-fixative for chromosomes permits a one-step difi"erential staining of tissues. The introduction of fast green and NaCl (to prevent overstaining by the former) provides a green counterstain for the reddish-brown chromatin. The intensity of this counterstain may be modified by varying the salt concentration (increas- ing the salt concentration reduces the intensity of green staining), but the method described here has proved satis- factory for most materials. The stain mixture may be used as a stain-fixative, as for dipteran salivaries, some plant materials, and for the study of isolated chromosomes and nuclei, or as a stain following other fixatives. Flood ma- terial for few minutes in following solu- tion: 27 ml. 1% orcein in 45% acetic acid, 3 ml. 1% fast green in 95% alcohol, 2 ml. 2M NaCl; cover with cover slip, press out on filter paper, if desired. Paraffin sections must be brought to water before staining Permanent mounts may be prepared by rinsing the stained material successively in 70%, 95%, 100% alcohol, xylene, and mounting in Clarite. Cytoplasm, col- lagen, and nucleoli are green, chromatin is reddish-brown (Kurnick, N. B., C^old Spring Harbor Symp. Quant. Biol., 1947, 12, 191; Kurnick, N. B. and Ris, Hans, Stain Tech., 1948, 23, 17-18). Acetone, see Dehydration of Tissues, as fixative for Phosphatases and Lipases. Acid Alcohol is used for the differentiation, or decolorization, of certain stains. It is usually made by adding 1 cc. hydrochloric acid to 99 cc. 70% ethyl alcohol. It is also employed for clean- ing cover glasses. Acid Alizarin Blue (1) G.R. (CI, 1048). An acid anthraquinone dye called for in Buzaglo's Method which the author pro- poses as substitute for Van Gieson. (2) B.B. (CI, 1063) likewise an acid anthraquinone dye little used, if at all. ACID ALIZARIN GREEN G ACID FAST BACILLI Acid Alizarin Green G (CI, 1049), a direct mordant dye of color fastness 1. Use for staining blue green and green algae and paraffin sections of animal tissues after mordanting in 1% aq. ferric alum is described (Emig, p. 63). Acid Blue B (CI, 736), an acid dye of light fastness 5 gives light, fugitive and in- distinct coloration of tissue (Emig, p. 52). Acid Blue G (CI, 712)— Brilliant Acid Blue V — an acid dye of light fastness 5 (Emig, p. 52). Acid Bordeaux, see Bordeaux Red. Acid Congo R, see Vital Red. Acid Dyes, see Staining. Acid Fast Bacilli. Of these the organisms of tuberculosis and leprosy are the most important. 1. In smears apply Carbol Fuchsin gently heat 3-5 min. or stain room temperature 15 min.; decolorize 95% ethyl alcohol containing 3% of cone, hydrochloric acid until only slight pink color remains; wash in water; counter- stain sat. aq. methylene blue or Loef- fler's Alkaline Methylene Blue; wash and dry. 2. In sections the organisms can be stained red in paraffin sections after almost any fixation (formalin-Zenker preferred). First color with Harris hematoxylin. Wash in water and per- haps decolorize a little in Acid Alcohol. Wash again. Stain with warmed carbol fuchsin 1 hr. or more. Decolorize in acid alcohol. Wash carefully in water plus few drops ammonia. 95% ale, abs. ale, xylol, balsam. A critique of the methods has been published (Fite, G. L., Am. J. Path., 1938, 14, 491-508). To color the organisms blue, fix 3-5 days or more in equal parts 10% formalde- hyde and 95% alcohol. Stain sections in new fuchsin 0.5 gm.; phenol crystals, 5.0 gm.; alcohol methyl or ethyl, 10 cc. + aq. dest. to make 100 cc. at 60° C. over night, 12-24 hrs. or at room tem- perature 24-48 hrs. Longer for M. leprae. Freshly distilled aq. formalde- hyde 5-30%, 5 min. (Note that this formalin must not be alkaline and that it is safer to have it faintly acidified.) 2% hydrochloric acid in 95% alcohol, 5 min. 1% aq. potassium permanganate 2-5 min. (until brown). 2% aq. oxalic acid, 1 min. Harris' hematoxylin 2 min. Stain in acid fuchsin, 0.1 gm.; picric acid, 0.5 gm.; aq. dest. to make 100 cc. Without washing, dehydrate in alcohol, clear in xylol and mount in balsam. Nuclei, brown; connective tissue fibers, red; muscle, yellow; acid fast bacilli, dark ultramarine blue. Good for photography (Fite, G. L., J. Lab. & Clin. Med. 1939, 25, 743-744; re- vised by G. L. Fite, U. S. Marine Hos- pital, Carville, La. May 13, 1946.). 3. Mr. J. M. Albrecht employs the following method in our laboratory. Deparaffinize 5-6 n sections of 10% formalin or Regaud fixed tissues. Wipe off excess water around sections and cover with strip of filter paper. Flood filter paper with carbol fuchsin (Phenol crystals, 8 gm.; basic fuchsin, 4 gm.; 95% ethyl alcohol, 20 cc; aq. dest., 100 cc). Steam for 3 min. and then allow to stand for 30 min. adding more stain if necessary. The filter paper prevents deposition of ppt. of dye on sections. Flush off stain with aq. dest. Partly differentiate in 1 cc. cone hydrochloric acid in 100 cc. 70% alcohol, sections be- coming deep pink. Wash in aq. dest. Stain Harris' Hematoxylin 10 min., wash in aq. dest. Complete differentia- tion of both fuchsin and hematoxylin in 50 cc. 70% ale -f 4-5 drops hydrochloric acid, sections becoming light pink. Wash in aq. dest. Neutralize in 6 drops cone ammonia + 50 cc. aq. dest. Wash, dehydrate, clear and mount as usual. 4. In frozen sections (Krajian, A. A., Am. J. Clin. Path., Techn. Suppl., 1943, 7, 45-47). Transfer frozen sections of leprous tissue to slides. Dehydrate, blot with filter paper, dip in celloidin. Blow over surface till dry. Wash in tap water. Apply Carbol Fuchsin steaming gently for 3 min. Pour off and wash in tap water. Differentiate with 1 gm. arsenic acid in 100 cc. 60% alcohol ap- plied by medicine dropper. Again wash in tap water and counterstain with Loeffler's methylene blue 2 min. Wash in tap water, dehydrate with 3 applica- tions of anhydrous isopropanol or absolute ethyl alcohol. Apply imme- diately equal parts anhydrous iso- propanol or abs. alcohol and beechwood creosote. Agitate slide removing ex- cess blue color. Blot with filter paper, clear with xylol and mount in damar. See Tubercle and Leprosy Bacilli, Fluorescence Microscopy, also paper by Richards, O. W., Kline, C. K. and Leach, R. E., Am. Rev. Tubere, 1941, 44, 255-266. Efficiency of Ziehl-Neel- sen and fluorescence techniques com- pared. The latter superior (Van Dyke, A. E., Am. J. Clin. Path., Techn. Suppl., 1943, 7, 6-8.) For acid fast bacilli in urine see Kelso, R. E. and Galbraith, T. W., Am. J. Clin. Path., Techn. Suppl., 1943, 7, 8-11. Less is known about the conditions that determine acid fastness than those which determine Gram positiveness (see Gram Stain). The facts are well stated for mycobacteria in general and ACID FUCHSIN ADENOSINASE especially for the Tubercle Bacillus by Dubos, R. J., The Bacterial Cell. Harvard Univ. Press, 1945, 460 pp. There is present in the tubercle bacillus mycolic acid which is acid fast even after isolation in the pure state; but the property of acid fastness is lost by the bacilli under conditions that do not destroy this acid. These conditions involve destruction or impairment of structure of the organisms by mechani- cal, chemical or enzymatic means. Apparently the cell surface must be intact. Dubos quotes Yegian et al. as showing that tubercle bacilli stained in absence of electrolytes are uniformly colored rods, that addition of electro- lytes causes a beaded appearance and that treatment with ethyl alcohol re- stores uniform solid staining to beaded organisms which means that the change from beaded to uniform state is a re- versible process. This dependence of microscopic appearance on experi- mental conditions of technique is ob- viously a matter of great consequence in leprosy as well as in tuberculosis. The investigator has to check carefully by study of living unstained bacilli. Acid Fuchsin (CI, 692) — acid magenta, acid rubin, fuchsin S, SN, SS, ST or S Ill- Commission Certified. Since this is a sulfonated derivative of basic fuchsin, and, because there are 4 possible pri- mary basic fuchsins, Conn (p. 118) points out that at least a dozen primary acid fuchsins are possible and samples are usually mixtures of several. Acid fuchsin is employed is so many ways that to enumerate them would be both futile and unnecessary. See New Fuchsin. Acid Green, see Light Green SF yellowish. Acid Green O, see Naphthol Green B. Acid Hemalum, see Hemalum. Acid Magenta, see Acid Fuchsin. Acid Orange II, Y or A, see Orange II. Acid Phosphatase, see Phosphatase. Acid Phloxine GR, see Chromotrope 2R. Acid Rubin, see Acid Fuchsin. Acid Violet. Several triphenyl methane dyes come under this heading. Conn (p. 132) says that the term "acid violet" is too indefinite for identifica- tion. This is unfortunate because dyes bearing this label have been used in several combinations as in Bensley's Neutral Safranin acid violet. Bailey, P., J. Med. Res., 1921, 42, 349-381 and Maurer, S. and Lewis, D. D., J. Exp. Med., 1922, 36, 141-156, working in Bensley's laboratory, used it for the pituitary. Acid violet is one of the stains employed by Weiss, E., J. Inf. Dis., 1928, 43, 228-231 to stain flagella and spirochetes (J. Lab. & Clin. Med., 1928-29, 14, 1191-1193). Acid Yellow, see Fast Yellow. Acid Yellow R, see Metanil Yellow. Acidity, see Hydrogen ion indicators. Acidophilic, see Staining. Acids, see under first name, Acetic Acid, Hydrochloric Acid, etc . Ackerman, see Auer Bodies. Acridine Dyes. As the name suggests they are formed from acridine which is re- lated to xanthene. Examples: acri- flavine, neutral acriflavine and phos- phine. Phosphine 3R is employed as a fluorochrome for lipids. Acridine Orange (CI, 788), a basic dye of light fastness 1 to 2. Gives clear brown or dark orange coloration of plant tis- sues of exceptional fastness. Tech- nique described (Emig, p. 55). Acridine Red 3B is, according to McClung, Microscopical Technique, 1950, p. 73, not an acridine dye but a pyromin closely related to Pyronin Y. It has been employed by Gomori, G., Am. J. Path., 1936, 12, 655-663 mixed with methyl green to reveal calcium salts and phosphatase activity. Acriflavine (CI . 790) . A yellow fluorchrome . It is useful as a vital stain for nuclei. Farr, R. S., Anat. Rec, 1946, 94, 16, has employed acriflavine hydrochloride to label transfused leucocytes and to determine how long they remain in the circulation. Actinomyces. Mallory's stain for actino- myces in sections (Mallory, p. 279). For the organisms, fixation in alcohol or in 10% formalin is preferable; but for the lesions, Zenker's fluid is better. Stain deparaffinized sections in Alum Hematoxylin 3-5 min. After washing in water stain in 2.5% aq. phloxine or in 5% aq. eosin in paraffin oven, 15 min. After again washing, stain in Stirling's or Ehrlich's aniline crystal violet (see Anilin Crystal Violet), 5-15 min. Wash in water and treat with Gram's Iodine, 1 min. Wash in water, blot and destain in aniline oil until no further color comes out. Rinse in xylol and mount in balsam. Branched forms, blue; clubs, pink to red. Actinosphaerium, see McClung, Microscopi- cal Technique, 1950, p. 469. Addis Count to provide quantitative data on number of red blood cells and casts in the urine is critically described by C. J. Gentzkow and H. A. Van Auken in Simmons and Gentzkow, p. 32. Adenosinase. A method for analysis of adenosinase in lymphocytes and poly- morphonuclear leucocytes (neutro- philes) is given by Barnes, J. M., Brit. J. Exp. Path., 1940, 21, 264-275. ADENYLPYROPHOSPHATASE AGONAL^CHANGES Adenylpyrophosphatase. The technique of localization of this inaportant enzyme in cytoplasmic granules has been de- scribed and used in extracts of chick embryos by Steinbach, H. B. and Moog, F., J. Cell and Comp. Physiol., 1945, 26, 175-183. These authors are, how- ever, not sanguine about the feasibility of its localization by histochemical methods (Science, 1946, 103, 144) as reported by Glick and Fischer, Science, 1945, 102, 429^30. However, Malngwyn- Davies, E. D. and J. S. Friedenwald, J. Nat. Cancer Inst., 1950, 10, 1379, recently reported at the Histochemical Society that specific localizations were achieved when unfixed frozen sections were incubated in muscle adenosine triphosphate substrates. Adermin, see Vitamin B6. Adhesiveness, or stickiness of cellular sur- faces is a phenomenon of great im- portance in connection with movement, phagocytosis embryological develop- ment and other processes. There is no standard technique to measure it, ex- cept in special circumstances as when it ia manifested by agglutination of bacteria and sedimentation of red blood cells. The way leucocytes stick to the endothelial wall of a small blood vessel, shown by Motion Pictures, is impres- sive. Adhesion tests have been intro- duced as means of diagnosis of various trypanosomes. A fine general discus- sion of this phenomenon is provided by Beams and King in Calkins, G. N. and Summers, F. M., Protozoa in Biologi- cal Research. New York: Colombia University Press, 1941, 1148 pp. Adrenal. For routine purposes fix in Zenker's Fluid and stain paraffin sec- tions with Hematoxylin and Eosin. There are many techniques for Lipids. The Chromaffin Reaction is often used for adrenalin but Cramer, W., J. Path. & Bact., 1937, 44, 633, considers black- ening with osmic acid vapor as more specific. Silver methods for vitamin C are difficult to apply but are appar- ently reliable. They are given under Vitamins. The Schultz cholesterol test gives excellent results. A selection may be made from several methods for Reticular Fibers. Corner, G. W., Con- trib. to Embryol., Carnegie Inst., 1920, 9, 87-93, employed for reticulum the Bielschowsky-Maresch silver method exactly as specified by Ferguson, J. S., Am. J. Anat., 1912, 12, 277-296. The Bodian protargol method for nerve fibers has been adjusted to the adrenal by MacFarland, W. E., and Davenport, H. A., Stain Techn., 1941, 16, 53-58, also Cajal's chloral hydrate method. If one contemplates ultracentrifugation and the demonstration of the Golgi apparatus consult Guyer, M. F., and Claus, P. E., Anat. Rec, 1939, 73, 17-27. Method proposed by Bennett, S. H., Am. J. Anat., 1940, 67, 151-227 for keto- steroid cortical hormone said by Go- mori, G., Proc. Soc. Exp. Biol. & Med., 1942, 51, 133-134 not to be specific but to indicate merely location of lipids having keto or aldehyde groups. A technique for microscopic study of living grafts of adrenal cortex (Wil- liams, O., Anat. Rec, 1945, 91, 307). Adrenalin, see Chromaffin Reaction. Aerosol, a detergent used in preparing bac- teria for staining (Sineszko, S. F., Science, 1942, 96, 589). Affixatives are materials used to fix sections to slides. See Albumen-Glycerin. Agar, as matrix for cutting plant material with freezing microtome (Evenden, W. and Schuster, C. E., Stain Techn., 1938, 13, 145^146). Lillie (p. 42) says that infiltration of tissues from water in 2% aq. agar at 55-60°C. for 2-4 hrs. is useful for holding friable tissues and exudates in place before cutting frozen sections. The Agar does not color appreciably with the usual stains. Age Changes are as manifold as life itself. Some are detectable by structural modifications while others can only be measured by decrease in performance. Many old tissues can easily be dis- tinguished from new ones as for example Bone. Some accumulate definite prod- ucts with age like Lipofucsin. The age of tissue and of cellular components, that is the time they endure, can be determined by attaching Tracer Sub- stances to them so that their rates of Replacement can be measured. With the passage of time colloids age, become less elastic and more granular. Old Elastic Fibers can be distinguished from young ones. Now that the ultra struc- ture of Collagenic Fibers has been re- vealed by the electron microscope we may hope for more accurate means of estimating their condition in relation to age. Numerous physical techniques, including the Polarization Optical Method, may well bring to light sig- nificant age changes. Obviously many methods of chemical analysis and of enzyme activity provide data on the modes of run down of vital activities. Aggeler, see Blood Platelets. Agonal Changes are particularly difficult to avoid in villi of small intestine. They are evidenced by a ballooning of the epithelial cap most marked when absorption of ordinary food stuffs is active. The ballooning phenomenon can be produced in the living animal by ALBERT'S STAIN ALDOLASE ligating arteries of supply or by em- ploying fixatives which induce forcible contraction of smooth muscle (Macklin, C. C. and M. T., Chapter on Intestinal Epithelium in Cowdry's Special Cy- tology, N. Y., Hoeber, 1932, 1, 235). Albert's Stain for Diphtheria Bacilli, which see. Albumen-Glycerin for mounting paraffin sections. Egg white 50 cc, glycerin 50 cc, sodium salicylate 1 gm. This is "Mayer's Albumen". Shake together and filter during several days. See also Starch Paste and Masson's Gelatin Glue. Alcohol. Unless indicated to the contrary the word "alcohol" as employed in this book refers to the ethyl variety. Alone it is a good fixative preliminary to tests for Amyloid, Copper, Fibrin, Glycogen, Gold, Hemofuscin, Hyaline, Iron, Lead, Palladium, Phosphatase, Potassium and Thallium, which see. It is also employed in the demonstration of Nissl bodies by Gallocyanin, of mucus by Mucicarmine, of proteins by the Ro- mieu Reaction, etc. In combination with other chemicals alcohol is also much used as a fixative, see Alcohol Formalin, Carney's Fluid and many others. Alcohol of 70% is a good preservative and celloidin blocks can be stored in it. Absolute alcohol is supposed to contain not more than 1% by weight of water. It is considered to be 100 per cent. A very rough test for absolute alcohol is to mix with it a few drops of turpentine. If it becomes milky it contains too much water. To make a lower per cent from a higher one by dilution take the number of cc. corresponding to the percentage required and add aq. dest. to make in cc. the percentage of the alcohol diluted. Thus to make 30% from 70% take 30 cc. of 70% and add aq. dest. to make 70 cc. Alcohol is the best dehydrating agent for tissues. It is sometimes not easy to purchase absolute alcohol so that it must be prepared. Take say 10 liters of 95% alcohol, add 400 gms. freshly ignited calcium oxide. Leave, with occasional shaking, 24 hrs. until most of the water is absorbed by the oxide. Pour off fluid (leaving oxide at bottom of con- tainer) and distill using appropriate precautions. Keep the "absolute" as nearly so as possible by using a tight glass stopper for the bottle, or in place of the stopper an absorption tube con- taining calcium chloride so that any water in entering air will be absorbed and will not reach the alcohol. See Dehydration, also Amyl, n-Butyl, Ter- tiary Butyl, Isopropyl, n-Propyl and Polyvinyl Alcohols. Alcohol -Formal in is a fixative containing 9 parts of absolute alcohol and 1 part of formalin. Since it penetrates quickly and dehydration can be commenced in absolute alcohol immediately after fixa- tion, skipping the lower grades of alco- hol, permanent preparations can be made within a few hours' time. For routine purposes 3-6 hrs. fi.xation will suffice but as a preliminary to Micro- incineration 24 hrs. is recommended. Alcohol-formalin is recommended for Fibrin, Glycogen, Indigo-Carmine stains and Peroxidase. It is employed with acetic acid in Bodian's Method for nerve fibers. Aldehyde Green (CI, 676a) — Aniline Green, Benzaldehyde Green— a basic dye of light fastness 4, employed as counter- stain for Biebrich Scarlet, Acid Fuch- sin. On xylene and sclerenchyma gives rather brighter shade than Malachite Green (Emig, p. 48). Aldehydes. The bound form of aldehyde has been called "plasmalogen". From this the loosely bound "plasmal" form is developed on treatment with mercuric chloride or by acid hydrolysis (the Schiff Reaction for aldehydes). This is the basis of the Feulgen reaction for Thymo- nucleic Acid. According to Oster, K. A. and Oster, J. G., J. Pharmacol, and Exper. Therap., 1946, 87, 306-312, the fuchsin sulphurous acid reagent em- ployed in this reaction when "true" is specific for aldehydes while other car- bonyl compounds sometimes give a "pseudo" reaction. Oster, K. A. and Mulinos, M. G., J. Pharmacol, and Exper. Therap., 1944, 80, 132-138 report that the purple of the "true" reaction can be decolorized with dilute sodium hydroxide and restored with hydro- chloric acid, whereas the reddish tint of the "pseudo" reaction cannot be restored in this way after decoloriza- tion (see Glick p. 65). See Thymo- nucleic Acid (desoxyribonucleic acid) and Carbonyl Compounds. Aldolase. Triose phosphate was used as a substrate for the Gomori phosphatase procedure by Allen, R. S. L. and G. J. Bourne, J. Exp. Biol., 1943, 20, 61-64. The phosphate ion liberated by enzymic activity was precipitated in an alkaline medium. The enzyme was not in- hibited by fluoride in concentrations which blocked alkaline phosphatase activity. The pH of the mixture was not stated, but was presumably about 9.5. The method was not completely satisfactory because of spontaneous precipitates; nevertheless, concentra- tions of enzyme were noted in striated and cardiac muscle, kidney and liver. Little or none was seen in the lung. ALGIRE ALPHAZURINE 2G Aldolase + Isomerase are known as Zymohexase. Algire, see Transparent Chamber Tech- niques. Alizarin (CI, 1027) a little used acid an- thraquinone dye. Alizarin No. 6, see Purpurin. Alizarin Blue RBN, see Gallocyanin. Alizarin Carmine, see Alizarin Red S. Alizarin Cyanine R (CI, 1050), an acid mor- dant dye which is not stable in solution, and on heating yields reddish ppt. (Emig, p. 64). Alizarin SX, or anthrapurpurin, use same as Alizarin Red S. Alizarin Green G (CI, 917), an acid mordant dye of light fastness 1. After mor- danting in 1% aq. ferric alum stain for 30 min. at 50°C. in 0.1 gm. of dye in 100 cc. 1% aq. ammonium acetate. The green color obtained is the clearest given by a mordant dye. Additional directions are supplied (Emig, p. 59). Alizarin Line Test for new bone and vitamin D (Martin, G. J., J. Lab. & Clin. Med., 1940,26,714-719). See Line Test. Alizarin Purpurin, see Purpurin. Alizarin Red S (CI, 1034) — alizarin red water soluble, alizarine carmine — Com- mission Certified. By far the most used of all the alizarin stains. An important ingredient in Benda Method. Much superior to Madder for the staining of bone and dentine laid down while it is in the circulation. Schour has employed it extensively. The technique is de- scribed in detail by him and his asso- ciates (J. Dent. Res., 1941, 20, 411-418). He employed an Alizarin red S (CI, 1034) obtained from Coleman and Bell Co. The effective dose for rat, rabbit, guinea pig, cat, monkey and human in- fant is between 50^100 mg. per Kilo, conditioned by species, age and weight. For newborn white rats he recommends 0.2 cc. 2% Alizarin and for rats weighing 100-200 gms. 5-I cc. given intraperi- toneally. Colors are retained in speci- mens fixed in 10% neutral formalin or in 95% ale. As in the case of Madder staining of bone, tissues can be cleared and examined as whole preparations, or ground sections can be prepared for microscopic study. Decalcification spoils the color. Age factor in alizarin staining (Ercoli, N. and Lewis, M. N., Anat. Rec, 1943, 87, 67). See Ossifica- tion and Line Test. Alizarin Red Water Soluble, see Alizarin RedS. Alizarin Sapphire BN (CI, 1054) of NAC, a direct mordant dye of light fastness 2 (Emig, p. 64). Alkali Blue 6 B (CI, 703), an acid dye of light fastness 4 to 5 and of little value for permanent preparations (Emig, p. 51). Alkali Green (CI, 665), an acid dye of light fastness 5 gives very fugitive pale dull green color (Emig, p. 47). Alkaline Methylene blue, see Loeffler's. Alkaline Phosphatase, see Phosphatase and Kidney. Alkalinity, see Hydrogen Ion Concentration. Allantoin, colorimetric method, Borsook, J. Biol. Chem., 1935, 110, 481-493. Allen's Fluids are modifications of Bouin's often containing urea. They are excel- lent for chromosomes. See McClung. Allergy, see Pollens. Allochrome Procedure, a differential method segregating tissues, collagen, reticulum and basement membranes into two groups (Lillie, R. D., Am. J. Clin. _Path._, 1951, 21, 484-488). The term is derived from G. allochroos, a different or changing color, since colla- gen and some related substances during the procedure changes from red to blue. See Lillie's paper for details of this Periodic Acid Schiff, picro-methyl blue technique. Alloxan Reaction. 1% alcoholic solution of alloxan gives red color with a aminoacids. Romieu (M., Bull. d'Hist. appl., 1925, 2, 185-191) employs a cold neutral solu- tion. Giroud (A., Protoplasma, 1929, 7, 72-98) uses heat but states that great care is necessary in interpretation. See Lison, p. 129. This reaction is described as follows by Serra, J. A., Stain Techn., 1946, 21, 5-18. Fix tissue as given under Nin- hydrin Reaction. "An alcoholic 1% solution of alloxan gives with amino acids and proteic compounds a pink coloration, after a long time at room temperature, or rapidly if the reaction is activated by heating in a boiling water bath. In our experience, this test is relatively insensitive; besides this, the coloring formed diffuses easily, so that the reaction can be in- distinctly localized. With fixed mate- rials the reaction is weak. "The test must be carried out in neutral solutions; this is attained by addition of a phosphate buffer, as de- scribed for the ninhydrin. This reac- tion is not specific for amino acids and proteins, as it is also given by com- pounds with free NH2 and perhaps SH groups (see Winterstein, 1933)." Almkvist-Christeller, see test for Mercury. Alpha Naphthol, see Oxidase. Alpha Particle, see Gross, J., Bogoroch, R., Nadler, N. J., and Leblond, C. P., Am. J. Roentgenol. Rad. Therap., 1941, 65, 420-458. Alphazurine 2G see Patent Blue. ALTMANN'S FLUID ALUMINUM CHLORIDE CARMINE Altmann's Fluid. Equal parts of 5% aq. potassium bichromate and 2% aq. osmic acid. Employed in his method as well as for staining with Copper Chrome Hematoxylin. It gives good surface fixation but penetrates very badly. Altmann's Method of anilin fuchsin and pic- ric acid for mitochondria. Fix small pieces not more than 2 mm. in diameter 24 hrs. in Altmann's Fluid. Wash for 1 hr. dehydrate, clear imbed in paraffin and cut sections i/i. Pass down to water. Stain in anilin fuchsin (20% acid fuchsin in anilin water) 6 min. Blot with filter paper. Differentiate and counter stain by flooding the sec- tions with 1 part sat. ale. picric acid and 2 parts aq. dest. Rinse rapidly in 95% ale, dehydrate in abs. ale, clear in xylol and mount in balsam. The mito- chondria are stained crimson against a bright j^ellow background. Altmann's magnificent original plates should be examined (Altmann, R., Die Elementar- organismen und ihre Beziehungen zu den Zellen. Leipzig: Veit Co., 1894, 160 pp.). If these are not available see Meves, F., Arch. f. mikr. Anat., 1913, 82, (2), 215-260. Altmann-Gersh frozen-dehydration method (Gersh, I., Anat. Rec, 1932, 53, 309- 337). — Account written by Dr. Gordon H. Scott, Dept. of Anatomy, Wayne University School of Medicine, De- troit, Mich. This method has proved to be of much value in the preparation of tissues for microchemical proce- dures. It has also been used as a pre- liminary treatment for tissues destined for examination by the electron micro- scope (Wyckoff, R. W. G., Science, 1946, 104, 21-26). Tissues are frozen in liquid nitrogen or in liquid oxygen and dehydrated in vacuo at low tem- peratures. The tissue sample remains frozen at such a temperature that little or no chemical change can take place. It is believed that the only significant revision in cellular organization takes place during the freezing process. This is occasioned by possible shifts in pro- teins, etc., during ice crystal formation. Some users of the method believe that it is possible to freeze small tissue samples at speeds which will actually prevent ice crystal formations. Efforts in this direction have been made by freezing in cooled iso-pentane (technical) (Hoerr, N. L., Anat. Rec, 1936, 65, 293- 317; Simpson, W. L., Ibid., 1941, 80, 173-189). For many reasons it has been found desirable to dehydrate at lower tem- peratures than were first thought neces- sary. Now the standard procedure is to dehydrate in vacuo from 40-65°C. Apparatus of special design has been constructed a number of times to meet various needs. In general the prin- ciples are the same. What is needed is a vacuum system with high pumping speed and with provision for keeping the frozen tissue at constant tempera- ture. Several of these have been de- scribed, each with its adaptation to the needs of the case. For general use in histochemistry the device described by Packer and Scott (J. Tech. Methods, 1942, 22, 85-96) and by Hoerr and Scott (Medical Physics, Otto Glasser, 1944, Year Book Pub- lishers) is both easy to operate and re- liable. It has the distinct advantage that tissues can be infiltrated with paraffinjwithout^exposure to air. This apparatus can also be used for the preparation of tissues for electron microscopy. For this use only the de- hydration device described by Wyckoff is probably more suitable. Recently developed equipment will permit drying frozen tissues in 5 hours. (Stowell, R. E., Stain Techn. 1951, in press) Alum. The alums are double salts of sul- phuric acid. Aluminum potassium sul- phate, or potassium alum, unless other- wise stated is the one used in making up hematoxylin solutions. Aluminum am- monium sulphate, or ammonia alum, should not be used as a substitute unless called for. Ammono-ferric sulphate, or iron alum is used as a mordant and differ- entiator in the iron hematoxylin tech- nique and for other purposes. The crystals are of a pale violet color. Their surfaces oxidize readily and become use- less. The surface should be scraped off. Only the violet crystals are of any use. Alum-Carmine (Grenacher). Boil 1-5% aq. ammonia alum with 0.5-1% powdered carmine. Cool and filter. Does not penetrate very well and hence is not suitable for staining large objects in bulk. But it is useful and does not overstain (Lee, p. 140). Alum-Cochineal, see Cochineal. Alum Hematoxylin. Many hematoxylin so- lutions contain alum, see Delafield's, Ehrlich's, Harris', Mayer's. Aluminium Chloride Carmine (Mayer). Dissolve 1 gm. carminic acid and 3 gm. aluminium chloride in 200 cc. aq. dest. Add an antiseptic as formalin or 0.1% salicylic acid. Employ in same way as carmalum. Gives blue violet color. Very penetrating but not so specific for chromatin as carmalum (Lee, p. 142). ALVEOLAR EPITHELIUM OF LUNGS 8 ALVEOLAR PORES Alyeolar Epithelium of Lungs 1. Gold sodium thiosulphate (Bensley, R. D. and S. H., Anat. Rec, 1935, 64, 41-49). Inject a mouse intravenously through the tail vein with 100 mg. of gold sodium thiosulphate in 1 cc. aq. dest. The mouse dies in about 20 min. from asphyxia. Fix pieces of lung in 10% neutral formalin, dehydrate with- out washing in water, clear and imbed in paraffin. Deparaffinise sections and stain in 1% aq. toluidin blue (tested for polychromatism) and examine in water. The epithelium is raised by in- crease in volume of ground substance which is stained metachromatically pink while the cells and their nuclei are blue. The color of the ground sub- stance can be changed to blue by alco- hol and back again to pink by water. To mount protect against reversing action of alcohol by treating with equal parts freshly prepared 5% aq. am- monium molybdate (Kahlbaum or Merck) and 1% aq. potassium ferro- cyanide. Dehydrate, clear in xylol and mount in balsam. (Revised by R. D. and S. H. Bensley, Dept. of Anatomy, University of Chicago, Chi- cago, 111., April 18," 1946.) 2. Silver nitrate (Bensley, R. L). and S. H., Anat. Rec, 1935, 64, 41-49). Use guinea pigs. Silver Citrate sol. (which see) is injected into lung substance by hypodermic syringe, the roots of the lung being first ligated, until the lung is moderately distended. Cut out pieces, fix in 10% formalin, imbed in paraffin or celloidin, section, develop with dilute photographic developer and counterstain or examine unstained. The margins of the cells are blackened. For the most delicate results a slow acting, fine grain developer such as the following should be used: phenyl hy- drazine hydrochloride, 1 gm., sodium sulphite (anhydrous), 10 gm.; aq. dest., 100 cc. Caution: Phenyl hydrazine hydrochloride is extremely toxic to some people producing skin reactions. (Revised by R. D. and S. H. Bensley, April 18, 1946.) Alveolar Fluid. Method for collecting, Terry, R. J., Anat. Rec, 1926, 32, 223- 304; 1936, 64, 75. Alveolar Foam Cells. — Written by C. C. Macklin, Dept. of Histological Re- search, The University of Western Ontario, London, Canada. November 28, 1951. — These represent nonphago- cytic pneumonocytes which have be- come free in the alveoli and air tract of the lungs. They may be obtained by the "gash-irrigation" and "wash- out" techniques (which see). Macklin found in them refractile vacuolelike bodies or vacuoloids (which see) readily demonstrable in fresh mounts by bright- or dark-field illumination (Proc 6th International Congress of Experimental Cytology, Stockholm, 1947, published 1949, pp. 383-387). In ordinary sec- tions these bodies appear empty or with a very small granule within them. When foreign particles appear in them they are known as "Dust Cells" (which see). Mitochondria in foam cells are mainly in the frothy perivacuoloidal cytoplasm (Macklin, C. C, Biol. Bull., 1949,96, 173-178). Alveolar Phagocytes of Lungs, see Dust Cells. Alveolar Pores of the lung.^Revised by C. C. Macklin, Dept. of Histological Research, The University of Western Ontario, London, Canada. November 28, 1951— Formalin (10%) and Zenker- formalin are among the fixatives sug- gested. The fixative is injected into the trachea or bronchus at a gravity pressure of 4-6 inches until the lungs are moderately distended. During this operation they are covered with physio- logical salt solution. The lungs are then immersed in fixative for days or even weeks. Slices about 1 cm. thick are cut, imbedded in soft paraffin and sections are made at 100/i or more. Resorcin-fuchsin and other stains may be used. The blood in the capillaries is a useful guide. The pores can be identified by their rounded edges (Macklin, C. C, Arch. Path., 1936, 21, 202-216). In lungs fixed by immersion of the flayed intact thorax (IIT) or perfusion of the pulmonary capillaries of the in- tact thorax (PIT) the pores, in thick sections, appear as short narrow tunnels with funnel-shaped entrances joining neighboring alveolar spaces. Their ex- tremely thin walls are composed of parts of the contiguous capillaries. (See "Fixation of the Uncollapsed Lung" and "Dust Cells".) Thus seen a pore is the empty sheath or vagina from which a process of a pneumonocyte (septal cell, alveolar wall cell, etc.) has been withdrawn. When such a process cannot be withdrawn because it is bul- bous the cell is seen as a dumbbell - shaped structure with the thin con- necting part in the pore. In its func- tional state this vagina is occupied by a part of a pneumonocyte, and the latter is thus placed where nutrition from the capillaries is constantly available. Any good fixative suffices to show pores by these methods. When pieces of lung tissue are fixed by immersion the ensuing contraction usually closes the pores to that they cannot be seen ALVEOLAR SIZE 9 AMLXE OXIDASE (Macklin, C. C, Jour, of Thor. Surg., 1938, 7, 53&-551, Macklin, C. C, The Lancet, Feb. 24, 1951, 432-435). See also Loosli, C. G., Arch. Path., 1937, 24, 743, and Loosli, C. G., Amer. J. of Anat., 1938, 62, 375. When the entire fresh collapsed lungs are fi.xed b}^ filling them with preservative via the trachea, or when parts of the lung are so filled via a bronchus, the alveolar walls are com- pressed and flattened, and here the pores appear as holes punched in thick paper; and if the distention has been great the morphology of the pore and its relation to the environing capillary is not obvious (Macklin, C. C., Trans. Roy. Soc. Can., Sect. V, 1934, 28: p. 28; J. Anat. [pt. 2] 1935, 69, 188; J. Assoc. Am. Med. Coll., 1935, 10, 265; Arch. Path., 1936, 21, 202). Pores are well seen "en face" in frozen sections which have been dried on the slide. The foregoing description refers to true pores. A second type of communi- cation between adjoining alveoli occurs in "medical emphysema" or alveolar ectasia and is due to the wasting of the alveolar sidewalls or bases. These openings may be very large and numer- ous, and the walls are then said to be riddled with them. Pathologists are familiar with this type of "false pore". The true type of pore is usually un- discernible in the collapsed lung tissue examined by pathologists. Alveolar Size, in the lungs. For techniques used in determining alveolar size in ten mammalian types see the papers of W. S. Hartroft and C. C. Macklin in the bibliography of the 26th Lewis Linn McArthur Lecture of the Frank Billings Foundation; by C. C. Macklin in Proc. of the Institute of Medicine of Chicago, 1950, 18, 78-95. Alveolus of the Lungs. The morphologj', in sections, can best be demonstrated in small animals by immersion of the fresh skinned intact thorax in a fixative, such as Bouin's fluid. See Fixation of the uncollapsed lung. Alzheimer's Modification of Mann's eosin- methyl blue for neuroglia and degenerate nerve fibers as given by Mallory (p. 245) is abbreviated. Fix thin slices, 14 days, in Weigert's Neuroglia Mordant -f 10% of formalin. Wash 8-12 hrs. in running water. Mordant lO^i frozen sections 2-12 hrs. in sat. aq. phosphomolybdic acid. Wash 2 changes aq. dest. Stain in Mann's Eosin Methyl Blue 1-5 hrs. Wash quickly in aq. dest. until color "clouds" are no longer given off. Treat with 95% alcohol until gray matter becomes light blue and white matter pink or bright red. Dehydrate quickly in absolute alcohol, clear in xylol and mount in balsam. Normal axis cylin- ders, purple or deep blue ; degenerating ones, red; neuroglia fibers, dark blue; and neuroglia cytoplasm, pale blue. Mallory states that change from blue to red staining of axis cylinders occurs as soon as 48 hrs. after experimental lesion. Amanil Garnet H., see Erie Garnet B. Amaranth (CI, 184) — azo rubin, Bordeaux, Bordeaux SF, fast red, naphthol red S, C or O, Victoria rubin O, wool red — An acid mono-azo dye used long ago by Griesbach, H., Zeit. wis. mikr., 1886, 3, 358-385 to color axis cylinders. Amebae. The techniques that have been and can be employed for the organisms are almost endless. In brief, these are their examination in the living condi- tion unstained using ordinary, phase and dark field microscopes, imple- mented or not by supravital stains. As a beginning, the method of Meyers is suggested: washed amebae in a clean vessel are allowed to settle to the bottom. They are then fixed in warm Bouin's Fluid, concentrated in a centri- fuge tube and stained with Grenacher's borax carmine and Indulin (Meyers, E. H., Trans. Am. Micr. Soc, 1933, 52, 58). For showing cytoplasmic com- ponents of Amoeba Proteus, see Mast, S. O. and Doyle, W. L., Arch. f. Pro- tistenk., 1935, 86, 155. To determine the density of amebae a starch Density Gradient is recommended by Lovtrup, S. C. rend. Lab. Carlsberg, Ser. Chim., 1950, 27, 137-144. For determination of permiability of nucleus see Monn6, L., Proc. Soc. Exp. Biol. & Med., 1935, 32, 1197. The technique for enucelation of fresh water amebae advised by Holter, H. and Kopac, M. J., J. Cell, and Comp. Physiol., 1937, 10, 423 is recommended. See Entameba. Amethyst Violet (CI, 847)— heliotrope B, iris violet — It is a basic azin dye of little importance to histologists. Amine Oxidase. Oster, K. A. and Schloss- man, N. C, J. Cell. Comp. Physiol., 1942, 20, 373-378. As explained by Glick, p. 93, the method is based on detection of aldehyde formed by amine oxidation. The Fuelgen sulphurous acid agent (see Thymonucleic Acid) is employed for microscopic visualization of aldehyde. Binding of naturally occurring aldehydes and "plasmal" is accomplished with bisulphite so that they do not invalidate the method. Because the color produced diffuses, localization is subject to criticism. Place frozen sections of fresh tissue in 2% aq. sodium bisulphite at 37°C., 24 hrs. wash thoroughly. Control sec- tions immersed in Feulgen agent should remain colorless showing that free AMIDONOPHTHOL GR 10 AMYL ACETATE aldehyde has been bound. Incubate these sections in 0.5% tyramine hydro- chloride in M/15 phosphate buffer pH 7.2 at 37°C. 24 hrs. Incubate other control sections in same solution minus tyramine. Immerse both test and con- trol sections in Feulgen agent. Ex- amine when quickly produced blue color seems to be at maximum. Foci of enzymatic activity, blue in con- venient contrast with reddish purple of "plasmal" (see Aldehydes). Amidonaphthol GR, see Azophloxine GA. Amino Acids, see Alloxan Reaction, also Schmidt, C. L. A., The Chemistry of the Amino Acids and Proteins. Spring- field, Charles C Thomas, 1938, 1031 pp. See paper chromatography added by Roberts. Aminoacridines, some are strong antiseptics, do not stain skin (Albert, A. and Ritchie, B., J. Soc. Chem. Ind., 1941, 60, 120). Amitosis is direct nuclear division by con- striction without formation of a chro- matin thread. No special technique required. Study of embryonic mem- branes and of bladder of mouse (Dogiel, A. S., Arch. f. Mikr. Anat., 1890, 35, 389-406) is suggested. Ammonia Carmine (Ranvier). A suspen- sion of carmine in water, with slight excess ammonia, is allowed to evaporate in air. If it putrefies so much the better. Dissolve the dry deposit in aq. dest. and filter (Lee, p. 145). Ammoniacal Silver for branchioles — Written by C. C. Macklin, Dept. of Histological Research, The University of Western Ontario, London, Canada. November 28, 1951 — This is useful to demonstrate the two types of epithelium of the finest bronchioles, as in the albino mouse. There are two stock solutions. Solu- tion 1: Five grams of silver nitrate are dissolved in 300 ccms. of distilled water and dilute aqua ammoniae is added until the precipitate is nearly redis- solved; filter; add water to make 500 ccms. Solution 2: One gram of silver nitrate is dissolved in a small quantity of water and poured into half a liter of boiling water. Rochelle salt (0.83 gm.) is dissolved in a small quantity of water and added to the boiling solution, which is then boiled for half an hour till a gray precipitate gathers at the bottom of the flask. Filter hot; add water to make 500 ccms. In using, equal parts of the two solutions are mixed. The separate solutions, kept in the dark, remain good for a month or two. The etherized animals are exsangui- nated, the lungs collapsed by carefully nicking the diaphragm and filled via the trachea with the ammoniacal silver solution. At the end of two minutes the lungs are emptied, refilled with 10% formalin, and the trachea is tied. The material is placed in 10% formalin for a day or more. Frozen, paraffin, or celloidin sections are cut and exposed to direct sunlight or weak photographic developer. Flattened frozen sections are useful in affording surface views of lengths of bronchiolar epithelium. The following description is from C. C. Macklin (Canad. Jour, of Research, D, 1949, 27, 50-58— Bibliography) : "Two definite types of cells, dark and light, are revealed in the terminal bronchiolar epithelium by this supravital silveriza- tion. The dark cells are ciliated and scattered among the light unciliated cells as singles, pairs, triads or larger groups to form a striking and charac- teristic surface pattern. The cuticles of the dark cells, far outreached by the neighboring light cells, appear en face as dense crowds of small uniform golden brown granules. Viewed laterally these show as pairs in vertical alignment, and make two layers with a thin lighter band between. The sides and bases are in- dicated by deposits of black grains. Near the sharply marked margin of the definitive epithelium the dark cells are shorter, more dispersed, and formed like truncated pyramids with narrow densely impregnated apices on some of which are single delicate points instead of discrete cilia. The light cells have bulging villuslike free ends and make flutings on the contiguous dark cells. Small silverized particles sparsely stipple their air surfaces, and rounded plaques crown their summits. A simi- lar dicellular picture is found in the terminal bronchiolar epithelium of the golden hamster." Ammonium Molybdate, as mordant for Mann's stain and Weigert-Pal (Perdrau, J. R., J. Path. & Bact., 1939, 48, 609- 610. Recommended by Bethe as a fixative for supravital methylene blue, see Lillie, p. 245. Amoeba, see Entameba. Amphinucleolus (G. amphi on both sides). A nucleolus which is double consisting of both acidophilic and basophilic parts, the former is usually a central core ana the latter plastered on its surface. Amphioxus, as an ancestor of vertebrates of interest to all, see the technique of Conklin, E. G., J. Morph., 1932, 54, 69. Amphophilic, see Staining. Amy! Acetate, as solvent for imbedding tissues (Barron, D. H., Anat. Rec, 1934, 59, No. 1 and Suppl., 1-3); as a clearing agent for embryological material (Drury, H. F., Stain Techn., 1941, 16, 21-22). AMYL ALCOHOL 11 AMYLOID Amyl Alcohol. Merck lists 3, commercial, normal and tertiary. It mixes with 95% alcohol and with xylol. HoUande (A. C, C. rend Soc. de Biol., 1918, 81, 223-225) was the first to recommend amyl alcohol as a substitute for absolute alcohol in the dehydration of specimens sta,ined by the Romanovsky and Giemsa techniques. Amyl Nitrite. McClung (p. 620) says that this may serve as a dilator of peripheral capillaries when a complete injection of small blood vessels is required. Add it to the ether at time of anesthetization. Amylase, micromethod for (Pickford, G. E. and Dorris, F., Science, 1934, 80, 317- 319). This was later used with marked success by Dorris (F., J. Exp. Zool., 1935, 70, 491-527) in a study of relation between enzyme production and histo- logical development of gut of ambly- stoma. An extract is made, adjusted to proper pH, applied to slides coated with a starch-agar solution and incu- bated. The slides are then washed, the coating fixed in formalin and colored with dilute iodine solution. Sites of amylase activity are clear or pink stain- ing spots. For necessary details, see author's description, van Genderen andEngel (H.andC., Enzymologia, 1938, 5, 71-80) localized this enzyme by analysis of horizontal sections through the intestinal wall. It was found that it is present in rabbits in maximum amounts in Brunner's glands. Holtfir and Dogle (C. R. Lab. Carlsberg, S6r. Chim., 1938, 22, 219-225) observed that in amebae it is concentrated in association with the mitochondria which they assume to be carriers of amylase. See Barnes, J. M., Brit. J. Exp. Path., 1940, 21, 264-275 for identification of amylase in lympho- cytes and polymorphonuclear leuco- cytes . Salivary amylase digests glycogen in sections, but does not alter mucus, cartilage matrix and other mucins which give similar histochemical reactions. Lillie, R. D., Stain Tech., 1947, 22, 67- 70, recommends a commercial prepara- tion of diastase for this purpose, but Dempsey and Wislocki prefer saliva, since the commercial preparations ap- parently contain traces of mucinase which attacks mucus, cartilage and mast cell granules. Amyloid (G. arnylon, starch and eidos, re- semblance), a substance which accumu- lates in pathological conditions in the tissue fluids between cells particularly in chronic infections. Methods for its detection are fully described by Mallory and Parker (McClung, pp. 417-419). From nimaerous tests the following are selected : 1. Iodine and sulphuric acid: Stain section lightly with Lugol's iodine. Place in 1-5% aq. or cone, sulphuric or hydrochloric acid. Color of amyloid changes quickly from red through violet to blue or it may become deep brown . 2. Methyl-violet: Treat frozen sections of fresh, formalin or alcohol fixed tissue with 1% aq. methyl violet, 3-5 min. Wash in 1% aq. acetic acid, and remove acid by washing carefully in water. Examine in glycerin or water. Amyloid is violet and tissue blue. Colors will be retained longer if sections are mounted in Levulose Syrup. 3. Iodine green: Fresh or hardened sections are stained 24 hrs. in 0.3% aq. iodine green. Wash in water and examine in water or glycerin. Amyloid is stained violet red and tissue, green. 4. Mayer's stain: Transfer paraffin sections immediately after cutting to 0.5% aq. methyl violet or gentian violet at 40°C. for 5-10 min. Rinse in water and differentiate in 1% aq. acetic acid for 10-15 min. Wash thoroughly in wa- ter. Change to ^ sat. aq. alum and wash it off in water. Place section on slide and let water evaporate. Remove paraffin, clear in xylol and mount in balsam. Crystal violet and iodine green can be employed in the same way. A Congo red test has been described (Taran, A., J. Lab. & Clin. Med., 1936- 37, 22, 975-977) and a polysaccharide has been isolated from amyloid bearing tissues which closely resembles chon- droitin-sulphuric acid obtained from infantile cartilage (Hass, G., Arch. Path., 1942, 34, 92-105). As pointed out by Highman, B., Arch. Path., 1946, 41, 559-562 the staining methods for amyloid are in general satisfactory when employed by skilled workers. However, when stained sec- tions are mounted in glycerin Apathy's syrup, or some such medium, they tend to fade quickly, or the stain diffuses out into the surrounding tissue, or mount- ing medium, and the nuclei are seldom sharply colored. Highman therefore recommends staining of deparaffinized sections of formalin fixed tissues in iron hematoxylin 5 min., washing in water, staining in 0.5% crystal violet or methyl violet in 2.5% aq. acetic acid, washing again in water and mounting in Lillie 's Apdthy's syrup modified by addition of 50 gm. potassium acetate or 10 gm. sodium chloride to 100 cc. of syrup. He also gives a technique for mounting in clarite. See Mallory-Heidenhain rapid one step stain. AMNIOTIC FLUID 12 AXILIN BLUE Amniotic Fluid, technique for study of ab- sorption from, Wislocki, G. B., BulL Johns Hopkins Hospital, 1921 32, 93. Anaplasma is a small spherical body found within red blood cells in anaplasmosis diseases. There are two types A margi- nale and A centrale depending upon whether the bodies are situated near the margin or in the centers of the cells. The bodies are supposed to be parasites consisting of nuclear material with little if any cytoplasm. Anaplasmosis is im- portant economically as a group of tick borne diseases of domestic animals. For demonstration stain blood smears by the methods of Giemsa or Wright. Anethol is anise camphor suggested as a medium in which to soak tissues before making frozen sections (Stephanow, Zeit. wiss. Mikr., 1900, 17, 181). Anethole Clearing Agent.— Written by Joan Haberman, Parkland, Washington. March 10, 1951 — Anethole is a useful agent in clearing segments of brain tissue because of its refractive index. The basic principle is that homogeneous tissue, which has been bleached and dehydrated, may be cleared by placing in an oil of similar refractive index was discussed by Groat, R. A., Stain Techn., 1941, 16, 111-117. Brain tissue consists primarily of protein and lipids and is therefore not homogeneous as are other types of tissue such as muscle. Since protein forms the framework of the brain, it necessitates that the lipids be removed to obtain a homogeneous tissue. The lipid composition of the brain tissue must be known in order to per- form an adequate extraction. Accord- ing to Koch, as quoted by A. P. Mathews, Physiological Chemistry. Baltimore: Williams and Wilkins Co., 1939, the lipid composition is as follows: phospholipids— 27.3% of dry matter; cerebrosides— 13.6% of dry matter; cholesterol— 10.9% of dry matter. The phospholipids, according to G. A. Hill and K. Kelly, Organic Chemistry. Philadelphia: Blakiston Co., 1943, are composed mainly of : lecithin— 48 p. p.m., soluble in ether and in alcohol; cepha- lin — 200 p. p.m., soluble in ether, in- soluble in alcohol; sphingomyelin— 56 p. p.m., insolublejin ether, soluble in hot alcohol. These are all insoluble in acetone. The cerebrosides are soluble in hot alcohol, insoluble in ether. Cholesterol can be extracted with ether and alcohol. (The data on solubilities are taken from Harrow, B., Textbook of Biochemistry. Philadelphia: W. B. Saunders Co., 1943). Brain tissue must first be dehydrated by using the alcohol series starting at 35% or higher if one wishes to risk more shrinkage and changing every few hours depending upon the size of the tissue. The lipids are then removed by placing the tissue in 100% alcohol at 60 degrees centigrade for 1-2 days with frequent changes in solution. The remaming lipids are extracted with ether for 2 days or longer depending upon the size of the tissue. The procedure results in a tissue that consists largely of pro- tein and gives a refractive index of 1.560 as determined on the Abb6 re- fractometer. By placing the tissue in an oil of very similar refractive index it will soon be- come transparent when brightly illum- inated. Anethole (C10H12O) having a re- fractive index of 1.5614 was found to be the oil of choice. It is colorless, very slightly soluble in water, soluble in alcohol and ether, and is liquid at ordi- nary temperatures. It can be obtained from the Eastman Kodak Company, Rochester, New York. After the tissue has been placed in anethole the container should be left uncovered so that the displaced ether may escape. The transparency of the tissue de- pends upon the completeness of the lipid extraction. Larger pieces of tissue such as 4 x 6 x 3 cm must be kept in 100% alcohol for a week and in ether for 1-2 weeks. Frequent changes of solution are advisable. This block of tissue will still appear only translucent when cleared in anethole. However, sections of § to 1 cm. in thickness cut from this block will be sufficiently clear to insure visualization of injected vessels etc. under the binocular with good illumination. It is probable that a large piece of tissue could be cleared satisfactorily if the alcohol and ether extraction were carried out by means of an intermittent siphoning distillation extractor. Angina, see Vincent's Angina. Angstrom Unit. lA = 0.1 m/* = O.OOOIm = 10-' mm. Anhydrase, see Carbonic Anhydrase. Anilin Blue Alcohol Soluble, see Spirit Blue. Anilin Blue, WS (CI, 707)— China blue, cotton blue, marine blue V, soluble blue 3M or 2R, water blue (Wasserblau)— A mixture of trisulphonates of di-phenyl rosanilin and tri-phenyl pararosanilin. Conn (p. 135) explains that this desig- nation (like acid fuchsin) applies not to a single compound but to a group of dyes. Anilin blue is, nevertheless, the best stain for Collagenic Fibers and is employed for many other purposes. ANILIN CRYSTAL VIOLET 1. Anilin Crystal Violet 1. Ehrlich's. Shake up 5 cc. anilin oil with 95 cc. aq. dest. Filter and to 84 cc. of filtrate addl6 cc. Bat. ale. crystal violet. Leave 24 hrs. before using. After about 10 days stain- ing potency decreases (Mallory, p. 89). 2. Stirling's. Crystal violet, 5 gm. ; abs. ale, 10 cc; anilin oil, 2 cc, aq. dest., 88 cc. Keeps well (Mallory, p. 90). See Anilin Crystal Violet and Gentian Violet. Anilin-Fuchsin Methyl Green method for mitochondria. This technique is based on Altmann's method. It was used by Bensley to stain tissues fixed in his Acetic-Osmic-Bichromate fluid. Cow- dry recommends instead fixation in the better penetrating Regaud's fluid. Fix small pieces in freshly prepared Regaud's fluid (3% aq. potassium bi- chromate 4 parts, commercial formalin 1 part). Ordinarily it is not necessary to neutralize the formalin before hand by saturating it with magnesium car- bonate. Keep in ice box and change the fluid every day for 4 days. Pour off fixative and mordant in 3% aq. po- tassium bichromate 8 days changing every second day. Wash in running water over night or in several changes of water. Dehydrate iu alcohol, clear in xylol, imbed in paraffin and cut sections about 4 fi thick. Pass mounted sec- tions through xylol and alcohol to water. Dry the slide with a cloth ex- cept area covered by sections. Pour on anilin acid fuchsin and heat to steaming over a spirit lamp. (To make this saturate 125 cc. aq. dest. with anilin oil by shaking the two together. Filter and add 15 gms. acid fuchsin to 100 cc. of filtrate. Allow to stand 24 hrs. before using. It lasts about a month.) Allow to cool and stain about 6 min. Pour stain back into bottle. Remove most of remainder, except from sections, with a cloth or filter paper. Rinse in aq. dest. about 1 min. Allow 1% aq. methyl green, added with a dropper, to flow over sections and counter stain them. This usually takes about 5 sec. but the time must be determined by trial. Wash off excess methyl green in 95% alcohol, dehydrate quickly in absolute, clear in toluol (or xylol) and mount in balsam. The mitochondria are stained crimson and the nuclei green. For colored illustra- tions see Cowdry, E. V., Contrib. to Embryol., Carnegie Inst, of Washing- ton 1917, No. 11, 27-43. If the methyl green does not stain intensely enough treat the sections, before coloration with fuchsin, with l%aq. potassium perman- ganate 30 sec. followed by 5% oxalic acid 13 ANTERIOR CHAMBER TRANSPLANTATION 30 sec. and wash in water. More methyl green can be retained by blotting the sections after staining in it with filter paper and by then passing directly to absolute alcohol. If the time of fixation and mordanting is reduced much below that specified the fuchsin itself may not color with sufficient intensity. Such preparations hold their colors for a year or more unless they have been unduly exposed to sunlight, or the balsam is acid. Anilin Fuchsin Picric Acid, see Altmann's method for mitochondria. Anilin Fuchsin Toluidine Blue and Aurantia, see Champy-Kull method for mito- chondria. Anilin Gentian Violet usually credited to Ehrlich. Rarely is its composition given exactly the same by any two people. The "emended formula" (Soc. Am. Bact.) is A: 2.5 gm. crystal violet (85 per dye content) -f 95% ethyl alco- hol, 12 cc. B: anilin oil 2 cc. -f aq. dest. 98 cc. (shake, leave few minutes, filter). Mix A and B. (McClung, p. 137.) Anilin Oil. A good product is easily obtain- able. It is much used in the making of stains (cf. anilin fuchsin) and to clear tissues from 95% alcohol and even sec- tions from 70%. Lee (p. 71) says that it should not be employed after fixation in osmic acid and that unless removed by chloroform or xylol it will give the tissues and mounting medium a brown coloration . Anilin Red, see Basic Fuchsin. Anilin-Safranin (Babes). Aq. dest., 98 cc. ; anilin oil, 2 cc. ; excess of safranin O. heat in flask in hot water bath at 70- 80°C. Cool, filter and use filtrate. This is an excellent nuclear stain, but it must be freshly prepared every month or so. It colors very promptly. Dif- ferentiation is in 95% ale or even in Acid Alcohol. Anilin Yellow, see McClung, 1950, p. 439. Anterior Chamber Transplantation — Written by Harry S. N. Greene, Labora- tory of Pathology, Yale University School of Medicine, New Haven. Sept. 11, 1951 — The anterior chamber of the eye possesses many advantages over other bodily regions as a transplanta- tion site. The technique of transfer is simple, a high percentage of takes is obtained, and the growing tissue can be followed by direct visual observa- tion or even subjected to microscopic examination. A further advantage de- rives from the fact that the chamber supports the growth of heterologous embryonic or cancer tissue whereas in other bodily sites such tissues generally fail to survive. The technique varies somewhat with ANTERIOR CHAMBER TRANSPLANTATION the size of the animal used. Animals larger than mice are immobilized on an operating board with tie strings in such a manner that the desired eye presents to the operator. The cornea is anaes- thetized by contact with a 5% aqueous solution of cocaine administered with a medicine dropper. An incision is made at approximately the midpoint of the upper border of the limbus utilizing a sharp double-edged corneal knife. The knife is directed slightly forward so that the blade enters the anterior chamber without damage to the iris. The pressure necessary to pierce the cornea is sufficient, even with a sharp knife, to rotate the eyeball beneath the lower lid and conceal the operative field. A short, quick thrust of the knife, however, results in an adequate opening and the temporary obscurity is inconsequential. Withdrawal of the blade is accompanied by the escape of a small amount of aqueous humor, but unless the iris has been cut by a mis- directed knife, there is no bleeding. Transfer of the tissue is effected by means of a trocar commensurate in diameter with the corneal incision. The trocar should be equipped with a tight-fitting plunger and a short bev- elled mouth with all edges filed to smoothness. A small fragment of tissue is placed in the mouth of the trocar and forced into the barrel, a step greatly facilitated by withdrawing the plunger to produce suction. The tip of the trocar is inserted through the incision a short distance so that the bore is entirely within the chamber and the fragment is expelled. It is important that all manual pressure about the eye and head of the animal be released before withdrawing the trocar to pre- vent escape of the fragment. Finally, the fragment is forced into the inferior angle of the iris by applying light pres- sure along the corneal surface with a blunt instrument. The incision is not closed. The knife should be sharp and of such width that a stab wound will admit the trocar. The use of a narrow knife necessitates side cutting to obtain a sufl&cient opening and this may be attended by iris damage with conse- quent bleeding. Pointed surgical blades or single-edged corneal knives are undesirable, for the triangular cut produced heals slowly and may result in herniation of the iris. The fragments of tissue to be trans- planted should be cut with sharp instru- ments and should not exceed 1 mm. in diameter. The careful selection of fragments is of the utmost importance 14 ANTERIOR CHAMBER TRANSPLANTATION in the heterologous transplantation of human cancer. In addition to essential parenchyma, all human cancers contain stroma and many are infiltrated with desmoplastic connective tissue. Such tissues are adult in nature and will not grow on heterologous transfer. In fact, their presence in quantity will give rise to a foreigh body reaction in the alien host and result in death of the trans- plant. It is essential, therefore, that selection be based on the content of tumor parenchyma and some knowledge of gross pathology is necessary for such a differentiation. Frozen sections ob- tained from different areas of the tumor mass are of considerable aid in some cases. It is obvious that necrotic tumor or normal tissue adjacent to the tumor are not suitable materials for transfer. The placing of the fragment in a wedged position in the inferior angle of the iris is important for early vasculari- zation. Occasionally, fragments so placed work loose shortly after opera- tion and an examination with the re- quired readjustment is desirable before returning the animals to the colony. Fixation occurs within a few hours and further check is unnecessary. A slight modification of the technique is desirable when applied to mice or to newborn animals of larger species. General anesthesia is preferable and may be effected with ether or nembutal. Both hands are employed in the opera- tion and preliminary loading of the trocar is necessary. The trocar is made by shortening the bevel at the tip of a 20-gauge hypodermic needle. A suit- able, tight-fitting plunger can be manu- factured or obtained simply be select- ing a wire stylet of proper size from the stock supplied with the needles and ap- plying a knob of plastic material to one end. The mouse is held loosely in the left hand and the lids of the right eye forced apart with the thumb and index finger. Slight pressure with the fingers causes the eye to protrude sufficiently to allow adequate exposure for the operation. An incision is made at the upper border of the limbus using a double-edged knife of the same caliber employed in larger animals. The trocar, held between the thumb and middle finger of the right hand, is inserted into the chamber through the incision and the fragment expressed by pushing the plunger with the index finger. In order to prevent extrusion and escape of the fragment through the incision, all pressure exerted on the animal by the left hand should be re- ANTHROCOSIS 15 ANTIGENS, HISTOCHEMICAL leased before withdrawing the trocar. Trouble may be encountered in pre- venting the escape of soft, slippery tissues such as embryonic brain. This difficulty may be circumvented by in- cising the iris as well as the cornea at the limbus and directing the trocar behind the superior half of the iris, through the pupil and into the inferior portion of the anterior chamber. With withdrawal of the trocar, the fragment is almost invariably caught at the pupillary border and retained in the chamber. The tissue concerned may be trans- planted immediately or stored at ice- box temperature for several days before use. It is essential that the material be free of infection and that surgical sterility be maintained throughout all manipulations. Aside from careful technique, the success or failure of anterior chamber transfer depends on the nature of the tissue used and the species of the re- cipient host. Adult, embryonic and cancer tissues grow on homologous transfer, while benign tumors and pre- cancerous tissues fail to survive, and heterologous transfer is successful only in the case of embryonic tissue and cancer. In the selection of recipient species for the heterologous transplan- tation of cancer, it should be noted that transfer between species with the same type of Vitamin C metabolism (man and guinea pig) is comparatively easy, while transfer between species with different types (man and mouse) is difficult. Takes are first recognized by increase in size and vascularization of the trans- planted fragment. The time required varies within wide limits; 1 day in the case of homologous embyronic tissues and 3 months in the case of a hetero- logous glioblastoma multiforme. In the former instance, the growing trans- plant may fill the chamber in a week while in the latter, 6 to 8 months may be required. Serial transfer is readily effected with fragments of the first generation growth. For details see Greene, H. S. N., Cancer Res., 1943, 3, 809-822; 1947, 7, 491-501, and Yale J. Biol. & Med., 1950, 6, 611-620. Anthracosis. The deposition of carbon, usually in lungs and mediastinal lymph nodes, distinguished by its resistance to solvents and bleaching agents and by its blackness. See Carbon. Anthrapurpurin, see Alizarin SX. Anthraquinone Dyes. Derivatives of an- thracene through anthraquinone. Acid alizarin blue GR and BB, alizarin, alizarin red S, purpurin. Antibiotics, influence on dehydrogenase. Systems of bacteria, see Triphenyl- tetrazolium Chloride. Anticoagulant Solutions have been very care- fully studied by Leichsenring, J. M., et al., J. Lab. & Clin. Med., 1939-40, 25, 35-44. They found that 1.6% potassium oxalate prepared from dried salt is most nearly isotonic for human blood. Win- trobe, M. M., Clinical Hematology, Philadelphia, Lea & Febiger, 1942, 792 pp. advises 0.06 gms. of ammonium oxalate and 0.04 gms. of potassium oxa- late for 5 cc. of blood. He dissolves 1.2 gm. ammonium oxalate and 0.8 gm. potassium oxalate in 100 cc. aq. dest. and adds 1 cc. formalin to prevent de- terioration. Then he measures out with a burette 0.5 cc. into each of the con- tainers and lets it dry before taking into each 5 cc. of fresh blood. Heparin is also advised but it is much more expen- sive. 0.075 gm. will prevent coagula- tion of 5 cc. of blood. See citrate. Antigens, Histochemical Identification Of — Written by A. H. Coons, Dept. of Bac- teriology and Immunology, Harvard Medical School, Boston, August 31, 1951 — The localization of antigenic substances in tissue cells can be carried out by the use of specific antibody con- jugated with fluorescein. The method furnishes a means for localization and identification with all the specificity of immune reactions. Suitably prepared tissue sections containing an antigenic substance which it is desired to study are flooded with a solution containing antibodies against the substance pre- viously conjugated with fluorescein isocyanate. The antibody molecules precipitate over those sites in the tissue section containing the specific antigen, the excess of fluorescent proteins is washed away, and the tissue section mounted in glycerol. When examined under the fluorescence microscope, the brilliant yellow-green fluorescence of fluorescein is visible over those areas where the immune reaction has taken place. The preparation and assay of immune sera is described in standard works on immunology. Two such recent ones are Kabat, E. A., and Mayer, M. M., Experimental Immunochemistry, Thomas, Springfield, Illinois, 1948; Boyd, W. C., Fundamentals of Im- munology, 2nd Edition, Interscience, New York, 1947. Whenever possible, it is best to start with a purified antigen, since antibodies against any impurities present in the material injected may be represented by antibody in the result- ing serum. These antibodies against biological impurities may react in tissue ANTIGENS, HISTOCHEMICAL 16 ANTIGENS, HISTOCHEMICAL sections with antigenic components other than that under investigation. However, it is possible to remove such interfering antibodies by absorbing the anti-serum with material containing the * antigenic impurities, but not contain- ing the antigen it is desired to study. Pains should be taken to secure as high a titer of antibody as is feasible. Pro- longed courses of immunization and the use of adjuvants may be necessary. Concentration of antibodies. It is often advantageous to concentrate the globulin fraction of such immune sera, either by precipitation of the globulins with half-saturated ammonium sul- phate, or the use of low temperature, buffers, and alcohol after the method of Nichol and Deutsch (J. Am. Chem. Soc, 1948, 70, 80). If ammonium sulphate is used, the ammonium ion must be carefully dialyzed from the final globulin solution as otherwise it will interfere seriously with subsequent procedures. Conjugation with fluorescein isocyanate. The derivatives of fluorescein leading to the isocyanate are not as yet com- mercially available. Fluorescein amine may be synthesized and converted to the isocyanate for conjugation to pro- tein by the procedures desci-ibed by Coons and Kaplan (J. E.xp. Med., 1950, 91, 1). Conjugation of antibody solution with fluorescein isocyanate. The protein con- tent of the serum or isolated globulin fraction to be conjugated must be de- termined. It should be at least 1.7%. A convenient amount of protein for one run is from 300 to 600 mgm. The amount must be known. Fix a small beaker in an ice-bath and equip it with good mechanical stirring. Put reagents by volume into it in the following order: Saline (0.9% NaCl) to make 100%. Carbonate - bicar- bonate buffer (0.5 M, pH9.0) 15% Dioxane (distilled from and stored over sodium) 15% Acetone 7.5% (minus 2 ml.) When the solution is 4°C. or below, and the isocyanate solution is ready, add Protein solution, such that the pro- tein concentration in the final mixture is 1%. For example, there are 30 ml. of a concentrated antibody solution con- taining 2.0% total protein. It is de- sired to conjugate 500 mgm. (25 ml.), holding the remainder for control pur- poses. The reaction mixture: Saline 6.75 ml. Buffer 7.5 ml. Dioxane 7.5 ml. Acetone 1.25 ml. Protein sol 25 ml. Total 48 ml. An amount of one of the two fluores- cein amine isomers such that there is 0.05 mgm. of amine per mgm. of protein (in the example, 25 mgm.) is dissolved in dry acetone and treated with phos- gene. This procedure should be carried out in a good chemical hood with forced exhaust. Phosgene is led out of the tank through concentrated sulphuric acid, thence to a vessel containing 15 ml. of dry acetone and fitted with a dropping funnel, thence to an empty vessel which serves as a trap, thence through 20% sodium hydroxide to destroy the excess phosgene, and finally through a trap with a controlled leak to a water suction pump. The whole reaction train should be maintained at a pressure slightly below atmospheric by means of the controlled leak. Phos- gene is turned on and allowed to bubble through to remove air and to saturate the acetone in the reaction flask. At the end of a few minutes, the amine dis- solved in 5 cc. of dry acetone is added slowly from a dropping funnel to the vessel containing the acetone saturated with phosgene. When all the amine- containing solution has been added to the reaction vessel, phosgene is allowed to continue bubbling through. During this time the color of the solution in the reaction flask slowly changes from a fluorescent green to a pale yellow. A small amount of heat is generated dur- ing the reaction of the amine with phosgene. At the end of about 15 min- utes the reaction flask is transferred to a vacuum still, immersed in a water bath at about 45°C., and the acetone boiled off under reduced pressure. Small pieces of dry anthracite can be used as antibumping chips. This step removes the phosgene still dissolved in the ace- tone. When the reaction flask is warm and dry, a greenish brown gum is vis- ible on the wall; this should be dis- solved in 2 ml. of acetone. This solu- tion is added in toto, drop by drop, to the stirred, chilled, buffered protein so- lution. (This additional acetone brings the total acetone concentration up to 7.5%.) Stirring is allowed to continue for 16 hours in the cold. The solution is poured into a cellophane sac and dialyzed against repeated changes of saline buffered with 0.01 molar phoa- AXTIGENS, HISTOCHEMICAL 17 ANTIGENS, HISTOCHEMICAL phate at pH 7.0 in the cold until the dialysate outside the sac shows fluores- cence of less than 1 part in 20 million. This can be roughly determined by eye using a known solution of fluorescein as a standard. The antibody solution may be further purified by one or more precipitations with half saturated ammonium sulphate followed by re-solution and dialj^sis, and by precipitation with cold acetone (Coons, et al., J. Immunol., 1942, 45, 159) or by 40% alcohol (Marshall, J. Exp. Med., 1951, 94, 21). Merthiolate (Eli Lilly Co.) should be added as a preservative (1:100 of a 1% solution). Despite these chemical purification procedures, substances remain in such fluorescein-protein conjugates which stain some tissue elements. It has been found necessary to shake such con- jugates with dry acetone-powder de- rived from animal tissues, usually liver powder of the species whose tissue it is proposed to study. Such powder may be prepared by homogenizing the tissue in a Waring blendor, washing several times with distilled water in the centrifuge, suspending the product in saline and precipitating it with four volumes of acetone. The acetone pre- cipitate can be harvested on a Buchner funnel and washed with dry acetone to remove water. Such a powder is added to a small aliquot of conjugate in the proportion of 100 mgm. of powder to each ml. of conjugate, the paste allowed to stand for an hour at room temper- ature, and the powder separated in the centrifuge. The yield is higher if the centrifugation is carried out in the cold at 18,000 rpm. Two such absorptions are often necessary to remove "non- specific staining." Merthiolate should be added again as above. The preparation of tissue sections for use ivith fluorescent antibody. The prob- lem of preparing tissue sections retain- ing the antigenic activity of the ma- terial sought varies with the antigen in question. The bacterial polysaccha- rides which have so far been studied survived fixation in Rossman's picric acid-alcohol-formalin followed by paraffin embedding. Such sections are deparafRnized and hydrated and then stained with the appropriate antibody solution. Care must be taken not to wash out the antigen during the pro- cedures preceding the flooding of the section with labeled antibody. In the case of bacterial polysaccharides, it is necessary to remove the picric acid in 70% alcohol, in which these polysac- charides are insoluble. In the case of less stable materials, for example proteins, fixation of the tissue-block is unsatisfactory; sections must be prepared from unfixed material and fixation carried out on the indi- vidual section. There are two methods available for the preparation of such sections from unfixed tissue, one that of Linderstr0m-Lang and Mogensen, the other that of Altmann-Gersh, and others. With either of these two methods, it is necessary to fix the tissue section in some appropriate reagent before applying labeled antibody solutions lest the material looked for be dissolved out of the section during exposure to conjugated antibody. A certain amount of experimentation is necessary with each new antigen in order to find the appropriate fixative. For mumps and influenza A virus and the virus of infectious canine hepatitis, acetone is satisfactory. This is used at room temperature for 15 to 30 minutes fol- lowed by drying of the section in an incubator. For proteins, 95% ethanol by volume (start with absolute ethanol) at 37°C. for 30 minutes is satisfactory. For ACTH in the anterior lobe of the hog pituitary, absolute methanol has been shown to be a satisfactory fixative (Marshall, J. Exp. Med., 1951, 94, 21). In general, these organic solvents are best removed by evaporation. These fixatives and exposure-times are cited as examples only. Any anti- gen-antibody system chosen for study must be investigated from this as well as from other points of view. The use of labeled antibody solutions on tissue sections. A small drop of fluorescein-antibody solution is placed over the tissue section, and allowed to react for from 10 minutes to 48 hours, depending on the system under study. Thirty minutes is usually satisfactory. During the reaction, evaporation must be minimized. Usually the reaction can be carried out at room temperature, although incubator temperatures or refrigerator temperatures can be em- ployed. Following exposure to the fluorescent antibody solution, the sec- tion should be placed in a Coplin jar containing 0.9% saline buffered at pH 7.0 with 0.01 molar phosphate, and washed with very gentle motion for 10 minutes. At the end of this time, the slide should be wiped dry except for the area of the section and the sec- tion itself mounted under a cover slip in glycerol containing a trace of buffer at pH 7 (commercial glycerol is slightly acid), and examined under the fluores- cence microscope. Control of specificity of staining. ANTIGENS, HISTOCHEMICAL 18 ANTIGENS, HISTOCHEMICAL Because of the ever-present hazard of "nonspecific" staining, careful controls of specificity must be carried out at every step. When the antigenic material sought is foreign to the tissue under investigation, control is relatively simple since the conjugate should fail to stain normal tissue sections. Im- munologically specific staining can be greatly diminished or altogether pre- vented by pretreatment of parallel sec- tions with unlabeled homologous anti- body, thereby blocking the antigen. However, replacement of unlabeled by labeled antibody occurs and the time of staining must usually be decreased in order to carry out this control suc- cessfully. Failure to stain the tissue under investigation with heterologous labeled antibody is also a suitable control, although the degree of purifica- tion of conjugates varies and it is diffi- cult to establish that any two conju- gates are strictly comparable. There are so many possible variations that controls appropriate to each situation must be carefully planned. Fluorescence microscopy. Fluores- cence microscopy is described in an- other section. The amount of de- posited antibody is quite small, and the fluorescence therefore faint. Light sources adequate for the study of brightly fluorescent materials present in high concentration are not appropri- ate for use with labeled antibody. Either a 10 amp. carbon arc or a high pressure water cooled mercury vapor arc (General Electric Co., AH -6) are satisfactory. At this level of bombard- ment, fluorescence of optical elements becomes important. It is necessary to use a quartz condenser since glass ones often fluoresce in the ultraviolet beam. Definition is improved at the cost of intensity by the use of a cardioid darkfield condenser. Photography. Findings revealed by this method can be recorded photo- graphically using 35 mm. film (either high speed panchromatic film or fast green-sensitive film, e.g., "Photoflure" Eastman). Exposure times vary with the amount of fluorescent antibody deposited and the autofluorescene of the background, varying at "high dry" (400 X) from 3 to 10 minutes. Focus- ing on ground glass is very difficult because of the low degree of intensity of the projected image, but can be carried out by the use of a magnifying lens focused on a clear area in the ground glass. Anyone attempting to carry out these procedures is advised to consult the original papers to which reference has been made (cf. Federation Proc, 1951, 10, 558). Frozen Sections by the Method of hinderstr^m-Lang and Mogensen (Compt.-Rend. Trav. Lab. Carlsberg, s6rie chim., 1938, 23, 27; Coons ,et al., J. Exp. Med., 1951, 93, 173). The prin- ciple of this method is quite simple. It involves the quick-freezing of tissues at low temperatures, and cutting them while frozen in a cryostat maintained at —20. This cold chamber should be equipped with adequate lighting, an insulated window through which the operator can see, and gloved armholes through which he can work. Sections are cut on a good rotary microtome, the knife of which is equipped with a glass guide to keep the sections from curling as they are cut. Convenient improvements in the method are de- scribed by Coons, et al. Tissues may be quick-frozen by placing thin slices on the side-walls of test tubes which are then stoppered and plunged but not submerged into alcohol cooled to the temperature of dry ice. They can then be stored in a deep freeze below — 20, or put in the cold cryostat for immediate sectioning. Long storage results in the growth of ice crystals. The tissue is mounted by putting a small drop of water on a previously chilled tissue holder and touching the tissue to it. Within a few seconds the tissue block will be tightly frozen to the holder. Care must be taken to avoid ice formation near the cutting area. The knife blade should be set at an angle of approximately 20° from the plane of movement of the tissue block. The glass guide should be ad- justed so that its upper edge is parallel to and at the height of the cutting edge of the knife. Each section as cut is removed with forceps from the knife blade, placed gently on a cold slide, and thawed by placing a finger under the section. It can then be dried in the air stream from a fan at room tem- perature. Appreciable movement of tissue components during this momen- tary thawing has not been troublesome, although it could be serious in some situations. An adhesive is necessary on the slides; the one in use by the authors quoted has been formalinized gelatin. Acid cleaned, dry glass slides are dipped in 0.5% gelatin, and placed on end to drain and dry. When dry, they are dipped in 10% formalin, drained, and allowed to dry. They can then be stored for long periods until required. Good sections 10 ju thick are easy to cut by this method, and with practice ANTIRACHITIC VITAMIN 19 ARGENTAFFINE REACTION sections about 4 ju thick can be cut as a routine. Antirachitic Vitamin, see Vitamin D3. Antiscorbutic Vitamin, see Vitamin C. Antimony Trichloride, see Carr-Price Re- action. Aorta, see Arteries and, for an account of technique for measuring elastic proper- ties, Saxton, J. A., Arch. Path., 1942, 34, 262-274. Aortic Paraganglion (Glomus aorticum). Technique for blood supply and innerva- tion is provided by Nonidez, J. F., J. Anat., 1936, 70, 215-224. Negative re- sults in application of the chromaffin reaction to the rabbit and guinea pig are described by the same author. Am. J. Anat., 1935, 57, 259-293. Carotid glomus is very similar. V. Apathy Syrup has been modified by Lillie, R. D. and Ashburn, L. L., Arch. Path., 1943, 36, 432-435. Dis- solve acacia (gum arable) 50 gm. and cane sugar 50 gm. in aq. dest. 100 cc. shaking frequently at 55-60°C. Add aq. dest. to make up original volume and merthiolate (sodium ethylmercuri- theosalicylate) 15 gm., or thymol 100 gm. to act as a preservative. This syrup is recommended as a mounting medium for frozen sections stained with Sudan III, or other alcohol soluble dyes. Aposiderin, see Lillie, p. 127. Aquax — Written by C. C. Macklin, Dept. of Histological Research, The Univer- sity of Western Ontario, London, Canada. November 28, 1951 — A water- soluble wax for embedding purposes produced by George T. Gurr, 136, New King's Road, London, S.W. 6. It has been found useful for the demonstra- tion of osmiophil granules in pneumo- pneumonocyies (which see) after levu- lose mounting (See Levulose Syrup). Its merit is the retention, in sections, of materials which tend to diffuse away in alcohol, clearing fluids and par- affin. Aqueous Humor, see Anterior Chamber of Eye. Arachnids, sectioning is facilitated by methods intended to soften Chitin. See also Fleas, Ticks. Archelline 2B, see Bordeaux Red. Argentaffine gastrointestinal cells (entero- chromaffin cells). Rare even in duo- denum. Occur singly, usually in deep- est parts of crypts and may be free from epithelium. Cytoplasmic argentaffine granules are of small size, often closely packed together and acidophilic. It is said that they cannot be found in bodies autopsied as late as 4-5 hrs. after death (Hamperl, H., Ztschr. f. Mikr.-anat. Forsch., 1925, 2, 506-535). Fluorescence Microscopy brings out the argentaffine cells sharply because the cytoplasmic granules fluoresce a yellow color. This color is not abol- ished by fat solvents and is attributed to pterins (McClung, Microscopical Technique, 1950, p. 682). See Eros, G., Zentralbl. f. Allg. Path. Anat., 1932, 54, 385; Jacobson, W., J. Path. &Bact., 1939,49, 1). Two specific methods are advised by Jacobson, W., J. Path. & Bact., 1939, 49, 1-19. For both fix in 10% formol- saline, or 10% neutral formol, dehydrate in alcohol, clear in cedarwood oil or in methyl benzoate -\- 2% celloidin and imbed in paraffin. In the first wash deparaffinized sections 10 mm. in 2 changes glass-dist. water. Transfer for 12-24 hrs. to Fon tana's sol. prepared by adding NH4OH to 5% AgNO, until ppt. is dissolved, then AgNOs drop by drop until fluid exhibits slight presistent opalescence. Wash in glass-dist. water, 1 min., 5% NajSjOs, 1 min. and tap water 10 min. Counterstain with car- malum. Dehydrate, clear and mount in balsam. Granules of argentaffine cells appear black. In the second more rapid method dissolve small amount p-nitro-methyloxybenzene diazotate in aq. dest. producing light yellow solution alkalinize with a little Li2C03. After about I5 min., when pH 10-11 is reached, color has changed to dark orange-yellow. Immerse sections brought down to aq. dest., in this 30-40 sec. Then wash in aq. dest., 1 min. Granules of argen- taffine cells appear dark red in yellow background. Counterstain with hema- lum if desired. Since Dawson, A. B., Anat. Rec, 1944, 89, 287-294 has found that a larger number of argentaffine cells are demons- trable in the rat's stomach by Bodian's technique than are reported after silver impregnations like those of Masson- Hamperl, it is important to try the Bodian Method in the manner suggested by Dawson. Sharpies, W., Anat. Rec, 1945, 91, 237-243 used the Bodian Method successfully in study of human stomach. Argentaffine Reaction. This, according to Lison (p. 147) is given by polyphenols, aminophenols and polyamines in ortho and para position. It is a reduction of ammoniated silver hydroxide into me- tallic silver. He recommends Masson's method for sections : Fix in Bouin's fluid or other fixative. Deparaffinize sec- tions and wash 2 hrs. in aq. dest. Treat for 3&-40 hrs. in Fontana's fluid in dark- ness and in a sheltered place. Wash in much aq. dest. Tone with 0.1% aq. gold chloride (few minutes). Fix in ARGINASE 20 ARGININE REACTION 5% aq. sodium hyposulphite. Counter- stain with alum carmine, mount in usual way. To make Fontana's fluid add ammonia drop by drop to 5% aq. silver nitrate until ppt. formed is ex- actly redissolved; then carefully drop by drop 5% aq. silver nitrate until appearance of persistent cloudiness and the liquid does not smell of ammonia. Decant before employing. See also Clara, M., and Canal, F., Zeit. f. Zellf. u. Mikr. Anat., 1932, 15, 801-808; Clara, M., Ergeb. d. Anat. u. Entw., 1933, 30, 240-340. Arginase. It is possible to localize arginase in the cytoplasm and nuclei of liver cells by Behren's technique (Zeit. Physiol. Chem., 1939, 258, 27-32). Finely ground tissue is dried to powder in frozen condi- tion. It is then suspended and cen- trifuged in different mixtures of benzene and carbon tetrachloride. The nuclei only are found in the lowest layer, next comes nuclear debris and above this cytoplasmic debris. His analysis showed arginase present in the same con- centration in the nuclei as in the cyto- plasm. Blaschko and Jacobson (Bourne, p. 217) remark that this is the first in- stance of the demonstration of an enzyme in the cell nucleus. Arginine Reaction. The method of Serra, J. A., Stain Techn., 1946, 21, 5-18 is detailed by him as follows: Prepare tissue as described under Ninhydrin Reaction. "1. Before the reaction the pieces or sections are hardened with 10% for- maldehyde during 12-24 hours, the formalin being afterwards well washed out. (If the fixative contains formalin this step can be omitted.) "2. Immerse the pieces for 15 minutes in a mixture consisting of 0.5 ml. of diluted a-naphthol ; 0.5 ml. of N NaOH ; and 0.2 ml. of 40% aqueous urea solu- tion. The diluted a-naphthol is pre- pared at the moment of use by diluting a stock solution (1% crystallized n- naphthol in 96% alcohol) 1 : 10 with 40% alcohol. The watch glass containing the liquids is placed in an ice-bath and the temperature of the reaction fluid inside it must be 0.5°C. "3. After 12-15 minutes add 0.2 ml. of a 2% solution of NaOBr. This re- agent is allowed to act for 3 minutes and the solution must be well stirred during this time. The 2% NaOBr must be freshlj' prepared by pouring 2 g. (or approximately 0.7 ml.) of liquid bro- mine into 100 ml. of 5% NaOH, with agitation and cooling. "4. Add another 0.2 ml. of 40% urea solution, stir, and immediately after- ward, "5. Add another 0.2 ml. of 2% NaOBr and stir well. The coloration attains its maximum after 3-5 minutes and would last only for a short time if it were not stabilized. To stabilize the coloration: "6. Take the pieces out of the reac- tion mixture and immerse in pure glycerin for 2-3 minutes and then trans- fer to fresh glycerin. Repeat the opera- tion another two or three times. The passage through 4 glycerin baths is sufficient to stabilize the coloration for some months, even if the pieces are left at room temperature. (We have not mentioned this improvement in any previous publication.) "Besides this procedure, which we may call the normal method, there is also another method which results in stronger colorations and very satisfac- tory preparations. To accomplish this, after step 5 the pieces are taken off the reaction liquid and immersed in NaOBr solution for not more than 3 minutes. Afterwards the coloration is stabilized in glycerin, as in the normal procedure. The pieces are mounted and observed in pure glycerin. "This reaction is specific for guani- dine derivatives in which only one H- atom of one amino group is substituted by a radical of the alkyl or fatty acid type. In proteic compounds it is specific for arginine. As all proteins hitherto analyzed possess arginine in their molecules, the reaction may be used to demonstrate the presence of proteins in general, other compounds with a reactive guanidine group being rare. The test may also be used to characterize the basic proteins." Arginine Reaction. The method of Thomas, Lloyd E., Stain Techn., 1950, 25, 143-148, has been modified as follows (unpub- lished) written byL. E. Thomas, Dept. of Biochemistry, University of Missouri, Columbia. July 8, 1951 — Bouin's fixa- tive has given the best results. Formal- dehyde (4%) is almost as good. Carnoy's (6:3:1) may be used. After fixation pre- pare paraffin sections. 1. Remove the paraffin from the slide with xylene and pass it through the alcohols to 70%. 2. Place the slide in a 0.3% aq. of 8-hydroxyquinoline (oxine) in a Coplin jar for 15 min. at room temperature. This solution is prepared by diluting with water a 1.0% stock solution of oxine in absolute alcohol. 3. Move the slide with a quick motion {not allowing it to drain) into a 0.15 N sodium hypochlorite solution (0.015 N with respect to potassium hydro.xide). This solution is prepared by standardiz- ARGON 21 ARTERIES ing Clorox or other suitable hypochlorite solution and using it as a stock solution. Leave the slide in the hypochlorite solution exactly 60 sec. at room temper- ature, holding it stationary. 4. Move quickly [without draining) into a solution containing per 100 cc: 15 gm. urea, 70 cc. tertiary butyl alcohol and potassium hydroxide to make the concentration of the latter 0.015 N. Move the slide gently in the solution for 10 sec, then transfer to another jar of the same solution for 2 min. 5. Transfer to 100% tertiary butyl alcohol, moving the slide gently for 10 sec, then place in a second jar of the same reagent for 3^ min. 6. Pass through three changes of xylene for 10 seconds, 1 minute and 2 min., respectively. 7. M.ount with Permount containing aniline. This reagent is prepared by dissolving 0.1 cc. of aniline in 100 cc. of xylene. One volume of this solution is mixed with four volumes of Permount. Reagents 2, 3 and 4 must be made fresh daily. It is best not to let any water accumulate in the tertiary butyl alcohol. The three arginine methods are all based on the Sakaguchi reaction, which, in biological materials, is specific for arginine, galegine and agmatine. The latter two are extremely rarely en- countered and so for most purposes, it is specific for arginine. The histo- chemical methods are specific for pro- tein-bound arginine, since free arginine is inevitably removed by the pro- cedures. Argon, see Atomic Weights. Argyrophilic Fibers. Because of their affin- ity for silver, Reticular Fibers are often called argyrophilic. Arneth Count of lobes of granular leucocytes as a basis for estimation of their rela- tive age. See Leucocyte Counts. Arsenic 1. Use 10% neutral formalin in aq. dest. after test with hydrogen sulphide shows absence of trace of metals. To 100 cc. add 2.5 gm. copper sulphate. Fix small pieces of tissue 5 days. Wash 24 hrs. in running water. Imbed in paraffin. Direct examination of section after removal of paraffin shows arsenic as well defined green granules of hydro- arsenite of copper (Scheele's green). If neutral acetate of copper is employed in place of the sulphate the green granules are of acetoarsenite of copper (Schweinf urth's green ) . 2. Fix pieces of tissue 12-24 hrs. in abs. ale 50 cc. ; chloroform, 50 cc. ; pure hydrochloric acid, 3 cc. saturatea by passage of pure hydrogen sulphide. In sections the arsenic ppt. appears as yel- low granules. Double coloration with hematein-eosin is possible. Both tech- niques have been devised by Castel (P., Bull. d'Hist. Appl., 1936, 13, 106- 112). He lias described the histologic distribution of the arsenic. See, how- ever, paper by Tannenholz , H . and Muir, K. B., Arch. Path., 1933, 15, 789-795 who employed a somewhat similar method ana were unable to conclude that the yellow crystals were in fact those of arsenic trisulphide. They considered them more probably a sulphur-protein combination. Consult the detailed account of Os- borne's method for arsenic given by Heuper, W. C, Occupational Tumors and Allied Diseases. Springfield: Thomas, 1942, 896 pp. (p. 50). This releates particularly to localization of arsenic in the skin. The distribution to the several tissues of radioactive arsenic injected intra- venously into rabbits as sodium arsenate has been investigated by duPont, O., Irving, A. and Warren, S. L., Am. J. Syph. etc., 1942, 26, 96-118. It is impor- tant to determine whether the results conform with those given by the micro- chemical techniques. Arsphenamines. The specificity of the silver reaction of Jancs6, N., Ztschr. f. d. Ges. exper. Med., 1929, 65, 98 is questioned by Gomori, G., J. Mt. Sinai Hosp., 1944-45, 11, 317-326 since it may demonstrate other reducing sub- stances beside the arsphenamines. Artefacts, see Artifacts. Arteries. If one wishes an elastic artery take a large trunk near the heart such as the aorta, innominate or subclavian; if, on the other hand, a typical muscular artery is required select one further afield like the radial or external carotid. Arterial walls are seldom examined microscopically in vivo because they are relatively large and difficult to get at without injury. An exception in man is the retinal artery which can be seen by ophthalmoscopic examination. To closely observe excised pieces of arteries is all too frequently neglected. The tissue elements are so tightly bound to- gether that to tease them apart for study at high magnification is rather unsatis- factory. However, when the adventitial adipose and connective tissue is stripped off from a fresh specimen, the remainder of the wall can very advantageously be made translucent by treatment with Eure glycerin for 1-2 hrs. as described y Winternitz, M. C, Thomas, R. M. and LeCompte, P. M. in their book "The Biology of Arteriosclerosis", Spring- field: Thomas, 1938, 142 pp. Since the color of the blood is preserved within ARTERIOLES 22 ARTIFACTS the intramural vessels their arrangement can be studied (see Vasa Vasorum). Fatty substances can also be located because they are not removed by the glycerin. Chief reliance is ordinarily placed in the appearance of arterial walls when seen in sections of fixed tissue. It is important to remember that, when carry- ing blood during life, the lumina are larger and the walls less folded than in the fixed condition. The difference has been graphically demonstrated by Gallo- way, R. J. M., Am. J. Path., 1936, 12, 333-336. His figures should be exam- ined. For routine purposes fixation in Formalin-Zenker followed by Mallory's Connective Tissue Stain supplemented by Resorcin Fuchsin or Orcein for elastic tissue is satisfactory. Special methods may be needed for Lipids ; and for minerals, see Calcium, Iron and Microincineration. Innervation, like- wise, is to be studied by methods em- ployed to demonstrate Nerve Endings in other tissues. See Vasa Vasorum. Much literature on techniques is given by various authors in Cowdry, E. V., Arteriosclerosis, New York: Macmillan, 1933, 617 pp. The investigation of arterial walls is apt to be one sided limited only to structure and composi- tion demonstrated microscopically. It is high time that these lines of study are supplemented by accurate meas- urement of the physical properties of pulse wave velocity, sound production, elasticity and so on of the same vessels by methods described by Bramwell in the above mentioned volume. Arterioles, capillaries and venules, in con- trast to the much larger arteries and veins, can readily be examined in experi- mental animals microscopically in the living state. Since they are linked together a single preparation by Sandi- son's rabbit ear method shows all three, or they may be viewed in the living tadpole's tail or other transparent tissue of lower forms. For convenience, how- ever, it seems best to briefly mention the microscopic techniques for each separately. There is much to choose from. Information is frequently de- manded on the condition of the arterio- lar walls. This can best be supplied by staining paraffin sections of Forma- lin-Zenker fixed material with Mal- lory's Connective Tissue stain or with Masson's Trichrome stain which is closely related to it. Weigert's Re- sorcin Fuchsin is satisfactory for elastic tissue. The Silver Citrate technique is capable of yielding valuable data on arte- rioles and capillaries. Because arte- rioles contain a higher percentage of muscle than any other blood vessel their appearance will vary greatly with the degree of contraction or relaxation of muscle. According to Kernohan, J. W., Anderson, E. W. and Keith, N. M., Arch. Int. Med., 1929, 44, 395-423 in fixed preparations from normal persons the average ratio of thi ckness of arteriolar wall to width of lumen is 1:2. Arteriovenous Anastomoses are direct con- nections between arteries and veins vrithout intervening capillaries. No special histological technique is required for their demonstration in sections but one should look for them where they are particularly numerous, as in rabbits at the tip of the nose (diameter, 80-100/i) and in humans in the palms of the hands, the soles of the feet and near the ends of the fingers where their diameter is about 35m (Grant, R. T. and Bland, E. F., Heart, 1930, 15, 385-411). The best way is to study them in vivo (Clark, E. R. and E. L., Am. J. Anat., 1934, 55, 407-467). Arteriosclerosis. The arteries in this condi- tion show changes well demonstrated by Mallory's Connective Stain and its modifications as well as by Weigert's Resorcin Fuchsin. In addition, tech- niques for Lipids, Calcium and Iron are indicated. Methods for the measure- ment of physical properties of arteries might well be applied to arteries most and least prone to develop arterio- sclerosis. These are summarized by Bramwell, C, in Cowdry's Arterio- sclerosis. New York: Macmillan Co., 1933, 617 pp. Artifacts. Webster defines an artifact as being "in histology, a structure or appearance in a tissue or cell due to death or to the use of reagents and not present during life." The degree of artifact is proportional to the difference between the structure existing normally in the living body and the structure in the condition directly studied. 1. In the case of living tissues, ob- served with blood and nerve supply intact, there is a possibility of artifact. It is at a minimum in the Rabbit Ear Chambers and rather more to be reck- oned with when tissues must be dis- placed in order to supply the necessary illumination. With increase in time modifications due to changes in light, temperature, hydrogen ion concentra- tion, etc. are likely to also increase. 2. In living cells removed from the body and examined in Tissue Cultures the possibility of artifact is again at a minimum ; but, though the cells in suc- cessive generations in suitable media go on living indefinitely, their environ- ments are different from those existing ARTICULAR NERVE TERMINALS 23 ATABRINE within the body. When after Vital Staining or Supravital Staining still living cells are examined in approxi- mately isotonic media, there is a grave danger of artifact if the study is pro- longed because the cells are slowly dying. 3. In fixed tissues the degree of di- vergence from the normal living condi- tion is obviously much greater than in the case of still living ones. However death has been sudden so that artifacts due to gradual death are eliminated. If the technique has been carefully stand- ardized the same fixative applied to the same type of cell in the same physiologi- cal state is likely to yield similar results. Among common artifacts are: 1. The shrinkage and increased affinity of cells near the surface for stains due to allow- ing the surface of the tissue to dry be- fore fixation. 2. The glassy appearance of nuclei and cytoplasm sometimes oc- casioned by overheating in imbedding or in spreading out sections. 3. Mate- rial within blood vessels faintly resem- bling organisms caused by coagulation of blood proteins. 4. Extraneous sub- stances either present in the albumen fixative used to mount the sections or deposited as dust from the air. Careful focussing is required. See Agonal and Postmortem changes, Ice Crystal Arti- facts. A paper by Bensley, R. R., Exp. Cell Res., 1951, 2, 1-9 on "Facts versus Artifacts in Cytology: the Golgi ap- paratus" is illuminating. See Agonal and Postmortem changes. Ice Crystal Artifacts, Normality, Nucroscopic. Articular Nerve Terminals. — Written by Dr. E. D. Gardner, Wayne University School of Medicine, Detroit. June 15, 1950— Gardner, E. D., Anat. Rec, 1942, 83, 401-419, adapted silver methods to the demonstration of nerve terminals in the knee joints of mice 1-60 days old. Subsequently, J. Comp. Neur., 1944, 80, 11-32, similar methods were applied to the knee joints of 33- and 46-day-old cat fetuses. The Bodian method was used, but the necessary protargol seems no longer to be avail- able. It appears, however, that similar results can be obtained with Romanes' staining method (J. Anat., 1950, 84, 104-115) . A variety of fixatives are satis- factory, particularly Bouin and acetic- formol-alcohol. Decalcify in 20% sodium citrate and 50% formic acid, equal volumes, prior to embedding. Sections are placed overnight at 56 °C. in the following solution: Distilled water 50 ml., 0.1% silver nitrate 2.9 ml., and 0.1% sodium chloride 1.0 ml. To this are added a few drops of very weak ammonia sufficient just to turn phenol red paper pale pink. Subsequent treatment is similar to that of the Bodian method. Silver methods are excellent for following nerves in thin, serial sections, for tracing nonmyeli- nated fibers and for small nerve end- ings. But large, proprioceptive endings such as occur in the posterior region of the knee joint capsule are best demon- strated by methylene blue. Excellent results may be obtained by using 0.05% methylene blue in normal saline. It may be perfused, injected into the joint cavity or pieces of capsule can be removed and placed in methylene blue at 37°C. for 10-15 minutes. They are then exposed to air for 10-15 minutes, placed in 8% ammonium molybdate in normal saline for at least 1-2 hours and then dehydrated rapidly in cold alcohols. Whole mounts are made after clearing in xylol. This method shows better than any other the grouping and distribution of large endings, and the preparations are also satisfactory for small fibers, particularly those in vascular plexuses. Artificial Fever, influence on adrenal (Bern- stein, J. G., Am. J. Anat., 1940, 66, 177-196). See Cramer, W., Fever, Heat Regulation and the Thyroid- Adrenal Apparatus. London: Long- mans, Green & Co., 1928, 153 pp. Asbestos, see Lillie p. 135. Ascorbic Acid. Colorimetric method of Lowry, O. H., Lopez, J. A. and Bessey, O. A., J. Biol. Chem., 1945, 160, 609- 615 for blood serum in volumes of 10 ix\ up. Pijoan, M. and Gerjovich, H. J., Science, 1946, 103, 202-203 advise cau- tion in use with tissues. See titrimetric technique of Click, D., J. Biol. Chem., 1935, 109, 433-436. See Vitamin C. Ascorbic Acid, see Vitamin C. Aspirated Sternal Marrow, method for preparing smears and sections (Gordon, H., J. Lab. & Clin. Med., 1940-41, 26, 1784-1788). Astra Violet, see Leishmania. Astrocytes. These cells make up one of the two divisions into which neuroglia is usually divided. They also pass under the names of "Classical neuroglia" and "macroglia." They are star shaped cells with processes radiating in all directions some of which form peri- vascular expansions on the surface of small blood vessels. The most selective methods for their demonstration usually involve some form of metallic impregna- tion. For details see Chapter by Pen- field and Cone in McClung's Micro- scopical Technique, 1950, p. 407 and Lillie's Histopathologic Technic, 1948, p. 237. In this book see Neuroglia. Atabrine, anti-malarial agent; fluorescence microscopical localization of atabrine ATHEROMA 24 AUER BODIES in the body (Jailer, J. W., Science, 1945, 102, 258-259. Atheroma, see methods for Calcium, CoUa- genic Fibers, Elastic Fibers, Lipids, Atomic' Weights. See G. E. F. Lundell, Chairman, Report of Committee on Atomic Weights. J. Am. Chem. Soc, 1949, 71, 1141-1142. International Atomic Weights 1949 Revision Actinium Ac 227 Aluminum Al 26.97 Americium Am [241] Antimony Sb 121.76 Argon A 39.944 Arsenic As 74.91 Astatine At [210] Barium Ba 137.36 Beryllium Be 9.013 Bismuth Bi 209.00 Boron B 10.82 Bromine Br 79.916 Cadmium Cd 112.41 Calcium Ca 40.08 Carbon C 12.010 Cerium Ce 140.13 Cesium Cs 132.91 Chlorine CI 35.457 Chromium Cr 52.01 Cobalt Co 58.94 Columbium (see Niobium) Copper Cu 63.54 Curium Cm [242] Dysprosium Dy 162.46 Erbium Er 167.2 Europium Eu 152.0 Fluorine F 19.00 Francium Fr [223] Gadolinium Gd 156.9 GalUum Ga 69.72 Germanium Ge 72.60 Gold Au 197.2 Hafnium Hf 178.6 Helium He 4.003 Holmium Ho 164.94 Hydrogen H 1.0080 Indium In 114.76 Iodine I 126.92 Iridium Ir 193.1 Iron Fe 55.85 Krypton Kr 83.7 Lanthanum La 138.92 Lead Pb 207.21 Lithium Li 6.940 Lutetium Lu 174.99 Magnesium Mg 24.32 Manganese Mn 54.93 Mercury Hg 200.61 Molybdenum Mo 95.95 Neodymium Nd 144.27 Neptunium Np [237] Neon Ne 20.183 Nickel Ni 58.69 Niobium Nb 92.91 Nitrogen N 14.008 Osmium Os 190.2 Oxygen O 16.0000 Palladium Pd 106.7 Phosphorus P 30.98 Platinum Pt 195.23 Plutonium Pu [239] Polonium Po 210 Potassium K 39.096 Praseodymium Pr 140.92 Promethium Pm [147] Protactinium Pa 231 Radium Ra 226.05 Radon Rn 222 Rhenium Re 186.31 Rhodium Rh 102.91 Rubidium Rb 85.48 Ruthenium Ru 101.7 Samarium 8m 150.43 Scandium Sc 45. 10 Selenium Se 78.96 Silicon Si 28.06 Silver Ag 107.880 Sodium Na 22.997 Strontium Sr 87.63 Sulfur S 32.066 Tantalum Ta 180.88 Technetium Tc [99] Tellurium Te 127.61 Terbium Tb 159.2 Thallium Tl 204.39 Thorium Th 232. 12 Thulium Tm 169.4 Tin Sn 118.70 Titanium Ti 47.90 Tungsten (see Wolfram) Uranium U 238.07 Vanadium V 50.95 Wolfram W 183.92 Xenon Xe 131.3 Ytterbium Yb 173.04 Yttrium Y 88.92 Zinc Zn 65.38 Zirconium Zr 91.22 A value given in brackets denotes the mass number of the most stable known isotope. Auditory System, see Ear. Auer Bodies. — Written by G. Adolph Acker- man, Hinsman Hall, Ohio State Uni- versity, Columbus 10, Ohio. May 24, 1951 — Auer bodies are rod-like struc- tures occasionally present in the cyto- plasm of leukemic myelocytes and monocytes; they are less than 1 micron in width and vary in length from 1-10 micra. Although the number of Auer bodies observed within a single leukemic cell is frequently limited to one or two, more than fifteen have been observed. Auer bodies are formed bj' the coas- cervation of the cytoplasmic granules in young leukemic cells. As the cell matures, the Auer bodies undergo dis- solution into cytoplasmic granules. AUER BODIES AUER BODIES 1. Unstained moisl preparations: A drop of blood or bone marrow is placed on a clean cover slip, inverted on a slide and rimmed with vaseline or suitable substance to prevent drying. The prepa- ration is examined using the phase microscope. Auer bodies appear as dis- crete cytoplasmic structures. 2. Supravital technic: The supravital technic is carried out in the same man- ner as indicated above (No. 1.) for un- stained moist preparations with the exception of the fact that the slide has been previously coated with neutral red and janus green. Preparations may be examined with the bright field, dark field or phase microscope. Under the bright field microscope, the Auer bodies appear as discrete structures which stain with neutral red. The color of the Auer bodies varies from a deep red (acidic reaction) in young cells to tan (less acidic reaction) in the more mature leukemic cells. The staining reaction of the Auer bodies with neutral red closely parallels that of cytoplasmic granules. Auer bodies stain blue-purple in supravital preparations prepared with brilliant cresyl blue (Dameshek, W. Arch. Int. Med., 1932, 50, 579). 3. Romanowsky stains: The Roman - owsky dyes may be used to demonstrate Auer bodies. Due to the solubility of the azurophilic component of the Auer body in water and alcohol, this technic is not as satisfactory as the supravital and the unstained moist technics. 4. Swlan black B technic: Dry blood and bone marrow films are fixed with either formalin vapor or with a 1% solu- tion of formalin in 95% ethyl alcohol for 20-60 seconds. The dried-fi.xed films are placed in a saturated solution of Sudan black B (National Aniline Co., New York) in 70% ethyl alcohol for 20-30 minutes. The solution of Sudan black must be prepared at least 2 weeks prior to using. The blood films are then dipped in 70% ethyl alcohol and rinsed in 50% ethyl alcohol and washed in dis- tilled water, dried and mounted with "permount." Auer bodies are sudano- philic and appear black or black-brown. This technic is an excellent method for demonstrating Auer bodies. 5. Periodic Acid-Schiff {PAS) re- action: Dried films are fi.xed with forma- lin vapor for 20-60 seconds. The fixed preparations are immersed in an aqueous 0.5% solution of periodic acid for 5 minutes, washed thoroughly with distilled water and immersed in Schiff's reagent 10 minutes, followed by 3 suc- cessive changes in sulfurous acid, 3-5 minutes each, washed in distilled water, dried and mounted with "permount." Auer bodies are PAS-positive appear- ing a moderate pink. 6. Toluidine blue ami Thionin: Dried films may be fixed with formalin vapor, 4% aqueous basic lead acetate or other suitable fixatives. Preparations are washed, dried and immersed in a dilute solution (0.2-0.5%) toluidine blue or thionin, washed in distilled water, dried and mounted with "permount." Auer bodies stain lavender following the use of this technic. 7. Plasinal reaction: Dried films are fixed with formalin vapor for 20-60 seconds, washed in distilled water and placed in a 1% aqueous solution of mercuric chloride for 5 minutes, washed in distilled water and placed in Schiff's reagent; the films are placed in three successive changes of sulfurous acid, washed in distilled water, dried and mounted with "permount." In staining control preparations, the same pro- cedure is followed with the exception of the fact that distilled water is sub- stituted for mercuric chloride. If the control exhibits a positive reaction, the length of exposure to Schiff's reagent must be reduced. Following the plasmal reaction, the Auer bodies appear a very pale pink, and may best be identified by means of the phase microscope followed by bright field observation of the stained bodies. 8. M. nmli reaction: Dried blood and bone marrow films may be fi.xed either with formalin vapor or 1% formalin in 95% ethj'l alcohol, washed with dis- tilled water and dried. The fi.xed films are immersed in a mixture containing equal parts of 1% alkaline solution of alpha-napthol and 1% aqueous solution of p-aminodimeth_ylanaline (Eastman Kodak, P2147) for 1-5 minutes, washed in distilled water and mounted in water. Auer bodies stain deep blue following the M. nadi reaction. Preparations fade rapidly. 9. Sato and, Sekeya Peroxidase Technic: Dried blood and bone marrow films are placed in a 0.5% aqueous solution of copper sulfate for 30 seconds, washed briefly in 50% ethyl alcohol and placed in a benzidine-hydrogen peroxide mix- ture (0.1 gm. benzidine in 100 ml. dis- tilled water and 2 drops of 3% hydrogen peroxide) for 2 minutes, washed with distilled water, dried and mounted with "permount". Counterstaining with 1% aqueous safranin is optional. Auer bodies are peroxidase-positive and ap- pear blue or j'ellow. Auer bodies are soluble in water, saline and many organic solvents and are readily digested bj' ribonuclease. Digestion by ribonuclease may be pre- AUERBACH'S PLEXUS 26 AZURE DYES vented by immersing the Auer bodies in an aqueous solution of lanthanum ace- tate for 2 hours prior to exposure to the enzyme. Wright's stain may be used to demonstrate the basophilic properties of the Auer rods. Auer bodies are not demonstrable following the Bauer- Feulgen, Lugol's aqueous iodine, Go- mori acid and alkaline phosphatase and lipase reactions. Auerbach's Plexus. Supravital staining by injecting methylene blue through the aorta is apparently improved by addition of hydrogen acceptors. Scha- badasch, A., Bull. d'Hist. AppL, 1936, 13, 1-28, 72-89, 137-151 advises 0.03- 0.05 gm. per liter of p-amidophenol, 0.02-0.07 of p-phenylenediamine, 0.02- 0.05 of pyrocatechine or 0.05-0.9 of resorcin. The methylene blue must be of high quality and free from metallic salts. He obtained in 5 min. intense staining of the plexus in a cat which re- ceived 1200 cc. of fluid of the following concentration : aq. dest., 1000 cc. ; NaCl, 7 gm. ; resorcin, 0.15 gm. and methylene blue (chlorzink free, Hoecht) 0.2 gm. Aural Smears, see Ear Smears. Auramin (CI, 655) — canary yellow, pyok- tanin yellow, pyoktaninum aureum — This basic diphenyl methane dye may be of use in fluorescence microscopy. Auramine O is Commission Certified. It is one of the substances which arrests mitosis in the metaphase, an action which has been carefully studied by Ludford, R. J., Arch. f. Exper. Zellf., 1935-6, 18, 411-441. Tubercle bacilli treated with auramin give golden yellow fluorescence (Hageman, P. K. H., Munch. Med. Woch., 1938, 85, 1066). Aurantia (CI, 12) — imperial yellow — An acid nitro dye employed in Champy- Kull method. Aurantia is explosive and it can cause severe dermatitis. All those using it should be warned of the danger. Aurin or rosolic acid (CI, 724). Autoradiography, see Radioactive Isotopes. Axenfeld Reaction. Giroud (A., Proto- plasma, 1929, 7, 72-98) : Add to prepara- tion few drops of formic acid, then 3-4 drops 0.1% aq. gold chloride and heat slowly. A rose color appears, then vio- let. Lison (p. 129) says that the reaction is very little characteristic of proteins since analogous reactions are given by creatine, urea, uric acid, glycogen. Its employment is contraindicated. Axis Cylinders. These are the cytoplasmic cores of the nerve fibers. Mitochondria can often be seen in them unstained and after supravital coloration with Janus Green. The best method to demonstrate mitochondria in fixed tis- sues is Anilin Fuchsin Methyl Green after Regaud fixation. Silver methods show Neurofibrils. Alzheimer's modi- fication of Mann's eosin-methyl blue method is recommended to show early degenerative changes . De Renyi , G . S . , Cowdry's Special Cytology, 1932, 3, 1370-1402 has fully described use of methods of microdissection. See Ama- ranth. Azan Stain, see Heidenhain's. Azidine Blue 3B, see Trypan Blue. Azidine Scarlet R, see Vital Red. Azins. Azin dyes are those formed from phenazin. Two benzene rings are joined by 2 nitrogen atoms forming a third ring. Examples : amethyst violet, azocarmine G, indulin alcohol and water soluble, Magdala red, neutral red, neutral violet, nigrosin water soluble, phenosafranin, safranin O. Azo Blue (CI, 463) — benzoin blue R and direct violet B — This acid dis-azo dye is one of those microinjected vitally into cytoplasm against the nucleus of amebae to ascertain whether the nucleus can be vitally colored (Monn6, L., Proc. Soc. Exp. Biol. & Med., 1934-35, 32, 1197-1199). Butt, E. M., Bonynge, C. W. and Joyce, R. L., J. Inf. Dis., 1936, 58, 5-9 report that azo blue can be substituted for India ink in the nega- tive demonstration of capsular zones about hemolytic streptococci. Azo-Bordeaux, see Bordeaux Red. Azocarmine G (CI, 828) — azocarmine GX, rosazine, rosindulin GXF — This basic azin dye is used in place of acid fuchsin in Heidenhain's Azan stain. Azocar- mine B is CI, 829. Azocarmine GX, see Azocarmine G. Azo Dyes. Chromophore — N=N — uniting naphthalene or benzene rings. See Mono-azo, Dis-azo and Poly-azo Dyes. Lipophilic substitutions in, and slight curative effect claimed in tuberculosis and leprosy (Bergmann, E., Haskelberg, L. and Bergmann, F., J. Am. Chem. Soc, 1941,63,2243. Azo-fuchsin. Seven are recognized in the Colour Index. Acid mono-azo dyes re- lated to Bordeaux red and orange G. Azolitmin, see Hydrogen Ion Indicators. Azophloxine G. A. — Fast crimson GR., Amidonaphthol G— an acid Azo dye. Azo Reaction for phenols. Formation of azo color by action of diazonium salt on tissue phenol (Lison, p. 140). See Lison, L., C. Rend. Soc. de Biol., 1933, 112, 1237-1239). Azo Rubin, see Amaranth. Azure Dyes. These are basic thiazin stains of great usefulness. The description given by Conn (pp. 76-80) should be consulted. It is here summarized. Azure I (Giemsa) is a trade name for a secret preparation apparently a variable AZURE OR TOLUIDIN BLUE-EOSIN 27 BACTERIA mixture of Azure A and B. Azure II is an intentional mixture, in equal parts, of Azure I and methylene blue. It is the main constituent of Giemsa's stain. 1. Azure A is asymmetrical dimethyl thionin and has been Commission Certi- fied for some time. It is considered as the most important nuclear staining component of polychrome methylene blue by MacNeal, W. J., J. Inf. Dis., 1925, 36, 538-546. This dye has been used as a nuclear stain following eosin and after phloxine, see Phloxine-Azure (Ilaynes, R., Stain Techn., 1926, 1, 68-69, 107-111). 2. Azure B is the tri -methyl deriva- tive of thionin. It is specified by Jordan, J. H. and Heather, A. H., Stain Techn., 1929, 4, 121-126 as a stain for Negri bodies. Roe, M. A., Lillie, R. D. and Wilcox, A., Pub. Health Reports, 1940, 55, 1272-1278 recommend its in- clusion in Giemsa's stain. 3. Azure C is mono-methyl thionin. French, R. W., Stain Techn., 1926, 1, 79 has described a method for its use followed by Eosin Y and orange II in staining sections of formalin fixed mate- rial; but Haynes, R., Stain Techn., 1927, 2, 8-16 doubts whether it is sig- nificantly better than Azure A and thionin. Azure or Toluidin Blue-Eosin. — Written by Dr. R. D. Lillie, Division of Pathol- ogy, National Institute of Health, Bethesda, Md. May 8, 1950.— Prepare a 1/1000 solution of Azure A, Azure C or Thionin (85-90% dye content) or a 0.15% solution of Toluidin Blue (60% dye content) and a 1/1000 solution of Eosin Y or Eosin B (a redder shade). Bring paraffin sections to water as usual, including an iodine, thiosulfate sequence for material fixed with mer- curic chloride mixtures. Stain 1 hour in stock azure, Toluidin Blue or Thionin 4 cc, stock eosin 4 cc, Mcllvaine buffer of desired pH level 2 cc, acetone 5 cc. and distilled water 25 cc. Rinse, de- hydrate with acetone, clear with 50:50 acetone xylene and 2 changes of xylene, mount in synthetic resin (polystyrene, permount, clarite, HSR or the like). The procedure has been used for techni- con staining. The stain mixture is made fresh weekly in this case. For neutral formalin or Orth fixa- tions, use pH 4.0-4.5, for acid formalin pH 4.5 is better, for Zenker or Helly pH 5.0, for Bouin pH 5.5-6.0 (less satis- factory than others as the picric acid seems to interfere), for Carnoy, alcohol and similar fluids 4.8-5.5. Color values are deep blue for nuclei, bacteria, and rickettsiae, violet to purple for mast cell granules and carti- lage matrix, lighter blues for cyto- plasms, varying pinks for muscle, ery- throcytes, fibrin, necrotic cytoplasm and oxyphil inclusion bodies. This has been modified somewhat from Histopathologic Technic, Lillie (Blakiston, Phila., 1948), which gives further details. Azure II Eosin and Hematoxylin (Maximow, A., J. Inf. Dis., 1924, 34, 549), gives, in addition to coloration of chromatin by hematoxylin, a granule stain some- thing like that provided by Giemsa's method. Make up: (1) azure II eosin: A. eosin water soluble yellowish, 0.5 gm.; aq. dest., 500 cc. B. azure II, 0.5 gm.; aq. dest., 500 cc. Mix 10 cc. A, 100 cc. aq. dest., and 10 cc. B. (2) hematoxylin (Delafield's) 1-2 drops, aq. dest., 100 cc. to make a pale violet solution. Formalin-Zenker fixed tissues (sec- tions, smears, spreads) are stained up- right in hematoxylin washed in aq. dest. and counter-stained with azure II eosin 24 hrs. each. Transfer to 95% ale, differentiate and dehydrate in abs. (2 changes); clear in xylol and mount in balsam. Care must be taken to use pure aq. dest. The proportions of A and B can be varied slightly to suit the tissue. In order to hold the azure II eosin colors the balsam should be neu- tral or nearly neutral as when Giemsa's stain is employed. To appreciate the beauty of this method see numerous colored illustra- tions marked "ZF, Ham, EAz"of agreat many organs and tissues by Maximow, A. Section on Bindegewebe und Blutbil- dende Gemebe in Mollendorff's Handb. d. mikr. Anat. d. Menschen, 1927, 2, (1) 232-583. Babes' see Anilin-Safranin. Bacillus Typhosus, technique for dark field study of flagella (Pijper, A., J. Path. & Bact., 1938, 47, 1-17). See 9 plates by author. Bacteria. Methods employed for the micro- scopic identification of bacteria and to demonstrate their structure are legion. The Committee on Bacteriological Tech- nique of the Society of American Bac- teriologists has prepared a useful leaflet entitled "Staining Procedures" pub- lished in Geneva, N. Y. (Fifth Edition 1934) to supplement their "Manual of Methods for the Pure Culture of Bac- teria" (1923). A detailed account of Bacteriological methods by H. J. Conn, F. B. Mallory and Frederic Parker, Jr., is contained in McClung's Microscopical technique to which reference should also be made. Bergey's "Manual of Determinative Bacteriology" (Balti- more: Williams & Wilkins, 1948), which BACTERIA. BIOCHEMICAL TESTS 28 BACTERIA. MEDIA is a key to identification of bacteria, is often useful. Motility, agglutination, lysis under influence of bacteriophage, ingestion by leucocytes and many other phenomena can best be observed by examination of living bacteria by direct illumination or in the darkfield. Smears, usually fixed by heat, are, however, most often used. A choice must be made from many well known stains including : Anilin Gentian Violet, Loeffler's Methylene Blue, Giemsa, Gram and Carbol Fuchsin. Others are best listed under the particu- lar structures to be demonstrated Spores, Flagella, Capsules. In some cases search for bacteria in Milk, Soil, Cheese, Sputum, etc. is indicated. When bacteria are so few in number that they may be missed, or large numbers are required separated from the tissues for chemical analysis, Concentration methods may be useful. Accurate localization of bacteria requires their study in sections. See Giemsa's stain, Gram-Weigert stain, Goodpasture's stain (!MacCallum's modification), Mal- lory's Phloxine-Methylene blue and Acid Fast Bacilli. The darkfield examination of stained preparations is said to be an advantage (Goosemann, C, J. Lab. and Clin. Med., 1935-36, 21, 421-424). Appearance when viewed at high magnification with electron microscope (Mudd, S., Polevitsky, K., and Anderson, T. F., Arch. Path., 1942, 34, 199-207). See Fluorescence micros- copy, Negative Strains, Dead bacteria, Tubercle bacilli, Leprosy bacilli, Mito- chondria and Bacteria in same cells, Rickettsia, Gonococcus, Diphtheria Ba- cilli, Bacterium Tularense, Bacterium Monocytogenes. Bacteria. Biochemical Tests. Given in greater detail by H. R. Livesay in Simmons and Gentzkow, 387-389. 1. Indicators of pH. Incorporate in basic culture of medium measured amounts of 0.02% aq. phenol red, 0.04% aq. bromcresol purple, or 0.1% aq. bromthymol blue. Their pH ranges and colors are given under Hydrogen Ion Indicators. 2. Indoltest. Use Bohme's reagents. To 5 day culture in 1% aq. peptone add 1 cc. ether, shake and settle. Let 1 cc. of following run down inside tube : p- dimethylaminobenzaldehyde, 4 gm.; 95% ethyl alcohol, 380 cc; cone, hydro- chloric acid, 80 cc. If after 1 min. no color develops add 1 cc. sat. aq. po- tassium persulf ate . Positive , pale pink to deep magenta. 3. Ilosvay's Nitrate reduction. To 5 day culture at 37°C. in broth + 0.1% HNO3 add 1 cc. of following solution. Dissolve 1 gm. a-naphthylamine in 22 cc. aq. dest. Filter and add 180 cc. of dilute acetic acid (sp. gr. 1.04). Then 1 cc. of sulfanilic acid (0.5 gm. in 150 cc. dilute acetic acid). Positive, pink, red or maroon; negative, no color. 4. Ammonia. To 5 day peptone water culture add 0.5 cc. Nessler's Re- agent. Positive, brown; negative, faint yellow. 5. Hydrogen sulfide. Inoculate or- ganisms on lead acetate agar made by sterilizing extract broth containing 4% peptone + 2.5% agar and equal volume 0.1% aq. basic lead acetate. Positive, brown or black; negative, no color. 6. Reductase. To a 24 hr. broth cul- ture add 1 drop 1% aq. methylene blue. Incubate at 37°C. Positive, complete decolorization; weakly positive, green color; negative, no decolorization. 7. Catalase. Pour 1 cc. HjOj over 24 hr. agar slant culture incubated at 37''C. holding tube on incline. Posi- tive, gas bubbles; negative, none. 8. Methyl red. To 4 day culture in glucose phosphate medium at 37°C. add 5 drops 0.04% methyl red in 60% alcohol. Positive, red; negative, yel- low. 9. Voges-Proskauer. To 4 day cul- ture in glucose phosphate medium at 37°C. add 5 cc. 10% aq. KOH. After 18-24 hrs. positive, pink fluorescence; negative, no color. 10. Oxidase. To surface of colony add loop full or 1-2 cc. fresh 1% aq. dimethylparaphenylenediamine hydro- chloride. Positive, color change from pink to maroon to black. Bacteria Flagella. Electron microscopic technique reveals the ultrastructure of bacterial flagella as composite struc- tures made up of a central coiled trypsin resistant filament and a peripheral sheath probably nonresistant (De Robertis, E. and Franchi, C. M., Exp. Cell Res., 1951, 2, 295-298). Bacteria. Media. The following are brief summaries of culture media as described by H. R. Livesay in Simmons and Gentzkow, 388-403. (Glucose phosphate. Witte or Difco proteose peptone, 0.5 gm.; K2HPO4, 0.5 gm.; glucose, 0.5 gm.; aq. dest., 100 cc; pH 7.5.) Meat extract broth (routine). Add to 1000 cc aq. dest., beef extract, 3 gm. ; peptone, 10 gm.; sodium chloride, 5 gm. Dissolve by stirring with heat (water bath 65°C.). Make up weight loss with aq. dest. and make pH 7.2-7.4. Boil over flame, cool to 25°C., again make up weight loss, clarify and check pH. Place in flasks or tubes, autoclave 15 lbs., 15 min. BACTERIA. MEDIA 29 BACTERIA. MEDIA Meat extract broth (for water anal- ysis). As above, using beef extract, 3 gm.; peptone, 5 gm.; aq. dest. 1000 cc. pH 6.4-7. Meat extract agar (routine). Dis- solve 20-30 gms. powdered agar in 1000 cc. meat extract broth stirring over flame and titrate to pH 7.4. Cool to 50°C., add stirred eggs, heat gently till egg material is firmly coagulated. Remove coagulum with fine wire mesh strainer, filter through cotton, make up filtrate to original weight with aq. dest. and make pH 7.2-7.4. Tubes or flasks. Autoclave 15 lbs., 15 min. Meat extract agar (for water anal- ysis). Add 15 gm. best quality agar to 1000 cc. of above meat extract agar and make pH 6.4-7. Meat infusion broth. Mix 500 gms. ground fat-free beef, or veal round, in 1000 cc. aq. dest in ice box 18-24 hrs. Heat over small flame in Arnold steri- liier, 1 hr., add 5 gm. sodium chloride and 10 gm. peptone. Dissolve our flame, filter, add aq. dest. to 1000 cc, titrate to pH 7.4, tube or flask, and autoclave 15 lbs., 15 min. Meat infusion agar. Add 20 gm. agar to 1000 cc. Meat infusion broth and continue as in making meat extract agar, pH to 7.4. Gelatin, Nutrient. Add 120 gm. gelatin to 1000 cc. meat extract broth in double boiler, weigh, dissolve by heat, titrate to pH 7.4 and add aq. dest. to make original weight. Add 1 egg clarified by mixture with small volume of aq. dest., heat slowly till egg is coagulated, filter through cotton and sterilize filtrate in 10 cc. portions in tubes in Arnold 20 min. 3 successive days. Huntoon's hormone. Add 500 gm. fresh finely ground beef heart, 10 gm. peptone, 5 gm. sodium chloride, 1 whole egg, 20 gm. agar (Bacto) to 1000 cc. aq. dest. in enamel-ware dish, heat and stir constantly. Make pH 8. Cover, place in Arnold 1 hr. Remove, separate clot from sides and return to Arnold IJ-hr. Remove, let stand in- clined, room temperature, 10 min. Remove clear part and filter it through fine wire sieve into tall cylinders. Al- low to stand 15-20 min. and skim off fat. Clear further by passing through glass, or asbestos wool, or by centrifug- ing. Tube in 10 cc. lots, sterilize in Arnold 30 min. on 3 successive days. Glucose agar. Add 10 gm. glucose to 1000 cc. meat extract or meat in- fusion agar and dissolve by slowly heating. Adjust pH to that of original agar. Pour in tubes, or flasks, and sterilize in Arnold 3 successive days. Blood agar. Add 5-10% of sterile defibrinated blood (preferably horse) to meat infusion or meat extract agar which first has been melted and cooled to 45°C. Pour into plates or into tubes and slant, then incubate to prove sterilit}'. Chocolate blood agar. Add 5% of sterile defibrinated blood to meat in- fusion agar at 50-55°C. mix avoiding bubbles, slowly increase to 75°C. Pour into plates, or into tubes and slant, then incubate to prove sterility. Serum agar. Add 100 cc. sterile normal horse serum to 1000 cc. melted meat infusion agar, pour into plates, or tubes, and slant, then incubate to prove sterility. Liver infusion agar (for Br. abortus). Mix 500 gm. ground beef liver with 500 gm. aq. dest. in cool place 24 hrs., strain through cheesecloth and collect 500 gm. resulting infusion (1) . Add 20 gm. agar and 500 gm. aq. dest. and autoclave 15 lbs. pressure, 30 min. (2). Dissolve 10 gm. peptone and 5 gm. sodium chlo- ride in No. 1, beef infusion (3). Add aq. dest to 2 and 3 combined to make up weight lost by evaporation, adjust pH to 7 and cool to 50°C. Add 10 gm. egg albumin (first dissolved in 10 cc. aq. dest.), heat to 100°C. IJ-hrs., strain through fine wire sieve, filter through clean glass wool, adjust pH to 7, tube in 15 cc. lots and autoclave at 15 lbs., 30 min. When required melt and pour plates, or make slants. Trypagar. Put 500 gm. fat free, finely ground beef or veal ground in 1000 cc. aq. dest. in container adding 20% aq. NaOH until slightly alkaline to litmus. Cook at 75°C., 5 min., cool to 37°C. and add 0.5 gm. trypsin (Bacto). Incubate 37.5°C., 5 hrs. If trypaniza- tion is complete 5 cc. liquid -|- 5 cc. in NaOH + 1 cc. dil. aq. CuSO^ will give pink color. If not incubate again 1 hr. and re-test. When complete, slightly acidify with glacial acetic acid, slowly bring to boiling point and hold 15 min. Filter through wet paper, add 20 gm. agar and 5 gm. sodium chloride. Dis- solve agar with heat, clear with an egg, adjust to pH 7.6 and autoclave 15 lbs., 15 min. Veal infusion brain broth (for Strep- tococci and anaerobes). With large bore pipette insert about 50 cc. ground fresh calf brain in bottom 200 x 25 mm. tube and add 35 cc. veal infusion broth pH 7.6. Autoclave 15 lbs., 20 min. Remove 10 cc. test reaction, pH 7.4- 7.6 being satisfactory, if a change has taken place adjust to pH 7.6 and esti- mate from titration of this 10 cc. amount needed to bring to this figure BACTERIA. MEDIA 30 BACTERIA. MEDIA bulk and correct the whole. Fill tubes with similar amounts, then incubate at 37°C. to prove sterility. Robertson's (for Anaerobes). To 500 gm. ground fat, fascia and blood vessel-free fresh beef heart, add 10 gm. peptone and 1000 cc. aq. dest., bring to boil and adjust to pH 8. Continue sim- mering 1^-hrs. and again adjust reac- tion. Separate broth from meat, place former in flasks, autoclave 15 lbs., 15 min. Dry meat on filter paper in oven 56°C. 48 hrs. Place desired amounts of meat plus 10 cc. broth in tubes. Autoclave cool, remove broth and re- titrate. Adjust to desired pH, finally fill tubes same quantity meat and broth and autoclave 15 lbs. 30 min. Final pH should be 7.4-7.6. Calcium carbonate broth (for Pneu- mococci). Dissolve 10 gm. glucose in 1000 cc. meat infusion broth by heating, make pH 7.6. Place clean marble chips (CaCOs) in bottom of tubes pour in broth, sterilize in Arnold 15 min. 3 suc- cessive days. Blood culture (Kracke). Add 500 gm. finely ground fat-free beef heart muscle to 1000 cc. aq. dest. in ice box over night. Press through 4 layers gauze cloth, heat extract to boiling, filter through small mesh wire gauze. Add 250 gm. ground beef brain to 500 cc, treat in same way but do not filter this suspension. Mix 800 cc. extract, 110 cc. suspension, 1 gm. sodium citrate, 10 gm. dextrose (Bacto), 10 gm. pro- teose peptone (Difco), 2 gm. disodium phosphate and 4 gm. sodium chloride and place 50 cc. lots in tubes or flasks. Autoclave 15 lbs., 20 min. Bile (For typhoid group). Combine 900 cc. ox bile, 100 cc. glycerol and 20 gm. peptone by heating over water bath. Pour in bottles or small flasks and autoclave. Brilliant green lactose bile. Dissolve 10 gm. peptone and 10 gm. lactose in 500 cc. aq. dest. add 200 cc. fresh ox bile, or 20 gm. dehydrated ox bile dissolved in 200 cc. aq. dest., the latter having pH 7.4. Add 13.3 cc. 0.1% aq. brilliant green and aq. dest. to make 1000 cc. Filter through cotton, place in fer- mentation tubes, sterilize after which pH by potentiometer (not colorimeter) should be 7.1-7.4. Le vine's eosin methylene blue agar (Standard for water analysis). Dis- solve 10 gm. peptone, 2 gm. K2HPO4, and 15 gm. agar in 1000 cc. aq. dest. by boiling. Add aq. dest. to compensate for evaporation and distribute meas- ured amounts in flasks. Immediately before use to each 100 cc. add 5 cc. 20% aq. lactose (sterile), 2 cc. 2% aq. eosin and 2 cc. 0.5% aq. methylene blue. Mix, pour into Petri plates, harden and incubate to prove sterility. Endo's (Standard for water analysis). Add 5 gm. beef extract, 10 gm. peptone and 30 gm. agar to 1000 cc. aq. dest. in container and weigh. Boil till dis- solved, restore lost weight with aq. dest., place in vessel with straight walls and autoclave 15 lbs., 15 min. Let agar harden, remove en masse to clean paper, cut away and discard debris from bot- tom. Melt clean agar, make pH 7.8- 8.2, pour in 100 cc. or larger amounts and autoclave 15 lbs., 15 min. To each 100 cc. of this stock agar add 5 cc. 20% aq. C.P. lactose (sterilized by fractional method), 0.5 cc. 10% basic fuchsin in 95% alcohol (from filtrate of super- natant fluid having let stand 24 hrs.). Mix carefully, pour into sterile Petri dishes, let agar set at room tempera- ture and harden over night in incubator. Check sterility. Agar, sodium desoxycholate. Dis- solve 10 gm. peptone in 1 Kg. water, bring to pH 7.3-7.5 with sodium hy- droxide, boil few minutes and pass through filter paper. Add 12-17 gm. agar. After soaking 15 min,, melt by boiling. To each 1000 cc. add 6 cc. 1 N sodium hydroxide plus ferric am- monium citrate, 2 gm. dipotassium phosphate and 1 gm. sodium desoxy- cholate. Titrate with phenol red in- dicator to pH 7.3-7.5 and add 3 cc. 1% aq. neutral red. Sterilize in flowing steam only sufficient to kill vegetable cells (15 min. enough for tubes with 10-15 cc. medium). Selenite-F enrichment. Use mono- sodium and disodium phosphates in exact proportions which experiment shows that with particular lot of pep- tone and brand of sodium selenite will give pH 7.0-7.1. Dissolve with heat 10 gm. these phosphates (anhydrous), 4 gm. this sodium hydrogen selenite (anhydrous), 5 gm. peptone, 4 gm. lac- tose in aq. dest. to make 1 Kg. Boil. Russell's double sugar agar. Mix 1000 cc. melted meat extract agar, 40 cc. 25% aq. lactose (sterile) and 4 cc. 25% aq. glucose (sterile) and adjust to pH 7.2. Add 50 cc. 0.02% aq. phenol red, filter if necessary, tube and autoclave 8 lbs., 25 min. Slant with deep butt. Check reaction of medium with known E. coli and E. iyphosa. Simmons' citrate agar. Dissolve 5 gm. sodium chloride, 0.2 gm. MgS04, 1.0 gm. (NH4)H2P04, 2.28 gm. sodium citrate (2H2O) in 1000 cc. aq. dest. and add 20 gm. agar. Heat to dissolve agar, make pH 7.2, and add 10 cc. 1.5% alcoholic bromthymol blue. Filter BACTERIA. MEDIA 31 BACTERIA. MEDIA through cotton, tube, autoclave 15 lbs., 15 min. Slant with deep butt. Check reaction of medium with known, E. coll., A. aerogenes, S. schottmuelleri and E. typhosa. Jordan's tartarate agar. Dissolve by heating 20 gm. agar, 10 gm. peptone, 10 gm. sodium potassium tartarate, 5 gm. sodium chloride in 1000 cc. aq. dest. Adjust pH to 7.4 and add 12 cc. 0.2% alcoholic phenol red. Tube in 10 cc. lots, autoclave 15 lbs., 15 imn. Check reaction of medium with known S. aertrycke, S. enteritidis, S. paratyphi and S. schottmuelleri. Lead acetate agar (for H2S test). To 100 cc. sterile meat extract agar add following sterile Seitz-filtered solutions: 4 cc. 25% aq. glucose, 4 cc. 25% aq. lactose and 1 cc. 0.5% aq. lead acetate. Tube aseptically and incubate to prove sterility. Check reaction of medium with known S. paratyphi and S. schott- muelleri. Dieudonne's alkaline blood agar (for Vibrio comma). Make 700 cc. nutrient agar and neutralize to litmus about pH 6.8. Ivlix 150 cc. defibrinated beef blood and 150 cc. in 1 AT KOH and steam in Arnold 30 min. Add this to blood agar in proportion of 3 to 7. Pour Petri plates, let harden uncovered (but pro- tected by paper) placing strips sterile filter paper between dish and protection to take up ammonia and moisture. Incubate 15 hrs. at 37°C. before use. Carbohydrate broth (for fermenta- tion tests). Inoculate 1000 cc. infusion broth with active E. coli and incubate 18 hrs. at 37.5°C. Boil few minutes to kill organisms. Put in large mortar 20-30 gms. purified talc. While grind- ing add broth and thoroughly mix. Pass through wet filter paper till clear. Titrate and adjust pH to 7.3. Weigh broth and add 1% of desired ferment- able substances dissolved in a little hot water. Then add 45 cc. 0.04% aq. bromcresol purple per liter. Sterilize in Arnold 20 min. on 3 successive days, or autoclave 7 lbs., 10 min. Lactose broth (Standard for water analysis). Add 0.5% lactose to nu- trient extract broth and adjust reac- tion to pH 6.4-7. Autoclave 15 lbs. 15 min. restricting total heat exposure to 30 min. Clark and Lubs. Dissolve 5 lbs. each of peptone, dextrose and dipo- tassium phosphate in 1000 cc. aq. dest. using heat. Filter through paper, add water lost, tube in 10 cc. lots, sterilize in Arnold 20 min., 3 successive days. Bendick's saccharose peptone-water (for Vibrio comma). Add 1 gm. an- hydrous sodium carbonate to 1000 cc. peptone solution neutralized to phenol- phthalein. Boil, filter and to filtrate add 5 gm. saccharose + 5 cc. sat. phe- nolphthalein in 50% alcohol. Tube 10 cc. lots, sterilize in Arnold 15 min., 3 successive days. Dunham's peptone solution (for indol test). Dissolve 10 gms. bacto-tryp- tone (Difco) + 5 gm. sodium chloride in 1000 cc. aq. dest. with heat. Make pH 7.6 and filter if necessary, tube 10 cc. lots, autoclave 15 lbs. 15 min. Nitrate broth (for nitrate reduction test). Dissolve 10 gms. peptone + 1 gm. potassium nitrate (nitrite-free) in 1000 cc. aq. dest. (ammonia-free) with heat. Filter through paper, tube 10 cc. portions and sterilize in Arnold 20 min., 3 successive days. Bromcresol purple milk. Remove cream and heat remainder in cylinder in Arnold 20 min. Again skim off fat and to each liter remaining add 40 cc. 0.04% aq. bromcresol purple. Tube 10 cc. lots, sterilize in Arnold, 20 min., 3 successive days. Prove sterility by incubation. Loeffler's (for C. diphtheriae) . Col- lect beef blood in large glass vessels and let clot without moving. Loosen clot from wall with sterile glass rod and place in refrigerator. To 3 parts clear serum removed by pipette add 1 part meat infusion broth containing 1% glucose pH 6.8-7. Mix by stirring, in- spissate on slant raising temperature gently to approximately 85°C. Main- tain temperature till coagulated firmly. Sterilize in Arnold 20 min., 3 successive days, paraffinize cotton plugs and test sterility. Hiss' serum-water (for fermentation tests). Add 3 parts aq. dest. to 1 part clear serum, mix, heat in Arnold 15 min. Add 1% desired carbohydrate dissolved in small quantity hot aq. dest. Add 50 cc. 0.02% aq. bromthymol blue to each 1000 cc, tube, sterilize in Arnold 20 min., 3 successive days and prove sterility by incubation. Glycerol agar. Add 30 cc. pure glycerol to 1000 cc. melted infusion agar, adjust to pH 7.2, tube, autoclave 15 lbs., 15 min. and slant. Petroff's (for M. tuberculosis). In- fuse 500 gm. beef or veal in 500 cc. 15% aq. glycerol. After 24 hrs. put in sterile press and collect extract in sterile vessel. Place washed eggs in 70% alcohol, 10 min. Take out with sterile tongs, flame and remove con- tents to sterile vessel. To 2 parts egg add 1 part meat extract. Add 1% alco- holic gentian violet to make final con- centration 1:10,000. Mix and continue as with Loeffler's medium. BACTERIA. MEDIA 32 BALSAM Cystine blood agar (for P. tularensis) . To 1000 cc. beef or veal infusion broth add 15 gm. agar, 10 gm. peptone and 5 gm. sodium chloride. Autoclave 15 lbs., 15 min. Before use add 1 gm. cystine (or cystine hydrochloride) and 10 gm. glucose. Dissolve by heating in Arnold and sterilize 30 min. Cool to 50°C. add 50 cc' sterile horse blood, tube aseptically in 10 cc. lots, slant and incubate to prove sterility. Noguchi's leptospira medium. Com- bine sterile 80°C. 0.9% aq. sodium chloride, 100 cc. fresh rabbit serum, 100 cc. 2% aq. agar (melted pH 7.4) and 10-20 cc. rabbit hemoglobin (1 part blood, 3 parts aq. dest.). Tube asepti- cally in 10 cc. lots. Incubate to prove sterility. Tryptone glucose extract milk agar. Combine 15 gm. agar, 3 gm. beef ex- tract, 5 gm. tryptone, 1 gm. glucose and 1000 cc. aq. dest. by boiling over free flame. Make up volume lost with aq. dest., adjust to pH 7, add 10 cc. skim milk, place measured volumes in flasks or tubes and autoclave 15 lbs., 15 min. Tellurite (for C. diphtheriae). Melt infusion agar, or 0.2% dextrose agar and cool to 50°C. To each 10 cc. add 1 cc. citrated, or defibrinated, blood -f 1 cc. sterile 2% aq. potassium tellu- rite, mix and pour into Petri dishes. Bismuth sulfite agar (Wilson and Blair for E. iyphosa). Mix 20 gm. agar, 5 gm. beef extract and 10 gm. peptone in sufficient hot aq. dest. to make 1000 cc. Dissolve by autoclaving 15 min. Store in refrigerator. (A). Dis- solve 6 gms. bismuth ammonium citrate scales in 50 cc. boiling aq. dest. (1), 20 gm. anhydrous sodium sulfite in 100 cc. boiling aq. dest. (2), and 10 gms. dex- trose in 50 cc. boiling aq. dest. (3). Mix 1 and 2, boil and add 10 gms. an- hydrous disodium phosphate while boiling. Cool and add 3. Add water to restore lost weight. Store in closely stoppered pyrex container in dark at room temperature (B). Dissolve 1 gm. ferric citrate in 100 cc. aq. dest. using heat and add 12.5 cc. 1% aq. brilliant green. Store likewise in pyrex vessel in dark. With 1000 cc. hot (A) thor- oughlj' mix 200 cc. (B) and 45 cc. (C). Immediatel}' pour into porous-top petri dishes each 15-20 cc. After 2 hrs. at room temperature store in refrigerator and use within 4 days. Chocolate agar (for Neisseria). Grind strips lean meat of 5-6 beef hearts. To each 500 gm. add 1000 cc. tap water, infuse in refrigerator over night, strain and press through course gauze. Add 10 gm. proteose peptone No. 3 (Difco) per liter, heat to 50°C. 1 hr. and boil 10 min. Strain through gauze, dissolve 5 gm. sodium chloride per liter and titrate to pH 7.6. Boil lightly 10 min. pour off measured quan- tities in flasks, autoclave 15 lbs., 15 min. Cool to 60°C., add 5% human or horse blood, heat slowly on water bath to 80-85''C. rotating to get even mix- ture. Cool to 55°C. and plate. Bacterial Pigments. These cannot be meas- ured microscopically but a method has been devised for doing so with spectro- photometer and photoelectric colorim- eter (Stahly, G. L., Sesler, C. L. and Erode, W. R., J. Bact., 1942, 43, 149-154). Bacterial Polysaccharides. Solutions of reduced bases and leuco bases of penta- and hexa-methyl triamino-triphenyl- methane and tetramethyl diamino- triphenylmethane and certain other triphenylmethanes react with staphylo- coccal polysaccharides and may be useful in their detection (Chapman, G. H. and Lieb, C. W., Stain Techn., 1937, 12, 15-20). Bacteriophage Localization by Electron Microscopy, see Hennessen, W., Zeit. f. wis. Mikr., 1951, 60, 172-180. Bacteriostatic Titration of Dyes. (Reed, M. V. and Genung, E. F., Stain Techn., 1934, 9, 117-128). Bacterium Monocytogenes. Intravenous injections of this organism in rabbits produce a marked increase in the num- ber of circulating monocytes and there- fore provide an important experimental method (Murray, E. G. D., Webb, R. H. and Swan, M. B. R., J. Path, and Bact., 1926, 29, 407-439). Bacterium Tularense in sections. Add 10 cc. sat. aq. nile blue sulphate and 6 cc. l%aq. safranin to60 cc.aq. dest. Stain sections over night. Wash quickly, dehydrate in alcohols, clear in xylol and mount (Foshay, L., J. Lab. & Clin. Med., 1931, 17, 193-195). Balances. Ordinary balances need no de- scription but for weighing very small amounts special balances are essential. See review of literature by Gorbach, G., Mikrochemie, 1936, 20, 254-336. The torsion balances of Roller-Smith Co., Bethlehem, Pa., are sensitive to ap- proximately 2 Atgm. The quartz fiber balances are still more sensitive. See Click, pp. 189-191. Balantidium. Celloidin embedding and sec- tioning (Scott, M. J., J. Morph. & Physiol., 1927, 49, 417). Balsam for mounting sections is usually satisfactory as purchased. To make, mix equal parts dry balsam and sodium bicarbonate and grind in mortar. Add sufficient xylol to make clear solution. BARBER AND KOMP 33 BASOPHILE LEUCOCYTE After few days filter and heat gently (avoiding flame) to bring to suitable consistency. The best mounting me- dium when neutrality is essential is Clarite or the cedar oil used for oil immersion objectives. The latter sets more slowly than balsam and it is ordi- narily not necessary to employ it. See Mounting Media. Barber and Komp thick film for malaria Plasmodia (Barber, M. A. and Komp, W. H. W., Pub. Health Rep., 1929, 44, 2330) is described by Craig, p. 290-291 as the most used and satisfactory of the thick film techniques. His account of the method abbreviated: Place large drop blood on clean slide and smear with needle over area about half size of that usually covered by a thin blood smear. Dry in incubator at 37°C., 1-1| hrs. Stain in 1 part good Giemsa and 6 parts neutral or slightly alkaline aq. dest., about 30 min. Partly decolorize in aq. dest. 5 min. (If films have back- ground deep blue and leucocytes almost black they may be worthless; but leav- ing in aq. dest. longer may help). Drain thoroughly, dry and examine. Barium, spectrographic analysis of, in retina (Scott, G. H. and Canaga, B., Jr., Proc. Soc. E.xp. Biol. & Med., 1940, 44, 555- 556). Barium chloride and formalin are advised as fi.xative for Bile Components. Barium sulphate emulsion injections are recommended by Woollard, H. H. and Weddell, G., J. Anat., 1934-35, 69, 25-37 to demonstrate arterial vascular patterns. The emulsion should be of such consistency that it cannot easily be forced beyond the small arterioles by a pressure of 1 .5 atmospheres. Fi.x tissues by hypodermic injection of formalin and subsequent immersion in it. Take x-ray photographs of the radiopaque barium. See Cretin's Test for Calcium and barium under Calcium 5. Bartholomew, see Gram Stains Mechanism. Bartonella, Try Giemsa's Stain for sections. Basal Bodies of cilia (Wallace, H. M., Science, 1931, 74, 369-370). Fix in Zenker (containing acetic) or in Zenker- formalin (90 cc. Zenkers + 10 cc. 10% formalin). Mount paraffin sections 5/x thick. After very light staining with hematoxylin and thorough washing in tap water dip in 0.5% aq. eosin (Grubler's wasserlich. If not available, use Eosin Y.) ^ min. and wash quickly in large volumes of water. Make up stain by adding 9 parts sat. aq. methyl violet (Grubler's 6B only. If not available, use CC. which is 2B.) to 1 part abs. alcohol 33 cc. ; aniline oil 9 cc. -f methyl violet in excess. Stain is best 3-8 days after mixing but the two solutions can be kept separately. After staining sections for 2 hrs. wash well in tap water, treat with Lugol's iodine 10-15 min. and repeat the washing. Blot with filter paper. Differentiate in 1 part aniline oil -f 2 parts xylol. Wash in several changes of xylol and mount in balsam. Basal bodies deep purple, nuclei dark blue. Good also for intra- cellular bacteria and fibrin. Basement Membranes of epithelia. Suggest Mallory-Heidenhain's connective tissue stain as modified by Schleicher, E. M., Am. J. Clin. Path.", 1943, 7, 3&^39 and Schiff Reagent method as detailed by McManus, J. F., Am. J. Path., 1948, 24, 643. Basic Brown, G, GX, or GXP, see Bismark Brown Y. Basic Dyes, see Staining. Basic Fuchsin — anilin red, basic rubin, and magenta (CI 676 or 677) — Commission Certified. The tri-amino tri-phenyl methane dyes bearing this name are mixtures of pararosanilin, rosanilin and magenta II in varying proportions. They are employed for a great many purposes. Basic fuchsin in a cytologi- cal technique for anterior pituitary is described bj' Faire, W. R., and Wolfe, J. M., Anat. Rec, 1944, 90, 311-314. New fuchsin (CI 678) is a different com- pound. It is the deepest in color of 4 dyes and pararosanilin is the lightest. Basic Lead Acetate used as fixative for Tissue Basophiles. Basic Rubin, see Basic Fuchsin. Basket Cell Arborizations, technique for, Cajal, S. R., Trav. Lab. Rech. Biol. Univ. de Madrid, 1926, 24, 217. Basophila Erythroblasts, see Erythrocytes, developmental series. Basophile Leucocyte (mast-leucocyte, blood mast cell). Least numerous granular leucocyte ; percentage about 0-1 ; slightly smaller (8-10m) than other types; nucleus spherical or slightly lobated, faintly staining and centrally placed; specific granules only slightly refractile, basophilic, large, variable and less numerous than in other types; function unknown. This cell is difficult to study in fresh preparations of pe- ripheral blood because it is so scarce. Smears colored by the usual methods (Giemsa, Wright, etc.) are satisfactory. The basophilic granules appear to be particularly soluble in water. Doan and Reinhart (C. A. and 11. L., Am. J. Clin. Path., 1941, 11, Tech. Suppl. 5, 1-39, with beautiful colored plates) recommend supravital staining with neutral red and janus green. There is difference of opinion as to whether the oxidase and peroxidase reactions are positive (Michels, N. A. in Downey's BASOPHILIC 34 BERBERIAN'S METHOD Hematology, 1938, 1, 235-372). See Tissue Basophiles. Basophilic, see Staining. Bauer Negative and positive substances, for method see Glycogen. Beams, see Ultracentrifuges. Bell's Method for fixing and staining of fats as described by the Bensleys (p. 114). Intracellular fats are mobilized by heat to form droplets which are chromated and later stained. Consequently the preparations show these fats, in addition to other microscopically visible fat, but not their true distribution in the cells. Fix for 10 days at 45-50 °C. in 10% aq. potassium bichromate 100 cc. + 5 cc. acetic acid. Imbed and make paraffin sections as usual. Pass them down to absolute alcohol. Stain with freshly prepared Sudan III 10 min. Rinse off in 50% alcohol and pass to water to arrest action of alcohol. Counter-stain with Delafield's Hematoxylin. Wash in water, differentiate in acid alcohol, wash in water again and mount in Glycerine Jelly. Benda's Method of crystal violet and alizarin for mitochondria. Fix in Flem- ming's fluid 8 days (see Flemming's Fluid). Wash in water 1 hr. Then half pyroligneous acid and 1% chromic acid, 24 hrs. 2% potassium bichromate, 24 hrs. Wash in running water 24 hrs. Dehydrate, clear, imbed in paraffin and cut sections at 4ju. Pass down to water and mordant in 4% iron alum 24 hrs. Stain amber-colored sol. sodium sulpha- lizarinate made by adding sat. ale. sol. to water, 24 hrs. Blot with filter paper and color in equal parts crystal violet sol. and aq. dest. (The sol. consists of sat. crystal violet in 70% ale. 1 part, ale. 1 part and anilin water 2 parts.) Warm until vapor arises and allow to cool 5 min. Blot and immerse in 30% acetic acid 1 min. Blot, plunge in abs. ale. until but little more stain is ex- tracted, clear in xylol and mount in balsam. The mitochondria are stained deep violet in a rose background. The colors are more lasting than in Altmann preparations. This is one of the classi- cal techniques of histology but it is difficult. For colored illustrations see Duesberg, J., Arch. f. Zellforsch., 1910, 4, 602-671. Benda's stain for fat necrosis. See Fisch- ler's modification. Bensley's Neutral Safranin. For mitochon- dria and secretion antecedents especially in the pancreas. Fix in 2.5% aq. potas- sium bichromate, 100 cc; mercuric chloride, 5 gms. 24 hrs. Wash, dehy- drate, clear, imbed and section. To prepare stain slowly add sat. aq. acid violet to sat. aq. safranin 0 in a flask until ppt. ceases when a drop of mixture on filter paper gives not an outside red rim of safranin but a solid neutral color. Filter. The filtrate should be as nearly as possible colorless. Dry ppt. on filter paper and make of it a sat. sol. in abso- lute alcohol. Pass sections through 2 changes each of toluol and absolute alcohol, then down through lower alco- hols to aq. dest. (Bleach chrome and osmium fixed tissues in permanganate and oxalic acid, as described under Anilin Fuchsin Methyl Green and mercury fixed ones in Lugol's solution, 10 sec. finally washing in aq. dest.) Dilute alcoholic neutral safranin with equal volume aq. dest. and stain 5min.- 2 hrs. Quickly blot with filter paper. Plunge into acetone and immediately pass to toluol without draining. Exa- mine and if further differentiation is needed treat with oil of cloves. If this is not enough rinse in abs. ale, flood momentarily with 95% ale. and pass back through absolute to toluol. Wash in 2 changes toluol and mount in balsam. This is a difficult method but the results are worth it. (see Bensley, R. R., Am. J. Anat., 1911, 12, 297-388). Benzamine Blue 3B, see Trypan Blue. Benzene-Azo-a-Naphthylamine. A mono- azo dye used by Carter, J. S., J. Exp. Zool., 1933, 65, 159-179 as a vital stain for Stenostomum. Benzo Blue SB, see Trypan Blue. Benzo New Blue 2B, see Dianil Blue 2R. Benzo Sky Blue, see Niagara Blue 4B. Benzoazurine G (CI, 502), a direct dis-azo dye of light fastness 4 sometimes polychromatic (nuclei red, cytoplasm blue to blue-violet). Applied after treating blue-green algae with copper sulphate, spores orange red, vegetative cells dark blue or violet (Emig, p. 41). Benzene ring compounds distinguished from pyrrols by Nitro Reaction. Benzoin Blue R, see Azo Blue. Benzopurpurin 4B (CI, 448) — cotton red, diamin red, dianil red. Sultan and direct red, all 4B — An acid dis-azo dye no longer used. Benzoyl Perioxide treatment is recom- mended by McClung, Microscopical Technique, 1950, p. 71 for revival of staining capacity of old smears and spreads. Benzyl Benzoate is employed in the Spalte- holz Method of clearing. Benzyl Violet. Conn (p. 132) states that this term relates to a group of violets which are pararosanilins, some acid and some basic, with benzyl substitution in one or more amino groups. Berberian's Method. Berberian, D. A., Arch. Dermat. & Syphil., 1937, 36, 1171- 1175, has developed a method for stain- BERBERINE SULPHATE 35 BILE ing fungi in epidermal scales and hair, which differentiates epithelial cells, blood cells, bacteria and 'mosaic fungi'. The following account was written by D. A. Berberian, American University of Beirut, Beirut, Lebanon, June 22, 1946: Fix small pieces of scales or hair on a slide with 50% aq. glacial acetic acid by drying in an incubator. Defat, clear, hydrate, and wash off the acid as follows: Cover the preparation with ether 2-3 times, 20-30 sec. each; flood twice with absolute acetone, 30-60 sec. each; and then flood consecutively with absolute, 95, 70 and 50 per cent alcohol. Stain for 3-5 min. with Martinotti's solution (aq. dest., 75 cc; lithium car- bonate, 0.5 gm.; and toluidine blue, 1 gm. After the stain dissolves, add 20 cc. glycerin and 5 cc. 95% alcohol). Wash gently in water and differentiate with 0.5% acetic acid. Dehydration is best carried out by 3-4 changes of absolute acetone kept 2-3 minutes each time. Pass through xylol and mount in Euparol or any other neutral mount- ing agent. Success of preparation depends largely on proper differentia- tion, dehydration and de-acidification. See Fungi. Berberine Sulphate. An alkaloid used as a fiuorochrome for malarial parasites (Metcalf, R. L. and Patton, R. L., Stain Techn., 1944, 19, 11-22). Bergamot Oil is sometimes used for clearing because it will mix with 95% alcohol. Berlin Blue is another name for Prussian Blue (a metallic pigment). It is em- ployed for microchemical detection of Iron. Kremer, Zeit. f. wiss. mikr., 1938, 54, 429-432 suggests proceeding as follows: Fix in absolute alcohol. De- paraffinize 10/x sections. Bleach in 3-5% H2O2 3-5 days. Wash carefully in aq. dest. Quickly darken in (NH4)2S. Transfer to K ferrocyanide and HCl. Iron gives blue color. Curiously enough when injection of blood vessels is demanded this mineral pigment is usually called for as Berlin blue. Thus the Bensleys (p. 153) give directions for making up Tandler's Berlin blue gelatin. Soak and melt 5 gms. pure gelatin in 100 cc. aq. dest. Add sufficient Berlin blue to give desired color and then 5-6 gms. potassium iodide and a crystal of thymol as a preservative. Inject this mass, which is fluid above 17°C. Fix tissues in 5% formalin which preserves it even through decalcifica- tion. Beryllium. In various forms and dosages in production of osteogenic sarcomata in rabbits (Hoagland, M. B., Grier, R. S., and Hood, M. B., Cancer Rea., 1950, 10, 629-635. Best's Carmine. Griibler's carminum ru- brum optimum, or some other good carmine, 2 gm. ; potassium carbonate, 1 gm.; potassium chloride, 5 gm.; aq. dest., 60 cc. Boil gently until color darkens, cool and add 20 cc. cone, am- monia. Allow to ripen 24 hrs. This is stock solution. Used to stain Glyco- gen. See Bensley, C. M., Stain Tech., 1939, 14, 47-52. Beta Particles, influence in making radio- autographs, see McClung's Microscopi- cal Technique, 1950, p. 707. Beyer Brown, a diazo dye, stains in aq. or alcoholic solution like a good Ehrlich's hematoxylin (H. G. Cannan, J. Roy. Micr. Soc, 1941, 61, 88-94). Biaxial Bodies, see McClung's Microscopical Technique, 1950, p. 605. Bichromate-Chromic-Osmic mixture, see Champy's Fixative. Blebrich Scarlet, water soluble (CI, 280) — croceine scarlet, double scarlet BSF. Ponceau B, scarlet B or EC — An acia dis-azo dye much used in histology. See Bowie. Blebrich Scarlet and Picro-Anilin Blue, as a differential stain for connective tissue and muscle (Lillie, R. D., Arch. Path., 1940, 29, 705). Deparaffinize sections of material fixed in formalin, Zenker's or Orth's fluid. Stain for 5 min. in following: Dissolve 1 gm. hematoxylin in 95% ale. and 4 cc. 29% aq. FeCla in 95 cc. aq. dest. -f- 1 cc. cone, hydrochloric acid. Mix and use while fairly fresh. Wash in tap water. Stain for 4 min. in 0.2 gm. Biebrich scarlet + 100 cc. 1% aq. acetic acid. Rinse again inaq. dest. Stain for 4 min. in 0.1 gm. anilin blue W.S. (CC.) + 100 cc. sat. aq. picric acid. Wash for 3 min. in 1% aq. acetic acid. Dehydrate in acetone or alcohol. Clear and mount in salicylic acid balsam. Connective tissue, glomerular basement membrane and reticulum, deep blue; muscle and plasma, pink; erythrocytes, scarlet. (Checked by R. D. Lillie, National In- stitute of Health, Bethesda, Md., April 22, 1946.) Bielchowsky Silver Methods. These are designed for the nervous system and consist essentially of formalin fixation, silver impregnation, washing, treating with ammoniacal silver solution, wash- ing and reduction in formalin. Several useful modifications are detailed by Addison (McClung, pp. 463-466). See Nervous System, Silver Methods. Bile. This frequently comes in for micro- scopic examination of centrifuged sedi- ment. Stitt (p. 761) says that one must be on the lookout especially for: (1) BILE CAPILLARIES 36 BIOLOGICAL STANDARDS Pus cells (neutrophiles), scattered through the specimen and bile stained, which, when occurring in fair numbers, indicate cholecystitis. Unstained pus cells associated with mucus are generally from the mouth. (2) Bile colored epi- thelial cells and cellular debris suggest chronic cholecystitis. (3) Cholesterin crystals are identifiable as opaque or translucent, flat, rhombic plates or irregular masses. (4) Large amounts of light brown granules or dark black- brown ppt. of calcium bilirubinate are suggestive of gall stones. (5) Tiny gall stones (bile sand) are identifiable by their concentric lamination. Negative findings are not, he is careful to point out, conclusive of absence of lesions. Bile Capillaries. 1. Hematoxylin staining. Clara, M., Zeit. f. mikr. Anat. Forsch., 1934, 35, 1-56 advises treatment of cel- loidin sections of pieces of liver fixed in Alcohol Formalin, formalin — absolute alcohol — acetic acid (20:80:1) and other mixtures by the Stolzner Holmer tech- nique and his own method. According to the former, mordant the sections in liquor ferri sesquichlorati (try 10% aq. ferric chloride) 30-45 min. Wash quickly in aq. dest. Stain in ripe 0.5% aq. hematoxylin, 20-30 min. Wash quickly in water. Differentiate in much diluted liquor ferri sesquichlorati. Wash again quickly in water. Blue with dilute aq. lithium carbonate. Wash in spring water (tap water will do ) . Dehydrate, clear and mount. According to Clara, mordant the sections in equal parts A and B at 40-50 °C. for 24 hrs. (A = potassium bichromate, 2.0 gm. ; chrome alum, 1 gm., aq. dest., 30 cc. B = ammonium molybdate, 2.5 gm.; chromic acid, 0.25 gm.;aq. dest., 100 cc.) Wash briefly in aq. dest. Stain in Kultschitzky's Hematoxylin. Wash in spring water. Dehydrate, clear and mount in balsam. See Clara's illustra- tions. 2. Rio Hortega silver carbonate method adapted by Mclndoe, A. H., Arch. Path., 1928, 6, 598-614. Fix small pieces normal human liver at least 20 days in 10% formalin. Heat gently but do not boil and cool several times thin frozen sections for 20 min. in silver bath until they are uniformly of a golden brown color. (To make the bath combine 30 cc. 10% aq. silver nitrate and 10 cc. sat. aq. lithium carbonate. Wash ppt. re- peatedly with doubly distilled water, decanting washings. Add 100 cc. doubly distilled water to ppt. Dissolve ^i of it by adding ammonia water drop by drop. Filter supernatant fluid into opaque bottle and store in dark where it can be kept 2-4 weeks. For use take 5 cc. of this stock solution and add 5 cc. aq. dest. and 2-3 drops pyridine.) Wash quickly in aq. dest. Place in 20% neutral formalin, 1 min. Fix in 2% aq. sodium thiosulphate, ^-1 min. Wash thoroughly in tap water, 2-3 days adding a little neutral formalin. Dehydrate in 95% and abs. ale, clear in carbol- xylol and mount in balsam. Canaliculi, black. Bile Components in hepatic cells. Place small pieces of liver in 3% aq. barium chloride for 6 hours ; fix 18 hours in 10% formalin; dehydrate rapidly in alcohol, clear in benzol and embed in paraffin. The bile components, precipitated by barium chloride, can be stained with acid dyes especially the acid fuchsin in Mallory's connective tissue stain (Fors- gren, E., J. Morph., 1929, 47, 519-529). Bile Pigments. Histochemical reaction. Fix in 10% formalin or in alcohol. Pro- longed fixation is contraindicated. Fix paraffin sections to slides with egg albumen. Deparaffinize and immerse in 2 or 3 parts Lugol's solution and 1 part tincture of iodine, 6-12 hrs. Wash in aq. dest. and cover with sodium hypo- sulphite (5% aq.) 15-30 sec. until de- colorized. Wash in aq. dest. and stain with alum carmine 1-3 hrs. Wash in aq. dest., dehydrate in acetone, clear in xylol and mount in balsam. Bile pigment granules emerald green (Stein, J., C. R. Soc. de Biol., 1935, 120, 1136- 1138). See Gmelin's Test. Bilharzial Cercariae. For intra - vitam staining examine in serum plus a little neutral red. For permanent prepara- tions fix in hot lactophenol (equal parts lactic acid, carbolic acid, glycerin and aq. dest.). Stain with alcoholic borax- carmine. Mount in following: dissolve by boiling gum tragacanth 3 parts and gum acacia 1 part in aq. dest. 100 parts. Add equal parts lactophenol and use filtrate. (Marshall, A., Lab. J., 1937, 7, 565-569). Bilirubin, a reddish bile pigment which is isomeric or identical with Hematoidin and which by oxidation can be converted into the green Biliverdin, see Bile Pigments, Urobilin and Van den Bergh Test. Biliverdin, a green bile pigment produced by oxidation of Bilirubin. See Bile Pigments. Bindschedler's Green (CI, 819). A basic indamin dye easily reduced to a sub- stituted diphenylamine. See use as a Redox dye in study of metabolism of tumor tissue (Elliott, K. A. C. and Baker, Z., Biochem. J., 1935, 29 (2), 2396-2404). Binnennetz, see Golgi Apparatus. Biological Standards. Vitamins, antitoxins, BIOTIN 37 BISMUTH PIGMENTATION hormones and other substances adopted internationally are critically considered by Irwin, J. O., J. Hyg., 1950, 48, 215- 238. Biotin, see Vitamins. Bird's Eye Inclusions. Some of these bodies, and the so-called Plimmer's Bodies, seen in cancer cells are ap- parently greatly enlarged Centrosomes. Methods and results are given by Le- Count, E. R., J. Med. Res., 1902, 7 (N.S. 2), 383-393. Birefringence, see Polarization Optical Method. Bismark Brown Y (CI, 331) — basic brown, G, GX, or GXP, Excelsior brown, leather brown, Manchester brown, phenylene brown, Vesuvin — A mixture of basic dis-azo dj'es of different shades. Quite widely employed, see Blaydes, G. W., Stain Techn., 1939, 14, 105-110 for use with plant tissue. See Weissen- berg's method, as described by McClung, 1950, p. 184, for the preservation in sections of Bismark brown employed for supravital staining of eggs. Bismiocymol (see Pappenheimer, A. M. and Maechling, E. H., Am. J. Path., 1934, 10, 577-588. Bismuth. Microchemical detection of : 1. Method of Christeller-Komaya. Make frozen sections of formalin fixed tissues. A = quinine sulphate, 1 gm.; aq. dest., 50 cc; nitric acid, 10 drops. B = potassium iodide, 2 gm., aq. dest., 50 cc. Immediately before use mix equal parts A and B and add 2 drops nitric acid, C.P. After treating sec- tions with this for 1 min. wash very quickly in 10 cc. aq. dest. + 2 drops nitric acid. Mount section on slicle. Dry, counterstain with gentian violet. Bismuth appears as dark brown grains (Lison, p. 98). See Komaya, G., Arch, f. Dermat. u. Syph., 1925, 149, 277-291 (good colored figures) and Califano, L., Zeit. f. Krebsf., 1927-28, 26, 183-190. 2. Another modification of the Komaya method is given by Castel, P., Arch. Soc. d. Sci. Med. et. biol. de Montpellier, 1934-35, 16, 453-456 as follows : Dissolve 1 gm. quinine sulphate in 50 cc. aq. dest. with aid of a few drops of sulphuric acid. Dissolve 2 gm. potassium iodide in 50 cc. aq. dest. Mix, apply to section, gives red ppt. of salts of bismuth in form of iodo- bismuthate of quinine or double iodide of bismuth and quinine. See Pappen- heimer and Maecnling's (Am. J. Path., 1934, 10, 577-588) study of nuclear inclusions in the kidney. 3. Wachstein, M. and Zak, F. G., Am. J. Path., 1946, 22, 603-611 have in turn improved the Castel method. See Glick, Techniques of Histo- and Cyto- chemistry, 1949, p. 31. Reagents: A. Modified Castel: After dissolving 0.25 gm. brucine sulphate in 100 cc. aq. dest. add 2 gm. aq. dest. Store in brown bottle and filter before using. B. Diluted Castel: One part A to 3 parts aq. dest. C. Levulose: Dis- solve 30 gm. in 20 cc. aq. dest. at 37°C. for 24 hrs. and add 1 drop of B. D. Counterstain: Add 1 cc. 1% aq. light green S. F. (Hartman-Leddon Co., Philadelphia) to 100 cc. of B and filter before use. Technique: Treat frozen sections or deparaffinized formalin fixed sections on slides for few sec. with several drops 30% hydrogen peroxide (Superoxol, Merck) thus removing black sulfide. Wash in tap water and treat with A 1 hr. Transfer to B and shake slightly to detach precipitates. Counterstain in Z) 4 min. Blot and mount in C. Bis- mith appears as an orange yellow de- posit. Bismuth Pigmentation. Histochemical identification as advised by Wachstein, M. and Zak, F. G., Am. J. Path., 1946, 22, 603-611 depends on ability of hydro- gen peroxide to decolorize bismuth sulfide instantaneously and of a slightly modified Castel reagent to change bis- muth sulfate into an orange red deposit. Treat deparaffinized, or frozen, sec- tions with few drops superoxol (30% hydrogen peroxide, Merck) from dark bottle kept in refrigerator. In a few seconds black of bismuth sulfide dis- appears. Wash thoroughlj' in tap wa- ter and place in Coplin jar containing modified Castel reagent made as fol- lows: Dissolve 0.25 gm. brucine sulfate (Merck or Eastman Kodak) in 100 cc. aq. dest. plus 2 or 3 drops concentrated sulfuric acid. Then acid 2 gm. potas- sium iodide, keep in a brown bottle and filrer before use. After 1 hr. transfer sections to another jar containing some of reagent diluted with 3 parts aq. dest., and shake gently to remove precipi- tates. Remove most of fluid from sec- tions by blotting and cover with levu- lose solution made by dissolving 30 gm. levulose in 20 cc. aq. dest. at 37°C. for 24 hrs. to which drop of diluted Castel reagent has been added. To counter- stain color 4 min. with freshly filtered 100 cc. nondiluted reagent plus 1 cc. 1% aq. light green S F (Hartman-Led- don Co.). Method also can be used for study of fresh tissues and gross specimens. Add cone, hydrogen peroxide drop by drop to pigmented area. Decolorization is rapid if bismuth sulfide is present. Wash thoroughly in running water to BISMUTOSE 38 BLOOD CELL VOLUME remove excess hydrogen peroxide. Ap- ply modified Castel reagent to surface and examine for orange ppt. See de- scription by authors of distribution of bismuth pigmentation in the tissues and comparison with other pigments. Bismutose, a compound of bismuth and albumen which on application becomes concentrated in the area of the Golgi apparatus (Kredowsky, Zeit. f. Zellf., 1931, 13, 1). Biuret Reaction. Described as follows by Serra, J. A., Stain Techn., 1946, 21, 5-18: Prepare tissue as described under Nin- hydrin Reaction. "The pieces are im- mersed in strong NaOH or KOH solu- tion in a watch glass and some drops of a 1% aqueous solution of CuSO^ are then added with stirring. A blue- violet coloration indicates the presence of peptides or proteins. "The reaction is given by the peptide linkage when the peptides are composed of at least three amino acids. The color is more reddish with the simpler peptides. For cytological or histologi- cal work, the reaction has the disad- vantage of requiring a strong alkaline reaction, which tends to dissolve the protoplasm. To avoid a serious dis- solution the tissues must be hardened, for instance with formalin (10% for- maldehyde during 24 hours, followed by a thorough washing). This reaction has also the disadvantage of being in- sensitive." Blastomeres of eggs, see separation of in McClung's Microscopical Technique, 1950, p. 557. Blastomycosis. The differentiation of Zy- monema (Blastomyces) dermatitidis , the cause of blastomycosis, from Crypto- coccus hominis, the cause of crypto- coccosis or torulosis, is best accom- plished by w^et India ink technique of Weidman, F. D. and Freeman, W., J.A.M.A., 1924,83, 1163. Stir suspected material in a drop of india ink, place on a clean slide and cover. Use a small drop so as to form a thin film. Work rapidly before the ink dries out. In blastomycosis the wall of the organism is thick and presents a double-contoured appearance. Cryptococciis hominis is surrounded by a thick mucoid capsule which, against a dark background, shows up as a clear halo surrounding the fun- gus. Spinal fluid usually dilutes the ink making a lighter background. See Fungi. Bleaching of tissue fixed in osmic acid mix- tures can be brought about by immers- ing in dioxan for as long as 3 daj^s. Sec- tions require only a few minutes (Asana, J. J., Stain Techn., 1940, 15, 176). Blendors, Micro-Waring for low temper- ature use (Sorof, S. and Cohen, P. P., Exp. Cell Res., 1951, 2, 299-300). Blood. Microscopically considered blood is the field of the hematologist (see Downey's Hematology, N. Y., Hoeber, 1938 in 4 volumes). Any conception of the formed elements of the blood is artificial and inadequate unless it is based upon knowledge of their appear- ance and behavior in vivo. To examine circulating blood in the web of a frog's foot is helpful but it is better to use mammals. In the latter, the methods devised by Covell and O'Leary for study of the living Pancreas are recommended for blood cells also. Probably the best technique is that of Sandison for direct e.xamination of contents of small blood vessels and capillaries in transparent chambers inserted into rabbits' ears. Living blood cells can be observed in vitro at high magnification in Tissue Cultures; but, of course, circulation is lacking. When blood cells are removed from the body and mounted on slides in approxi- mately isotonic media, they can be studied for a short time before they be- come seriously injured and die. Examination in the dark field and after Supravital Staining may be helpful. It is important in interpreting the results to remember that the conditions are very abnormal, that the cells are often more flattened than in vivo, and that the actual speed of movement is not that seen, but is that observed di- vided by the magnification because the distance travelled per unit of time naturally appears greater than it actuallj^ is. The motion picture tech- nique has great potentialities. Examination is usually limited to fixed and stained Blood Smears but valuable data can also be secured from sections. Normal values for blood cells during first year of life (Merritt, K. K., 1933, 46, 990-1010). For details, see Blood Pro- tein (coagulated). Bone Marrow, Chylo- microns, Erythrocytes, Erythrocyte Counts, Fibrin, Hematoidin, Hemato- porphyrin, Hemofuscin, Hemoglobin, Hemosiderin, Leucocytes, Leucocyte Counts, Lymphocytes, Monocytes, Platelets, Parhemoglobin, Reticulo- cytes, Sulfmethemoglobin. Blood Agar, see Bacteria, Media. Blood Cell Volume. Dry Evans Blue (Merck) at 100 °C. Dissolve 400-800 mg. in 1 liter aq. dest. Put 0.5-1 cc. in tube 3-4 cc. capacity and evaporate to dryness at 70 °C. Collect blood to contain 2.0-2.5 units heparin or 0.2% ammonium oxalate. Centrifuge and transfer 1 cc. plasma to tube containing dye. Remove 0.1 cc. dyed plasma to BLOOD FLOW 39 BLOOD PLATELETS 9.9 cc. saline in photoelectric colorimeter tube. Make blank without plasma. Compare in Evelyn or Klet-Summerson colorimeter using filter to pass only light of about 620 m/x. Calculate as directed for the colorimeter (Shohl, A. T. and Hunter, T. H., J. Lab. & Clin. Med., 1941, 26, 1829-1837). See also earlier cell opacity method (Shohl, A. T., J. Lab. & Clin. Med., 1939-40, 25, 1325- 1332). Blood Flow, technique for local measure- ment of, using radioactive sodium (Semple, R., McDonald, L. and Ekins, R. P., Am. Heart J., 1951, 41, 803-809). Blood Grouping technique does not properly come in the scope of this book; but since it is involved in fundamental medical and biological problems the following leading reference is given: Schiff, F., and Boyd, W. C, Blood Grouping Technic. New York: Interscience Pub- lishers, Inc., 1942, 248 pp. Blood Platelets— Written by Paul M. Ag- geler, University of California Medical Center, San Francisco 22, California. November 15, 1951 — It is believed by most authorities today that the plate- lets originate from megakaryocytes, chiefly in the bone marrow but perhaps also in the lungs. The platelets are thought to be detached fragments of the cytoplasm of mature megakaryocytes. In man the platelets usually vary be- tween 2 and 3 microns in length, al- though microplatelets of less than one micron and macroplatelets as long as 25 to 50 microns have been observed. They vary in thickness from 0.5 to 1.0 micron. In rapidly fixed blood they usually assume the shape of an oval disc or lentil although a variety of forms may be encountered. In unfixed blood, even in the presence of an isotonic anticoagulant many degenerative forms may occur. These may appear shrunken or "exploded" and there may be numer- ous spinelike projections from the sur- face of the platelet. In dry smears of imperfectly fixed blood stained with Wright's stain the platelets appear to be divided into two zones: the clear blue hyalomere and the chromomere made up of purple staining granules. This separation into two zones is prob- ably an artefact produced by changes in distribution of the granular material of the platelet after leaving the circula- tion, for when blood is rapidly fixed the granules are evenly distributed through the body of the platelet. Macroplatelets found in the blood in periods of abnormal blood regeneration often take a deeper stain and the granules are coarse and do not show, even in slowly dried preparations, the clear separation between hyalomere and chromomere. Platelets are found in the circulating blood, particularly in the capillaries of the liver and lung, in the bone marrow and in the spleen, both in the sinuses and between the cells of the pulp. They are not found in the lymph or in the thoracic duct. More platelets are found in arterial than in venous or capillary blood. Physiological decreases in the plate- let count are said to occur during the first day of menstruation and increases have been found after violent exercise and following a change to a high alti- tude. An increased platelet count (thrombocytosis) may be found in in- fectious diseases, trauma, fractures, asphyxiation, surgical operations, acute blood loss, chronic myelocytic leu- kemia, Hodgkin's disease and erythre- mia. A decreased platelet count (thrombocytopenia) is the basic defect in idiopathic thrombocytopenic purpura and also occurs secondarily in certain acute infectious diseases of the blood and blood forming organs, diseases of the spleen, allergies, sensitization reac- tions to certain drugs and chemicals, and following the use of certain toxic agents such as benzol or ionizing radia- tions. The platelets are thought to survive in the circulation of the normal subject for from three to five days. There is recent evidence to suggest that in idiopathic thrombocytopenic purpura they are destroyed at a much more rapid rate. The reduction of the plate- let count in this disease had previously been thought to be due either to failure of production of the platelets or to abnormally rapid removal from the circulation by phagocytosis in the spleen. The mechanism of reduction of the platelet count in secondary thrombocytopenic purpuras may be: (1) destruction of megakaryocytes as in ionizing radiation; (2) splenic in- hibition of maturation of megakaryo- cytes as in congestive splenomegaly or; (3) a direct inhibition of maturation of megakaryocj'tes as in sensitivity reactions to drugs. The platelets are concerned in the coagulation of the blood, in retraction of the blood clot, in the formation of thrombi and in the maintenance of capillary continuity. The most signifi- cant property of platelets is the readi- ness with which they agglutinate in shed blood or when exposed to a for- eign surface. Agglutination is gener- ally followed by fusion and lysis. Direct Methods: The principle of all BLOOD PLATELETS 40 BLOOD PLATELETS direct methods of platelet counting consists in the accurate dilution of a measured quantity of blood with a suitable fluid. The diluted blood is placed in a counting chamber and the number of platelets in a circumscribed volume is counted. These methods of counting platelets involve the use of the same apparatus as is employed in the erythrocyte count. The Thoma diluting pipette is generally used. It consists of a capillary tube graduated in tenths, which opens into a bulb con- taining a glass bead. The bulb, when filled to the mark 101 above it, will hold 100 times the quantity of fluid contained in the 10 divisions of the capillary tube. The counting chamber is a heavy glass slide, with two central ruled platforms. They are separated from each other by one moat, and from elevated shoulders on each side by transverse moats. These lateral shoul- ders are so ground that a cover-slip resting on them lies exactly 0.1 mm. above each platform. A ruled area of 3 mm. by 3 mm. (9 sq. mm.) is engraved on each side. This area is divided into 9 large 1 mm. squares. The central square is divided into intermediate- sized squares of 1/25 sq.rnm., each of which is further divided into smaller squares of 1/400 sq. mm. In making a count, blood is drawn by suction into the Thoma pipette to the 0.5 mark. The blood adhering to the outside of the pipette is wiped off and the diluent is drawn in until it fills the bulb and reaches the mark 101. While drawing in the diluent, the pipette is revolved between the finger and the thumb, in order to mix the blood thor- oughly with the diluent. After the desired quantity of solution has been drawn into the pipette, it is held hori- zontally and shaken for one-half niin- ute, or it may be placed in a mechanical shaking device, in order to secure thorough mixing. The coverglass is next placed on the chamber. Several large drops of fluid are expelled from the pipette and discarded, and a small quantity of diluted blood is allowed to run under the coverglass. The plat- form should be completely covered with fluid, and none should run over into the moats. The preparation is allowed to stand 10 minutes to allow settling of the cells, then the number of platelets seen in the entire central 1 mm. ruled area is counted. This number is multiplied by 2000 [1/10 (depth) X 1/200 (dilu- tion)]. There may be errors due to uncleanli- uess of the glassware, inaccurate calibration of pipettes or chambers, imperfect dilution, failure to mix the blood and diluting fluid thoroughly, imperfect filling of the chamber and improper sampling, such as might occur if a free flow of blood is not obtained from the finger or ear when capillary blood is employed, or from improper sampling if venous blood is used. The above sources of error can be avoided by an experienced technician, so that their contribution to the inaccuracy of the platelet count is minimal. How- ever, there are additional sources of error when this method, which was originally devised for erythrocyte counting, is used for the enumeration of platelets. Olef, I. (J. Lab. & Clin. Med., 1935, 20, 416) has listed the fol- lowing: 1) clumping of the platelets during drawing of the blood into the pipette, 2) the impossibility of visualiz- ing the smaller platelets with the high dry lens, 3) the failure to visualize some of the platelets in the 100 micron space that exists between the bottom of the counting chamber and the over- lying coverglass, 4) the sticking of the platelets to the side of the pipette and to the parts of the hemacytometer, and 5) the confusion of foreign particles or precipitates with platelets. Platelet clumping, the adhesion of platelets to glassware and the occur- rence of morphological changes in the platelets can be prevented only by the immediate mixing of the blood with a diluting fluid which contains both an anticoagulant and a fixative. It is not generally appreciated that de- generative changes occur quite rapidly in platelets, even in the presence of an anticoagulant, unless a fixative is also incorporated in the diluting fluid. The anticoagulants which have been employed are sodium, potassium or ammonium oxalate, sodium citrate, sodium metaphosphate, sodium sulfate, magnesium sulfate, heparin, peptone and gelatin. Of these, 3.8 per cent sodium citrate appears to be the most satisfactory, since it is isotonic with the blood plasma, does not form crystals or precipitates with the plasma, and is not highly conducive to bacterial growth. The fixatives most commonly employed are mercury bichloride, osmic acid, solution of formaldehyde, and methyl or ethyl alcohol. Of these, the solution of formaldehj^de is prefer- able because it does not produce clump- ing of the erythrocytes, does not form protein precipitates with the plasma, and does not produce secondary changes in the platelets. In order to avoid hemolysis from the formation of formic BLOOD PLATELETS 41 BLOOD PLATELETS acid, the diluting fluid should be made fresh every week and only 38 per cent neutral formaldehyde, U.S. P. should be employed. A concentration of 0.1 per cent of formaldehyde in the dilut- ing fluid is sufficient. Higher con- centrations may hemolyze the erythro- cytes and form precipitates with the plasma. It is generally' conceded that the in- corporation into the fluid of a dye, such as brilliant cresyl blue, methyl violet, methylene blue, toluene red, crystal violet and nile blue, is superfluous, since it does not assist in the differenti- ation of platelets from artefacts and may even itself be the source of arte- facts. Diluting fluids containing no anticoagulant, such as Tyrode's or Ringer's solution, produce many arte- facts and, in addition, cause rapid disintegration of platelets unless a fixative, such as solution of formalde- hyde, is incorporated in the solution. A satisfactory diluting fluid, therefore, is the one proposed by Rees, H. M., and Ecker, E. E. (J. A. M. A., 1923, 80, 621) without the brilliant cresyl blue dye. This solution is prepared by adding 0.22 ml. of neutral formalde- hyde (38 per cent U.S. P.) to 100 ml. of 3.8 per cent sodium citrate. The solution should be kept in a well-stop- pered bottle in a refrigerator and should be filtered each time just before use. Tocantins, L. M. (Arch. Path., 1937, 23, 850) also recommends sterilizing the solution in order to reduce the num- ber of artefacts caused by bacterial contamination. Despite the use of a satisfactory dilut- ing fluid, the adhesion of platelets to each other and to the walls of the pipette is almost impossible to avoid when capillary blood is used. Drawing the diluting fluid to the 0.5 mark on the pipette and subsequently drawing the head of the column of fluid to the 1.0 mark while aspirating the blood into the pipette is unsatisfactory, since there is little opportunity for the blood to mi.\ with the diluting fluid within the narrow confines of the capillary stem of the pipette. Furthermore, even the very short time required to draw the exact quantity of blood into the pipette and to mix it with the dilut- ing fluid is sufficient in many instances to allow marked clumping of the plate- lets to occur. The clumping of plate- lets can best be avoided by mixing the blood with the diluting fluid before it is drawn into the capillary pipette. This can be accomplished by the use of venous blood. The blood may be drawn into a silicone-lined syringe and then immediately transferred to a tube con- taining the diluting fluid or, preferably, a given amount of blood is drawn into a syringe containing a measured amount of diluting fluid. Needles varying in size from No. 18 to No. 26 have been employed without any apparent effect on the quality of the prep.-iration. A satisfactory method for obtaining ve- nous blood is as follows. Place exactly 1 cc. of diluting fluid in a 5 cc. syringe. Attach a sterile dry No. 24 needle and aspirate 1 cc. of blood from the vein, without stasis, by withdrawing the plunger to the 2 cc. mark. Withdraw the needle from the vein and aspirate the blood contained in it into the syr- inge. Remove the needle and thor- oughly mix the diluted blood in the syringe and expell it into a collecting bottle. It is important to use a syr- inge whose plunger and needle fit snugly, in order to avoid the leakage of any air into the syringe while aspirat- ing the blood. A 1 to 200 dilution of the blood can be made by drawing this mixture to the 1.0 mark on the Thoma pipette and subsequently filling it to the 101 mark with the diluent. If this procedure is carried out carefully, it is unnecessary to resort to methods, such as those suggested bv Aynaud, M. (Compt. Rend. Soc. Biol., 1910 68, 1062) and Preiss, W. (Zeitsch. Ges. Exp. Med., 1932, 84, 1932) in which no attempt is made to obtain an accurate initial dilution in the syringe, and con- sequently the final dilution of blood in the counting chamber must be deter- mined b}' establishing the ratio of an erythrocyte count done on the platelet preparation with an independent eryth- rocyte count done in the usual manner. There is still another limitation in the accuracy of the direct platelet count, imposed by the relatively small concentration of platelets in the count- ing chamber. The dilution of 1 part blood in 200 parts of fluid is required because with any greater concentration the platelets would be obscured by the erythrocytes. However, since there are only appro.ximately 5 per cent as many platelets as erj^throcytes present, the statistical error is much greater than that of the erythrocyte count, even if all the platelets in the entire 1 square mm. central ruled area are counted. Attempts have been made to overcome this difficulty by increas- ing the concentration of platelets in preparations from which the erythro- cytes have been eliminated. In some methods, such as that of Brecher, G. and Cronkite, E. P. (J. Applied Phj's- iol., 1950, 3, 365) the erythrocytes are BLOOD PLATELETS 42 BLOOD PLATELETS hemolyzed. However, the use of hemo- lytic diluting fluids, such as potassium cyanide, urea, or 1 per cent ammonium oxalate is to be discouraged since hemolyzed erythrocytes may be the source of artefacts. Furthermore, if such diluting fluids contain no fixative, dissolution of some of the platelets may also occur. Another means of attaining a higher concentration of platelets in the counting chamber is to do the platelet count on plasma. In Reimann's modification of Thorn- sen's method (J. Exper. Med., 1924, 40, 553) 0.9 CO. of blood is drawn into a tuberculin syringe containing 0.1 cc. of 10 per cent sodium citrate. The needle is removed and the open end of the syringe is closed with a piece of rubber tubing fastened to the barrel with rubber bands. The plunger is removed and the syringe is placed in a vertical position. After sedimenta- tion has occurred, a 1 to 20 dilution of the supernatant plasma is made with physiologic sodium chloride solution. The platelets in the mixture are then counted in the counting chamber in the usual manner. If the platelets seen in five intermediate sized squares (80 small squares) are counted, the number is multiplied by 1000 [1/5 (area) X 1/20 (dilution) X 1/10 (depth)], in order to find the number of platelets per cubic millimeter of plasma. A further procedure has been designed to determine the number of platelets per cubic millimeter of blood. The syringe containing the blood is centrifugalized for 20 minutes at 2000 r.p.m. The relative amount of plasma and packed cells is recorded and the number of platelets per cubic millmeter of blood is determined thus: Number of platelets per cubic milli- meter of blood Number of platelets per cubic milli- meter of plasma Amount of plasma Total amount mixture of To the result, 10 per cent of the total number is added to offset the original dilution in the syringe. The determi- nation of the platelet count in plasrna by this method has the advantage of increasing the concentration of plate- lets per unit volume in the counting chamber approximately ten times. However, there is some question as to the stability and uniformity of the sus- pension of platelets in plasma during the time required for sedimentation of the erythrocytes. Furthermore, the determination of the relative plasma and packed cell volumes by the method given is inadequate, since even at a centrifugalization speed of 3000 r.p.m. at least 15 per cent of the plasma is retained in the packed cell mass. In order to avoid the errors inherent in the Thomsen method and still in- crease the concentration of platelets in the counting chamber, methods have been devised for eluting the platelets from the whole blood. In the method of Villarino and Pimentel (Am. J. Clin. Path., 1942, 12, 362) a 1 to 2 dilu- tion of venous blood with Aynaud's fluid is made in the syringe. Four cc. of pooled eluate from 0.2 cc. of this mixture are obtained by four separate centrifugations for 1 minute at 1500 r.p.m. Only an insignificant number of platelets can be recovered by further washing. A similar method has been devised by Scheff, G. I. and Ralph, P. H. (Am. J. Clin. Path., 1949, 19, 1113) using an angle centrifuge at a speed of 700 r.p.m., and dark field illumination. Although it would ap- pear that practically all of the plate- lets are recovered by these methods since only an insignificant number can be recovered by further washing and only a very few can be found in stained smears of the washed sediment, there is some question as to whether some of them may not have been destroyed during the process of repeated centri- fugalization. Regardless of the source of the speci- men, type of diluting fluid or concentra- tion of platelets per unit volume of fluid in the chamber, there are certain difficulties in differentiating platelets from artefacts when high dry magnifica- tion and bright field illumination are used. Under these circumstances, a platelet is defined simply as "a small refractile body". According to Tocan- tins, L. M. (Arch. Path., 1927, 23, 850) "Only forms from 1 to 3 microns or larger in size, rod or comma-like if seen sidewaj^s, and thin, translucent and disclike if flat on the floor of the count- ing chamber, should be counted. Gran- ules 0.8 micron in diameter or smaller, jerkily moving about more or less ac- tively, globules of oil, irregularly shaped debris floating on the upper layers of the fluid, strings of cocci, and other minute objects may be dis- tinguished from platelets after a little practice. The error of counting too few platelets may be equaled only by the error of counting every particle in the field as a platelet." That experi- enced observers differ significantly, even when using the same method, is shown by the following results of cu- taneous platelet counts on normal adult subjects, using the Rees and Ecker method. BLOOD PLATELETS 43 BLOOD PLATELETS Author Number of Subjects Mean Stand- ard Error of Mean Stand- ard Devia- tion Tocantins, L. M., Arch. Path., 1937, 23,850 40 64 80 250,000 409,000 241,000 7458 8500 68,500 68,000 50,000 Aggeler, P. M., How- ard, J. and Lucia, S. P., Blood, 1946, 1,472 Sloan, A. W., J. Clin. Path., 1951,4, 37 The author (Aggeler) is aware of the fact that the platelet counts reported by him and his associates could be higher than those observed by Tocan- tins and Sloan because certain arte- facts were consistently counted as platelets. On the other hand there is no proof that the latter authors did not mistake some of the platelets for artefacts. It is begging the point to demonstrate that any single observer may obtain consistent results. This means only that he is constant either in identifying platelets as platelets, platelets as artefacts, or artefacts as platelets. It does not prove that the particular observer is always identify- ing platelets as platelets and artefacts as artefacts. Furthermore, the fact that several observers using the same naethod may obtain reasonably con- sistent results does not prove the valid- ity of the method, since both observer may be committing the same error. The ultimate cause for the great varia- tion in the reported results of different observers using the same method is that platelets cannot be distinguished from artefacts with certainty in any method in which the magnification and resolution is not considerably better than that which can be achieved with the high dry lens and bright field il- lumination. Some improvement can perhaps be gained by the use of dark field illumination, as recommended by Scheff and Ralph (Am. J. Clin. Path., 1949, 19, 1113) or by the use of phase contrast illumination, as advocated by Brecher, G. and Cronkite, E. P. (J. Applied Physiol., 1950, 3, 365). Nothing is gained by the use of ocu- lars giving a magnification greater than lOX since, in the absence of any mprovement in resolution, the in- creased magnification only serves to increase the confusion. Attempts have been made by Helber, E. (Arch. f. Klin. Med., 1904, 81, 316) and by Maixner and Von Decostello (Med. Klin., 1915, 11, 14) to increase magnifica- tion and resolution by the use of the Zeiss D water immersion lens. With this equipment, magnification of lOOOX was attained. Approximately the same magnification with better resolution can be achieved with an oil immersion lens. However, with these lenses, it is necessary to use a specially constructed counting chamber of 0.02 mm. depth and a cover slip not more than 0.25 mm. in thickness. Because of the reduced depth, a 1 to 40 dilution of blood is used in order to obtain the same num- ber of cells per unit area of the counting chamber as are present when using a 1 to 200 dilution in the 0.1 mm. depth chamber. The author and his associ- ates have found no difficulty in dis- tinguishing artefacts from platelets with a chamber and coverslip of this type* used in conjunction with a Spen- cer binocular microscope equipped with 1.8 mm., 97X (oil immersion) N.A. 1.25, medium dark contrast phase ob- jective, standard condenser lower ele- ment, standard N. A. 1.25 condenser top element, 97X annular diaphragm and lOX Hugenian eyepieces. Even the morphologic characteristics of small platelets were clearly visible. Most of the platelets appeared as flat, round or oval discs, about 2 microns in diameter and 0.5 microns thick. The cytoplasm showed a uniform, fine granulation. Some of the platelets had a slightly irregular shape, most had a single short slightly curled filament, others had more than one filament, and a few had none. Occasionally two platelets at- tached by a long streamer of cytoplasm were observed. Rarely was an "explo- sive" degeneration form of platelet seen, and none of the platelets had the multiple sharp spinelike projections which always appear when no fixative is employed in the diluting fluid. The principal artefacts observed were bits of amorphous debris which could be distinguished by their irregular shape and heavy granulation; erythrocyte fragments, which were colored yellow, had no granulation and were intensely refractile; and clumps of bacteria, which were identified by their regularly spaced heavy granules separated by fine dark filaments. Unfortunately, while the problem of differentiation of platelets from artefacts is solved by this systern of microscopy, extreme differ- ences in the platelet counts on aliquots of the same venous blood specimen were encountered, so that the advantage of • Manufactured by the American Optical Co., Buf- falo, N. Y., through the courtesy of Mr. C. E. Guellich, Manager of Product Salea, Scientific Division. BLOOD PLATELETS 44 BLOOD PLATELETS better visualization of the platelets was nullified. The variations in the platelet count appear to be due to differences in the volume of fluid contained in the chamber caused by upward lifting or downward bending of the coverslip. Before this technique can be perfected, it will be necessary to develop methods for insuring scrupulous cleanliness of the glassware. In addition, a radical change in the design of the chamber to prevent downward bending of the cover- slip due to intense capillary attraction will be required. With regard to the established direct methods of platelet counting, there- fore, it appears that most of the sources of error can be minimized, except that of accurate identification of the plate- lets. This error can be made a constant for a given method by an experienced observer, but may lead to large in- consistent variations in platelet counts done by inexperienced individuals. Indirect Methods: One of the principal advantages of indirect methods of platelet counting is that microscopic objectives giving higher magnification and greater resolution can be employed. Furthermore, the blood can be mixed immediately with the diluting fluid and the platelets fixed before it is neces- sary for them to come in contact with any foreign surface, except perhaps momentarily with the skin or a veni- puncture needle. In all indirect methods of platelet counting unknown quantities of blood and diluent are mixed. The number of platelets seen per 1000 erythrocytes is multiplied by the number of thousands of erythro- cytes found in an independent erythro- cyte count done in the usual manner. There are three methods of determining the ratio of platelets to erythrocytes: in a counting chamber, a wet slide preparation or a dry smear. Methods, such as that of Kemp, G. T. and Calhoun, H. (Brit. Med. J., 1901, 2, 1539) in which capillary blood is em- ployed and the ratio of erythrocytes to platelets is determined in the count- ing chamber, are subject to the same error involved in identifying the plate- lets as is inherent in all direct methods of platelet counting. Methods such as those of Aynaud, M. (Compt. Rond. Soc. Biol., 1910, 68, 1062) and Preiss, W. (Zeitsch, Ges. Exp. Med., 1932, 84, 810) referred to above, in which venous blood is used are sometimes referred to as indirect methods. They are not, since in these methods, the ratio of the erythrocyte count in the platelet preparation to the erythrocyte count done in the usual manner only serves to establish the dilu- tion of blood employed in the plate- let count. The number of platelets counted per unit volume is an absolute not a relative value. The additional step of establishing the ratio of the two erythrocyte counts could be avoided by accurate measurement of the degree of dilution of the blood in the syringe. These methods are also subject to the errors involved in identifying platelets with the high dry lens in the standard counting chamber. Some of the objections to the indirect method of platelet counting in which the ratio of platelets to erythrocytes is determined in a wet preparation or a dry smear have been summarized by Tocantins (Arch. Path., 1937, 23, 850). 1) The mixture of blood and diluting solution is seldom, if ever, uniform and not the same each time. 2) Platelets and erythrocytes are not distributed evently through the preparation, since no provision is made for shaking before counting. 3) The method has defects intrinsic in any determination done indirectly, that is, in relation to another equally changeable element. 4) The greatest source of error, however, is in the assumption that platelets and erythrocytes keep an even proportion in numbers toward each other between the two main steps of the method. The markedly different physical proper- ties (adhesiveness, specific gravity, size and others) of platelets and eryth- rocytes lead to continuous changes in this ratio. 5) The proportion of plate- lets to erythrocytes varies at the same time in different portions of the circula- tion and this variation is even more marked within short spaces of time in capillaries than venules. The term "indirect platelet count" has come to imply that the count is done on capillary blood. Curiously enough it seems never to have occurred to anyone to do an indirect platelet count on accurately diluted venous blood. This would overcome some of Tocantins' objections since it would allow for a constant uniform dilution of the blood, shaking of the diluted blood before mixing and the determina- tion of the erythrocyte count on the same specimen as is used for the plate- let count. Olef (J. Lab. & Clin. Med., 1935, 20, 416) has also raised objections to the technique employed in certain indirect methods of platelet counting. In those in which a drop of blood is allowed to fall into the diluting fluid on a glass slide or contained in a special vessel (Pratt, J.H.J. A. M. A., 1905, 45, 1999). BLOOD PLATELETS 45 BLOOD PLATELETS the undiluted blood is allowed to come into contact with the surface of the skin and with the external air, thus al- lowing the platelets to clump. In methods in which the finger is punctured through a drop of fluid (Fonio, A., Deutsche Ztschr. f. Chir., 1912, 117, 176) the first drop of blood must be used. This procedure is said not to yield accurate counts because the blood contains disintegrated products of crushed and injured cells and is also diluted by an admixture of Ij'^mph. With regard to methods in which the blood and diluent are mixed on the finger, Olef stated, "This technic in- volves a number of inaccuracies. Dur- ing the process of stirring the mixture there is unavoidable contact of blood and skin with resulting destruction and clumping of platelets. If the blood is flowing freely, as it should if a correct count is to be obtained, the blood and diluting fluid form a very large drop in which it is rather difficult to obtain a uniform distribution of the blood and which frequently rolls off the finger, especially in women in whom the surface at the tip of the finger is small. Fur- thermore, freely flowing blood mixed with only one drop of preserving fluid yields preparations too thick for ac- curate platelet counting. Dameshek does not stir the blood-diluent mixture at all ; he places a drop of the preserving fluid over the puncture wound after the first drop or two of blood has been wiped away, than allows some blood to escape into the overljdng drop of diluent and by applying a cover slip to the mixture carries some of it away. This pro- cedure is inaccurate because the plate- lets, being very light, quickly rise to the surface of the drop of diluting fluid before the considerably heavier eryth- rocytes have become uniformlj^ dis- tributed. The fluid on the cover slip, therefore, contains a relatively larger number of platelets than red cells." Olef has also pointed out that all glass- ware must be scrupulously clean since hemolysis may occur with soiled glass- ware. For use in wet preparations, he advocates a 1 to 5 dilution of blood, since in very thin preparations both the platelets and erythrocytes are likely to be destroyed, whereas in thick preparations the erythrocytes may ob- scure some of the platelets. Olef's ob- jection to dilutions of blood of greater than 1 to 5 on the grounds that this maj' cause dissolution of the platelets is not valid if a fixative is contained in the diluting fluid. In Olef's method, the first drop or two of blood is wiped away. A drop of diluting fluid is then placed over the puncture wound before the blood reaches the surface of the skin, and the hand is quickly turned over so that the palmar surface is directed downward. After a sufficiently large drop has es- caped, the entire mixture is applied to the surface of a small quantity (three to four drops) of diluting fluid contained in a paraffin cup. The entire drop on the finger, consisting of approximately equal parts of blood and diluent, drops off into the cup. The contents of the cup are then stirred gently with a wooden applicator, the end of which is coated with paraffin. The mixture is allowed to stand for a minute or two, stirred again, and then is transferred by means of a clean paraffin-coated ap- plicator to a glass slide. Usually three preparations are made. A coverslip is placed over each drop and after the preparations have been allowed to stand for ten to fifteen minutes, a relative platelet-erythrocyte count is made, using the oil immersion lens. While this method has certain advantages over other indirect methods, it does not overcome the principal objection raised by Tocantins, i.e., that the platelets and erythrocytes may not keep an even proportion in numbers toward each other between the two main steps of the method. That they do not can be easily demonstrated by counting both the erythrocytes and platelets in successive microscopic fields in different parts of the same preparation. It will be found that the platelets maintain a fairly even distribution despite large differences in the erythrocyte concentration. This leads to variation in the ratio of plate- lets to erythrocytes in different parts of the same preparation. The observer must arbitrarily choose areas to be counted, but because of unconscious bias in selection he will tend to count only those which he thinks have an average distribution of erythrocytes. However, in this type of preparation it is impossible to determine what the average distribution is. This bias in sampling greatly increases the sub- sampling error. The uneven distribu- tion of erythrocytes occurs regardless of whether the blood is capillary or venous, or whether it has been thor- oughly shaken in a pipette or simply mixed with a stirring rod before the preparation is made. This maldistribu- tion of erythrocytes cannot be avoided since it is caused by physical phe- nomena which occur during the very act of placing the coverslip on the drop of blood. Dry slide preparations made from a BLOOD PLATELETS 46 BLOOD PLATELETS TABLE 1 Characteristics of various direct methods of platelet enumeration . •-» 1 ^ ■0 P4 (3 (L, Cb W w « H X w if 5 J3 (U V 4.J 3 td _ O a. M ^ .£) a c3 11 T3 a to % T3 2j a d a o 5? d K '•t-J a 'a J3 CO i r& lo" C3 (J o o 0) )-4 (J J^ 2 Pi H H P3 H > W a 1. Diluting fluids With dye X Without dye. X X X X X X X With fixative. X X X X Without fixa- tive X X X X With hemo- lytic prop- erties X Without hemolytic properties... X X X X X X X 2. Source of speci- men Capillary blood X X Venous blood. X X X X X X 3. Type of prepa- ration counted Diluted whole blood X X X X X Plasma X Eluate from whole blood X X 4. Counting cham- ber em- ployed Standard 0.1 mm. depth. X X X X X X Special 0.02 mm. depth X X 5. Microscope lens employed High dry X X X X X X X Water immersion. X 6. Microscope illu mination employed Bright field.. X X X X X X Dark field... X Phase contras t X 1 to 2 dilution of blood, stained in the usual manner, and examined with the oil immersion lens have the advantage of a somewhat more uniform distribu- tion of erythrocytes and platelets, but there is question as to whether some of the platelets and/or erythrocytes may not be destroyed during the process of making the smears. The above multiplicity of methods, and numerous other variations not men- tioned, is not the result of idle inven- tion but rather of a persistent effort on the part of each investigator to over- come the errors inherent in the methods of his predecessors. In many instances however, the successful solution of one difficulty has only served to give rise to other sources of error. The im- possibility of achieving a uniform dis- tribution of erythrocytes and platelets makes it unlikely that a satisfactory indirect method can be devised. On the other hand the inadequate visualiza- tion of platelets with the high dry lens makes all direct methods in which the standard 0.1 mm. depth counting cham- ber is employed unreliable. The only solution to the problem appears to be the perfection of a technique for using an oil immersion lens in conjunction with a chamber of 0.02 mm. depth and phase contrast lighting. There is, at the present time, no standard procedure for the enumeration of blood platelets. Numerous methods have been devised, which vary in the following manner: 1) Manner of count- ing— direct or indirect; 2) type of dilut- ing fluid — with or without a variety of dyes, fixatives and anticoagulants — with or without hemolytic properties; 3) source of specimen — capillary or venous blood; 4) material on which the count is made — whole blood, plasma or eluate from whole blood; 5) type of counting chamber — standard 0.1 mm. depth or special 0.02 mm. depth with thin coverslip; 6) microscopic mag- nification and resolution — 430X (high dry) to lOOOX (water or oil immersion) ; 7) microscopic illumination — bright field, dark field or phase contrast (see tables 1 and 2). Reports of average normal platelet counts in man have varied from 200,000 per cu. mm. (1) to 800,000 per cu. mm., "J. A. M. A., 1923,80,621. * .\rch. Path., 1937, 23, 850. « S. Gynec. et Obst., 1927, 15, 436. ^ J. Applied Physiol., 1950, 3, 365. ' Exper. Med., 1924,40, 553. /Am. J. Clin. Path., 1942, 12. 362. 9 Am. J. Clin. Path., 1949, 19, 1113. * Arch. f. Clin. Med., 1904, 81, 316. BLOOD PLATELETS 47 BLOOD SMEARS TABLE 2 Characteristics of various indirect methods of platelet enumeration u -a a a Wv. Hi -a 6 a •a a a o Q < o 'c (2 m rtpq 1. Diluting fluids With dye X X X X X Without dye. X X X With fixative. X X X Without fixa- tive X X X X X With anti- coagulant... X X X X X X Without anti- coagulant. . . X X 2. Source of speci- men Capillary blood X X X X X X X Venous blood . X 3. Type of prepa- ration counted Dry smear (stained)... . X Wet prepara- tion X X X X X Counting chamber X X 4. Microscope lens employed High dry X X X Oil immersion X X X X X 5. Microscope illumina- tion em- ployed Bright field... X X X X X X X X Dark field.... Phase contrast « Arch. Path., 1937, 23, 850. *J. A. M. A., 1923,80,621. ' J. A. M. A., 1905, 45, 1999. ''Arch. Int. Med., 1932, 50, 579. • Deutsche Ztschr. f. Chir., 1912, 117, 176. /J. A. M. A., 1921,76,427. ePrit. Med. J., 1901,2, 1539. ''Lancet, 1929, 1,992. depending upon the methods employed. The range of reported normal platelet counts in man has varied from a mini- mum of 130,000 per cu. mm. (2) to a maximum of 900,000 per cu. mm. (3) Since all methods so far devised are subject to inherent errors, there is no sound basis for a choice between the mutually exclusive standards of normal- ity which have been reported.* Blood Protein. Coagulated blood protein within the vascular lumina of stained sections of fixed tissues is an artifact in the sense that its appearance has been greatly modified by the technique. It is sometimes made up of particles of quite uniform size and has been mistaken for masses of microorganisms ; but it docs not exhibit both acidophilic and basophilic staining reactions sug- gestive of cytoplasmic and nuclear components. Blood Smears. These should be made on slides rather than on cover glasses for several reasons. A larger film of blood is thereby provided for examination. Smears on slides are easier to make and to handle. They can be studied without covering them whereas a smear on a cover glass cannot be moved about on the stage of the microscope unless it is mounted smear side down on a slide. The colors are often more permanent in smears on slides which are not covered with cover glasses. A good way is to spread a thin film of immersion oil over them. This dries much more quickly than balsam or any other medium under a cover glass. Slides of good quality with ground edges and scrupulously clean are neces- sary (Cleaning Glassware). A finger tip or ear lobule is first cleaned with 95% alcohol. As soon as the surface^ has dried a small puncture is made with a previously sterilized needle. Special needles with lance shaped cutting ends are better than ordinary pointed ones. A small droplet of blood should appear on slight pressure. The first is wiped away with sterile gauze and the second and following ones are used. Unless the blood is very strongly pressed out, the differential count of white cells will not be affected. Some advise holding the fingers in hot water beforehand to produce a temporary hyperemia in them but this is seldom advisable. A droplet of size sufficient to produce a smear of the desired thickness (determined by trials ) should be touched to the surface of a slide about 3 cm. from one end conven- iently placed on a table. Immediately the end of a second slide, with its edge • Original observations and confirmation of published data contained in tliis paragraph were done under a con- tract (No. AT-ll-l-GEN-10, Project 2), between the United States Atomic Energy Commission and the Uni- versity of California. BLOOD SPECIES CHARACTERISTICS 48 BODIAN METHOD squarely across the first slide is brought in contact with the blood on the remote side of the drop from the nearest end of the first slide. The blood spreads quickly along this edge toward the sides of the slide on the table which is steadied with the left hand. The end edge of the second slide is slowly but steadily pushed the length of the first slide and the blood is drawn out in a thin layer after it. The angle of inclination of the second to the first slide determines the thickness of the smear. It is well to make the first smear at an angle of about 45 degrees; increase it for a thicker smear and decrease it for a thinner one. In the making of smears it is important to have plenty of elbow room. To rnake good smears is a fine art and a credit to the individual. Blood smears, whether simply dried by waving in air or thereafter fixed by gently heating, retain their staining properties for a few days but they should be colored without undue delay. However they can be kept unstained or stained if protected by dipping in melted paraffin (Queen, F. B., Am. J. Clin. Path., Techn. Suppl., 1943, 7, 50). It is both wasteful and undesirable to cover the whole slide with stain. Part of the slide will have to be used for record written with a diamond pencil. Therefore draw two lines across the slide near each end with a wax pencil or a piece of paraffin. The stain added with a dropper will cover only theinter- vening part. For stains see Giemsa, Wright, Ehrlich, Oxidase, Peroxidase and Gordon's Silver Method. For electron microscopy of blood cells, see Bessis, M., Blood, 1950, 5, 1083-1098. Blood Species Characteristics. References to the literature on the blood of many different kinds of animals and data on their differential counts, total counts, hemoglobin concentrations and so on are often found of great service (Win- trobe, M.M., Clinical Hematology. Philadelphia: Lea & Febiger, 1942, 703 pp.). Blood Vessels. These comprise structures of different sorts, existing in a wide variety of environments, which can be investigated from many angles. Con- sequently to present examples of avail- able techniques under the expected headings involves a lot of mind-reading. The blood vessels of the Skin are of course the most accessible. Detailed methods for their direct and indirect study are presented by Sir Thomas Lewis (The Blood Vessels of the Skin and their Responses. London: Shaw & Sons, 1927, 322 pp.). But to microscopically examine all the blood vessels of any particular organ is not possible in the living state because of lack of accessibility, thickness and other mechanical obstacles. Resort is therefore made to various devices for viewing the vessels by themselves unobscured by surrounding tissue. The unwanted tissue is removed by corrosion when the vessels are demonstrated by Neoprene injection. It is simply passed over when x-ray photographs are examined after the vascular injec- tion of radiopaque substances like Bismuth Sulphate and Diotrast. It is rendered transparent when the vessels are filled with easily visualized materials such as Carmine or Berlin Blue, or is relatively colorless after their walls are selectively stained by Janus Green, Silver Citrate or Silver Chloride Di- chlorfluoresceinate. See red lead and glue method for blood vessels of nerves (Epstein, J., Anat. Rec, 1944, 89, 65- 69). Though the larger blood vessels are too thick and cumbersome for micro- scopic study in vivo, this is not so with the smaller ones. Indeed excellent moving pictures can be made of them. A film entitled "Control of Small Blood Vessels" by G. P. Fulton and P. R. Lutz of Boston University is very help- ful. The supravital method of studying Nerve Endings with methylene blue must be combined with careful dissec- tions (Woollard, H. H., Heart, 1926, 13, 319-336) in order to gain an impression of the innervation of blood vessels. See Arteries, Arterioles, Capillaries, Sinus- oids, Venous Sinuses, Venules, Arterio- venous Anastomoses, Veins, Vasa Vasorum, Valves, Perfusion. See Quartz Rod Technique. Bodian Method. For staining nerve fibers in paraffin sections (Bodian, D., Anat. Rec, 1937, 69, 153-162; MacFarland, W. E. and Davenport, H. A., Stain Tech., 1941, 16, 53-58). The following details of this very useful technique have been supplied by Dr. J. L. O'Leary. Fix by vascular perfusion, with 80% alcohol containing 5% formol and 5% acetic acid, or by immersion in 10% formalin or Bouin's fluid. For boutons terminaux, perfuse tissue with 10% chloral hydrate and extract tissue with alcohol for several weeks. Run paraffin sections (15/i or less) to aq. dest. Place in 1% Protargol (Winthrop Chemical Co.) with 4-6 gms. of metallic copper per 100 cc. (This can be used only once.) Wash in redistilled water 1 change. Transfer for 10 min. to : hydro- chinone, 1 gm.; sodium sulfite, 5 gm.; aq. dest., 100 cc. Wash in redistilled BOEDEKER'S METHOD 49 BONE water 1 change. Tone in 1% gold chlo- ride with 3 drops of glacial acetic acid per 100 cc, 5-10 min. Wash in re- distilled water 1 change. If sections do not have a light purple color place in 2% oxalic acid until the entire section has the slightest blue or purplish tinge. Pour off as soon as tissue gets slightly blue. Remove residual silver salts in 5% sodium thiosulfate 5-10 min. Wash, dehydrate, clear and mount. Note : the Coplin jars used must be cleaned in Cleaning Fluid. The Bodian method has been adjusted for the demonstra- tion of melanin by Dublin, W. B., Am. J. Clin. Path., 1943, 7 (Technical Sec- tion), 127. Boedeker's Method, see Enamel matrix. Bogoroch, see Radioautographic Technique. Boling, see Teeth, Decalcification. Bollinger Bodies, see Borrel Bodies. Bone. A good account of methods is provided by Shipley (McClung, pp. 344-352). Examination without decal- cification involves the cutting and grinding of thin sections. The instru- ments used by dentists for the making of sections of undecalcified teeth are of the greatest service and should be pur- chased or borrowed. If they are not available Grieves' method for dental tissues is suggested. In order to de- termine the structure of bone with organic material removed, Shipley ad- vises cutting away all soft parts after which the bone may or may not be split. Place in tap water, or in a 2% aq. gelatin, to which a loop full of culture of B. coli has been added. After 5-6 days wash in running water 24-48 hrs. in a stink cupboard. This will dissolve and wash away all organic material. Sterilize the bone by boiling or immersion in alcohol. Saw into sections, grind these to the necessary thinness and polish. De- hydrate in ether. Dry thoroughly and mount in balsam. Routine examination includes some method of fixation, de- calcification and staining. Hematoxylin and eosin are recommended, likewise phosphomolybdic acid hematoxylin and Mallory's connective tissue stain. For different structural components special techniques are reauired. Bone corpuscles may be isolated by putting a thin section of bone in concentrated nitric acid for a few hours to a day. Then place the section on a slide, cover. Pressure on the cover glass will squeeze out ellipsoidal bone cells with their processes (Shipley). Bone lamellae ca,n be peeled off easily when decalcified bone has been allowed to simmer in water for several hours (Shipley). Lacunae and canaliculi. The easiest method is to impregnate sections of ground bone with 0.75% aq. silver nitrate for 24 hours. Wash, polish the sections on a fine hone to remove preci- pitated silver, dehydrate in alcohol, clear in xylol and mount in balsam. The lacunae and canaliculi appear black in a yellowish brown background. To impregnate thin sections with acid fuchsin, dry them after extraction with alcohol. Place them in watch glasses in a 20% aq. sol. of acid fuchsin in a desiccator connected with a suction apparatus. Extract air for about an hour and close the dessicator. After 24 hrs. the solution will have dried. Rub off ppt. on a hone, pass through xylol and mount in damar or balsam (Shipley). Linings of lacunae and canaliculi. (Schmorl's method modified by Ship- ley.) Employ a fixative not containing mercury. Decalcify in Miiller's fluid, wash in running water, embed in cel- loidin and section not over 10 microns. Stain in thionin solution alkalinised by 2 drops ammonia. Transfer with glass needle to sat. aq. phosphotungstic or phosphomolybdic acid. Leave until blue, gray or green. Place in water until sky-blue. Ammonium hydroxide 1 cc. and aq. dest. 10 cc, 3-5 min. Several changes 90% alcohol. 95% ale. Clear in carbolxylol and mount in damar (or balsam). This method is suggested for bones of children. Processes of young osteoblasts in grow- ing bone. Shipley suggests following treatment of slices of bone of a rickety animal. 4% aq. citric acid 20^30 min. in the dark. Rinse in aq. dest. 1% aq. gold chloride in the dark, 20-30 min. 3% formic acid in the dark, 48 hrs. Rinse in aq. dest. and preserve in pure glycerin. Make frozen sections, mount in glycerin and ring with damar, balsam, paraffin or cement. Keep specimens in dark when not is use. To determine relative age of deposi- tion the following method has proved useful in senile osteoporosis. Saw sec- tions of bone not more than 0.5 cm. thick and fix in 4% formalin 2-4 days. Decalcify in 6% isotonic formalin, 40 cc, 85% formic acid, 60 cc, and sodium citrate, 5 gm. changing every second day for, say, a week, that is until they become flexible and can be penetrated by a fine needle. Embed in celloidin (slow method). Prepare stain by dis- solving 30 gm. potassium alum in 1 liter hot water and by adding 1.5 gm. hema- toxylin crystals. Cool and add 1 gm. chloral hydrate. Ripening in sunlight to rich dark color is hastened by addition of crystal of potassium hydroxide. Stain celloidin sections about 2 days BONE MARROW 50 BOTANICAL TECHNIQUE checking by microscopic examination until some areas are definite violet azur, others lighter or colorless. Wash in tap water 24 hrs. Stain in 100 cc. aq. dest. + 2-3 drops 1% aq. eosin 1-2 days (uncolored areas become dark rose color). Dehydrate, clear in xylol and mount in balsam. Old bone azur; new- bone bright rose (Belloni, L., Arch. Ital. Anat. e Istol. Path., 1939, 10, 622) . See Madder staining of new bone, Alizarin Red S staining of dentine, various tests for Calcium, and Ossifica- tion, Line Test for vitamin D potency. Polarized light is excellent for the demonstration of bone camellae. The micro-diffraction technique per- mits obtaining diffraction patterns of small areas such as a single Haversian system. Using it Enstrom, A. and Zetterstrora, R., Exp. Cell Res., 1951, 2, 268-274, found that the orientation of mineral salts is relatively unchanged in different physiological conditions. Bone Marrow. Microscopic examination of bone marrow in vivo has not been achieved because of the obvious techni- cal difficulties involved. The best that can be done is to study still living cells removed from bone marrow unstained or supravi tally stained. The methods are essentially the same as for blood. From humans samples can be obtained by sternal puncture (Young, R. H. and Osgood, E. E., Arch. Int. Med., 1935, 55, 186-203, and many others). Pri- mitive cells of the erythrocytic and leucocytic series can only be identified when hemoglobin and specific granules respectively appear within them. Mi- crochemical tests for Hemoglobin should be more used. For the granules the methods of Giemsa, Wright, Ehrlich and others are the best available. Special techniques have been described for Megakaryocytes particularly in rela- tion to platelet formation. The normal megakaryocyte concentration is as- pirated human bone marrow is described by Ebaugh, F. G. Jr. and Bird, R. M., Blood, 1951, 6, 75-80. To demonstrate the vascular pattern special methods are required (Doan, C. A., Johns Hop- kins Hosp. Bull., 1922, 33, 222-226). To reveal the nerve supply is par- ticularly difficult. Glaser (W., Ztsch. f. Anat. u. Entw., 1928, 87, 741-745) has described a fine network accompanying the vessels but Doan and Langworthy (Downey, p. 1852) were less successful. Sternal bone marrow during first week of life (Shapiro, L. M., and Bessen, F. A., Am. J. Med. Sci., 1941, 202, 341- 354). Bone marrow of normal adults (Plum, CM., Acta Med. Scand., 1941, 107, 11-52). See chapters by Sabin and Miller and by Doan in Downey's Hand- book of Hematology, New York, Hoeber, 1938, 3, 1791-1961 for details. A method for studying numerical and topographic problems in the whole femoral marrow of rats and guinea pigs, with the use of undecalcified sections (Mayer, E. and Ruzicka, A. Q., Anat. Rec, 1945, 93, 213-231). A technique for the quantitative estimation of mast cells in bone marrow is advanced by Mota, Ivan, Blood, 1951, 6, 81-83. Borax Carmine (Grenacher). Make con. sol. of carmine in borax (2-3% carmine in 4% aq. borax) by boiling for 30 min. Allow to stand 2-3 days with occasional stirring. Dilute with equal volume 70% ale, again allow to stand and filter. Much used for staining tissues in bulk. They are colored for days if necessary, transferred directly to acid ale. (70% ale. 100 cc, hydrochloric acid 4 drops) in which they assume a bright red trans- parent appearance. Then wash in alco- hol, mount as whole specimens or embed in paraffin and cut sections. Borax carmine can also be employed to stain sections (Lee, p. 146). Borax Ferricyanide, see Weigert's. Bordeaux, see Amaranth. Bordeaux Red (CI, 88) — acid Bordeaux, archelline 2B, azo-Bordeaux, cerasin R, fast red B, BN or P — An acid mono-azo dye very widely employed in histology. Bordeaux SF, see Amaranth. Boron, see Atomic Weights. Borrel Bodies (Bollinger bodies) in fowl pox. References to earlier staining methods and directions for applying the microin- cineration technique with figures show- ing the comparative results are given by Danks, W. B. C, Am. J. Path., 1932, 8, 711-716. See microincineration of Mol- luscum bodies (Van Rooyen, C. E., J. Path. & Bact., 1939, 49, 345-349). Borrelia Vincenti, see Vincent's Angina. Borrel's Stain. Fix in osmic acid, 2 gm.; platinum chloride, 2 gms. ; chromic acid, 3 gm. ; glacial acetic acid, 20 cc. and aq. dest., 350 cc. for 24 hrs. Wash in run- ning water several hours. Dehydrate, clear, embed and section. Stain sections in l%aq. magenta Ihr. Then in sat. aq. indigo-carmine, 2 parts and sat. aq. picric acid, 1 part. Wash in ale, dehy- drate, clear and mount. The above has been partly taken from Lee's Vade Mecum, p. 433. Other more convenient fixatives will do equally well. The stain has been used for the Borrel bodies in fowl pox. Botanical Technique. Many of the methods used in animal histology are applicable also in plant histology and vice versa. Details are given in a chapter by W. R. BOUIN'S FLUID 51 BRAZILIN-WASSERBLAU Taylor in McCIung, p. 155-245. See Plants. Bouin's Fluid. Sat. aq. picric acid, 75 cc; formalin, 25 cc; acetic acid, 5 cc. For mammalian tissues fix 24 hrs., wash in water, dehydrate and embed in the usual way. This is the most generally useful of all fixatives containing picric acid. Almost any stain can be used after it. The picric acid need not be altogether washed out because it serves as a desir- able mordant. Giemsa'a stain gives good coloration of protozoan parasites after fixation in Bouin's fluid (Cowdry, E. V. and Danks, W. B. C, Parasitology, 1933, 25, 1-63) . The use of this fixative is specified under Argentaffine Reaction, Bodian's Method, Elementary Bodies, Foot's Method, Gold, Johnson's Neu- tral Red Method, Laidlaw's Method, Liebermann-Burchardt Reaction, Mas- son's Trichrome, Purkinje Cells, Tape- worm Proglottids, etc. It is a fixative rapidly gaining in popularity and there are naturally many modifications. The application of Davenport's silver tech- nique to Bouin fixed tissues is described by Foley, J. O., Stain Techn., 1938, 13, 5-8. The cytological action of Bouin's fluid has been investigated at the University of Pennsylvania. Three formulae are particularly recommended by McClung and Allen (McClung, p. 561). (1) Allen's fluid: sat. aq. picric acid, 75 cc; formalin C.P., 15 cc. ; glacial acetic acid, 10 cc; urea, 1 gm. (2) The same plus 1 gm. chromic acid. (3) The original formula plus 2 gms. urea and 1.5 gms. chromic acid. For details regarding use in study of cell division, shrinkage, etc. see Allen, Ezra, Anat. Rec, 1916, 10, 565-589. Bourne, see Golgi Apparatus, Mitochondria. Boutons Terminaux. For this special type of nerve ending the methods given under Nerve Endings are useful, partic- ularly that of Bodian. These terminal buttons or swellings can be visualized and their behavior determined in living tadpoles by techniques introduced by Speidel, C. C, J. Comp. Neurol., 1942, 76, 57-73. Several special methods for their demonstration in fixed tissues are recommended by Gibson (McClung, 1950, pp. 389-398). Distinction be- tween normal and degenerating in the inferior olive of the cat made by silver methods (Blackstad, T., Brodal, A. and Walberg, F. Acta Anat., 1951, 11, 461-477). Bowie's Ethyl Violet-Biebrich Scarlet stain for pepsinogen granules (Bowie, D. J., Anat. Rec, 1935-36, 64, 357-367). Dis- solve 1 gm. Biebrich scarlet in 250 cc. aq. dest. and 2 gms. ethyl violet in 500 cc. Filter the former through a rapid filter paper into a beaker and then the latter into the same beaker. The end point of neutralization is when a little of the mixture placed on filter paper does not show any scarlet color. Collect the ppt. of neutral dye by filtering and dry it. To make stock solution dissolve 0.2 gm. in 20 cc. 95% alcohol. To make staining solution add 1-5 drops to 50 cc. of 20% alcohol. Stain paraffin sections of Regaud fixed gastric mucosa in this for 24 hrs. Wipe dry around edges and blot with smooth filter paper. Differ- entiate by covering section with equal parts clove oil ancl xylol. This takes about 30 min. and should be observed under microscope. Pass through several changes of xylol, rinse in benzol and mount in benzol balsam. Pepsinogen of pepsin-forming cells, violet ; and parietal cells, red. Bowie also makes a crystal violet-orange G stain which does not differ materially from Bensley's Neutral Gentian. Brandt's glycerin jelly. Melted gelatin, 1 part; glycerin I5 parts plus few drops carbolic acid to serve as a preservative. See Kaiser's glycerin jelly under gly- cerin. Bryan, see Ear Cell Smears, Nasal Cell Smears. Brazilin (CI, 1243) is a substance produced from red wood of Brazil. Its formula is like that of hematoxylin minus 1 hydroxyl group and in its use, as well as its origin, it resembles hematoxylin. Ripening may be required for both. Thus we have an iron brazilin method (Hickson, S. J., Quart. J. Micr. Sci., 1901, 44, 469-471) and O'Leary's Bra- zilin for myelin sheaths. See also Brazilin-Wasserblau technique of Bensley. Brazilin-Wasserblau for secretion ante- cedents of thyroid gland (Bensley, R. R., Am. J. Anat., 1916, 19, 37-54) as described later by the Bensleys (p. SO) is : To make up the Brazilin stain dis- solve 0.05 gm. in a little aq. dest. with aid of heat and add this to 100 cc. 1% aq. phosphotungstic acid. Ripen by addition of 2 drops hydrogen peroxide. Solution should not be employed after 3 days. Run paraffin sections of forma- lin-Zenker fixed thyroids down to aq. dest., mordant briefly in a fresh aq. ammonium stannic chloride, and stain in above solution 1 or more hrs. Wash in water and treat for 1-5 min. with aq. dest., 100 cc. + 1.0 gm. phosphomolyb- dic acid and 0.2 gm. Wasserblau (anilin blue). Wash quickly in water, dehy- drate in absolute alcohol, clear and mount. See colors in R. R. Bensley's plate. Nuclear chromatin, red; secre- BRAZILWOOD 52 BUFFERS tion antecedents in pale blue droplets; mitochondria, reddish-purple; connec- tive tissue, blue; erythrocytes, orange- red; etc. Brazilwood. The true brazilwood is of the tree, Caesalpina echinata and its varie- ties. It yields a dye stuff formerly much used after an aluminum mordant for fabrics, except silk, to which it gave a bright red color. After potassium bichromate as a mordant the color ob- tained was purple red. The term "brazil" is from the arable word "braza" meaning fiery red. Leggett writes that increased use of brazilwood in Europe resulted from the delivery of Asiatic brazilwood directly to Lisbon made possible by Vasco da Gama's dis- covery of an all water route from India around the Cape of Good Hope and, further, that three years later a Portu- guese expedition bound for India missed the mark and landed on the north east- ern bulge of South America where the voyagers found many brazilwood trees so they called the land "Terra de Brazil" (Leggett, W. F., Ancient and Medieval Dyes, Brooklyn: Chemical Publishing Co., Inc., 1944, 95 pp.). Breast, see Mammary Glands. Brilliant Alizarin Biue(CI, 931), a basic dye of light fastness 3 to 4. Gives darker color than New Methylene Blue (Emig, p. 61). Brilliant Blue C, see Brilliant Cresyl Blue. Brilliant Congo R, see Vital Red. Brilliant Congo Red R, see Vital Red. Brilliant Cresyl Blue (CI, 877)— brilliant blue C, cresyl blue 2RN or BBS- Commission Certified. This basic oxa- zin dye is used for making Platelet Counts and for many other purposes. Brilliant Dianil Red R, see Vital Red. Brilliant Fat Scarlet B, see Sudan R. Brilliant Green (CI, 662) — diamond green, ethyl green, malachite green G, solid green JJO — Commission Certified. This di -amino tri-phenyl methane dye is used to color culture media. Brilliant Pink B, see Rhodamine B. Brilliant Ponceau G, see Ponceau 2R. Brilliant Purpurin R (CI, 454). An acid dis-azo dye. Conn (p. 62) says that this is the dye which Gutstein, M., Zeit. f. Ges. Exp. Med., 1932, 82, 479-524 called "brilliant purpur R" and used as a vital stain for yeasts. Brilliant Vital Red. Use in determination of plasma volume is justified, since the dye is not taken into the erythrocytes (Gre- gersen, M. I., and Schiro, H., Am. J. Physiol., 1938, 121, 284-292). See Vital Red. Brilliant Yellow (CI, 364), an acid dis-azo dye of light fastness 3 apparently of little use as a stain for paraflRn sections. In acid solutions colors resinous tissues bright yellow, and in alkaline solutions, blue-green algae a clear yellow (Emig, p. 39). Bromcresol Green. See Hydrogen Ion Indi- cators. Bromcresol Purple. See Hydrogen Ion In- dicators. Bromine. According to Lison (p. 110) bromine has not been investigated histo- chemically in animal tissues. For its detection in plants consult Mangenot, H. G., Bull. d'Hist. Appl., 1927, 4, 52-71. Bromphenol Blue. See Hydrogen Ion Indi- cators. Bromphenol Red. See Hydrogen Ion Indi- cators. Bromthymol Blue. See Hydrogen ion Indi- cators. Bronchial Aspirates, see Papanicolaou Tech- niques. Bronchiolar Epithelium — Written by C. C. Macklin, Dept. of Histological Re- search, The University of Western Ontario, London, Canada. November 28, 1951— For the dark and light cells, as revealed by supravital silverization, see Ammoniacal Silver. For a means to demonstrate the continuation of the network of surface silver lines from the bronchiolar epithelium to the alveolar walls, in en face views, see Silver Linea- tion. Mitochondria in the "villus" cells are abundant and often apparently merged (Macklin, C. C, Anat. Rec, 1949, 103, 550; Rev. can. de Biol., 1949, 8, 328; and Proc. Instit. of Med. of Chicago, 1950, 18, 78-95— the 26th Lewis Linn McArthur Lecture). They are well demonstrated by Altviann^s method of anilin fuchsin and picric acid (which see). Tumor formation in the bronchiolar epithelium of rats that have been subjected to urethane (which see) has been described by Rosin (Cancer Res., 1949, 9, 583). Bronz Bromo, see Eosin Y. Brown Salt R, see Chrysoidin Y. Brownian Movement. Calculation of cyto- plasmic viscosity through measurement of displacement of particles in Brownian movement gives results not very differ- ent from determinations by the centrif- ugation method (Danielli in Bourne, p. 31). Brucella Ring Test, see Triphenyltetra- zolium Chloride. Buffalo Garnet R, see Erie Garnet B. Buffers. For many purposes it is essential to use solutions buffered at a certain pH. Details concerning numerous buffers are given by Clark, W. M., The Determina- tion of Hydrogen Ions. Baltimore: Williams & Wilkins, 1928, 717 pp. BUNDLE OF HIS 63 CADMIUM French, R. W., Stain Techn., 1930, 5, 87-90 (see correction, 1932, 7, 107-108) recommends Sorensen's phosphate mix- tures and Palitzsch's borax-boric acid mixtures each over certain ranges of pH. He emphasizes the fact that the addition of buffer salts is known to have a decided influence in some cases on the behavior of the dyes irrespective of pH. See also Clark and Lubs BuflFers, also Veronal Acetate BuflFers. Petrunkevitch, A., Anat. Rec, 1937, 68, 267-280 explains that aqueous solu- tions of stains at certain pH's are more selective than alcoholic ones and that the greatest differentiation is obtained with the former ones with pH suit- ably adjusted by addition of HCl or NaOH. Next in desirability come stains dissolved in acetate, phosphate and borate buffers. Citrate buffers are in his experience less suitable because a more diffuse staining results while phthalate buffers should not be used. He gives specific directions for the preparation of solutions at pH of maxi- mum staining of acid fuchsln, aniline blue, aurantia, benzoazurine, eosin Y, light green, metanil yellow, methylene blue, orange G, toluidin blue, Wrights stain and eosin methylene blue. For safranin O, see Sawyer, C. H., Stain Techn., 1940, 15, 3-7 and for hema- to.xylin, malachite green and eosin Y, Craig, R. and Wilson, C, ibid, 1941, 16, 99-109. Levine, N. C, ibid, 1940, 15, 91-112 contributes useful data on buffered stains in relation to isoelectric point of cell components. Obviously the maximum intensity of staining depends not only on pH but also on properties of substances stained and their treatment from beginning to end of the technique. Lillie, R. D., Stain Techn., 1941, 16, 1-6 employed McU- vaine citric buffers in order to improve Romanowsky staining (see Toluidine Blue Phloxinate) after various fixatives. See McJunkin-Haden Buffer. Use of buffered thionin as Nissl stain (Windle, W. F., Rhines, R. and Rankin, J., Stain Techn., 1943, 18, 77-86). For buffering in connection with silver impregnation see Davenport, H. A., McArthur, J. and Bruesch, S. R., Stain Techn., 1939, 14, 21-26; Silver, M. L., Anat. Rec, 1942, 32, 507-529. When accuracy is essential check the actual pH of the solution to which buffers have been added by the glass electrode method which anyone can learn to use in a few hours and which gives the answer very quicklv. See Hydrogen Ion Indicators. Bundle of His, see Todd, T. W., Cowdry's Special Cytology, 1932, 2, 1173-1210. Burettes. For accurate volume determina- tions use Microliter Burettes. Burns. Methods of experimental produc- tion, vital staining with trypan blue, and histological changes (Ham, A W., Ann. Surg., 1944, 120, 689-697. Butter Fat, reactions in tissue to fat stains after various fixations (Black, C. E., J. Lab. & Clin. Med., 1937-38, 23, 1027-1036). Butyl Alcohol, see n-Butyl and Tertiary Butyl. Buzaglo's Connective Tissue Stain. (Bu- zaglo, J. H., Bull. d'Hist. Appl., 1934, 11, 40-43). This method is intended to replace that of Van Gieson. Solutions required: (1) Gallocyanin (Hollborn, 2264). Boil 0.1 gm. in 100 cc. 5% aq. chrome alum for 10 min. After cooling make up to 100 cc. with aq. dest., filter and adci a little formalin to filtrate. (2) Orcein (Hollborn, 2466). Dissolve 1 gm. in 100 cc. acid alcohol (70% alcohol, 100 cc. + 1 cc. hydrochloric acid stand- ard). (3) Acid alizarin blue (Hollborn, 2559). Boil for 10 min. 5 gm. in 100 cc. 10% aq. aluminum sulphate. After cooling make up to 100 cc, filter and add formalin. (4) Alizarine-viridine (Holl- born, 2035). Dissolve 0.2 gm. in 100 cc. aq. dest. acidulated to pH 5.8 with hydrochloric acid. He advises fixation in formalin, Maximow's fluid, Susa or Hoffker (of which he does not give composition). Pass sections (presum- ably paraffin) down to aq. dest. Stain nuclei in gallocyanin as deeply as pos- sible 5 times, 24 hrs. Rinse twice in aq. dest. Stain elastic fibers in orcein, then aq. dest., 3 times, 5 min. Stain muscle in acid alizarin blue, 7 min., aq. dest. twice. Differentiate in 5% aq. phosphomolybdic acid 25-30 min., aq. dest. twice. Stain collagen in alizarine viridine 7 min. Blot with 4 layers filter paper. 95% ale 96% ale Carbol- xylol, 2 changes xylol. Balsam. Nu- clei, dark blue ; elastic fibers, red brown ; muscle and epithelium, pale blue violet ; collagen, mucus, cartilage, shades of §reen; myelin sheaths, rose; axis cylin- ers, dark blue; erythrocytes, red brown . Cabot Rings. Ring-shaped formations in erythrocytes which color red with Wright's Blood Stain. The color in the concavity of the ring is the same as that in the rest of the cell. Oc- casionally observed especially in per- nicious anemia and lymphatic leukemia. Cadmium. The chloride is employed in fi.xation of Golgi apparatus prior to silver impregnation (Aoyama, F., Zeit. wiss. mikr., 1929, 46, 489-191). See comment by Baker (Bourne, p. 19) on this and use by Ciaccio of cadmium nitrate to CAJAL'S 54 CALCIUM render phospho- and galactolipines less soluble. Bourne (p. 106) refers to Joyet-Lavergne's claim that cadmium lactate reacts with glutathione in the cell producing a cadmium glutathione compound which is microscopically visible. Cajal's. Properly the name should be listed as Ramon y Cajal. 1. Brom-formol- silver method for neuroglia. Details supplied by Dr. J. L. O'Leary. Fix small fresh pieces, 3-15 days, in: aq. dest., 85 cc; formalin, 15 cc; ammo- nium bromide, 2 gm. Cut 25/i frozen sections and return to: aq. dest., 50 cc; formalin, 6 cc; ammonium bromide, 3 gm. for 4-6 hrs. at 30-38°C. or for 8-10 hrs. at room temperature. Wash for a few seconds in aq. dest. Place in the following fluid in a porcelain dish and heat over the flame: aq. dest., 10-15 cc; ammoniacal silver oxide, 5 cc; pyridine C.P., 4-5 drops. (To prepare silver oxide solution: Take 10 cc. 10% silver nitrate, add 12 drops 40% NaOH. Col- lect the ppt., wash 5-6 times with aq. dest., then add ppt. to a beaker con- taining 60-70 cc. aq. dest. Redissolve with least quantity of ammonia neces- sary. If too much ammonia is added, results are bad.) Remove when sec- tions have reached a tobacco brown color. Wash through 2 changes aq. dest. not more than 5 sec. in all. Re- duce in 5% formalin for 2-3 min. Tone with 0.2% aq. gold chloride and fix in 5% aq. sodium hyposulfite. After washing carry to 95% alcohol, carbol- xylol, xylol balsam. See Microglia and Oligodendroglia. 2. Chloral hydrate method as de- scribed by Willard, D. M., Quart. J. Micr. Sci., 1935-36, 78, 475-485 for innervation of adrenal. Fix for 24 hrs. in : chloral hydrate, 2.5 gm. ; 95% alcohol, 40 cc. ; aq. dest., 40 cc. ; pyridine, 20 cc Wash in aq. dest. until smell of pyridine disappears. 97% alcohol, 24 hrs. Wash again in aq. dest. and transfer to 2.5% aq. silver nitrate at 37°C. for 9-12 days (longer times better for nerve cells). Wash for 1 min. in aq. dest. Reduce for 12-24 hrs. in: hydroquinone, 1 gm.; neutral formol, 10 cc; aq. dest., 90 cc. Dehydrate rapidly, embed in paraffin and cut 15-30m sections. Nerve fibers, black; background, yellow. Cajal Silver Methods. These depend mainly on silver impregnations reduced by photographic developers such as hydroquinone. They have all been very greatly improved by a preliminary fixation and in other ways and have played a leading r61e in neurology. See Ranson pyridine method and other modifications given by Addison (Mc- Clung, pp. 452-463). Many techniques spring from a combination of Cajal and Bielchowsky methods. Calcareous deposits. Vital staining with Alizarin Red S (Ham, A. W., Arch. Path., 1932, 14,613-626). Calciferol, see Vitamin D2. Calcium. There is no absolutely specific microchemical test for calcium in sec- tions. A critical account by Cameron (G. R., J. Path, and Bact., 1930, 33, 929-955) affords instructive reading. 1. von Kossa test. Sections are trans- ferred from aq. dest. to 10% silver nitrate and exposed to bright light for 30 min. or more. Wash carefully in aq. dest. Mount in glycerin, or dehydrate clear and mount in balsam. Inorganic material in most cases calcium phos- phate or carbonate is deep black. See comments of Gomori, G., J. Mt. Sinai Hosp., 1945, 11, 317-326. Test has been adapted to investigation of bone by McLean, F. C. and Bloom, W., Anat. Rec, 1940, 78, 333-359. 2. Alizarin. Sections from aq. dest. are stained in 1% aqueous alizarin S (sodium alizarin sulphonate) or in 1% alcohol tetra-hydroscy-anthraquinon (or anthrapurpurin) for an hr. or more. They are then differentiated in 1 part concentrated ammonia and 9 parts absolute alcohol. This is followed by rapid washing in acid alcohol (hydro- chloric acid 5 cc, 95% alcohol 95 cc). It may be desirable to alternate alkali and acid alcohols 2 or 3 times. Wash thoroughly in aq. dest. ; dehydrate clear and mount. The alizarin forms a fast compound with earthy salts especially calcium more easily in young than in old bones. Substances may exist in the tissues that inhibit the combination (see Bone, Madder staining). 3. Hematoxylin. This is not, as is generally supposed, a stain for calcium though it may color calcium as well as other materials when mordanted with chromium salts or alum. According to Cameron, in bone, "staining with hematoxylin is dependent on the essen- tial ground substance and the presence of certain heavy metals especially iron chromium and aluminum ; it has no direct relation to calcium salts." He thinks that areas of pathological calci- fication which stain deeply with alum hematoxylin do so because of the pres- ence of free iron. 4. Fluorescence x-radiation. Used for thin sections of undecalcified bone. It is not feasible to magnify much but the method is said to be almost specific for calcium (Dershem, E., Proc Nat. Acad. Sci., 1939, 25, 6-10). 5. Cretin, A., Bull. d'Hist. Appl., CALCOZINE RED CG 55 CANNULAS 1924, 1, 125-132 has proposed a blue color reaction with trioxymethylene and gallic acid. In comparison with calcium strontium and barium show green, mag- nesium rose and iron brownish violet. See Lillie p. 251 for details. 6. With magnesium, but free from all other minerals in muscle, by electron microscope (Scott, G. H. and Packer, D. M., Anat. Rec, 1939, 74, 17-45). 7. Sulfuric and oxalic acid. Lillie (p. 251) refers to the granular, opaque and white appearance of unstained calcium deposits. If the material is mounted under a cover glass in water and sulfuric acid is drawn through by removal of water from one side with filter paper the deposits dissolve but colorless mono- clinic calcium sulphate (gypsum) crys- tals are formed while if 5-10% oxalic acid is used typical cubic calcium oxalate crystals appear. Carbonates, on the contrary, are dissolved by run- ning through acetic acid with the forma- tion of gas bubbles. Phosphates are dissolved by the acetic acid without gas bubbles. The development of these crystalline calcium salts is of course specific. 8. Microincineration. To distinguish between the dense white ashes of cal- cium and magnesium it is suggested that a "microdrop" of 0.1 N hydrochloric acid be added, plus a similar drop of 0.1 N sulphuric acid in order to produce needle like calcium sulphate crystals (Moreau, P., Bull, histol. appl. physiol. et path, et tech. microscop., 1931, 8, 245-248). As follow up on the above microchemical methods a curvette colorimetric by Sendary, J. Jr., J. Biol. Chem., 1942, 144, 243-258; 1944, 152, 539-556 and a titrimetric technique by Sobel, A. E. and Kaye, I. A., Ind. Eng. Chem., and Ed., 1940, 12, 118-120 are possibilities. By the latter as little as 4 Mgm. of calcium can be measured in a 5 ml. burette. See Glick, p. 273. Calcozine Red CG, see Rhodamine CC. Camphor, see Sandarac. Camsal is a mixture of camphor and salol used by McClung in making San- darac. Canada Balsam, see Balsam. Canary Yellow, see Auramin. Cancer. Because cancer and other malig- nant tumors can develop in so many organs and tissues that contain inter- mitotic or reverting postmitotic cells (Cell Classification), techniques de- signed to compare the malignant cells with their non-malignant prototypes are altogether too numerous to mention. They will be found under the several tissues: Pancreas, Connective Tissue and so forth. There is no known technique which will reveal a structure or a substance in cancer cells wholly absent in normal cells of the sort from which the particu- lar cancer cells have originated. Neither can the reverse be demon- strated, that is something absent in cancer cells and present in normal ones. Available methods are only capable of demonstrating quantitative differences in properties exhibited by normal and malignant cells. Properties of cancer cells have been systematically reviewed by Cowdry, E. V., Arch. Path., 1940, 30, 1245-1274. Yet the Dopa Reaction is of service in diagnosis of Melano- carcinoma. Frequently it is helpful to excise can- cers and transplant them into other situations such as the Anterior Chamber of the Eye where they can conveniently be studied. The Tissue Culture method is of great service, likewise Motion Pictures made of cancer cells. The most recently developed line of investigation is by Radioactive Isotopes. See also Papanicolaou Techniques. Candida Albicans. Method for demonstrat- ing this parasite with fat soluble dyes in frozen sections by Fuentes, C, J. Bact., 1946, 51, 245-246. Cannulas. Glass cannulas are required for insertion into blood vessels in the Per- fusion technique. To make one of about the size for guinea pig's thoracic aorta file and break 6 mm. outside, 4 mm. inside diameter soft glass tubes into pieces about 15 cm. long. (Pyrex of this size will do. It requires a little more heating.) Take one of these, place middle in gas flame rotating it so as to heat it evenly. When fairly soft remove from the flame, draw the ends apart to a distance of about 50 cm. and hold until solid. File and break in the middle. With a little practice this will give two tubes, each tapering evenly from the 6 mm. outside diameter to about 2-3 mm. within a distance of approximately 3 cm. Next bring the tube where it has a diameter of 2-3 mm. near to a fine flame, like that of a small alcohol lamp. Let it get soft and pull just enough to produce a slight narrow- ing to be used later to prevent the thread employed to tie the cannula in the vessel from slipping. Then fracture with file and break off the thin end about 4 mm. beyond the constriction and distant from the wide part of the tube. If this break can be made at an acute angle to the length of the tube, so much the better; because then one rim of the small end of the tube will project out beyond the rest which will facilitate its insertion into the vessel to be cannu- CAPILLARIES 56 CAPRI BLUE lated. When the break is made across the tube, at right angles, the rim on one side can be ground down on a water stone so as to produce a similarly projecting lip. In either case it is necessary to remove sharp cutting edges from both ends of the cannula by smoothing in a flame. The 6 mm. wide body of the cannula should be 3-4 cm. long for con- venient attachment of rubber tube. Obviously larger cannulas are required for larger vessels. Those for Micro- injection are very much smaller, made of hard glass and do not require to be tied in. Capillaries. In living humans these can best be seen in the skin by the method of Capillaroscopy. Render the epider- mis at the root of the finger nail trans- lucent by addition of a drop of highly refractive oil and examine directly at fairly high magnification the capillary loops in the dermal papillae. It is possible to record their changes by making moving pictures through a long period of time. See review by Wright, I. S. and Druryee, A. W., Arch. Int. Med., 1933, 52, 545-575. See also Gingiva. In living mammals the most favorable site in which to watch capillaries at high magnification is in the transparent chambers of the Sandison's Technique. For shorter periods they can be studied in the displaced but living pancreas by the methods of Covell, W. P., Anat. Rec, 1928, 40, 213-223 and O'Leary, J. L., ibid, 1930, 45, 27-58. Some changes in Permeability of living capillaries are evidenced by the trypan blue capillary permeability test. If microdissection is intended and a shift to the tongues and nictitating membranes of frogs is made consult Zweifach, B. W., Anat. Rec, 1934, 59, 83-108, and Am. J. Anat., 1937, 60, 473-514. The results have been recorded in moving pictures. Supravital staining of the tissues just mentioned with janus green (Bensley, R. R., and Vimtrup, B., Anat. Rec, 1928, 39, 37-55) affords beautifully clear views of the muscular elements of arterioles grading into capillaries. See Perivascular Cells, Rouget Cells. For investigations on the topographic arrangement of capillaries arterial injec- tions with Carmine, Berlin Blue or some other easily recognizable material followed by clearing by the Spalteholz method may be helpful. When however any fluid is injected, under great pres- sure, into a fresh, relaxed tissue that can easily swell there is a chance that an exaggerated idea of the capillaries will be conveyed. In resting muscle for instance a large proportion of the capillaries are collapsed (Krogh). The structure of the endothelial capillary wall is relatively uncompli- cated. The outlines of the endothelial cells are nicely revealed in pink by the Silver Chloride Dichlorfluorescineate technique or in black by simply treating with silver nitrate. Nuclear and cyto- plasmic structure can be brought out by methods used for other tissues. Nerve fibers closely accompany most capil- laries. The existence of actual nerve endings on the wall is debated. The most convincing looking preparations of human tissues have been secured by Stohr, Ph., Zeit. f . Zellf . u. Mikr. Anat., 1926, 3, 431-448 who employed a modifi- cation by Gros of the Bielchowsky silver technique (see particularly his Fig. 2). See Sinusoids. Capillaries of brain. Lepehne-Pickworth Eeroxidase method simplified by Camp- ell and Alexander (Mallory, p. 257). Fix for 1-3 weeks in 10% formalin. To make required solution dissolve 0.1 gm. benzidine in 0.5 cc glacial acetic acid and add 20 cc. aq. dest. Dissolve 0.1 gm. sodium nitroprusside in 10 cc. aq. dest. and add benzidine solution. Add aq. dest. to 100 cc. In case a ppt. forms filter it out. Solution must be fresh. Cut frozen sections 200-300/:( and wash in aq. dest. H hr. Change to above described solution for 5 hr. at 37 °C. agitating often. Wash in aq. dest. 10 sec. and transfer to 100 cc. aq. dest. + 2-3 drops 30% hydrogen peroxide for i hr. at 37°C. shaking frequently. Wash in aq. dest. and dehydrate in 70%, 95% and absolute alcohol. Clear in xylol and mount in balsam. Blood ves- sels black in almost colorless back- ground. This method has the advantage of not involving vascular perfusion. See comparison of injection and red cell staining methods for quantitative study of capillaries of central nervous system (Drummond, S.P., Anat. Rec, 1944, 89, 93-106). Microinjection of capillaries, Chambers, R. W. and Kopac, M. J., McClung's Microscopical Technique, 1950, p. 530. Capillary Fragility Tests. Discussion (Gold- man, L. and Corrill, E. M., J. Invest. Dermat., 1945,6, 129-147). Capillary Respirometry. The development of the techniques is described bv Tobias, J. M., Physiol. Rev., 1943, 23, 51-75. A differential respirometer is described by Cunningham, B. and Kirk, P. L., J. Gen. Physiol., 1940, 24, 135-149. Whole problem is discussed by Glick, pp. 314-326. Capri Blue (CI, 876), a basic dye of light CAPSULE STAIN 57 CARBOL-THIONIN fastness 3. 0.1 gm. in 95 cc. aq. dost. + 5 cc. 5% aq. ammonium alum + 0.5 cc. acetic acid stains plant tissues blue or black. Can be employed in prefer- ence to Cyanine. Should stain well after Erythrosin (Eniig, p. 58). Capsule stain. 1. Hiss' method for smears (McClung, p. 145). Dry organisms in ascitic or serum medium on slides. Stain, slightly heated in 5-10 cc. satu- rated ale. gentian violet or basic fuchsin made up to 100 cc. aq. dest., few sec. Wash off dye with 20% aq. copper sul- phate crystals. Dry by blotting. See also: Huntoon, F. M., J. Bact., 1917, 2, 241. See Pasteurella. 2. W. H. Smith's method for sections (Mallory, p. 275). Cover deparaffin- ized sections of Zenker fixed material with Anilin Crystal Violet (either Ehrlich's or Stirling's). During few seconds warm by passing slide through flame 2 or 3 times. Wash in Gram's Iodine solution followed by formalin (commercial). Decolorize in 95% ale. Quickly wash again in Gram's iodine. Cover with aniline green eosin and heat as before. To make this shake 1 part aniline green with 200 parts 3-6% aq. eosin yellowish W.S. and after 1-2 hrs. remove ppt. bj' filtering. Wash in aq. dest. Dehydrate in 95% and abs. ale, clear in xylol and mount in balsam. Bacterial capsules, red; Gram positive bacteria, blue. Mallory says that a stronger iodine may be desirable (iodine, 1 gm., potassium iodide, 2gm. ;aq. dest., 100 cc.) and that the times must be suited to each preparation. 3. Churchman's (S. Bayne-Jones in Simmons and Gentzkow, p. 385). Flood air-dried films with Wright's stain and leave until almost evaporated to dryness. Original blue of stain is replaced by pinkish color. Wash quickly in water, or in Clark and Lubs buffer pH 6.4-6.5. Do not blot but dry with fan. Body of organisms, blue; capsular material, purplish-pink; often surrounded by capsular membrane or peripheral zone, deep purplish-pink. Capsule Substance. This obviously is un- der investigation in many sorts of cells and the methods introduced for one kind may well be of service for others. See Cell Membrane for physical proper- ties, thickness, etc. See Adhesiveness and Acid Fast Bacilli. Under Gram Stains is a description of the mechanism of their action which includes data ob- tained by use of the enzyme, ribonu- clease, on the nature of walls of Gram positive bacteria. Under Enzymes, see enzymatic destruction of capsules of pneumococci. Carbanthrene Blue GO (CI, 1113), Carban- threne Brilliant Orange RK, Carban- threne Jade Green (CI, 1101), Carban- threne Red BN (CI, 1162) Carbanthrene Red BN (CI, 1162) and Carbanthrene Violet 2R (CI, 1104) all of NAC are referred to by Emig, p. 64. Carbohydrates, see Starch. Carbol-Anilin Fuchsin methylene blue method for Negri bodies (Goodpasture, E. W., Am. J. Path., 1925, 1, 547-582). Fix in Zenker's fluid 24 hrs. (not Helly's fluid). Color for 10-30 min. in mixture made by adding 1 cc. of pure phenol and 1 cc. of anilin oil to 100 cc. of stock 0.5% basic fuchsin in 20% alcohol. Wash in running water, blot with filter paper and decolorize with 95% alcohol until sec- tions become pink. Then wash in water and stain with Loeffler's methylene blue, 15-60 sec. Wash again in water. Dehydrate and destain for few sec. in absolute alcohol, clear in xylol and mount in balsam. Negri bodies, crim- son; background, blue. Also excellent for Borrel Bodies. Carbol-Crystal Violet. Because the solu- tions as prescribed in Nicolle's original formula for carbol gentian violet tend to gelatinize, the following formula is recommended by Conn, H. J., Stain Techn., 1946, 21, 31-32: Mix solution of 0.4 gm. crystal violet C. C. in 10 cc. 95% ethyl alcohol with solution of 1 gm. phenol in 100 cc. aq. dest. Carbol-Fuchsin, The original formula of Ziehl has been much modified. Ziehl- Neelsen is sat. ale. basic fuchsin, 10 cc. ; 5% aq. carbolic acid, 90 cc. Verhoeff (F. H., J.A.M.A., 1912, 58, 1355) advises basic fuchsin, 2 gm.; abs. ale, 50 cc; melted carbolic acid crystals, 25 cc. McClung (p. 136) suggests mixing 10 cc. 3% basic fuchsin (90% dye content) with 95 cc. 5% aq. phenol. The im- portant thing is the character of the fuchsin not its concentration relative to carbolic acid. Carbol-fuchsin is em- ployed in stains for Acid Fast Bacilli. DeipoUi, G. and Pomerri, G., Mon. Zool. Ital., 1938, 49, 123-124 have ad- vised its use as follows for Nissl Bodies. Fix small pieces in 95-98% alcohol or in 10% formalin water or in physiological saline 24 hrs. or longer. Stain deparaf- finized sections 3-4 min. in carbol- fuchsin (basic fuchsin, 0.2 gm.; cone, phenol, 1 cc; 95% ale, 2 cc. ; aq. dest. 20 cc.) 2.5 cc. ; aq. dest., 100 cc. ; glacial acetic acid, 0.5 cc. Wash rapidly in aq. dest. and destain in: aq. dest., 100 cc; formalin, 1 cc. ; glacial acetic acid, 1 cc. Wash in aq. dest., dehydrate in alcohols, clear in xylol and mount in neutral balsam. Nissl bodies and nucleoli dark red, rest unstained. Carbol-Thionin, see King's. CARBOL-XYLOL 58 CARBOWAX EMBEDDING Carbol-Xylol. Xylol saturated with car- bolic acid crystals. After using it for clearing celloidin sections, wash quickly in xylol before naounting them in balsam. Carbon from inspired air occurs abundantly in lungs and bronchial lymph nodes. It may be transported to the great blood filters (spleen and liver) where it is distinguishable by its black color and by its insolubility in cone, sulphuric acid which dissolves all other body pigments. Fine suspensions of carbon are of great service as vital stains to demonstrate phagocytosis. See Hig- gins' Ink and Lampblack. Carbon Monoxide, Measurement of, see Scholander, P. F., and Roughton, F. J. W., J. Biol. Chem., 1943, 148, 551- 563, or Glick, p. 334. Carbonic Anhydrase. This can be localized in the oxyntic (or parietal) cells of the fundus of the stomach. Davenport, H. W., Am. J. Physiol., 1940, 128, 725-728; 129, 505-514 employed an adaptation of Linderstr0m-Lang's tech- nique and observed that in rats and cats the parietal cells contain 5 to 6 times as much of the enzyme as red blood cells while the peptic cells are free from it. A microspectroscopic method for demon- stration of carbonic anhydrase within erythrocytes depends on the action of methemoglobin as an indicator which changes both its color and pattern of absorption spectrum with change of pH from 6.5-9.5 (Keilin, D. and Mann, T., Nature, 1941, 148, 493-496). For data on the distribution of this enzyme in lower forms, see Blaschko and Jacobson (Bourne, p. 200). Carbonyl Compounds, water insoluble alde- hydes and ketones, see critical state- ment by Glick, pp. 69-72. Bennett, H. S. (Am. J. Anat., 1940, 67, 151-228) regarded his phenylhydrazine reaction for carbonjd compounds as indicative of ketosteroids in the adrenal cortex. Gomori, G. (Proc. Soc. Exper. Biol. & Med., 1942, 51, 133-134) however does not agree unless there is additional evi- dence. Glick is of the same opinion that in the absence of such evidence the carbonyl reactions only indicate lipid aldehyde or ketone. According to Albert, S. and Leblond, C. P. (Endo- crinology, 1946, 39, 386-400) it is plasmal- ogen instead of ketosteroid which is demonstrated by the phenylhydrazine reaction. 1. Bennett's phenylhydrzine reac- tion. Place frozen sections of fresh tissue from microtome into M/10 ace- tate buffer, pH 6.0-6.5. If sections of fixed tissue are employed place in water. Add 1% iodine in ale. drop by drop till pale straw yellow color persists. Let stand 15 min. Add 1% aq. sodium thiosulphate drop by drop till color is lost and a small amount more is added. Let stand 5 min. Wash sections re- peatedly in aq. dest. Transfer sections to buffered phenylhydrazine solution just prepared by mixing equal volumes of 2% aq. phenylhydrazine hydro- chloride and the acetate buffer and by removing oxygen by gently bubbling through carbon dioxide for 15 min. This solution is to be poured into glass- stoppered bottles so that with the sec- tions added no air bubbles remain under the stopper. Control slides are taken through the same procedures but minus the phenylhydrazine treatment. 2. Albert and Leblond's 2,4-Dinitro- phenylhydrazine reaction. Saturate 2,4-dinitrophenylhydrazine (No. 1866 Eastman Kodak Co.) in 30% ale. and bring pH to neutrality by addition of 0.2 N sodium acetate. This is the re- agent. Fix tissue in formalin neutra- lized with magnesium carbonate for 48 hrs. and wash in running water for 24 hrs. Cut frozen sections 10-15 n and transfer to 17% ale. 4 hrs. Place them in the reagent over night and wash in 17% ale. 20 min. Change to aq. dest. and mount in glycerol gelatin (see Glychrogel). Yellow color indi- cates positive reaction. 3. Seligman, A. M. and Ashbel, R. (Cancer, 1951, 4, 579-596): Frozen sec- tions, 10 to 20 M, are cut from formalin- fixed tissues and attached to slides by air-drying, after which the formalin is washed out wath several changes of water. Sections are incubated for one or more hours at room temperature in a 0-1% solution of 3-hydroxy-2-naph- thoic acid hydrazide in 50% aldehyde- free alcohol and 5% acetic acid. Re- move e.xcess reagent by washing for 2 hrs. in 50% alcohol followed by 2 hrs. in several changes of water. A blue pigment is produced at the sites of carbonyl reactivity by immer- sion in a solution prepared from equal volumes of absolute ale. and an aqueous solution containing two parts water and one part 1/15 M phosphate buffer (pH 7-2), followed by the addition of tetrazotized o-dianisidine in powered form (50 mg. for 50 cc. of solution). The development of a blue color reaches a maximum in one or two min. The sections are washed in several changes of water (acidified with a few drops of acetic acid) and are mounted in glycerogel. Carbowax Embedding — Written by Dr. H. I. Firminger, Pathology Section, National Cancer Institute, Bethesda, CARBOWAX EMBEDDING 59 CAREY'S Maryland. February 9, 1951 — Carbo- wax (Carbide and Carbon Chemical Division, 30 East 42nd Street, New- York 17, N. Y.), a water soluble wax, is an excellent embedding medium for tissues. It circumvents the long de- hydration process necessary for em- bedding in paraffin or celloidin and yet sections prepared by this method show excellent preservation of cytological de- tail. Sections of Carbowax embedded tissue are easy to cut at from 1 to 10 ^i in thickness which makes this embed- ding method well suited both for the study of minute cytological details and for routine histological examinations. In contrast to paraffin and celloidin embedded tissues, lipids can be studied in sections of the same tissue block (Firminger, H. I., Stain Tech., 1950, 25, 121-123). Any type of fixation may be used and almost all staining pro- cedures can be performed on Carbowax sections with the exception of osmic acid. Staining with osmic acid is better after the conventional methods. Carbowax is not miscible with fats and penetrates adipose tissue, brain or spinal cord only very slowly. Such tissues can sometimes be embedded after long periods of infiltration in the oven with resultant distortion or by previous removal of lipid with fat sol- vents. Other disadvantages are the difficulty in mounting ribbons on the slide because of the solubuility of the Carbowax in the aqueous bath used for floating the sections, and the difficulty of making sections stick on the slide during the staining procedure. Blank et al. (Blank, H. and McCarthy, P. L., J. Lab. and Clin. Med., 1950, 36, 776- 781) recommend placing sections on an aqueous bath containing gelatin and potassium dichromate, floating sec- tions onto a clean slide and drying thoroughly. An alternate procedure which avoids exposure to chromates (undesirable for certain staining pro- cedures) is to cover the sections with thin colloidin prior to staining. Blank has also suggested mounting ribbons directly on a slide wet with the gelatin- dichromate solution. The solution recommended by Blank et al. for floating and affixing sections to slides is made by dissolving 0.2 gm. of potassium dichromate and 0.2 gm. of gelatin in 1000 cc. aq. dest. The mix- ture is boiled in daylight for five minutes, cooled and filtered. Embedding in Carbowax ordinarily employs a mixture of Carbowax "4000" and Carbowax "1500". The exact pro- portions of the components must be varied to suit climatic conditions. Pure Carbowax "4000" may be neces- sary in hot climates. In cooler climates one can use a mixture composed of 85 gms. of Carbowax "4000" and 15 gms. Carbowax "1500". For use, the Carbo- waxes are combined in the proper pro- portions, heated to 175°C. for 30 seconds and placed in an oven at 56°C. This solution should be kept in the oven. If the temperature of the oven should drop and the Carbowax mixture solidifies, it requires reheating above 56°C. to remelt it. To embed: 1. Place fixed tissue in small Stender dish containing the above Carbowax mixture in oven at 56°C. 1-3 hrs. 2. Agitate dish and contents every 10 min. to aid penetration. All tissues, including lung tissue, should sink before further embedding. 3. After sufficient infiltration remove tissue and place in fresh mixture in another Stender dish. 4. Cover dish and place in icebox to harden at 5°C. to avoid crystal forma- tion. 5. Trim block with hot knife. 6. Block on hot object holder; then cool object holder with ice, taking care that water or ice do not come in con- tact with block. 7. Cut sections (1 to 10 n). If satis- factory ribbons are not obtained the procedure is not working properly. 8. (a) Float sections on a water bath at room temperature to which 2 drops of a detergent or the first ribbons of Carbowax have been added to prevent "explosion" of the sections by the sur- face tension. Stain sections before mounting on slide; or, float sections onto the slide from the water bath, cover carefully with a few drops of a mixture of equal parts of absolute al- cohol and ether. Pour off excess and cover with thin collodion; harden in 70% ale. or water. Stain. (b) Or, place sections on a bath con- taining 0.02 gm. % gelatin and 0.02 gm. % potassium dichromate. Float onto clean slide, permit to drj^ thoroughly and stain. Carey's method for motor end plates is an adaptation for his study of their ameboid motion (Carey, E. J., Anat. Rec, 1941, 81, 393-413) of Wilkinson's (H. J., Med. J. Austral., 1929, 2, 768-793). Modifica- tion of Ranvier's gold chloride technique. — Written by the late Dr. E. J. Carey, Dept. of Anatomy, Marquette Uni- versity School of Medicine, Milwaukee, Wis. 1. Remove any muscle from rat or chameleon from its origin to insertion while the animal is under ether ornemo- butal anesthesia. Using a very sharp CAREY'S 60 CARMINE DUSTING knife cut the muscle quickly into pri- mary pieces, 0.5 cm. long, and 0.5 cm. thick, following the long axis of the muscle fibers. Then cut the primary pieces longitudinally into thin strips 1 to 2 mm. wide. 2. Soak strips in freshly prepared filtered lemon juice for 5 to 10 min. until they become clear or translucent. Rinse in cold tap water 4 to 5 times. 3. Place strips in 1% aq. gold chloride at 30°C. using at least 10 times the volume of gold chloride solution to each volume of muscle. While muscle is in gold chloride solution, stir at least once a min. The time for the optimum im- pregnation of gold varies in the different muscles of the same animal at a rela- tively constant rate, for example, the sternocleido-mastoid muscle of the normal rat requires 16 min.; the pec- toralis major, adductors of the thigh, and biceps femoris, 13 min.; and the gastrocnemius, tibialis anterior, and the intercostal muscles 10 min. After these muscles have been in the gold chloride solution for the proper length of time, they assume a yellowish-tan color and have a firm consistency. It is highly important that this variability in the reaction of different muscles in the same animal to gold impregnation be realized. This may have been one of the factors that led to the discarding of the gold technique because it could not be rigidly standardized. 4. Pour off gold chloride solution and rinse the tissue with tap water until the water remains clear. Then place muscle in 25% aq. formic acid in the dark 16 to 24 hrs. Too little time gives incomplete reduction of the gold and too long time excessive softening and maceration. 5. Quickly rinse in tap water 5 or 6 times to remove as much of the formic acid on the surface of the muscle as possible. Even small amounts of for- mic acid in the preserving fluid may cause ultimate maceration of the tissue. 6. Store the muscles until they are teased in a mixture of § glycerine and I5 70% alcohol. (The muscles have been preserved in a good condition for teasing in this mixture for 7 years.) 7. To tease the muscle cut from one edge with a flat bladed teasing needle a piece 1 mm. thick and the full length of the muscle fiber of short muscles. The edge of the teasing needle may be flat- tened by hammering the needle after it has been placed in a Bunsen flame until the needle is red hot. Orient this strip of muscle in a drop of glycerine on a clean, 1x3 slide. Gently add a clean cover slip. Lightly press down with the teasing needles, using a gentle lateral movement at right angles to the long axis of the muscle fibers. The muscle fibers, by this means, are gently rolled out so that the preparation is one muscle fiber thick. Check with micro- scope. Such a preparation will keep without any sealing of the cover slip for at least 7 years. Any of the usual cements, however, used for glycerine mounts, may be used to make the prepa- ration permanent. We have success- fully used clarite. 8. When cross or longitudinal sections are desired reduce the gold by placing muscle in a mixture of formalin 10% for its hardening effect, and in formic acid 3% for the reduction of the gold. The gold may, likewise, be reduced by strong electric light for 16 to 24 hrs. The rou- tine method for celloidin embedding is then used. After the tissues have been cut in sections, the nuclei can be coun- terstained by various techniques. Carmalum (Mayer). Dissolve, if necessary with heat, 1 gm. Carminic acid and 10 gms. ammonia alum in 200 cc. aq. dest. Filter and to filter add 1 cc. formalin as a preservative. The tissues stained should not be alkaline (Lee, p. 141). Carmine has been very widely used as a stain. Most of the formulae for stain- ing of fixed tissues were proposed 40 or more years ago chiefly by Ranvier and Mayer. Now aniline dyes are more popular but carmine is still of great use for staining small animals m toto, for staining tissues in bulk which are later sectioned, as the best counterstain for blue vital dyes like trypan blue, as the most specific stain for Glycogen and for Mucus in the form of mucicarmine, for coloring gelatin used to inject blood vessels and as a vital stain. Karsner, H. T. and Swanbeck, C. E., J. Med. Res., 1920, 42, 91-98 employed 15-25 cc. of fairly thick suspension for intra- pleural injections in cats. At present carminic acid is available and can be employed instead of powdered carmine. The only advantage is that the acid is of more uniform composition. See Aceto- carmine (Schneider), Alum Carmine (Grenacher), Aluminum Chloride-Car- mine (Mayer), Ammonia Carmine (Ranvier), Best's Carmine for glycogen, Borax Carmine (Grenacher), Carma- lum (Mayer), Lithium Carmine (Orth), Mucicarmine for mucus, Para-Carmine (Mayer), Picro-Carmine (Ranvier). Many more carmine combinations are given by Lee (pp. 139-149). Carmine Dusting of the Lungs— Written by C. C. Macklin, Dept. of Histological Research, The University of Western Ontario, London, Canada. November 28, 1951 — Mice or other mammals are CARMINE-GELATIN INJECTIONS 61 CARR-PRICE REACTION exposed in a closed compartment to air laden with the dust of dry powdered carmine. Agitation is by fan or blast from air-main. Atropinization facili- tates entry into the lung alveoli. One hour suffices to mark the alveolar phago- cytes (phagocytic pneumonocytes — which see) with red particles. The cytological picture varies with the time elapsing after cessation of the dusting (Macklin, C. C, The Lancet, Feb. 24, 1951, 432-435). See Dust Cells. Carmine-Gelatin Injections of blood vessels. Methods have been reviewed by Moore, R. A., J. Tech. Methods, 1929, 12, 55- 58. He proposes a more accurate technique for preparation of the gelatin mass. Allow 80 gms. gelatin to take up 200 cc. cold water and heat to complete the gel . Suspend 20 gms . carmine in 100 cc. water and add ammonia until dis- solved. Mix the gelatin and carmine solutions and add 15 gms. potassium iodide to reduce gelation point to less than 25°C. Place in water bath at 25 °C. and immerse a prepared platinum electrode in it. Pass electrolytic hydro- gen from a tank over the electrode and agitate the gelatin with a motor stirrer. Read electrical potential by balancing against a standard cell. Add acetic acid cautiously until reading of voltage corre- sponds to pH 7.2. Two other techniques are listed by Moore: 1. Dissolve 40 gms. carmine in 40 cc. strong ammonia and add water. Allow to stand 12-24 hrs. and filter through paper. Boil filtrate until it is ammonia free. Precipitate the carmine as a colloidal gel by adding 95% alcohol. Filter, wash well with alcohol and dry material collected. Dissolve 2 gm. in 5 cc. water and add 5 cc. 100 percent gelatin in water thus making the product 20% carmine and 50% gelatin (Bensley, R. R., personal communication to Dr. R. A. Knouff). 2. Triturate 40 gms. carmine Merck NFIV with 40 cc. strong ammonia and add water to 200 cc . After standing 24 hrs. filter through paper. Boil filtrate down to 100 cc, add water to 200 cc. and repeat. Add 70 gms. gelatin dissolved in water and make up with water to 1 liter (MacCallum, D. B., Am. J. Anat., 1926,38, 153-175). Carmoisine, see Chromotrope 2 R. Carnoy-Lebrun fixative for insects and ticks. Equal parts chloroform, absolute alcohol and acetic acid saturated with mercuric chloride. See Slifer-King Method. Carnoy's Fluid in abs. ale, 6 parts; chloro- form, 3 parts; and glacial acetic acid, 1 part. Also known as Van Gehuchten's mixture. A very quick fixative. Do not wash in water but in 95% ale. It is employed for many purposes. See Fibrin, Foot's Method, Glycogen Neu- rofibrils. Carotene (Carotin), put green leaves in sat. aq. KOH, 1 part; 40% ethyl alcohol, 2 parts and tap water 3 parts in wide mouthed bottle with tight glass stopper to prevent absorption of CO2 from air or seal with vaseline. Keep several days in dark until tissue is yellow and fluid is green. Change pieces to aq. dest. several hours. Remove small pieces, dry on slide with filter paper. Add 1 drop cone. H2SO4. It turns green, then blue. Under microscope carotene crystals appear dark blue (Steiger, A., Microkosmos, 1941, 8, 121-122). Carotene is a precursor of Vitamin A. Carotinalbumins. Combinations of caro- tinoid pigments with protein. Rather uncommon. As an example Lison (p. 245) cites the blue carotinalbumin in the carapace of the lobster which on boiling is split into a protein and a red carotinoid. Carotinoids. Pigments which are non- saturated and nonnitrogenous hydro- carbons. Entirely different chemically from fats, they are nevertheless only present in vivo as solutions within lipoids. They generally appear yellow, orange or brown in unstained frozen sections mounted in syrup of levulose. Lison (p. 244) indicates that tissues con- taining these pigments can sometimes be embedded in paraffin, because they are only slowly soluble in cold alcohol. They are however more quickly soluble in chloroform, acetone petroleum ether and toluol. According to Lison (p. 245) they are always easily identifiable by the fact that when treated with concen- trated sulphuric acid they turn intense blue before being destroyed. Treated with solution of iodine-iodide (say Gram's, Lugol's) they give a black green or brown color. When treated with solution of chromic acid they lose their color more or less quickly. See Lipids, tabular analysis, also Carotin. Carr-Price Reaction for vitamin A. When frozen sections of liver are plunged directly into a solution of antimony trichloride in chloroform and immedi- ately examined therein mitochondria take bright blue color which fades within 30 min. (Bourne, G., Austral. J. Exp. Biol. & Med. Sci., 1935, 13, 238-249). Antimony trichloride is said not to be specific for vitamin A since it also gives blue color with carotinoid pigments (Bourne, p. 106). Sterols yield by this reaction a red color (Raoul, Y. and Meunier, R., J. Pharm. Chim., 1939, 29, 112-118). CARRUTHERS 62 CASPERSSON Carruthers, see Oxidation Reduction Poten- tial, Vitamins. Cartesian Diver Manometry, see detailed description of apparatus and technique by Glick, pp. 342-393. Cartilage. This is one of the most awkward tissues of the body to examine in the living state because of the mechanical difficulties involved in separating its component parts sufficiently thinly for examination at high magnification in approximately isotonic media. But the differentiation of cartilage in tissue cultures has been studied to advantage (Fell, H. B., Arch. f. exper. Zellf., 1929, 7, 390-412) and an account of the direct investigation of living cartilage in Sandison transparent chambers in- serted in the ears of rabbits (Clark, E. R., and E. L., Am. J. Amt., 1942, 70, 167-200) sounds very promising. The varieties of cartilage (hyaline, articular, elastic and fibrous) depend upon the quantitative and qualitative differences in the three chief components — cells, fibers and ground substance. When the cartilage is fixed to bone, which is also to appear in the sections, it is obviously necessary to employ decalcification, see Bone. Otherwise cut thin slices, 2-4 mm. thick, and fix by immersion. Fixation by perfusion is not a great help because cartilage is practically avascular. The choice of fixatives and stains will depend upon what it is desired to demonstrate. For routine purposes Zenker's Fluid is satisfactory followed by coloration of paraffin sections with Hematoxylin and Eosin or Mallory's Connective Tissue stain. But many prefer Celloidin sec- tions. Resorcin Fuchsin is recom- mended for the elastic fibers of the matrix. Since the fibers are somewhat obscured by the ground substance in hyaline cartilage dark field and polarized light may be useful as employed by Lubosch, W., Zeit. f. mikr. Anat., Forsch., 1927, 11, 67-171. A paper by Dawson, A. B., and Spark, C, Am. J. Anat., 1928, 42, 109-137 also contains useful information. If it is desired to show the Golgi apparatus in the cells follow the technique used by Fell, H. B., J. Morph., 1925, 40, 417-459. See Chondriotin Sulphuric Acid and Phos- phatase as components of cartilage. The specific staining of cartilage cells with crystal violet has been reported by Hass, G. M., Arch. Path., 1942, 33, 174-181. The characteristic basophilia of the ground substance is the basis for the following excellent method for the demonstration of cartilage in whole mounts. Van Wijhe^s methylene blue (Noback, G. J., Anat. Rec, 1916-17, 11, 292-294). This, by demonstrating cartilage in blue in transparent whole mounts, supple- ments very nicely the vital coloration of growing bone by Madder feeding or Alizarin injections. Use embryos, or bones of young animals like rats or mice, long bones, ribs, chrondocranium, etc. Fix in 10% formalin a day or more. 1% hydrochloric acid in 67% alcohol several days or a week. Same solution + 0.25% methylene blue or toluidin blue 1 or 2 weeks until thoroughly stained. De- colorize in Acid Alcohol. Change alco- hol when it becomes much colored or every 1 or 2 days. Continue until only the cartilage retains deep blue color. Wash several days in 82% ale. Dehy- drate in 95% and abs. Equal parts abs. and benzene. Benzene change twice. Leave in this or mount in xylene damar which is better than balsam because of its light color. R(^ioactive gold distribution in carti- lage (Ekholm, R., Acta Anat., 1951, Suppl. 15 to 11, 75 pp., a first rate study especially on the knee joint). Cartilaginous Skeleton of mammalian fe- tuses. A modification of the Wijhe, Lundvall and Schultze techniques used in the Department of Embryology, Carnegie Institution of Washington is given by Miller, C. H., Anat. Rec, 1921, 20, 415-419. Wash formalin fixed material over night in water plus few drops ammonia. Transfer to 70% alco- hol and leave 7-14 days changing alcohol daily for first five. Stain for 3-10 days in: toluidin blue (Grubler), 1 gm.; 70% alcohol, 400 cc; and hydrochloric acid, 4 cc. Decolorize for 7-10 days until decolorizer is but slightly tinged with the dye in: 70% alcohol, 100 cc. plus hydrochloric acid, 1 cc. Then 80% and 95% alcohol, 3 days each. Transfer to 2% potassium hydroxide, in aq. dest. and leave 2-3 days until cleared. Change to 20, 40, 60, and 80% glycerin in aq. dest. 2 days or more in each. Store or mount in pure glycerin plus few crystals of thymol. Obviously length of times depends chiefly upon size of specimen. This staining of cartilage with toluidin blue can be combined with the coloration of bone with Alizarin Red S to make very contrasty prepara- tions (Williams, T. W., Stain Techn., 1941, 16, 23-25). Carycinel Red is l-amylaminoanthraqui- none, an oil soluble dye, recommended by Lillie, R. D. Stain Techn., 1945, 20, 73-75 as a stain for fat which it colors deep red. Employ as described for Coccinel Red. Caryospora, see Coccidia. Caspersson, see Absorption Spectra. CASEATION 63 CELL MEASUREMENT Caseation (L. caseus, cheese). This change follows local Necrosis. It is charac- terized by grayish or light yellow cheesy masses of tissue which look amorphous and have lost their original structure. Identification is morphological. Almost any good staining method is satisfactory. In some cases fibrin is present. Cason, see Mallory-Heidenhain Rapid One- Step Stain for Connective Tissue. Catalase. Method for demonstration in elementary bodies of vaccine virus (Macfarlane, M. G., and Salaman, M. H., Brit. J. Exp. Path., 1938, 19, 184; Hoagland, C. L. et al., J. Exp. Med., 1942, 76, 163-173). See Holter, H. and Doyle, W. L., J. Cell Comp. Physiol., 1938, 12, 295-308. Cataphoresis. Most solid particles sus- pended in water move under electric stress. A positively charged one moves toward the cathode and a negatively charged one toward the anode. Micro- cataphoretic cells are employed to de- termine and measure the movement which obviously has an important bearing on bacterial agglutination. Electrophoresis is a better term than cataphoresis. (Holmes, H. N. in Glas- ser's Medical Physics, 257-263) see Coagulation. Cataract, see Optic Lens. Cathepsin. A method for analysis of cathepsin in lymphocytes and poly- morphonuclear leucocytes (neutro- philes) is given by Barnes, J. M., Brit. J. Exp. Path., 1940, 21, 264-275. Cebione, see Vitamin C. Cedar Oil, see Clearing, Immersion Oils and Mounting. Celestin Blue B (CI, 900)— coreine 2R— A basic quinone-imine dye employed by Proescher, F. and Arkush, A. S., Stain Techn., 1928, 3, 28-38 and by Lendrum, H. C, J. Path. & Bact., 1935, 40, 41&- 416 as a nuclear stain. Cell Classification according to manner of life. lutermitotic cells live from the mitosis which gives them birth to the mitosis by which they divide to produce two other cells. They thus cease life as individuals by division not by ageing, degeneration and death. There are 2 kinds of intermitotic cells: First, the vegetative intermitotics some of which continue a sort of vegetative life con- stituting a reservoir of undifferentiated cells on which the body can draw in some cases as long as it lives. They are found in the epidermis bone marrow and other places. Second, the differ- entiating intermitotics, which exist in series, one building up a certain degree of differentiation, which, when it di- vides, it passes on to its daughter cells. The progeny of these daughter cells differentiate still further and pass on this higher level of specialization to their successors. Good examples are myeloblasts and myelocytes in leuco- cytogenesis. But the first differentiat- ing intermitotic in any line of differen- tiation is produced by division of a vegetative intermitotic. One of the daughter cells of this division, or in some instances both daughter cells from mitosis of a dividing vegetative inter- mitotic, achieve no further differentia- tion than their parent cells, for otherwise the reservoir of vegetative intermitotics would not be maintained but would differentiate itself out of existence. Postmitotic cells, on the other hand, are cells whose lives are postmitotic in the sense that they perform their duty, age and die. They are the culminations of the various lines of differentiation. Again, two sorts are recognizable: First the reverting postmitotics, which are capable of full functional activity and usually go on to death, yet, on occasion, some of which can revert and divide. Hepatic and renal cells are examples. Second, the fixed postmi- totics, which are different insofar that they are incapable of mitosis so that aging and death is for them inevitable as for instance nerve cells of adults, sperms and polymorphonuclear neutro- phile leucocytes. In contrast with the other 3 kinds these fixed postmitotics have lost the potentiality of malignant transformation (Cowdry, E. V., Prob- lems of Aging. Baltimore, Williams & Wilkins, 1942, 626-629). Cell Components can be examined by tech- niques too numerous to list including Staining, Supravital and Vital Staining, Impregnation, Microdissection, Micro- manipulation, Microinjection, Centrif- ugation, many Microchemical Reac- tions, and Indicators by at least 6 differ- ent kinds of Microscopes. Methods for many of these components are given under Capsule Stains, Mitochondria, Zymogen, Nissl Bodies, etc. Cell Division, see Mitosis, Amitosis and series of papers on chemistry of cell division (Mauer, M. E. and Voegtlin, C, Am. J. Cancer, 1937, 29, 483-502). Cell Enlargement, see Giant Cells. Cell Injury detected by fluorescence (Herick, F., Protoplasma, 1939, 32, 527-535). See Dead Cells. Cell Measurement, The Elliptometer — Written by Dr. J. D. Hamilton, Dept. of Medical Research, University of Western Ontario, London, Canada. February 13, 1951 — The geometric shape of the section of many histological structures is circular or elliptical. The CELL MEASUREMENT 64 CELL MEASUREMENT cross sections of cell nuclei, the bound- aries of many cells such as sea urchin eggs, nerve cells, the central zone of cells undergoing division, ovarian fol- liculi, glomeruli, blood vessels and ducts, may all be described in terms of their geometrical parimeters. A quantitative measurement of the area, eccentricity, and geometric center of these structures may often be made by means of an elliptometer (Hamilton, J. D. and Barr, AL L., Stain Tech., 1948, 23, 123). Essentially the elliptometer method provides a means of fitting an ellipse of light to a camera lucida drawing of the cell or structure. Alternatively in principle, the camera lucida drawing board may be replaced by a ground glass screen and an ellipse of light of suitably controlled intensity, angle, and size matched to the structure as viewed directly in the ocular of the microscope. In both methods, by direct graphic means, the axes of their equivalent ellipse or ellipse of best fit is then determined. In the instrument a variable circular iris such as a camera diaphragm, is used to define a cone of light arising from a point source. Point sources of light provided by a zirconium or con- centrated arc lamp are quite suitable and make possible the construction of the apparatus without the use of a con- densing lens system to define a point source. The cone of light falls upon a screen upon which is mounted a grid of graph paper. An alternative to the above design uses a lens between the illuminated diaphragm and the screen. The lens provides an image of the diaphragm in the perpendicular plane of the screen. The screen maj^ be rotated by means of an attached arm which rotates in a sleeve bearing lying parallel to the hori- zontal lines of the grid. By tilting the screen out of the vertical plane it is thus possible to create a family of ellipses. The minor axes will be con- trolled and fixed by the diameter of variable diaphragm. The points at which the ellipse touches the horizontal and vertical grid lines may be deter- mined and the axes calculated by addi- tion. In practice the camera lucida draw- ing is placed flat on the screen and the elliptometer aperture and screen angle adjusted to give the best fit. The two ends of the major axes are marked on the drawing for future reference, and the grid values noted. Derived Measurements: a) Area: If A, and B, are the major and minor diam- eters of the ellipse then the area is K TT A,B,/4 n^ where K is a propor- tionately constant depending on the grid system used and the magnifica- tions entering into the camera lucida tracing. In our experience with motor nerve cells (Barr, M. L. and Hamilton, J. D., J. Comp. Neur., 1948, 89, 93) the areas determined by elliptometer and planimeter measurements agree within five per cent on an absolute basis in comparing individual cells, and the statistical parameters of populations examined by the two methods of meas- urement are of equal value when con- trol and experimental populations are compared. Area measurement using the elliptometer are more rapid and less fatiguing than planimeter measure- ments. b) Center and Foci: The center and foci of elliptic sections may be readily determined by using a rule or by graphic methods. If the plane of section is transverse, sagittal, or coronal, a refer- ence line running dorsal-ventral or antero-posterior or medio-lateral may be drawn through the center of the elliptic figure. The orientation of the ellipse or the location of inclusions or other features of the figure, may then be measured with respect to the axes of the body as a whole. By this type of orientation and definition for ex- ample, it has been possible to show that the axone hillock of motor cells of the dorso-lateral group of the ventral horn arise predominantly in the ventral medial quadrant of the cell. c) Eccentricity: The eccentricity is the ratio of the minor to the major axis B/A. The eccentricitj^ of indi- vidual sections may possess some value as a descriptive measurement. The average eccentricity of populations may be of value in following the time course of changes of shape, as for example in hypertrophy, cell division, or blastula development. d) Cell Shape and Value: Consider an ellipsoid having major, intermediate, and minor diameters, A, >B, -^ C, lying at random in space related to the plane of section. The eccentricities of sections drawn from an ellipsoidal population, homogenous in respect to shape (but not necessarily size) will range from a maximum C/B (which may be unity if the body is an ellipse of "revolution) to a minimum C/A. This distribution of eccentricities is true even if the plane of section does not pass through the center of the el- lipsoid. The eccentricity data may be used to secure an estimate of the shape and CELL MEMBRANES 65 CELLOIDIN IMBEDDING volume of the average body. Accord- ing to experience, eccentricities are placed in several classes, from 1.0 to 0.4, say, with class intervals of 0.2. From the first class, 1.0 to 0.8, an esti- mate of the eccentricity C/B can be made, since ellipses of this class are primarily of the type C, B, particularly if B and C are appro.ximately equal and somewhat less than A. Similarly eccen- tricities of the last class interval 0.6 to 0.4 will provide an estimate of C/A. Combining these two estimates the average ratio B/A may be determined. The minor axes of sections of the first and last eccentricity groups are representative of the C or minor axes of the space figure. Hence an estimate of the average value of C may be made, and using the eccentricitj^ ratios al- ready determined the average values of A and B can be calculated. The aver- age volume V equals iir ABC can then be calculated. Cell Membranes do not require any special technique for their demonstration. Al- most any good fixative will do and they can be stained a host of different colors. There is however some difference in the interpretation of what we see with the microscope. The essential component of the walls of all cells is called the plasma membrane. This conditions per- meability and its integrity is essential to the life of the cell. It is said to con- sist of a continuous layer of lipoid molecules (phosphatides, sterols, fats) not more than 2-4 molecules thick on which proteins are adsorbed, the lipoids give permeability and the proteins elasticity and great mechanical strength. The evidence is critically presented by Danielli (Bourne, pp. 68-98). He says that it is improbable that the lipoid layer is ever thicker than 10 m/x and that the whole membrane is between Ifx and 1 m/i thick. Consequently in many cases we cannot expect to visualize the plasma membrane itself directly with visible light because the theoretical limit of visibility is a particle size of 0.25m. However the position of the plasma membrane is made clear by the difference in properties of the cytoplasm which it limits and the fluid without and also in the dark field by the light reflected from its surface. In addition it is often backed internally by a thin layer of cytoplasmic cortex (ectoplasm) which is typically free from cytoplasmic granules. The plasma membrane may be supplemented externally by special membranes such as the myelin sheaths about nerve fibers. There are many special techniques for its investigation. Some are briefly referred to under Lysis, Permeability, Surface Tension and Wetting Properties, Nuclear Mem- brane, PinocytosiB. Cell Shape. The shape of epithelial cells, and of all cells for that matter, is deter- mined by perfectly definite causes. Obviously those suspended in fluid tend to be spherical (lymphocytes) unless their internal organization conditions some other shape (erythrocytes) . Con- tact with a surface generally promotes flattening on that surface. Epithelial cells are sessile. The study of their rnorphology is not complicated by mo- tility. When disposed in a single layer and subjected to lateral pressure from their neighbors they take a distinctive shape which has been analyzed in a convincing way by F. T. Lewis (Am. Scientist, 1946, 34, 357-369, and many earlier papers). In sections of the laj^er parallel to the surface it may be seen that most of the cells are six-sided, or hexagonal. They form a mosaic, the character of which can easily be re- membered by students forced to dream of the benzene "ring" with its 6 carbon atoms. By drawing many such chemi- cal symbols side by side a similar mosaic is formed. As Lewis points out, the intersections are three-raj'ed not four- rayed as might be the case if the cross- sections were squares. Mechanically this is a great advantage. When the de- ithelium is stratified provision must be made for contact with cells on all sides. Nature adheres to the same three-rayed intersection and molds the cells in that shape which provides the smallest sur- face area for closely crowded bodies. Lewis found that this could be deter- mined mathematically as a 14-sided figure and by careful reconstruction of actual cells proved that they were all primarily tetrakaidecahedral in shape. Examination of his clear illustrations will be more helpful than pages of de- scription. The same architectural prin- ciples apply to many other cell aggregates, like fatty tissue for ex- ample. No longer is the histologist justified in vaguely referring to such cells as polyhedral. Evidently in the construction of epithelial surfaces the cells are fitted together in a much more effective way than bricks in the building of a wall. Except for the reference, the above paragraph is quoted from the Second Edition of Cowdry's Histology, Philadelphia : Lea & Febiger , 1938. See technique for three-dimensional study of cell shape in plants, Holtzman, D. H., Am. J. Bot., 1951, 38, 221-234. Celloidin Imbedding. Celloidiu is a kind of generic term covering various cellu- lose compounds, nitrocellulose, soluble CELLOIDIN IMBEDDING 66 CELLOIDIN INJECTIONS gun cotton, etc., employed for imbed- ding. The collodions are solutions of pyroxylin made as specified in the U.S.P. Pyroxylin U.S.?. XI consists chiefly of cellulose tetranitrite (Merck Index, p. 465). Obviouslj'^ a purified, nonexplosive form of pyroxylin is necessary. There are several in the market of which Parlodion (Mallinck- rodt) is the one used in our laboratory. The Bensleys (p. 37) use as celloidin "RS 5 sec. low viscosity nitrocellulose 30 per cent solvent in absolute alcohol" obtained from the Hercules Powder Co., Gillespie, N. J._ To make 20% stock solution they dissolve 140 gms. nitro- cellulose in 250 cc. ether and 210 cc. absolute alcohol. This requires 4-5 days shaking occasionally. It is diluted with ether alcohol to make 10 and 5% solutions respectively. Nitrocellulose is much used especially in neurological technique. It is abbreviated L.V.N. Some advantages over "celloidin" are claimed for it by Davenport, H. A., and Swank, R. L., Stain Techn., 19^4, 9, 137-139. Celloidin imbedding is less popular than it used to be owing to certain advantages of Paraffin Imbedding rein- forced by the mania for speed. But celloidin imbedding is in some respects superior. It yields sections in which the affinity of the tissue components for dyes is often greater. Clearing of the tissue in xylol and similar fluids is not required and it need not be subjected to heat. The tissue usually shrinks less and seldom becomes so brittle. Brain specimens can easily be cut in celloidin even after long mordanting. When sections are required of large pieces of tissue in which cavities, such as the lumina of the paranasal sinuses, alter- nate with stout bony walls this method is indicated because the celloidin in the spaces gives more support than paraffin (see also Double Imbedding). The slow method, which is the best, requires for tissue slices not more than 5 mm. thick, at least 1 day each in 95% alcohol, absolute alcohol, and in half absolute and ether. This is followed by 1 day in thin celloidin (about 4% dried strips of celloidin — Parlodion, Mal- linckrodt— dissolved in equal parts absolute alcohol and ether) and 1 or more weeks in thick 8% celloidin. The tissue, with some celloidin about it, is then mounted on a fiber block, hard- ened in chloroform 1-2 hrs. and stored in 80% alcohol. Mallory (p. 60) gives the following as a rapid method. Fix thin tissue pieces 12-18 hrs. in Formalin-Alcohol. Then 95% alcohol, 2 changes, 2 hrs.; absolute alcohol, 2 changes, 3 hrs.; alcohol -ether, 3 hrs. ; thick celloidin 12-15 hrs. ; mount and harden in chloroform, 1 hr.; 80% alcohol. A still quicker technique has been proposed (Richardson, G. D., J. Tech. Meth., 1934, 13, 81) : To make celloidin solution, add 1100 cc. absolute ethjd alcohol to 8 oz. celloidin (dried in air) and leave over night. Add 1100 cc. ether. Let stand several days. It is ready when celloidin is dissolved. Fix tissue in 10% formalin, 2 hrs.; acetone, 2 hrs.; oil of cloves ^-2 hrs. or until clear; celloidin 6 hrs. at room temperature or ^-3 hrs. in water bath at 55 °C. (being careful to keep away from flame). Block and harden in chloroform j-2 hrs. Another so called hoi celloidin method is proposed with all steps in the tech- nique at an elevated temperature (Koneff, A. A., and Lyons, W. R., Stain Techn., 1937, 12, 57-59). Fix pieces not thicker than 2-3 mm. in 10% neutral formol, Bouin or Siisa. Wash in aq. dest. several changes (1 hr. each) at room temperature. Dehydrate at 50°C. 70, 80, 95 and abs. ale. 2 changes ^ hr. each. Equal parts abs. ale. and ether 1 hr. Infiltrate at 56 °C. in (1) 10% nitrocellulose (R.S. ^ second, viscosity ■ia~z^> Hercules Powder Co.) in equal parts abs. ale. and ether, 1 hr. (2) 25% in 45 cc. ale. + 55 cc. ether, over night. (3) 50% in 40 cc. ale. + 60 cc. ether 2-3 hrs. Then transfer tissue to micro- tome block moistened with ether-alcohol. Add 50% nitro-cellulose and the tissue. Harden in 2 changes chloroform during 1 hr. Then pass through 3 changes 80% ethyl alcohol and cut. The authors mention fixation in "Carnoy II" and removal of mercury with iodized alcohol in case a fixative containing mercuric chloride was employed. Obviously every precaution must be taken to avoid explosion. Store celloidin blocks in 80% ale. See special methods for imbedding Teeth and Bone. Celloidin Injections of lungs. For smaller vessels and bronchi use : acetone, 100 cc. ; celloidin, 4 gm.; and camphor, 3 gm. For larger vessels and bronchi employ : acetone, 100 cc, sheet celloidin, 20 gms., and camphor, 15 gm. In place of sheet celloidin old x-ray films can be used if first the emulsion is removed by washing in warm water and they are then dried and cut into strips. If colors are desired employ oil paints. If Roentgenograms are to be made of the corrosion specimens add 10-12% sodium iodide or barium sulphate to a 30% suspension. In case of the vessels wash out blood first by CELLOIDIN SECTIONS 67 CENTRIFUGATION forcing physiological saline solution into vena cava thence through right heart and via pulmonary arteries to lungs evacuating by pulmonary veins. Allow injected lung to stand in running water over night thus hardening celloidin. Immerse in concentrated hydrochloric acid to digest away tissues leaving celloidin cast. This usually takes 24 hrs. Wash thoroughly in gentle stream of water. Mount dry or mount wet in solution made up as follows : Boil for 10 min. 100 cc. aq. dest. + 20 cc. glycerin. When cool add formalin to 2% and filter until clear (Marquis, W. J., J. Tech. Methods, 1929, 12, 59-64). See illus- trations of Marquis and arrangement of pressure bottles. A celluloid corrosion technique for the kidney is described by N. W. Baker, J. Tech. Methods, 1929, 12, 65-68. Celloidin Sections. Cut side of celloidin block to smooth plane surface. Moisten this and surface of microtome block holder with alcohol-ether. Add drop thick celloidin. Press together, harden in chloroform and cut in 80% alcohol on a sliding microtome with knife at an angle. Keep surface of knife and block wet with 80% alcohol from overhead dropping bottle. (A method has been described for treating block with cedar oil and cutting dry with rotatory micro- tome, Walls, G. L., Stain Techn., 1936, 11, 89-92). Sections are usually cut at a thickness of 10-16 p. (It is possible to arrange the sections serially but it is a tedious business. If serial sections are needed, paraffin should be selected in place of celloidin.) The sections un- mounted can be stained without remov- ing the celloidin after which they are to be dehydrated and cleared before mount- ing. The object is not to remove the celloidin but to soften it. The following mixture is recommended by Lee (p. 108) in place of xylol, toluol or benzol : creo- sote, 40 cc. ; Bergamot oil, 30 cc. ; xylol, 20 cc. and origanum oil, 10 cc. Cellosolve is ethylene glycol monoethyl ether. It mixes with water, acetone, alcohol, ether and dissolves many oils, waxes, etc. Employed by Lendrum (A.C., J. Path. & Bact., 1939, 49, 590- 591). Cellulose, microchemical reaction for. Solu- tion A : Dilute 20 cc. of 2% iodine in 5% aq. potassium iodide with 180 cc. aq. dest., add 0.5 cc. glycerin and mix by shaking. Solution B : Saturate 15 cc. aq. dest. with lithium chloride at 80°C., cool and use supernatant solution. Tease out section or fibers. Apply 2-3 drops "A" by glass rod and leave 10 sec. Blot with filter paper and dry. Add drop "B", cover and examine. Cellu- lose blue, green, yellow depending upon its source (Post, E. E. and Laudermilk, J. D., Stain Techn., 1942, 17, 21-26. See Polysaccharides. Cements. W. C. Tobie (in Simmons, and Gentzkow, p. 356) gives two useful types : Vacuum wax for ordinary vacuum seals not subjected to high temperature is made by melting together equl parts of beeswax and rosin. It is pliable and easily removed with hot water. Acid resisting cement is made by mixing asbestos powder and sodium silicate solution (water glass) into a paste of desired consistency. Will dry in 24 hrs. For ringing specimens mounted in glycerin, etc. see Kronig's Cement and Mounting Media. Centigrade temperature to Fahrenheit 1. Above 0°C. multiply by 9, divide by 5, add 32. Example: 37°C. = 37 X 9 = 333 -^ 5 = 66.6 -f 32 = 98.6°F. 2. Between -17.77 and 0°C. multiply by 9, divide by 5 subtract from 32. Ex- ample: -12°C. = 12 X 9 = 108 -T- 5 = 21.6; 32 - 21.6 = 10.4°F. 3. Below -17.77°C. Multiply by 9, di- vide by 5, subtract 32. Example: -18°C. = 18 X 9 = 162 -^ 5 = 32.4 - 32 = 0.4°C. Central Body, see Centrosome. Centrifugation. To even sketch in outline the techniques that come under this heading is difficult because the centrifu- gation of so many materials and tissues is helpful and the instruments vary from simple hand driven machines to power- ful ultracentrifuges which may weigh several tons and which certainly require experts to care for them. See Svedberg, T. and Pedersen, K. O., The Ultra- centrifuge, Oxford, Clarendon Press, 1940, 478 pp. The centrifuge has long been of help in the displacement of certain com- ponents of cells (especially marine eggs) in order to determine their functional r61es. It has also proved invaluable in the investigation of cytoplasmic and nuclear Viscosity, which see. In recent years centrifugation has opened a new chapter in microchemistrj' by the part which it has played in the collection of cellular components in sufficient volume for analysis. Pioneer work was done with the liver. The Bensleys (p. 6) give instructions which are in part as follows. First perfuse the abdominal organs of a guinea pig with about 1000 cc. 0.85% aq. sodium chloride (see Perfusion). This removes a good deal of the blood. Excise liver and grind up thoroughly in a mortar. Place the resulting thick fluid in large centri- CENTRIFUGE MICROSCOPE 68 CENTROSOMES fuge tubes, add about twice the volume of 0.85% aq. sodium chloride and balance the tubes with more as may be necessary. If complete separation of mitochondria is desired centrifuge for 1 min. at 3000 r.p.m. which results in stratification. In first and lowest stratum, at the bot- tom of the tubes, will be found liver cells, cell debris and connective tissue ele- ments ; in the second, nuclei and red blood cells; in the third mitochondria and small cell fragments; and in the fourth and uppermost, free fatty drop- lets. The materials in any of these layers can then be collected by drawing up in a pipette, suspended again in salt solution and purified by further cen- trifugation. For the isolation of ellipsin (structural protein) and mitochondria see Bensley, R. R. and Hoerr, N. L., Anat. Rec, 1934, 60, 251-266 and 449-455. Since it is in the mitochondrial fraction resulting from centrifugation that vitamin A is found the Goerners have greatly ex- tended the usefulness of the method in a series of studies on tumors (Goerner) A., J. Biol. Chem., 1937-38, 122, 529- 538 and A., and M. M., ibid, 1939, 128, 559-565). The technique has been further improved by Claude (A., Sci- ence, 1938, 87, 467-468 ; Cold Spring Har- bor Symposia on Quantitative Biology 1941, 9, 263-270) who used 18000 r.p.m. See, particularly, standardized tech- niques in his 1941 paper. Beams, H. W. and King, R. L., Anat. Rec, 1940, 76, 95-101, and in a series of other papers, have greatly contributed to the use of ultracentrifugation in the solution of biological problems. See Lucas, A. M., Am. J. Path., 1940, 16, 739-760 on intranuclear inclusions. Centrifuge Microscope. By this ingenious combination of microscope and cen- trifuge it is possible to observe living cells with the highest dry objectives while they are actually being cen- trifuged. Cells or organisms to be examined are placed in isotonic media of appropriate density in special slides constructed so that the centrifugal force derives them into approximately the focus of the objective. The clear- ness of the 2 dimensional image is not conditioned by the speed of rotation. The slide is fixed into the centrifuge head remote from the axis of rotation. Strong light is focussed by condensing lens from above onto the slide. A prism below the slide in the centrifuge head directs the light toward the axis of rotation directly through an objective. When received at the axis of rotation it is directed upward by reflecting prisms into an ocular in position above the center of the centrifuge head. Not only can stages in displacement of intra- cellular components be watched but permanent records are easily made in the form of motion pictures. Since its introduction by Harvey and Loomis in 1930 several structural improvements have been achieved. A commercial design is made by Bausch and Lomb Optical Company (Harvey, E. N., in Glasser's Medical Physics 1944, 147). Centriole, see Centrosome. Centrosomes (G. Kentron, center; soma, body), sometimes called a "central body", is a minute spherule which is a dynamic center of some sort involved in cell division. It is sometimes called a centriole though Conklin (Cowdry's General Cytology, pp. 542 and 544) says that a central body, the centriole, appears within the centrosome during mitosis. When the centrosome is double, that is consists of two minute bodies side by side, it is designated a diplosome. About the centrosome, or diplosome, there is usually a clear area which is known as a centrosphere. The centro- some, or centriole plus the clear area is called the cytocentrum. For terminology see Wilson, E.B., The Cell. New York: Macnaillan Co., 1925, 1232 pp. For functional significance see Fry, H.J., Biol. Bull., 1929, 57, I3I7I5O. Giant centrospheres in degenerating cells are described by Lewis and Lewis (Cow- dry's General Cytology, p. 427) and multiplication of centrioles in striated muscle tumors by Wolbach, E. B., Anat. Rec, 1928, 37, 255-273. Centrosomes are not easily demon- strated in tissue sections. The tech- nique originally used by Heidenhain (Arch. f. mikr. Anat., 1894, 42, 665) ap- pears to be the best. It consists of fixation in a Sublimate Acetic, or Sub- limate Alcohol Acetic, and of staining the sections 24 hours in a dilute aq. sol. of Bordeaux red or of anilin blue fol- lowed by iron hematoxylin in the usual way. The centrosomes are stained black or gray with a tinge of red or blue. In glandular epithelial cells look for them in the cytoplasm between the nucleus and the lumen. To reveal centrosomes in non-dividing nerve cells is difficult, probably because 1 they are seldom present. Hatai (S., J. Corap. Neurol., 1901, 11, 25) was able to stain them in certain nerve cells of adult rats. He fixed in sat. mercuric chloride in formalin, 30 cc; glacial acetic acid, 50 cc. and physiological salt solution, 15 cc. for 6-12 hrs., then washed, 4-5 hrs. in running water, im- bedded in paraffin, stained in sat. aq. toluidin blue or thionin, dehydrated, CEPHALIN 69 CHAMPY-KULL'S METHOD cleared and mounted the sections. Rio Hortega (P., Trab. Lab. Invest. Biol. Univ. Madrid, 1916,14, 117)has obtained beautiful silver preparations of centro- somes. Addison (McClung, 1950 p. 378) advises fixation in Flemming's Fluid or in Allen's chromic-urea modification of Bouin's Fluid followed by staining with Heidenhain's Iron Hematoxylin. A detailed investigation of the effects of a great many fixatives on the mitotic figure in chaetopterus eggs has been made by Fry (Fry, H.J. Biol. Bull., 1933, 65, 207-237). He concluded (1) that acetic acid, picric acid, formaldehj'de and alcohol and certain combinations of them are most useful as fixatives (2) that anesthetics like chloroform and ether and inorganic fixatives are to be avoided; (3) that the fixatives must be diluted to about 10% of the original con- centration with aq. dest. or better with sea water. Comparable information for human tissues is lacking. Cephalin, a phosphatide, is a compound of phosphoric acid, glycerol, 2 fatty acid molecules and amino ethyl alcohol. It differs also from lecithin in being only very slightly soluble in alcohol, see Lipoids. Cerasin R, see Bordeaux Red. Cerasln Red, see Sudan HL Cerebrosides are galactosides, that is com- pounds of fatty acid, galactose and sphingosine, without phosphorus, sol- uble in benzene, pyridine and hot alcohol and almost insoluble in ether, see Lipoids. Cerebrospinal Fluid. Total cell count is best made in a Fuchs-Rosenthal count- ing chamber. In making smears for the differential count it may be neces- sary first to add a little albumin fixa- tive to the slides to get the cells to stick (C. J. Lind in Simmons and Gentz- kow, p. 91). Ceresin Imbedding. Ceresin is purified ozokerite, a mixture of hydrocarbons, with melting point 61-78°C. used as a substitute for beeswax and for other purposes. Waddington, C. H. and Kriebel, J., Nature, 1935, 136, 685 ad- vise for hard objects like feathers addi- tion of ceresin to a paraffin of slightly lower melting point than that usually employed. The whole, when cooled, has a very fine texture. See the methyl benzoate celloidin ceresin method of 'Espinasse for imbedding hard objects in a suitable condition for sectioning as described by Lee (p. 96) and Waterman, H. C, Stain Techn., 1939, 14, 55-62. Ceresin can be obtained from Shell Oil Co., melting point, 82-85°C. Cerium, see Atomic Weights. Ceroid. This is a wax like endogenous pig- ment (G. keros, wax -f eidos, resem- blance). It is greenish yellow to golden brown in color, fluorescent, acid fast, stains with fat dyes as well as with methyl green, and is relatively insoluble in fat solvents. Ceroid has oeen sepa- rated from the tissues of rats, collected en masse and analyzed by Moore, T. and Wang, Y. H. (Brit. J. Nutr., 1947, I, 53-64). Mason, K. E. and Emmel, A. F. (Anat. Rec, 1945, 92, 33-59) con- sider its demonstration in muscle to be reliable indication of vitamin E deficiency. Grenados, H., Mason, K. E. and Dam, H. (Acta Path., 1947, 24, 86-95) present evidence in rats of a metabolic relationship between vitamin E and unsaturated fatty acids. A help- ful summary of the distribution of ceroid pigment in human tissues is supplied by Pappenheimer, A. M. and Victor, J. (Am. J. Path., 1946, 22, 395-412). The properties of ceroid pig- ment in relation to experimental necro- sis in rats and mice, as revealed by many histochemical techniques, are presented by Lee, C. S. (J. Nat. Cancer Inst., II, 339-347). The important point is made that there are easily noticeable differences between the ceroids of these two species. An interesting technique for the study of ceroidlike substances is to produce them in vitro and in vivo from certain lipids and erythrocytes as has been done by Hartroft, W. S. (Science, 1951, 113, 673-674). Cerotine Ponceau 3B, see Sudan IV. Cerulein MS (CI, 783) — Anthracene Green, Coerulein MS — a mordant dye of light fastness 3 to 4 gives unsatisfactory coloration of animal tissues. Direc- tions for plants (Emig, p. 55). Cervical Swab-Smears, see Paparnicolaou Techniques. Cesares-Gil flagella stain evaluated, Thatcher, L. M., Stain Techn., 1926, 1, 143-144. Cesium, spectrograph c analysis of, in retina (Scott, G. H. and Canaga, B., Jr., Proc. Soc. Exp. Biol. & Med., 1939, 40, 275). Cestoda, see Parasites, Taenia. Cevitamic Acid, see Vitamin C. Chalkley ratio method for determining rela- tive volumetric composition of tissue sections (Chalkley, H. W., J. Nat. Cancer Inst., 1943, 4, 47). Chambers, see Micromanipulation. Champy-KuH's Method of anilin fuchsin, toluidine blue and aurantia for mito- chondria. Fix in Champy's fluid (3% potassium bichromate, 7 cc. ; 1% chromic acid, 7 cc. ; 2% osmic acid, 4 cc.) 24 hrs. Wash in aq. dest. Place in pyrolig- neous acid, 1 part and 1% chromic acid, 2 parts 20 hrs. Wash aq. dest. 30 min. ; CHAMPY'S FLUID 70 CHLORAZOL PAPER BROWN B mordant 3% aq. potassium bichromate, 3 days. Wash running water 24 hrs., dehydrate, clear, imbed and section at 4ju. Remove paraffin from sections. Stain with anilin acid fuchsin (acid fuchsin 10 gms., anilin water 100 cc.) heated over spirit lamp and allow to cool 6 min. Rinse in aq. dest. Counter- stain in 0.5% aq. toluidine blue 1-2 min. Rinse inaq. dest., then 0.5%aurantiain 70% alcohol 20-40 sec. Differentiate in 95% alcohol, dehydrate, clear and mount. Mitochondria red, nuclei blue and ground substance yellow. Champy's Fluid is 3% potassium bichro- mate, 7 parts ; 1% chromic acid, 7 parts ; and 2% osmic acid, 4 parts. It is an excellent fixative for cytologic details. Charcot-Leyden's Crystals. Octahedral phosphate crystals found in stools of persons infected with Endameba his- tolytica and in a variety of other condi- tions. See description and illustration by Craig, p. 58. Cheese. Bacteria in, see Hucker, G. J., N. Y. Agric. Exp. Sta. Tech. Bull. 1921,87(McClung, p. 147). Chelidonium, see Fluorescence Microscopy, shows "secondary" fluorescence. Chicago Blue, see escape from venules after intravenous injection (Smith, F. and Rous, P., J. Exp. Med., 1931, 54, 499- 514). Chick, see Chorioallantoic Membrane. China Blue, see Anilin Blue. Chitin. This is a keratoid, or keratin-like, substance which is an important part of the exoskeleton and hard parts of invertebrates (G. chiton, a coat). It is soluble in hot concentrated sulphuric and hydrochloric acids; relatively in- soluble in alcohol, ether, dilute acids and alkalies. There are several color reactions for chitin. Lillie (p. 147) applies to chitin, thoroughly washed after treatment with potassium hydrox- ide, 33% aq. zinc chloride to which 3-5 drops cone. aq. iodine potassium iodide have been added to each 10 cc. Chitin becomes brown on the surface, violet within. Chitin is colored red violet by a solution containing: iodine, 50 mg., potassium iodide 50 mg., calcium chloride 16 gm. and aq. dest. 4 cc. 1. A method for softening of chitin in formalin fixed insects (Murray, J. A., J. Roy. Micr. Soc, 1937_, 57, 15). Fix primarily in 10% formalin in 0.8% aq. sodium chloride, or indefinitely. Fix sec- ondarily and dehydrate in equal parts absolute alcohol, chloroform and glacial acetic acid + corrosive sublimate to satu- ration (about 4%). Warm together equal parts chloral hydrate and phenol until they fuse and form an oily liquid which is fluid at room temperature . Leave speci- mens in this 12-24 hrs. or longer. Clear in chloroform, xylol or carbon disul- phide. Imbed in paraffin. 2. According to Hennings (see Lee, p. 597) fixation of insects in the following mixture softens the chitin sufficiently to permit the making of paraffin sections: nitric acid, 16 cc. ; 5% aq. chromic acid, 16 cc; sat. corrosive sublimate in 60% alcohol, 24 cc; sat. aq. picric acid, 12 cc; and abs. ale, 42 cc. Fixation is 12-24 hrs. followed by washing in iodine alcohol. An older method is to soften chitin by treatment with a solution of hypochlorite of soda (Lee, p. 249). See Diaphanol, N. Butyl Alcohol, In- sects, and Ticks. Chloral Hydrate, as a fixative for peripheral nerves (Bank, E. W. and Davenport, H. A. Stain Techn., 1940, 15, 9-14). Chloral hydrate is also recommended as a macerating medium for the separation and isolation of epithelial and lining cells by the Bensleys (p. 5). Accord- ing to their instructions remove small pieces alimentary tract of pithed or freshly killed frog and leave them in 5% aq. chloral hydrate 12-48 hrs. Then tease with fine needles and examine. See Cajal's chloral hydrate method. Chlorazol Black E (CI, 581) of British Dye- stuffs Corporation — Erie black G X 00 (National Aniline and Chemical Com- pany), Pontamine black E (I. E.Du Pont deNemours & Co.) — an acid poly-azo dye. First described as a new biological stain by Cannan (H. J., Nature, 1937, 139, 549). Review of its uses (Cannan, H. J., J. Roy. Micr. Soc, 1941, 61, 88-94). As a vital dye (Baker, J. R., Nature, 1941, 147, 744). Stains chro- matin black, cytoplasm greenish gray after Zenker fixation (Darrow, M. A. Stain Techn., 1940, 15, 67-^8). As an acetocarmine auxiliary stain for chro- mosomes (Nebel, B. R., Stain Techn., 1940, 15, 69-72) . As a simple connective tissue stain (Levine, N. D., and Morril, C. C, Stain Techn., 1951 16, 121-122) it is excellent for arterial elastic tissue. Chlorazol Blue 3B, see Trypan Blue. Chlorazol Fast Pink used as anticoagulant in experiments designed to influence growth of transplants of lymphosar- comas (Williams, W. L., Cancer Re- search, 1946, 6, 344-353). Toxicity as an intra vitam stain is presented by Wil- liams, W. L. and Hodge, H. C, Anat. Rec, 1943, 87, 181. Chlorazol Paper Brown B. A sat. aq. sol. of this dye has been employed to stain plant tissues. It is differentiated in 1% aq. nitric acid. Acetone is used for dehydration and Euparal for mount- ing (Verdcourt, B., Stain Techn., 1947, 22, 155-156). CHLORAZOL PINK Y 71 CHLORIDE Chlorazol Pink Y, see Thiazine Red R. Chloride. In 1908 Macallurn reviewed the older literature and described his silver test for chloride (Macallum, A. B., Ergeb. d. Physiol., 1908, 7, 552-652). The possibility, which has not yet been finally answered, is that at some stage in the technique there is a shift in the position of chloride. The mere applica- tion of the silver reagent may conceiv- ably withdraw chloride from the cell. For these reasons prior treatment of the tissue by the Altmann-Gersh freezing and drying method which reduces the chance of movement of chloride to a minimum is recommended. 1. Gersh (Gersh, I., Anat. Rec, 1938, 70, 311-329) gives details of the proced- ure on which the following instructions are based. Tissues frozen in liquid air, dried in vacuum, embedded in paraffin and sectioned at 15m are mounted near one edge on chemically clean large cover slips by simply pressing down with a finger, just melting over a flame and pressing down again. Immerse cover- slips with attached sections in anhydrous petroleum ether (b.p. 20-40°C.) freshly distilled over sodium in a watch glass covered by another at all times except during actual manipulations. This re- moves the paraffin. Remove and burn off the ether quickly by a flame and allow to cool to room temperature. Then treat two coverslips with attached sections differently. A. Cover for few seconds with drop of 60% aq. silver nitrate diluted with sufficient quantity of cone, phosphoric acid to prevent precipitation of rather large concentrations of phosphates and then saturate with silver chloride. After filtering 2-3 drops aq. dest. are added to every 10 cc. before using. B. Cover similarly with: 60% aq. silver nitrate saturated with silver phos- phate and silver chloride and dilute after filtering in the same way. Decant fluids from both coverslips. Add to each 1 drop chemically pure glycerin and mount with section plus glycerin down on chemically clean slides. Expose both to carbon arc radiation for same length of time but at a distance not to warm the specimens. Examine immediately the reduced silver by direct illumination or in the dark field. A. shows specifically only the chloride and B. the same amount of chloride plus maximal concentrations of phosphate and some carbonate. 2. Dichlor fluorescein method (Bens- ley, R. D. and S. H., Anat. Rec, 1935, 64, 41-49). For the lung of a rabbit. Inject 1% aq. dichlorfluorescein intra- venously until the animal becomes quite yellow. Then kill it and inject 10% aq. silver nitrate or Silver Citrate solution either intratracheally or di- rectly into the lung substance by a hy- podermic syringe until the lung is moderately distended. In about 20 min. the color reaction reaches its maximum. The silver chloride becomes pink owing to adsorption of the dichlor- fluorescein on the positively cliarged silver chloride molecule. Then fix pieces of lung in 10% neutral formalin and make frozen sections. Examine immediately for best color reaction. Dehydrate the sections, clear in absolute alcohol and iso-safrol and mount in bal- sam. The color reaction is not perma- nent but is masked and finally lost by the browning and blackening of the sil- ver. It is not a true microchemical test; but it does detect the presence of chlorides though they are mobilized by the silver and tend to move to the per- iphery of the cell. The alveolar epi- thelial cells are outlined by pink stip- pling and their cytoplasm is also stippled and the nuclei are richly stippled. Mesothelial and endothelial cells are brilliantly and completely outlined in pink. The technique was first sug- gested by David M. Ritter. The location of chloride is a matter of great importance. Lowry, O. H. and Hastings, A. B. in Cowdry's Problems of Ageing, Baltimore: Williams & Wil- kins, 1942, 936 pp. cite the following as evidence for the extracellular position of chloride in skeletal muscle : (1) Direct microscopic studies show- ing that chloride is exclusively extra- cellular (Gersh, I., Anat. Rec, 1938, 70,311-329). (2) Perfusion experiments showing that chloride can be removed without apparently affecting the intracellular phase (Amberson, W. R. et al.. Am. J. Physiol., 1938, 122, 224-235). (3) Variations in amount of chloride and in acid base balances of tissues can only be accounted for by assuming an extracellular position for chloride (Hastings, A. B. and Eichelberger, L., J. Biol. Chem., 1937, 117, 73-93). (4) Isolated tissues equilibrated in vivo against solutions of varying chloride concentrations retain chloride in pro- portion to the concentration in the medium but at a very much lower level (Fenn, W. O., Cobb, D. M. and Marsh, B. S., Am. J. Physiol., 1934, 110, 261- 272; Eggleton, M. G. and P. and Hamil- ton, A. M., J. Phvsiol., 1937, 00, 167- 182). (5) Conclusion that in many tissues for all practical purposes all radioactive sodium and radioactive chloride remain CHLOROPHENOL RED 72 CHLOINESTERASE outside the cells (Manery, J. F. and Bale, W. F., Am. J. Physiol., 1941,132, 215-231; Manery, F. W. and Haege, L. F., ibid, 134, 83-93). See, however, Heilbrunn, L. V. and Hamilton, P. G., Physiol. Zool., 1942, 15, 363-374 for demonstration of chloride in muscle fibers. If chloride is always extracellular in all tissues it is possible accurately to meas- ure the amount of extracellular fluid and a new chapter in histochemistry is opened. Lowry and Hastings give an example. If rat muscle is found to contain 10.5 milliequivalents of chloride per kilogram of tissue and the serum of the same animal 105.2 milliequivalents of chloride per kilogram of serum water, in view of the Donnan effect on chloride distribution it can be calculated that a kilogram of extracellular fluid contains 109.7 milliequivalents of chloride. Con- sequently the sample of muscle contains 10 5 rrr^ X 1000 = 96 gms. of extracellular fluid per kilogram. When the extra- cellular fluid contains coUagenic and elastic fibers, collagen and elastin must be determined and the necessary correc- tions made as well as for blood and fat when these are present. When the in- tracellular phase is chiefly composed of a single type of cell as in skeletal or cardiac muscle the further evaluation of intracellular components is not diffi- cult. Taking every known precaution, evidence can apparently be collected of the relative composition of extracellular and intracellular phases. If it is desired to determine chloride in very small amounts of fluid, as in the analysis of glomerular urine, a method described by Westfall, B. B., Findlev, T. and Richards, A. N. (J. Biol. Chem., 1934, 107, 661-672) is sug- gested. Glick (p. 200) says this per- mits chloride determination in a frac- tion of a m1 of fluid containing 1 Mgni. or less of the choride. The technique of Sendroy, J. (Jr., J. Biol. Chem. Chem., 1942, 142, 171-173) is adequate for 10 (mI of serum. There are in addi- tion several titrimetric methods for chloride which should likewise be con- sidered as possibilities. See Glick, pp. 281-283. Chlorophenol Red. See Hydrogen Ion Indi- cators. Chlorophyll. The green pigment of plants is a mixture of 2 substances chlorophyll a and b, of which many derivatives are known. In man several fluorescent chlorophyll porphyrins are identifiable in feces and urine. A detailed account of chlorophyll is provided by Rothe- mund. P., in Glasser's Medical Physics, 1944, 154-180. Chloroplasts. Isolation and collection en masse from spinach leaves by centri- fugation (Menke, W., Zeit. f. Physiol. Chem. , 1938-39, 257, 43. See Glick. Chloroprene, see Neoprene. Chlorothymols, as preservatives of gelatin, glues, starches, etc. (Law, R. S., J. Soc. Chem. Ind., 1941, 60, 66). Chocolate Blood Agar, see Bacteria, Media. Cholesterol (esters) = cholesterides. In unstained frozen sections mounted in syrup of levulose they show no color of their own; but the Liebermann-Bur- chardt Reaction in frozen sections of formalin fixed tissue is positive. Digi- tonine Reaction in similar sections yields a complex in which the esters, if present, will color with Sudan III and lose birefringence in polarized light. See Lipids tabular analysis, see Schultz test for cholesterol and its esters. Technique for determination in buffer is given by Nieman, C. and Groot, E. H., Acta Physiol, et Pharmacol. Neerland., 1950, 1, 488-501. Cholesterols (free). In unstained frozen section mounted in syrup of levulose, they show no color of their own. Lie- bermann-Burchardt Reaction in frozen sections of formalin fixed tissue is posi- tive : blue, purple or violet then becom- ing green. Digitonine Reaction in simi- lar sections yields strongly birefringent cr3-stals and rosettes which do not stain with Sudan III. See Lipids, tabular analysis. Choline. See Florence's Reaction for Semi- nal Stains. Choline Deficiency. Use of fluorescence microscopv in (Popper, 11. and Chinn, H., Proc. Soc. Exp. Biol. & Med., 1942, 49, 202-204). See Vitamin B complex. Cholinesterase — Written bj' E. W. Demp- sey, Dept. of Anatomy, Washington University, St. Louis, February 26, 1951 — Important since it is implicated in nervous transmission. It is present in high concentration in the neuromuscu- lar junction and in the electric organ of eels, and has also been detected in the axis cj4inder of nerves. Anfinsen, C. B., O. H. Lowry and A. B. Hastings (J. Cell, and Comp. Physiol., 1942, 20, 231-237) have developed a method whereby the same section of rat brain cortex can be stained for microscopic examination and thereafter used for enzyme measurement. Recently, a number of techniques have been pro- posed for its detection on slides. Go- mori, G. (Proc. Soc. Exp. Biol, and Med., 1948, 68, 354-358) reported that long-chain fatty acid esters of choline were hydrolyzed by tissue sections, and CHONDRIOSOMES 73 CHOR'S MODIFICATION that the fatty acids could then be visualized. However, hydrolysis of such substrates is slow and probably caused by a non-specific esterase. Koelle, G. B. and J. S. Friedenwald (Proc. Soc. Exp. Biol, and Med., 1949, 70, 617-622) proposed the use of acetyl- thiocholine iodide as a substrate, and obtained beautiful localizations within the central nervous system. These au- thors have also differentiated between the true and pseudocholinesterases by means of specific inhibitors (J. Nat. Cancer Inst., 1950, 10, 1364). Seligman, A. M., M. M. Nachlas, L. H. Man- heimer, O. M. Friedman and G. Wolf (Ann. Surg., 1949, 130, 333-341) have proposed a naphthyl derivative of ace- tylcholine as a substrate. After hydro- lysis, the naphthyl moiety is visualized by diazotization. Chondriosomes, see Mitochondria. Chondriotin Sulphuric Acid. Present in cartilage and bone, stains metachro- matically with basic dyes, described in detail by Lison, L., Arch, de biol., 1935, 46, 599-668. See Muccproteins. Chorioallantoic Membrane. 1. Vital stain- ing of virus lesions in membrane (Cooke, J. V. and Blattner, R. J., Proc. Soc. Exp. Biol. & Med., 1940, 43,255-256). Place 1 cc. 0.5% aq. trypan blue directly on membrane through window in shell. Rotate egg gently and return to incu- bator, 10-30 min. Small lesions require longer time to stain than large ones. Remove membrane, wash it gently in physiological saline and fix flat in 10% formalin, a few minutes. Make up glycerin jelly by soaking 5 gms. gelatin in 44 cc. aq. dest. Then add 50 cc. glycerin and 1 cc. phenol. Heat gently and stir. Flatten membrane on a 2 X 2.5 in. slide, warm glycerin jelly to about 70°C. Add drop by drop to mem- brane until well covered. Flame a cover glass and apply with slight pressure until it has begun to set. Remove hardened jelly around edges and seal with balsam. Foci of virus increase are sharply marked by clumps of deep blue stained cells. 2. Cultivation of microorganisms. The membrane has been shown to be an excellent medium for the cultivation of viruses by Goodpasture, E. W., Wood- ruff, A. M. and Buddingh, G. J., Am. J. Path., 1932, 8, 271-282 and many others. Its usefulness has been extended to Rickettsiae and spirochetes by Good- pasture, E. W., Am. J. Hyg., 1938, 28, 111-119, to fungi by Moore, M., Am. J. Path., 1941, 17, 103-125 and to acid- fast bacteria by Moore , M . , Am . J . Path . , 1942, 18, 827-847. This method of inocu- lation has the advantage over laboratory animal inoculation in that lesions will develop in the former within 5-8 days as compared to weeks or months in the latter; most organisms will produce defi- nite and usually characteristic lesions in the chick membrane, whereas they may have no effect on experimental animals, often requiring human subjects; and because the lesions are so readily visible and traceable the chlorioallantois serves well as a means of virulence deter- mination. The technique is essentially that of Goodpasture and Buddingh (E. W.and G. J., Am. J. Hyg., 1935, 21, 319-360) with some slight changes. Fertile eggs are incubated 12 days in an electrical thermostat-controlled incubator regu- lated to maintain a temperature of 98°F. The eggs are turned twice daily. A cm. square window is cut in the shell above the embryo, exposing the chorioallantoic membrane. The position of the em- bryo is determined by candling. The membrane is then inoculated directly with the fungus and the window is cov- ered with a sterile coverslip and sealed with a paraffin-vaseline mixture (9 parts vaseline, 1 part paraffin). After inocu- lation, the eggs are set in a bacteriologic incubator and maintained at a tempera- ture of approximately 33°C., without turning. The membrane is watched daily through the window. When the inoculated area has shown marked change, the shell is cut below the window and the membrane exposed. The chorioallantois is cut with a pair of fine curved-end scissors, removed, fixed in Zenker's solution (with 5% glacial acetic). After washing, dehydrating, clearing in xylol, and imbedding in paraffin, it is sectioned and stained. Various staining techniques can be used depending on the organism inoculated. In general, for fungi, Loeffler's meth- ylene blue and eosin have given satis- factory results. For experimental tech- nique of growing mouse sarcoma in chorio-allantoic membrane, see Jacoby, F. , McDonald, S. and Woodhouse.D. L., J. Path, and Bact., 1943, 55, 409-417. Chor's Modification of Ranson's pyridine silver method was worked out in our laboratory to show alterations in motor end plates in biceps and triceps of mon- keys in experimental poliomyelitis (Chor, H., Arch. Neurol. & Psychiat., 1933, 29, 344-357). Fix in 1% ammonia water (28% Merck) in 95% alcohol for 24 hrs. Wash in aq. dest., ^ hr. Pyri- dine, 48 hrs. Wash in 8 changes aq. dest. during 24 hrs. 2% aq. silver nitrate in dark at room temperature, 72 hrs. Reduce 6-8 hrs. or over night in : pyro- gallic acid, 4 gm.; aq. dest., 95 cc; formalin, 5 cc. Dip in water and trans- CHROM BLUE GCB 74 CHROMOPHIL fer immediately to 95% alcohol for a few seconds. Place tissue on slide with longitudinal markings of fibers visible. Add a second slide and squeeze gently. Trim edges with sharp knife so that neat, flat blocks result. 95% alcohol, 30 min. Absolute alcohol, 2 changes, over night. Xylol, 10-12 hrs. until blocks are clear. Imbed in paraffin 8 hrs. changing re- peatedly each hr. for first five. Cut serial sections 10/it. Mount in neutral balsam. Nerves, dark brown or black; muscle and connective tissue, yellow. Chrom Blue GCB, see Gallocyanin. ChromaflBn Reaction (chromic salts + L. ajjinis, akin). Brown coloration when treated with fixatives containing bi- chromate. In adrenal medulla adrenalin is revealed by this brown color but the reaction can also be elicitated by po- tassium iodate and is not altogether specific for adrenalin. Lison (p. 147) advises fixation in Formol-Miiller or in 6% potassium iodate containing 10% of formol. After the usual fixations chro- maffin substances can be demonstrated simply by treating the sections for a few hours with 3% aq. bichromate or iodate of potassium (Lison). See Vulpian Re- action and Osmic Acid. Chromatin Filaments. The studies of Claude, A. and Potter, J. S., J. Exper. Med.,1943,77, 345andof Mirsky, A. E., and Pollister, A. W., Biological Sym- posia., 1943, 10, 247-260 indicate that chromatin is almost wholly made up of fibrous nucleoprotein. By extraction, precipitation and centrifugation the chromatin filaments can be collected. They are of very uniform diameter, like chromosomes, are very resistant to deforming mechanical injury, can easily be stained with acetocarmine are given a positive Feulgen reaction. Mirsky and Pollister favor the view "that chromatin is largely, if not entirely, a complex of highly polymerized desox- yribose nucleic acid with a basic protein of either the protamine or histone type." Chromatin Stains. The most specific stain for basic chromatin is methyl green. Bismark brown is less so. Safranin is useful for chromatin if a red coloration is desired as in the safranin-light green combination. Tests for Iron and Thymo- nucleic Acid are listed separately. See Idiochromatin, Linin, Chromosomes and Nucleolus. Chromatolysis of nerve cells investigated by absorption spectra of Nissl bodies (Gersh, I., and Bodian, D., Biological Symposia, 1943, 10, 163-184). Chromatophores. These, when present in the dermis, are also called melanoblasts, see Dopa Reaction for their demon- stration. Chrome Violet CO (CI, 727). A carboxyl derivative of pararosolic acid. Chromic Acid is purchased as the red crys- tals of chromic anhydride which dissolve easily in water forming chromic acid. The crystals should be kept in a bottle with closely fitting glass stopper because they are highly deliquescent. Alone in very dilute solution chromic acid is helpful in Maceration. When applied in aqueous solutions of about 1% to a slice of fresh adrenal it produces a brown color in the medulla known as the chromaffin reaction. In mixtures with other chem- icals it was more used as a fixative 50 years ago than today but in Perenyi Fluid it is recommended strongly by Lee (p. 32) for embryos, segmenting eggs, etc. It is also a component of Flem- ming's fluid. Chromidial Substance, a designation often applied to basophilic cytoplasmic ma- terial supposed to be of nuclear origin and therefore to resemble theextranu- clear chromatin (chromidia) of protozoa. It is nongerminal chromatin or tropho- chromidia in contrast to germinal or idiochromidia (G. idios, individual, one's own). See Nissl bodies. Chromolipoids. In contrast to the caro- tinoids, which are hydrocarbons, the chromolipoids are fats or derivatives of fats themselves colored. They occur frequently especially in nerve cells, in- terstitial cells of the testicle and in the adrenal, and are easily distinguishable from carotinoids because they do not give the color reactions with sulphuric acid and iodine-iodide. From melaninsthey are to be distinguished by not dissolving in alkalies, by staining with sudanand scharlach and by not reducing am- moniacal silver nitrate. The following method of Hueck is given by Lison : Stain with nile blue. Treat the sections for 24 hrs. with aq. dest. oxygenated 3% (= commercial hydrogen peroxide diluted with 12 volumes water). This leaves the chromolipoids blue, themela- nins decolorized. Lison concludes that distinction from pigments of hema- togenous origin is not so easy because some chromolipoids contain iron. See Lipids, tabular analysis. Chromophil (G. chroma, color and phileo, I love), a loose term applied to almost any granule, cell, or tissue which has a pronounced affinity for stains. Baso- philic cytoplasmic materials in gland cells and in nerve cells (Nissl bodies) are sometimes called chromophil, moreover chromophil reaction is unwisely used to designate the chromaffin reaction of epinephrin producing tissues. CHROM-OSMIC-ACETIC 75 CHROMOSOMES Chrom-Osmic-Acetic fixative, see Lillie's. Chromosomes. — Written by A. R. Gopal- Ayengar, Barnard Free Skin and Cancer Hospital, St. Louis, Sept. 10, 1946— (Now Pata Memorial Hospital, Bom- bay) . These are discrete bodies usually constant in number in the cells of a given species and frequently having distinctive structure into which the chromatin material of a nucleus re- solves itself during the mitotic process (see Mitosis). From a chemical stand- point the chromosome is a protein fiber like silk or hair, presumably depending on a peptide chain linkage — C — C — N — . On this structural framework the per- manent hereditary units, the genes (which may be considered as the atoms of heredity), are located at definite loci. In a sense, therefore, the chromo- some may be considered a giant mole- cule (Darlington, C. D., Nature, 1942, 149, 66-69, Astbury, W. T., Proc. 7th Int. Genet. Congress., 1939 (Camb.), 1940, 49-51). It is generally stated that the chromosomes of sperm cells consist of basic proteins, such as pro- tamines or histones, in combination with highly polymerized desoxyribose nucleic acid (Mirsky, A. E., Advances in Enzymology, 1943, 3, 1-34). Smear-Squash technique. The rapid and spectacular advances in our knowl- edge of cytology and cytochemistry during the last fifteen years have greatly altered our ideas of chromo- some structure and behavior. Progress in this direction has been possible through the introduction of newer tech- niques, such as smears and squashes, which have largely supplanted the classical methods of paraffin sectioning. In order to obtain the most satisfactory results, it is imperative to employ cyto- logical techniques calculated to bring out fine structures and details as quickly and accurately as possible. Most of the smear-squash methods, de- spite their extreme simplicity, give superior results because of instantane- ous fixation and rapidity of staining and finishing schedules. Moreover, it is possible to examine thin layers of cells or even isolated cells and their parts. They are especially useful in the anal- ysis of chromosome complexes and associations and in the exact establish- ment of numerical relations. Squash preparations have special advantages. Individual chromosomes can be recog- nized more readily and easily, inter- relations between chromosomes become clearer, the preparations are almost two-dimensional, which is of undoubted value in chromosome measurement since it obviates errors of measurements due to differential focussing of three dimensional structures. However, there are a few disadvantages resulting from the disturbances of the natural relationships of the chromosomes. These are more than offset by the merits of the method. Of course a uniform application of the same technique is not likely to prove suitable for every kind of material, but the general principles are valid, requir- ing a few modifications depending upon the particular type of material. No single method can be recommended which would prove adequate for all stages of development. The methods that have proved particularly satisfac- tory and have given the best results are the Feulgen-smear-squash technique of Coleman, L. C, Am. J. Bot., 1940, 27, 887-895; Coleman, L. C, Genetics, 1943, 28, 2-8; Hillary, B.B., Bot. Gaz., 1939, 101, 276-300; Hillary, B.B., Ibid, 1940, 102, 225-235; Heitz, E., Ber. d. biol. Ges., 1936, 53, 870-878; Darlington, C. D., and LaCour, L. F., The Handling of Chromosomes, 1942. New York: The Macmillan Company; and McClin- tock, B., Stain Tech., 1929, 4, 53-56. In my experience of making prepara- tions for the study of somatic and meio- tic chromosomes, the method given in the schedule here has proven most satis- factory. It is essentially a modifica- tion of the technique developed by Dr. Coleman and his associates of the Uni- versity of Toronto and may be applied to a wide variety of materials, both plant and animal. The tissues are fixed in Carnoy, one of the Navashin modifications or Flemming-Heitz. One of the best fixatives for small mam- malian tissues is that given by LaCour, L. F., Proc. Roy. Soc. Edin. B., 1944, 62, 73-85. It is a mixture of methyl alcohol (15 parts), 5% acetic acid (5 parts), formalin (1 part), and water (5 parts). The proportions may need to be varied according to the material. For a study of the morphology of mouse chromosomes the liver tissue of a new- born mouse is particularly good. Chromosome structure. The nuclear cycle, whether in plants or animals, is an alternation between two stable states — spiralization and despiralization. The metaphase chromosomes usually repre- sent a state of maximum spiralization and the chromosomes in the resting nucleus, the maximum of despiraliza- tion or minimum of spiralization. For demonstration of the spiral structure of the chromosomes and unravelling of the coils, fixation should be preceded by some form of pretreatment. Differ- ent methods such as hydration, dessi- CHROMOSOMES 76 CHYLOMICRONS cation, exposure to acid vapors or sub- jection to dilute solutions of alkalis, treatment with dilute solutions of salts of strong alkalis and weak acids such as KCN, NaCN, in fact anything that tends to change the pH, have been em- ployed to bring out the real structure (Nebel, B. R., Zeitschr. Zellf. u. Mikr. Anat., 1932, 16, 251-284; Kuwada, Y. and Nakamura, T., Cytologia, 1934, 5, (2), 244-247; Sax, K., and Humphrey, L. M., Bot. Gaz., 1934, 96, 353-362; Huskins, C. L., and Smith, S. G., Ann. Bot., 1935, 49, 119-150; LaCour, L. F., Stain Tech., 1935, 10, 57-60; Oura, G., Zeit. f. Wiss. Mikr., 1936, 53, 36-37; Kuwada, Y., Shinke, N., and Oura, G., Zeit. f. Wiss. Mikr., 1938, 55, 8-16; Cole- man, L. C, and Hillary, B. B., Am. J. Bot., 1941, 28, 464-469; Gopal-Ayen- gar, A. R., Genetics, 1942; Coleman, L. C, Genetics, 1943, 28, 2-8; Ris, H., Biol. Bull., 1945, 3, 242-257). Some of the best results have been obtained by treatment with K or NaCN 21-^ to 21-6 mol. solutions for periods varying with the material. Stain with aceto-carmine, acetic orcein, acetic lacmoid or Feulgen. If Feulgen is used a counter stain with fast green in acetic acid may be used if desired. The cells are squashed on the slide after staining. The amount of pressure needed is determined by experience. The following schedules of treatment for mouse chromosomes may be applied mutatis mutandis in the study of chromosomes from other tissues. Fix pieces of liver from a newborn mouse in a mixture of methyl alcohol- formalin-acetic acid of LaCour for 15 min. Wash in 70% ale. Transfer small piece of material on to a slide and add first drop of acetocarmine or acetic orcein and then coverslip. Gently tap with the rubber tipped end of a pencil until the cells are loosened up and are more or less one layer in thickness. Squeeze out gently the excess of stain. Apply pressure on the coverslip with thumb or by carefully rolling a round edged pencil over it, taking care to see that the coverslip does not slide during the process. The amount of pressure needed is judged by experience. If air bubbles get in add a drop or two of the stain at the edge of coverslip and repeat the process if necessary. Seal edge of coverslip with beeswax and vaseline. If it is desired to make slides permanent follow McClintock's method (McClin- tock, B., Stain Tech., 1929, 4, 53-56). For Feulgen-squash preparations fix material as in the preceding outline. Wash in water thoroughly. Hydrolyse in N.HCl at 60°C. for 6-8 min. Trans- fer to Leuco-basic fuchsin for 20 min. to ^ hr. Pour off stain and add SO2 — water and allow it to remain for 3 min. Change 2-3 times. Place a small piece of material in a drop of 45% acetic acid on a slide and add a coverslip. Gently tap and squeeze out excess of stain as described above. Flatten out the cells by applying pressure with thumb or by rolling a round edged pencil over the coverslip. Transfer slide into large petri dish containing dioxan until cover- slip floats off. The cells will adhere either to the coverslip or slide. Use dioxan balsam as mounting medium. Chlorazol black E + acetocarmine (Nebel, B. R., Stain Techn., 1940, 15, 69-72). Fixation in cold Flemming's fluid plus urea (Hance, R. T., Anat. Rec, 1917, 12, 371-382). Microincin- eration of (Barigozzi, CI., Bull. d'Hist. Appl., 1938, 15, 213-219). Method of localization of genes by experimental deletions, distribution of protein and nucleic acid, classification, etc. (Pain- ter, T. S., J. Roy. Micr. Soc, 1940, 60, 161-176). Feulgen stain for chromo- somes (Mensinkai, S. W., J. Roy Micr. Soc, 1939, 59, 82-112). Aceticorcein is advocated as a new stain-fixative for chromosomes (LaCour, L., Stain Techn., 1941, 16, 169-174). Demonstra- tion of alkaline phosphatase in chromo- somes (Krugelis, E. J., J. Cell. & Comp. Physiol., 1942, 19, 376-379). Chromotrope 2R (CI, 29) — acid phloxine GR, chromotrope blue 2R, fast fuchsin G,XLcarmoi3ine6R — An acid mono-azo dye employed by Lendrum, A. C, J. Path. & Bact., 1935, 40, 415-416 in a study of breast carcinoma and skin lesions as counterstain for celestin blue. Chromotrope Blue 2R, see Chromotrope 2R. Chrysamine G (CI, 410) an acid dis-azo dye of light fastness 5 of no value as a tissue stain (Emig, p. 40). Chrysoidin Y (CI, 20)— brown salt R, dark brown salt R — A basic mono-azo dye suggested by Conn (p. 46) as a substi- tute in some techniques for Bismark brown. Used as stain for mitochondria and Golgi apparatus viewed in polarized light (Monne, L., Protoplasma, 1939, 32, 184-192). Chryosomonadina. Fixation and staining for, Doflein, F. (Arch. f. Protistenk., 1922, 44, 149), also Wenrich, D. H. and Diller, W. F., in McClung's Microscopi- cal Technique, 1950, p. 470. Chrysophenine (CI, 365), a direct dis-azo dye of light fastness 4 to 5, for paraffin sec- tions too light and fugitive a color (Emig, p. 39). Chylomicrons (lipomicrons). These tiny fatty droplets are easily demonstrated by CIACCIO 77 CILIA dark field examination of blood of a person or animal fed butter or cream. The increase begins about 1 hr. there after and reaches a maximum at 4 hrs. after which the number of chylomicrons de- clines. By contrast a carbohydrate meal of rice and sugar or a protein meal of whites of boiled eggs and salt does not result in an increase. For details see Gage, S. H. and Fish, P. H., Am. J. Anat., 1924-25, 34, 1-86; also, Had- jiolofT, A., Bull. d'Hist. Appl. 1938, 15, 81-98. Ciaccio, methods for lipoids. One of the simplest is : Fix small pieces 2 days in : 5% potassium bichromate, 80 cc; for- malin, 30 cc, acetic acid, 5 cc. 3% potassium bichromate for 5-8 days. Running water 24 hrs. Ascending alco- hols, 24 hrs. Abs. alcohol 2 hrs., xylol, 1 hr., xylol -paraffin at 60°C., 1 hr. Paraffin 1-1 J hrs. Pass sections down to 70% alcohol, stain ^1 hr. at 30 °C. in: 80% alcohol, 95 cc, acetone 5 cc. saturated at 50°C. with sudan III then cooled and filtered. Rinse in 50% alco- hol, wash in water, counterstain with hemalum. Mount in syrup of Apathy (or glycerin). Lipoids yellow orange. Lison (p. 206) questions specificity for lipoids and gives in addition, with useful comments, several other methods of Ciaccio. Cilia — written by Alfred M. Lucas and Miriam S. Lucas, U. S. Regional Poultry Research Laboratory and Biological Science Department, Michigan State College, East Lansing, Michigan. Au- gust 10, 1951 — Ciliary activity can be studied in isolated cells, in rows of cells, and in broad epithelial surfaces. Cilia and flagella are found throughout most of the animal kingdom except in nema- todes and arthropods. They perform many functions, such as locomotion of whole organisms, driving fluids through tubes, and propelling sheets of mucus across epithelial surfaces. The simplest method of study is to collect isolated cells by gently scraping a ciliated surface and observing the vibrations of the cilia in a suitable fluid on a slide under the microscope. However, numerous special techniques are required to answer problems relat- ing to the shape of cilia during active and passive phases of their cycle, ciliary coordination and how much work they can do. The beats of cilia may oc- casionally be counted by eye; for ex- ample, Lucas, A. M., and Doublas, L. C. (Arch. Otolaryng., 1935, 21, 285- 296) reported 2.2 to 5.5 vibrations per second for cilia on epithelium of the turtle trachea, at about 30°C. More rapid vibrations require special devices. Martius (Arch. f. Physiol., 1884, 456- 460) who was the first to use the strobo- scope, found 10 to 17 vibrations per second for the cilia of the frog's pharynx and values as high as 32 to 42 vibrations per second are given by Plammond, J. C. (Science, 1935, 82, 68-70) for the membranelle of the protozoan, Episty- lis. The use of the stroboscope and moving pictures have been the methods most used, not only to determine the frequenc}'^ of vibrations but also the succession of shapes taken by the cilia or flagella during the cycle of vibration. Martius emphasized a fundamental cau- tion in using the stroboscope that one should reduce the speed until all mul- tiples of the ciliary frequency have been eliminated. Hammond, on the other hand, recommends using a known har- monic of the frequency. The shapes taken by vibrating cilia and flagella have often been subject of study. One of the simplest methods is to reduce the rate of movement by cooling the fluid in which they are beating. Gray, J. (Proc Roy. Soc, B, 1930, 107, 313-332) and Jenni- son, M. W., and J. W. M. Bunker (J. Cell, and Comp. Physiol., 1934, 5, 189-197) used both photographic and stroboscopic methods to study the form of the isolated, giant compound ab- frontal cilia of the mollusc gill. It is a peculiarity of this cilium to swing usu- ally through an arc of only 90° instead of 180° as characteristic of most cilia and thus it may be atypical material for study. Gray noted that in these cilia the active stroke was longer than the recovery stroke. Brown, H. P. (Ohio J. Sci., 1945, 45, 247-301) has given an extensive analysis of types of ciliary and flagellar movement. He tested his theories in various practical ways and one was the construction of a mecha- nical model which would swim through the water. It was composed of a test tube which contained a strong bent wire holding a rubber band. The rubber band as it unwound rotated a wire pass- ing through a cork stopper. The free end of the wire revolving in the water propelled the tube. Couch, J. N. (Am. J. Bot., 1941, 218, 704-713) recommends the use of dark field to study movement of individual cilia and flagella. The three common types of dark field, the use of a disc stop below the ordinary condenser, the Zeiss cardioid condenser and even mov- ing the diaphragm laterally to give oblique light worked satisfactorily. With dark field are often produced sha- dow effects of one cilia lying above and across others, which sometimes re- CILIA 78 CILIA semble cross-striations and may be con- fusing. Metachromism is shown best by cili- ated cells arranged in rows. The most studied examples are the lateral cilia of the mollusc gill, especially Mytilus and Modiolus, because in these species the gills are primitive and simple. The location and shape of the cells are shown in Modiolus (Lucas, A. M., J. Morph., 1931, 51, 147-193; and J. Morph., 1932, 53, 265-276). Atkins, D. (Quart. J. Micr. Sci., 1938, 80, 331-344) has made a comparative study of these cells for many families and genera of lamelli- branchs. Gray, J. (Proc. Roy. Soc, B. 1930, 107, 313-332) observed that the waves were a succession of spikes and semicircles and with the stroboscope, which caused them to appear to stand still, he measured the relative time con- sumed by the active and recovery phases of the beat. The ratio was about 1 to 5. A photographic set-up for obtaining a permanent record of coordination of lateral cilia has been described in some detail by Lucas, A. M. (J. Morph., 1932, 53, 243-263). However by this method stroboscopic effects of the movie camera shutter may give false impressions of the rate of wave propagation. The most commonly used material for studies on ciliary activity is the frog's pharyngeal membrane, yet it is poorly adapted for practically all the problems for which it has been used as will appear later. Many earlier workers set up recording devices by which records of ciliary activity were recorded on smoked kymograph drums; the cilioscribe, devised by Dixon, W. E., and Inchley, O. (J. Physiol., 1905, 32, 395-400) is a good example and still has merit for classroom demonstration. The apparatus consists of a vertical spindle against which is gently pressed the ciliated surface of a strip of frog epithelium. The cilia cause the spindle to rotate and this in turn moves a light weight drum on which an interval timer marks rate of movement. The chief difficulty is the variable amount of mucus which collects on the spindle interferes with its smooth rotation. Many investigators have used the ciliated epithelium of the frog's pharynx as a delicate indicator to show the re- sponse of living tissue to a wide variety of chemical substances, therapeutic compounds, and biological extracts, yet it was not recognized until the work of McDonald, J. R., Leisure, C. E., and Lenneman, E. E. (Am. Acad. Ophthal. and Oto-Laryng., 1928, 318-354) that the cilia of the frog's pharynx are under definite nervous regulation. A few years later Seo, A. (Jap. J. Med. Sc. Ill Biophysics, 1931, 2, 47-75) estab- lished clearly that cilia respond to stimuli through the glossopharyngeal nerves and that the motor fibers are carried along the palatine nerves. Methods which had been used thus far involved placing fluids or particles on the surface but two years later Lu- cas, A. M. (Arch. Otblaryng., 1933, 18, 516-524) devised a method whereby ciliary activity could be followed di- rectly under very low powers of the microscope focusing on the high-lights reflected from the surface of the moving ciliary waves. Application of this method the same year to the frog (Lucas, A. M., Proc. Soc. Exp. Biol, and Med., 1933, 30, 501-506) demon- strated conclusively that the cilia in the frog's mouth are normally not ac- tive and are completely at rest. They become active when particles, no matter how fine and light they may be, are added to the surface; even the addition of clear saline solutions has the same effect. Thus, the normal baseline for ciliary activity in the frog's pharynx is zero instead of some positive value derived by the use of foreign stimulating agents. Recently Steward, W. C. (Am. J. Physiol., 1948, 152, 1-5) although recognizing these facts, sought to estab- lish some arbitrary baseline values for cilia which had been stimulated by discs and weights placed on the mucous sur- face. His experiments gave a mean velocity of 0.75 mm/sec. but he found that even very light stimuli on the membrane, well removed from the route covered by the test object, would in- crease the velocity 142 to 415 per cent. Lucas, A. M. (Am. J. Physiol., 1935, 112, 468-476) designed a moist chamber in which threshold levels of the nerve- cilia and nerve-muscle preparations could be determined simultaneously. He found that the threshold current which would stimulate the cilia by way of the palatine nerve was 15.4 times stronger than that required to activate the muscles of the toe. The behavior of of excised mucous membrane is quite similar to that found in vivo, the cilia are normally quiescent and become ac- tive only when stimulated. What is needed is a different biologi- cal material for studies where chemicals are to be tested in which the cilia beat incessantly, as in the oviduct of birds. This was one of the materials chosen when ciliary studies were just beginning (Purkinje and Valentin, Muller's Arch. Anat. and Physiol., 1834, 1, 391-400). They used macerated black pigments CILIA 79 CILIA of the retina suspended in fluid to fol- low ciliary movement. The oviduct or trachea of turtles or other reptiles would be useful where it is not conven- ient to control the temperature of tis- sues from a warm blooded animal. The ciliature of the avian and reptilian oviducts has been described by Parker, G. H. (Phil. Trans. Roy. Soc. B, 1931, 219, 381-419). Umeda, T. (Acta Der- matologica, 1929, 6, 629-646) used ox trachea which provides large ciliated areas. He employed it to study the effects of temperature, sugar solutions, alkaloids, alcohol, x-ray, and ultra- violet light radiation. The underlying tracheal cartilage was removed with the ciliated epithelium in pieces about 2 x 3 cm. and pinned out beneath a layer of Ringer's solution at 38°C. It was fixed at a slant of 10 to 15 degrees to facili- tate the movements of millet seeds placed on the surface. There is ob- viously need for someone to recheck the reactivity of the frog's pharyngeal cilia against several other kinds of ciliated epithelium and sort out the effects due to the type of biological material used from the real effects of chemicals or other agents being tested. In the frog's pharynx and in the ten- tacle of the snail (Merton, H., Pfluger's Arch. Ges. Phys., 1923, 198, 1-28), nerves are responsible for activation of ciliary movement; whereas, in the Ctenophore swimming plates (Gothlin, G. Fr., J. Exp. Zool., 1920, 31, 403-441) and the velar cilia of the nudibranch veliger (Carter, G. S., Brit. J. E.xp. Biol., 1926, 4, 1-26) the nerves are re- sponsible for inhibition of ciliary ac- tivity. The procedures used by these authors are well adapted to differentiate between inherent ciliary activity and nerve regulation. In this same cate- gory should be included the work of Copeland, M. (Biol. Bull., 1922, 42, 132-142) who studied the r61e played by nerves in regulating the ciliary ac- tivity on the foot of the snail. Polarity and ciliary reversal have been problems of considerable academic interest. Coonfield, B. R. (Biol. Bull., 1936, 70, 460-471) described his opera- tive techniques for reversing rows of swimming plates in Ctenophores and Twitty, V. C. (J. Exp. Zool., 1928, 50, 319-344) and later Luther, W., (Roux Arch. Entw. Organ. 1934, 131, 532-539) reported the relatively simple methods used in reversing small segments of the ciliated epidermis of the amphibian larva. Induction effects on ciliary po- larity have been worked out recently by Twitty, V. C. and Bodenstein, D. (J. Exp . Zool . , 1941 , 86, 343-379) . Reversal of tracheal epithelium in dogs was ac- complished by Isayam, S. (Zeit. f. Biol., 1924, 82, 155-156) who found no reversal of direction of ciliary beat. Ciliary reversal is a well known reaction in some ciliated protozoa, such as Para- mecium and minimal techniques are required for study of its physiology (Oliphant, J. F., Physiol. Zool., 1942, 15, 443-452). Ciliary reversal in Meta- zoa is rare indeed. Parker, G. H. (Am. J. Physiol., 1905, 14, 1-6) employed simple techniques to demonstrate its existence in the labial cilia of sea- anemones. Equally simple procedures were followed by Matthews, S. (J. Exp. Zool., 1928, 51, 209-262) to demonstrate that the seeming reversal of the cilia- ture of the pelecypod palp did not actu- ally exist. Atkins, D., (J. Marine Biol. A. United Kingdom, 1930, 16, 919-970) observed permanent, natural reversal in frontal cilia of the gill filaments of Mytilus following injury. Numerous investigators have pre- sented diagrams of the direction of cili- ary movement in a field such as Irving, L. (J. Exp. Zool., 1924, 41, 115-124) who plotted the circulation of fluids and particles within the coelom of the star- fish. Another good example is the work of Atkins, D. (Quart. J. Micr. Sci., 1936-1937, 79, 181-308, 339-373, 375-421) who worked out carefully the direction of ciliary movements in a great many molluscs and evolved a phylogenetic tree based, in part, on the lateral- frontal cilia (Quart. J. Micr. Sci., 1938, 80, 345-436). Also the study made by Meyer, A. (Biol. Zentralbl., 1936, 56, 532-548) on Nephthys hombergli illus- trates a careful approach to this type of problem. Barclay, A. B., Franklin, K. J., and MacBeth, R. G. (J. Physiol., 1937, 90, 347-348) observed that mucus is moved in a clockwise direction up through the mammalian trachea. Hilding, A. (Arch. Otolaryng., 1932, 15, 92-100) plotted the direction of cili- ary movement in the human nose by watching the direction of drainage of the mucous sheet with a speculum after dusting with face powder and observed in the posterior two-thirds of the nose a new mucous layer about every 10 minutes and about once an hour in the anterior third. Lucas, A. M. (Am. J. Anat., 1932, 50, 141-177) and Lucas, A. M. and Douglas, L. C. (Arch. Oto- laryng., 1934, 20, 518-541) used carbon particles to plot the drainage pattern for monkey, rat, mouse, rabbit, cat, cow, and sheep. The potential error resulting from following mucous flow rather than direct observation of cilia is shown on some wound experiments CILIA 80 CILIA on the frog's palate by Lucas, A. M. (Arch. Otolaryng., 1933, 18, 516-524). Hill, L. (Lancet, 1928, 2, 802-805) had previously shown that cuts across the trachea arranged in alternating fashion completely stopped the flow of mucus and also showed a high sensitivity to ultra violet light. A combination of both direct and indirect techniques gives the most accurate results. The double nature of the mucous layer and the three ways in which ciliary move- ment and mucous flow could act to- gether or independently were suggested by Lucas, A. M., and Douglas, L. C. (Arch. Otolaryng., 1934, 20, 518-541). Most ciliary activity can be defined in terms of work. Bowditch, H. P. (Boston Med. and Surg. J., 1876, 15, 159-164), one of the first to study this problem, permitted a sheet of ciliated epithelium from the frog to climb up an inclined plane set at various angles and carrying various loads. He cooncluded that cilia are capable of doing only about one-thirty-fifth the work that the heart does in pumping blood. Parker, G. H. (J. Exp. Zool., 1914, 16, 443-446) placed a tube in the osculum of the sponge and ascertained that the pres- sure developed in the system by the collar cells was only a few millimeters but that the volume turn-over was large. A similar technique was applied by Hecht, S. (J. Exp. Zool., 1916, 20, 429-434) to the large Bermuda ascidian. A 100 gm. animal could circulate about 173 liters of sea water in a day. Hecht gives his technique for getting the glass tube into the excurrent siphon. Galt- soff, P. S. (Bull. U. S. Bur. Fish. 1928 (1930) 44, 1-39) has gone farther than anyone thus far in designing apparatus to measure rate of flow of water due to ciliary action. He used the oyster and collected data on feeding and effects of temperature and seasonal and diurnal cycles. Hilding, A. C. (Trans. Am. Acad. Opth. and Otolaryng., 1944, 367- 378) found that the trachea of the hen was particularly well suited to measure the positive and negative pressures de- veloped at the two ends of the tube. He collected mucus from other hens and used it to form a plug at the lower end of the trachea being tested. A negative pressure of 34 mm. and a posi- tive pressure of 32 mm. of water was developed at the two ends of the tube. The techniques used are simple but effective. The procedures devised by White, H. L. (Am. J. Physiol., 1929, 90, 689-704) to measure the pressure developed by the nephostomal cilia in the Necturus kidney is a good example of how the microscopic methods can be applied. In general, in vivo studies of ciliary movement have been rather few but a nice technique was used by Ernst, A. M. (Arch. Internat. Pharmacodyn. et de Th^rap., 1938, 58, 207-212) in which periodic roentograms were made of the cat's thorax to show the rate of elimination of "Neobar" powder (Merck) which had previously been blown into the trachea and bronchial spaces. Normally the trachea was cleared in an hour and from this base- line the effect of various anesthetics could be determined. X-ray radiation in itself has very little effect on ciliary activity as shown by Goldhaber, A., and Black, A. (Proc. Soc. Exp. Biol, and Med., 1941, 48, 150-151) who ob- served that the oral epithelium of the toad transplanted as a tissue culture required 1,000,000 to 1,400,000 r to stop ciliary activity. The essential details of their x-ray set-up are given. Numerous papers mention the pres- ence of cilia lining various parts of the central canal of the nervous system and suggestions have been offered concern- ing the function they perform but only one author (Chu, H. Y., Am. J. Physiol., 1942, 136, 223-228) to our knowledge has selected a suitable material (trans- lucent stages of amphibian larva) and thus been able to study this activity in situ. He followed the movements of naturally occurring pigment granules within the ventricles and also injected red cells. Satisfactory methods for examination of ciliary movement in tubular organs such as the lungs are not always easy. SoUmann, T., and Gilbert, A. J. (J. Pharmacol, and Exp. Therap., 1937, 61, 272-285) filled the excised organ with 10 per cent gelatin in Ringers and then chilled it in iced Ringers. Free-hand sections were pinned on rings of cork and studied in shallow dishes contain- ing Ringer's warmed to 37 °C. Cilia were vigorously active up to about 8 hrs. A method for testing the phagocytic properties of ciliated epithelium was employed by Ropes, M. W. (Contrib. to Embryol. #128, Carnegie Inst., Wash., 1930, 22, 77-90). He used the tracheal mucosa of the rabbit. In- haled carbon particles were taken up by the cytoplasm and could be recognized in the living ciliated cells. The older editions of Lee's Vade Mecum suggest boric acid methods for maceration of ciliated epithelia. This technique has been omitted from some of the more recent editions but is men- tioned again in the last edition (11th). The method was revived by Goodrich, CILIA 81 CILIA E. L. (Quart. J. Micr. Sci., 1942, 83, 245-258) as a new technique with slight modifications. Lucas, A. M. (J. Morph., 1931, 51, 147-193) used sodium borate to which was added a trace of iodine or eosin. The cells swelled as they separated. Poska-Teiss, L. (Zeit. f. wiss. Mikr., 1934, 51, 238-243) used Ranvier's 1/3 alcohol method to isolate the cells. Loginoff, W. J. (Anat. Anz., 1911, 38, 353-361) who worked with horses, cows and sheep, discovered that preliminary fixation of ciliated cells of the trachea for 30 min. in 1% formalin followed by 1/3 alcohol for 24 hrs. gave well preserved cells which could be stained under the coverglass with picro- carmiue. In vitro explants of ciliated epithelium show, eventually, dedifferentiation and loss of cilia. More often short term studies on surviving cells have been resorted to. Isolated cells from the frog's palate under tissue culture condi- tions beat continuously since they are no longer regulated by the nervous sys- tem and are suitable for testing pur- poses (Ishikawa, S., ActaDermat., 1927, 9, 339-364). Verne, J. (Compt. Rendu Assoc. Anat., 1932, 27, 564-578) using well known tissue culture techniques cul- tivated lung tissue of 15 to 17 day old chick embryos. In this case the cil- iated epithelium did not undergo meta- plasia but remained active. Proetz, A. W., and Pfingsten, M. (Arch. Otolar- yng., 1939, 29, 252-262) combined cul- ture of guinea-pig nasal epithelium with motion picture photography. Umeda, T. (Acta Dermat., 1930, 1, 13-38) tested some 700 dyes for their vital staining characteristics and found that thionin blue and Nile blue gave satisfactory results with frog ciliated epithelium. Vital methylene blue has frequently been used to stain lining ciliated cells. Carter, G. S. (Brit. J. Exp. Biol., 1926, 4, 1-26) observed that it was the region of the ciliary rootlets which showed granules staining strongly with this dj'e and Coonfield, B. R. (Biol. Bull., 1936, 70, 400-471) found the greatest staining in Nephthys where the rootlets converged near the nucleus. Most of the usual histologic or cyto- logic techniques will demonstrate cilia. Cilia are stained chiefly with acid dyes but may hold Heidenhain's iron hema- toxylin if the tissues have been only lightly destained with iron alum. When staining the very short sensory ciliary hairs on olfactory epithelium, the iron hematoxylin without counter stain or only a light counter stain makes them stand out quite well. A cuticular border is present on some types of ciliary cells and absent on others. It is refractile to most stains and oftentimes appears as a very nar- row clear space between the line mark- ing the surface of the cuticle and the underlying cell membrane. If the over- lying mucus is stained with mucicar- mine, the outer surface of the cuticle is clearly delimited. Frequently, the lower margin of a nonciliated striated cuticular border looks like a row of basal bodies and Heidenhain's iron hema- toxj'lin gives the best differentiation. Likewise it is the stain usually chosen to show flagellate diplosomes and stages in ciliogenesis. Ciliary rootlets are as difficult to demonstrate in the stained as in the living condition. In order to see them clearly it is important to select the right kind of cell — one which has a clear protoplasm containing a minimum of pigmented opaque or refractile gran- ules. Even living unstained ciliated cells of the mammalian nose when iso- lated on a slide show clearly the root- lets in the narrow clear zone of proto- plasm just beneath the cell membrane and in relatively transparent cells they can be observed converging toward the nucleus as an inverted truncated cone. In the latero-frontal ciliated cells of the mollusc gill the cilia form two rows transversely across the surface of the cells so that the rootlets lie in rows one behind the other and under these condi- tions they show up very easily. Grave, C, and Schmitt, F. O. (J. Morph. and Physiol., 1925, 40, 479-515) recom- mended the Ehrlich-Biondi triple mix- ture as a counterstain to Heidenhain's iron hematoxylin and this works very well although sometimes more than one attempt is necessary to obtain a satis- factory mixture of Ehrlich-Biondi stain. Considerable latitude is permitted in the use of fixatives. Allen's Bj and Bu are good fixatives although the alveolar appearance that picric-containing fixa- tives give to the appearance of the cyto- plasm may be a disturbing factor. Del- linger, O. P. (J. Morph., 1909, 20, 171- 209) made an informative comparative study of the fixatives and the basic con- stituents which go into them. He found that HgCli emphasized fibrillar struc- tures and enhanced the staining and so fixatives such as Zenker's, with formalin or acetic acid, are often used for preser- vation of ciliated tissues. These are vigorous fixatives and may result in some cell distortion. Bellinger recom- mended 0.4 to 2% osmic acid as best for cilia. Thanhoffer, L. von. (Zeit. Anat. Entwickl., 1929, 90, 713-724) employed CILIA 82 CILIA Heidenhain's "Susa mixture" in his study of ciliogenesis. Mihalik, P. von. (Anat. Anz., 1935, 79, 259-268) preferred the "Susa mixture" for the study of intracellular ciliated cysts in the rabbit oviduct. Meves, Fr. (Arch. mikr. Anat., 1897, 50, 110-114) used Hermann's osmic mixture for the study of sperm flagella of the salamander. Kindred, J. E. (J. Morph. and Physiol., 1927, 43, 267-297) recommended Meves' fluid as giving the most constant results in his investigation of ciliogenesis in the pharynx of the frog. Flemming's fluid also gave good results but he did not find 2% osmic acid satisfactory. Ap- plication of Da Fano's modification of Cajal's silver method by Cowdry, E. V. (Anat. Rec, 1921, 22, 289-299) revealed flagella present on the thyroid follicles of the dogfish. The blackened flagel- lum showed up nicely against a yellow- ish background. The collars and fla- gella of sponge choanocytes are difficult to preserve for microscopic study. Rob- bertsou, M. and Minchin, E. A. (Quart. J. Micr. Sci., 1910, N.S._55, 611-640) found Hermann's fluid satisfactory and stained with Heidenhain's iron hema- toxylin and light green. Cilia and flagella of protozoa can be demonstrated in fresh preparations by first introducing strands of some ma- terial such as cotton fibers or frayed lens paper beneath the cover glass which will limit activity of the organisms. Maier, H. N. (Arch. f. Protist., 1903, 2, 73-179) drew 1% osmic acid and sub- sequently 5% soda solution beneath the cover glass to study cilia and this method is still used. Noland, L. E. (Science, 1928, 67, 535) used a gentian violet stain (20 mg. gentian violet to 1 cc. aq. dest ; plus 80 cc. sat. aq. phenol ; 20 cc. of 40% formaldehyde; 4 cc. gly- cerin) which he mixed with a drop of ciliates or flagellates in culture. Don- aldson, R. (Lancet, 1917, 2, 571-573) demonstrated protozoa in feces by using 0.1 to 0.5% aq. of eosin, or with iodine solutions of various strengths, or with a mixture of the eosin and dilute iodine. These solutions are also good for dem- onstrating cilia and flagella. Fixation of flagella by cupro-picro- formol acetic mixture and staining by eosin and phosphomolybdic acid ac- cording to A. C. Hollande's (Arch, de Zool. exper. et gen., 1920, 59, Notes et Revue, 75-77) method is satisfactory. This technique is described in detail by Wenrich, D. H. (McClung's Micr. Tech- nique, 1937, Hoeber, p. 547). Relief staining demonstrates ciliary rows and other surface markings on protozoa: B. M. Klien's silver method (Zool. Anz., 1926, 67, 160-162) opal blue, china blue, and other stains dried on the protozoa (Bresslau, E., Arch. f. Protist., 1921, 43, 467-480) and uigrosin (Coles, A. C, Watson's Micr. Rec, 1927, 23-25). Owen, H. M. (Trans. Amer. Micr. Soc, 1947, 66, 50-58) maintains that the "brush" or "flimmer" effect produced on flagella of bacteria by Loeffler's stain is an artifact due to interaction of heavy metals and mordant. He recom- mends a fixative which does not produce the "brush" on flagella (10 parts, 2% osmic acid to 1 part 20% formalin). Ciliogenesis can be observed occur- ring in a number of protozoa. Lucas, M. S. (Arch. f. Protist., 1932, 77, 407- 423) used vom Roth's platinic chloride fixative followed by Heidenhain's iron hematoxylin stain on smears of Cya- thodinium from the guinea pig intestine, demonstrated formation of new basal bodies and cilia within cysts which later evert upon the protozoan's surface. When cilia or flagella are to be studied under the electron microscope, it is necessary, of course, that they be dried under high vacuum. This may intro- duce distortion. A critique on the ad- vantages, limitations, and possible arti- facts of electron microscope techniques has been given by Williams, R. C. (Growth Symposium, 1947, 11, 205-222). Baylor, M. R. B., Nalbandov, A., and Clark, G. G. (Proc. Soc. E.xp. Biol, and Med., 1943, 54, 229-232) studied the sperm head and axial filament. They observed that fresh sperm, dried and dehydrated showed a fraying out of the axial filament into its component fibrils whereas stained sperm did not and they conclude that the stain is sufficiently adhesive to prevent the separation. Brown, H. P. (Ohio J. Sci., 1945, 45, 247-301), in his study of flagella, gives his technique for preparation of his material in great detail, including the results from his tests on numerous fixa- tives and stains suitable for use with the electron microscope. Jakus, M. A. and Hall, C. E. (Biol. Bull., 1946, 91, 141- 144) studied the trichocyst and cilia of Paramecium and used the shadow-cast- ing technique. Microdissection has been used oc- casionally to study the mechanisms of ciliary movement and coordination. The techniques of microdissection are well known from various books and articles. It was used by v. R6nyi, G. (Zeit. f. Anat. u. Entwick, 1926, 81, 692-709) to determine if cilia, separated from their basal bodies, were contrac- tile. Carter, G. S. (Proc. Roy. Soc. B, 96, 115-122) made use of this tech- CINEPHOTOMICROGRAPHY 83 CLEARING nique to demonstrate that the latero- frontal cilia of Mytilus are compound and that the separate components can beat independently of the others. Peterfi, T., and Woerderman, M. W. (Biol. Zentralbl., 1924, 44, 264-268), Grave, C. and Schmitt, F. O. (J. Morph. and Physiol., 1925, 40, 479-515), Lucas, A. M. (J. Morph. 1931, 51, 147-193) and Worley, L. G. (J. Cell, and Comp. Physiol., 1941, 18, 187-197) employed this method to gain information con- cerning ciliary coordination. Cinephotomicrography. A convenient illus- trated account of apparatus and meth- ods is given by Tuttle, H. B., in Glas- ser's Medical Physics, 183-194. See Motion Pictures. Cinnamon Oil (Cassia oil) resembles clove oil and is particularly recommended by Lee (p. 70) for clearing. Two kinds are given in Merck Index. The U.S.P.XI variety contains 80-90% cinnamalde- hyde. Citrate of sodium can be used as an antico- agulant in the proportion of 18 cc. of 2% aq. sodium citrate to 100 cc. of blood. Clarite X (Neville Co., Pittsburg) 60% in toluol is suggested as substitute for balsam owing to its neutral reaction, lack of yellow color and quickness oi hardening. Clarite, also called Nevil- lite V, is useful if added to paraffin when one wishes to obtain thin sections when it is not convenient to imbed in a very high melting point paraffin. Wehrle, W., Stain Techn., 1942, 17, 131-132 ad- vises imbedding in a mixture of 90% paraffin (m.p. 53°C.), 5% bleached bees- wax and 5% clarite and the elimination of electrical charge when ribbons are cut by a spark-coil device described by Blandau, R. J., Stain Techn., 1938, 13, 139-141. Clark and Lubs Buffers (Clark, W. M. The Determination of Hydrogen Ions, Balti- more: Williams & Wilkins, 1928, 717 pp.). Prepare: (1) A solution contain- ing M/5 boric acid and M/5 potassium chloride made by dissolving 12.368 gms. of H3BO3 and 14.912 gms. of KCl in aq. dest., and diluting to 1 liter. (2) A M/5 sodium hydroxide (carbonate free) solution by dissolving 50 gms. of NaOH in 50 ml. (cc). aq. dest. in a Pyrex flask. Let stand overnight to allow the sodium carbonate to settle, or filter through a Gooch or sintered glass cruci- ble. (Exclude air to prevent formation of more carbonate by atmospheric CO2.) Keep the strong alkaline solution in a paraffin-lined glass bottle. Dilute with aq. dest. which has been boiled to re- move the excess CO2 so that the solution is about 1 N. Then make an approxi- mately M/5 solution of the alkali which can be accurately standardized against potassium acid phthalate. To make buffer of the desired pH add to 50 cc. of (1 ) M/5 HjBC-KCl the desig- nated amount of (2) M/5 NaOH and dilute to 200 cc. with aq. dest. Or combine the two in similar proportions but in larger amounts to minimize error in measurement. pH CO. of M/6 NaOH 7.8 2.61 8.0 3.97 8.2 6.90 8.4 8.60 8.e 12.00 8.8 16.30 9.0 21.30 9.2 26.70 9.4 32.00 .6 36.85 .8 40.80 10.0 43.90 Cleaning Glassware. Pulverize 20 gms. potassium bichromate. Dissolve this in 200 cc. aq. dest. with aid of a little heat. Add slowly 20 cc. sulphuric acid C.P. Before treating beakers, graduates, bottles, etc. with this acid cleaning solu- tion first wash them in soap and hot water. Rinse in water to remove the soap. Leave in cleaning solution 2 hrs. or more. Rinse in running tap water and dry with opening downward on drying racks as in biochemical laboratories if possible in a dust free cupboard. For new slides and cover glasses wash in the same way and after final rinsing in tap water store in fresh 95% alcohol in covered dishes until they are required for use when they should be wiped with gauze. For old slides and cover glasses soak in xylol to permit separation and removal of most of balsam. Then leave in waste alcohol several days. Soak for a day or more in strong soap solution. Wash in running water. Clean in clean- ing solution. Wash in water and store in 95% alcohol. Unless strict economy is necessary it is hardly worthwhile to use slides and covers twice especially when the former have been marked with diamond pencils. Clearing is a process in microscopic tech- nique which is required in three different situations. 1. As the step following dehydration in paraffin imbedding. The tissue be- comes translucent but this is not the essential feature of the process. What is necessary is for the alcohol, which is not a paraffin solvent, to be removed by the clearing agent before the tissue is infiltrated with paraffin. Consequently the agents must mix freely with alcohol on the one hand and with paraffin on the CLOTH RED B 84 COAGULATION other. Of them xylol is by far the most widely used and rightly so. Two changes of half absolute alcohol and xylol within 1 hr. and 2 changes of xylol within the next 3-4 hrs. are usually sufficient for slices of tissue 4-6 mm. thick, but the time should not be extended beyond that needed to attain translucency be- cause so doing causes a hardening and a shrinkage of the tissue. Several other substances can be used in place of xylol. Cedar wood oil is ac- cording to Lee (p. 80) the very best clearing agent for paraffin imbedding. It penetrates rapidly, does not make the tissues brittle, and, when not entirely displaced by paraffin, does not seriously interfere with sectioning. First treat the tissue with ^ absolute and xylol for about 2 hrs. The time required in the oil of cedarwood is however a little longer than in the case of xylol used alone, say 12 hrs. Some recommend 2 changes of xylol (about 30 min.) after the oil of cedarwood before entering ^paraffin and cedarwood oil. Methyl benzoate is now quite popular. Pass the tissue from absolute alcohol through 2 changes of pure methyl ben- zoate within 12-24 hrs. When it has been definitely cleared remove benzoate by 2 changes of benzol (^-1 hr.) before passing into paraffin, or half benzol and paraffin. Chloroform penetrates pxaorly and should not be employed unless called for. It has the further disadvantage that unless completely removed in the paraf- fin bath, it will make the final paraffin block soft and unfit for cutting. The usual practice is to clear very small pieces for about 12 hrs. in 2 changes, or as long as may be necessary to make them transparent, and in the imbedding to use 4 changes of paraffin. A more rapid method is to pass di- rectly from the fixative, Bouin or forma- lin, without washing, to 3 changes of pure dioxan within 4 hrs. and thence into 3 changes of paraffin as advised by Graupner, H. and Weissberger, A., Zool. Anz., 1931, 96, 204-206. Stowell, R. E., Stain Techn., 1941, 16, 67-83 confirms and extends earlier work of Seki which shows that, although xylol shrinks tissues more than dio.xan, in placing in hot paraffin, the final shrink- age is greater in tissues after dioxan. When great haste is necessary Mallory (p. 54) suggests acetone \-2 hrs. ; benzol, 15-30 min. ; and paraffin 3 changes, 30-90 min. The shrinkage, however, is very marked and it would probably be better to use Frozen Sections. By the Altmann-Gersh technique, which is at once very time consuming and very valuable for special purposes, fixation, alcoholic dehydration and clear- ing can be side stepped and the dried tissue directly impregnated with paraffin. 2. As the step following dehydration of sections before mounting. The clear- ing is of course easier and much quicker owing to the thinness of the tissue. Again xylol comes first and will probably not be displaced though some prefer toluol. It is not necessary to protect against shrinkage and brittleness. When desired, abs. ale. can be omitted and the clearing be done from 90 or even 80% ale. with terpineol, clove oil, anilin oil, beech wood creosote, Bergamot or some other substance. 3. As a means of rendering clearly visible certain structures in embryos or whole tissues. Clearing is generally done by the Spalteholz method. See Cartilaginous Skeleton and Ossification centers. When glycerin mixtures are employed as Mounting Media they also clear the tissues. See Groat, R. A., Stain Techn., 1941, 16, 111-117 for clear- ing tissues with mixtures of tributyl and tri-o-cresyl phosphates. Cloth Red B (CI 262). A sulfonated azo dye. For formula and influence on mouse tumors, see Stern, K., Cancer Res., 1950, 10,565-570. Cloudy Swelling. This is a marked swelling and granulation of the cytoplasm of cells. It is sometimes observed post- mortem in acute febrile conditions especially in the kidneys, liver and m5'o- cardium. An almost meaningless syno- nym, often used, is Parenchymatous Degeneration. The extent of cloudy swelling that may occur in vivo and from which the cells may recover is not known. The fatty droplets present can be demonstrated in Sudan stained frozen sections of formalin fixed mate- rial. Special stains for Fibrin, Myo- fibrils and Mitochondria may be de- sirable. Coacervates (L. acervus, a cloud or swarm) are masses of particles clumped together (but encased in a little water) by a change in their electrical charge while in colloidal suspension in water or by dehydration with resultant loss of loosely bound water. Ilirsch (G. C, Form und Stoffwechsel der Gol- gi-Korper. Protoplasma Monographs, Berlin, 1939) has likened the Golgi apparatus to a coacervate. See Bensley, R. R., Anat. Rec, 1937, 69, 341-353 for critical consideration of suggestion that mitochondria are coacervates. Coagulation. A phenomenon frequently encountered in the case of blood and lymph is of wide occurrence and is in- COBALT NITRATE SILVER 85 COLLAGEN IC FIBERS fluenced by many factors. Small quan- tities of many electrolytes cause coagu- lation of colloids. Some ions are much more powerful in this respect than others and certain "protective col- loids" such as gelatin protect colloidal suspensions against coagulating action of electrolj'tes. Peptization is dis- integration of the coagulum into col- loidal particles. Water is employed as a peptizing agent in dissolving glue, agar, and similar materials (Holmes, H. N., Glasser's Medical Physics, 257- 263). Cobalt Nitrate Silver for Golgi Apparatus. Cocarboxylase, see Method of Westen- brink, H. G. K., Enzymologia, 1940, 8, 97-107, capable of determining as little as 0.01 fjL gm. of carboxylase. Coccidia. These sporozoa include many parasites of great importance not only to physicians and veterinarians but also to cj'tologists who are interested in their intracellular behavior. Conse- quently the volume by Becker, E. R., Coccidia and Coccidiosis of domesti- cated, game and laboratory animals and of man. Ames: Collegiate Press, Inc., 1934, 147 pp. will contain numerous helpful leads on the coccidia of the digestive tracts of vertebrates and in- vertebrates. See also McClung, 1950, p. 455. Coccinel Red is 1, 5-diamylaminoanthra- quinone, an oil soluble dye, recom- mended by Lillie, R. D., Stain Techn., 1945, 20, 73-75 as a stain for fat which it colors deep orange red. Make up stock solution of 4.2% in absolute (99%) isopropanol. Dilute this down to 30 or 40% isopropanol with water and treat frozen sections of normal cat kid- ney and human adrenal with resulting solution for 10-20 min. This solution is only usable for several hours. Coc- cinel red is a good counterstain after hematoxylin. Coccinine (CI, 120), an acid monoazo dye, light fastness 3 to 4, which colors sec- tions pale pink not equal to Biebrich Scarlet (Emig, p. 31). Cochineal (CI, 1239). This crimson dye was in use by the Aztecs before the Spanish conquest. It is derived from an insect which feeds on a cactus. So highly prized was it that Montezuma took as yearly tribute from the State of Huaxyacas (Now Oaxaca) 20 sacks of cochineal. The invading Spaniards were not slow to note the superiority of cochineal over Kermes, the crimson stain in use at home (1523 A.D.). Charles V of Spain commanded Cortez to inform him immediately "whether what has been reported is true that Kermes were to be found in abundance in New Spain and, if so, could be sent with advantage to Spain". So coch- ineal figured largely in the Aztec tributes to Cortez and the industry became a Spanish monopoly. In 1858 A.D. aniline red became a competitor, depressed the sales of cochineal, which, latter as a commercial dye, was defi- nitely replaced when azo dyes came into the market about A.D. 1880. (Leggett, W. F., Ancient and Medieval Dyes. Brooklyn: Chemical Publishing Co. Inc., 1944, 95 pp.). See Kermes, Lac. In microscopy cochineal is used mostly for staining in tola of small in- vertebrates. Mayer's alcoholic cochi- neal is a popular preparation made, ac- cording to Lee (p. 149), by powdering 5 gm. cochineal with 5 gm. calcium chloride and 5 gm. aluminum chloride to which 100 cc. 50% alcohol and 8 drops of nitric acid (sp. gr. 1.20) are added. Heat to boiling point, cool, shake oc- casionally during several days and filter. Before staining bring objects to 70% alcohol, destain if necessary in 70% alcohol containing 0.1% hydrochloric acid. Dehydrate, clear and mount in balsam. Nuclei are colored red and other structures a variety of colors from red to deep purple. In some respects it is better than carmine. Neither fade. Cochlea, see Ear. Coelestin Blue, see Skyblue. Coeline, see Skyblue. Coeruleum, see Skyblue. Colchiceine, different from colchicine, see Ludford, R. J., Arch. f. exper. Zellf., 1935-36, 18, 411-441. Colchicine, see Mitosis Counts. Collagenase. An enzyme which specifically destroys collagenous fibers. Bidwell, E. and W. E. van Heyningen, Biochem. J., 1948, 42, 140-151, report on its isola- tion and purification. Collagenic Fibers — Written by A. I. Lans- ing, Dept. of Anatomy, Washington University, St. Louis 10, Mo. October 5, 1951 — These are widely distributed in the body, occur in large bundles in tendons and are best viewed in spreads of loose connective tissue. Unlike elas- tic fibers which are yellow and highly refractile, coUagenic fibers are white and poorly refractile, form broad wavy bands which do not branch, have great tensile strength, and are attacked by dilute acids and alkalis. On boiling they yield gelatin. The fact that they pass from the gel to the sol state on alkalinization or warming is the basis for methods of separating epidermis from dermis. Collagenic fibers are readily digested by pepsin in acid solu- tion but resist trypsin. A specific col- lagenase has as yet not been found. COLLAGENIC FIBERS 86 COLOR PRESERVATION Amino acid analyses (see Elastic Fi- bers) indicate an absence of aromatic amino acids, an abundance of proline, hydroxylysine and particularly hy- droxyproline, the latter being almost a diagnostic feature. The isolectric point of collagen is about pH 7.0. Although tinctorial reactions would imply that reticulum and coUagenic fibers differ, recent studies indicate that reticulum is but a finer unit of collagen. Both have a marked affinity for aniline blue. They differ in that reticulum is strongly argyrophilic while collagenic fibers are not; the former is weakly acidophilic (in H & E) while the latter takes up much eosin. Electron microscopy and x-ray dif- fraction studies have done much to re- veal the submicroscopic structure of col- lagenic fibrils (Gross, V., J. Gerontol., 1950, 5, 343). These are apparently composed of parallel polypeptide chains approximately 10 A° in diameter which are bonded together laterally. Elec- tron microscopy reveals considerable fine structure in fibrils. There is a regular periodicity of 640 A° with much intra-period detail. A protein termed procollagen may be prepared by extraction with citrate at pH 4. Such procollagen can form fibrils with regular periodicity by the addi- tion of monovalent salts or mucoprotein (Highberger, J. H., J. Gross and F. O. Schmitt, Proc. Nat. Acad. Sci., 1951, 37, 286). The best stain for collagenic fibers in sections after Zenker fixation is aniline blue in Mallory's Connective Tissue Stain and in Masson's Trichrome Stain. Phosphomolybdic Acid Hematoxylin also gives a fine coloration of collagenic fibers. See Van Gieson, Buzaglo, Bieb- rich Scarlet and Picro-Aniiin Blue of Lillie and Curtis' Substitute for Van Gieson. Lillie, R. D. (J. Tech. Methods, 1945, No. 25, 45 pp.) has performed a very useful service in testing the effective- ness of a large series of dyes as colla- genic fiber stains in the Van Gieson, Mixed Masson-Van Gieson and Masson- Mallory procedures. He found the best to be Naphthol blue-black (CI, 246), Fast Green FCF, Acid Fuchsin (CI, 692), Methyl Blue (CI, 706), Anilin Blue (CI, 707), Wool Green S (CI, 737) and Volamine R (CI, 758) . For photometric histochemical determination see Sto- well, R. E., J. Invest. Derm., 1945, 6, 183-189. The technique of microincineration as adapted to collagenic fibers is de- scribed by AUara, E., Bull. d'Hist. AppL, 1938, 15, 220-242. See Tendons. Collargol, as negative stain for spirochetes (Harrison, Brit. Med. J., 1912, 2, 1547). Collodions. There are several. See U.S.P. XI. All are solutions of Pyroxylin. Colloxylin, see Pyroxylin. Colophonium, usually dissolved in turpen- tine is employed to mount sections. Not advised. Color Estimation. Accuracy in reporting differential stains and micro-chemical reactions yielding colors is highly de- sirable. The same holds for colors determined by naked eye inspection. A monograph, Ridgway, R., Color Standards and Color Nomenclature, Washington, D. C, 1912 with 53 colored plates, is the accepted standard for comparison. In general, however, it is desirable to achieve some measure of uniformity by limiting oneself when- ever possible to use of the terms recom- mended in the National Formulary VII. Washington: American Pharmaceutical Association, 1942, 690 pp., a publication which is available in most medical libraries: pink yellow greenish blue red olive-brown blue reddish orange greenish yellow purplish blue reddish brown olive bluish purple orange-pink yellow-green purple orange olive-green reddish purple brown yellowish green purplish pink yellowish orange green red-purple yellowish brown bluish green purplish red blue-green For accurate measurement of color employ Photoelectric Colorimeter or Photoelectric Microphotometer. See Hemoglobin Estimation. Color Index, p. xxvii. Color Preservation in museum specimens. Fix 24 hrs. or less in 10% formalin. Wash in running water 3-6 hrs. Stand in 2% aq. ammonia 5-10 min. which hastens return of original colors. Run- ning water another hour. Mount for permanent exhibition in mixture made as follows: Filter a sat. sol. antimony trioxide in aq. dest. (about 5 gm. per liter). To each 1000 cc. filtrate add 100 gm. potassium acetate, 100 gm. chloral hydrate and 50 cc. glycerin. Stir until completely dissolved (Meiller, F. H., J. Tech. Methods, 1938, 18, 57-58). Mallory (p. 380) recommends for this purpose the methods of Kaiserling and Jores. There are 3 Kaiserling solutions : 1. For fixation: Formalin, 40 cc; tap water, 2000 cc; potassium nitrate, 30 gm. and potassium acetate, 60 gm. Small specimens require 1-14 days. Large ones can be more uniformly fixed by vascular Perfusion. Sometimes it is advisable to inject fixative into central COLORS 87 CONCENTRATION parts of the tissue with a hypodermic syringe and long needle. Do not use too much pressure and be careful not to let any of the fixative spurt back into one's face. Before the next step wash in running water for about 12 hrs. 2. For color restoration: Place the tissue in 80% ethyl alcohol for 10-60 min. and watch for optimum coloration. If left too long in the alcohol the colors fade. Rinse in water and transfer to No. 3. 3. For final preservation: Change to glycerin, 500 cc. ; 1% aq. arsenious acid, 200 cc; tap water, 2300 cc; potassium acetate, 250 gms.; thymol, 2.5 gm. To obviate difficulty of dissolving the arsenious acid and to sterilize add 25 gms. arsenic trioxide to 2500 cc. water + the thymol crystals first ground up in a mortar and place in steam sterilizer for 6 hrs. Then add other substances. There are 2 J ores solutions. 1. For fixation: Chloral hydrate, 50 gms.; artificial Carlsbad salts (sodium sulfate, 22 gm.; sodium bicarbonate, 20 gm. ; sodium chloride, 18 gm. ; potassium nitrate, 38 gm.; potassium sulphate, 2 gm.), 50 gm.; formalin, 100 cc ; tap water, 1000 cc Allow to act 2-14 days depending on size, wash 12 hrs. in run- ning water. 2. For final preservation: Potassium acetate, 300 gm., glycerin, 600 cc; aq. dest., 1000 cc. Mai lory suggests fixation in Jores' first solution and preservation in Kaiser- ling's third solution. Colors, Interference, see Interference Colors. Columbium, see Atomic Weights. Concentration. 1. Tubercle bacilli in spu- tum. Nagy (A.H., J. Lab. & Clin. Med., 1939, 25, 67-71) having critically evaluated several techniques recom- mends Pottenger's Dilution-Flotation method. Shake equal parts sputum and 0.5% aq. sodium hydroxide for 10 min. Digest in water bath at 56°C. for 30 min. Add 1 ml. (= 1 cc.) hydrocarbon (gasoline or xylene), then 200 ml. aq. dest. and shake 10 min. Allow hydro- carbon to collect at top 15-20 min. Take up hydrocarbon layer in rubber bulbed pipette. Keep in vertical posi- tion until supernatant fluid separates from hydrocarbon, 5-10 min. Make smears from hydrocarbon and dry. Remove hydrocarbon by washing with ether. Stain with carbol fuchsin 3 hrs. or longer. Decolorize with acid alco- hol 30 sec. or less. If further decolor- ization is required employ 10% aq. sodium sulphite. Counterstain with 1% aq. picric acid or with methylene blue. The concentration of bacilli is about 33 times. Perhaps a modification of the method could be used for leprosy or- ganisms in emulsions of tissues. See also Pottenger, J. E., Am. Rec Tuberc, 1939, 40, 581. Concentration of tuber- cle bacilli in spinal fluids (Hanks, J. H. and Feldman, H. A., J. Lab. & Clin. Med., 1939, 25, 886-892). It is often necessary to concentrate for micro- scopic study objects which are not present in abundance and which might otherwise be overlooked. See exami- nation of Feces for ova of parasites, of Urine for sediment. 2. Leprosy bacilli for chemical analy- sis. Ra void's method for leprosy bacilli can perhaps be used for others. Rela- tively large masses of bacilli-laden cells are dissected away from neighboring uninvolved tissue and from necrotic tissue when present in the centers of the nodules. They are placed in a Wueller press without addition of any fluid. On exertion of pressure many of the cells are ruptured and the tissue fluid, together with cytoplasm, nucleo- plasm and some entire cells, passes through minute holes in the press and is collected, leaving most of the fibrous elements behind. Then a little saline solution is added and the material is ground up in sand and made up to a volume of about 50 cc. The sand is allowed to sediment out at the bottom of a centrifuge tube. The supernatant fluid is then centrifuged at low speed (300 r.p.m.). This throws all the rest of the debris to the bottom while the bacilli remain in suspension. The supernatant fluid, containing the bacilli, is again decanted and centrifuged at high speed (3500 r.p.m.) in an angle centrifuge for 1 hr. The supernatant fluid is discarded and the pasty material at the bottom of the tube, made up of bacilli, is diluted and washed by re- peated centrifugation in some experi- ments with saline solution and in others with distilled water. Beginning with a large nodule or with several small ones it is a simple matter to collect in 4 or 5 hrs. billions of bacilli. The pasty bacterial mass can be desic- cated and weighed in grams. For our experiments we used only the wet bacilli. When viewed en masse they appear dense white with a faint shade of gray. They are not yellow or even yellowish. Examination of a thick smear, made after washing in saline, shows myriads of bacilli without any trace of cellular material. The bacilli retain to a remarkable degree their characteristic morphology, as seen in sections and in smears of fresh tissue, and their acid-fast properties are not CONDENSER MANOMETERS 88 COPPER interfered with. After washing in distilled water until the supernatant fluid gave no precipitate when added to an aqueous solution of silver nitrate, the bacilli do not fuse together but still remain discrete bodies though their shape is different. (Cowdry, E. V., Ravold, A. and Packer, D. M. Proc. Soc. Exp. Biol. & Med., 1939, 41, 341- 345). See Floatation Techniques for in- testinal parasites. Condenser Manometers in differential pres- sure measurements (Hansen, A. T. and Warburg, E., Acta Physiol. Scand., 1951,22, 211-215). Congo Blue 3B, see Trypan Blue. Congo Corinth G or GW, see Erie Garnet B. Congo Red (CI, 370). Synonyms: Congo, cotton red. A, B or C, direct red C, R or Y. An acid dis-azo dye which is an excellent indicator and a useful stain. Matsuura, S., Fol. Anat. Jap., 1925, 3, 107-110 has obtained very fine coloration of the skin which he has illustrated in colors. Congo red is the basis of Kra- jian's stain for elastic fibers. See also Blackman, V. H., New Phytol., 1905, 4, 173-174 (uredineae); Merton, H., Arch. Protistenk., 1932, 76, 171-187; Cumley, R. W., Stain Techn., 1935, 10, 53-56 (negative stain for bac- teria), etc. Connective System. Provides both for the binding together of parts and for their separation one from another by capsules, membranes and other structures (see Cowdry, p. 429-466). It ranges all the way from Loose Connective Tissue and Fatty Tissue through Fibrous Connective Tissue and Tendons to Cartilage and Bone. Neuroglia is a special form of it. In general there are three components. Fibroblasts, Fibers and Tissue Fluid (ground substance). Cells of hematogenous and lymphatic origin may be present since blood vessels and lymphatics run in connective tissue pathways. See techniques under these headings, also Masson's Trichrome Stain, Mallory's Connective Tissue Stain, Phosphomolybdic Acid Hema- toxylin, Van Gieson, Buzaglo, Mallory- Heidenhain rapid one step stain, etc. Connective Tissue Cells, preservation of trypan blue and neutral red in those of loose connective tissue. Inject sub- cutaneously 5 cc. fresh sterile 1% aq. vital trypan blue (Coleman and Bell Co.) into a mature white rat weighing about 90 gras. and wait 48 hrs. Make up 0.02% certified neutral red (National Aniline in 0.9% NaCl). After slight etherization exsanguinate the animal. Inject neutral red into subcutaneous tissue of groin in several places near original puncture. After 3-5 min. re- move small blobs of edematous tissue. Tease these out on clean slides with aid of needles and filter paper. When corners are dry spread is ready for direct observation under cover glass or for fixation. Make up 10% formalin. Test it by addition of a drop or two of neutral red. If this turns orange add a little N/10 HCl until it becomes red. Fix in this formalin over night or for several days. Rinse in aq. dest. Coun- terstain in 1% fast green F.C.F. (War- ner-Jenkinson Co.) in 2% aq. acetic acid for ^1 min., pass through suc- cessive changes dioxan, 3-5 min. each. Agitate slightly. Mount in dioxan employing medium hardened diaphane (Will Corp.), redissolve in dioxan or pass through xylol and mount in balsam. Avoid alcohols. Note blue granules in macrophages and fine red granules in mast cells (Snook, T., Stain Techn., 1939, 14, 139-142). See Connective System. Contraction Bands, or waves, demonstration of in smooth muscle. Remove intestine of freshly killed cat, expose to air of room or rub with blunt end of scalpel. When preparations are made numerous con- traction bands will be seen. Contrast with this intestine of cat killed with chloroform and not excised until rigor mortis begins in which muscle fibers are fully extended (Dahlgren, McClung, p. 430). Coons, see Antigen Localization. Copper. 1. Microchemical tests. Fix in formalin or alcohol, use same hema- toxylin or methylene blue stain as for Lead. With former copper hemofuscin is blue and hemosiderin (iron pigment) is black, while with latter copper pig- ment is pale blue and the iron pigment uncolored (Mallory, F. B. and Parker, F., Jr., Am. J. Path., 1939, 15, 517-522). See also Okamoto, K., Utamura, M. and Mikami, G., Acta Sch. Med., Univ. Imp. in Kioto., 1939, 22, 335-360 (il- lustrated in colors); Mendel, L. B. and Bradley, H. C, Am. J. Physiol., 1905, 14, 313-327 (bromine test for) ; Claude, A., Cold Spring Harbor Symposia on Quantitative Biology, 1941, 9, 263-270 (copper of respiratory pigment) ; Hoag- land, C. L. et al., J. Exper. Med., 1942, 76, 163-173 (copper and other substances in virus elementary bodies). Water- house, D. F., Bull. Council for Scientific and Industrial Research, Melbourne, Australia, 1945, No. 191, 20 pp.: add 1 drop 0.1% aq. sodium diethyl dithio- carbamate to fresh tissue followed by 1 drop cone, hydrochloric acid which yield yellow product with copper. Iron interferes with this result but the sen- sitivity for copper is much greater than COPPER CHROME HEMATOXYLIN 89 CORONARY ARTERIES for iron (quoted from Click p. 23). When search is necessary for traces of copper without need for microscopic localization an emission spectrograph may give the information qucikly, see Histospectrography. If quantitative determinations of copper in small amounts of tissue are required use the polarographic technique elaborated by Carruthers, C, Indust. and Engin. Chem., 1945, 17, 398-399. Details for determination of copper in epidermis are given bj^ Carruthers, C. and Sunt- zeff, v., J. Biol. Chem., 1945, 159, 647- 661. 2. As vital stain. Intravenous in- jections of colloidal solutions of copper in rabbits are described by Duhamel, B. G., C. rend. Soc. de Biol., 1919, 82, 724-726. Copper Chrome Hematoxylin (Bensley's) for mitochondria. Fix very small pieces in Altmann's Fluid or in Acetic-Osmic- Bichromate fixative of Bensley 12-24 hrs. Wash, dehydrate, clear, imbed in paraffin ana cut sections at 4 or 5 mi- crons. Deparaffinize. Sat. aq. copper acetate, 5 min. Several changes aq. dest., 1 min. 0.5% aq. hematoxylin, 1 min. After rinsing in aq. dest. pass to 5% aq. neutral potassium chromate, 1 min. which should turn sections dark blue-black. If they are only light blue, rinse in aq. dest. again place in copper acetate and repeat if necessary several times until no increase in color is ob- tained. Wash in aq. dest. and treat for few sec. with copper acetate. Wash in aq. dest. and differentiate under the microscope in Weigert's borax-ferri- cyanide mixture (borax, 1 gm. ; potas- sium ferricyanide, 1.25 gm.; aq. dest. 100 CO.) diluted with twice the volume aq. dest. Wash in tap water, 6-8 hrs. Dehydrate, clear and mount in balsam. The mitochondria appear a beautiful deep blue against a yellowish back- ground. It is important to have good, ripe hematoxylin. It is usually made by dilution from a 10% sol. in abs. al- cohol. This method of staining should be tried after fixation in Regaud's fluid. Coproporphyrin of megaloblasts in embryos, see Porphyrins. Coproporphrin III can be isolated from human tubercle bacilli by chromatographic and fluorescence analysis (Crowe, M.O'L., and Walker, A., Brit. J. Exp. Path., 1951, 32, 1-6). Coreine 2R, see Celestin Blue B. Corinth Brown G, see Erie Garnet B. Coriphosphine O (CI. 787). An acridine dye used as a fluorchrome (Metcalf, R. L. and Patton, R. L., Stain Techn., 1944, 19. 11-27). Corn Blue B, see Victoria Blue R. Corn Blue BN, see Victoria Blue B. Cornea. This is a difficult tissue to prepare in stained sections because of its curva- ture and avascularity. A valuable sil- ver method is minutely described by Pullinger, B. D., J. Path, and Bact., 1943, 55, 97-99. Fix anterior and posterior surfaces in 10% aq. neutral formalin before ex- cision of eye, if possible, by flooding anterior surface with fixative and by injecting fixative into anterior chamber through a hypodermic needle at the same time withdrawing fluid from the chamber likewise bj' hypodermic. Re- move eye, inject fixative into vitreous at same time removing fluid from it. Leave in fixative 24 hrs. Excise cornea along corneo-scleral margin, detach iris, ciliary body and lens. Fix latter separately and cornea for further 3 days, 4 altogether. Indicate location future sections by nick in opposite edge. Transfer cornea to aq. dest. avoiding metal instruments then and thereafter. Wash and leave over night in aq. dest. + 3 drops ammonia (S. G. 0.88) per, say, 50 cc. After washing in 2 or 3 changes aq. dest., and pouring off last aq. dest., filter onto cornea through cotton wool moistened with aq. dest. 20 cc. del Rio- Horiega's solution. To make this add 300 cc. 5% aq. sodium bicarbonate to 100 cc. 10% aq. silver nitrate in brown glass stoppered bottle. Add few drops ammonia waiting each time for smell of ammonia to disappear until almost but not all ppt. is dissolved. They add 250 cc. aq. dest. Place container with cornea plus fil- tered solution in incubator at 37°C. 4 hrs. Pour off solution and wash cornea in several changes aq. dest. Then reduce by pouring onto cornea 10% aq. neutral formalin, 15 min. Cut away "dome" of cornea with knife and support its concave surface with the lens, freeze and section at about 15 /x. parallel to surface. Take sections into aq. dest.; mount at once in glycerin jelly or pass through alcohols to abso- lute, clear in creosote and mount in balsam. Collagen pale yellow, nuclei and cytoplasm well shown and espe- cially Descemets membrane. Cornyebacterium Diphtheriae. Evaluation of methods for staining metachromatic granules (Morton, H. E., Stain Techn., 1942, 17, 27-29). See Gobar, M. A., J. Bact., 1944, 47, 575, also Diphtheria Bacilli. Coronary Arteries. Their distribution may be demonstrated by injection of the easily recognizable fluids listed under Blood Vessels. Owing however to their great importance it is well to mention two special adaptations of the said fluids. CORPORA AMYLACEA 90 GROSSMAN'S Gross (L., The Blood Supply of the Heart in its Anatomical and Glinical Aspects. New York: Iloeber, 1921) employed injections of barium sulphate suspensions in gelatin followed by x-ray photographs; while Spalteholz (W., Die Arterien der Herzwand, etc., Leipsig: Herzel, 1924) used injections of cinnabar and other pigments likewise in gelatin which were later cleared by his method. Ehrich, Ghapelle and Gohn (W., C., and A. E., Am. J. Anat., 1931, 49, 241-282) found the latter technique preferable. Celloidin injections also give good results. Histological demon- stration of the blood supply of the coronaries is described under Vasa Vasorum. Corpora Amylacea, see Prostate. Corpuscles, see those of Pacini, Meissner, Krause, etc. Corrosion Preparations. In them the struc- tures to be demonstrated are left while all the surrounding tissue is corroded and washed away, for instance Celloidin and Neoprene injections. Corrosive Sublimate, see Mercuric Chlo- ride. Corti, organ of, see Ear. Cortin (interrenalin), hormone of adrenal cortex. Cotton Blue, see Anilin Blue, Methyl Blue. See Fungi. Cotton Corinth G, see Erie Garnet B. Cotton Red, see Safranin O. Cotton Red, A, B, or C, see Congo Red. Cotton Red 4B, see Benzopurpurin 4B. Covell, see Ear. Cover Glasses, see Cleaning. Creatine and Creatinone, see colorimetric methods of Borsook, H., J. Biol. Chem., 1935, 110, 481-493 and of Sure, B. and Wilder, V. M., J. Lab. Clin. Med., 1941, 26, 874-878. Cresol Red. See Hydrogen Ion Indicators. Creosote (Beechwood) is an important clearing agent for celloidin sections. It is a mixture of phenols, mainly guaiacol and creosol. Cresyl Blue 2RN, or BBS, see Brilliant Cresyl Blue. Cresyl Violet — cresylecht violet (cresyl fast violet) — Commission Certified. A basic oxazin dye. A technique for its use (or that of toluidin blue) in studies on the cytoarchitectonics of the nervous system is proposed by Landau, E., Bull. d'Hist. Appl., 1934, 11, 44^6. As a stain for nerve cells in celloidin sections (Tress, G., and M., Stain Tech., 1935, 10, 105-106). Wash low viscosity nitrocellulose (celloidin) sections of 10% formalin fixed tissues in aq. dest. Stain for 30 min. at 50°C. in cresyl violet, 0.5 gm.; aq. dest., 100 cc. ; glacial acetic acid, 4 drops (filtered before using). Wash in aq. dest. Differentiate in 70% alcohol until most of stain leaves cel- loidin. Completely immerse for 2-5 min. in: chloroform, 60 cc; abs. ale, 10 cc; and ether, 10 cc. Almost no destaining of cells occurs but stain is removed from background. Differen- tiate in 100 cc. 95% ale + 4 drops 1% aq. hydrochloric acid but stop while cells are a little darker than desired. Neutralize sections in 90% alcohol + a little sodium bicarbonate. Wash in 95% alcohol to remove the bicarbonate. Complete dehydration in 2 changes n butyl alcohol. Clear in 4 changes xylol and mount. See Kallichrom. Note: There are two different dyes sold as cresyl violet: (1) The CC. product (Nat. Anilin, Mfgrs.; see Conn, 1940, p. 93) which is good in biopsy work; (2) The Grtibler product (also sold by Coleman and Bell, but not on the market during the war) which is needed in neurological work, cf. Tress, above. Cresylecht Violet, intensification of meta- chromatic properties (Williams, B. G. R., J. Lab. & Clin. Med., 1934-35, 20, 1185-1187). A new domestic cresylecht violet has been made available by Cole- man and Bell and is stronglj'- recom- mended by Banny, T. M. and Clar, G., Stain Techn., 1950, 25, 195-196. Cretin's Test, see Calcium. Crime Detection Techniques. These are of course legion. Many of them are mi- croscopic and involve identification of materials. See for example, Hair, Semen Stains and Hemoglobin. In re- spect to the latter the object is to deter- mine whether blood is human by pre- cipitin tests and to which group it be- longs by detection of agglutinins as is well described by Schiff, F. and Boyd, W. C, Blood Grouping Technic New York: Interscience Publishers, Inc., 1942, 248 pp. The identification of metals, such as chips from a razor blade, by spectroscopic examination is often conclusive, see Histospectrography. Cracks in metal surfaces can be de- tected with astonishing delicacy by the Magnaflux. An interesting elementary account of Crime Detection Techniques is provided by Hoover, J. E., Scientific Monthly, 1945, 60, 18-24. Croceine Scarlet, see Biebrich Scarlet, water soluble. Grossman's modification of Mallory's con- nective tissue stain (Grossman, G., Anat. Rec, 1937, 69, 33-38). Deparaf- finize sections of Zenker fixed material. Lugol's iodine, 5 min. Rinse in 70% alcohol, several changes. Wash 10 min. in running water. Overstain nuclei in Mayer's acid Hemalum or Weigert's Iron Hematoxylin. Wash in running CRYOSTAT 91 CURTIS' SUBSTITUTE water 10 min. Stain for 1 min. or more in: acid fuchsin (C.C.), 1 gm.; orange G (CO.), 0.4 gm.; aq. dest., 300 cc; thymol, 0.2 gm.; glacial acetic acid, 3 cc. Rinse in aq. dest. Decolorize in fresh 1% aq. phosphotimgstic or phos- phomolybdic acid until arterial media is red and adventitia is colorless. Rinse very quickly in aq. dest. Counterstain in 2% aq. anilin blue, W.S. (CC.) 100 cc. + glacial acetic acid, 2 cc. or in 1% aq. light green SF yellowish (CC.) 100 cc. + glacial acetic acid, 1 cc. Rinse in aq. dest. Decolorize in 1% acetic acid under microscope. Rinse in aq. dest. Dehydrate in 3 changes abs. ale. Clear in 3 changes .xylol and mount. Like original method but nuclei brown or black and collagen blue or green de- pending on counterstain. Cryostat. Written by Dr. Gordon H. Scott, Dept. of Anatomy, Wayne University, School of Medicine, Detroit, Mich. — This apparatus is one which is designed to dehydrate tissues at low tempera- tures. A detailed description has been given by Packer and Scott (J. Tech. Methods, 1942, 22, 85-96) and by Hoerr and Scott (Medical Physics, Otto Glas- ser, 1944, Year Book Publishers). Tis- sues frozen in liquid air or nitrogen are placed in a chamber which is connected with a fast pumping vacuum system. Water vapor which is released from the tissues is trapped by P2O5 as well as by a cold trap. In the Packer-Scott ap- paratus the relative amount of water vapor in the system is determined by its flow past a spaced pair of ionization gauges between which is placed the P2O5 trap. When these gauges are in balance it is assumed water vapor is no longer being released in quantity, and therefore the tissues are dry. As soon as the tissues, placed on a container of solid out-gassed paraffin, are dry, the paraffin is melted and the tissues are infiltrated. This latter procedure is accomplished without the necessity of breaking the vacuum. This small but important step provides tissues which have been frozen-dried and pre- pared for cutting without their having been partially rehydrated by exposure to air at ambient pressure and temper- ature. See Altmann-Gersh frozen de- hydration method. Cryptococcus Hominis, see Blastomycosis. Cryptosporidium, see Coccidia. Crystal Violet as vital stain for fibroblast nuclei (Bank, O. and Kleinzeller, A., Arch. exp. Zellforsch., 1938, 21, 394- 399). See Anilin Crystal Violet and Gentian Violet. Crystal Violet-Acid Fuchsin. This is one of R. R. Bensley's neutral stains espe- cially advocated for the demonstration of secretion antecedents in gland cells. The technique is described by the Bensleys (p. 97). To make stain add filtered sat. aq. acid fuchsin to similar solution crystal violet until precipita- tion is complete. Collect ppt. on fdter paper, wash through once with aq. dest. Dry and dissolve in absolute alcohol to saturation. For staining add 5 cc. of above stock solution to 45 cc. 20% alcohol made from absolute. In this color paraffin sections of Formalin- Zenker fixed material for 5 min. Blot with filter paper in one hand and add with other hand absolute alcohol from a pipette, flood with absolute. Blot immediately. Add few drops clove oil. When differentiation, observed under microscope, is optimum transfer to pure benzol and mount in balsam. Crystal Violet and Alizarin, see Benda's Method for Mitochondria. Crystals. These are encountered in many forms, see Charcot-Leyden, Ice, Sul- fonamides, Hemin, Florence, Virchow's, Spermin, Lubarsch, Neumann's, Teich- mann's, Mineral residue of Microincin- eration, Polarization Optical methods. Numerous Microchemical Reactions especially for minerals, yield crystal- line materials. Fat crystals are often distinctive as for beef, duck, lard, etc. (Schneider, A. The Microbiology and Microanalysis of Foods. Philadelphia: P. Blakiston's Son & Co., 1920, 262 pp.) . Study of crystals is really a problem for experts. For the best techniques con- sult Section I on "Identification" in Bunn, C. W., Chemical Crj^stallog- raphy, Oxford University Press, 1946, 234 pp. Comparison of the crystals to be diagnosed with some of the 234 figures in the book may result in prompt recognition. Culture Media, see Bacteria, Leishmania, Protozoa, Tissue Culture, Trypano- somes, NNN Medium. Curcumine, see Brilliant Yellow. Curettings, gelatin method for rapid frozen sections of (Meeker, L. II., J. Techn. Meth. & Bull. Int. Assoc. Med. Mu- seums, 1936, 16, 41-42). Curtis' Substitute for Van Gieson stain as modified by Leach, E. H., Stain Techn., 1946, 21, 107-110. Use any desired fixative. Bring sections to water and treat with iodine and hypo (sodium thiosulfate) if necessary. Stain for 5- 10 min. in Weigort's hematoxylin. To make this mix (just before use) 1 part 1% hematoxylin in absolute alcohol with 1 part of mixture containing 30% aq. ferric chloride 4 cc, cone, hydrochloric acid, 1 cc. 2% acetic acid 100 cc. and add 2 parts aq. dest. Wash for 5 min. in CYANOCHIN 92 DARK-FIELD MICROSCOPE running water. Stain 2-4 min. in Cur- tis' substitute: 2% Ponceau S, CI, 282, (National Aniline) 5 cc; sat. aq. picric acid, 95 cc. ; 2% acetic acid, 2 cc. Rinse in 96% alcohol, dehydrate, clear and mount. Chromatin, black; cytoplasm, yellow; collagen and reticular fibers, red. Red and yellow colors are said to be purer than those given by the Van Gieson technique and too heavy staining with red is less likely. In original account volumes are given in ml. which are of practically the same value as cc. Cyanochln of Bresslau, E., Arch. f. Protis- terik., 1921, 43, 467, see Cyanosine. Cyanosine, see Phloxine B. Cyclohexanone has been recommended for dehydration and clearing instead of absolute alcohol and xylol by Bourdon, P., Bull. d'Hist. Appl., 1942, 19, 55. After dehydrating tissue in 95% alco- hol, 12 hrs.; pass to cyclohexanone, 4 hrs.; then to another lot of cyclo- hexanone, 2 hrs.; and impregnate with paraffin 2 baths 2 hrs. or le.ss each. For pieces more than 3 nmi. thick longer times are necessary. This saturated cyclic ketone has density similar to water, mixes with organic solvents and paraffin and does not harden tissue. From Review by Jean E. Conn in Stain Techn. Cyclospora, see Coccidia. Cytocentrum, centrosome plus centrosphere. Cytochrome. This is the name given by Keilin (D., Proc. Roy. Soc, 1925, B, 98, 312-339) to hemin compounds of a reddish color which occur in oxidized or reduced condition in almost all living cells. Blaschko and Jacobson (Bourne, p. 192) have summarized our knowledge about them. They say that the red color of cytochrome can be observed when a slice of brain tissue, from which the blood has been carefully washed out, is suitably illuminated by transmitted light. A thick suspension of yeast and the thoracic muscles of insects are also recommended as material. There are 4 cytochromes : a, b, c and as recog- nizable spectroscopically. Cytochrome Oxidase. Cytochrome is ox- idized by cytochrome — oxidase which is identical with indophenol o.xidase and Warburg's respiratory enzyme. See study of cytochrome oxidase-cyto- chrome system in kidne}^ (Flexner, L. B., J. Biol. Chem., 1939, 131, 703-711). See Oxidase. Cytolipochrome Pigment, see Lillie, p. 127. Cytophaga Group of organisms, enrichment cultures, pure culture techniques, methods of examination and identifica- tion (Stanier, R. Y., Bact. Rev., 1942, 6, 143-197). Cytoplasmic Inclusions caused by viruses. They are more diversified in size, shape and chemical composition than the Nuclear Inclusions. Frequently, as in the case of large Negri Bodies, they contain both acidophilic and basophilic components (Trachoma Bodies). Gly- cogen tests for Trachoma inclusion bodies are described by Thygeson, P., Am. J. Path., 1938, 14, 455-462. The techniques mentioned for Nuclear In- clusions may be employed. See de- scription by Goodpasture, E. W. and Woodruff, A. M., Am. J. Path., 1930, 6, 699-711 ; 713-720 of the reactions of fowl-pox inclusions to potassium hy- droxide and other chemicals and the nature of the particles. See also Borrel, Guarnieri and KurlofiT bodies. Rickett- sia are not to be listed as cytoplasmic inclusions but Giemsa's stain is the best for them. In plant cells, as in animal ones, cer- tain cytoplasmic inclusions are indica- tive of virus action. They are of two sorts: (1) X bodies, which are rather amorphorus structures, and (2) crystal- line inclusions. The latter are best seen in the dark field and in polarized light and are made up chiefly of virus. For technique employed to demonstrate the relationship of virus to inclusion and a critical review of the whole prob- lem of plant viruses, see Bawden, F. C. Plant Virus Diseases, Waltham: Chronica Botanica Co., 1943, 294 pp. Cytosiderin Pigment, Lillie, p. 127. Dahlia, see Hofmann's Violet. Dahlia B, see Methyl Violet. Damar is gum damar dissolved in xylol and used to mount sections. D'Antoni's Iodine solution. First make standardized 10% aq. potassium iodide. Adjust by specific gravity method to exact 10% concentration. To 100 cc. of 1% aq. potassium iodide made from it add 1.5 gm. powdered iodine crystals. Allow to stand for 4 days before using. Recommended for staining intestinal protozoa (D'Antoni, J. S., Am. J. Trop. Med., 1937, 17, 79-84). See McClung, 1950, p. 450. Dark Brown Salt R, see Chrysoidin Y. Dark-Field Microscope (From account in Cowdry's Histology, 1950). In dark- field microscopy the light comes in at an angle, and does not enter the ob- jective lens at all. It may be thought of as oblique illumination carried to the limit of obliquity. If there is nothing in the field of view the background will of course be perfectly dark; but the presence of objects will result in the reflection of some light laterally into the objective lens. Since only a minute fraction of- the illumieation beam of DARK-FIELD MICROSCOPE 93 DAVENPORT'S light will go toward the formation of an image this system will require a very strong light source as well as a dark- adapted eye for observation. If a spe- cial dark-field condenser is not avail- able, one can be easily improvised by adding a central opaque stop just be- low the regular condenser in the fol- lowing manner: Select an objective of not over 0.66 N. A. and a condenser of at least 1.25 N. A. and set up the illumination as for bright field, with the slide oiled to the top of the condenser. Stop down the iris diaphragm until it just encircles the bright central disk which is seen through the tube with ocular removed. Now cut from black paper or thin metal a disk of diameter equal to the diameter of this opening in the diaphragm and cement it to a piece of glass that fits the insertion slot under the condenser. This central stop should block all light from entering the objective if it is properly centered below the iris open- ing. Next open the diaphragm com- pletely, thus permitting a hollow cone of light to focus on the specimen but not to enter the objective. If the microscope is already supplied with a dark-field stop it should be tested in this manner. The great advantage of dark-field microscopy is that it enables one to per- ceive the presence of particles far be- low the theoretical limit of resolution. Even though a particle may be much smaller than the wave length of light it does scatter incident light laterally into the objective and is imaged as a rather fuzzy point of light. We have here an exact analogy of the situation whereby we can "see" dust particles dancing in a strong beam of light when it enters a darkened room. Most of these particles are of a size far below the limit of visual resolution. Another advantage is that small, transparent objects, like chylo- microns, can be seen in dark-field whereas they are invisible in the glare of bright-field illumination. For the examination of ordinary his- tological sections little is to be gained by dark-field observation. Neverthe- less Ralph, P.H. (Stain Tech., 1942, 17, 7-10) recommends dark-field examina- tion in the study of stained blood smears. It is most helpful however in the search for very small, scattered bodies in a more or less empty medium, such as chylomicrons or spirochetes in blood plasma. Dark field examination is the standard technique for the study of microincineration preparations. The cytological results of 20 years of of microincineration have been summa- rized by Policard, A. (J. Roy. Micr. Soc.,1942, 42, 25-35). The source of the light must be very, very strong. To obtain the best results do not rely on improvised, or home made, equipment. A special condenser is needed. Probably the highest ob- jective that can be usefully employed 18 a 3 mm. oil immersion objective with iris diaphragm. See Microincineration, Spirochetes, Chylomicrons, etc. Davenport's 2-hour method for staining nerve fibers in paraffin sections with protargol. 1946 modification written by Dr. H. A. Davenport of original (Davenport, H. A., McArthur, J., and Bruesch, S. R., Stain Techn., 1939, 14, 21-26). Fix for 1 to 3 days in: Form- amide (Eastman Kodak Co.), 10 cc; paranitrophenol, 5 gm.; 95% ethyl alco- hol, 45 cc; aq. dest., 45 cc. Transfer thru graded alcohols to absolute, then either n-butyl alcohol or xylene and embed in paraffin. Sections are cut and mounted in the usual manner, paraffin removed and the slides run thru graded alcohols to dist. water. Impregnate for 1 hr. at 58-^2 °C. in a 5% aq. silver nitrate. Rinse in 3 changes of aq. dest. with 20-30 sec. allowed for each change. The rinse water should cover the slides completely, each slide sepa- rate (not back to back) and the water discarded with each change to prevent carry-over of silver nitrate into the protargol. Place the slides in 0.2% protargol (Winthrop Chemical Co.) for 1 hour at room temperature. Rinse quickly (2 sec.) in aq. dest. and reduce for 1 to 2 min. in the following mixture : Sodium sulfite, 5 gm. ; Kodalk (Eastman K. Co.), 0.5 gm.; hydroquinone, 1 gm.; aq. dest., 100 cc. Wash in running tap water several minutes and rinse once in dist. water. Tone in 0.1% aq. gold chloride for 5 to 10 min. Wash again for about 1 min. and reduce in 1% aq. oxalic acid for 10-20 sec. Rinse and place in hypo (10% aq. sodium thio- sulfate) for about 1 min. Wash in run- ning water, dehydrate and cover. Notes: If the stain is too dark, try any or all of the following modifications : rinse longer after the protargol, use 0.1% protargol, omit the oxalic acid re- duction after gold toning. If too pale: double the concentration of the pro- targol, double the time of either or both silver impregnations, omit rinsing after protargol, double the concentration of kodalk in the reducer, lengthen the time of reduction in oxalic acid. The technic is suitable for mammalian cen- tral or peripheral nervous tissue, but for sympathetic fibers in intestine and uterus a moderate degree of success has DEAD BACTERIA 94 DECALCIFICATION been obtained with material fixed in Bouin's picric-formalin-acetic (aq.)- Use clean glassware and fresh solutions! Dead Bacteria. To distinguish from living try: 1. Proca-Kayser stain (Gay, F. P. and Clark, A. R., J. Bact., 1934, 27, 175- 189). Fix bacterial smear by drying and flaming. Stain 3-5 min. in Loef- fler's alkaline methylene blue. Wash quickly and stain in Ziehl-Neelsen's carbol fuchsin only 5-10 sec. Wash and dry. Living bacteria blue, dead ones purple to red. 2. Neutral red (Knaysi, G., J. Bact., 1935, 30, 193-206). Add a little neutral red to the medium. Escherichia coli and Schizosaccharomyces pombi are considered dead when tinged even slightly by the stain. 3. Decolorization (Prudhomme, R. O., Ann. Institut Pasteur, 1938, 61, 512- 518). Living bacilli separated from all tissue decolorize solutions of 1-naphthol- 2-sodium sulphonate-indo-2-6-dibrom- phenol, O-cresol-2-6 dichlorophenol and 0-chlorophenol-indo-2-6-dichloro- phenol. Bacilli killed by 100 °C. for 15 min. do not decolorize them. The value of these methods is questionable. Dead Cells. Often it is very difficult to say whether a particular cell was dead or alive when the preparation was made. The appearance of nuclei in Postmortem Degeneration may be a clue. Evans and Schulemann (H. M. and W. Science, 1914, 39, 443-454) remarked upon the extraordinary rapidity with which dead cells take in vital benzidine dyes and the diffuse, uniform coloration that ensues. In cells supravitally stained with neutral red Lewis and McCoy (W. H. and C. C, Johns Hopkins Hosp. Bull., 1922, 33, 284-293) employed the follow- ing criteria for death: " (1) loss of color from the granules and vacuoles; (2) diffuse pink staining of the cytoplasm and nucleus; (3) the appearance of a sharp and distinct nuclear membrane and a change in texture of the cyto- plasm and nucleus." Using dark-field illumination W. H. Lewis (Anat. Rec, 1923, 26, 15-29) observed the appear- ance in dying cells of certain very small brightly shining (white) bodies which he called d or "death granules." These were first in Brownian movement which soon ceased. To quote Lewis: "During the period when the cells were dying, spherical blebs often appeared on both the flat and rounded cells. These were pale grayish sacs with very thin walls and fluid contents in which va,rying numbers of small white granules in ac- tive Brownian motion were seen. The blebs varied in size and were occasion- ally as large as a contracted cell. Sometimes the blebs were so crowded with granules that they were milky in appearance. Frequently one would burst, freeing its granular contents into the surrounding fluid medium where they showed Brownian motion until they settled down on the slide." Luyet's (B., Science, 1937, 85, 106) method for the differential staining of living and dead plant cells may prove of value for animal cells also. He has written the following account: A piece of the lower epidermis of the scale of the onion bulb is peeled off and placed, cutin side down, on a sHde. A drop of a .5 per cent, slightly alkaline, aqueous solution of neutral red is deposited on the piece of epidermis and left there for 2 minutes; then it is blotted off and re- placed by a drop of a .4 per cent potas- sium hydroxide solution, which is imme- diately removed (also with a blotter) ; then the preparation is washed with tap water. The living cells take with that treatment a bright cerise red color, while the dead cells are of an intense orange yellow. The contrasts are vio- lent. There are intermediate tints which correspond to the dying cells. See Necrosis, Necrobiosis, Survival of tissue. Decalcification. The removal of calcium so that bony tissues can be cut in sections. There are many methods almost all of which involve acid treatment. It is generally better to apply the de- calcifying agent after fixation, particu- larily so when the agent is a poor fixative. The volume of decalcifier should be about 100 times that of the tissue. The usual, crude, way of testing the progress of decalcification is to stick a fine needle into the bone being careful to avoid the area that will be cut in sections; but less objectionable methods can be used, see Teeth, Decalcification. Saturated aq. sulphurous, 5% tri- chlorlactic, 5% hydrochloric and equal parts of 1% hydrochloric and 1% chromic acids are all fairly good de- calcifiers. Lactic, acetic, phosphoric and picric acids are usually unsatis- factory. Shipley (McClung, p. 347) recommends slow decalcification by long immersion in Muller's Fluid through liberation of small amounts of chromic acid from the bichromate. The bones of an adult rat require 21-30 days. The process can be hurried somewhat by using an incubator at 37°C. Adequate decalcification is de- tected by slight bending of the bone or DEGENERATION 95 DEHYDRATION by the needle method. Over decal- cification is not likely. For rapid decalcification he advises using sat. aq. phloroglucin to which 6-30% Nitric Acid is added. A some- what slower formula is : nitric acid, 5 cc; phloroglucin, 70 cc; 95% ale, 1 cc; and aq. dest., 30 cc. The phloro- glucin allows use of stronger acids. 1-2% aq. hydrochloric acid decalcifies quickly but it causes the tissue to swell. Formic Acid 1-5% in 70% alcohol is, according to Shipley, the best decal- cifying agent for large masses of bone. With 5%, the decalcification is com- pleted in 4-5 days. Use 70% ale. not water, to wash out the acid. Kramer and Shipley devised a Magne- sium Citrate method of decalcification in neutral solutions. To make the de- calcifier dissolve 80 gms. citric acid in 100 cc. hot aq. dest., add 4 gms. magne- sium oxide and stir until completely dissolved. If the magnesium oxide contains carbonate it should be freshly ignited. Cool and add 100 cc. ammonium hydroxide (density 0.90) and aq. dest. to make 300 cc. Allow to stand 24 hrs. and filter. Titrate filtrate with hydrochloric acid to about pH 7.0- 7.6 and add equal volume aq. dest. In decalcifjdng, this reagent should be changed every 3 days. A dog's rib is decalcified in approximately 15 days. After decalcification, by whatever method, the bone, or the area of calcifica- tion, must be thoroughly washed to remove the decalcifer and imbedded in paraffin or celloidin. Some investiga- tors prefer the latter but celloidin sections are not so easily handled. See Bones, Teeth. The ion exchange resin technique for bone is new and interesting (Dotti, L. B., Paparo, G. P. and Clarke, B. E., Am. J. Clin. Path., 1951, 21, 475-479). Try keeping tissue in cork stoppered bottle containing 10 gms. of the resin (Win-3000 supplied by Winthrop- Stearns, Inc. 1450 Broadway, New York 18) in 80 cc 10-20% formic acid for 1-4 days. The optimum time should be determined for the particular kind of bone and the size of piece. The authors supply helpful figures illustrating the dependence of intranuclear details on time of decalcification. Degeneration. Because the structural or- ganization of various sorts of cells is, like their function, so very different the types of degeneration leading to death are also different at least in many of their aspects. See Nerve Fiber Degeneration, Cloudy Swelling, Necro- sis, Caseation, Parenchymatous Degen- eration, Postmortem Changes. Dehydration is the removal of water from a tissue preliminary to clearing and paraf- fin or celloidin imbedding. This is routinely done by treating the tissue after Fixation and Washing by passing it through a series of ethyl alcohols of increasing concentration. Usually the percentages are 30, 50, 70, 80, 95 and absolute. The time depends upon the size and kind of the tissue and the sort of fixative. For slices of tissue less than 3 mm. thick the dehydration can be accomplished in 6-12 hours. The alcohols for large slices fixed say in Zenker's fluid are ordinarily changed every morning and evening, but it is not desirable to leave them in absolute alcohol very long because it makes them brittle. Three to 6 hours should be sufficient. Tissues fixed in alcoholic solutions take a shorter time to de- hydrate. After fixation in alcohol- formalin or in Carnoy's fluid the tissue can be dehydrated and partly washed in several changes of absolute alcohol skipping the lower grades of alcohol entirely. When, for some reason, it is desired to eliminate treatment with absolute alcohol the tissues can be passed directly from 95% alcohol into Aniline Oil (say 30 min.) which is itself later removed, at least partly, in 5-10 minutes by washing in 2 changes of chloroform. Clearing is continued in chloroform for imbedding in paraffin, or the tissue may be passed from 95% alcohol, even from 80%, into Terpineol and cleared in half terpineol and xylol. Still another way to avoid absolute alcohol is to transfer from 95% alcohol to Bergamot Oil which serves as a clearing agent. Several substitutes for ethyl alcohol as a dehydrating agent are available. Acetone is the best known. This is strongljr recommended by R. D. Lillie, p. 43. Four changes of acetone each lasting 40 minutes are suggested for routine work but this can be reduced to 4 changes each 20 min. Thereafter pass, the blocks of tissue to a paraffin solvent such as benzene, toluene or xylene before placing in melted para- ffin. The schedules which he provides (p. 46) for dehydration, clearing and infiltrating with paraffin are useful. See Cellosolve. Dioxan will not only take the place of the alcohol but also that of the clear- ing agent so that it is possible to greatly' simplify the technique and make the sequence: fixative to dioxan to paraffin. See Dioxan and note as to possible danger to those using it. Cellosolve has also been proposed as a dehydrating agent. Lee (p. 64) says that it is ex- DEHYDROGENASE 96 DESOXYRIBONUCLEIC ACID pensive, inflammable and quickly takes up water from the air. Wtiether it is injurious when breathed remains to be determined. On the whole it ap- pears that little is to be gained by such substitutes. However, Cyclohexanone deserves further trial. If alcohol must be avoided it is always possible to fix in formalin and to use frozen sections. By the Altmann-Gersh technique the tissues are dehydrated in vacuo while still frozen. Dehydrogenase — Written by E. W. Demp- sey, Dept. of Anatomy, Washington University, St. Louis. February 26, 1951 — Sometimes used synonomously with Oxidase, since it is an enzyme which catalyzes the transfer of hydro- gen from one substance to another, and therefore operates to reduce one ma- terial and simultaneously to oxidise another. A variety of dehydrogenases are recognized, depending upon the sub- strate which acts as the hydrogen donor (e.g. succinic, malic, lactic dehydrogenase). Dehydrogenases have been measured in slices or homogenates of tissues, particularly with reference to malignancy (Greenstein, J. P., Bio- chemistry of Cancer, New York: Aca- demic Press, 1947, 389 pp. ) and to changes in the reproductive cycle, a review of which is presented in Chapter 4 of The Enzymes, New York: Academic Press, 1950, Vol. 1, Part 1, 724 pp. Methylene blue and other dyes have been used as hydrogen acceptors in the dehydrogenase systems. The rate of decolorization of the dye serves as an index of activity. Recently, tet- razolium salts, which form colorless solutions but which upon reduction are converted into colored, insoluble formazans, have been used to localize the enzymes in tissue sections or blocks. Rutenberg, A. M., R. Gofstein and A. M. Seligman, Cancer Research, 1950, 10, 113-121 present a review of the use of tetrazolium salts and methods for studying both non-specific and spe- cific dehydrogenase systems in living tissue and in frozen sections. These methods are still somewhat lacking in histological precision, but are provid- ing interesting data showing altered concentrations of the enzyme in states of different physiological activity (Zweifach, B. W.,'M. M. Black and E. Shorr, Proc. Soc. Exp. Biol, and Med., 1950, 74, 848-854). See Succinic De- hydrogenase and Triphenyltetrazolium Chloride. Dehydropyridines. Warburg noted a marked whitish fluorescence in ultraviolet light. Blaschko and Jacobson (Bourne, p. 196) report that the pyridines do not show this fluorescence and that the small gran- ules that exhibit it in sections of living liver tissue may well be dehydropyri- dines. Their brilliant white fluores- cence quickly fades. Delafield's Alum Hematoxylin. To 400 cc. sat. aq. ammonia alum add 4 gms. hematoxylin dissolved in 25 cc. 95% ale. Leave exposed to air and light 4 days. Add 100 cc. methyl ale. and 10 cc. glycerin; filter. Filtrate will slowly ripen. To hasten ripening add 10 cc. hydrogen peroxide. Delta Dye Indicator, see Nitrazine. Dempsey, see Phosphatases, Esterases, En- zymes, Dehydrogenase, Nucleases. Dempster, see Shaddow Casting. Density Determinations. Technique de- scribed for amebae is by use Starch Density Gradient (L0vtrup, S., C. rend. Lab. Carlsberg, S6r. Chim., 1950, 27, 137-144). Dental Enamel, see Enamel. Dentin. Can be studied in ground sections of undecalcified teeth as well as in paraffin and celloidin sections of de- calcified ones (see Teeth). For the latter Hematoxylin and Eosin, Mal- lory's Connective Tissue stain and many others can be applied as in the case of decalcified bone. Hanazawa's (Dent. Cosmos, 1917, 59, 125) methods for the minute structure of dentin are given in detail by Wellings, A. W., Practical Microscopy of the Teeth and Associated Parts, London: John Bale, Sons & Curnow. Ltd., 1938, 281 pp. Dentin can be advantageously ex- amined after vital staining with Alizarin Red S. Its pH can be estimated (Grossman, L. I., J. Dent. Res., 1940, 19, 171-172). For determination of rate of mineral replacement see Radio- active Phosphorus; for Korff's fibers, see Teeth, Developing; and for nerve endings, see Teeth, Innervation. Desmoglycogen, see Glycogen. Desoxyribonuclease is an enzyme for de- grading desoxyribonucleic acid. It has been used for histochemical observa- tions (Catheside, D. G., and Holmes B., Symp. Soc. Exp. Biol., No. 1, Nucleic Acids, 1947, 225-231, Cambridge University Press) to remove this type of nucleic acid from cells. Desoxyribonucleic acid. Method for deter- mination in Isolated Hepatic Nuclei — Written by Joseph A. Falzone, Dept. of Anatomy, Washington University, St. Louis 10, Mo. October 26, 1951— This technique may be applied, with slight modifications to a wide variety of tissues and organisms, but is here de- scribed for use with rat liver. In prin- ciple, the nuclei of a tissue homogenate are isolated and washed by centrifu- DESOXYRIBONUCLEIC ACID 97 DESOXYRIBONUCLEIC ACID gation, counted in an hemocytometer chamber, and the Desoxyribonucleic Acid (DNA) extracted with hot per- chloric acid and determined by the Dische (diphenylamine) reaction. By this means, the DNA content of a known number of nuclei is determined, making it possible to calculate the av- erage DNA content per nucleus (Boivin, A., Vendrely, R., and Vendrely, C, Compt. rend. Acad, sci., 1948, 226, 1061-1063. Isolation, Counting, and Washing of Nuclei: Anesthetize the rat, expose ab- dominal and thoracic viscera, clamp the inferior vena cava just below the liver, sever the portal vein, and perfuse the liver through the hepatic vein or thoracic inferior vena cava, using about 50 ml. of cold normal saline, followed by 30-50 ml. of cold 2% citric acid. Remove the liver, which will weigh about 10 gm., and dissect away from it any adhering diaphragmatic and con- nective tissues. Frozen liver may be used, but nuclear yield is reduced, and increased cellular debris makes counting difficult. The following steps are to be carried out in a cold room at 5°C. After taking a specimen for histological control, ho- mogenize remainder of the liver in about 50 ml. cold 2% citric acid (Cunningham, L., GrifRn, C., and Luck, J. M., Gen. Physiol. 1950, 34, 59-63) using a Waring Blendor with metal head equipped with an ice-brine cooling jacket. The cold room alone does not prevent heating, but with a cooling jacket, the tempera- ture should not exceed 5°C. Homogenize for 5 minutes, adding a few drops of capryl alcohol to prevent foaming. The yield is 50-70 ml. of a 20% ho- mogenate, which is filtered through four layers of cheesecloth. Microscopic ex- amination at this stage reveals a uni- form suspension of relatively undis- torted nuclei, without any lumped or intact cells. Nuclei which are spun down for analy- sis cannot be counted directly, due to clumping, but are estimated by first counting the whole homogenate then subtracting from this value the numbers remaining in the various supernates after centrifugation, as described below. Thoroughly shake the homogenate, withdraw four 5 ml. aliquots, and dilute each with 10 ml. 2% citric acid. These are the counting dilutions, each of which is counted twice in the red cell space (0.1 mm.') of an hemocytometer chamber. Eight to ten individual counts, totaling 3000-5000 nuclei, should keep the error within 5%. Fifty ml. graduated conical centrifuge tubes are used in the following steps. Remove four 10 ml. homogenate ali- quots (each containing about 200,000,000 nuclei as determined above) and carefully layer each over 20 ml. of a solution containing 2% citric acid and 10% sucrose. Centrifuge for 20 min. at about 500 x G. By use of this layering technique nuclei are both washed and isolated in a single step (Wilbur, K. M., and Anderson, N. G.. Exp. Cell Res. 1951, 2, 47-57). Decant and keep this supernate. Re- wash sediment (containing nuclei) in 35 ml. 2% citric acid, centrifuging as above. Combine supernates 1 + 2 from each aliquot, measure volumes, and count nuclei without diluting, if more than 10 per chamber (0.9 mm.^) are present. Extraction of DNA: A recent modi- fication of the Schneider procedure is suggested (Schneider, W. C, Hoge- boom, G. H., and Ross, H. E., J. Nat. Cancer Inst., 1950, 10, 977-982). Ex- tract sediment once with cold 12% perchloric acid and twice with cold 6% perchloric acid, centrifuging for 15 min. at 500 X G each time. This removes nucleotides and sucrose. Combine these supernates (3, 4, + 5), measure volumes, and count nuclei as above, adding this value to that of supernates 1-1-2, and subtracting the total from that for the whole homogenate. Super- nates 3, 4 + 5 should not be mixed with 1 + 2 because cytoplasmic proteins in the latter would be precipitated, with clumping of nuclei. Extraction of phospholipids with ab- solute alcohol may be omitted in work- ing with liver and some other tissues. It is necessary for brain. Extract sediment with 30 ml. 6% perchloric acid at 90°C. for 15 minutes, cool, and centrifuge. Wash sediment with 10-15 ml. cold 6% perchloric acid and combine these supernates (6 + 7) as the DNA extract. This may be stored for 24 hrs. in a deep freeze with little change but longer storage (several weeks) produces unreliable results. Determination of DNA: The Dische reagent should be prepared with di- phenylamine that is colorless or nearly so, with a well defined melting point at 53°C. Otherwise this compound must be recrystallized before use. Dische Blank Reagent Dische Glacial acetic acid 100 ml. 100 ml. Concentrated H2SO1 2.75 ml. 2.75 ml. Diphenylamine 1.0 gm. — Following the Seibert procedure (J. DESOXYRIBONUCLEIC ACID 98 DEUTERIUM Biol. Chem. 1940, 133, 593-604) set up 10 8" X 1, pyrex tubes as below. 1 3 ml. DNA extract from aliquot 1 + 6 ml. blank Dische 2 3 ml. DNA extract from aliquot 2 + 6 ml. blank Dische 3 3 ml. DNA extract from aliquot 3 + 6 ml. blank Dische 4 4 ml. DNA extract from aliquot 4 + 6 ml. blank Dische 5 3 ml. DNA extract from aliquot 1 + 6 ml. Dische reagent 6 3 ml. DNA extract from aliquot 2 + 6 ml. Dische reagent 7 3 ml. DNA extract from aliquot 3 + 6 ml. Dische reagent 8 3 ml. DNA extract from aliquot 4 + 6 ml. Dische reagent 9 3 ml. 6% perchloric acid + 6 ml. blank Dische 10 3 ml. 6% perchloric acid + 6 ml. Dische reagent Heat tubes 10 minutes at 100°C., cool to room temperature and read optical density (D) at GOO m/i. Read unknowns against corresponding blanks set at zero, as below: 5 10 C D C D — = Corrected DNA concentration of 1 ® tube 5 a in Q J) Q J) = Corrected DNA concentration of tube 5 against tube 1 as a blank tube 6 against tube 2 as a blank tube 7 against tube 3 as a blat (°t) tube 8 against tube 4 as a blank ( D — I / 10\ tube 10 against tube 9 as a blank 1 D — I The nucleic acid concentrations (C) corresponding to the five D readings are then obtained from a standard curve. The latter is constructed in the usual manner from at least 20 points, by subjecting various concentrations of a standard DNA to the Dische pro- cedure and plotting the resulting D readings against these concentrations. The solvent (O.OIN NaOH or hot 6% perchloric acid) plus Dische reagent is used as a blank. Beer's law should be closely obeyed in the useful range. The DNA concentration correspond- ing to the D reading of tube 10 against tube 9 ( C D^ ) is the correction for color developed by the reagents alone and is subtracted from the concentra- tions found for the unknowns. tube 6 etc. The volumes of DNA extracts are measured and the total DNA per aliquot thus found. This value is divided by the number of nuclei per aliquot (ho- mogenate nuclei minus supernate nu- clei) to obtain the average DNA content per nucleus, most conveniently ex- pressed in fifig (mgm. X 10 — 9). This will usually range from 5-10 nixg., de- pending upon the purity of DNA em- ployed in constructing the standard curve. The values of the four aliquots should agree within 5%. If an efficient means could be found of separating the various nuclear types of a given tissue, the value of this technique would be greatly enhanced. A method for determination of De- soxyribonucleic Acid in isolated nuclei of tumor cells (Dounce, A. L., J. Biol. Chem., 1943, 151, 235-240). Same as DNA and Thymonucleic Acid. The structure of this acid in relation to the cytochemical significance of methyl green-pyronin staining is carefully considered by Vercauteren, R. , Enzymologia, 1950, 14, 134-140. See Nucleic Acid. Destin's fixative. 1% aq. chromic acid, 99 cc; formalin, 6 cc; glacial acetic acid, 2 cc. After standing for a few days it becomes green when it can be used. Detergents, see discussion of cutaneous detergents by Lane, C. G. and Blank, I. H., J.A.M.A., 1942, 118, 807-817. See Aerosol. Deuterium is heavy hydrogen. It is an iso- tope having atomic weight of 2.0135 and the symbol H^. Schoenheimer, R., Harvey Lectures, 1937, 32, 122-144 em- ployed deuterium combined with oxy- gen as heavy water H|0 to mark fatt}'- acids. In his experiments on mice, held on a carbohydrate diet plus heavy water the fatty acids of the body are replaced by new fatty acids containing deuterium. The rate of replacement of fatty acids can therefore be deter- mined. For further experiments along this line see Symposium on Interme- diate MetaboUsm of Fats. Biological Sj'mposia Lancaster: Jaques Cattell Press, 1941. Leading references on deuterium: Cope, O., Blatt, H. and Ball, M. R., J. Clin. Invest., 1943, 22. 111-115; Flexner, L. B., Gellhorn, A. and Merrell, M., J. Biol. Chem., 1942, 144, 35-40; Stern, K. and Dancey, T. E., DEUTROPLASM 99 CENTRIFUGATION OF PARTICULATES Proc. Soc. Exp. Biol. & Med., 1941, 48, 619-620. Deutoplasm, see Paraplasm. Diacetin (glycerol diacetate) use in flatten- ing paraffin sections (Carleton, H. M. and Leach, E. H., J. Path. & Bact., 1939, 49, 572-576). Diamin Red 4B, see Benzopurpurin 4B. Diamine Bordeaux CGN, see Erie Garnet B. Diaminoacridines have marked affinity for nuclei in vivo. They can be visualized by their fluorescence in near ultraviolet light. Their localization resembles the chromatin pattern as revealed by "nu- clear" dyes. These compound are ap- parently not toxic because regeneration of rat liver cells while they are still within the nuclei takes place at the same rate as in controls (DeBruyn, R. S., Anat. Rec, 1950, 108, 279-307). Di-Amino Tri-Phenyl Methane Dyes. Ex- amples : brilliant green, fast green FCF, light green SF yellowish and malachite green. Diamond Green, see Brilliant Green. Diamond Green B, BX or P Extra, see Malachite Green. Diamond Fuchsin, see Basic Fuchsin. Dianil Blue H3G, see Trypan Blue. Dianil Blue 2R (CI, 265)— benzo new blue 2B, direct steel blue BB, naphthamine brilliant blue 2R — Conn (p. 63) gives the same formula for this acid dis-azo dye as that supplied by Corner, G. W. and Hurni, F. H., Am. J. Physiol., 1918, 46, 483-486 and Sutter, M., Anat. Rec, 1916, 16, 164-165 for dye employed by them in study respectively of corpora lutea and mammary glands but these authors do not employ the name : dianil blue. Dianil Red 4B, see Benzopurpurin 4B. Dianthine B, see Erythrosin, bluish. Diaphane for mounting Giemsa preparations (Coulston, F., J. Lab. & Clin. Med., 1940, 26, 869-873). Diaphanol is according to Lee (p. 598) the trade name for a mixture, formerly obtainable from Leitz, produced by passing chlorine dioxide vapor into ice cold 70% acetic acid. It should be fresh. He advises against attempts to make it and outlines its use in the soft- ening of Chitin. Rinse well fixed tissues in 63% alcohol and transfer them to diaphanol until they are softened and bleached. If the diaphanol becomes discolored, repeat. Transfer to 63% alcohol, dehydrate, clear in tetralin (if not available, benzol) and imbed in paraffin. See use of diaphanol in demonstrating Melanins. Diazin Black, see Janus Black. Diazin Green, see Janus Green B. Diazo Reaction. Serra, J. A., Stain Techn., 1946, 21, 5-18 gives the technique as follows : Prepare tissue as described under Ninhydrin Reaction. "Treat the pieces for 2-3 minutes with a saturated aqueous solution of sodium carbonate; afterwards add some drops of the diazo reagent and stir the liquid well. Ob- serve in glycerin. (The coloration de- velops rapidly and lasts for some days.) Preparation of the diazo-reagent: into a 50 ml. flask immersed in an ice bath, pour 1.5 ml. of a sulphanilic acid solu- tion (dissolve 0.9 g. of pure sulphanilic acid in 9 ml. of concentrated HCl and add water to 100 ml.); add 1.5 ml. of a 5% aqueous solution of NaN02, shaking the flask meanwhile. After 5 minutes in the ice bath add, also while shaking, another 6 ml. of nitrite. After 5 min- utes fill up to 50 ml. with cooled dis- tilled water. The reagent must be prepared every day and kept in the ice chest. "The reaction gives an orange or yel- low color with the histidine and the tyrosine of the proteins." Dichlorofluorescein. Structure of, Milligan, R. F. and Hope, F. J., J. Am. Chem. Soc, 1945, 67, 1507-1508. Dientamoeba fragilis. Technique of stain- ing and points to be considered in diag- nosis (Hood, M., J. Lab. & Clin. Med., 1939-40, 25, 914-918). Diethylene Dioxide = Dioxan. Diflferential Centrifugation of Cell Particu- lates— Written by Joseph A. Falzone, Department of Anatomy, Washington University Medical School, St. Louis. November 27, 1951 — Since the pioneer- ing work of R. R. Bensley and N. Hoerr (Anat. Rec, 1934, 60, 449-455), this technique has proven one of the most versatile and direct in the armamen- tarium of cytology. In essence it con- sists of a rather drastic mechanical disruption of large numbers of cells in various media, separation of the result ing particulates by centrifugation, and determination of chemical components and enzymatic activities in these frac- tions. There are almost as many varia- tions in the technique as investigators, each variation having its peculiar merits or limitations, the one chosen depend- ing upon the particular cell organ- elle or chemical data desired. The obvious and inherent weakness of these methods is their tendency to produce morphological and chemical artefacts. For example, when a tissue is homogenized in an aqueous medium, enzymes and other substances may be lost by denaturation or solution, or what is worse, adsorbed to particulates that never contained them in life. Large particles may fragment and sedi- ment with an unrelated small granule CENTRIFUGATION OF PARTICULATES 100 CENTRIFUGATION OF PARTICULATES fraction. With nuclei, at least, these ob- stacles are in part overcome by the M. Behrens technique (Ztschr. f. physiol. Chem., 1932, 209, 59) by which tissue is frozen and dehydrated at the outset, fractionation being accomplished in non- polar organic media. The factor of tonicity is poorly under- stood. A solution isotonic to eryth- rocytes may not be so to mitochondria or nuclei. Mitochondria eventually swell and burst in distilled water, but appear well preserved in almost syrupy sucrose solutions. The review of W. C. Schneider and G. H. Hogeboom (Cancer Res., 1951, 11, 1-22) should be consulted in this respect. Nuclei, on the other hand, do not appear to behave as osmometers. Hydrogen ion concentration, of course, is an important variable and should be adjusted according to experi- mental aims. In using citric acid media, for example, we find a number of EH dependent effects. At pH 4 or elow (2% citric acid) nuclei are readily isolated in bulk, morphologically free of cytoplasm, with no tendency to clumping, and with an apparently com- plete complement of desoxyribonucleic acid (DNA) . At pH 4-6, agglutination of cytoplasmic particles produces nu- clear clumping. At pH 6, nuclei are again dispersed, and contain, in addi- tion to DNA, some enzymatic activity. Above pH 6.5, they disintegrate com- pletely, in contrast to cytoplasmic com- ponents, which are better preserved in nearly neutral media (Bounce, A. L., Ann. N. Y. Acad. Sci., 1950, 50, 982- 999). It might appear that ideal media would duplicate the tonicity, pH, and electrolyte pattern of cytoplasm, and some attempts in that direction have been made (Wilbur, K. M. and Andre- son, N. G., Exp. Cell Res., 1951, 2, 47-57). However, the most versatile media employed to date have been simple iso- or hypertonic sucrose solutions, without buffers or electrolytes. These media, especially if hypertonic, yield excellent morphological preservation of all components, including nuclei, which resemble those of living cells in their optical homogeneity (Schneider and Hogeboom, Ibid.). One wonders if this is really an advantage in the determina- tion of nuclear nucleic acids, where acid precipitation may be desirable, but the question is not fully answered. Bearing on this problem is the fact that many such unbuffered homogenates are slightly acid, due probably to glycoly- sis, but that liver appears to be an exception in this respect (Wilbur and Anderson, Ibid.). The separation of cell components by centrifugation has relied more upon difference in particle size, with resulting difference in velocity of fall, than upon any variation in specific gravity. This would be expected from a consideration of Stokes' Law, which states that the velocity of particle fall is proportional to the square of its radius, but only directly proportional to the density difference between particle and medium. Moreover, we are dealing with semi- permeable or frankly porous cell parti- cles, whose density is altered with that of the medium, so that very dense (and correspondingly viscous) fluids must be employed to effect separation by flotation. In these cases we may be roughly measuring 'dry weight densi- ties', as in the Behrens procedure. An obvious exception to these considera- tions is that of lipid-rich constituents, which migrate centripetally. As a result, whole cells are commonly sedimented at very low speeds, nuclei at approximately 5(X) x g mitochondria at 2000-20,000 x g, and microsomes at 20,000 to over 100,000 x g. The "super- nate" is the remaining non-sediment- able fraction, and with the preceding particulates, completes the list of usually studied components. Others, such as chromosomes and melanin gran- ules, will not be considered here. The time of centrifugation or field required varies with the viscosity of the medium, the time ranging from a few minutes to several hours. A layering technique, as emphasized by Wilbur and Anderson (Ibid.), is the most efficient means of centrifugation, as the mean distance of particle fall is both increased and nearly equalized. This naturally has the effect of both isolating and washing the most rapidly sedimenting fraction at a single step. From this and the above considerations it is obvious that a more efficient centri- fuge would be one constructed to hold longer tubes. With such an instru- ment, employing tubes only double the length of standard models, an even further fractionation of particulates might be achieved, with diminished loss due to washing. Separation of nuclear types suggests itself, and some en- deavors in that direction are promising (Marshak, A., Cancer Res., 1950, 10, 232). Below is a brief outline of some of the more popular and useful techniques, listed according to homogenizing fluids employed. Centrifuging media are us- ually similar to these, or made slightly CENTRIFUGATION OF PARTICULATES 101 DIGITONINE denser by addition of sucrose, if layer- ing is employed. Temperature should be kept at 5°C. or below throughout. No attempt at completeness has been made, and some interesting methods, including ultrasonic and enzymatic cell disruption, are omitted. 1) Citric acid, low pH (Bounce, Ibid; Cunningham, L., Griffin, A. C. and Luck, J. M., J. Gen. Physiol., 1950, 34, 59-63). Procedure: Tissue homogenized sev- eral minutes in cold 2% citric acid in a Waring Blendor. The latter should be equipped with a cooling jacket. Particulates obtained: Nuclei. Morphology: Reasonably good. Nuclei plump and unwrinkled, but with fine internal granulation. No clumping or cytoplasmic tags. Value: Simplicity and use of large volumes of tissue, if desired. Nuclei readily counted and DNA per nucleus determined by the Dische or other suitable analytical technique. The method of choice for this purpose, but of little other value. Limitations: Water soluble sub- stances, including much protein and probably RNA, are lost; enzymes inactivated. 2) Citric acid, controlled pH (Bounce, Ibid.). Procedure: Enough O.IM citric acid is added dropwise to partially homog- enized tissue in ice water to give a pH of 6.0. Used with a low speed Waring Blendor or glass homogenizer. Particulates obtained: Nuclei. Morphology: Similar to procedure 1. Value: Suitable for BNA and many enzyme studies. Limitations: Similar to 1, except that apoenzj'mes are retained. Co- enzymes must often be added in de- terminations of their activities, how- ever. Adsorption effects must be controlled by repeated washing of nu- clei. 3) Sucrose, near pH 7 (Schneider and Hogeboom, Ibid.). Procedure: Small amounts of tissue homogenized by ground glass appara- tus or other "gentle" mechanical means, in a medium of neutral but unbuffered iso- or hypertonic (usually 0.88 M) sucrose. iProbably best to avoid NaCl and other electrolytes for general purposes. Particulates obtained: Nuclei, mito- chondria, microsomes, supernate. Also melanin granules and particulate glycogen. Morphology: Excellent, with pres- ervation of all particulates in a form resembling that in living cells. Mitochondria filamentous, at least in hypertonic sucrose, and stainable with Janus Green B; nuclei homo- geneous with several nucleoli. Value: Versatility, due to the large number of fractions obtained. Suit- able for determinations of enzymes, nucleic acids, proteins and lipids of these fractions. The method of choice for microspectrophotometry of stained nuclei, as optical homogeneity is a definite prerequisite for valid results with this method. Limitations: Solution and adsorp- tion effects again come into play, but many artefacts can be partially ruled out. Nuclei probably not so readily obtained in bulk as with methods 1) and 2). 4) Behrens technique (Behrens, Bounce; Schneider and Hogeboom, Ibid.). Procedure: Tissue frozen, dried in vacuo, ground in ball mill to disrupt cells, and centrifuged in non-aqueous media, usually hydrocarbons. Particulates obtained: Nuclei. Morphology: More seriously dis- torted than with any other proce- dure; nuclear membranes wrinkled. Contamination with cytoplasm must be appraised from smears stained with both acid and basic dyes. Value: Probably the most widely applicable method for nuclei. Pro- cedure of choice for water soluble substances, i.e., coenzymes, vitamins, free amino acids, etc. Results with total protein and RNA also appear the most reliable of any method. Suitable also for BNA and many enzyme studies. Limitations: Procedure is laborious. Lipids, lipases, and many other en- zymes removed or destroyed. This brief summary can only suggest the variety of applications of cell frac- tionation to cytology, as new ones are continauUy uncovered. For example, B. L. Opdyke has recently described a valuable technique for the isolation of keratohyaline granules of epidermis, using isotonic saline and a Waring Blendor for homogenization. The par- ticles are sedimented at 25,000 x g and appear to have all the morphological and staining characteristics of these granules (See Keratohyaline Granules, procedure for isolation). Differential Leucocyte Count, statistical study of uniformity in (Klotz, L. F., J. Lab. & Clin. Med., 1939, 25, 424^34). Diffraction Methods for measuring diameter of red blood cells (Haden, R. L., J. Lab. & Clin. Med., 1937-38, 23, 508-518). Digitonine reaction of Winaaus for free DILATOMETRIC TECHNIQUES 102 DIRECT GARNET R cholesterol. This has been adapted to histochemical use by Brunswick and by Leulier and Noel (A., and R., Bull. d'Hist. Appl., 1926, 3, 316-319). Lison (p. 211) recommends a slight change. Immerse frozen sections of formalin fixed tissue in 0.5% digitonine in 50% ale. for several hrs. Rinse in 50% ale, then in water and mount in Apathy's syrup or glycerin gelatin. With crossed nicols (polarizing microscope) one observes appearance of needles or rosettes of the complex cholesterol- digitonide. To resolve this complex stain with sudan. The esters will color and lose their birefringence while the cholesterol will remain uncolored and retain birefringence. Dilatometric Techniques. These have been adjusted so that they will give the fine quantitative measurements needed in histochemistry by Linderstr0m-Lang, K Nature, 1937, 139, 713. He reported especially ultra-microestimations of peptidase activity. Di-Nitrosoresorcinol test for iron, see Iron. Diodrast, trade name for an organic iodine preparation recommended by Gross, S. W. (Proc. Soc. Exp. Biol. & Med., 1939, 42, 258-259) for injection into com- mon carotid with later x-ray photo- graphs of the vascular tree. For visual- ization of ventricular chambers and large arteries see method of Ponsdome- nech, E. R. and Nunez, V. B., Am. Heart J. 1951, 41, 643-651. Dioxan is diethylene dioxide. It mixes with water, ethyl alcohol, many clearing agents and paraffin (slightly). McClung (p. 39) recommends its use to replace ordinary agents like xylol. Dioxan fumes are said to be dangerous to laboratory workers so that it should be used under a hood or in a well ventilated room with container covered when not in use (Magruder, S. R., J. Lab. & Clin. Med., 1937-38, 23, 405-411). For fixation the following mixtures are recommended (McClung, p. 39) : (1) Sat. aq. picric acid, 5 parts; glacial acetic, 1 part; dioxan, 4 parts. (2) Sat. picric acid in dioxan , 4 parts ; glacial acetic, 1 part; absolute alcohol, 4 parts. Graupner and Weissberger (von H. and A., Zool. Anz., 1933, 102," 39-44) suggest: dioxan 80%, methyl alcohol 20%, paral- dehyde 2%, and acetic acid 5%. See Clearing, Pituitary. See as ingredient of Lison's gh'cogen method; also dioxan imbedding of Pituitary. A method for the dehydration, puri- fication and clarification of dioxan so that its use in tissue technique can be continued has been described by Hall, W. E. B., Am. J. Clin. Path., 1943, 7 (Technical Section), 98-100. Dipeptidase can be localized in chief cells of stomach. See review of methods (Gersh, I., Physiol. Rev., 1941, 21, 242-266). Di-Phenyl Methane Dyes. Of these only auramin need be referred to. Diphosphopyridine Nucleotide, see tech- nique of Anfinsen, C. B., J. Biol. Chem., 1944, 152, 285-291. Diphtheria Bacilli. 1. Neisser's stain (Stitt, p. 863). A = methylene blue, 0.1 gm.; 95% ale, 2 cc; glacial acetic acid, 5 cc; aq. dest., 95 cc. B = Bismark brown, 0.2 gm. ; aq. dest. (boil- ing) 100 cc. Dissolve and filter. To stain smear pour on A , 30-60 sec. Wash. Then B, 30 sec. Wash in water, dry and mount. Bacilli brown with dark blue dots at either end. Better results can be secured by adding 1 part of crystal violet (Hoechst) 1 gm.; 95% ale, 10 cc; aq. dest., 300 cc. to 2 parts of A before using. Chrysoidin 1 gm. in hot aq. dest. 300 cc. is more satis- factory counterstain than Bismark brown. Most American brands of crys- tal violet are satisfactory. 2. Pander's stain (Stitt, p. 863). Toluidin blue (Grubler) 0.02 gm.; aq. dest., 100 cc; glacial acetic acid, 1 cc ; abs. ale, 2 cc Add small amount to fixed film on cover glass. Invert and mount on slide. Diphtheria bacilli recognizable by metachromatic granules intensely stained, diphtheroids by their strong color in contrast with ordinary cocci and bacilli the bodies of which are only faintly blue. 3. Laybourn's modification of Albert's stain (Stitt, p. 863). A = toluidin blue, 0.15 gm. ; malachite green, 0.2 gm. ; glacial acetic acid, 1 cc. ; 95% ale, 2 cc, aq. dest., 100 cc. B = iodine crystals, 2 gm.; potassium iodide, 3 gm.; aq. dest., 300 cc. Let both stand 24 hrs. and use filtrate. Apply A to heat fixed smears 3-5 min. Wash in water. Apply B for 1 min. Wash, blot and dry. Granules of diphtheria bacilli, black; bars, dark green; inter- mediate parts, light green and all three in sharp contrast. Diplosome, a double centrosome. Direct Black, see Chlorazol Black E. Direct Fast Orange (CI, 326)~Erie Fast Orange (NAC), Erie Fast Scarlet YA (NAC) — a direct disazo dye of light fastness 3 (Emig, p. 38). Direct Fast Scarlet 4 BS (CI, 327)— Pont- amine Fast Scarlet 4 BS of DuPont — , a disazo direct dye of light fastness 3, can be employed instead of carmine as a general stain. Details of use in study of plant and animal tissues are de- scribed (Emig, p. 38}. Direct Garnet R, see Erie Garnet B. DIRECT GREEN B 103 DUCTS Direct Green B (CI, 593)— Diazine Green B — a direct disazo dye of light fastness 3 to 4. Recommended as counterstain for Crocein Scarlet 7 B of invertebrates or paraffin sections, time 5 min. (Emig, p. 43). Direct Green G (CI, 594)— Alkali Green D— a direct disazo dye of light fastness 3 to 4. Formula for blue green algae and whole mounts is given (Emig, p. 43). Direct Red 4B, see Benzopurpurin 4B. Direct Red, C, R, or Y, see Congo Red. Direct Sky Blue, see Niagara Blue 4B. Direct Steel Blue BB, see Dianil Blue 2R. Direct Violet B, see Azo Blue. Direct Violet C, see Erie Garnet B. Dis-Azo Dyes. Azo blue, benzopurpurin 4B, Biebrich scarlet, Bismark brown Y and R, brilliant purpurin R, Congo red, dianil blue 2R, Erie garnet B, Niagara blue 4B, orseillin, trypan blue, trypan red, sudan III, sudan IV, vital new red, vital red, etc. Dissociation, see Maceration. Distrene 80 is a polysterene which forms a water clear solution in xylol. It is recommended by Kirkpatrick and Len- drum (J. and A. C, J. Path, and Bact., 1939, 49, 592-594) as a naounting medium giving good preservation of color in microscopic slides. See also Kirk- patrick, J. and Lendrum, A. C, J. Path. & Bact., 1941, 53, 441. Dominici's Stain, see Eosin-Orange G and Toluidin blue. Donaldson's lodine-Eosin Method, see lo- dine-Eosin. Dopa, Oxidase Reaction for Melanoblasts (Laidlaw, G. F., Anat. Rec, 1932, 53, 399-407). Dopa is short for 3.4-dihy- droxyphenylalanin, a substance which when applied in a certain way picks out the melanoblasts by blackening them. Use frozen sections of fresh material or of tissues fixed 2 to 3 hours but not longer in 6% formalin. Rinse 4 or 5 seconds in aq. dest. and immerse in buffered dopa. (To make dopa stock solution dissolve 0.3 gm. dopa powder- manufactured by Hoffmann-La Roche, Nutley, New Jersey— in 300 cc. cold aq. dest. Keep in refrigerator and dis- card when solution becomes dark red. To make buffers dissolve 11.87 gms. di- sodium hydrogen phosphate (Na2HP04 + 2H2O) — or what would be better 9.47 gm. anhydrous Na2HP04— in 1000 cc. aq. dest. and 9.08 gms. anhydrous potassium dihydrogen phosphate (KH2PO4) in an equal amount aq. dest. Immediately before use buffer to pH 7.4 by adding 2 cc. potassium phosphate solution, and 6 cc. sodium phosphate solution to 25 cc. dopa solution). The reaction is slow for 3-4 hours at room temperature. If solution becomes sepia brown it is likely to overstain. Observe under microscope. Wash in aq. dest., dehydrate and counterstain if desired with alcoholic crystal violet, clear and mount in balsam. Melan- oblasts should be black. This much used method has been criticized by 11. Sliarlit et al. (Arch. Dermat. and Syph., 1942, 45, 103-111) chiefly on the ground that the incuba- tion for 3 hrs. at room temperature may itself increase the amount of melanin present which happened in their ex- perience at 37 °C. See also remarks by Blaschko and Jacobson (Bourne, p. 198) on specificity of the reaction. It is given by phenoloxidases but thus far they have not been found in mam- malian tissues. Dorothy Reed Cells, see Reed-Sternberg Cells. Double Green, see Methyl Green. Double Imbedding. To facilitate section cutting by making a celloidin block firmer, harden first in chloroform vapor, then in chloroform, transfer to benzol until it becomes transparent and in- filtrate with 38°C. paraffin (Lee, p. 104). See Fleas, Another method of double imbedding is that of Peterfi (T., Zeit. f. wiss. mikr., 1921, 38, 342-345). As employed in this laboratory it is as follows : Make 1% and 3% solutions of celloidin in methyl benzoate which take about a month. Foursome 1% into a dish. Add absolute alcohol containing the tissue which gradually sinks down into the celloidin. Transfer tissue to 3% solu- tion, 48-96 hrs. Drop tissue directly into benzol for a few hrs. Then infiltrate and imbed in 40 °C. paraffin about 12-24 hrs. Double Scarlet BSF, see Biebrich Scarlet, water soluble. Downey's Fluid, see Megakaryocytes. Ducts. These structures lead (L. ducere) the products of glands to the site of discharge. They are of considerable variety. Ordinarily they are easily identified by their morphology in hema- toxylin and eosin preparations. But special techniques are required for their visualization in whole mounts of some glands. In the pancreas for example the system of small ducts (ductules) can easily be demonstrated by perfusion of the pancreas with pyronin — one of the many methods discovered by R. R. Bensley. Proceed as described under Perfusion using a solution made up by adding 10 cc. of 1% aq. pyronin to 1000 cc. 0.85% aq. sodium chloride. When the pancreas has assumed a rose red color the optimum intensity of which DUGGINS 104 DUST CELLS must be determined by trials, remove a piece of it, tease out a small lobule and examine under low power mounted in 0.85% aq. sodium chloride. The com- plicated system of ducts should be sharply delineated by their deep rose red color in an almost colorless background. If there is any question of their identification examine the original figures of Bensley, R. R., Am. J. Anat., 1911, 12, 297-388. A double staining of ducts and Islets of Langerhans can be obtained by perfusing in the same way with pyronin solution to 1000 cc. of which 6 cc. 1% aq. neutral red has been added. The islets appear yellow red in contrast to the rose red ductules. See, in addition, ducts in whole mounts of Mammary Glands and in sections of Submaxillary Glands which are of par- ticular interest in detecting the action of salivary gland viruses. Duggins, see Hairs Duodenal Fluid. Microscopic study must be prompt because of the presence of cytolytic engymes. Examine sediment after centrifugal concentration as in the case of urinary sediments. Epithelial cells from the entire alimentary tract leading to and including the duodenum may be present, generally bile stained, also a few neutrophiles. A great in- crease in both or either may indicate inflammatory lesions. A polarizing mi- croscope is helpful, but not essential, in recognizing cholesterol crystals as thin, flat, colorless fragments with chipped edges. The more irregular the crystals the more significant they are of calculi formation. Bilirubin is easily detected as amorphous amber, brown or black material and calcium bilirubinate as bright yellow granular deposits. See Gentzkow and Van Auken in Sim- mons and Gentzkow, p. 63. Duodenal Drainage Smears, see Papani- colaou Techniques. Duran-Reynals, see Spreading Factors. Dust Cells of the Lungs— Written by C. C. Macklin, Dept. of Histological Re- search, The University of Western On- tario, London, Canada. November 28, 1951 — These phagocytes develop in the alveolar walls of the lungs of man and other mammals from the primitive pneumonocy tes to arrest particles of car- bon and other particulate matter which elude the apprehensional mechanism of the respiratory tract; and to dispose of them by conveying them to the ciliary escalator which evacuates them. Col- lapse of the lungs dislodges them from their normal position in the alveolar wall (Macklin, C. C, Lancet, Feb. 24, 1951, 432-435) where they are wedged in between capillaries, whence they derive their nutriment. Thus the ordi- nary human lung material available to pathologists is unfavorable in that it shows phagocytes lying loose which normally are tethered. If the skin and outer musculature be removed from a fresh intact thorax of a small animal such as the mouse and it be at once im- mersed in a fixing fluid such as Regaud or Bouin's, the lung is quickly fixed in its normal uncollapsed condition. Pen- etration is facilitated by exposing the lower surface of the diaphragm, but this must not be breached. Sections from such material show as many as 99% or more of the dust cells morphologically affixed to the alveolar walls, while only 1% or less are free, and these are re- garded as spent and on their way to the exterior. If, however, fresh normal mouse lungs are collapsed and promptly filled with fixing fluid via the trachea, and cut into sections, it is found that only about 5% of the dust cells remains fixed in the alveolar walls, the re- mainder now appearing free. An er- roneous picture of the relation of the dust cells to the alveolar walls is thus presented. That collapse alone dis- lodges many of the phagocytes formerly affixed in the alveolar walls is shown by finding undue numbers of free dust cells in sections from pieces of lung tissue which have been fixed by immersion. For larger lungs the method of perfusion of fixing solution through the pulmo- narvvasculature of the unopened thorax (Hartroft, W. S., Anat. Rec, 1942, 82, 419) also showed the majority of alveo- lar phagocytes affixed to the walls rather than free. Thus dust cells are funda- mentally mural phagocytes, and are regarded as originated ultimately from endodermal epithelium (Macklin, C. C, Trans. Roy Soc. of Canada, Sect. V, 1946, 40, 93-111) . The presence of fluid in the alveoli favors a shedding of mural phagocytes from their walls. No ev- dence of dust cell entry into the con- nective tissue or lymphatic channels of the lung was found, contrary to the opinion of many pathologists. On the other hand, dust cells are often found stuck in the mucus overlying the epi- thelium of the bronchioli and bronchi, and these are probably wornout cells being carried to the glottis by ciliary movement (Macklin, C. C, Can. J. of Res., D, 1949, 27, 50-58; Macklin, C. C, Proc. Instit. of Med. of Chicago, 1950, 18, 78-95). They are recoverable from sputum, and then have been re- ferred to by the unsuitable term "heart failure cells" (which see). In the mouse most alveolar phago- cytes are in alveolar angles and have DYES 105 EAR processes inserted into tunnels encircled by capillary loops; but a lesser number, resting on connective tissue, occupy sockets. After brief treatment of fresh lungs with ammoniacal silver solution, many of the dust cells are blackened and their mode of insertion into mural vaginae are made clear (Macklin, C. C, Anat. Rec, 194S, 100, 693). After with- drawal of such processes these vaginae or tunnels, now vacated, have become pores (Macklin, C. C, J. of Thor. Surg., 1938, 7, 536-551). These mural phagocytes may have as many as four functional air faces or particle receptors of varying area. These often conform to the contour of the alveolar wall, but, in presumably active cells, project into the air space. The edges may extend upon adjacent capillaries and have been mistaken for respiratory squames by some workers. They are sister cells of the alveolar granular cells (septal cells, niche cells, etc.) and the term "pneumonocyte" (which see) includes both types. Dust cells may be recovered from fresh lungs by the "gash-irrigation" and "wash-out" methods (which see), and studied in fresh mounts or after being dried and stained like a blood smear. Mito- chondria are scarce or absent in them. One hour after e.xposure to carmine particles in the inspired air they con- tain much carmine, which is almost completely absent on the remaining alveolar wall surface, and almost 100% are inserted into the walls. One week after such dusting, however, only 35% are mural phagocytes, the rest being free. Since dust cells are constantly being lost to the body they must be replaced correspondingly by the mul- tiplication of sister cells remaining in the walls. Dust cells often show bi- zarre shapes and a common one is that of a dumb-bell in which the middle narrow bridge occupies a pore. Such cells are probably not in transit from one avleolus to another. Histocj^tes of the lung connective tissue often pick up dust particles which have passed the inefficient surface guards of the broncho-alveolar system. These are regarded as quite different from the endodermal alveolar phagocytes. Great amounts of such aberrant dust may accumulate and may give rise to disease processes. Dyes, see "standardization of Stains" pp. xxvii-xxx of this book and Staining. Dysentery, see Endamoeba. Dysprosium, see Atomic Weights. Ear— Written by W. P. Covell, Dept. of Anatomy, Washington University, St. Louis 10, Mo. October 26, 1951— Micro- scopic examinations of the ear are nearly always made on sections. This is understandable, but it is possible that the study of still living tissues, removed by careful and minute dissections, is a field of considerable promise. The close apposition of epithelial and nervous components to bone necessitates de- calcification except in the case of young embryos. The frequent use of celloidin in place of paraffin for imbedding is occasioned bj^ the wide range of diver- sity in resistance of the organ to the microtome knife, fluid containing lumina being surrounded bj' hard dense bone. The histological techniques actually in use for the ear are fewer in number and more limited in range than those employed for most other parts of the body. The difficulty experienced in obtaining fresh and normal adult specimens has turned investigation toward human fetuses and the ears of experimental animals. The commonly used fixatives are either Zenker's fluid, with or without acetic acid, Zenker-formol, 10% forma- lin, Wittmaack's solution and Heiden- hain-Susa. Mygind, S. H. and cowork- ers (Acta oto-laryng., 1945, 33, 273- 280) prefer the Wittmaack's for fixation of hair cells and use the latter for study of such structures as the stria vascularis. The best results are to be obtained by the use of animal material for which prompt fixation by perfusion methods has been done. Kristensen, H. K. (Acta oto-laryng., 1945, 33. 225- 244) recommends the use of a 6% gum arable in physiologic saline for intravital perfusion to precede the fixative. Iso- lated temporal bones placed in fixative are prone to show autolytic changes in end organs and ganglion cells in one-half hour following death. The lit- erature is filled with autolytic changes described as specific pathologic altera- tions due to drugs, toxins, poisons, and so forth. Actually many of these are the result of poor penetration of fixa- tives and elapse of time between au- topsy and fixation. In an attempt to overcome the slow penetration of a fixa- tive and prevention of artefacts Guild made use of water from which gases had been exhausted as the medium for fixatives, decalcifying solutions and al- cohols. A variety of decalcificants have been used with the view to preserving finer cell structures of the soft tissues, viz: Formic, trichloracetic, and trichlora- lactic acids, long immersion in MuUers' fluid and so forth. Nitric acid in 2 to 5% concentration is generally used for human temporal bones. The lower con- EAR 106 EAR centration while it takes longer to act is less likely to overdecalcify. A few investigators recommend the use of 3% nitric acid in water and a constant tem- perature (37^°C.) to hasten the proce- dure. Degree of decalcification is us- ually judged by probing with a needle, or a simple test with an indicator such as phenol red. Most small animal bones are decalcified in about 4 to 10 days. However, human temporal bones vary considerably and may take as long as 6 to 8 weeks with weekly change of the solution. After decalci- fication, thorough washing for 24 hours in running water is necessary following which neutralization in 5% sodium sul- fate may be used and washing in run- ning water repeated. In order to preserve cytologic detail attempts have been made to circumvent decalcification in strong acids. Small laboratory animals may be perfused with Regaud's solution and following fixation mordanted in potassium bi- chromate for a considerable length of time. The blocks can be embedded in paraffin and sections made although de- calcification is usually incomplete. Mitochondria in hair cells, stria vas- cularis and spiral ganglion cells can be studied by this method. In his study of kittens, young rabbits, dogs and rats, Van der Stricht, O., Contrib. to Embryol., Carnegie Inst., 1920, 9, 109-142 fixed isolated cochleas in 5% aq. trichlorlactic acid, Bouin's and Zenker's fiuids, mordanted for "many weeks" in 70% alcohol + a few drops of iodine solution. After the last 2 fixatives he completed decalcification in 2% nitric acid in 70% alcohol. Be- fore imbedding in paraffin he stained with Borax Carmine and he colored the sections with Iron Hematoxylin, Congo Red and Light Green. Directions will be found in his paper for the demonstra- tion of mitochondria in the sustentacu- lar and hair cells. A differential stain for hair cells is described by MacNaugh- ton, I. P. J., and Peet, E. W., J. Laryng. and Otol., 1940, 55, 113-114 with a fine colored figure of the results. Celloidin is generally used for im- bedding animal and human material. It is not ideal since it is difficult to handle, takes considerable length of time to infiltrate and is expensive. Various nitrocellulose samples have been tried for small blocks of bone with success but usually centers of large blocks, particularly human temporal bones, do not become sufficiently hard. Concentrations of celloidin usually ranging up to 15% are employed in suc- cessive steps. While the material is in 8%, 10% and 15% celloidin negative pressure is used in moderate amounts to insure infiltration of celloidin into chambers of the inner ear. This should be done with considerable caution as rupture of the delicate membranes may result. When the specimen is ready for embedding it is amply covered with 15% celloidin and allowed to remain in the refrigerator (the lower temperature pre- vents excessive bubble formation) until it can be blocked. Blocking of the ma- terial is important and depends upon whether vertical or horizontal sections through the cochlea are desired. This is readily determined by such land- marks as the eminentia arcuata, and external and internal auditory meati. Sections of large blocks are usually cut at 10 to 15 micra in thickness on a sliding microtome. Every section is numbered and kept for further study if necessary, while every tenth or every twentieth section is put aside for stain- ing as a "tracer" series. The nerves of the tympanic mem- brane were successfully stained intra- vitally by the use of methylene blue; Wilson, J. G., J. Comp. Neurol, and Psychol., 1907, 17, 459-468. Peripheral endings of the cochlear nerve were stained with 1:5000 methylene blue in isolated pieces of the fresh membranous cochlea removed under the dissecting microscope; Covell, W. P., Ann. Oto., Rhino., Laryngo., 1938, 47, 62-67. The peripheral fibers of the cochlear nerve have recently been demonstrated by Fernandez, C. (Laryngoscope, 1951, 59, (in press)) using Bodian and Cajal silver Methods. The course of the fibers were traced in pieces removed by dissection and mounted on slides. A method for study of Wallerian de- generation in the cochlear nerve by use of the Swank-Davenport osmic mix- ture is described by Rasmussen, G. L. (Abstr., Anat. Rec, 1950, 106, 120). Ross, E. L. and Hamilton, J. W. (Arch. Otol., 1939, 29, 428-436) allowed mer- curochrome to remain in the middle ear cavities of dogs for 20 minutes to 2 hours, fixed the mercurochrome in acid solution, decalcified the bones, and studied the distribution of the dye in frozen sections of the cochlea. The pas- sage of fluorescein after arterial injec- tion was observed by Gisselsson, L. (Acta oto-laryng., 1949, 37, 268-275) in the cochlea. Trypan blue has been utilized to ascertain the effects of trauma on scala media cells of the cochlea (Lurie, M. H., Ann. Otol., Rhin., Laryng., 1942, 51, 712-717). The capil- lary areas of the cochlea have been further studied in animals by Smith, EAR SMEARS 107 EAR SMEARS C. (Laryngoscope, 1951, 59, (in press)). The precipitation of Prussian Blue in the small vessels or the lead chromate method of Williams, T. W. (Anat. Rec, 1948, 100, 115-125) gave satisfactory preparations. A method for graphic reconstruction of the organ of Corti was introduced by Guild, S. R., Anat. Rec, 1921, 22. 141- 157. This method has been used for ascertaining damage to the organ of Corti and for measurements of the length of the organ of Corti in man by Hardy, M., Am. J. Anat., 1938, 62, 291- 311. A simple technique for measuring the length of the basilar membrane is reported by Keen, J. A., J. Anat., 1939- 40, 74, 524-527. To study the mode of vibration of the basilar membrane B^k^sy, G. v., J. Acous. Soc. Am., 1948, 20, 227-241, used fresh human temporal bones and suspended fine silver crystals in the cochlear duct to visualize the transparent memarane. Various methods of reconstruction have been employed particularly for study of development of the ear. See Bast, T. H., Arch. Otol., 1932, 16, 19-38 and others. Casts of the labyrinth have been made of a number of different materials including Wood's metal, wax, rubber and so forth. Cummins, H., J. Comp. Neurol., 1924-25, 38, 399-459 used mercury for this purpose. With monomeric methyl methacrylate (Pera- pe.x) Gray, 0., J. Laryng. and Otol., 1948, 62, 308, developed a method for making an accurate anatomic cast of the laby- rinth. See Endolymph. Ear Smears — Written by Marian Pfingsten Bryan, Dept. of Otolaryngology, Wash- ington University, St. Louis 10, Mo. September 20, 1951 — Cytological studies of aural exudates are valuable in help- ing to understand the patterns of disease pertaining to the middle ear, mastoid and external canal of the ear. The smear technique, more fully appreci- ated now, although stressed by Dean, L. W. (J. A. M. A., 1932, 99, 543-546), yields reliable information. When this is accurately correlated with clinical symptoms it is of diagnostic value. With repeated consecutive smears the course of an inflammatory reaction in the middle ear can be charted and the bacteria generally observed. The se- verity and type of infection is often indicated by the number and variety of inflammatory cells. The response of the individual, as evidenced by the number and type of phagocytes is im- portant. The phagocytic power of the polymorphonuclear neutrophilic leuco- cytes along with the monocytes and other cellular forms can be evaluated. These, with other distinguishing cy- tological features, are evident in the following-categories of aural disease: chronic and acute suppurative otitis media, cholesteatoma of the middle ear and mastoid, acute and chronic mas- toiditis, external otitis and carcinoma. Studies of these aural secretions have been sparsely scattered through the literature. Ts'en, Shih-Ping (China Med. J., 1926, 40, 136) reported seven aural smears with case histories and suggested that the differences in cytology were due to the duration of the discharge. He noted that in chronic cases the neutrophiles were more degenerated than in acute ones. Since then it has been found that chronic suppurative otitis media can readily be differentiated from acute suppurative otitis media by smears. The amount of fatty squamous epithelial degenera- tion is of importance. This type of degeneration is rarely found in acute otitis media. The cytology in these cases was studied by Dean, L. W. Jr. and Pfingsten, M. G. (Ann. Oto., Rhinol. and Laryngol., 1933, 42, 484-496). Chronic suppurative otitis media is also characterized by excessive numbers of mixed bacteria and the neutrophiles usually exhibit marked degeneration. If there is an acute exacerbation of a chronic condition, the typically chronic picture is seen plus the acute one, in which the neutrophiles are many, well preserved, and often exhibit phagocytic activity as evidenced by containing bacteria. In acute otitis media all gra- dations of monocytes are found from those characteristic of the circulating blood to other larger mononuclears, but in chronic otitis media the larger ones are rare. In some cases of chronic otitis media an increase of eosinophiles has been observed in the exudates. Dohlman, F. G. (Nord. Med. Tidskr., 1943, 17, 224) worked on this problem. In 178 cases of varying types of otitis he found in 99 of them some increase in eosino- philes. Koch, Hjalmar (Acta Otolaryn- gol., 1947, Supp. 62) has also made a complete and detailed study concerned with the finding of eosinophiles in the ear discharges from cases of chronic otitis media. He stated that in the 210 chronic ears examined, there were 52 cases which exhibited an increase in eosinophiles. He concluded that in the eosinophile cases the healing time of the lesion was lengthened and that they were characterized by a very vis- cous secretion varying from clear glass to serum-like in appearance. Hansel, F. K., Allergy of the Nose and Para- EAR SMEARS 108 EAR SMEARS nasal Sinuses; St. Louis: C. V. Mosby Co. 1936, 383 pp.) believes that increase of eosinophiles in affections of the middle ear is due to the chronicity of the infection rather than to an allergic condition comparable to that which appears in the nose. Eosinophiles are frequently noted where there is a con- tinual irritation factor. In aural polyps there may be eosinophilic infiltration in the tissue and if discharge is present eosinophiles may be seen. Proetz, A. W. (Ann. Otol., Rhin. and Laryngol., 1931, 40, 67) reported in detail an asthmatic infant who had attacks of otitis media accompanied by asthma. The secre- tion from the ear did not contain eo- sinophiles. The problem merits careful consideration and further investigation. Considerable significance in the past has been attached to the microscopic findings of cholesterol crystals and fatty degenerated epithelium in chronic mid- dle ear suppurations. Some have re- garded these cytological findings as diagnostic of cholesteatoma of the mid- dle ear and mastoid. Particular care must be taken in these conditions when studying the cytology of the ear, as it has been shown by Dean, L. W., Jr. and Pfingsten, M. G. (Ann. Otol., Rhinol. and Laryngol., 1933, 42, 484-496) that characteristic cholesterol crystals and positive chemical tests for cholesterol can be found in acute otitis media and in external otitis as well as in chronic otitis media. Normal cerumen from the ear may contain more amorphous and crystalline cholesterol than choles- teatoma. It is thus important that the ear canal be thoroughly cleansed and that the exudate studied, be taken from the point of perforation of the drum membrane. It would seem that the finding of cholesterol crystals along with fatty degenerated epithelium in aural secretions is not alone diagnostic of cholesteatoma, but may be sugges- tive in the light of the clinical symp- toms. Cholesterol crystals show double re- fraction of polarized light, so are best observed in unstained direct smears of secretions with a microscope equipped with polarizer and analyzer. Since lipoid substances are the only ones, so far as is known, that doubly refract polarized light, this method is simple and convenient for detecting cholesterol in its crystalline state. When the Nicol prisms of the polarizing apparatus are crossed the field appears dark, but cholesterol crystals, when present, are brightly illuminated against the dark background. They are seen in the char- acteristic flat rhomboid plates, often with irregular edges. The crystals, be- ing very fragile, may be broken into fragments lacking true crystal forma- tion. What the actual association of the crystal formation may be to the fatty degenerated epithelial cells is not known. Cholesterol is also evident in the secretions of infected ears and in ceru- men in the form of liquid crystals ob- served only with a polarizing micro- scope. They appear as small luminous spheres with a black maltese cross superimposed on each one. The phe- nomenon is caused by a molecular for- mation of cholesterol esters that takes place in a liquid medium. The maltese crosses are the result of interference lines of light rays passing through the crystals. They are sometimes seen within large phagocytic cells. Nothing is known of their significance except that their presence indicates choles- terol. In mastoiditis the smear is equally useful in helping to evaluate whether the condition is acute or chronic. Some consider the clinical signs and symp- toms of the patient sufficient, but in these serious diseases every laboratory aid available is needed to facilitate diagnosis. In acute suppurative mas- toiditis, the secretion, if there is ample opportunity for it to drain through the tympanic membrane, may contain large lipoid bone phagocytes, which hint at bone destruction. These same cells are usually found at mastoidectomy in cell pockets of diseased bone containing purulent material where the bone is actively being destroyed. They were first observed in the ear and mastoid by Pfingsten, M. G. in 1934 by using the supravital staining technique as developed by Sabin, F. R. (Bull. Johns Hopkins Hosp., 1923, 34, 277-288). Their identification was reported at that time to the clinical conference for Otolaryngology, Barnes Hospital. A complete report concerning these cells, with case histories, before and after the use of antibiotics, is to be published this year by Bryan, M. P. and Bryan, W. T. K. Such cells are best studied in the liv- ing condition since stains and fixations dissolve the lipoid substance within the cell leaving large empty vacuoles in the cytoplasm giving it a foamy appearance. The cell membrane in ei- ther the fixed or living condition may be indistinct and irregular. The nu- cleus is usually eccentric and sometimes double. The cells are for the most part spherical varying in size from 20- 42 microns in diameter. In comparison EAR SMEARS 109 EAR SMEARS to the size of the cell the nucleus is small and ovoid, often obscured by the lipoid globules which completely fill the cytoplasm. Pseudopodia may be present but active motility has never been observed. They stain positively with Sudan III in the fresh condition. The stain dissolves the globules and the cytoplasm fills with the orange dye. These large phagocytes, when not filled with lipoid material, may be seen to phagocytose cellular debris, red blood cells and entire neutrophiles as well as bacteria. All of the cellular elements should be taken into consideration in order to evaluate the inflammatory response. The display of polymorphonuclear neu- trophilic leucocytes is important. It can be demonstrated that 45-65% of these cells will engulf carmine particles suspended in tyrode or neutral saline solution. If the same preparation is counterstained with dilute neutral red the identical vacuoles containing car- bon will react to neutral red dye, in- dicating that neutral red staining is a fairly accurate criterion for judging phagocytic activity. Hesse, Herr (Ztschr. f. Hals, Nasen u. Ohrenh., 1927-1928, 18, 377-382) studied fresh exudates from middle ear suppurations and distinguished phagocytic and non- phagocytic leucocytes, but he stated that ceil death occurred very rapidly. For staining he used the Synderhel- mishe dye, a preparation of trypan blue and Congo red. The dye has been used here and found to be very toxic for the cells. It does not give sufficient time for observation in the living state. He made no mention of the giant bone phagocytes. Other cells encountered in acute mas- toiditis include monocj^tes, lympho- cytes, eosinophiles and basophiles. Many variations in the mononuclear cells are found. Besides the typical blood monocytes there are gradual tran- sitions between these cells and the giant phagocytes. Often it is advisable to distinguish between these cells when they are not engorged with lipoid ma- terial, but yet are larger than the typical monocyte with many varying shades of neutral red vacuoles and a smaller ovoid eccentric nucleus, rather than the typical horse-shoe nucleus of the monocyte. These are termed "transitional mononuclears", or cor- respond in appearance to the "clasmato- cytes" in the classification of Sabin, F. R., Doan, C. A. and Cunningham, R. S. (Contrib. to Embryol., 1925, 16, 127-162). The lymphocytes may show slight motility in fresh preparations and sometimes a few neutral red in- clusions. If Janus Green is used the mitochondria may be observed. Eosin- ophiles, if seen in these secretions, may exhibit motility in the living state but are never seen to store particulate matter. In acute mastoiditis there are rarely more than a few seen. Baso- phile cells are also noted in many cases. They are usually few in number and their significance is not known. In contrast to the various cells as- sociated with active mastoiditis, there are those observed in chronic mas- toiditis, namely, large numbers of de- generated neutrophiles, fattj^ degen- erated squamous epithelial cells and masses of mixed bacteria. Bacteria are numerous along with cellular debris. Giant phagocytes are rarely found in the chronic condition unless there is an acute exacerbation of the infection. Many other cellular details require to be correlated with the symptoms of the patient in these diseases. Cytological studies aid in differentiat- ing the many types of external otitis. Senturia, B. H., Matthews, J. I. and Adler, B. C. (The Laryngoscope, 1950, 60, 543-550) have made important con- tributions showing that the smear gives information as to the causative agents in external otitis, whether they are bacilli, cocci or fungi. Examination of the smear is helpful in distinguishing between suppurative otitis media and external otitis (except the circum- scribed type). Senturia states that the ear with a hidden tympanic perforation and a secondary diffuse external otitis may be difficult to differentiate from a diffuse external otitis with exudate cov- ering the tympanic membrane. Cyto- logical examination usually makes the difference clear. Otitis media with per- foration shows neutrophiles, lympho- cytes, mucus strands, with a few bac- teria and epithelial cells. Diffuse external otitis shows a predominance of epithelial cells and bacteria, with per- haps an occasional leucocyte or mucus strand in the secretion. The most striking finding in the exudates of acute and diffuse external otitis is the absence of neutrophiles. In regard to the early diagnosis of malignant and benign tumors of the middle ear and external canal, the cytology of the discharge should be studied carefully with the staining tech- nique developed by Papanicolaou, G. N. (Science, 1942, 95, 438-439). This is a reliable aid in conjunction with biopsy and it is especially important if biopsy is not possible. Fortunately carcinoma of the middle ear is somewhat EAR SMEARS 110 EHRLICH'S TRIACID rare . House , H . P . (Ann . Otol . , Rhinol . and Laryngol., 1949, 58, 789-797) re- viewed the literature and reported that 201 authentic cases of the disease have been reported. He discussed two cases in which the technique of Papanicolaou aided in diagnosis and indicated, that since the majority of malignancies of the middle ear are superimposed on chronically discharging ears, the method should prove of value in the early diagnosis of carcinoma of the middle ear. Diamont, M. (Acta Otolaryng., 1941, 29, 77-79) pointed out the diffi- culty even with the Papanicolaou stain of differentiating clinically between granulations in chronic otitis and early malignancy. Smears, however are very useful in these cases in efforts to follow the cytology of the lesions after opera- tive procedures. Subsequent biopsies are not always feasible. The smear technique causes no inconvenience to the patient. It is reliable in the evalua- tion of x-ray and radium therapy since the effects of such treatments are re- flected in the cellular response. It seems important, in order to ob- serve all of the cellular details possible, to use the method of Papanicolaou as well as a good polychrome stain, such as Wright's or Hansel's, especially in the chronic ear conditions, so that the exudate can be studied in both stains. Details of the light staining with Wright's and of the use of buffer solu- tion are described in the staining of Nasal Smears. Short drying of the slide in air, rather than flaming, is recommended for maximum cellular de- tail. Whenever possible, in order to complete the cytological picture, it is interesting to use the supra-vital tech- nique on living fresh material. There are vast differences in appearance be- tween living and stained cells so that accurate cellular differentiation be- comes a complicated problem. In order to insure good results from any of the staining techniques, a fresh sampling of the discharge is imporatnt. It has often been found satisfactory to use a frontal sinus silver cannula with rubber bulb to obtain the secretion as close as possible to the point of perforation of the drum membrane. The discharge may then be released and spread evenly and thinly on a clean glass slide. At mj^ringotomy it is taken directly follow- ing the incision of the drum membrane. Since there are a number of reliable staining methods that are adaptable to the study of aural exudates, the knowledge gained thereby can be cor- related with the clinical symptoms of the patient. This laboratory aid is important in otologic examination and stimulates an awareness of the ever changing pathological processes. Earle, see Tissue Culture. Ectoplasm. Cytoplasm lying immediately internal to the plasma membrane. It is usually gelled, and, being free from various formed bodies present in the endoplasm, has a clear hyaline appear- ance. Egg, inoculation of hen's eggs, see Chorio- allantoic Membrane. Egg of hel- minths, see Floatation Techniques. Transplantation of living fertilized eggs, see account of Placenta. Ehrllch-Biondi Stain, known also as the Ehrlich-Biondi-Heidenhain mixture, is one of the classical stains. Add 20 cc. sat. aq. acid fuchsin and 50 cc. sat. aq. methyl green to 100 cc. sat. aq. orange G agitating the fluid while doing so. Add 60-100 cc. aq. dest. The diluted mixture should redden slightly if a little acetic acid is added. A drop placed on filter paper should be bluish green at the center and orange at the periphery. If there is an outside red zone too much fuchsin has been used. Stain sections of sublimate fixed tissues 12-24 hrs. Do not wash in water but dehydrate quickly. Clear and mount. This stain gives beautiful results when properly employed but it is fickle. Many helpful suggestions are given in Lee, p. 179. Ehrlich's Acid Hematoxylin. Dissolve 2 gm. hematoxylin in 100 cc. 95% alcohol and add; aq. dest., 100 c; glycerin, 100 cc; ammonium (or potassium) alum, 3 gm., glacial acetic acid, 10 cc. Ripen by exposure to air (but not dust) 2 or 3 weeks, or immediately by addition of 0.4 gm. sodium iodate. Ehrlich's Aldehyde Reagent. 2 gms. para- dimethj^lamino-benzaldehyde in 100 cc. 20% aq. hydrochloric acid. See Uro- bilin. Ehrlich's Triacid blood stain. This, also, is one of the classic stains, now seldom used. It contains methyl green, orange G and acid fuchsin; but methyl green is a basic dye so that it is not made up of three acid dyes. Ehrlich explained that it is so called "because in it all the three basic groups of the methyl green are combined with acid dye-stuffs" (Lee, p. 167) with which modern chemists do not agree. Air dried smears are fixed by heat (110°C) about 2 min.; stained in triacid (Griibler) 5 min. ; washed in aq. dest. until no more color is extracted and dried with smooth filter paper. Said to color neutrophile granules and leave azur granules unstained. EIMERIA 111 ELASTICA-TRICHROME STAIN Eimeria, see Coccidia. Einarson, see Gallocyanin-Chromalum Staining of Basophilic Cell Structures. Elacin, see Elastic Fibers. Elastase. An elastolytic enzyme from pan- creas has been reported by Bal6, J. and I. Banga, Biochem. J., 1950, 46, 384- 387. This enzyme renders elastic tis- sue soluble, without the formation of amino acid residues. Consequently, it apparently depolymerizes elastin. Since elastic tissue is highly insoluble, this enzyme should prove most useful both for chemical and histological in- vestigations. Elastic Fibers — Written by A. I. Lansing, Dept. of Anatomy, Washington Uni- versity, St. Louis 10, Mo. October 5, 1951 — Viewed in fresh unstained spreads of Loose Connective Tissue these fibers are generally yellow and are more highly refractile than coUagenic fibers. They are optically homogeneous, branch re- peatedly to form networks, are of vari- able thickness and do not swell in dilute acids. Elastic fibers are resistant to both pepsin and pure trypsin and are slowly digested in commercial grade trypsin. The latter may be due to contamination by elastase, a new, rela- tively specific enzyme which solubilizes elastic fibers (Bal6, J. and Banga, I., Biochem J., 1950, 46, 384). Elastic fibers are also solubilized by prolonged boiling in 0.25 N oxalic acid (Adair, G. S., Nature, 1951, 167, 605). Elastin may also be distinguished from collagen by its amino acid compo- sition. As indicated in the accompany- ing table, collagen is very rich in hy- droxyproline while elastin has only small amounts. Not all elastins have the same amino acid compositions. Ligamentum nuchae (cow) differs sig- nificantly from elastin of human arteries which differ from one another. Indeed age influences the amino acid composi- tion of elastin from human aortas. Age influences many of the properties of elastic tissue. Senile elastic tissue of skin is often called elacin. This material is not unlike elastic tissue in old arteries. Age differences in elastic tissue may be summarized as follows: Elastic fibers are readily demon- strated in sections by the use of one of several fairly specific stains including Weigert's Resorcin-Fuchsin, Verhoeff's Hematoxylin Stain, Unna's Orcein Method, Krajian's Congo Stain. After oxidation elastic fibers are colored red by the Schiff Reagent. Victoria blue and Basic Fuchsin also effectively stain elastic fibers. When viewed with polarized light elastic fibers are not birefringent but become so when stretched. This would suggest that, although the fibers are optically homogeneous, they possess internal structural orientation. Elec- Amino Acid Composition of Collagen and Elaslin* Amino Acid Total N Glycine Alanine Leucine Isoleucine Valine Phenylalanine. .. Tyrosine Tryptophan Serine Threonine Cystine Methionine Proline Hydroxyproline . Lysine Hydroxylysine. . Arginine Histidine Aspartic acid .... Glutamic acid. . . Amide N •CO c4 i-i O V a-*-* < "2 flla SI? a o < SfS lastin (Liga Nuch so •52 sU o"-^ ^t ssy U W w w < & rt a o a 3 C.S gN per 100 gN 18.6 26.2 9.5 3.22 2. IS 3.4 4.2 1.4 0.0 3.4 2.4 0.0 0.8 15.1 14.0 4.5 1.3 8.8 0.8 6.3 11.3 0.66 Total found 119.00 17.1 15.71 29.9 26.10 18.9 23.18 8.7 4.52 4.0 2.10 17.4 13.00 5.0 1.73 1.61 1.45 0.01 0.06 .82 0.29 .96 0.65 0.15 0.06 0.03 0.06 17.0 10.10 2.0 — 0.39 0.49 0.89 1.78 0.07 0.15 0.63 0.38 2.1 1.83 0.04 2.90 — 90.83 15.56 21.30 21.58 4.76 2.33 11.50 1.97 1.76 0.24 0.70 1.13 0.10 0.35| 9.20i 1.17 4.35 0.75 l.Il 3.01 2.86 16.00 32.00 4.78 2.28 12.9 90.17 11.0 0.62 1.75 1.51 • Adapted in part from Gross: J. Gerontol., 1950, 5, 343. t Bowes, J. H., and Kenton, R. H.: The amino acid composition and titration curve of collagen. Biochem. J., 1948, 43, 358-365. t Neuman, R. E.: Amino acid composition of gela- tins, coUagens and elastins from different sources. Arch. Biochem., 1949, 24, 289-298. § Lan.sing, A. I., Roberts, Eugene, Ramasarma, G. B., Rosenthal, Theodore B., and Alex, Morris: Changes with age in amino acid composition of arterial elastin. Soc. Exp. Biol. Med., 1951, 76, 714-717. tron microscopy has so far failed to reveal internal structure. Elastic fibers partially digested with elastase do re- veal the presence of intertwined fibrils. Elastic Properties, see Surface Tension. Elastica-Trichrome Stain. In order to demonstrate elastic fibers with equal clearness to the smooth muscular and collagenic fibers, especially in the walls ELASTIN 112 ELECTRON MICROSCOPY of blood vessels, a useful combination of Weigert's elastic tissue stain and Masson's trichrome stain has been worked out by Mendeloff, J., Am. J. Clin. Path., 1943, Tech. Suppl. 7, 65. Deparaffinize sections in usual way, wash thoroughly in water and stain in Weigert's Resorcin Fuchsin mixture for 60 min. Wash quickly in Acid Alcohol, dehydrate and differentiate in abs. ale. till section is only faintly red. Pass Some Properties of Human Arterial Elastin Young Senile Physical Straight, anasto- Frayed, fragmented, appear- mosing threads or thin strands and ance ribbons granules Glistening, refrac- Tend to clump, yel- tile, water-clear low, dull Tinctorial 1. Not stained by 1. Take up hema- hematoxylin toxylin 2. Red color with 2. Yellow - orange Congo Red with Congo Red 3. Resorcin-fuchsin, 3 Stain more orcein "Van densely with re- Giesen" positive sorcin, etc. Chemical 1. Mineral -free 1. Severely mineral- 2. Phosphorus-free ized. Contains as 3. Amino acids: much as 14% cal- a) trace of as- cium partic acid 2. Large amounts of b) 1% glutamic phosphorus acid 3. Amino acids: a) appreciable amounts of as- partic acid b) 4% glutamic acid Physical Dry isolated elas- Dry isolated elastin. tin, has specific has specific gravity gravity less than greater than 1.30 1.30 through 70% ale. to aq. dest. and stain in Harris' Alum Hematoxylin 8 min. Differentiate in water 5 min. Stain in Ponceau acid fuchsin mixture (see Mas- son's Trichrome) 5 min. Wash thor- oughly and place in 3% aq. phospho- tungstic acid, 10 min. Wash again thoroughly in water and stain with light green. Transfer directly to 1% acetic acid, 3 min. Do not wash but dehydrate, clear and mount in Gum Damar. Elastic tissue, blue -black; smooth muscle, red; collagen, green. Elastin, see Elastic Fibers. Electric Tissues of fishes, methods for are given by Dahlgren (McClung, 1950, p. 343). Electrical Resistance and capacity or Impedence. By employing alter- nating currents of varying frequencies figures for apparent resistance and capacity can be obtained. Red cells, yeast cells, ova etc. have been investi- gated. The technique is not micro- scopic but the data have an important bearing on structure. In view of the wide variety of cells studied it is inter- esting, as Danielli remarks (Bourne, p. 42), that a definite pattern should emerge of a cell plasma membrane only 10~*-10~' cm. in thickness corresponding to a specific resistance of lO^^-lO" ohms. Electrodes. See the several varieties de- scribed in full with literature references and diagrams by Click, pp. 183-188. Electromagnet Technique to determine elas- ticity (Heilbroun, A., Jahrb. wiss. Bot., 1922, 61, 284), employed by Seifriz, W. and Hock, C. W. (Paper Trade J., 1936, 102, 36) and described by Cham- bers, R. W. and Kopac, M. J. in Mc- Clung's Microscopical Technique, 1950, p. 542. Electron Microscopy — Details originally Erovided by Dr. W. L. Simpson; revised y Dr. T. B. Rosenthal, Dept. of Anat- omy, Washington University, St. Louis. June 6, 1951. 1. Transmitted electron beam type. The relationship of the wave length (X) of light employed and to the nu- merical aperture (N.A.) of a lens system as expressed in the relation R.P. equals T;^—r- proved for many years an ap- parently insurmountable limitation to the biologist's desire to investigate di- rectly minute structure of cells and tissues. Even when ultraviolet light of 2250A was employed the limit of resolution was 0.08 y. in & system of N.A. 1.40. With visible light the limit was approximately 0.2 y.. On the as- sumption that the angle of visual acuity is 1 minute of arc, the greatest mag- nification that was practical with visi- ble light ranged from 1750 to 2100 times. There is, of course, no limitation to the actual magnification that may be achieved. Increases beyond the limit mentioned, however, do not reveal new structures. As long as this was true there seemed no hope of direct confir- mation of the amazing findings made possible by such new methods as x-ray diffraction, ultracentrifugation studies, chemical studies of virus structure, and polarized light methods. Small wonder is it then that the biologist has grasped with enthusiasm at the possibilities of visualizing ultra- microscopic structure by means of devices that have grown from the fertile ELECTRON MICROSCOPY 113 ELECTRON MICROSCOPY field of electron optics. Of most general interest is the electron microscope. With this instrument, using the same equation for resolving power, it should be possible to reach a resolution of at least O.OOl/x- Thus, an improvement of at least 200 times over the limit with visible light might be achieved. The practical limit on magnification has been placed at from 70,000 to 100,000 times. Historically the electron microscope is now twenty-four years old. Busch described the first such instrument us- ing magnetic lenses (Busch, H., Arch, f. Elektroteknik, 1927, 18, 583-594). Though many improvements were made in design it was not until 10 years later that the instrument reached the point of being of practical use. Chiefly through the work of Ruska and Borries (numerous papers, 1934-1940) the in- strument was developed to the state that it is in at present. In this country an e.xcellent instrument, capable of giving high resolution has been de- veloped and commercially marketed by the Radio Corporation of America. The apparatus is described by Zwory- kin, V. K. (Science, 1940, 92, 51-53). In this instrument electrons emitted from a hot wire filament are accelerated by a potential of 30,000 or more volts. This beam is condensed and passed through the object which is carried on a wire screen supported nitro-cellulose film. The transmitted electron beam is focussed in a greatly enlarged image by means of two magnetic lenses. The image can be seen on a fluorescent screen or photographed on a sensitive plate. The conditions under which good re- sults are obtainable are rather stringent. The ideal object must be very small, or capable of being minutely divided with- out losing its identity; it must maintain its form on drying in vacuum; and it must be rigid enough to resist the heat and disruption caused by exposure to the electronic bombardment. Rela- tively few biological structures fall into this category; hence early studies were confined to bacteria, viruses, connective tissue fibers, blood cells, spermatozoa, etc. The great need in histological in- vestigation with the electron micro- scope has been a method for uniformly cutting tissues no thicker than 0.1 ^ and about 1 mm* or larger in area. With these dimensions a section will yield acceptable electron micrographs. Such a method seems to have been found in a modification of the conven- tional microtome whereby the advance of the block is reduced about tenfold, either mechanically (Baker and Pease, J. Appl. Phys., 1949, 20, 480) or by means of a thermal expansion device (Newman, Borysko, and Swerdlow, Anat. Rec, 1949, 105, 267). The tissue must be embedded in celloidin or plas- tic, but a special blade is probably necessary for the best results. Since 1950 publications have appeared with electron micrographs of nearly all tis- sues of importance. See Excerpta Med- ica, Sect. I, for listings and abstracts. Histologists have also borrowed spe- cial methods from metallurgists for investigation of hard, opaque structures like bone, tooth, hair, keratinized epi- thelium, etc. These methods involve the casting of very thin replicas of surface details. Additional contrast is gained by the ingenious process of "shadow casting", either on the original specimen or on its replica. A very thin layer of metal is deposited over the object in a vacuum by evaporation from a hot filament. When done at an acute angle, the elevated portions of the object shadow the depressed areas, thus giving a striking three-dimensional pic- ture (Williams and Backus, J. Appl. Phys., 1949, 20, 98). Methods have also been developed for true stereoscopic images (Heidenreich and Matheson, J. Appl. Phys., 1944, 15, 423). See Shad- dow Casting. The old technique of selective stain- ing has been combined with this newest of procedures in histology. It has been shown that the salts of heavy metals, such as osmium and phosphotungstate, are deposited in a highly localized way on certain protein structures, e.g. mus- cle fibrils, thus enabling visualization of practically molecular details (Hall, Jakus, and Schmitt, J. Appl. Phys., 1945, 16, 459). With the proper selection and com- bination of histological methods now on hand the electron microscopist should have little difficulty in studying any problem in morphology. A practi- cal handbook for the guidance of novice and expert has been prepared by the Royal Microscopical Society: "The Practice of Electron Microscopy", edited by D. G. Drummond and pub- lished as'Part I of vol. 70, 1-141, 1950 of the J. Roy. Micro. Soc. Papers dealing with details of manipulation are to be found regularly in the Journal of Ap- plied Physics. 2. Emission electron type. The earli- est description of an electron micro- scope in this country was of an entirely different type from the new R.C.A. mi- croscopes that give such prodigious magnifications. McMillan and Scott (J. H. and G. H., R.S.I. , 1937, 8, 288- ELECTROPHORESIS 114 ELEMENTARY BODIES 290) published an account of an electron microscope of simple design that used as a source the electron emission of heated sections of tissues. These were accelerated by a potential of 1000 to 2000 volts, focussed by a magnetic lens onto a fluorescent screen. An improved design (Scott, G. H. and Packer, D. M., Anat. Rec, 1939, 74, 17-29) makes pos- sible magnifications of at least 150 times. By certain modifications the magnification can be increased con- siderably. It is feasible with this in- strument to obtain photographs that record the precise localization of cal- cium and magnesium salts in tissues. Scott and Packer {ibid, 31-45) showed that the calcium and magnesium of skeletal muscle was confined almost en- tirely to the muscle fibers themselves, and that in contracted fibers a great concentration of magnesium appeared in the contraction nodes. Tissues to be studied with this tech- nique must be preserved in a manner that permits no redistribution of min- erals. The satisfactory method is that of Altmann-Gersh. Electron microscopic technique sup- plements histospectrography by pre- cisely locating certain elements within tissues and is very useful in conjunction with the technique of microincineration as a means of identifying certain com- ponents of the ash seen in sections. Electrophoresis. Most particles suspended in water carry electricity. If placed in an electric field those possessed of positive charge move toward the cath- ode and those with a negative charge toward the anode. Obviously there- fore the nature of the charge and the speed of movement can be determined by microscopic study of particles sus- pended in fluid in what is known as a micro-electrophoresis cell. Types of cell and precautions to be observed in their use are described by Moore, D. H. and Abramson, H. A. Glasser's Medical Physics, 403-407. Their account of the "moving boundary" method of electro- phoresis and of the Tiselius apparatus is clear and to the point. This latter method, in contrast with the micro- scopic one, affords a technique of great accuracy and sensitivity for separating concentrations and purifying submicro- scopic components in blood serum and other complete liquids. Eleidin (G. elaia, oil) gives to the stratum lucidum its clear, glassy appearance. It may be a dissociation product of keratohyalin. There has been no great improvement on the specificity of the older methods. Mallory (p. 260) gives the method of Buzzi (1889), first cau- tioning that fixation must be in formalin, Orth's or Bouin's fluid. Stain frozen sections of 10% formalin fixed tissue in sat. aq. picric acid (approximately 1.2%) 5 min. Rinse in aq. dest. and counterstain for 1 min. in 1% aq. nigro- sin. Wash in water and then in 95% ale. (Skip absolute) Clear in ter- pineol or origanum oil. Mount in bal- sam: keratin, bright yellow; eleidin, blue black. Ranvier's Picro-Carmine gives a fine red staining of eleidin. See finger Nails. Elementary Bodies are the smallest particles of viruses. Those of certain viruses are large enough for direct microscopic examination in suitably stained prepara- tions which usually show also the larger Inclusion Bodies if these are present. Various methods designed for Rickettsia are usually satisfactory. Many special techniques have been proposed of which 2 follow : 1. Methyl violet or Victoria blue for smears (Gutstein, M., J. Path. & Bact., 1937, 45, 313-314). Dry smears on perfectly clean slides in air or incu- bator. If necessary remove excess protein by rinsing in physiological saline solution followed by aq. dest. Fix in methyl alcohol 1 hr. Stain in either of 2 ways: (1) Place slide in Petri dish. Mix equal parts 1% aq. methyl violet and 2% aq. NaHCOs. Filter imme- diately onto the slide, cover dish and incubate at 37 °C. 20-30 min. Rinse in aq. dest., dry and mount in cedar oil or liquid paraffin. Elementary bodies light violet. (2) Same except filter onto slide equal parts (a) Victoria blue 4R 1 gm., ale. (abs.) 10 cc. and aq. dest. 90 cc. and (b) 0.02% aq. KOH and leave at room temperature over night. Ele- mentary bodies of vaccinia and other viruses dark blue. 2. Methyl blue acid fuchsin for sec- tions (Nicolau, S. and Kopciowska, L., C. r. Acad. d. Sci., 1937, 204, 1276-1278). Fix in alcoholic Bouin's fluid. Stain 4-5 micron paraffin sections 30-60 min. in: methyl blue (Griibler) 1.5 gm., aq. dest. 65 cc, methyl alcohol 35 cc, glyc- erin 5 cc, 3% aq. oxalic acid 5 cc. Wash well in aq. dest. and change to absolute alcohol. Stain 20 min. in: acid fuchsin 1.5 gm., aq. dest. 100 cc, 3% aq. oxalic acid 2 cc Wash directly in absolute alcohol and mount in the usual way. Small particles in cells associated with following viruses : herpes, Borna, Zoster, rabies and pseudo-rabies are stained bright red. A summary of methods for demon- strating elementary bodies is given by Seiffert, G., Virus Diseases in Man, Ani- mal and Plant. New York : Philosophi- ELLIPSIN 115 ENAMEL cal Library, Inc., 1944, 332 pp. Under favorable conditions some kinds of of elementary bodies are visible at high magnification unstained by both direct and dark field illumination. Supra- vital stains such as brilliant crcsyl blue, neutral red, methylene blue and azur II are recommended. Before staining smears, fixed in a variety of ways, pre- treatment with 2.5% aq. potassium per- manganate or 2% aq. chromic acid is advised. Giemsa stain gives good re- sults but the methods of Paschen, Moro- sow and Herzberg are suggested by Seiffert. The fluorescence technique of Hagemann consists of staining thin air dried smears with 1 gm. primuline dissolved in 1000 cc. aq. dest. + 20 cc. pure phenol for 15 sec. washing in aq. dest. and observation in ultraviolet light by fluorescence microscope. New methods for the collection and purification of elementary bodies permit their direct examination at very high magnifications with the electron micro- scope (von Borries, E. G., Ruska, E. and H., Klin. Woch., 1938, 17, 921 ; Green, R. H., Anderson, T. F., and Smadel, J. E., J. Exp. Med., 1942, 75, 651-656) and their chemical analysis for vitamin catalysts, copper and enzymes (Hoag- land, C. L., Ward, S. M., Smadel, J. E., and Rivers, T. M., J. Exp. Med., 1942, 76, 163-173). See fluorescence of ele- mentary bodies (Turevich, E. I. ab- stracted in Stain Techn., 1941, 16, 182.) Ellipsin is structure protein of cells. Meth- ods for its isolation from liver cells of rabbit and guinea pig by grinding fresh tissue, washing, centrifugation and so on are fully described by Bensley, R. R. and Hoerr, N. L., Anat. Rec, 1934, 60, 251-266. Embedding, see Imbedding. Embryo Chick, see Chorioallantoic Mem- brane. Embryological Methods. In general the techniques which give good results with adult tissues are also satisfactory for embryos ; but there are differences as for example in silver impregnations. More- over greater care is necessary to avoid too sudden changes in the fluids used. Helpful suggestions are given in Mc- Clung, pp. 279-286. Application of trichrome staining methods to embryos (Baxter, J. S., J. Anat., 1940-41, 75, 137-140). See demonstration of Car- tilaginous Skeleton, Ossification and Spalteholz method. Technique for handling chick embryos (Adamstone, F. B., Stain Techn., 1931, 6, 41-42). Block staining of nervous tissue of em- bryos with silver (Davenport, II. A., Stain Techn., 1934, 8, 143-149). Emission Electron Microscopy, see Electron Microscopy. Enamel (dental). This can best be studied in ground sections of Teeth. 1. Cuticle. Wash and brush tooth in tap water. 4% neutral formalin, 24 hrs. Wash tap water, 24 hrs. Mallory's anilin blue (0.5% aq.) 24 hrs. Again wash and brush in tap water. 10% aq. hydro- chloric acid, 10 min. As enamel is dis- solved delicate opaque white membrane appears. Tease membrane off onto slide coated with egg albumen (Albumen- Glycerin). Blot with filter paper. 5% aq. sodium thiosulphate or bicarbonate 10 min. Wash in tap water 10 min. Dehydrate in alcohols, clear in xylol and mount in gum damar (McClung, p. 371). 2. Rods. Macerate tooth in 5-10% aq. hydrochloric acid for 24 hrs. Re- move a little softened enamel and examine (McClung, p. 372). See Chase, S. W., Anat. Rec, 1927, 36, 239-258. 3. Organic Matrix. Boedeker's method abbreviated from McClung (p. 372). Dehydrate small piece (0.5- 1 mm. thick), free from dentin, through alcohols 10 min. each. Methyl alcohol 1-2 hrs. Decalcify in celloidin mixture (parlodion, DuPont) made by dissolving sufficient in methyl ale. C.P. to give thick syrupy solution. To 150 cc. of this add drop by drop constantly stir- ring nitric acid C.P., 10 cc. -f methyl ale. 40 cc. Keep tissue in this mixture in glass dish with air tight cover. Or- ganic matrix appears as brown, spongy material in 10-12 hrs. care being taken to leave the dish stationary. After decalcification is completed, 2-7 days, uncover and permit celloidin to harden. Cut out specimen with narrow margin of celloidin. 70 and 40% ale. 1-2 hrs. each. Aq. sol. alum, 24 hrs. Running water, 6-12 hrs. Ascending alcohols to 95% 1-2 hrs. each. Anilin oil, 6-12 hrs. (becomes brown and transparent). Equal parts anilin oil and chloroform, 6-12 hrs. Imbed in paraffin not over 52°C. Mount 3-10^ sections, dry and treat with xylol 3 min. Dissolve cel- loidin in ether-alcohol. Abs. ale. 1 min. Descending alcoliols to water. Stain in Iron Hematoxylin. Improved results may be obtained by slow decalcification and concurrent fixa- tion as described b.y Sognnaes, J. Dental Res., 1949, 28, 558-564. Teeth are placed in celloidin bags filled with 10% formalin and immersed in a solution of 5 gm. trichloroacetic acid, 2^ gm. po- tassium bichromate in 100 cc. distilled water. Ten centimeter of formalde- hyde is added to the solution. Decalci- fication of enamel is completed after ENDAMOEBA 116 ENTEROCHROMAFFIN CELLS one week in a refrigerator at a tempera- ture of 1° C. Decalcification of dentin is completed by placing the bag in 5% trichloroacetic acid. Not more than two weeks should be required. Washing and dehydration in ethyl alco- hol and butyl alcohol is carefully ac- complished with tooth still in the bag. Imbed in paraffin, section and stain by ordinary methods. Constant pressure during decalcification may further im- prove results (Sognnaes, R. F., J. Den- tal Res., 1948, 27, 609-622). 4. Cape-Kitchin celloidin decalcifica- tion method. Cut DuPont's parlodion into small cubes and dissolve in acetone free methyl alcohol making thick solu- tion. To 200 cc. add 90 cc. methyl alcohol constantly stirring and 9 cc. nitric acid, sp. gr. 1.42. Follow decalci- fication of enamel in this mixture be- tween crossed nicols of polarizing micro- scope with 24 mm. objective. Double refraction disappears with decalcifica- tion (Bodecker, C. F., J. Dent. Res., 1937, 16, 143-150). 5. Permeability. When the apex of a tooth is immersed in strong alcoholic solution of fuchsin -f NaCl the enamel becomes stained (v. Beust, T., Dental Cosmos, 1912, 54, 659). Another way is to test for penetration of lead, boron and other easily recognizable chemicals (Howe, P. R., Dental Cosmos, 1926, 68, 1021-1033). After intraperitoneal in- jections of trypan blue blue coloration can be observed in developing enamel only (not adult) as well as in dentin of dogs (Gies, W. J., J. Nat. Dent. Assoc, 1918, 5, 529-531). Marshall (J. S., J. Dent. Res., 1921, 3, 241-255) employed Naphthamine brilliant blue similarly as a vital stain. See Dentin, vital staining. A "Triple Embedding" tech- nique is described by Brain, E. B., J. Roy. Micr. Soc, 1950, 70, 313-316. Endamoeba, see Entameba. Endocervical Smears, see Papanicolaou Techniques. Endolymph. To demonstrate its circulation employ method used by Guild, S. R., Am. J. Anat., 1927,39, 57-81. Introduce solution of potassium ferrocyanide and iron ammonium citrate into cochlear ducts of living guinea pigs under anes- thesia. Kill at intervals up to 48 hrs. Excise tissue and fix in acid fluid which precipitates Prussian Blue wherever the solution has circulated. Endometrial Smears, see Papanicolaou Techniques. Endospore stain for bacteria in blood smears. Smear, air dry and fix by flaming 3 times. 5% aq. malachite green 5 min., wash in tap water 10-20 sec. 0.5% aq. safranin, 10 sec, wash quickly, dry and examine (Bruner, D. W. and Edwards, P. R., 3. Lab. & Clin. Med., 1939, 25, 543-544). Enrichment techniques, see Concentration. Entameba. Craig (p. 35) gives a useful table of diagnostic features of intestinal amebae in man; also, on p. 55, a list of objects that may be mistaken for amebae in unstained and stained prepa- rations; and details as to media for cul- tivation of which the Boeck and Doboh- lav media and the simpler Craig media are the most helpful. This genus includes E. histolytica, the cause of amebic dysentery and E. coli and E. gingivalis, two apparently harmless commensals. The technique is essentially the same for all three. In searching for E. histolytica or E. coli take a small amount of fresh feces, mix with physiological saline solution and examine directly. Recognize amebae by large size and movements if slide is kept warm. E. histolytica frequently contains erythrocytes. Mallory (p. 296) advises mixture with Gram's Iodine solution to demonstrate glycogen if present, or mixing with drop 1-2% formalin, then treatment with drop 2% acetic acid and coloration with 1 drop 1% aq. neutral red. E. gingivalis is to be found in decayed teeth. Only E. histolytica extensively invades tissues. 1. To make permanent smear prepara- tions (Mallory, p. 296) fix thin smear while moist in 95% alcohol, 1 part, and sat. aq. corrosive sublimate, 2 parts, for 15 min. Wash for few sec. in water and cover with 1% alcoholic iodine for 3 min. Wash in aq. dest. until iodine color is extracted. Wash again and stain with Phosphotungstic Acid Hema- toxylin, 30 min. Wash in water, dehy- drate in 95 and abs. alcohol, clear in xylol and mount in balsam. Nuclei and ecto- sarc, deep blue; cytoplasm, bluish. 2. To stain differentially in sections (Mallory, p. 297). Fix in 95% or abs. ale, and make paraffin or celloidin sec- tions. Stain in 0.25% aq. thionin 3-5 min. Differentiate in 2% aq. oxalic acid, i-1 min. After washing in water, dehydrate in 95% and abs. ale Clear in xylol and mount in balsam, except for celloidin sections which require clearing in terpineol, or origanum oil, after 95% ale. Nuclei of amebae brownish red, those of all other cells, blue. See lodine-Eosin stain and Walker's Method. Enterochromaffin Cells. Perhaps the best technique is Bodian's protargol method as described by Dawson, A. B. and Barnett, Julia, Stain Techn., 1944, 19, 115-118. For the influence of pilo- carpin on enterochromaffin cells see Hamperl, II., Ztschr. f. Mikr. Anat. ENTOMOLOGICAL TECHNIQUES 117 EOSIN -ORANGE G Forsch., 1925, 2, 506-535. See Small Intestine. Entomological Techniques, see Mosquito, Ticks, Insects, Arachnids, Parasites. Enzymes — Written by E. W. Dempsey, Dept. of Anatomy, Washington Uni- versity, St. Louis. February 26, 1951 — Their name is legion. At present only a few can be localized with any degree of histological precision, but the number of histochemical methods available is increasing rapidly. There are no bet- ter examples of felicitous association between histological and biochemical methods. Four principal kinds of tech- niques are employed for localization: (1) By spectrographic identification in the tissues — especially the metal-con- taining Cytochromes and Peroxidase. (2) By close comparison of enzymic activity with cellular composition of tissues. See Glick for detailed meth- ods, derived from the Linderstr0m- Lang procedures — Amylase, Pepsin, Peptidase, Esterase, Protease, Cho- linesterase, Lipase, Urease, Carbonic Anhydrase, etc. (3) By separation of cellular components, such as nuclei, granules and mitochondria, from homo- genates of tissue. Such separation is accomplished by differential flotation or centrifugation, and is followed by esti- mation of the enzymic activity of the purified component fractions. Argi- nase, Adenylpyrophosphatase, Phos- phatase, etc. have been investigated by such methods. (4) By the develop- ment of characteristic insoluble prod- ucts within tissues or cells — Aldolase, Acid and Alkaline Phosphatase, Cholin- esterase. Cytochrome Oxidase, Dehy- drogenase, Dopa Oxidase, Esterase Glucuronidase, Lipase, Peroxidase, Phosphamidase, Sulfatase. Enzymes are coming into their own as technical tools. Purified enzymes may be used to destroy certain tissue components. Ribonuclease selectively destroys basophilic substances in cyto- plasm and nucleus, Desoxyribonuclease similarly attacks nuclear chromatin. Hyaluronidase solubilizes certain meta- chromatic ground substances, whereas other mucoid materials react only with more potent Mucinases. The connec- tive tissue fibers have long been charac- terized by their digestibility in Pepsin and trypsin, and recently an Elas- tase has been prepared. A purified Collagenase has been reported. The solubility of glycogen in solutions of salivary Amylase is an old histochemical procedure. The effects of Lysozyme and other enzymes on the capsules of pneumococci and upon the Gram stain has been summarized by DuBos (The Bacterial Cell, Harvard Univ. Press, 1945). Eosin B or bluish (CI, 771)— eosin BN, BW, or DHV, eosin scarlet, eosin scarlet B, imperial red, nopalin G, saffrosin, scarlet J, JJ, V — Dibrom derivative of dinitro-fluorescein. Chemistry of (Holmes, W. C, Melin, C. G. and Paterson, H. R., Stain Techn., 1932, 7, 121-127). There are several fluorescein dyes and guidance may be needed in the choice of the one best suited for a par- ticular purpose. Conn, H. J. and Holmes, W. C, Stain Tech.. 1926, 1, 87-95 ; 1928, 3, 94-104 have made a study of color, acidity and chemical structure and Conn (p. 145) gives further data. Their color increases in depth in this order: eosin Y, ethyl eosin, eosin B, erythrosin B, phloxine and rose bengal. This increase in color is proportional to increase in number of hologen atoms. Their acidity increases in a different order: rose bengal, phloxine, erythrosin, eosin Y and eosin B. (1) When the eosin is to follow in alcoholic solution a basic dye always in aqueous solution (cf. hemato.xylin) the more acid and lighter colors are recommended (eosin Y, ethyl eosin and eosin B. (2) When it is to precede in aq. solution a basic dye (cf. methylene blue) also in aq. solution, use phloxin or erythrosin (see phloxine-methylene blue). Eosin lOB, see Phloxine B. Eosin BN, BW, or DHV, see Eosin B or bluish. Eosin J, see Erythrosin, bluish. Eosin-Methyl Blue, see Mann's. Eosin-Methylene Blue has been employed in many combinations for years. But when the acid dye is applied first, phloxine is preferred to eosin. See therefore Phloxine Methylene Blue. Eosin-Orange G — Toluidine Blue for bone marrow, spleen and connective tissue (Dominici, M. C. rend. Soc. biol., 1902, 54, 221-223). Stain eosin-orange G (eosin B. A. of Hollborn or eosin yellow- ish of American manufacturers 0.5 gm. ; aq. dest., 100 cc; orange G. 0.5 em.) 7 min. Rinse quickly in aq. dest. Counterstain in 0.5% aq. toluidin blue 20-30 sec. Rinse again aci. dest. Dif- ferentiate in 95% ale, dehydrate in abs., clear in xylol and mount in balsam. Instead of eosin, 0.5% aq. acid fuchsin gives a little sharper contrast. In place of toluidin blue 0.1% Azur A can be employed to advantage. Phloxine- orange G can be tried as a substitute for eosin-orange G. (phloxine 0.12 gm., aq. dest. 100 cc, orange G, 0.3 gm.). The crucial point is the differentiation in 95% ale. This should be quickly checked EOSIN SCARLET 118 EPIDERMIS under the microscope until the time has been determined. Eosin Scarlet, see Eosin B or bluish. Eosin Scarlet B, see Eosin B or bluish. Eosin Y or yellowish (CI, 768). Tetrabrom fluorescein with some mono- and di- brom compounds. This is the usual kind of eosin employed. Eosin Y and thionin as substitute for Wright's stain (Saye, E. B., Am. J. Clin. Path., Tech. Suppl., 1943, 13, 12). Eosinophile Leucocyte (acidophilic or coarsely granular leucocyte). Can easily be examined while still living in mounts of fresh blood. The dark field is useful. Most frequently studied in Blood Smears, which see. Mitochon- dria are readily stainable with Janus Green. For occasional presence of basophile granules and pigment see Downey, H., Folia Haemat., 1915, 19, 148-206. Techniques for rapid experi- mental increase of eosinophiles in circulating blood are described by Banerji, N., Am. J. Med. Sci., 1933, 186, 689-693; Chillingworth, F. P., Healy, J. C. and Haskins, F. E., J. Lab. and Clin. Med., 1933-34, 19, 486-494; Hajos, K., Nemeth, I., and Enyedy, Z., Zeit. f. d. ges. Exper. Med., 1926, 48, 590-592. Variations and errors in eosinophile counts of blood and bone marrow are described by Best, W. R. and Samter, M., Blood, 1951, 6, 61-74. Epidermis. This can be studied in situ with the dermis, see Skin, or it can be examined in 3 ways apart from the dermis. 1. Isolated pieces. Examination of scrapings of the epidermal surface is of limited usefulness in special cases. To cut away a few of the deeper cells, sepa- rate them by teasing and to study them in the still living state with or without supravital stains is not particularly helpful. But their microdissection is capable of giving important data on cellular consistency and connections (Chambers, R. and deRenyi, G., Am. J. Anat., 1925, 35, 385-402 and Than- hoffer, L., Zeit. f. Anat. u. Entw., 1933, 100, 559-562). Their cultivation is possible, see Tissue Culture. 2. Whole mounts for microscopic study (Cowdry's Histology, p. 530). Place excised fresh skin in 1% acetic acid in ice box for 12-36 hrs. depending upon size, age and region. Wash in tap water, 5 min. Pin skin down with epidermis up and cover with water. Strip off epidermis as a compete sheet. Wash in aq. dest., 5 min. Stain in Harris' hematoxylin, 20 min. Wash in aq. dest., 1 min. Differentiate in 50 cc. 70% alcohol plus 3 drops hydrochloric acid until epidermis becomes light pink color. Treat with 50 cc. aq. dest. plus 6 drops ammonia until it becomes blue. Wash in aq. dest. 5 min. several changes. Dehydrate in 50, 70, 95 and 2 changes of absolute alcohol, 10 min. each. Clear in 2 changes xylol, 1 hr. each and mount in balsam inner side up. If the skin is hairy, before excising it, remove hair with scissors and electric razor or depilatory solution. Hair fol- licles and sebaceous glands, unless par- ticularly large, generally remain at- tached to the epidermis, but the coiled bodies of the sweat glands are too deeply situated to come off with it. Conse- quently only their straight ducts are to be seen. Before dehydration, in the above technique the sebaceous glands can be sharply counterstained with Sudan IIL Such whole mounts of epidermal sheets are of value insofar that their study gives a concept of the morphology of the epidermal covering of the body which can be obtained in no other way. For the counting of mitoses they are far better than sections and have been extensively employed for this purpose by Dr. Cooper and her associates in The Barnard Free Skin and Cancer Hospital. See her latest paper (Cooper, Z. K. and Reller, H. C, J. Nat. Cancer Inst., 1942, 2, 335-344). Since the mucous membrane covering the nasal septum can be similarly prepared as a whole mount it is likely that the method may be of service in the study of other sheets of epithelial cells. 3. Sheets of epidermis for chemical analysis. Until very recently the handicap experienced in chemical analy- sis of the skin has been the difficulty of separating epidermis and dermis by themselves for analysis . All data on the epidermis are of doubtful value because variable amounts of dermis have been included. The method of obtaining pure epidermis by dilute acetic acid separation is unsatisfactory for numer- ous reasons. Baumberger, J. P., Sunt- zeff, V. and Cowdry, E. V., J. Nat. Cancer Inst., 1942, 2, 413-424 have discovered that dilute alkali will serve as well as dilute acetic but this also is objectionable from the chemical point of view. They therefore advance a heat method. Place excised skin with dermis down on warm plate such as is used for mounting paraffin sections. Apply temperature of 50°C. for 2 min. which loosens the epidermis so that it can be easily pushed off with a blunt instrument. Separation is more diffi- cult when temperature is over 51 °C. Epidermises removed in this way for a time continue to consume oxygen and EPINEPHRIN 119 ERYTHROCYTES are very suitable for chemical analysis. They have been used for epidermal iron and ascorbic acid by Carruthers, C. and Suntzeff, v., J. Nat. Cancer Inst., 1942, 3, 217-220, and for total lipid-protein nitrogen ration by Wicks, L. F. and Suntzeff, v., 3, 221-226. _ Epinephrin (adrenin, adrenalin), hormone of adrenal medulla. Erbium, see Atomic Weights. Erhlicki's Solution. Potassium bichromate, 2.5 gm.; copper sulphate, 1 gm.; aq. dest., 100 cc. Used for hardening nervous tissues. Erie Fast Red F D (CI, 419) of NAC is a direct disazo dye of light fastness 3 to 4. Resembles Congo red insofar that wash- ing in water, or in 95% alcohol, takes all color out of paraffin sections. In alkaline solutions it colors blue-green algae deep red to reddish brown (Emig, p. 40). Erie Fast Rubine B cone. A sulfonated azo dye. For formula nad influence on mouse tumors, see Stern, K., Cancer Res., 1950, 10, 565-570. Erie Fast Yellow WB, see Titan Yellow. Erie Garnet B (CI, 375). — amanil garnet H, Buffalo garnet R, Congo corinth G or GW, corinth brown G, cotton corinth G, diamine Bordeaux CGN, direct garnet R, direct violet C — an acid dis- azo dye used for staining frozen sections (Geschickter, C. F., Stain Techn., 1930, 5, 81-86). Erie Violet BW (CI, 387) of NAC is an acid disazo dye of light fastness 2 to 3. Directions for use in making prepara- tions of animal and plant tissues are described (Emig, p. 40). Erie Violet 3R (CI, 394) of NAC is a direct disazo dye of light fastness 3 not as satisfactory for microscopic work as Erie Violet BW (Emig, p. 40). Eriochrome Azurol V (CI, 720), a mordant dye of acid fastness 3 to 4. Gives color like that of Niagara Sky Blue. Direc- tions for use (Emig, p. 52). Eriometer, apparatus advocated by Em- mons, W. F., Quart. J. Med., 1927, 21, 83 to measure mean diameter of erythrocytes. See Erythrocytometer. Erythroblasts, see Erythrocytes, Develop- mental Series. Erythrocyte Counts do not fall in the scope of this book. It is sufficient to state that they are going out of fashion be- cause of the large experimental error involved and since it is so easy to detect variations in sliapc, size and maturity of erythrocytes in smears and to measure hemoglobin content of blood by hemo- globinometers. Blum, L. L., Am. J. Clin. Path., 1945, 15, 85 has introduced a rabid photoelectric technique for estimating the number of erythrocytes. See Retic- ulocytes, Erythrocytes. For chemical and physical studies erythrocytes are particularly adapted, because they can be collected in enormous numbers free from other kinds of cells and from intercellular substances. In order to determine marked differences in size and shape and hemoglobin content examination of fresh blood with direct illumination, or in the dark field, is probably the best procedure. An interesting photographic method for the stereoscopic visualiza- tion of the shape of erythrocytes has been described and illustrated by Haden, R. L., J. Lab. & Clin. Med., 1936-37, 22, 1262-1263. For more accu- rate techniques see Wintrobe, M. M., Clinical Hematology, Philadelphia : Lea & Febiger, 1942, 792 pp. A new aniso- cytosis index is proposed by van den Berghe, L., and Weinberger, E., Am. J. Med. Sci., 1940, 199, 478-481. The refractile body of Isaacs (R., Anat. Rec, 1925, 29, 299-313) can also be well studied in fresh blood. See Flagella. Smears, colored by Giemsa or Wright's stain, are satisfactory for Howell-Jolly bodies, Cabot rings, basophilic stippling and polychromalophilia. For resistance to hemolysis in hy potoni c sodium chloride solutions, see Daland, G. A., and Worth- ley, K., J. Lab. & Clin. Med., 1934-35, 20, 1122-1136. A lysolecithin fragility test is described by Singer, K., Am. J. Med. Sci., 1940, 199, 466-477. For microfragility tests see Kato, K., J. Lab. & Clin. Med., 1940, 26, 703-713 and for basophilic erythrocytes of the newborn see McCord, C. P., and Bradley, W. R., Am. J. Clin. Path., 1939, Tech. Suppl., 2, 329-338. A thorough investigation of erythrocytes in fetus and newborn has been made by Wintrobe, M. M. and Schumacker, H. B., Jr., Am. J. Anat., 1936, 58, 313-328. A simple method for determination of specific gravity of erythrocytes is described by Reznikoff, P., J. Exper. Med., 1923, 38, 441-444. After hemolysis the stroma remains and can be studied microscopically or chemi- cally. Lipid analyses are particularly significant (Erickson, B. N., et al., J. Biol. Chem., 1937-38, 122, 515-528). Isolation and collection en masse of nuclei of chicken erythrocytes by Dounce, A. L., and Lan, T. H., Science, 1943, 97, 584-585. Experiments have been made with radioactive iron as a means of tagging red blood cells (Cruz, W. O., Hahn, R. F., Bale, W. F. and Balfour, W. M., Am. J. Med. Sci., 1941, 202, 157-162) which open up a new field for study of age changes because the cells are ERYTHROCYTES, DEVELOPMENTAL 120 SERIES ESTERASES thereby dated. Stratification of con- tents of erythrocytes by ultracentrifu- gation (Beams, H. W., and Hines, E. H., Anat. Rec, 1944, 89, 531). Special meth- ods are given under Hemoglobin, Flagella, Reticulocytes, Cabot Rings, Jolly Bodies, Pencil Red Cells, and Target Cells. For red fluorescent erythrocytes in anemia, see Seggel, K. A., Ergeb. d. inn, Med. u. Kinderh., 1940, 58, 582. Erythrocytes, Developmental Series. The technique employed apparently makes a great deal of difference in the conclu- sions reached. See Cowdry's His- tology, 1938 p. 99. 1. Maximo w and Bloom employing mainly permanent preparations list: Hemocytoblasts: "... large (up to 15m) ameboid, non-granular basophil cells of lymphoid nature." Occur ex- tra vascularly. Basophil erythroblasts : The youngest erythroblasts, characterized by the intense basophilia of their cytoplasm. Also called megaloblasts , but "this term is misleading because it was first used for the erythroblasts of pernicious anemia which are cells of quite different nature." Polychromatic erythroblasts: So-called because after "fixation and staining with the Romanowsky mixture, especially in dry smears, the protoplasm has a mixed color varying from purplish-blue to lilac or gray." This is due to the presence of two substances, a basophile material and hemoglobin. Orthochromatic erythroblasts or normo- blasts: These are smaller "and only slightly larger than the mature, non- nucleated erythrocytes." Since the basophile substance diminishes and the hemoglobin increases, the protoplasm becomes acidophilic "and stains a bright pink with the Romanowsky mixture." They continue to divide mitotically for an unknown number of generations until the nucleus disappears. 2. Sabin and associates relying chiefly on supravital stains list : Endothelial cells: Occur in special "erythrogenic capillaries." Megaloblasts: "... a daughter endo- thelial cell which starts to synthesize hemoglobin." "The megaloblast has maximum basophilia, a moderate num- ber of rod-shaped mitochondria, a trace of hemoglobin, and a nucleus with a minimum of chromatin and conspicuous nucleoli." Early erythroblasts: "The young ery- throblast represents a growth phase, with less rapid division, for the cell is much larger than the megaloblast; it contains the maximum number of mito- chondria. The amount of hemoglobin is still small, but sufficient to give a poly chroma tophilia , predominately basophilic in methylene blue-azur. The nucleus has a marked increase in chromatin." Late erythroblasts: This cell "is inter- mediate in size between the early erythroblast and the definitive red cell. The nucleus has lost the nucleoli but still has massive chromatin. . . . The increase in hemoglobin is marked and in fixed films the cytoplasm is more acidophilic." Normoblasts: "The stage of the nor- moblast is defined as a nucleated red cell after its last cell division. It has a small pyknotic nucleus ready for extrusion or fragmentation." Erythrocytometer for measuring the diam- eter of red blood cells. Pijper, A., Med. J. South Africa, 1919, 14, 472 and Lan- cet, 1935, 1, 1152, deserves great credit for the discovery independently of Thomas Young (1813) of a technique for the measurement of small objects utilizing the principle of diffraction and Zeiss has manufactured an instrument on his specifications. Another, the Haden-Hausser erythrocytometer, is made by C. A. Hausser and Son and is sold by Arthur H. Thomas Co., Phil- adelphia (Haden, R. L. J. Lab. & Clin. Med., 1939-40, 25, 399-403). Erythrosin B, see Erythrosin, bluish. Erythrosin BB or B extra, see Phloxine. Erythrosin, bluish (CI, 773)— dianthine B, eosin B, erythrosin B, iodeosin B, pyro- sin B — Fluorescein with 2 iodine atoms. See Eosins. Escherichia Coli, see Triphenyltetrazolium Chloride. Ester Wax. An embedding medium de- signed especially to promote ribboning of sections. It is made up of diethylene glycol distearate, 73 gm.; ethyl cellu- lose, 4 gm.; stearic acid, 5 gm.; castor oil, 8 gm.; and diethylene glycol mono- stearate, 10 gm. (Steedman, H. F., Quart. J. Micr. Sci., 1947, 88, 123-133). Esterases. — Written by E. W. Dempsey, Dept. of Anatomy, Washington Uni- versity, St. Louis. February 26, 1951^ Strictly speaking, these are enzymes which hydrolyze the ester linkage de- rived from any acid, organic or inor- ganic. Thus, the phosphatases, cho- linesterases, glucuronidase, lipase, and sulfatase properly belong among the esterases. However, ordinary usage restricts the term to the enzymes hydrolyzing carboxy esters, particu- larly those of fatty acids. These es- terases apparently exhibit some sub- strate specificity; at least, the enzymes hydrolyzing esters of short-chain fatty ETHYL EOSIN 121 EYES acids appear to differ from the true lipases, which act upon long-chain substrates, and from cholinesterase. Gomori, G. (Proc. Soc. Exp. Biol, and Med., 1945, 58, 362-364, and ihid, 1949, 72, 697-700) has devised a method for localizing lipase. Nachlas, M. M. and A. IM. Seiigman (J. Nat. Cancer Inst., 1949, 9, 415-425) believe the histochemical methods do not differen- tiate a true lipase, but only a nonspe- cific esterase. They describe a new and vivid method for this enzyme, based upon the hydrolysis of the acetyl ester of naphthol and staining the liberated naphthol by diazotization. Seiigman, A. M., M. M. Nachlas, L. H. Man- heimer, O. M. Friedman and G. Wolf. (Ann. Surg., 1949, 130, 333-341) describe the development of specific methods for a number of hydrolytic enzymes. Ethyl Eosin (CI, 770). The ethyl ester of eosin Y. Sold often as alcohol soluble eosin. See Eosins. Ethyl Green (CI, 685) . This is, Hke methyl green, prepared from crystal violet but differs from it insofar that an ethyl group is added instead of a methyl one. For most purposes it is a satisfactory sub- stitute for methyl green. Ethyl Purple 6B, see Ethyl Violet. Ethyl Violet (CI, 682)— ethyl purple 6B— It is he.xaethyl pararosanilin, a basic dye employed by Bowie, D. J., Anat. Rec, 1924, 29, 57 to make a neutral stain with biebrich scarlet for staining islets of Langerhans of fish. Kernohan, J. W., Am. J. Clin. Path., 1931, 1, 399-403 has used in Heidenliain's modification of Mallory's ethyl-violet orange G after formalin fixation. Ethyl Violet-Biebrich Scarlet, see Bowie's stain for pepsinogen. Ethylene Glycol Mono-Ethyl Ether = Cellosolve. Euchrisine, see Acridine Orange. Eunematoda, see Parasites. Euperal is, according to Lee (p. 227), a mix- ture of camsal, eucalyptol, paraldehyde and sandrac, n = 1.483 of two sorts colorless and green. Since the green one contains a copper salt it strengthens hematoxylin stains. Euporium, see Atomic Weights. Evans Blue (T. 1824 Eastman Kodak Co.). Used clinically in man for estimation of blood volume. Vital staining of malig- nant tumors in man (Brunschwig, A., Schmitz, R. L., and Clarke, T. H., Arch. Path., 1940, 30, 902-910). It is not taken in by red cells and hence is valuable for the determination of plasma volume (Gregersen, M. I., and Schiro, H., Am. J. Physiol., 1938, 121, 284-292. See Blood Cell Volume. Excelsior Brown, see Bismark Brown Y. Excretion contrasted with secretion (Cow- dry's Histology, p. 259). Exfoliative Cytology, see Papanicolaou Techniques. Exogenous Pigments, classified by color, Lillie, p. 134 Extracellular fluid or phase, see Chloride. Exudates, see Agar infiltration to hold materials in place, also Papanicolaou Techniques. Eyes. Techniques easily used for other parts of the body require special care in the case of the eye. When sections through the entire eye are required it is important to see that the fixative chosen penetrates properly and that the normal shape of the organ is retained. Fixation by vascular injection may be helpful but it is not sufficient because so much of the eye is avascular. After removal of the eye from the orbit, whether previously injected or not, and after the dissecting away of unwanted muscular and other tissues, it should be immersed in the fixative. This will harden the outer coats somewhat. After a few minutes small amounts of the fi.xative should be injected by a hypodermic syringe into both chambers choosing locations not in the plane of the proposed sections and providing opportunity for fluid also to leave. Then, with a sharp razor blade, a deep cut should be made to permit free entrance of the fixative. After several hours, more of the tissue on either side of the plane should be cut away. Im- bedding in celloidin by the rapid method is preferable to paraffin since it affords much needed support to the less dense parts. Orientation for sectioning is also easier in celloidin because one can see through it fairly well. If, on the other hand, preparations are needed of small parts of the eye these parts should be carefully dissected out and the paraffin technique employed. Much time will be saveu by following the excellent suggestions made by S. L. Polyak, The Retina. Univ. of Chicago Press, 1941, 607 pp. and by G. L. Walls (Stain Techn., 1938, 13, 69-76). Dr. Polyak in a letter dated April 19, 1946 calls attention to the advisability of soaking celloidin blocks in oil as first described by Apdthy, S., Zeit. f. wis. Mikr., 1912, 29, 464. The same method is well presented by Kranse, R., Enzyk. d. Mikr. Technik., 3rd edit., 1926, 1, 281. For the investigation of perme- ability, oxidation-reduction potential, enzyme sj'stems, and such properties, see Friedenwald, J. S. and Stiehler, R. D., Arch. Ophth., 1938, 20, 761-786. Useful data are to be found in Kurzes FAHRENHEIT TEMPERATURE 122 FECES Handbuch der Ophthalmologie (Schieck and Bruckner, Berlin: Julius Springer, 1930, 1, 882 pp.) The Anterior Chamber is a favourite site for tissue trans- plantation. Frozen sections of bird's eyes. (Oak- ley, C. L., J. Path. & Bact., 1937, 44, 365-368). Fix in 10% formol saline 4 days, in Miiller's fluid, 6 weeks in incu- bator, or, in case speed is necessary, in Perdrau's fluid 4 days. Incise large eyes to aid penetration. Wash in run- ning water at least 24 hrs. because formalin and bichromate should be com- pletely removed. Cut eye in half being careful not to disturb various structures. 12.5% gelatin + 1% phenol over night, 25% 24 hrs. at 37 °C. Employ at least 25 cc. for each half eye. Mount with cut surface down in dish containing 25% melted gelatin. Set overnight in run- ning water or in icebox (not refrigera- tor). Cut out block, trim away excess gelatin. Harden in large amount 10% formalin, 2-3 days, store in 4% formalin. Before freezing soak 15 min. in tap water. Freeze slowly, over-freeze and then stain usual methods but carefully avoid strong alcohols. They will stand 70% and 1% HCl provided washing in water has been thorough. Use glycerin jelly for mounting. Fluorescence Mi- croscopy of the eye is very revealing, see Evans, J. N. and Singer, E., Arch. Ophthal., 1941, 25, 1007. Fahrenheit Temperature to Centigrade. Use the following relation: 8 (F°- 32) =0° 302 °F ± I (302 - 32) = a (270) = 160° C. .5°F ± a (5 - 32) = J (- 27) = - 15°C. - 13°F ± § (- 13 - 32) = S (- 45) = - 25°C. Fallopian Tubes (oviducts, uterine tubes). References to many techniques will be found in C. G. Hartman's chapter in Allen, Danforth and Doisy's Sex and Internal Secretions. Baltimore: Wil- liams and Wilkins, 1939, 1346 pp. Falzone, see Desoxyribose Nucleic Acid. Farrant's Medium. Gum arabic, 30 gm.; glycerin, 30 cc; arsenous oxide (arsenic trioxide), 0.1 gm.; aq. dest., 30 cc. There are many types of this medium differing slightly in composition, see Gray, P. and Wess, G., J. Roy. Micr. Sci., 1950,70,287-291. Fast Acid Blue R (CI, 760). An acid xan- thene dye. Conn (p. 143) says that it is almost the same as violamine 3B which contains small amount of a red dye. See Romell, L. G., Stain Techn., 1934, 9, 141-145 under Soil, bacteria. Fast Acid Green N, see Light Green SF yellowish. Fast Blue B, OB, R, etc., see Indulin, water soluble. Fast Blue 3R, see Naphthol Blue R. Fast Crimson GR, see Azophloxine GA. Fast Fuchsin G, see Chromotrope 2R. Fast Green FCF. Commission Certified. Closely related to Light Green SF yellowish and recommended as a sub- stitute because it fades less. Fast Oil Orange II, see Oil Red O. Fast Phosphine NAL, see Rheonine A. Fast Red, see Amaranth. Fast Red B, BN or P, see Bordeaux Red. Fast Violet, see Gallocyanin. Fast Yellow (CI, 16)— acid yellow, fast yellow FY, G, S, BG, etc.— An acid mono-azo dye. Employed by several investigators, see use by Wallart, J. and Houette, C, Bull. d'Hist. Appl., 1934, 11, 404-407 in rapid trichrome hematox- ylin, acid fuchsin fast yellow method. They used "Jaune solide G or GG (Ciba). Fasting. Structural changes in human di- gestive tract (Cowdry's Histology, p. 305). Fat Blue B, see Victoria Blue B. Fat Blue 4R, see Victoria Blue 4R. Fat Ponceau, see Oil Red O. Fat Ponceau, see Sudan IV. Fat Ponceau G, see Sudan III. Fat Ponceau R or LB, see Sudan IV. Fats, see Lipids. Fatty Acids, see Lipids, examination with polarized light, also lack of specificity of blue color with Nile Blue Sulphate. A review of the method of tagging fatty acids with radioactive isotopes is given by Bloor (W. R., Physiol. Rev., 1939, 19, 557-577). Feathers, see Ceresin imbedding. Feces. 1. To demonstrate ova of parasites (Mallory, p. 301). If they cannot be seen when a small bit of feces is mixed with water on a slide attempt to concen- trate them. To a small amount of feces add sufficient sugar solution (common granulated sugar, 500 gm., water, 360 cc, phenol, 1%) to almost fill tube. Cover and gently mix contents. Centrifuge at 1000 r.p.m. 5-6 min. Remove ma- terial from surface in wire loop and examine microscopically for ova. Another method is to use hypertonic salt solution in proportion to feces of not more than 20:1 in the same way, remov- ing large particles as may be necessary before centrifuging. 2. To find segments and whole adult worms. Wash feces in small amount water through medium mesh screen, collect and examine at low magnification. For identification consult a text book of parasitology. See Floatation Techniques, Intestinal Protozoa. FELL 123 FISCHLER'S Fell, see Organ Culture in Vitro. Ferments, see Enzymes. Ferric Chloride-Osmic Acid for demonstra- tion of Golgi apparatus (Owens, H. B. and Bensley, R. R., Am. J. Anat., 1929, 44, 79-100). Fix and impregnate each piece of tissue 7-10 days at 37 °C. in ferric chloride, 0.05 gm. ; 2% osmic acid, 10 cc. Ferrihemate, see Hematin. Fettblau -braun, -griin, -orange, -rot and -Schwartz. These are lipid stains of Hollborn. For use of hydrotropes (Hadjioloff, A., Bull. d'Hist. Appl., 1938, 15, 37-41). Feulgen Reaction, see Thymonucleic Acid. Fiberglass, see Glass Cloth. Fibers. Many are recognized. See Nerve, Collagenic, Reticular, Elastic, Neu- roglia. Muscle fibers are given under Muscle. Fibrils. These are really small fibers many of which are intracellular. See Neuro- fibrils, Myofibrils, Epidermal Fibrils, Fibroglia, Myoglia. Fibrin. Usually easily identifiable in Hem- atoxylin and Eosin preparations. Wei- gert's (1887) standard differential stain for fibrin may be used as advised by Mallory (p. 193). Paraffin sections of material fixed in abs. alcohol, Carnoy or Alcohol-Formalin can be employed. If the fixative contains chrome salts (Zenker, Helly) treat first with 0.25% aq. potassium permanganate, 10 min., then 5% aq. oxalic acid, 20 min. and wash in aq. dest. Stain nuclei with Lithium Carmine. Mix 3 cc. of A: abs. ale, 33 cc; anilin oil, 9 cc. saturated with methyl violet (crystal violet) with 27 cc. of B: sat. aq. methyl violet. Stain 5-10 min. Drain and blot. Treat sections with Gram's Iodine, 5-10 min. Drain and blot. Differentiate in equal parts anilin and xylol drop by drop until purple ceases to be removed. Blot and remove anilin with xylol. Mount in balsam. Fibrin blue-black, nuclei red. Fibroblasts. There is no specific stain for fibroblasts. In fresh spreads of Loose Connective Tissue they are fairly con- spicuous elements identifiable by their large usually slightly kidney shaped nuclei (possessed generally of a single nucleolus) and tapering cytoplasmic processes devoid of specific granulations. In sections less cytoplasm is seen and it may be impossible in some cases to identify the nuclei with assurance. Recognition is mainly by position and the exclusion of other possibilities. View the beautiful colored plates of Evans, H. M. and Scott, K. T., Contrib. to Embryol., Carnegie Inst., 1922, 47, 1-55 for a comprehensive picture of the responses of fibroblasts to vital stains. Pure strains of fibroblasts can easily be cultured, their behavior watched and their nutritional and other requirements investigated. See Tissue Culture. Fibroglia Fibrils. Mallory's Phosphotungs- tic Acid Hematoxylin stain for. Fibrous Connective Tissue. Since this is much denser than Loose Connective Tissue the method of making spreads is not feasible. It can best be examined in sections of Zenker fixed material colored by Mallory's Connective Tissue Stain supplemented by specific stains for Elastic Fibers. Figge, see Porphyrins, Hematoporphyrins. Filament-Nonfilament Count. Neutro- philic leucocytes are divided into two classes: filament, in which nuclear seg- ments are connected by delicate strands consisting apparently of nuclear mem- brane only and nonfilament in which there are no filaments the strand being so coarse that it may be resolved into nuclear membrane plus nuclear con- tents. The former are mature and the latter are less differentiated cells. Ac- cording to Pepper, O. H. and Farley, D.L., Practical Hematological Diagnosis, Philadelphia, Saunders, 1933, 562 pp., 8-16%of neutrophiles are normally non- filament cells. A shift to the right is a decrease in this percentage. The count is easier to make than the Arnett or Schilling count and is probably of equal value. See also Nonfilament-Filament Ratio. Filterable Viruses, see Victoria Blue 4B and Virus. Filters. Choice and use of the various types of filters employed in the study of viruses and bacteria are well described by J. R. Paul (Simmons and Gentzkow, 584-586). There are 4 principal kinds. Berkefeld. German, from diatoma- ceous earth. V. pores 8-l2fi; N, pores 5-7 fi; and W, pores 3-4^. Mandler. American modification of Berkefeld but made of kioselguhr, as- bestos and plaster of Paris. Corre- sponding grades of porosity are styled "preliminary", "regular" and "fine." Chamberland, French, from unglazed porcelain, in 9 grades of porosity. Seitz. Made of asbestos, in 2 grades K (coarse) and E. K. which filters out ordinary bacteria. Elford. Made of collodion. Firminger, see Carbowax Embedding. Fischler's modification of Benda's stain for fatty acids and soaps (Fischler, F., Zentralbl f. Allg. Path. u. path. Anat., 1904, 15, 913-917) lias been severely criti- cized by Lison (p. 203) who concludes that it is of no microchemical value. Mallory (p. 120) has, however, given a somewhat different description of the FITE 124 FIXATION technique. He explains that since the Na and K fatty acid salts (soaps) are soluble in formalin, it is necessary to change them into insoluble Ca soaps by saturating the 10% formalin fixative with calcium salicylate. Comparison of stained sections of such material with others fixed simply in formalin shows the presence and absence of the fatty acid salts (soaps) . Calcium soaps can be dis- tinguished from falty acids because they resist solution in a mixture of equal parts abs. ale. and ether or in hydrochloric acid whereas the fatty acids are soluble in this mixture and calcium in hydro- chloric acid. The method, as detailed by Mallory, is : Mordant frozen sections of 10% formalin fixed material in sat. aq. copper acetate (12.5%), 2-24 hrs. at room temperature. Wash in aq. dest. Stain 20 min. or more in Weigert's he- matoxylin made up by mixing 1 gm. hematoxylin dissolved in 10 cc. abs. ale. with 1 cc. sat. aq. lithium carbonate (about 1.25%) plus 90 cc. aq. dest. several days before use. Differentiate in Weigert's borax-potassium ferri- cyanide, (2.5gms.ferricyanideand 2gm. borax plus 100 cc. aq. dest.) much diluted until red blood cells become decolorized. Wash thoroughly in aq. dest. Mount in glycerin jelly or glyc- erin. Fatty acids deep blue black. Fe, Ca and hemoglobin may also be stained. To stainneutralfats inaddition stain with scarlet red after washing out Weigert's fluid, rinse in 70% ale. and in water and mount in glycerin. Fite, see his method for Acid Fast Bacilli. Fixation by immersion is usually the first step in making permanent preparations. Compared with the direct microscopic examination of still living cells removed from the body and placed in approxi- mately isotonic media, it has both ad- vantages and disadvantages. Among the first is the fact that the normal form relations of the tissue components are more faithfully preserved in large pieces by fixation; because it is not necessary to separate the tissue by teasing, or in some other way, into sufficiently small or thin pieces for microscopic study. Moreover, by fixation, the cells are suddenly and uniformly killed, so that the changes resulting from unfavorable fluid environment outside the body, leading slowly or quickly to injury and death, are not encountered. The chief objection to fixation is that the structure is very definitely modified therebj* and care must be exercised in reaching con- clusions as to living tissues from the study of fixed ones. It is important to restrict these structural changes to those inseparable from the action of the fixative itself, and of the subsequent technique under the most favorable conditions. Reduce to a minimum the time in which these complicating alterations can occur by prompt fixation. Remove the tissue from an animal under general anesthesia, or immediately after it has been killed, by a method unlikely to injure the tissues. In the case of human tissues removed at operation one should be on the look out for complicating factors. If the tissue is collected at autopsy the autopsy should be made at the earliest possible moment after death. See Postmortem Changes. If delay is unavoidable, keep the body,orthe tissue, inaniceboxto reduce the speed of chemi- cal change. In case an excised tissue cannot be immediately fixed, place it in a covered glass container with some cot- ton moistened with physiological saline solution. Do not put it in the solution. Keep the container likewise at a low temperature. Carefully avoid injury to the tissue from any cause. Letting its surface dry during removal from the body, or at any time before fixation, produces Artifacts. So also does mechanical manipulation. If forceps must be used, do not pinch the part of which the preparation is to be made. It is better to lift the tissues. Scissors tend to squeeze the tissue, but it is necessary to cut with them in some cases. The ideal way is to cut with a sharp razor blade. This is easy with the liver, kidney, brain and other more or less compact organs, but the sweep of a razor blade tends to draw the tissue and cause displacement, especially when the specimen is heterogeneous, some parts being loose connective tissue, others muscle, others gland, etc. When feas- ible, cut the tissues into slices and lift them into the fixative. For fixatives that penetrate easily (formalin, Zenker's and Bouin's fluids, etc.) make the slices 4-6 mm. thick. For the poor penetrators, in which osmic acid is the principal ingredient (Bensley's A.O.B., Flera- ming's fluid, etc.), the slices must be not more than 2 mm. thick. In the case of surface tissues (skin, gastric mucous membrane, bladder wall, etc.) fix a strip, flattened on the surface of a piece of wooden tongue depressor or stiff paper card. A volume of fixative at least 20 times that of the tissue fixed is required. Agitate the bottle slightly to prevent the tissue from sticking to the bottom and to ensure penetration from all sides. It may be desirable to inject the fixa- tive via a large artery supplying the tissue to be examined. This eliminates mechanical injury to the tissue before FIXATION 125 FIXATIVES fixation, preserves gross form relations better and is suggested when sections are required of large specimens. Before in- jecting the fixative wash out some of the blood by Perfusion with physio- logical salt solution, or at least let the blood drain out from the veins, because, if all is left in, it may clog the arteries and block the entry of the fixative. After fixation by vascular injection it is customary to cut, with a razor blade, suitable slices and to continue the fixa- tion by immersion. Obviously such tissues should not be employed for micro- chemical analyses because there is a danger of washing out chemical sub- stances. Clearly, also, the speed of fixation depends upon the degree of vas- cularity. For avascular tissues such as epidermis, cornea and cartilage fixation by injection is not recommended. After the tissues have hardened a little by immersion in or injection with the fixative, it may be helpful to remove them from the fi.xative and trim them with a razor blade so that their size and shape will be almost what is needed when they are finally cut into sections. The slices should have smooth upper and lower surfaces including an area which will yield sections that will fit nicely under a 22 x 22 mm. cover glass unless larger covers are to be used. The shape should be rectangular with opposite edges parallel. In general it is well to have two longer parallel edges and two shorter ones, because a square surface is not so convenient to section as an oblong one. However one must bear in mind exactly what one wishes to demonstrate. This making of uneven surfaces smooth does however introduce an experimental error; because, where much is shaved off, the fixation has penetrated less than where little or no tissue has been re- moved. After trimming return tissues to a fresh supply of fixative. Tissues fixed in poor penetrators should not be trimmed. The time of fixation depends upon the tissue, the fixative and the purpose in mind. In general, 24 hrs. is suitable. Some fixatives, particularly those con- taining potassium bichromate and/or osmic acid, are not very stable and for this reason should be renewed. The fixative deteriorates less quickly if the fixation is carried out at a low tempera- ture in an ice box. The speed of fixation is probably also somewhat diminished. The effect of pH on chromium fixatives has been studied by Zirkle (C, Proto- plasma, 1928, 4, 201-227). See results obtained by adding Wetting Agents and Hydroxybenzene Compounds to fixa- tives. Fixation involving Decalcifica- tion and Mordanting are special cases described under these headings. For choice of fi.xative see Fixatives. After fixation Washing may be neces- sary, or Mordanting. The tissue may be prepared as a Whole Mount, or Frozen Sections may be made, or it may be dehydrated, cleared and imbedded in Paraffin or dehydrated and imbedded in Celloidin for Sectioning. Fixatives. The number from which to choose is enormous but the number actually employed is comparatively small. Formalin unquestionably heads the list as being used for a far greater Acetic osmic bichro- Hischler mate Kleinenberg Alcohol (ethyl) Kolatohew Allen Lactophenol Barium chloride and LUlie formalin Mann Basic lead acetate Marchi Bouin Maiimow Cadmium chloride Mercuric chloride Carnoy Methyl alcohol Carnoy-Lebrunn MuUer Champy Orth Chloral hydrate Osmic acid Destin Parabenzoquinone Diaphanol Perenyi Dioxan Petrunkevitch Downey Regaud Erlicki Rabl Ferric chloride-osmio Schandian acid Silver nitrate Flemming Formalin Susa Formalin-Zenker Tellyesnicky Giemsa Weigert Gilaon Van Gehuchten HeUy Zenker Hermann Zweibaum variety of purposes than any other fixa- tive. It penetrates well and is an ex- cellent preservative. It is the only satisfactory fixative for use before the cutting of frozen sections and as a pre- liminary to certain microchemical re- actions. Alcohol comes next in variety of services performed but unfortunately it brings about considerable shrinkage. Both formalin and alcohol are frequently combined with other ingredients. For routine purposes Zenker's Fluid, either alone or with formalin, is perhaps the most popular fixative. Tissues so fixed give better contrasts of acidophilic and basophilic components than are obtained after fixation in formalin or alcohol by themselves. Bouin's Fluid is also an excellent fixative for general use and is being employed with increasing frequency. It is particularly advocated by dermatologists. Regaud's Fluid is FLAGELLA 126 FLOATATION TECHNIQUES the fixative of choice for mitochondria because it penetrates so much better than Osmic Acid containing fixatives. No important new fixatives have recently been devised. In making the selection one is natu- rally guided by data concerning the structures which it is desired to demon- strate (see Nerve Endings, Mitochon- dria, etc.) or the substances to be re- vealed (Lead, Copper, Oxidases, Lipids, etc.) or the techniques that seem best adapted to the purpose in mind (Mal- lory's Connective Tissue stain, Wei- gert's Method, etc.). Some of the more important fixatives are listed, further data being given under each heading. Flagella. 1. Of bacteria. Loeffler's stain. Mordant in fresh 20% aq. tannic acid, 10 cc; sat. aq. ferrous sulphate, 5 cc; 3-5% basic fuchsin in 95% ale, 1 cc. gently heated, 1 min. Rinse in water stain with slight heat in Carbol Fuchsin 1 min. wash and dry. For other flagella stains see discussion in McClung (pp. 143-145) and Shuuk, I. V., J. Bact., 1920, 5, 181 ; Galli-Valerio, B., Centralbl. f. Bakt. Orig., 1915, 76, 233; Gray, P. H., J. Bact., 1926, 12, 273. See technique for darkfield study of flagella (Pi j per, A., J. Path. & Bact., 1938, 47, 1-17). 2. Of erythrocytes (Oliver, W. W., J. Inf. Dis., 1934, 55, 266-270). Add 1 mg. hirudin to 2-3 cc. sterile Ringer's solu- tion in small, sterile test tube. Draw up about 0.5 cc. into a sterile Pasteur pipette fitted with rubber bulb. Apply to drop fresh normal blood from finger. Suck up quickly into pipette and expel into test tube. Incubate at 37 °C. 40-50 min. which promotes flagella production. Add small drop to clean slide held at 40° angle. After the drop has rundown slide, let dry completely in horizontal position at room temperature. Mor- dant in fresh 10% aq. tannic acid, 50 cc. ; sat. aq. ferrous sulphate, 25 cc. and sat. ale. basic fuchsin, 5 cc. which is poured on slide and warmed slightly 20 min. Wash thoroughly in running tap water and dry. Flood with fresh Ziehl-Neelsen (1 gm. fuchsin, 10 cc. alcohol -j- 90 cc. 5%aq. phenol acid) 20 min. not warmed. Wash carefully in running water, blot dry and examine with oil immersion. It will be helpful to examine Oliver's illustrations. (Revised by Wade Oli- ver, Dept. of Bacteriology, Long Island Medical College, Brooklyn, N. Y., 1946). The interpretation of observations on bacterial flagella offers many pitfalls. Dubos, R. J., The Bacterial Cell. Harvard Univ. Press, 1945, 460 pp. calls attention to their fineness, the slight affinity of their substance for stains, the use of mordants which ad- here to their surface increasing their apparent diameter when stained, and the fact that mechanical agitation alone is sufficient to detach them from the cells. By thus releasing flagella sufiicient flagellar material can be col- lected for immunological study and the action of flagellar antibody on mobile flagella can be followed microscopically. Dubos remarks that the amounts of flagellar material available are too small to permit chemical analysis but we may hope that techniques both of collecting material and of analysis will be so improved as to make this feasible. He refers to numerous papers on elec- tron microscopic examination of flagella as revealing structural details pre- viously unknown. Details in the structure of flagellae are revealed by Electronmicroscopy. See Brown, H. P., Ohio J. Sci., 1945, 45, 247; and DeRobertis, E., and Franchi, C. M., Exp. Cell Res., 1951, 2, 295-298. See also Cilia and Polysac- charides. Flagellates, intestinal. Those commonly found in man are, according to Craig, p. 115, Giardia lamblia, Chilomastix mesnili, and Trichomonas hominis; less frequently seen are Embadomonas in- testinalis and Enteromonis hominis. Stains much the same as for Endameba and Leishmania. See Craig for choice of suitable culture medium. Flame Photometer. Use of this instrument in the analysis of biological materials is critically presented by Wallace, W. M. et al., J. Lab. & Clin. Med., 1951, 37, 621-629. Flavins under fluorescence microscope show green fluorescence in liver tissue. See Riboflavin. Fleas, see method of double imbedding for (Lee, p. 598). Flemming's Fluid. Weak: 0.25% chromic acid, 0.1% osmic acid and 0.1% glacial acetic acid in aq. dest. Strong: 1% chromic acid, 15 cc; 2% osmic acid, 4 cc. ; glacial acetic acid, 1 cc. These are classic fixatives now not much used. The Bensleys (p. 45) advocate same ingredients differently made up. A: 1% aq. chromic acid, 11 parts; glacial acetic acid, 1 part; and aq. dest., 4 parts. B : 2% osmic acid in 1% aq. chromic acid. Immediately before use, mix 4 parts of A with 1 part of B and employ a volume ten times that of the tissue. Fix 2-72 hrs. and wash in water 24 hrs. See Safranin-Gentian Violet and Orange G method. Mitosis, Benda's Method. Floatation Techniques. Many methods are available for separating helminth eggs from feces for microscopic examination. FLORENCE'S REACTION 127 FLUORESCENCE MICROSCOPY They are floated out by the use of hyper- tonic salt and other solutions, some- times with the aid of centrifugal force (E. C. Faust, in Simmons and Gentz- kow, p. 684). Florence's Reaction. The standard test for choline in seminal stains. As described by Pollak, O. J. Arch. Path., 1943, 35, 140-196: Place one drop semen, or of aqueous extract of seminal stain, on slide. Add drop of reagent (Pot. iodide, 1.65 gm.; iodine, 2.54 gm.; aq. dest., 30 cc), cover and examine micro- scopically. Dark brown, rhombic crys- tals appear, about 25m long and Sm wide with bifurcated ends resembling swal- low tails and Teichmann's hemin crys- tals. In polarized light these show double contours. Fluids. Samples of body fluids are often presented for microscopic examination. In a human being containing, say, 100 lbs. of water the}^ are naturally of great variety even under normal conditions. Abnormal fluids are usually described as transudates or exudates. The for- mer compared with the latter are mainly filtrates, are more watery, have lower specific gravity, less albumin, no bacteria and are the result of mechani- cal forces rather than inflammation. See: Aqueous humor Intracellular phase Cerebrospinal Pericardial Duodenal Peritoneal Endolymph Pleural Extracellular phase Sjiiovial Tissue Fluoran Derivatives. As explained by Conn (p. 144) fluoran is not a dye but a prod- uct of phthalic anhydride containing a xanthene ring and a lactone ring with introduced hydroxyl groups and halogen atoms in particular positions. Ex- amples : eosin B and Y, erythrosin bluish and yellowish, ethyl eosin, fluorescein, mercurochrome 220, methyl eosin, phloxine, phloxine B, rose bengal. Fluorescein (CI, 766) is simplest fluoran dye. It stains very poorly but is highly fluorescent. Its sodium salt is called uranin. Fluorescence Microscopy. Details pro- vided by Dr. W. L. Simpson of The Barnard Free Skin and Cancer Hospital. Supplemented by Dr. T. B. Rosenthal, Dept. of Anatomy, Washington Univer- sity, St. Louis. June 6, 1951. Fluorescence is the property, pos- sessed by many substances, of convert- ing short wavelengths of light into longer wavelengths. In the field of microscopy those structures and sub- stances are of most interest that convert ultraviolet light into light of the visible spectrum, since it is only these sub- stances that can be observed directly. Though fluorescence microscopes de- signed for this type of observation have been available commercially for many years, their use has been limited until recently by their relatively high cost and by the apparent failure of biolo- gists to appreciate the possibilities of this type of observation. Recent tech- nological developments in the glass and electric lamp industries now make it possible to assemble an apparatus for fluorescence microscopy at a cost well within the budget of most labora- tories. Evidence of heightened interest in this field is found in the numerous papers concerning fluorescence micros- copy within the past 10 years. Al- though several reviews of the subject already exist (Haitinger, M., Flu- orescenz-Mikroscopie, Akademische Verlagsgesellschift, Leipzig, 1938; Ham- perl, H., Virchows Arch. f. path. Anat., 1934, 292, 1-51; Sutro, C. J., Arch. Path., 1936, 22, 109-112; and McClung's Handbook of Microscopical Technique, New York, Paul B. Hoeber Inc., 1937), the technique will be described as it can be used with an assembly of low cost apparatus available in the United States at the present time. Apparatus required: 1. An intense source of ultraviolet light that is rich especially in the region from 300 to 400 millimicrons. Certain electric arcs using electrodes of special metal alloys (the Haitinger Arc, C. Reichert — Vienna) have been developed for this purpose. More easily avail- able, low in cost, and having an intense output in the desired region, are the medium pressure mercury vapor arcs in capillary quartz tubes (the A H 4 lamp of the General Electric Company or Westinghouse Electric Co. and lamps made by Hanovia Chemical Co., etc.). 2. Filters that remove all or nearly all of the visible light. A considerable selection of glass and liquid filters may be used for this purpose. Since most of the so-ca,lled ultraviolet filters pass also a certain amount of red light, supplemental blue filters must be used with them. A solution of copper sulfate in a cell or tube of quartz, or of ultra- violet transmitting glass, is satisfactory and readily available. A combination of Shott glass filters U G 2 and B G 14 are recommended by Jenkins (R., Stain Techn., 1937, 12, 167-173). Corning Filters ^5840, 5860, or 5874 used with a copper sulfate solution are satisfactory in our experience. An entirely liquid filter, using solutions of cobalt sulfate and nickel sulfate, is described by Backstrom (H. L. J., Arkiv. for Kemi. FLUORESCENCE MICROSCOPY 128 FLUORESCENCE MICROSCOPY Mineralogi Och Geologi, 1940, 13A, 1-16). 3. Condensing lenses, if used at all, must be of quartz or ultraviolet trans- mitting glass. 4. A quartz prism or mirror of polished metal having a high reflecting power for ultraviolet. Aluminum and magne- sium-aluminum alloys are best for this. By mounting the microscope and light source horizontally this item can be eliminated. 5. An ordinary microscope that is fitted with a substage condenser of quartz or ultraviolet transmitting glass. Since the ultraviolet light has served its purpose when it has reached the tissue, ordinary glass objectives and eyepieces are used. With some older objectives the balsam of the lenses fluoresces in ultraviolet and causes an unpleasant diffuse light to appear in the microscope that masks the fluorescence of the tissue. This may be eliminated with a darkfield stop that prevents direct rays of ultra- violet light from entering the objective. Newer lenses are free from this fluores- cence and may be used without a dark- field stop. This is desirable since it permits the utilization of a greater por- tion of the light that strikes the con- denser. Popper has reported that the fluorescence of Vitamin A can be ob- served with an ordinary microscope with glass condenser. Ordinary optical glass transmits sufficiently far into the near ultraviolet that this type of apparatus might be successfully used for strongly fluorescent substances. 6. Slides for the specimens of ultra- violet transmitting glass. (Corex D glass slides, obtainable from Corning Glass Co. are suitable.) 7. An eyepiece filter that excludes ultraviolet light with a minimum ab- sorption of visible light. This may be of glass (Leitz ultraviolet protecting filter no. 8574 A, Corning Glass Works filters no. 3389 or 3060) or, simplest and cheapest, a circle of Wratten 2A gelatin filter cut to fit within the eyepiece (the Wratten 2 filter is not suitable since it fluoresces itself in ultraviolet light). 8. Non-fluorescent media for mount- ing the section to be examined. Me- dicinal mineral oil, or glycerin is suit- able. If immersion lenses are to be usep a non-fluorescing immersion medium must be employed. Sandlewood oil has been recommended for this purpose. Preparation of tissues: Hamperl (loc. cit.) recommends that tissues for fluores- cence examinations be fixed only in a dilute solution of formalin, since metal containing fixatives destroy the fluores- cence of some substances. A 5-10% solution of U.S. P. formalin in aq. dest. is ordinarily employed. Tissues should should not be fixed longer than 24 hrs.; certain components of tissue acquire abnormal fluorescence if the time of fixation is prolonged. If fats and other alcohol soluble substances are to bo ex- amined, i.e., vitamin A, polycyclic organic carcinogens, etc., frozen sections must be made. If these substances are not of interest, the tissue may be de- hydrated, cleared, and imbedded in paraflan in the usual manner. High quality reagents are required, because the impurities found in many organic substances themselves fluoresce. All paraffin must be removed since this too fluoresces. The section can be cleared in anhydrous glycerin or pure medicinal mineral oil. Gelatin and celloidin are not recommended for imbedding because of their fluorescence. Two types of fluorescence may be pro- duced in tissues with this type of appa- ratus. The first is that seen in tissues that have been subjected to no special treatment and is due to the presence of fluorescent substances in the tissues themselves. This is termed "primary" fluorescence or natural fluorescence and is exhibited by many substances found in animal organisms. In most tissues there are present sufficient quantities of these materials to permit the observer to recognize the general structure of the tissue without recourse to stained con- trol sections studied with transmitted visible light. Hamperl {loc. cit.) de- scribes, in considerable detail, the natural fluorescence of many human tissues. Jenkins (loc. cit.) summarizes the findings in the most common animal tissues. Cornbleet and Popper (T.and H., Arch. Dermat. & Syph., 1942, 46, 59-65) review the natural fluorescence of human skin. Popper and his co- workers have contributed a series of papers on the fluorescence of vitamin A in animal tissues (Popper, H., J. Mt. Sinai Hosp., 1940, 7, 119-132. Arch. Path., 1941, 31, 766-802; Popper, H. and Brenner, S., J. Nutrition, 1942, 23, 431- 443; Popper, H. and Ragins, A. B., Arch. Path., 1941, 32, 258-271). Simp- son and Cramer (W. L. and W., Cancer Research, 1943, 3, 362, 515, 604) have used the method to follow the distri- bution and persistence of methylcholan- threne in skin. Fluorescence color of various tissues and of drugs in tissue sections is described by Helander, S., Acta Physiol. Scand., 1945, 10, Suppl. 29, 103 pp. Another kind of fluorescence is the "secondary" fluorescence that appears in certain components of the tissue after FLUORESCENCE MICROSCOPY 129 FOOT'S METHODS sensitization with dyes and plant ex- tracts. This extends considerably the range of fluorescence microscopy and has been developed chiefly by Haitinger (loc. cit.) in conjunction with Hamperl and Linsbauer. Various fluorescent al- kaloids, azo dyes, primulins, auramine, berberine sulfate, chelidonium, rhubarb extracts, etc., are selectively absorbed by certain parts of the cell and cause them to show characteristic fluorescences in ultraviolet light. Such substances are called fluorochroraes. Sections of tissue are immersed in such substances for a short period of time before being examined. Examples of the use of these fluorochromes are found in papers by Haitlinger (loc. cit.),. Jenkins (loc. cit.), Clark and Perkins (W. M. and M.E., J. Am. Chem. Soc, 1932, 54, 1228-1248), Lewis (M. R., Arch. f. exp. Zellf., 1935, 17, 96-105) and Popper (H., J. Mt. Sinai Hosp., 1940, 7, 119-132). A good account of fluorescence microscopy of insects is given by Metcalf, R. L.. and Patton, R. L., Stain Techn., 1944, 19, 11-27. Noteworthy applications have been made in the use of acridine derivative dyes. Krebs and Gerlach (Am. J. Roentgenol. Rad. Therap., 1951, 65, 93-98) have demonstrated that acridine orange, C.I. 788, is a sensitive indicator of the viability of cells following ther- mal and radiational damage. The same dye was used by Zeiger and Har- ders (Z. Zellforsch., 1951, 36, 62-78) for flurochroming of nerve tissues. In a comprehensive investigation of 24 diaminoacridine stains by DeBruyn, Robertson and Farr (Anat. Rec, 1950, 108, 279-307) it was found that nuclei of many tissues and organs were vitally fluorochromed without perceptible toxic or degenerative effects. Primulin is recommended by Schmidt-LaBaume and Jager (Arch. f. Dermat. Syph., 1939, 179, 531) in an ingenious method for visualizing sur- face detail of the epidermis. A most interesting method of demon- strating the distribution of the right and left coronary arteries is given by Peterson and Gibson (Med. Radiogr. Photogr., 1951, 27, 14-17), who inject the vessels with plastics containing red and green fluorescing pigments. The heart is then encased in a plastic shell, following which the tissues are digested by KOH. Finally, the shell holding the arterial cast is filled with the transparent plastic. Under ultra- violet illumination the arterial system is revealed with extraordinary clarity. The use of fluorescein dyes in the diag- nosis of malignancy with special refer- ence to tumors of the central nervous system is described by Hubbard, T. B. and Moore, C. E., J. Nat. Cancer Inst., 1949, 10, 303-314 (good bibliog- raphy). The Diaminoacridines are im- portant vital stains for nuclei in the sense that they accumulate within nuclei without toxic action and can there be revealed b}' fluorescence microscopy (de Bruyn,P. P. H., Robert- son, R. C. and Farr, R. S., Anat. Rec, 1950, 108, 279-307). See Vitamin A, Tubercle Bacillus, Cell Injury, Ura- nium, Porphyrins, etc. Fluorescence Spectra. The technique in some detail is described for 3:4-Benz- pyrene by Hieger, I., Am. J. Cancer, 1937, 29, 705-714 who thinks that the photographs of the spectra can well be studied by simple visual examination. Fluorescent Blue, see Resorcin Blue. Fluorescent X. A special type of reduced neutral red (Clark, W. M. and Perkins, M. E., J. Am. Chem. Soc, 1932, 54, 1228-1248) employed for tissue cultures (Lewis, M. R., Arch. f. exp. Zelf., 1935, 17, 96-105). Fluorine, see Atomic Weights. Fluorochromes. See Fluorescence micros- copy. de Fonbrune pneumatic micromanipulator can be obtained from Aloe Scientific, 5655 Kingsbury, St. Louis 12, Mo. Foods. The examination of foods to ascer- tain their suitability for human con- sumption involves not only organolep- tic tests (use of unaided senses, sight, smell, taste, etc.), but direct micro- scopic examination and certain cul- tural, experimental feeding, and other tests. The techniques for adultera- tions, bacteria, fungi, crystals, spores, parasites and so on are usually the routine ones. However, much time will be saved by knowledge as to what to look for in each case, how to look and the best means of making the observa- tions accurately quantitative (Schnei- der, A., The Microbiology and Micro- analysis of Foods. Philadelphia: P. Blakiston's Son & Co., 1920, 262 pp.). Foot's Methods. 1. Rapid silver impreg- nation of reticular fibers (Foot, W. C, J. Tech. Meth., 1929, 12, 117-119). Fix in 10% formalin (not necessarily neutral), Zenker's, Bouin's or Helly's fluids, 24 hrs. Make paraffin sections. Remove mercury, if present, with iodine. Treat with 0.25% potassium permanga- nate, 5 min. and 5% oxalic acid, 10 min. Wash in aq. dest. Impregnate 15 min. in following silver solution at 50°C. : Add 40 cc 5%, aq. NajCOa to 10 cc 10% aq. AgNOa. Let precipitate settle. De- rant supernatant fluid. Make up to 50 cc. with aq. dest. Shake, repeat sett- FORAMINIFERA 130 FORMALIN ling and decanting. Dissolve ppt. in just sufficient NH4OH, added drop by drop, to almost completely dissolve it leaving a few gray granules. Heat to steaming to drive off excess NH3 and cool to 50 °C. Reduce in 1% formalin 2 min. Wash in tap water. Tone2min. in 0.2% aq. gold chloride. Wash. Tone in 5% aq. sodium thiosulphate. Coun- terstain with hematoxylin-Van Gieson. Reticulum, black; collagenic fibers, Vermillion; cytoplasm, yellow; and nuclei, brown. (2). Silver method for nerve fibers in paraffin sections (Foot, N. C, Am. J. Path., 1932, 8, 769-775). This is a modification of Cajal's technique. Fix in fresh Carney's Fluid for 24 hrs. Transfer to absolute alcohol for 1-2 hrs., clear in chloroform and imbed in par- affin. Remove paraffin from sections in usual way. Treat with 2 parts pyridine and 1 part glycerol for 1-12 hrs. Wash in 95% alcohol and then in aq. dest. to remove most of pyridine. A trace re- maining does no harm. Immerse in 10% aq. silver nitrate at 37 °C. for 12 hrs. or so covering container to avoid evapo- ration. Wash in 2 changes aq. dest. Place in 5% aq. neutral formalin con- taining 0.5% pyrogallol in which sections become yellow-brown, 20 min. Wash under tap. Tone in 1:500 aq. gold chloride, 5 min. (Nuclear precision is improved and glacial impregnation is made less intense if 2% glacial acetic is added to gold solution). Then place in 2% oxalic acid containing 1% neutral formalin for 5 min. Wash at the tap and transfer to 5% aq. sodium thiosulphate for 5 min. Finally washagain in running water, dehydrate, clear and mount. Foot also recommends Rogers' technique practically as given by him (Rogers, W. M., Anat. Rec, 1931, 49, 81-85) The idea of intensifying the gold toning with oxalic acid he credits toLaidlaw, G. F., Am. J. Path., 1929, 5, 239-247. See general remarks on Silver Methods. Foraminifera, see Carpenter, W. B. The Microscope and its Revelations, Lon- don, 1901. Formaldehyde is a gas (HCOH) soluble to 40% in water producing a solution which is termed commercial formalin or for- mol. See Formalin. Formalin (Formol) is a 37% aq. solution of the gas, formaldehyde. Solutions em- ployed as fixatives and preservatives are made in terms of the percentage of formalin, not of formaldehyde. Thus, a 10% solution of formalin (formol) is by convention 10 cc. of formalin plus 90 cc. of water. It is not however 10% for- maldehyde. (Obviously to dilute 10 cc. cone, hydrochloric acid with 90 cc. of water would not give 10% hydrochloric acid because cone, hydrochloric acid is not 100%, so that this practice cannot be extended.) Formerly it was necessary to neutralize commercial formalin in various ways, and it still is for special purposes. The best way to obtain neutral formalin is to distil. Atkins (Lee, p. 61) advises addition of borax to the diluted formalin until it shows a good red color with phenolphthalein or slaty blue with thymol blue. Others simply add a little calcium, sodium, magnesium or even lithium carbonate. Obviously the addition of such minerals greatly complicates the problem when formalin is employed with alcohol as a fixative preliminary to microincinera- tion. Lillie (p. 28) specifies a neutral buffered formaldehyde solution (pH 70) as follows: 37-40% formaldehyde solution, 100 cc; water, 900 cc; acid sodium phosphate, monohydrate, 4 gm. ; anhydrous disodium phosphate, 6.5 gm. Constant pH is desired. Unless neutral formalin is specified and the manner of neutralization, it is best simply to use the fairly pure product which now can readily be obtained. Experiments by Davenport, H. A., Stain Techn., 1934, 9, 49-52 show that as a neurological fixative slightly acid formalin is somewhat better than neu- tral formalin. A few of the manj^ de- mands for formalin as a fixative will be found under: Alizarin red S Alveolar pores Amyloid Argentaffine cells Arsenic Articular nerve ter- minals Bile pigments Bismuth Bodian Bone Buzaglo Cajal's brom-formol Cartilage Chitin Chloride Chorioallantoic mem- brane Christeller-Koyama Chromaffin reaction Color preservation Connective tissue cells Dopa oxidase Fatty acids Fluorescence micros- copy Foot Frozen sections Giemsa staining GUa staining Gomori Gordon Grieves Johnson's neutral red Kinney Krajian Liebermann - Burch- ardt Madder staining Mallory's connective tissue stain Microglia Mucus Nile blue sulphate Perdrau Pia mater Romieu Schultz cholesterol Sebaceous glands Silver citrate Smith- Dietrich Spirochetes Vorhoeff Weigert-Pal Weil Wilder FORMALIN-ALCOHOL 131 FROZEN SECTIONS In combination with other reagents formalin is also a good fixative cf. For- malin-Zenker in which formalin is sub- stituted for acetic acid, Bouin, Regaud's Fluid and many others. Since, how- ever, formalin is a strong reducing agent mixtures of which it is a part are un- stable so that it must be added immedi- ately before use. As Mallory (p. 40) points out, formalin also has certain disadvantages. It is inferior to alcohol as a preservative for iron and other pigments. It often changes the color of bile concretions from yellow to green and it may produce in the tissues a trouble- some brown-black finely divided crystal- line precipitate from laked hemoglobin. He advises removal of this precipitate by treating sections for 30 min. in 75% alcohol, 200 cc; plus 25-28% ammonia water, 1 cc. (Schridde's method), or for 10 min. in 80% alcohol, 100 cc. plus 1% aq. potassium hydroxide, 1 cc. (Vero- cay's method) after either of which they are washed thoroughly in water before placing in 80% alcohol and staining. When employed as a preservative con- centration of formalin should be 4%. Formalin-Alcohol, see Alcohol-Formalin. Formalin-Zenker. Zenker's fluid modified by substituting 5% formalin in place of the 5% acetic acid. It is also known as Helly's fluid and Zenker-formol. This is one of the three major routine fixatives the others being Zenker and Bouin. See Acid Fast Bacilli, Alveolar Pores, Arteries, Basal Bodies, Brazilin- Wasserblau, Mucus, Goodpasture's Method, Methyl-Green Pyronin. In some cases 10% formalin is inserted instead of 5%. Formalose see Formalin. Formamide of Eastman Kodak Co. is a sub- stance, called a "modifier", which when added in 10% to 50% alcohol improves fixation and staining of peripheral nerve (Bank, E. W. and Davenport, H. A., StainTech.,1940, 15, 9-14). Formazan, see Triphenyltetrazolium Chlo- ride. Formic Acid, see Decalcification. Formol is a synonym for formalin. Formol-Miiller, This is 1 part of formol to 10 parts MuUer's fluid. Formol-Nitric fixative. 3 parts 10% for- malin and 1 part 10% nitric acid. This has, according to McClung, proved very valuable for chick embryos. Formol-saline is the fluid resulting when formalin is diluted with isotonic salt solution (presumably 0.85% aq. sodium chloride) instead of with aq. dest. It is not advised as a fixative. Formvar, a plastic occasionally employed to make supporting films in electron microscopy. It is made by Shawinigan Products Corporation, New York, N. Y. Fowl Pox, see Borrel Bodies. Fractionation, see collection of cell com- ponents under Centrifugation. Fractures. Vital staining with Alizarin Red S (Schour, et al., J. Dent. Res., 1941,20,411-418). Fragility Tests. Micro for erythrocytes (Kato, K., J. Lab. & Clin. Med. ,1940, 26, 703-713. See Capillary Fragility Tests. Freezing. Details on the formation of ice within cells are given by Chambers, R. and Hale, H. P., Proc. Roy. Soc, B, 1932, 110, 336. See Revival after. Freezing and Drying, see Altmann-Gersh. Freifeld's Blood Stain. To 20 cc. tap water add 7 drops Ziehl's Carbol Fuchsin and 5 drops 1% aq. methylene blue. Stain methyl alcohol fixed blood smears 1 hr. Shows clearly basophilic granules of toxic neutrophiles (Naegeli, O., Zlutkrankheiten und Blutdiagnostik, Berlin, 1931, p. 19, quoted from Mc- Clung, 1950, p. 225). Frozen Sections. These are of great value when preparations must quickly be made and when methods of alcoholic dehydration before sectioning are contra- indicated. They are specified elsewhere in this book under several headings including : Amyloid Lipase Cholesterol Lipids Digitonine reaction Microglia Dopa oxidase Millon's reaction Gold Oxidase Indigo-carmine Pepsin Krajian'a Congo stain Spirocheta pallida Liebermann-Burchardt Urease To make the sections take recently excised still living tissue, or better fresh tissue fixed for about 30 min. in 10% formalin. First freeze a little water on the block of a freezing microtome. Then add the tissue and freeze it too plus a drop more of water. Allow block to thaw to optimum consistency, cut sections 15-50m thick as desired, and remove them with a camel's hair brush from the microtome knife to formalin, water or physiological saline. When many are required, it may be necessary to freeze several times as the tissue be- comes too soft. If thinner sections are wanted resort to Gelatin Imbedding before sectioning. For quick staining Thibaudeau, A. A., J. Lab. & Clin. Med., 1933, 19, 204-209 advises that sections of formalin fixed tissue be stained in Harris' hematoxylin 5-15 sec, rinsed in aq. dest., blued in aq. dest. -f few drops NH4OH, passed up through 70, 85 and 95% alcohol counterstained in absolute alcohol and FROZEN SECTIONS 132 FROZEN-DEHYDRATION METHOD eosin (5 sec), cleared in carbol xylol, blotted with filter paper and mounted in balsam. Proescher, F., Proc. Soc. Exp. Biol, and Med., 1933, 31, 79-81 recommends pinacyanol as giving ex- cellent color contrasts. Perhaps the simplest method advised by the Bens- leys (p. 138) is to stain the sections in Goodpasture's Acid Polychrome Meth- ylene Blue (which see) 1 min. or longer, wash and mount in aq. dest. This colors nuclei dark purple and connective tissue bright rose red. But methylene blue is less permanent than hema- toxylin. Adamstone and Taylor (Stain Techn. 1948, 23, 109-116) have devel- oped a modified technique useful for histochemical and routine studies. For reticular and collagenic fibers in frozen sections proceed as follows (Krajian, A. A., Arch. Path., 1933, 16, 376^378) : After fixation in 10% for- malin, cut sections 5-10 microns and wash in aq. dest. Then 10% aq. NH«OH at 60°C., 15 min. Wash in 3 changes aq. dest. and place in 0.3% KMn04 for 5 min. Rinse in aq. dest., decolorize in 1.5% oxalic acid until brown color has entirely disappeared. Wash 4-5 times in aq. dest. and soak in 5% AgNOj at 60°C. for 1 hr. Wash twice in aq. dest. Transfer to ammoni- acal silver sol. (to make add 6 drops 10% NaOH to 8 cc. 10% AgNO,. Then add freshly prepared 10% NH4OH drop by drop until almost entirely clear. Dilute to 28 cc. with aq. dest.) 16 min. at 60°C. Wash 3 times quickly in aq. dest. Change to 30 cc. formaldehyde + 70 cc. aq. dest. 1-3 min. at 60 °C. Wash in tap water. Mount on slide. Dehydrate with a little absolute alcohol and blot into position. Dehydrate more, blot, 3 changes equal parts anilin oil and xylol, xylol, balsam. Reticular fibers jet black, collagenic ones dark brown. For serial sections of brain (Marshall, W. H., Stain Tech., 1940, 15, 133-138) fix slices 24 hours or longer in 10 or 15% formalin and then treat them with a 20-30% alcohol or in 15% formalin in 20% alcohol. The object of the alcohol treatment is to avoid formation of hard and brittle ice crystals which fracture the sections as they are made. Cut tissue into blocks about 1.0 cm. thick. Place on a CO2 ice freezing disc which has been covered by a piece of wet blot- ting paper. (In our laboratory we use a regular COj gas freezing disc which has been adapted to a precision sliding microtome.) Freeze the block of tissue slowly throughout. The proper degree of freezing depends on the thickness of the sections to be cut. Marshall recom- mends a paraffin knife, 20-30° angle with block, knife set in a line perpendicular to the direction of motion. Remove cut sections by a camel's hair brush to 50% alcohol and keep them in serial order. Mount sections serially on slides coated with Albumen-Glycerin. Smooth out wrinkles and flatten sections by gentle pressure with blotting paper moistened with 50% alcohol. Remove slides to a 38 "C. oven for 4-6 hrs. when they are ready for staining. (In some cases it may be inadvisable to press the sections flat upon the slide. Thin sections re- quire less drying than thick ones. In any case until one has gained confidence in the use of the technique, the sections should be observed at intervals in the 38 °C. oven. At the least sign of exces- sive drying (whitening of parts of the section) the sections shoula at once be removed to the stain.) The Cresyl Violet method of Tress and Tress is recommended. Frozen-Dehydration Method for histologi- cal fixation — Written by Normand L. Hoerr, Department of Anatomy, West- ern Reserve University School of Medi- cine, Cleveland, Ohio. November 28, 1951 — This Method has distinct ad- vantages over the usual methods of preparation of tissues for microscopy, particularly for histochemical studies and for microincineration. Tissues are frozen as rapidly as possible and then maintained below the freezing point while being dried in vacuo. The method changes tissues little or not at all chemically, and physically only by the removal of water. If the freezing of a block of tissue can be effected rapidly enough the ice crystals which form during the freezing out of free water will be below microscopic size. The constituents of the tissue are then not displaced to an extent appreciable by microscopic examination. To pre- vent displacement of any constituent of the tissue after freezing, it is neces- sary that the dehydration be carried out below the eutectic point of the tissue. There is no way of knowing what the eutectic point of such a com- plex mixture of substances as obtains in animal tissues may be but, since in tertiary and quaternary systems the eutectic point is usually lower than the eutectic points of mixtures of any two of the substances, it is reasonable to assume that the absolute depression of the freezing point in tissues may be well below — 54.9°C. (the eutectic point of aqueous calcium chloride solu- tion). From our experience, we would say that better cytologic appearance of the tissues — i.e., an appearance com- paring most favorably with the appear- FUCHSIN NB 133 FUNGI ance of living tissues — is obtained by dehydrating at temperatures below — 3(J°C. If the freezing can be effected rapidly enough, the constituents of a tissue are not appreciably changed morphologically. Die Elementarorganismen und ihre Beziehungen zu den Zellen, Altmann, R., /Leipzig, Veit and Co., 1890, intro- duced the method of freezing tissues and dehydrating them over sulfuric acid in vacuo at temperatures below -15°C. Gersh, I. (Anat. Rec, 1932, 53, 309-337) overcame many early diffi- culties and elaborated the technic so that good histologic fixation could be procured on a number of tissues. Packer, D. M. and Scott, G. H. (J. Tech. Methods, 1942, 22, 85-96) have overcome, by a newly designed cryo- stat, many of the dehydration difficul- ties. The frozen dehydration method has been of value in the preliminary fixation of tissues for a study with the electron microscope of the localization of their contained salts by microincin- eration. It has been used for the pre- liminary preparation of tissues in the study of mitochondria and Nissl sub- stance, of secretion in the stomach and the thyroid gland, and in a histochemi- cal study of the Golgi apparatus. More extended discussions of the process of freezing and of the subsequent process of dehydration, with considerations of the advantages and limitations of the method are given by Hoerr, N. L. (Anat. Rec, 1936, 65, 293-295) and Simpson, W. L. (Anat. Rec. 1941, 80, 173-189). A number of cryostats have been designed recently, including those by Stowell, R. E. (Stain Technology, 1951, 26, 105-108) Emmel, V. M. (Anat. Rec, 1946, 95, 159-175) Scott, G. H., and Hoerr, N. L. (Medical Physics, 1950, 2, 292-296) Wang, K. J., and Grossman, M. I. (J. Lab. and Clin. Med., 1949, 34, 292-296) and Mendelow, H., and Hamilton, J. B. (Anat. Rec, 1950, 107, 443-451). A good commercial design is available from Scientific Specialties Corporation, Cambridge, Mass., or Euclid Glass Engineering Laboratory, 11310 Wade Park Avenue, Clevelend, Ohio. Fuchsin NB, see New Fuchsin. Fuchsin S, SN, SS, ST or S III, see Acid Fuchsin. Fungi. — Written by Morris Moore, Barnard Free Skin and Cancer Hospital, St. Louis, Mo. November 10, 1951. 1. Skin scrapings and hair. The usual method is to mount the material in an alkali — either sodium hydroxide (NaOH) or potassium hydroxide (KOH). The latter is preferable and should be used in a 10-30% solution. For rapid work 40% is employed but this tends to swell and disintegrate the fungi. A weak solution takes longer to clear the skin. The skin usually clears in 5 min. to 2 hrs. in concentrations of 10-30%. A little heat helps. Use sub- dued light in order to avoid high lights. The fungus is clearly discernible against the irregular nondescript background of skin which is usually clear. Dip infected hairs taken from scalps, par- ticularly those that are oily, in ether or in alcohol (absolute alcohol is preferable to 95%) for a moment in order to get rid of the oil which often simulates spores in sliape and size. Adamson (H. G., Brit. J. Dermat., 1895, 7, 201-211, 237-244) has recom- mended clearing with 5-10% KOH and staining by the Gram method. Chal- mers and Marshall (A. J. and A., J. Trop. Med. Hyg., 1914, 17, 256-265, 289-291) suggest soaking scales in 40% KOH for some hours in a watch glass in an in- cubator at 40 °C. Transfer specimens to watch glass containing 15% alcohol for 30 min., remove to slide, allow alcohol to evaporate and dry over flame ; stain with Anilin-Gentian Violet for 20 min. Treat with Gram's Iodine for 3 min.; decolorize with anilin oil, 30 min.; stain in concentrated alcoholic eosin, 1 min.; wash off eosin with anilin oil or clove oil ; treat with xylol and mount in balsam. Priestley (H., Med. J. Australia, 1917, 2, 471-475) recommends lacto- phenol (lactic acid, 1 part; phenol, 1 part; glycerol, 2 parts, aq. dest., 1 part) for clearing instead of 40% KOH ; or chloral hydrate crystals, 2 parts; lactic acid, 1 part; phenol crystals, 1 part, may be used. For thick material Langeron suggests : chloral hydrate crystals, 40 gm. ; phenol crystals, 40 gm.; lactic acid (U.S.P.), 20 gm.; and sodium salicylate, 10 gm. Slight heat facilitates clearing. To stain, Priestley recommends treatment with chloroform to remove the fat; boiling, 2-3 min., with formic acid ; washing for a few minutes in water and staining with Sahli's methylene blue : after which the tissue is to be washed, differentiated with alcohol if necessary, dehydrated, cleared and mounted in balsam. Bachman (R. W., Arch. Dermat. & Syph., 1920, 1, 50-54) recommends the following procedure : Place scrapings in a drop of water on a cover slip, tease thoroughly with a dissecting needle, dry over a flame but do not scorch. Stain for 2 min.; decolorize in 95% al- cohol, 15-30 sec; immerse in aq. dest., 15-30 sec. ; pour off excess, dry by heat, FUNGI 134 FUNGI and mount in balsam. Spores and mycelium, blue; scrapings, yellow. The dye is sat. ale. gentian violet, 2.5 parts; aq. dest., 17.5 parts; orange G solution, 9 parts; acetic acid, 1 part; 95% ale, 5 parts. The orange G solu- tion is orange G, 2 parts; 95% ale, 20 parts; water, 80 parts. Decolorize with 10-20% KOH. The hydroxide method of examination is simple and often rapid, but unless used by one familiar with it the results may be misleading. There is danger of confusion with structures which Becker and Ritchie (J. W. and E. B., Arch. Dermat. & Syph., 1940, 22, 790-802) have indicated as resembling yeast cells. These artifacts may be removed by treating the material progressively with absolute alcohol, ether, absolute and 95% alcohol. They have been termed 'mosaic fungus' and have been reported by Greenwood and Rockwood (A.M. and E. M., Arch. Dermat. & Syph., 1930, 21, 96-107) as degenerate fungi. In fact they are cholesterol crystals. The use of dyes eliminates in great measure such artifacts. However, the use of dyes is not practical with thick sections for which recourse must be had to the hydroxide method. When the scrapings or scales are thin, or when sputum, pus or exudate are ex- amined, a 1% aq. methylene blue and glycerin can be used as follows : One drop of the 1% solution of methylene blue is placed on a clean slide and the material is stirred within it, allowed to stand for approximately 2 min. when a clean cover slip is placed over the mix- ture and pressed down to flatten out the material and to express the excess solu- tion. The superfluous stain is taken up by filter paper. A drop of glycerin is then placed along one edge of the cover slip and allowed to seep under, dis- placing the stain and giving a clear back- ground to the stained material. The fungus appears bright blue. The lactophenol-cotton blue technique was developed in the French labora- tories using the formula of Amann (J., Zeit. Wiss. Mikr., 1896, 13,18-21). Lac- tophenol consists of phenol crystals, 20 gm.; glycerin, 40 gm.; lactic acid, 20 gm. andaq. dest., 20 gm. Cotton blue (anilin blue, China blue) is a mixture of the trisulphonates of tri-phenyl para- rosanilin (C.I. 706) and of di-phenyl rosanilin. Place a drop of the cotton blue (0.5% aq.) on the slide ; stir up the material within it and allow to stand for about 2 min. Add cover slip and press down to squeeze out any excess dye, which is taken up by filter paper. Add a drop of lactophenol to the edge of cover slip and allow it to replace the cotton blue which dries out. The stain may be rapidly replaced by holding a bit of filter paper at the edge of the cover slip op- posite the lactophenol. The cell wall stains lightly as compared with the darkly colored central portion of the fungus. The tissue elements also stain light blue. Swartz and Conant (J. H. and N. F., Arch. Dermat. & Syph., 1936, 33, 291-305) have modified the lactophenol and cotton blue procedure. First put a few scrapings in 5% aq. potassium hydroxide, heat somewhat and wash in water. Place material in a drop of the combined cotton blue (0.5%) and lacto- phenol. The fungi stain a darker blue than the tissue cells. Schubert M., Dermat. Wchnschr., 1937, 105, 1025-1029) has modified the Swartz-Conant technique. Soak the scales in 2% KOH for 30 min. or until they appear glassy and then wash in aq. dest. 2-10 hrs. Transfer small particles to a slide and add 1 or 2 drops of following stain : cotton blue, 0.25 gm. ; lactic acid, 10 gm.; phenol crystals, 10 gm.; and aq. dest., 20 gm. The fungi appear dark blue while the epidermal cells stain lightly. See also Berberian's Method. 2. Sputum, pus and exudates: Exam- ine for fungi after mounting directly on a slide after mixing in 20% KOH or on stained smears. The latter are not very satisfactory because smearing tends to disturb the arrangement of the cells but they are useful for detection of mycelium. Many contaminating or- ganisms are generally present in these exudates unless material is secured from fresh lesions opened aseptically. Sev- eral examinations may be necessary since the organisms in exudates are seldom numerous. The hydro .xide usually dis- solves most of the tissue elements and the fungi stand out as refractile bodies. Several of the staining methods em- ployed in the study of hair and scrapings may be used. Of these, the methylene blue and glycerin method is best but the lactophenol-cotton blue technique is likewise advised. 3. Vesicles, blister fluid, spinal fluid or urine: These can also be directly examined. But vesicle, or blister, fluid yields only a small amount of material and for best results, the methylene blue-glycerin method or the lactophenol- cotton blue technique is advised. Urine, or spinal fluid, should be con- centrated by centrifugation before examination. The same staining pro- cedures are advocated. See Blasto- mycosis. FUNGI 135 FUNGI 4. Skin: Unna, Jr. (P., Dermat. Wchnschr., 1929, 88, 314-321) advises the following modification of the Pap- penheim-Unna, Sr. method for stainins fungi in skin. Fix in absolute alcohol, then run through the alcohols to xylol and imbed in paraffin. Cut sections at 10/x, stain with pyronine-methyl green (pyronine, 9 parts; methyl green, 1 part; 96% alcohol, 90 parts; glycerol, 100 cc; 0.5% phenol to make 1000 cc), 5-10 sec; rinse in water; dry with absolute alcohol; and mount in balsam. Fungi, rubin red; leukocytes, green to blue green ; nuclei of cells of basal horny layer of the epidermis, red. Fungi in tissue can be easily stained by Iron-Hematoxylin and eosin. The fungous elements take the hematoxylin stain nicely, although some difficulty may be encountered in distinguishing spherical cells or spores from tissue elements. The Gram method of stain- ing for bacteria has been used with a measurable amount of success since fungi are, in general, gram -positive. Unna's Alkaline Methylene Blue (Unna, P., Monatsh. f. prakt. Dermat., 1891, 13, 225-237, 286-311), although recommended for staining plasma cells and as a general stain in combination with phloxine or eosin, has been espe- cially adapted for staining organisms in the stratum corneum. It consists of methylene blue, 1 gm.; potassium carbonate, 1 gm.; and aq. dest., 100 cc. The solution stains better after ripening for a week or two and should be diluted 1 to 10 or 1 to 5 before use. Malcolm Morris (Mallory, F. B. and Wright, J. H., Pathological Technique, Philadelphia and London, 1924, p. 175) in staining various parasites of the skin, avoids the use of potassium hydrate. Place skin in ether, or in 1:1 alcohol- ether; stain for 5-30 min. in 5% gentian violet in 70% alcohol. Then pass through iodine solution, 1 min.; anilin, or anilin plus 2-4 drops of nitric acid; anilin; and xylol (2 changes) to balsam. 5. Other tissues: A number of methods listed for staining Bacteria in tissue can be used successfully for fungi. Mallory's Connective Tissue stain is good for Cryptococcus hominis in brain tissue. Fungus cells, red ; thick mucoid capsules, light blue. TheGram-Weigert staining method is also excellent. Organisms, deep violet; nuclei, blue to violet; connective tissue, red. Actinomyces in sections may be stained successfully with Alum-Hema- toxylin followed by strong eosin. Mal- lory (p. 279) lists 2 methods of which the following gives good results with paraffin sections of formalin or Zenker fixed tissue. Stain in alum-hematoxy- lin, 3-5 min.; wash in water; stain in a 2.5% a,q. phloxine or 5% aq. eosin, 15 min. in the paraffin oven; wash in water; stain in Anilin Crystal Violet (try Stirling's), 5-15 min.; wash in water; treat with Gram's Iodine solu- tion, 1 min.; wash in water and blot with filter paper; differentiate in several changes of anilin until no more color comes off; rinse in several cliangea of xylol and mount in balsam. The branched organisms stain blue while the hyaline sheaths ("clubs") become pink to red. The Hotchkiss-McManus or Periodic Acid-Schiff Stain (Kligman, A. L. and Mescon, H., J. Bact., 1950, 60, 415-421) may be used to find fungi in tissue or sections. Tissue fixed, embedded in paraffin and sectioned in usual manner. After deparaffinizing, sections rinsed in absolute alcohol; washed in distilled water; immersed in 1% aq. periodic acid C.P. (Eimer & Amend) 5 min.; washed in tap water, 10 min.; placed in Schiff reagent, 10 to 15 min. ; transferred to 1 normal HCl, 5 min.; washed in tap water, 10 min.; counterstained with 1% aq. light green, for sec. ; dehydrated, cleared and mounted. Fungi stain red to purple. Tissue cell nuclei do not stain. Schiff reagent prepared as follows: Dissolve 0.5 gm. basic fuchsin (Cole- man & Bell) by passing over it 100 cc. boiling water; cool to 50°C.; filter and add 10 cc, 1 normal HCl and 0.5 gm. potassium metabisulfite dry reagent (Eimer & Amend) to filtrate. Solu- tion becomes colorless to pale straw colored by standing in dark overnight. Add 0.25 to 0.5 gm. activated charcoal, shake well and filter immediately to completely decolorize. Refrigerate in tightl}^ stoppered bottle. For smears or nails: Use thin smears and fine nail scrapings. Material made to adhere to slide with egg albumen. Coplin jar or drop bottle technique employed. Immerse material in 95% alcohol, 1 min.; cover with 5% aq. periodic acid, 3 minutes; wash in run- ning water. 2 min.; stain in Schiff re- agent, 5 min.; rinse in running tap water, 1 min.; dehydrate in 95% then absolute alcohol and two changes xylol, 1 min. each. Mount in clarite. See Polysaccharides. After the fungi have been successfully cultivated on the various mediums recommended (Moore, M., Arch. Dermat. & Syph., 1936, 34, 880-886) they can be examined microscopically by transferring part of the growth with a sterile platinum or nichrome wire to FURFURAL 136 G ALLOC YAN IN -CHROMALUM STAINING a clean slide. This should be done fently to avoid destruction of the ungous growth. The fungus is teased apart gently in one of several fluids such as water, alcohol, alcohol and glycerine (equal parts) or other mount- ing fluids. Water has a high surface tension and causes disruption of the growth; while alcohol evaporates rap- idly and must be replaced. The following solution serves well : 2% potassium acetate, 50 cc; glycerin, 20 cc; and 95% alcohol, 30 cc. The preparation is examined with reduced light. The preparations may be stained using one of the several methods listed : lactophenol-cotton blue ; methylene blue-glycerin; or Giemsa-glycerin. See Chorioallantoic Membrane, Actlno- mycetes. Furfural. Has been suggested but is not recommended as substitute for formal- dehyde (Stowell, R. E. and Stokes, J. M., J. Tech. Meth. and Bull. In- ternal. Assoc. Med. Museums, 1944, 24, 25-30). Fuscin (L. fuscus, dusky), a dusky pigment of retinal epithelium usually present in crystalline formations made up of albuminous cores, which determine their shape, plus the adsorbed fuscin material. A relationship to melanin is debated but the pigment is very resistant to chemical attack. It can, however, be bleached completely when exposed to light in vitro. For details see Arey, L. B. in Cowdry's Special Cytology, 1932, 3, 1218. Fustics. "Young" fustic is a stain obtained from the smoke tree, Rhus cotinus of West Indies and South America giving colors from bright yellow to dark olive now seldom used by dyers. "Old" fus- tic is obtained from a tree of the mul- berry family, Chlorophora tinctora, which grows in the same countries. It is chiefly employed for woolens giving shades of lemon and old gold (Leggett, W. F., Ancient and Medieval Dyes. Brooklyn: Chemical Publishing Co. Inc., 1944, 95 pp.). Gadolinium see Atomic Weights. Galliamine Blue (CI. 894) can be employed in place of hematoxylin as an iron lake stain for nuclei. Gallein (CI, 781), a mordant dye of light fastness 1. Use as solution 0.5 gm. in 100 cc. of either 1% aq. anmionium acetate or 0.1% sulphuric acid. Small invertebrates should be previously mordanted, 30 min. in 1% aq. ferric ammonium sulpha e and rinse in aq. dest. before staining for 1 to 2 min. in the solution at 50°C. Color blue black. If copper sulphate is employed for mor- dant color is hematein purple. In paraffin sections of animal tissues nuclei color blue black in 15 to 20 sec. at 50°C. Directions are also given for plant tis- sues and Blue-green algae (Emig, p. 54-55). Gallium, see Atomic Weights. Gallocyanin-Chromalum Staining of Baso- philic Cell Structures — Written by Ldrus Einarson. Normal-Anatomisk Institut. Aarhus Universitet, Aarhus, Denmark. February 27, 1951. — Baso- philia is an essential property common to some most important cell structures, and the study of the nature as well as the attainment of a method for quanti- tative estimation of basophily have been among the outstanding general problems in histology and cytology. Here, the nerve cells make an especially favourable object on account of their high degree of cytoplasmic basophily due to the Nissl substance, which con- tains nuclein, acid and basic proteins respectively (Einarson L. J. comp. Neur., 1935, 61, 101-133). An accurate estimation of basophily can be achieved by staining with dis- solved colour lakes of some synthetic dye-stuffs (Ranvier, 1875; Grenacher, 1879; Rawitz, 1896; Becher, 1921), and the staining by gallocyanin-chromalum affords the most accurate representative of this principle of staining by inner- complex dye-metal salts (Einarson, L., Acta path. & Microbiol. Scand., 195, 28,82-102). The staining solution is made as fol- lows: 5 gm. chromalum puriss. is dis- solved in 100 cc redistilled water, then 0.15 gm. gallocyanin (Griibler-HoU- born or Nat. Aniline Division, New York) is added and the whole is mixed by shaking the bottle. The mixture is warmed up gradually and boiled for 5 min. After cooling at room tempera- ture, filtration through filter paper and addition of redistilled water through the filter until the volume again is 100 cc, the solution is ready for use; its pH is 1.64. Staining time is 48 hrs at room temperature. After staining washing in aq. dest., alcohols, xylol balsam. The process by which the lake is formed consists in the formation of a soluble lake-cation (reddish), a slightly soluble lake-sulphate (blue) and a non- soluble lake-hydroxide; the latter is formed by an inmiediate hydrolysis of some of the lake under formation. After the solution has been prepared no further hydrolysis occurs until 4-5 weeks later (late hydrolysis), and a gradual sedimentation of lake-hydrox- ide takes place; at the same time the staining power of the solution be- GALLOCYANIN-CHROMALUM 137 STAINING comes weaker. Thns, for safety the solution should not be used for more than 3 weeks. Lake-sulphate and lake- hydroxide are the main constituents of the precipitate filtered off the solution before use. Although such inner-com- plex salts all have very low solubilities in water, the lake-sulphate, however, reamins dissolved in a sufficient amount to become dissociated into the mono- valent lake-cation and an anion ac- cording to the equation: (gallocyanin Cr(H20)4)2 S04^ 2 galloc ^ Cr(H20)4+ + SO4 In the lake-cation, Cr(H20)6+++ is attached to one gallocyanin molecule by two valency bonds to its OH and COOH, and by two coordinating bonds to its =0 and/0\ through substitu- tion of two HoO-molecules from the internal sphere of the Cr-atom. The designations =0 and /0\ are used for the sake of brevity. =0 means the 2-carbonvl-O, and /0\ means the oxazin-0 of the chromophor-group). The reddish lake-cations, the actual staining compound, unite progressively and selectively with the nucleoproteins of the fixed cell structures to form a blue salt of the lake-cation and the tissue. This proceeds until maximum occupa- tion has taken place; then no further attachment of the stain to the baso- philic cell structures is possible. When the staining has attained a certain in- tensity it will not be further increased, even if the tissue be left in the staining solution for a considerable length of time; in this respect the staining is independent of the staining time {ex- quisite progressivity) ; no other histo- logical staining known to me possesses this quality in the same degree. Owing to the great stability of the staining it is also completely unaffected by alcohol and xylol during the procedure of de- hydration and mounting. In consid- eration of these qualities the staining intensity depends directly on the in- herent capacity to bind the stain, pos- sessed by the living cell at the moment of fixation. Briefly the staining consists of : 1. A specific binding of the lake- cation to the nucleic acids, in which it reacts with the phosphoric acid groups to form a lake-salt of nucleic acid; within the range from pH 0.83 to 4.35 this specific binding always takes place by means of the free -f-valency of the lake-cation, regardless of the type of nucleic acid. 2. An adsorption of lake-sulphate to the tissue, i.e., an unspecific co- staining of non-basophilic structures, G ALLOC Y AN IN -CHROMA LUM STAINING which increases directly with the pH of the dye-solution; it mainly takes place by the basic 7-(CHj)2N-group of the dye molecule; by staining at a suffi- ciently low pH the co-staining is re- duced to a minimum. 3. A binding of the lake-cation to the proteins of the basophilic structures, which, however, only takes place in a definite range of pH on the alkaline side of the iso-electric point of the pro- teins and in fact is unimportant; on the acid side of the iso-electric point the lake-cation combines with the nucleic acids alone. As the lake compounds stabilize (buffer) the staining solution towards acids and alkalies its pH can be varied simply bv adding 1 to 10 cc of N/1 HCl and' N/1 NaOH respectively to each 40 cc. of stock solution. In the range from pH 1.50 to 1.80 the specific staining reaches its maximum intensity at the same time as the co-staining re- mains completely negligible; pH 1 .64 represents the optimum acidity for the specific attachment of the lake-cations to the nucleic acids, the relative quantity of which can be estimated by photometric or densitometric measurements. The in- crease of the co-staining first sets in at pH 1.80 to reach its maximum between pH 3.4 and 3.5 whereupon every stain- ing fades away; at pH 4.27 it is barely visible. The co-staining at pH 2.90- 3.42 may be used for demonstrating axons and neuroglia (Einarson & Ringsted 1938, p. 43). After the addi- tion of alkali the solution should not be used longer than 4-8 days. The usual basic blues preferably stain proteins and other amphoteric electro- lytes which display ''facultative baso- phily",\.e., under another set of condi- tions they are less basophilic or even acidophilic; they are amphophilic sub- stances, and the staining is a mere ad- sorption of the dye. Gallocyanin-chromalum indicates the degree of "genuine basophily" which depends directly on the quantity of nucleic acids present; genuine baso- phily is not in the same way dependent on the external conditions as the facultative one, and its changes reflect the decomposition during neuronal function or regeneration and the sub- sequent repair of cytoplasmic nucleic acid from the nucleus (Einarson, L., Am. J. Anat., 1933, 53, 141-176). Gallocyanin (CI. 883)-alizarin blue RBN, chrom blue GCB and fast violet. It is a basic oxazin dye. Gamma = 1000 mg. or 0.001 mg. GARDNER 138 GELATIN E^IBEDDING Gardner, see Articular Nerve Terminals. Garven's Gold Chloride method for nerve endings in muscle (Garven, H.S.D., Brain, 1925, 48, 380-441). This is Fis- cher's modification of Ranvier's tech- nique as used in Golgi's Laboratory. Immerse small pieces of tissue in 25% aq. pure formic acid and tease a little to assure penetration 10-15 min. Blot with clean cloth. Place in 1% aq. gold chloride just sufficient to completely cover tissue and shake. Avoid all iron instruments. Cover dish with blue or yellow glass. Leave 20 min. Blot with clean cloth and repeat above treat- ment with formic acid and gold leaving this time in latter 24 hrs. in absolute darkness. Repeat still again. Pass to glycerin and leave in closed vessel in ordinary light. The sharpness of the intensely purple black nerves in a lightly colored background increases with time. Small pieces can then be transferred to aq. dest. and the indi- vidual fibers separated. This is facili- tated by dissociation in dilute nitric acid. Wash and make final mounts in glycerin. The author used panniculus carnosus of hedgehog, striated muscle of frog and lizard, extrinsic eye muscle of rabbit and human pectoral muscle. Gas Analysis. — Techniques are given in de- tail by Glick, pp. 313-412. Gash-Irrigation recovery method for lung cells (GI).— Written by C. C. Macklin, Dept. of Histological Research, The University of Western Ontario, London, Canada. November 28, 1951. — Fresh collapsed mouse or other mammalian lung is cut through a drop of physiologi- cal salt solution, blood serum or other suitable liquid, inverted and drained onto a slide. It is then covered and ex- amined as a fresh mount, or spread, dried, stained and mounted like a blood smear. Liberated phagocytic and gran- ular pneumonocytes (dust and foam cells — which see) are thus obtained (Macklin, C. C, Proc. 6th Intern. Con- gress of exper. Cytol., Stockholm, 1947; published 1949, 383-387; Macklin, C. C, The Lancet, Feb. 24, 1951, 432-435). Gastric Contents. Examine microscopically material obtained by stomach tube after test meal as described by Stitt (p. 753). Look for mucus, epithelial cells, leu- cocytes, Gram positive bacilli in smears. Gastrointestinal Tract. Immediate fixation is desirable because postmortem changes occur especially quickly. Do not wash first with water but with physiological saline or with the fixative itself. It may be desirable to place the excised pieces, with peritoneal surface down, on wooden tongue depressor or stiff paper. Some flattening is required. The mucous surface must not be allowed to dry. See Small and Large Intestine. See Papanicolaou Techniques. Gautheria Oil used to be employed as a clearing agent. It has been displaced _ by the artificial oil, methyl salicylate. Geiger Counters are instruments for the counting of electrons which provide quantitative data of great importance in this electron age. A concise descrip- tion of the history of counter develop- ment and of the Geiger-Miiller type is supplied by Rovner, L. in Glasser's Medical Physics, 487-495. Gelatin-Carmine injections, see Carmine Gelatin injections. Gelatin Glue, method of mounting sections, see Masson's. Gelatin Imbedding and Sectioning. This is used when sections are required of loose, friable tissues which easily fall apart. Since the imbedding is directly from water, no alcoholic or other dehydration is required. Probably the best method is that of Zwemer (R. L., Anat. Rec, 1933, 57, 41-44), devised primarily for the study of adrenal lipoids. Wash material fixed in formalin or other fluid in water, 4 hrs. 5% gelatin in incubator at 35-37 °C. 24 hrs. 10% gelatin at same temperature, 12-16 hrs. Imbed by placing in 10% gelatin in Petri dish in refrigerator. Cut out blocks of tissue and fix in 10% formalin several hours to make gelatin insoluble in water. In this formalin solution tissues can be preserved indefinitely. Before section- ing rinse block in water and trim. Freeze with CO2 until block is uniformly white. Allow to thaw until knife cuts easily. Sections as thin as 5 microns can be obtained. Float onto slide in aq. dest. Drain off excess water and run a drop or two of 1% gelatin under setion. Again drain off excess. After heating in drying oven at 33-37°C. place slide in 10% formalin for 10 min. to fix gelatin. In this formalin solution the mounted sections can be stored. Stain sections in usual way with Sudan, Nile Blue Sulphate, Osmic Acid, Laidlaw's Silver Method, and mount in Gly- chrogel. Wright's method as described by Mallory (p. 34) is much quicker and is recommended for fragmented tissues such as those from curettings. Make a 10% solution of gelatin in warm aq. dest. and while still fluid add 0.5% carbolic acid. Do not overheat. The tissue, unfixed or fi.xed, preferably in 10% formalin, is "dried" and placed in a small "pool" of gelatin liquified by heat on a or slide in a glass vessel. This is allowed to solidify in the ice box for GELATIN MEDIA 139 GIEMSA'S STAIN 2 hrs. or more. If necessary, store gelatin blocks in 10% formalin. Cut out block containing the tissue, freeze and section. Float sections from water onto slide well coated with albumen- glycerin and spread. Remove excess of fluid and cover with piece of thin cigarette paper. Blot with fine filter paper till cigarette paper is partly dry. Cover cigarette paper with equal parts anilin oil and oil of cloves for few seconds. Drain and peel off cigarette paper. Remove oil by washing in 95% alcohol and pass to water when sections are ready for staining. Mallory suggests methods for Amyloid, Fat and staining with Hematoxylin and Phloxine for general purposes. Gelatin Media, see Bacteria, Media. Gelatin-Ringer electrode vessel, modifica- tion by Kriest, A. C, Acta Physiol, et Pharmacol. Neerland., 1950, 1, 32-34. Gentian Blue 6B, see Spirit Blue. Gentian Violet. The problem afforded by this dye, like many others, has been attacked by the Stain Commission. The stain thus referred to has no con- stancy. Originally it was a mixture in about equal parts of dextrin and methyl violet, the latter itself a mixture in widely varying proportions of tetra-, penta- and hexa-methyl pararosanilins. Later were placed on the market methyl violets with and without dextrin and crystal violet (the hexa methyl com- pound) all under the label of Gentian violet. As Conn (p. 124) advises the term Gentian violet should be elim- inated and crystal violet used wherever in the past the former has been specified. See Neutral Gentian, Methyl Violet, Crystal Violet. Geranine G (CI, 127). An acid thiazole dye employed in fluorescence studies on account of color imparted by it under ultraviolet illumination (Conn, p. 70). Germanium, see Atomic Weights. Giant Cells. There is no special technique for their demonstration. Since the features usually employed in classifica- tion are size and nuclear detail and arrangement, Hematoxylin and Eosin, or Iron Hematoxylin the latter followed by various counter stains as for Acid Fast Bacilli are recommended. The following is a much abbreviated classi- fication of Giant Cells from Cowdry's Histology 1938 Edition : 1. Megakaryocytes of bone marrow, granules in cytoplasm, best demon- strated by Giemfsa's Stain. 2. Foreign body gianl cells formed probably by a fusion of cells of mesen- chymatous origin, perhaps of non- granular leucocytes, in response to foreign materials of many kinds — tubercular giant cells, foam cells in leprosy, lympsocystic giant cells of fish (Weissenberg), and possibly Reed- Sternberg cells in Hodgkin's disease. 3. Osteoclasts (polj^karyocytes) of bone marrow and Langhans' giant cells of placenta are normal inhabitants of these organs. Myeloplague and Myelo- plax are other terms for osteoclast. Chorioplague is a plate like giant cell of the chorion. See original account for lack of specific properties of so- called Langhans' cells which designa- tion should be abandoned. 4. Epithelial giant cells are clearly of epithelial origin. Found in epidermis in chicken-pox and other diseases, oc- casionally in the liver and in kidney in many conditions. Often show nuclear irregularity and evidence of nuclear budding. 5. Hypertrophied cells can be either normal to meet physiological demands, as enormously enlarged smooth muscle cells of pregnant uterus, or due to vari- ous pathological conditions. Mauth- ner's Giant Cell in the fish brain is al- ways of tremendous size in adults. Giemsa's corrosive sublimate fixative. Sat. aq. corrosive sublimate 2 parts, absolute alcohol 1 part. Giemsa's Stain. 1. For hlood or bacteria in smears. Fix air dried smears in methyl alcohol in a covered dish 3-4 minutes. Remove and blot dry. Di- lute stock solution of Giemsa in propor- tion of 1 drop to 1 cc. aq. dest. and stain for 15 minutes. Then wash in aq. dest., blot and dry. If a precipitate is formed in the smear by the stain, invert the slide, support both ends, and the stain will adhere like a hanging drop, kept away from the ends by lines ruled in wax or paraffin. The pH of the aq. dest. used to dilute the stain may be altered by adding very dilute acid or alkali. Optimum pH of 6.4 is given by the McJunkin-Haden buffer. This may be used as diluting medium in place of aq. dest. Usually the azurophile are stained more distinctly and the neutro- phile granules less sharply than by Wright's stain. Bacteria and intra- cellular protozoa are better colored than by Wright's stain. The May-Giemsa, and Jenner-Giemsa and the panchrome stains of Pappenheim are important modifications. They are listed sepa- rately. Present situation concerning Giemsa's stain is that American products give equally good results with thin films but the German product appears to be better for thick ones (Conn, H. J., Stain Techn., 1940, 15, 41-43). GIEMSA'S STAIN 140 GLIA STAINING WITH ANILIN DYES 2. For sections. Much depends upon the choice of fixative. Formalin, gener- ally employed in 10% solution, acts as a sort of mordant for the blue component so that the blue coloration is particularly strong. Fixation in Regaud's gives good results particularly with Rickettsia, Zenker's fluid is recommended by Wolbach. When this is used it is neces- sary to remove the mercuric chloride by treating the sections with Lugol's solu- tion. They are then washed in 95% alcohol and the last traces of iodine are extracted by 0.5% aqueous sodium hyposulphite for 10-15 min. The hypo- sulphite in turn is washed out in run- ning water about 5 min. and rinsing in aq. dest. See Cowdry's colored figures of Rickettsia, J. Exper. Med., 1925, 42, 231-252. Bouin's fluid (75 cc. saturated aq. picric acid, 26 cc. commercial formalin and 4 cc. glacial acetic acid) is suggested for intracellular protozoa (East Coast fever parasites) by Cowdry and Danks (Parasitology, 1933, 25, 1-63) because after Giemsa staining it gives the chromatin a desirable purple color (see colored plate). Stain sections placed vertically in staining jars in 1.5 cc. Giemsa 's solution plus 50 cc. aq. dest., changed during the first hour, overnight. Dif- ferentiate in 95% alcohol, dehydrate quickly in absolute alcohol, clear in xylol and mount in balsam. If the sections are not blue enough add 1-2 drops 0.5% sodium bicarbonate and 1.5 cc. methyl alcohol to the stain; or remove excess of mordanting potassium bichromate from Zenker fixation by rinsing 1 min. in 1% potassium per- manganate followed by 5% oxalic acid 4 min. and thorough washing in aq. dest., or do both. If on the contrary they are too blue mordant in 5% potas- sium bichromate 15 min., rinse in aq. dest. until no more yellow is removed and stain; or add a little colophonium to the alcohol used in differentiating and dehydrating of the sections, as advised by Wolbach, or again do both. Usually Giemsa's stain gives satis- factory results without any special pre- cautions. The difficulty is that the colors fade quite rapidly particularly when the balsam is noticeably acid and when the sections are left in direct sunlight. Their period of usefulness can be extended by mounting in cedar oil, used for oil immersion objectives, instead of in balsam. Try Clarite. If a variety of fixatives is employed it may be necessary to suit the stain to the fixative by use of buffers, in which case see Lillie, R. D., Stain Techn., 1941, 16,1-6. To demonstrate the "nucleoids" of bacteria in smears the technique of C. F. Robinow published as Addendum to Dubos, R. J., The Bacterial Cell. Harvard Univ. Press, 1945, 460 pp. is suggested. Fix smears in osmium tetroxide vapor, treat 7-10 min. with N/1 HCl at 60°C. and color with Giemsa's solution. Bj' this method nu- cleoids are stained whereas similar bacteria not treated with the acid are uniformly colored by Giemsa. Robi- now prefers this staining of nucleoids by Giemsa after hydrolysis to the Feul- gen technique. Gilson's Fluid. Nitric acid (sp. gr. 1.456), 15 cc; acetic acid, 4 cc; mercuric chloride, 20 gm.; 60% ale, 100 cc; aq. dest., 880 cc. Used mostly for inverte- brates. Gilson's Mixture is equal parts chloroform and cedar oil. Gingiva. Capillaroscopy of (McClung, Mi- croscopical Technique, 1950, 328); Eo- sinophile leucocytes in (Orban, B., J. Dent. Res., 1940, 19, 537-543.) Glacial Acetic Acid, see Acetic Acid. Gland Cells contrasted. Endocrine, exo- crine, apocrine, merocrine, holocrine, serous, zymogenic and mucous (Cow- dry's Histology, p. 257). Glass Cloth, as a substrate for tissue cul- ture, Warner, D., Hanawalt, C. and Bischoff, F., J. Nat. Cancer Inst., 1949, 10, 67-74. Glass Electrode. Sisco, R. C, Cunning- ham, B. and Kirk, P. L., J. Biol. Chem., 1941, 139, 1-10 have devised an open cup variety described with diagrams by Click, pp. 183-184. See Claff and Swen- son glass capillary electrode and the Pickford sealed-in capaillary glass electrode also described by Click. Glia Staining with Anilin Dyes (Proescher, Fr., Stain Techn., 1934, 9, 33-38). Fix in 10% formalin or in 90% alcohol followed by formalin. Wash frozen sections, 10-15 microns thick, in aq. dest. Stain in sat. aq. victoria blue B (not filtered but poured off from the undissolved dye), 14-24 hrs. Wash quickly in aq. dest., mount with glyc- erin-albumen, blot and dry in air. Treat with ultraviolet light 30 min. Pass to N/20 iodine few sec. Remove iodine, blot, dry, destain in xylol- anilin, clear first in clove oil, then xylol, mount in balsam. Glia blue, nerve cells lightly stained, connective tissue metachromatic violet or colorless. Instead of ultraviolet light stained sections can be treated with 0.5% potassium bichromatic for 30 min. In place of victoria blue, methyl violet 2B, ethyl violet or crystal violet can be employed. GLICK 141 GLYCOGEN Glick, see Linderstr0m-Lang, Kaj, u., and Holter, Heinz, Histochemical Advan- ces. Glomus. Aortic and carotid, see Aortic Paraganglion. Glucose Agar, see Bacteria Media. Glutathione. Demonstrated by Nitro- prusside Reaction. Inhibiting factor in Vitamin C silver test. Glycerides, see Neutral Fats. Glycerine. Much used in histological tech- nique in the making up of stock solu- tions of hematoxylin, like Delafield's, in Albumen-Glycerin used for mounting paraffin sections, etc. It serves as an excellent clearing agent for the walls of large Arteries so that the intramural vessels can easily be distinguished by the blood in them. With potassium hydrate it is employed to clear speci- mens in the demonstration of Ossifica- tion centers. As a mounting medium for frozen sections glycerin is invaluable. In the form of Brandt's glycerin jelly (which see) glycerin is specified in the technique for Sebaceous Glands and many other structures. To make Ilai- ser's glycerin jelly (Mallory, p. 100) soak 40 gms. gelatin in 210 cc. aq. dest. for 2 hrs. Add 250 cc. glycerin, stir and heat gently 10-15 min. Keep in ice box and melt before use. The 5 gms. carbolic acid crystals specified in Kaiser's formula has unfortunately, according to Mallory, a deleterious influence on alum hematoxylin prepara- tions. See also Glychrogel and Lactophenol. Glychrogel, as a mounting medium for teased preparations, Marchi stained sections, gelatin sections, etc. To make 100 cc. dissolve 0.2 gm. chrome alum (potas- sium chromium sulphate) in 30 cc. aq. dest. with aid of heat. Add 3 gm. Knox granulated gelatin in 50 cc. hot aq. dest. Add 20 cc. glycerin with constant stirring and warm. When thoroughly mixed add crystal of camphor (Wotton, R. M. and Zwemer, R. L., Stain Techn., 1935, 10, 21-22). For use in mounting nematodes (Wotton, R. M., Stain Techn., 1937, 12, 145-146). Glycogen, the 3 chief microchemical meth- ods have been critically studied by C. M. Bensley (Stain Techn., 1939, 14, 47-52). This account follows her pre- sentation. Since glycogen is labile, immediate fixation of very small pieces of tissue (2-3 mm.) and agitation of the fixative are necessry. She recom- mends 9 parts absolute ethyl alcohol -f 1 part commercial formalin (i.e. 37% formaldehyde) neutralized with MgCOj. If desired the alcohol in this fixative can be saturated with picric acid. After fixation for say 24 hrs. wash in aboslute alcohol, embed in the usual way in par- affin (carefully avoiding overheating) or in celloidin. 1. Best's carmine. Griibler's car- minum rubrum optimum or some other good carmine 2 gm., potassium carbonate 1 gm., potassium chloride 5 gm., aq. dest. 60 cc. Boil gently until color darkens, cool and add 20 cc. con- centrated ammonia. Allow to ripen 24 hrs. This is stock solution. Mount paraffin sections, bring down to aq. dest. Stain nuclei with hematoxylin as in the H. and E. technique. Transfer to fresh stain (stock solution 10 cc, 15 cc. cone, ammonia and 30 cc. pure methyl alcohol) for 20 min. Rinse in 3 changes methyl alcohol, dehydrate in acetone, clear in toluol and mount in balsam. Glycogen brilliant red. 2. Iodine (Gage). Mount paraffin sections as before, being again careful to avoid unnecessary heat, and bring down to water. Lugol's aq. iodine 10-15 min. Blot with filter paper and dry in air. Mount in yellow vaseline as advised by S. H. Gage (J. Comp. Neur., 1917, 27, 451-465) with minimum of heat. Glycogen reddish brown. 3. Bauer -Feidgen. To make Feulgen reagent dissolve 1 gm. basic fuchsin in 100 cc. aq. dest. by heat. Filter while warm and add when cool 20 cc. normal HCI. Add 1 gm. NaHSOs. Allow to rest 24 hrs., when it should be of pale straw yellow color. Treat deparaffinized sections with 4% chromic acid for 1 hr. or with 1% chromic acid over night. After washing in running water 5 min., place in Feulgen reagent 10-15 min. Rinse IJ min. in each of 3 changes of molecular sol. NaHSOs 1 part and tap water 19 parts. Wash in running water 10 min. Counterstain nuclei with hematoxylin if desired. Dehydrate, clear and mount in balsam. Glycogen deep reddish violet, nuclei lavender. See Polysaccharides. Control. Prepare at same time some sections of liver rich in glycogen. Be- cause glycogen is quickly removed by salivary digestion, when sample sections are brought down to aq. dest., spit on them and allow to rest 15-30 min. chang- ing saliva several times. Wash thor- oughly in water at body temperature to remove mucus and stain by either of the 3 above mentioned techniques. If the material is then absent in such sections and present in other similarly stained and not digested, it is evidently glycogen. Fixation by the freezing and drying method is even better than with the alcohol, picric, formalin mixture because it is quicker and there is less GLYCOGEN 142 GOLD chance for displacement of glycogen in the cells. See also for glycogen staining of Trachoma inclusions Thygeson, P., Am. J. Path., 1938, 14, 455-462. Glycogen is immobilized in its natural position within the cells by the Freezing and Drying technique (Altmann-Gersh). Compare figures 3 and 4 of Bensley and Gersch (R. R. and I., Anat. Rec, 1933, 57, 205-215) showing results by this and other methods. A new ammoniacal silver nitrate method for glycogen is described by Mitchell, A. J., and Wislocki, G. B., Anat. Rec, 1944, 90, 261-266. To pre- pare silver solution dissolve 1 gm. silver nitrate in 10 cc. aq. dest. and add 11 drops 40% aq. potassium hydroxide. Dissolve ppt. by adding 26% ammonia drop by drop and make up with abs. ale. to 100 cc. Allow to stand over night before use. Fix livers of guinea pigs and placentas of same and other animals for 6-12hrs. in sat. picric acid in abs. ale, 90 cc. and neutral formaldehyde, 10 cc. Wash in abs. ale. several times likewise in chloroform and abs. ale. Transfer to chloroform and embed in paraffin. Place sections in 0.25% aq. potassium permanganate, 5-10 min.; rinse in aq. dest. 1-2 min., decolorize in 5% aq. oxalic acid, 5 min. and rinse again in aq. dest. Place in 2% aq. silver nitrate, 12-24 hrs., transfer to ammoniacal sil- ver nitrate, 15-30 min., rinse in 4% neutral formalin, 5-20 sec. and in run- ning water, 1 min. Fix in 5% aq. sodium thiosulphate, 5-10 min. After washing in running water, 1 min., counterstain in paracarmine (Mayer), dehydrate, clear in xylol and mount in balsam. Glycogen, dense black corre- sponds with that shown by Bauer- Feulgen technique. Excellent illus- trations. The recently developed techniques of Gomori (Am. J. Clin. Path., 1946, 16, 177) and Hotchkiss (Arch. Biochem., 1948, 31, 131) can be used on tissue sec- tions or blood smears (Gibb, R. P., and Stowell, R. E., Blood, J. Hematology, 1949, 4, 569-579). The evaluation of methods for the histochemical study of glycogen by Carpenter, A.M., Polon- sky, B. and Mesiten, M. U. (Arch. Path., 1951, 51, 480-485) may be help- ful. The best way to separate out glycogen en masse is by centrifugal isolation as employed by Lazarow, A. (Anat. Rec, 1942, 84, 31-50; Biol. Symposia, 1943, 10, 9-26) for suspensions of fragmented liver cells. Colorimetric methods for glycogen may afford valuable evidence. See Boettiger, E. G. (J. Cell. Comp. Physiol., 1946, 27, 1-8) and Van Wagten- donk, W. J., Simonsen, D. H. and Hack- ett, P. L. (J. Biol. Chem., 1946, 163, 301-306) and the critical discussion by Click, p. 247. See Heatley, N. G. and Lindahl, P. E. (Proc. Roy. Soc, B, 1937, 122, 395-402) for separation of desmo- and lyoglycogen. Glycol Stearate. As an imbedding medium (Cutler, O. L, Arch. Path., 1935, 20, 445-446). Pass up through alcohols to equal parts 95% ale. and glycol stearate in incubator at 56''C. 12-24 hrs. Pure glycol stearate at 56°C. 24 hrs. Imbed as in paraffin. Glucuronidase. An enzyme, widespread in occurrence in the mammalian organism, which hydrolyzes esters of glucuronic acid. Glucuronides are important de- toxification products. Chemical meth- ods for identification of glucuronidase are available (see Fishman, W. H., Chapter 18, The Enzymes New York: Academic Press). 1950 Vol. 1, part 1, pp. 635-652, Friedenwald, J. S. and B. Becker (J. Cell, and Comp. Physiol., 1948, 31, 303-309) have described a method for localizing glucuronidase on tissue sections, and Seligman, A.M., M. M. Nachlas, L. H. Manheimer, O. M. Friedman and G. Wolf (Ann. Surg., 1949, 130, 333-341) describe a method involving the hydrolysis of the beta glucuronide of beta naphthol. The liberated naphthol is converted to a dye by diazotization. Glyoxal. As substitute for formaldehyde in tissue fixation (Wicks, L. F. and Sunt- zeff, v.. Science, 1943, 98, 204; Stowell, R. E. and Stokes, J. M. J. Tech. Meth. and Bull. Internat. Assoc. Med. Mu- seums, 1944, 24, 25-30). Concentra- tions 2-6% produce less shrinkage and give better cytoplasmic preservation than 4% formaldehyde. Glyoxal is only recommended as general substi- tute for formaldehyde when latter is not available. Gmelin's test for bile pigments. On addi- tion of nitric acid containing a little nitrous acid, color changes to green, then red and finally blue observable under microscope. Gold, microchemical detection of: 1. Method of Borchardt. Modified by Michaelis, O., Biochem. Zeit., 1930, 225, 478-488. Treat sections of formalin or alcohol fixed tissues for 15 min. in a boiling water bath or for 12-24 hrs. at 40 °C. with 5% aq. silver nitrate. Remove ppt. from section with 20% aq. nitric acid. Gold appears as black granules (Lison, p. 100). 2. M.ethod of Okkels, H., C. rend. GOLD 143 GOLGI APPARATUS Soc. Biol., 1929, 102, 1089-1091. Simply produce gold salt in sections by exposing for at least 12 hrs. to sunlight or to ultraviolet lamp for same time (Gau- thier-Villars, P., C. rend. Soc. de Biol., 1932, 109, 197-198). Lison (p. 100) explains that whatever the technique used it is necessary to prove that the black granules are gold by their insolu- bility in concentrated acids, solubility in aqua regia (equal parts nitric and hydrochloric acids) and solubility in solutions of potassium or sodium cya- nide. 3. Method of Roberts, W. J., Bull. d'Hist. Appl., 1935, 12, 344-361. Fix tissues in 20% neutral formalin or in Bouin's fluid. Avoid fixatives contain- ing a metal. Wash thoroughly in water. Make paraffin or frozen sections. The latter has the advantage of speed. Make 2 solutions : A. Add 2 gm. silver nitrate pure for analysis to 100 cc. 10% gum arable in the dark immediately before use. B. Add 1 gm. hydroquinone pure to 100 cc. 10% gum arable the day before use. Take off the frozen sections in aq. dest. Mix 2 cc. A and 2 cc. B, add 1-3 drops 5% citric acid, shake 30 sec. Leave sections in this mixture 5-10 min. Then without first washing plunge into 5% aq. sodium hyposulphite for a few minutes. Wash thoroughly and mount. Gold in cells is covered with black deposit of reduced silver. Said to be more sensitive method than spectrographic analysis. See author's illustrations. 4. A technique for demonstration of gold in abs. ale. or neutral formalin fixed tissues, based upon reaction with p - Dimethylaminobenzylidenrhodanin is described by Okamoto, K., Akagi, T. and Mikami, G., Acta. Scholae Med. Univ. Imp. in Kioto, 1939, 22, 373-381. 5. Tin chloride method (Elftman, H. and Alice G. Stain Techn., 1945, 20, 59-62. After rats and guinea pigs are injected intraperitoneally with aqueous yellow gold chloride fix by injection of neutral formalin through heart. Make paraffin sections. Pass down to water in usual way. Place slides in mixture of 10 parts stock 5% aq. SnCl2-2H20 (with some pieces metallic tin added to retard oxidation) and 1 part cone. HCl (mixture prepared and filtered just be- fore use) in incubator at 56°C. for 24 hrs. Wash several changes aq. dest. before dehydrating clearing and mount- ing in damar. Presence of gold indi- cated by particles exhibiting purple of Cassius grading into brown. Colloidal gold in red, blue and black may likewise occur. To eliminate disadvantages of occasional precipitates of tin unrelated to gold and possible confusion with bile pigments and others the following tech- nique is proposed by these authors. 6. Fix in neutral formalin, bring down mounted sections to water. Place in 3% HjOs in incubator at 37°C. for at least 24 hrs. better 3 days. Wash in aq. dest. Run up and mount in damar. Gold thus reduced to metallic form shows range of colors, rose chiefly grad- ing into purple, blue and black. 7. Christeller, E., Verh. deutsch. Path. Ges., 1927, 22, 173 reports, as de- scribed by Gomori, G., J. Mt. Sinai Hosp., 1944-45, 11, 317-326, demonstra- tion of gold salts by reduction to metal- lic gold with SnCh. Similar to No. 5. 8. For micro-determination of gold in biological fluids and tissues, see Block, W. D., Ann. Rheumatic Dis., 1944-45, 4, 39-42. Use of this tech- nique provides a good check on above described microchemical methods. Radioactive gold, distribution of within cartilage of knee-joint, Ekholm, R., Acta Anat., 1951, Supp. 15 to II, 75 pp. Gold Chloride for nerve endings, see Craven's and Carey's methods. Gold Orange, see Orange IL Gold Orange MP, see Methyl Orange. Gold Particles. The particles of gold are held in colloidal state by the protective colloid, sodium lysalbinate, and are employed to stimulate macrophage pro- duction by intravenous injections in animals (Simpson, M. J., J. Med. Res., 1922,43,77-144). Goldman, see Iron Hematoxylin Single Stain. Golgi Apparatus — Written by Geoffrey Bourne, London Hospital Medical Col- lege, London, England. November 5, 1951 — -Most recent books which give details of techniques for demonstration of the Golgi apparatus preface their descriptions with the statement that, as some doubt exists as to the nature of the apparatus, it is difficult to describe techniques for demonstrating it. This doubt is still unresolved and various authors hold widely divergent views as to the structure of the apparatus. These may be summarized as follows: Gatenby (IVIicrotomist's Vade Me- cum, nth Ed. 1950) believes that the original conception of the Golgi appara- tus in vertebrate cells as a juxta-nuclear argentophil network is still correct. Baker's view (Quart. J. Micr. Sci. 1944, 85, 1-71 — a modification of that expressed by Parat, M. Arch. d'Anat. Micr., 1928, 24, 73) is that the apparatus is composed of a series of neutral red staining vacuoles more or less sur- GOLGI APPARATUS 144 GOLGI APPARATUS rounded by a zone of dense phospholipid material and in some cases embedded in a zone of diffuse phospholipid. Palade and Claude (J. Morph. 1949, 85, 71) claim that the Golgi apparatus is composed solely of neutral red staining vacuoles which possess a membrane con- taining an appreciable proportion of lecithin and that the classical Golgi apparatus is formed by myelin figures produced by the action of fixatives on these vesicles. Bensley (Exp. Cell. Res. 1951, 2, 1-19) contends that the Golgi apparatus is composed of vesicles or canals containing a watery solution in which various substances, e.g., polysaccharides are dissolved (Gersh, I., Arch. Path. 1949, 47, 99-109). The canalicular conception of the Golgi ap- paratus is also claimed by Gatenby and Moussa for the mammalian neurone. Against these views we must put the fact that no canals have been seen in cells by numerous workers using the phase contrast microscope and as Hibbard, H. (Quart. Rev. Biol. 1945, 20, 1-19) has pointed out Lewis has never seen anything resembling the classical apparatus in a life-time of study of tissue culture cells. Most workers on the Golgi apparatus have tended to range themselves behind one or other of these views. It should be realized, however, that all these points of view about the structure of the ap- paratus simply continue the con- troversy which has gone on almost since its discovery. The canalicular concep- tion of the apparatus originated in 1902 with Holmgren's Trophospongium, the neutral red vacuole theory found its origin in Parat's 1928 work and has seen some extension in Cowdry's labora- tory by the work of Cowdry, E. V. and Scott, G. H. (Arch. Inst. Pasteur de Tunis, 1928, 233), Covell, W. P. and Scott, G. H. (Anat. Rec, 1928, 38, 377), and of course the argentophil network theory originated with Golgi himself in 1898 (Arch. Ital. de Biol., 30, 60) and in more recent years has been supported bj'^ Ludford and particularly by Gatenby. So, it is apparent that our knowledge of the nature of the Golgi apparatus has remained more or less in the same state as it was 30 or 40 years ago. In view of this discrepancy of views techniques wall be given for demon- strating the classical Golgi net and also for demonstrating the apparatus as discrete bodies. The Classical Golgi Apparatus: Even if the Golgi network is an artefact as some workers believe the conventional Golgi preparation does give us informa- tion about its position in the cell and whether the Golgi material is present in small or in large amount. Golgi's original technique involved the fixation of tissue in a mixture con- taining potassium bichromate and osmic acid followed by impregnation with silver. The apparatus with this technique appears jet black against a yellowish background. It is a con- spicuous structure consisting of an intricate network of anastomosing strands. This network may closely en- velop the nucleus, be concentrated to one side of it or else be scattered rather diffusely throughout the cytoplasm. In glandular cells the apparatus grows in size with the development of secre- tory granules and strands from it ramify between the various granules. Kopsch, F. (Sitzungsber. K. Akad. Wissensch, 1902, 40, 929) showed that the Golgi apparatus can be blackened by prolonged treatment with 2% osmic acid. On this affinity for both silver and osmium all the conventional meth- ods of demonstrating the Golgi appara- tus are based. Few cytological re- actions are more fickle and incon- stant; but when, after many attempts, the technique is successful, convincing and very beautiful preparations result. Mitochondria can be stained supra- vitally by some vital dyes but no vital dyes will show up the reticular Golgi apparatus, a fact which is taken by some authors to indicate the non-exist- ence in vivo of such networks. How- ever, neutral red will stain spheres in the region of the apparatus. With both silver and osmium methods considerable experimentation is neces- sary in order to obtain the best results. The factors to be varied are principally the composition of the fixative and im- pregnating substance and the time during which they are allowed to act. During impregnation it is always ad- visable to keep the tissues in the dark and instructions as to temperature re- quirements should be carefully fol- lowed. When either the silver or osmium solution becomes blackened it should be renewed. It is important for the beginner to start with the most favorable material. The spinal ganglion cells of young mammals such as the rabbit are perhaps the best for this purpose. The acinous cells of the pancreas are also recommended but are somewhat more difficult to handle. All the methods of impregnation outlined below frequently bring to light the mitochondria also. GOLGI APPARATUS 145 GOLGI APPARATUS Osmic Acid Technique: 1. Mann-Kopsch method (Lee's Mi- crotomists Vade Mecum, Uth Ed. 1950, edited by J. Bronte Gatenby and H. W. Beams). Fix in Mann's fluid (equal parts of 1% osmic acid and sat. sublimate in normal saline), for J-3 hrs. Wash in aq. dest. 15-30 min. 2% osmic acid room temperature 10-14 days. Wash in ruiming water 3 hrs. or more. Dehydrate clear and embed. In the sections Gatenby was able to extract the blackening step "by step with turpentine and thus to improve considerably the preparations. 2. Liidford's modification (Ludford, R. J., J. Roy. Micr. Soc, 1926, 107). Fix mammalian and avian tissues in Mann's corrosive osmic solution 18 hrs. Wash aq. dest. 30 min., 2% osmic at 30°C. for 3 days. Water at 30°C. for 1 day, dehydrate, clear, embed in paraffin. A useful variant is to fix in the same way, and wash in aq. dest. Then osmicate at 35°C. for 3 daj'S, first day in 2% osmic, second in 1% and third in 0.5%. Leave in water for 1 day at 35°C. He recommends various counter stains. See Lee's 11th Ed. (pp. 404- 410) for a discussion of osmication methods; also Owens, H. B. and Bensley, R. R. (Anat. Rec, 1929, 44, 79) for a careful study of factors in- fluencing the osmic acid changes and for their ferric chloride osmic method. 3. Sjovall's method (Sjovall, E., Anat. Hefte, 1906, 30, 261-291). Fix in 10% formalin 8 hrs. Wash in aq. dest. 2% osmic acid at 35°C., 2 days. Dehy- drate, clear and embed. 4. Kolatchew's method (Nassonov, D. N., Arch. f. Mikr. Anat., 1924, 103, 437). Fix in 3% aq. potassium bi- chromate, 10 cc; 1% chromic acid 10 cc; and 2% osmic acid, 5 cc. for 24 hrs. Wash in running water 24 hrs. 2% osmic acid 40°C. 8 hrs. 3-5 days at 35°C. Wash in aq. dest., dehydrate, clear and embed. Osmic methods sometimes im- pregnate mitochondria as well as the Golgi material particularly if the period in osmic acid is prolonged, care must be taken therefore in interpreting results. Silver Nitrate Methods: Tissues from young animals usually respond best to silver methods but ganglia from older animals respond very well. 1. Aoyaina's method (Baker, J. R., Cytological Technique, 3rd Ed., 1950, p. 194). Fix tissues in Aoyama's fixa- tive (cadmium chloride 1 gm., neutral formalin 4%, 15 cc, aq. dest. 85 cc.) for 4 hrs. Wash with aq. dest. and then place in silver nitrate solution for 12-14 hrs. Wash with aq. dest. and then place in silver nitrate solution for 12-14 hrs. After 8-12 hrs. (kept in dark or in diffuse light). Wash with aq. dest. and place in Aoyama's reducer (Hj'dro- quinone 1 gm.. Neutral Formaldehyde 40% 15 cc, aq. dest. 80 cc, anhydrous sodium sulphite 0.15 gm.) for 5 hrs. Leave for I hr. in running water and then place in 50% alcohol overnight. Dehydrate and embed. 2. Cajal's method (Carleton, H. M., J. Roy. Micr. Soc, 1919, 321-329). This is one of the many methods devised by Cajal. It is recommended for embryos and young animals. Fix in uranium nitrate, 1 gm., formalin 15 cc, and aq. dest. 100 cc, 8-24 hrs. Wash quickly in aq. dest. 1.5% aq. silver nitrate 24^8 hrs. Rinse in aq. dest., hydroquinone 2 gm., formalin 6 cc, aq. dest. 100 cc, anhydrous sodium sulphite 0.15 gm. 12 hrs. Wash in aq. dest., dehydrate quickly, clear, embed and section. 3. Da Fano's method (Da Fano, C., J. Roy. Micr. Soc, 1920, 157-161). Here the uranium nitrate is replaced by co- balt nitrate. In other respects the technique is similar. Da Fano has, however, so carefully attempted to con- trol troublesome experimental condi- tions that the various steps are given in detail. Fix in cobalt nitrate 1 gm., aq. dest. 100 cc, formalin 15 cc 6-8 hrs. The formalin need not be neutralized unless it is strongly acid. For embryos and delicate tissues where there is dan- ger of shrinkage reduce formalin to 6 cc. Cartilage and small pieces of tissue (not more than 3 mm.) fix half fixation time. Hollow organs (e.g. stomach and intestine) fix 1 hr. then cut into smaller pieces. Spinal cord, cerebellum and cerebrum of adults fix 8-10 hrs. (fixation should never go beyond 24 hrs.). Testicle, inject fixa- tive through aorta then immerse testicle in fixative. Wash quickly in aq. dest. and impregnate in 15% aq. AgNOs 24-48 hrs. Very small fragments im- pregnate in 1% AgNOs, 2% for tissues containing much fat and for spinal cord. Impregnation normally satis- factory at room temperature. If un- satisfactory at 35°-37°C. Wash rapidly in aq. dest. and cut down tissues again to a thickness of 2 mm. or less. Reduce in Cajal's hydroquinone mixture, above mentioned, 12-24 hrs. Wash in aq. dest. i hr. Cut with freezing micro- tome or embed in wax. Golgi ap- paratus dark brown or black against a yellow background. Tone sections with gold to clear preparation. Pass to water. Then 0.1-0.2% gold chloride 2 GOLGI APPARATUS 146 GOLGI METHOD, QUICK hrs. Counterstain, dehydrate, clear and mount. It is of interest that all silver prepara- tions depend upon the treatment of the tissue by fixatives which contain the salt of a heavy metal. Silver, M. L. (Anat. Rec, 1942, 82, 507-529) has pointed out that silver micelles are not adsorbed on to the Golgi material un- less the cells have been treated with salts of heavy metals — hence the pres- ence of metals like cobalt and ura- nium in the fixatives used for silver techniques. Stains: 1. Baker's Sudan Black Method (Baker, J. R., Quart. J. Micr. Sci., 1944, 85, 1-71). Fix small pieces of tissue in formol calcium (formalin 40%, 10 cc, calcium chloride, anhydrous 10% aq. 10 cc, aq. dest. 80 cc.) for 3 days, embed in gelatine. Harden block in formalin-cal- cium-cadmium (formalin 10 cc, calcium chloride, anhydrous 10% aq. 10 cc, cadmium chloride, 10% aq. 10 cc, aq. dest. 70 cc.) and then wash 3-4 hrs. in running water. Section on freezing microtome at 15m. Sections when fixed to the slide are placed in filtered for- malin-calcium-cadmium solution until ready for staining. Then wash slides for 3 min. in running water, pass through 50% and 70% ale to a sat. solu- tion of Sudan black in 70% ale, leave for 7 min. Pass through 3 lots of 50% ale, rinse in aq. dest., counterstain, mount in glycerine jelly or Apathy's medium. This method does not demonstrate the Golgi apparatus as a network but as a series of discrete bodies, a form which is claimed by some authors to represent more nearly the true form of the Golgi apparatus in living cells. Many of the vesicles which are demonstrated by this method also stain with neutral red. 2. Baker's Neutral Red Method (Baker, J. R., Quart. J. Micr. Sci., 1944, 85, 1-71). This is a vital method. Cells are separated by teasing in a salt mix- ture (sodium Chloride, 0.7% aq. 100 cc, calcium chloride, anhydrous, 10% aq., 0.2 cc). To 3 drops of solution containing a suspension of cells add 3 drops of a solution made up of Neutral red 0.1% aq., 1 cc. and sodium and calcium chloride solution (as above) 9 cc. Final concentration of dye is 0.005%. Mix cell suspension with dj^e by sucking up with pipette and pass out again. Leave preparation (covered) for 20 min. Put 2 drops of mixture on the glass of a compressorium in such a way as to include some air. Cover edge of compressorium with soft paraffin (e.g. Vaseline). Examine under im- mersion lens. Golgi Cox Method for adult nervous system. — Written by J. L. O'Leary, Dept. of Neuropsychiatry, Washington Uni- versity School of Medicine, St. Louis 10, Mo. May 8, 1950.— Fix pieces 3-6 mm. thick in following fluid : add 20 cc 5% aq. potassium bichromate to 20 cc 6% aq. mercuric chloride. Dilute 16 cc. 5% aq. potassium chromate with 40 cc. aq. dest. and add this to the first two. Do not agitate but leave in fixative until scum forms on surface, usually after 1^-2 months. When im- pregnation is nearly complete, wash rapidly, dehydrate through graded alco- hols and imbed in low viscosity celloidin (see Celloidin Imbedding). Cut cel- loidin sections serially at 80 to 120 microns. Arrange in serial order on slides (80% alcohol). Blot sections dry and cover immediately with 1% celloidin. When somewhat dry, bring slides with sections to water. The sections on each slide may thereafter be treated as a unit. Run sections from water into a saturated solution of sodium sulfite. They rapidly turn a yellow gray. Wash over night and de- hydrate through graded alcohols to ab- solute. Coat with the following var- nish, applying it repeatedly in thin even layers, and allowing each to dry par- tially before applying the next (san- darac, 75 gm.; camphor, 15 gm.; turpentine C.P., 30 cc; oil of lavender, 22.5 cc; abs. ale, 75 cc; add castor oil, 7 drops. Mixture dissolves very slowly). Since sections are somewhat opaque, the varnish must dry for several days until abs. ale has evaporated. Golgi Methods. Fundamentally these are different from both the Cajal and Biel- chowsky techniques which were later developments. They depend upon a preliminary fixation in a potassium bichromate solution often containing formalin and sometimes other sub- stances such as osmic acid. The silver is selective tending to impregnate a few cells completely which become black- ened when it is reduced. Except for the occasional demonstration of the Golgi Apparatus these methods do not reveal details of the inner structure of nerve cells like Neurofibrils and Nissl Bodies. They are of great service in the demon- stration of many non-nervous tissue components, the parietal cells of the stomach, bile canaliculi of the liver, Rouget or perivascular cells, etc. Golgi Method, Quick. For brains of new- born animals, and of those 1 day to 30 days old. — Written by J. L. O'Leary, Dept. of Neuropsychiatry, Washing- ton University School of Medicine, St. Louis 10, Mo. May 8, 1950.— It is es- GOLGI, METHOD 147 GOMORI'S METHODS sential to determine the age of the animal at which the cell or fiber selected for study is reaching maturity. For example, if new born kittens are chosen, and the area striata is the object of study, the best impregnations of enter- ing fibers are obtained at 12 to 15 days after birth ; of short axon cells, at 18 to 21 days; and of pyramids at 21 to 24 days. Cut slices of brains 3-4 mm. in thick- ness by quick cuts of a sharp scissors. Fix in: potassium bichromate, 10 gm.; osmic acid, 1 gm.; aq. dest., 330 cc. Time of fixation must be determined for each part of the CNS studied. In general the older the animal, the longer it is. After fixation, blot blocks of tissue on filter paper and transfer to a bottle containing f% aq. silver nitrate. After 24 hrs. the reaction is complete. Imbed in celloidin. Subsequent treat- ment is very important. Place block in 95% ale. for about 5 min., remove and blot dry. Place block on paraffin disc mounted on a block holder in the orienta- tion desired for cutting. Using a hot teasing needle, melt paraffin around the block so as to fasten block to paraffin. Be sure that melted paraffin does not creep up on the block. Use knife at 45° angle to the block. Cut serially 80-100ai. Place each section as cut in order in 95% ale. using Petri dishes. Be sure not to miss first and last section of the block for these are often more valuable than the entire remainder of the block. Using a spatula, transfer to another 95% ale. after 5 min. After another 5 min. transfer to oil of cloves, arranging in serial order, by placing each section as it enters oil of cloves near the edge of the Petri dish so that it adheres to the edge. When all sections are transferred the group will be placed around the circumference of the Petri dish. As the sections start to retract from the edge, begin to arrange them in the usual order for serial sections. After clearing (clove oil 5 to 10 min.) transfer in serial order to slides. Blot off excess of clove oil and apply xylol, blot off xylol similarly and apply a thin layer of Damar, using the drop method. Let the slide dry on an even surface adding more Damar as necessary to keep sections protected. Golgi, Method (Porter, R. W. and Daven- port, H. A., Anat. Rec, 1949, 103, 583). Radical departure from prior methods because silver impregnation precedes the potassium bichromate solution. Fix 48 hours at 25°C. in 90 cc. 0.5 aq. AgNOj, 10 cc. formalin, 0.5 cc. Pyridine. Mix in order given, disregarding slight turbidity. Colorimetric test of fixing solution with bromcresol purple should show 5.5 to 6.0. Fixation can be done by perfusion, or only by immersion. Cut slices to thickness of 0.5 to 1.0 cm. After fixation rinse blocks with dis- tilled water and place in 2.5% (aq) po- tassium bichromate to which 1 cc. of 1% osmic acid is added for each 100 cc. Leave there 3 to 5 days. Dehydrate quickly through alcohols and xylol to soft paraffin. Sections should be cut 50 to 100 microns thick. Gomori's Methods For Reticulum and Acid Phosphatase. 1. Silver impregnation of reticulum (Gomori, G., Am. J. Path., 1947, 13, 993-1001). Treat deparaffinized sec- tions of formalin fixed material with 0.5-1% aq. potassium permanganate. 1-2 min. Rinse in tap water and decolorize in 1-3% aq. potassium meta- bisulphite, 1 min. Wash for several minutes in running tap water. 2% aq. iron ammonium sulphate (violet crystals), 1 min. Wash in tap water few minutes and then pass through 2 changes aq. dest. Impregnate for 1 min. in following solution: To 10% aq. silver nitrate add g to J of its volume of 10% aq. potassium hydroxide. While shaking add strong ammonia drop by drop until ppt. is completely dissolved. Add carefully silver solution drop by drop as long as resulting ppt. easily disappears on shaking. Finally add equal vol. aq. dest. Can be kept 2 days in stoppered bottle. Rinse in aq. dest., 5-10 sec. Reduce in commercial forma- lin diluted 5-10 times with tap water. Wash under tap few min. Tone in 0.1-0.2% aq. gold chloride, 10 min. 1-3% aq. potassium metasulphite for 1 min. Fix in 1-2% aq. sodium thio- sulphate (hyposulphite) for 1 min. Wash under tap, dehydrate, clear and mount. Reticulum black. Note author's figures of sarcomata (Revisde by G. Gomori May 7, 1946). 2. For Acid phosphatase — Written by G. Gomori (University of Chicago. May 7, 1950— see Stain Techn., 1950, 25, 81. 1. Fix thin slices of tissues in ice cold acetone for 24 hours. 2. Change acetone at room tempera- ture twice for the next 24 hours. 3. Two changes of benzene, 45 min. each. 4. Embed in paraffin (not abo\'e 56°C. and preferably below), 2 changes, 30 to 45 min. each. 5. Cut sections. Float them on luke- warm (30°C.) water. 6. Carry sections through xylene and 2 alcohols to dist. water. 7. Incubate in the following solutions GOMORI'S METHODS 148 GORDON'S SILVER METHOD for U to 24 hours at 37°C.: Molar acetate buffer pH 5* 2.5 ml. 5% lead nitrate 1.5 ml. Dist. water 40.0 ml. 2% Na glycerophosphatet 7.5 ml. • 100 cc. of 13.6% CHiCOONa-3HiO plus 60 cc. 6% acetic acid, t Commercial grade (mixture of alpha and beta salts) • Shake well, heat to ±60°C. for about 10 min.; filter. 8. Rinse sections thoroughly first in dist. water and afterwards in 2 to 3% acetic acid, followed again by dist. water. 9. Immerse sections in a solution of yellow ammonium sulfide (1-12 drops to a Coplin jar) for 1 minute. 10. Wash. Counterstain as desired. For Alkaline phosphatase: 1. Fix thin slices of tissues in 80% al- cohol (or absolute acetone). Dehy- drate in 95% and absolute alcohol (or 2 changes of absolute acetone), embed through benzene or xylene in paraffin. Cut sections around 6 micra thick. 2. Run slide through xylene and 2 al- cohols to distilled water. Incubate for 1 to 2 hr. at 37°C. in the following mix- ture: 2% sodium glycerophosphate. 25 cc. 2% sodium barbital 25 cc. Distilled water 50 cc. 2% calcium chloride 5 cc. 2% magnesium sulfate 2 cc. Chloroform a few drops This solution will keep in the ice box for months. 3. Rinse slide thoroughly in repeated changes of distilled water. 4. Immerse slide for 3 minutes in a 1 to 2% solution of some cobalt salt (chloride, acetate, sulfate). 5. Wash thoroughly under the tap. 6. Immerse slide for 2 minutes in a dilute solution of yellow ammonium sul- fide (1-12 drops to a Coplin jarful of distilled water). Wash under the tap. 7. Counterstain as desired; dehy- drate, clear and mount. Attention is called to the earlier demonstration of phosphatase in bone by Robison (R., Biochem. J., 1923, 17, 286-293) and to recent discussion by Blaschko and Jacobson (Bourne, pp. 217-221). The distribution of phos- phatase in some normal tissues is indi- cated in colors by ICabat, E. A. and Furth, J., Am.J. Path., 1941, 17, 303-318. For phosphatase in elementary bodies of vaccinia virus, see Macfarlane, M. 0., and Salaman, M. H., Brit. J. Exp. Path., 1938, 19, 184; Hoagland, C. L. et al., J. Exp. Med., 1942, 76. 163-173. See Kidney. Gonococcus, methyl green-pyronin stain. To 10 cc. absolute methyl alcohol add 1 gm. methyl green (dye content_60%) and 0.2 gm. pyronin (bluish certified). Add 100 cc. 2% aq. phenol and shake 2 hrs. per day for 2 days in a mechanical shaker. Filter and add 20 cc. glycerin, C.P. to filtrate. Fix smears by passing slides lengthwise through flame 4 or 5 times. Add stain immediately and warm to slight steaming. Wash off stain 20-50 sec. Dry and examine. Gonococci, deep red ; other bacteria except these of Neisseria group pale purplish or barely noticeable; nuclei of pus cells green in soft pink or rose cytoplasm (Walton, S. T., J. Lab. & Clin. Med., 1938-39, 24, 1308-1309). Goodpasture's Method as modified by Mac- Callum for bacteria in sections (McClung, p. 152). Fix in Zenker's fluid or in formalin Zenker. Stain thin paraffin sections 10-30 min. in: 30% ale, 100 cc; basic fuchsin, 0.59 gr.; anilin oil, 1 cc; phenol crystals, 1 gm. Wash in water. Differentiate in forma- lin (37% solution of formaldehyde) few seconds until bright red color changes to rose. Wash in water. Counterstain in sat. aq. picric acid 3-5 min. until sections become purplish yellow. Wash again in water. Differentiate in 95% ale. until red reappears and some of it as well as of the yellow is washed out. Wash in water. Stain in Stirling's gentian violet (gentian violet, 5 gms.; 95% ale, 10 cc; aniline oil, 2 cc; aq. dest., 88 cc.) 5 min. or more. Wash in water. Gram's iodine solution (iodine, 1 gm.; potassium iodide, 2 gms.; aq. dest., 300 cc.) 1 min. Blot dry. Clear in equal parts aniline oil and xylol until no color is removed. Clear in 2 changes xylol and mount in balsam. Gram- negative bacteria, red; gram-positive ones, blue; tissue red and blue; fibrin deep blue. See his Polychrome Methyl- ene Blue and Carbol-Anilin Fuchsin Methylene Blue. Gopal-Ayengar, see Chromosomes, Hyal- uronic Acid. Gordiacea, see Parasites. Gordon's SilverMethod. For blood smears, also shows parasites, Gordon, H., J. Lab. & Clin. Med., 1936-37, 22, 294- 298. Dry smears of blood or bone marrow in air and fix in 10% formalin. Wash in water. 2.5% aq. iron alum 10 min. or more. 4 changes aq. dest. Dip in 1% aq. gelatin -f 1 or 2 drops 2% sodium carbonate and drain. Wash quickly in aq. dest. Impregnate 5-15 min. in silver solution (Add strong ammonia drop by drop to 5 cc. of 10.2% GOSSYPIMINE 149 GRAM'S STAINS aq. silver nitrate until ppt. is dissolved. Add 5 cc. 3.1% aq. soaium hydroxide and redissolve ppt. with strong ammonia. With aq. dest. dilute to 100 cc). Wash in aq. dest. at 60 °C. Re- duce in: 10% formalin 90 cc. + 2.5% iron alum 10 cc. Wash in tap water, dehydrate in alcohol, clear in xylol and mount in balsam. Gossypimine, see Safranin O, Grafts. Intracoelomic of eye primordium, Joy, E. A., Anat. Rec, 1939, 74, 461- 486. See Transplantation. Gram, see Weight measurements. Gram's Iodine Solution. Iodine, 1 gm.; potassium iodide, 2 gm.; aq. dest., 300 cc. A stronger solution may be desirable with only 100 cc. aq. dest. Gram-Pappenheim stain as modified for smears and paraffin sections (Scudder, S. A., Stain Techn., 1944, 19, 39-44). Gram's Stains for bacteria: 1. In smears. Hucker modification (McClung, p. 138). Stain 1 min. in equal parts A and B : A = crystal violet (85% dye content, 4 gm.; 95% ale, 20 cc.) B = ammonium oxalate, 0.8 gm.; and aq. dest. 80 cc. After washing in water immerse in: iodine, 1 gm. potas- sium iodide, 2 gm., aq. dest., 300 cc. 1 min. Then wash in water and dry by blotting. Decolorize 30 sec. in 95% ale. gently moving. Blot and counter- stain in: 10 cc. sat. safranin in 95% ale. and aq. dest. 100 cc. Wash and dry. Kopeloff-Beerman Modification (Mc- Clung, p. 139). Stain 5 min. or more in: 1% aq. gentian or crystal violet, 1.5 cc. mixed before use with 0.4 cc. 5% aq. sodium bicarbonate. Rinse in iodine solution made by dissolving 2 gm. iodine in 10 cc. normal sol. sodium hydroxide and adding 90 cc. aq. dest. and stand 2 min. or more. Blot dry. Add 100% acetone drop by drop with specimen tilted till no more color is removed, less than 10 sec. Dry in air. 0.1% aq. basic fuchsin, 10-30 sec. Wash in water and dry. Weiss Modifica- tion (Weiss, E., J. I^ab. & Clin. Med., 1940-41, 26, 1518-1519). Make thin, uniform smears and fix over flame. Cover slide with 3% gentian violet in 20% alc^ 3-5 min. Wash in warm water. Cover 3-5 min. with iodine, 20 gm. ; potassium iodide, 40 gm., aq. dest. 300 cc. Wash with warm water. De- colorize in acetone and wash imme- diately in water. Counterstain quickly in 2% basic fuchsin in 95% ale. Wash in water, drj-^ and examine. The use of colloidal iodine has been suggested to improve the reaction be- tween bacteria and stain (Lyons, D. C, J. Lab. & Clin. Med., 1936^37, 22, 523-524). Methods for preparing col- loidal iodine are described by Chandler and Miller (W. L. and E. J., J. Phys. Chem., 1927, 31, 1091-1096). 2. In sections. Grarn-Weigert method (McClung, p. 152). Fix in Zenker's fluid. Stain paraffin sections lightly in alum hemato.xylin and wash in running water. 1% aq. eosin, 1-5 min., followed by washing in water. Stain ^-1 hr. in anilin methyl violet made by mixing 1 part of A with 9 of B : A. abs. ale. 33 cc. ; aniline oil, 9 cc. ; methyl violet in excess. B. Saturated aq. methyl violet and wash in water. Lugol's iodine 1-2 min. and wash in water. Blot; dehydrate and clear in equal parts aniline oil and xylol several changes. Wash with xylol and mount in balsam. Glynn's method. (Glynn, J. H., Arch. Path., 1935, 20, 896-899). To make stain triturate 1 gm. crystal violet and 1 gm. phenol crystals in mortar and add 10 cc. absolute alcohol. Before using dilute 10 times with aq. dest., allow to stand 2 days and filter. Stain deparaffinized sections of Zenker (less acetic), Bouin, Helly or 10% formalin fixed material for 2 min. Drain off but do not wash. Add Gram's iodine, 1 min. Differentiate in acetone until no more color is given off, 10-15 sec. Wash in aq. dest. Counterstain in 0.05% basic fuchsin in N/500 hydrochloric acid (see Normal Solutions). Drain, do not wash, apply 1% aq. trinitrophenol, j-l min. Wash in aq. dest. Dehydrate and differen- tiate in acetone 10-15 sec, clear in xylol and mount in balsam. Gram -f bac- teria, violet; Gram—, red; nuclei, light red; cytoplasm, yellow. 3. For organisms in frozen sections by Krajian, A. A., Arch. Path., 1941, 32, 825-827. Stain 7-10m frozen sec- tions for 2 min. in Harris' alum hema- toxylin. Wash in tap water till blue and destain quickly by dipping 5 to 7 times in acid alcohol. Rinse in tap water and apply following solution for 3 min. — copper sulfate, 7 gm.; zinc sul- fate, 4 gm. dissolved in 100 cc. aq. with aid of heat. Pour off and apply 0.3 gm. brilliant green in 10 cc. above copper zinc mixture for 5 min. Rinse in water and fortify with 5% aq. ammonium ni- trate for 1 min. Rinse in tap water and stain with carbol fuchsin (Ziehl-Neel- sen) for 2 min. Rinse in tap water, blot and apply dioxane for 2 min. Pour off and add equal parts creosote and .xylol, changing tliis mixture and agitating to promote even differentiation until back- ground appears clear red. Clear in pure xylol (2 min.) and mount in damar. Gram positive organisms bluish green; gram negative ones red. The Gram staining technique and GRAM STAINS MECHANISM 150 GRAM STAINS MECHANISM the interpretation of the findings has been concisely presented by Dubos, R. J., The Bacterial Cell. Harvard Univ. Press, 1945, 460 pp. The Gram + bac- teria differ from the Gram — ones in being more acidic and perhaps in pos- session of lipids with higher content of unsaturated acids. Their Gram posi- tiveness depends on intactness of their cell walls, for erosions of the walls make them Gram negative. When the outer layer of the cell walls is removed by extraction with bile salts they become Gram negative. The Gram positive property can be restored by "replating" the bacteria with the extract of the outer layer. The outer layer appar- ently contains a protein ribonucleate complex, for Gram positive organisms can be made Gram negative by action of the enzyme, ribonuclease. The quality of the cell membrane conditions not only the entry and retention of stains but the whole manner of life of the cells. See Cell Membrane, Acid Fast Bacilli, and Dead Cells. Gram Stains Mechanism — Written by James W. Bartholomew, Dept. of Bacteriology, University of Southern California, Los Angeles 7, Calif. July 9, 1951 — The literature concerning the mechanism of the Gram stain is voluminous and makes very interesting reading although the exact mechanism is still unknown. The laboratory tech- nician need not be too concerned with the mechanism. It is important for the technician to realize that the tech- nique involved is differential due to relative rather than absolute differences in the organisms studied. Thus, re- gardless of whether one uses the Hucker, Kopeloff-Beerman, Weiss, or other modification of the Gram pro- cedure, the dependability of the results obtained are greatly influenced by the experience and skill of the operator. One method should be adopted, and practiced, until proficiency and self assurance are obtained. Gram differentiation is mostly due to one fact. Gram-positive cells resist decoloration after treatment with dye and iodine to a much greater extent then Gram-negative cells. The cause of this resistance has been attributed to 1) cell membrane permeability char- acteristics, 2) the presence of certain unique chemical compounds, 3) a low isoelectric point of the cell protein, and 4) the presence of a morphological struc- ture, the Gram-positive cortex. Each of these theories, by itself, fails to ex- plain certain experimental data. It is certain that the mechanism of the Gram stain is very complex and several of the above factors probably combine to give the differentiation effect. Cell mem- brane permeability is hard to ignore since it is well known that rupturing the cell wall immediately results in a Gram-positive cell staining Gram-nega- tively. The importance of a chemical factor has been well established through the easily demostrable dependence of the Gram-positive state on the pres- ence of magnesium ribonucleate in the cell. The isoelectric point theory ap- Elies to the general staining phenomena, ut on close inspection looses its sig- nificance as a prime factor in the mecha- nism of the Gram stain. The morpho- logical concepts must await further development in our knowledge of bac- terial cell morphology, and better cell sectioning methods, before the con- tribution of morphological factors to the Gram staining mechanism can be determined. Currently, an appealing explanation based on published literature could be written as follows. Cell membrane permeability to iodine in alcoholic solu- tion is of first importance. Magnesium ribonucleate and possibly other com- pounds such as lipoproteins are im- portant in that they influence the cell membrane permeability. Gram-posi- tive cells are less permeable to iodine in alcoholic solution than are Gram- negative cells. On performing the Gram stain a reaction between the dye and iodine, and also possibly the cell protein, takes place within the cell. The alcohol, which is added next, enters the cells and dissociates the dj'^e-iodine precipitate, or dye-iodine-cell protein complex, which has been formed. Since the alcoholic iodine permeates fastest through the cell membrane of the Gram- negative cells the precipitate or complex are dissociated more rapidly in them than in the Gram-positive cells. Hence, the Gram-negative cells are the first to declorize. The counter stain also has a certain decolorization action and it must be employed if a true Gram stain differentiation is desired. While this concept of the Gram stain mechanism has not been proven, it has the happy quality of combining chemi- cal concepts with cell membrane perme- ability and it does not rule out possible morphological factors. In the case of the often observed Gram-positive bodies within the cell, a more strictly chemical concept would have to be used to explain them. As is often the case, the segregation of cells into two distinct groups, the Gram-negative and the Gram-positive, does not coincide with the state actually GRAM-TWORT STAIN 151 HAIRS existing in nature. There are many intermediates between the two, and this fact should be kept in mind by the technician. Attempts to create termi- nology for intermediate groups have not been well received and the classifi- cation as it now stands is extremely useful and comparitively simple. Fur- ther information is supplied by Bartholomew, J. W. and Umbreit, W. W., J. Bact., 1944, 48, 567; Bartholo- mew, J. W. and Mittwer, T., Stain Tech., 1949, 25, 103-110; Dubos, R. J., The Bacterial Cell. Cambridge: Har- vard Univ. Press, 1946, pp. 72-85; Knaysi, G., Elements of Bacterial Cytology. Ithaca: Comstock Pub. Co., 1951, pp. 267-278; Mittwer, T., Bar- tholomew, J. W., and Kallman, B. J., Stain Tech., 1950, 25, 169-179. Gram-Twort Stain. For study of Gram- positive and Gram-negative bacteria in sections (Ollett, W. S., J. Path. & Bact., 1947, 59, 357). Twort originally used a neutral red-light green mixture. Ollett, W. S., J. Path. & Bact., 1951, 63, 166 supplies further details. The stain advised is approximately as fol- lows depending on dye content of the samples employed. For stock solution 0.2% ale. neutral red, (CI no. 825 9 ml. + 0.2% ale. fast green FCF 10 ml. For use dilute 1 vol. stock solution with 3 vols. aq. dest. Employ as counter- stain in Gram's method as described (Ollett, 1947) and mount sections in L. P. M. which is Lustron (L 2020), apparently the same as Distreme 80 crystal clear 100 gm. dissolved in Di- butyl phthalate 50 ml. + monochlor- benzene 300 ml. Gray, R, B, BB, see Nigrosin, water soluble. Green PL, see Naphthol Green B, Greene, see Anterior Chamber Transplanta- tion. Gregarines. Technique given by McCIung, Microscopical Technique, 1950 p. 455. Fix in picric acid mixtures. Stain smaller ones in Heidenhain's Iron Hematoxylin and larger ones with Hemalum. For Golgi bodies see Joyet- Lavergne, P., C. R. Soc. de Biol., 1926, 94, 830. Grenacher, see Alum Carmine, Borax Carmine. Grieves' method for undecalcified dental tissues and bone as outlined by Shipley (McClung, p. 345) is: Fix small pieces in 10% formalin 24-36 hrs. or any other desired fixative. Wash in running water 24 hrs. Then pass through 2 changes of aq. dest. 1 hr. each. Dehydrate through ascending alcohols beginning with 50% ale. Equal parts abs. ale. and chloroform, 2 hrs. Chloroform, 2 hrs. 5% sol. of rosin in chloroform, 2 hra. 10% sol. rosin, 2 hrs. Sat. sol. rosin until it becomes transparent. Imbed in melted rosin using one after another the rosins in 3 small glass dishes on a heated copper bar, 1 min. each. The chloroform carried over evaporates. The rosin containing the tissue is al- lowed to cool. The block is ground very thin by hand on a carboriundum stone and polished on a fine hone all grinding being done under luke warm water. The smooth surface is then mounted on a slide with a little melted rosin after which the surface is ground and polished in the same waj' and the section is ready for mounting or for staining. Gross Specimens, see Color Preservation. Ground Substance (intercellular), see Tis- sue Fluid. Growth. Many techniques are now avail- able for the measurement of growth of tissues. Increase in number of cells can be revealed by mitotic counts (Mitosis). The amount of bone or of dentine laid down while Alizarin S or Madder is in the circulation can be estimated. The amount of radioactive isotropes accumulated is a third method (see Radioactive Phosphorus) if the amount increases per unit of time while elimination of the nonradioactive ele- ment in question remains the same. Valuable histochemical methods are given by Lowry, O. H. and Hastings, A. B., in Cowdry's Problems of Ageing, Baltimore : Williams and Wilkins, 1942, 936 pp. Gout, see Urates. Guanin appears as white granules in retinal tapetum of certain animals including nocturnal ones. Decreases in amount in regions containing more fuscin. For details see Arey, L. B. in Cowdry's Special Cytology, 1932, 3, 1218. Guanine, see microxide test under Purines. Guarnieri Bodies, cytoplasmic inclusions in smallpox and vaccinia. See Inclusion Bodies and Cowdry, E. V., J. Exper. Med., 1922, 36, 667-684 for supravital staining with brilliant cresyl blue. For sections Giemsa's stain is excellent. Gum Damar, see Damar. Gypsum test, see Calcium 7. Habermann, see Anethole Clearing Agent. Hafnium, see Atomic Weights. Hairs — Written by Mildred Trotter and Oliver H. Duggins, Dept. of Anatomy, Washington University, St. Louis, May 8, 1951 — The hair shaft (above the sur- face of the skin), the hair root (below it) and the hair follicle (encasing the root) call for somewhat different techniques. The shaft may be examined in a dry mount after first washing thoroughly HAIRS 152 HARRIS ALUM HEMATOXYLIN and repeatedly in ether-alcohol, or the shaft and root can be cleared and mounted in balsam for repeated study. In case it is too highly pigmented to Eermit a clear view of its structure first leach with hydrogen peroxide. Indi- vidual cells of the shaft can be isolated by maceration in 40% aq. potassium hydrate. Scale counts are made readily after a dry mount has had applied a drop or two of a glycerine-alcohol mixture at the ends of the specimen. This mix- ture progresses along the shaft by capillary attraction thus bringing into relief the free borders of the scales (Trotter, M. and Duggins, O. H., Am. J. Phys. Anthrop., in press). Deter- mination of the cuticular scale pattern may be made after partially embedding the hair in a glycerine jelly (Eddy, M. W. and Raring, J. C, Proc. Acad. Sci., 1941, 15, 164-168). Study of the cortex (fusi and pigment granules under very high power) and medulla (when present with its clumps of pigment) requires clearing by immersing in some oil the refractive index of which is approxi- mately the same as that of the hair (Hausman, L. A., Sci. Month., 1944, 59, 195-202). The refractive index of hair may be determined to the greatest accuracy by the double-variation method using both the Becke line and half shadow tech- niques (Gibb. T. R. P., Jr., Optical Methods of Chemical Analysis, pp. 249-250, McGraw-Hill, 1942). Cut hairs into a number of 1 mm. lengths, agitate for 30 minutes in ether-alcohol and mount in oil of a similar refractive index on a temperature cell under a polarizing microscope. True index is found only at crossed Nicols. It is suggested that the index be taken both perpendicular to the lower Nicol (90°) and parallel to the lower Nicol (0°) and that the lower index (90°) be sub- tracted from the higher index (0°) in order to determine the birefringence. The Becke line method may be followed by the half shadow method for greater accuracy. The limit of accuracy will be approximately +3 in the third place. In addition, it is suggested that the use of the phase microscope for such deter- minations may increase the accuracy to the fourth decimal place. Cross sections of a large number of hairs (appro.ximately 150) may be made at one time with very little preliminary preparations by using the "Dr. J. I. Hardy Thin Cross-Section Device" (Gosnell Mfg. Co., Washington, D. C). The root and the follicle are to be seen in most sections of hairy skin and require no special technique unless one wishes to study the follicles attached to whole mounts of epidermis. In order to study the growth of a given hair or to determine the cyclic activity of its follicle it is convenient to place a small tattoo mark with India ink in the skin near the mouth of the follicle (Trotter, M., Am. J. Phys. Anthrop., 1924, 7, 427-437). Distribution of alkaline phosphatase in growth of hair follicle (Johnson, P. L., Butcher, E. O. and Bevelander, G., Anat. Rec, 1945, 93, 355-361). For further details see Trotter, M., Chapter on Hair in Cowdry's Special Cytology, 1932, 1, 40-65. Cleaning and mounting of individual hairs (Duncan, F. W., J. Roy. Micr. Soc, 1943, 63, 85-88. Microphotography of keratin fibers of hairs (Stoves, J. L., J. Roy. Micr. Soc, 1943, 63, 89-90). The less pigment in the hair, the greater the fluorescence, so that gray hair is clear white. Hair containing tricophyton or microsporon fluoresces bright green. See Kinnear, J., Brit. Med., J., 1931, 1, 791-793 on diagnosis of ringworm. Halides, microscopic localization in tissues by precipitation methods (Gersh, I. and Stieglitz, E. J., Anat. Rec, 1933, 56, 185). Hal ©meter, apparatus designed by Eve, F. C, Lancet, 1928, 1, 1070 to measure mean diameter of erythrocytes, see Erythrocytometer. Halowax, see Paraffin Sections. Hanging Drop preparations are mostly em- ployed in the examination of living bacteria and protozoa. A drop of the fluid is simply attached to the under surface of a cover glass which is mounted over a depression in a slide. Equally satisfactory results can usually be obtained by simply mounting under a cover glass on an ordinary slide unless the greater depth of the hanging drop is required. When in Microdissection it is necessary to get at the cells from the under surface of the cover glass special chambers and hanging drops are em- ployed. Harderian Glands, fluorescence in mice (Strong, L. C. and Figge, F. H. J., Science, 1941, 93, 331). Technique for rat is given by Grafflin, A. L., Am. J. Anat., 1942, 71, 43-64. Harleco Synthetic Resin is recommended by McClung, Microscopical Technique, 1950, p. 24 as a mounting medium some- what similar to Clarite. It can be ob- tained from Hartman-Liddon Co., 5821 Market Street, Philadelphia 39, Pa. Harris Alum Hematoxylin. Dissolve 1 gm. hematoxylin in 10 cc absolute alcohol and 20 gms. ammonium or jrotassium HEART 153 HEMATOCRIT alum in 200 cc. aq. dest. the latter with the aid of heat. Mix the 2 solutions, bring quickly to boiling and add 0.5 gm. mercuric oxide. Solution turns purple. Cool quickly in cold water bath. Mal- lory (p. 72) recommends adding 5% of acetic acid. Heart, see Coronary Arteries, Myocardium. Pericardium, Purkinje Cells and Fibers. Technique and results of "electron histology" of the heart are presented by Kisch, B. and Bardet, J. M., E.xp. Med. & Surg., 1951, 9, 1-47. Heart Failure Cells— Written by C. C. Macklin, Dept. of Histological Re- search, The University of Western Ontario, Loudon, Canada. November 28, 1951. — This inapt designation, used by pathologists, applies usually to modified human pneumonocytes which have become free in the lung alveoli, or which are affixed to the moving mucous sheet of the air tract in transit to the mouth region, or which have been hawked up in the sputum. The cells may thus be examined in fresh mounts with or without vital or supravital staining; or in stained dried smears. They may also be fixed, embedded and cut into thin sections (Macklin, C. C, The Lancet, Feb. 24, 1951, 432-435). See Dust Cells. Heavy Water is water in which deuterium, the heavy hydrogen isotope H^, has taken the place of ordinary hydrogen. See Deuterium which is used as a tracer substance. Heidenhain's Azan Stain (Heidenhain, M., Ztschr. f. vyiss. Mikr., 1915, 32, 361-372). The following details are from Lee (1928, p. 279) : Color sections 1 hr. at 55°C. in 2% aq. azocarmine plus 10 drops glacial acetic acid in small staining jar. Wash in water. Differentiate in 96% ale. 100 cc. plus anilin oil 0.1 cc. until cytoplasm becomes pale pink and nuclei clear red. To hurry differentiation add 2 drops anilin oil. Rinse in 96% ale. containing few drops acetic. Put in 5% aq. phosphotungstic acid about 2 hrs. until connective tissue is com- pletely decolorized. Wash rapidly in water. Stain ^-3 hrs. in following solu- tion diluted with equal or double parts aq. dest.: anilin blue (water sol. Griib- ler) 0.5 gm.; orange G, 2 gm.; acetic acid, 8 cc; aq. dest. 100 cc. Examine staining under microscope. Wash in water, dehydrate in abs. ale, clear in xylol and mount in balsam. This is a very useful stain. See also McGregor, L., Ajn. J. Path., 1929, 5, 545-557 for use of this technique particularly as applied to normal renal glomerules. Under Islets of Langerhans is given use of a slightly modified azan method by Gomori. Heidenhain's Iron Hematoxylin, see Iron Hematoxylin. Heinz Bodies. These spherical bodies are sometimes seen in erythrocytes espe- cially when examined in the dark field or when colored with Azur 1. They have been referred to as Substantia Meta- chromatica Granularis and B-substance. The best way to demonstrate them is to use the technique of Figge, F. 11. J., Anat. Rec, 1946, 94, 17. Give 0.3% aq. sulfanilamide to mice as drinking water. Within 4-6 days these bodies will appear in at least 90% of erythrocytes whence they are cast out into the plasma. They are most readily seen in unstained, unmounted blood smears. They dis- appear when studied in oil, balsam or other mounting media. Heinz bodies are granules of heme-containing pro- tein denatured by this drug within the cells. They are not produced by sod- ium sulfathiazole. The Heinz Body phenomenon in erythrocytes is dis- cussed in detail by Webster, S. H., Blood, 1949, 4, 479-504. Helianthin, see Methyl Orange. Heliozoa, techniques for, see McClung, Microscopical Technique, 1950, p. 468, also Rumjantzew, A., and Wermel, E. Arch. f. Protistenk., 1925, 52, 217. Heliotrope B, see Amethyst Violet. Helium, see Atomic Weights. Kelly's Fluid is Zenker's fluid in which 5% formalin is substituted for 5% acetic acid. Helminthosporia. Stain for nuclei in (Par- ris. G. K., Phytopathology, 1944, 34, 700). Helvetia Blue, see Methyl Blue. Hemalum (Mayer's) Hematin, 1 gra.; 90% ale, 50 cc. ; aq. dest., 1000 cc. ; ammonia alum, 50 gms.; thymol, 1 crystal. Keeps better after adding 20 cc. glacial acetic acid and making Acid Hemalum. A good nuclear stain when diluted with aq. dest. 1:20. The above formula has been modified by Lillie (R. D., Stain Techn., 1942, 17, 89-90): hematoxylin, 5 gm.; sodium iodate (NalOs), 1 gm.; ammonia alum (A1NH4(S04)2 + 12 IIjO), 50gm.;aq. dest., 700 cc, glycerol, 300 cc, glacial acetic acid, 20 cc. No ripening is necessary. Stain sections formalin fixed material, 2-5 min. Blue 2-10 min. in tap water. Counterstain in 0.2% aq. eosin Y. Dehydrate clear and mount as usual. This method is quick and gives a sharp stain. Hematin, identified by luminescence with Luminol. Do not confuse with hema- tein, see Hematoxylin. Hematocrit, a tube used to concentrate red HEMATOIDIN 154 HEMOCHROMOGEN CRYSTAL TEST blood cells by centrifugation and to measure their volume, see Ponder, E. in Glasser's Medical Physics, 597-600. Hematoidin (hematin + G. eidos, appear- ance). An iron free pigment produced by phagocytic digestion of erythrocytes or in clots and old hemorrhages, chemi- cal composition similar or identical with Bilirubin. Seen as red or orange rhombic plates or radiating yellow needles, insoluble in ether, water and soluble only with difliculty in alcohol, easily soluble in chloroform. Gives positive Gmelin's test. Hematoporphyrin (G. haima, blood -{- porphyra, purple).— Written by Frank H. J. Figge, Dept. of Anatomy Univer- sity of Maryland Medical School, Baltimore, Md. Contrary to a deeply rooted misconception, this substance is not the pigment as it occurs in hemo- globin, but is artificially produced by the drastic decomposition of hemo- globin in concentrated strong acids. Since it does not occur in nature, such terms as "hematoporphyrinuria" are obsolete. In addition, protoporphyrin, which is the true, unaltered, pigment found in heme compounds, is not ex- creted as such by the Iddney. Proto- porphyrin is heme minus iron and has two vinyl group side-chains. Hemato- porphyrin is heme minus iron, plus two hydrogen and two hydroxy! groups. Hematoporphyrin is soluble in water, ether, alcohols, dilute alkalies, and acids. For references and additional informa- tion, see PorpJiyrins. Hematoxylin is the most useful of all dyes in animal histology and pathology (Gr. haimatodec, blood like + Xylon, wood). It is an extract of logwood {Haernatoxy- lon campechianum) and is marketed in crystalline form. When the crystals are first dissolved in water or alcohol it is not an energetic stain; but requires to be "ripened" before it can be used to advantage. Ripening is brought about by the formation of oxidation products. Consequently it is recommended that solutions be exposed to light and ajr. Hematein (not hematin — a blood pig- ment) is the oxidation product which yields a fine deep blue coloration and is the one most desired. It can be pur- chased. To make up solutions of hematein instead of hematoxylin is logically sound but there is no way to prevent further ripening (oxidation) with the development of other browner unwanted products and precipitation of dyes. Therefore it is good practice to begin with hematoxylin, to let it ripen naturally over a fairly long period of time or to ripen almost immediately by adding about 5% hydrogen peroxide, or 5% of 1% aq. potassium permanganate. 10% solution of hematoxylin in 96% or abs. ethyl alcohol should always be kept on hand. It attains maximum ripening in about one year, but must be kept in a stoppered bottle for otherwise the alcohol will evaporate. It is diluted to 0.5% of hematoxylin with aq. dest. for the Iron Hematoxylin technique. See also Delafield's, Ehr- lich's, Harris' and Mayer's hema- toxylin solutions, likewise Azure 11 eosin and Hematoxylin. Addition of a drop of Tergitol No. 7 to hematoxylin solution will greatly increase speed of staining but has no other advantage (McClung 1950 p. 136). Hematoxylin and Eosin is rightly the most used of all staining methods. If the tissues have been fixed in a fluid con- taining mercuric chloride such as Zen- ker's fluid deparaffinize sections and treat with dilute iodine in 70% alcohol for 1-2 min. Wash in aq. dest., bleach in 5-10% aq. sodium hyposulphite to remove iodine and wash again in aq. dest. Stain with Harris' Hematoxylin (full strength) for 12-15 min. Blue in tap water or in aq. dest. + few drops sat. aq. lithium carbonate, 5-10 min. Stain in 0.2% aq. eosin, 1 min. Rinse in aq. dest. and 95% alcohol. Dehydrate in absolute alcohol, clear in xylol and mount in balsam. Nuclei, deep blue; cytoplasm, pink. In place of Harris' alum hematoxylin, which we use, Delafield's Alum Hematoxylin or Ehr- lich's Acid Hematoxylin may be em- ployed. The Bensleys (p. 73) dilute 1 part of the last named with 2 parts cold sat. aq. ammonium alum and 4 parts aq. dest. Nuclei, dark blue; cytoplasm, collagenic fibers, erythro- cytes, pink; smooth muscle, lavender. 0.2% aq. erj'throsin can take the place of the eosin but the advantage is ques- tionable. Hemin Crystal Test for blood pigment, Teichmann (Stitt, p. 698). Dissolve in 100 cc. glacial acetic acid, 0.1 gm. of KI, of K Br and of K CI. Add few drops to suspected material on a slide and cover. Gently warm until bubbles begin, then slowly cool and examine for typical dark brown crystals. The test is not very sensitive but positive result is conclusive. Hemochromatosis, clinical test for, see Iron. Hemochromogen Crystal Test. Donogdny (Stitt, p. 698). Mix 1 drop of suspected fluid, of pyridin and of 20% aq. NAOH on a slide and allow to dry. Radiating crystals appearing within several hours indicate presence of hemochromogen. HEMOCYTOBLASTS 155 HERMANN'S FLUID Hemocytoblasts, see Erythrocytes, develop- mental series. Hemoflagellates on tissues may be demon- strated by many methods. (McClung, Microscopical Technique, 1950, p. 464). Hemofuscin. Mallory's fuchsin stain. Fix in Zenker's fluid, alcohol or 10% forma- lin. Stain nuclei in paraffin or celloidin sections with Iron Hematoxylin. Wash thoroughly in water. Stain 5-20 min. in : basic fuchsin 0.5 gm., 95% ale. 50 cc. and aq. dest. 50 cc. Wash in water. Differentiate in 95% alcohol, dehydrate in abs. ale, clear in xylol and mount in balsam in the case of paraffin sections. Celloidin sections are to be cleared in terpineol or origanum oil after 95% ale. Nuclei blue, hemofuscin granules bright red, hemosiderin and melanin unstained (Mallory, p. 136). Hemoglobin, histochemical test (Ralph, P. H., Stain Techn., 1941, 16, 105-106). Flood dried blood smear with 1% benzidine in absolute methyl ale, 1 min. Pour off and replace with 25% superoxol in 70% ethyl ale, 90 sec. Wash in aq. dest., 15 sec. Dry and mount in neutral balsam. Hemoglobin dark brown. Goulliart, M. C. rend. Soc. Biol., 1941, 135, 1260-1262 adds to frozen sec- tion or dried smear a drop from a bottle containing glacial acetic acid to which has been added less than a week ago a few crystals of potassium iodide. After about 30 min. examination with a polarizing microscope shows tiny boat shaped birefringent crystals of proto- iodoheme which later change into Teichmann crystals. Dunn, R. 6., Arch. Path., 1946, 41, 676, 677. Dissolve 1 gm. cyanol (Na- tional Aniline Division, Allied Chemical and Dye Corporation, 40 Rector Street, New York) in 100 cc. aq. dest. Add 2 cc. glacial acetic acid. Boil gently and blue color will disappear. Keep for several weeks. Immediately before using filter 10 cc. and add 2 cc. glacial acetic acid and 1 cc. commercial 3% hydrogen peroxide. Treat frozen or paraffin sections of tissue fixed in 4% formaldehyde buffered to pH 7.0 after rinsing in water with this fresh cyanol mixture, 3-5 min. Rinse in water, counterstain with 0.1% safranin in 1% glacial acetic acid. Wash in water, dehydrate, clear and mount in Clarite. Hemoglobin, blue; nuclei, red and cyto- plasm, pink (from Click, p. 63). Hemoglobin Estimation is done by compar- ing blood with a colored paper scale or by a more accurate scale in a hemoglobinom- eter. The experimental error is at least 5%. Staining reactions for hemo- globin within cytoplasm (Kindred, J. E., Stain Techn., 1935, 10, 7-20). Hemolysis. Methods for measuring the velocity of hemolysis depend on the fact that red blood cell suspensions as they hemolyse become more and more translucent. Techniques differ merely in the ways of measuring the trans- mitted light. Simple visual photom- eters and photoelectric ones are de- scribed by Ponder, E. Glasser's Medical Physics, 605-612. The same authority explains the "equilibrium methods" for measuring the amount of hemolysis which has taken place if the process has been arrested. One of these is to count the cells remaining, another to deter- mine the amount of hemoglobin set free, etc. Hemophilus Pertussis. Staining of cap- sules in air dried smears with 5% aq. phosphomolybodic acid. Growth on a special medium is advised (Lawson, G. McL., J. Lab. & Clin. Med., 1939-40, 25, 435-43S). Hemosiderin, soluble in acids and other reagents used in histological technique. After formalin fixation the order of decreasing removal is oxalic, sulphuric, nitric, formic and hydrochloric. Speeu of solution is but little influenced by age of pigment (Lillie, R. D., Am. J. Path., 1939, 15, 225-239). See Iron, Di- nitrosoresorcinol method. To demonstrate hemosiderin micro- scopically pour on deparaffinized sec- tions of freshly fixed tissue 1 part of fresh 2% aq. potassium ferrocyanide and 3 parts 1% aq. hydrochloric acid heated to 60°-80°C. Thoroughly wash in several changes of water. Counter- stain in 0.1-0.5% basic fuchsin in 50% alcohol, 5-20 min. Wash in water. Pass through 95% and abs. alcohol and xylol and mount in balsam. Nuclei and hemofuscin, red; hemosiderin, blue (J. E. Ash in Simmons and Gentzkow, p. 744). See Iron and Hemofuscin. Heparin. A method for the histological demonstration of heparin has been de- scribed by Jorpes, E., Holmgren, H. and Wilander, O., Ztsch. f. mikr. anat. Forsch., 1937, 42, 279-301. It is based on evidence that Tissue Basophiles contain this substance. See also Anti- coagulants. Heptaldehyde. An agent said by Strong, L. C, Am. J. Cancer, 1939, 35, 401-407, to produce liquefaction of spontaneous mammary tumors of mice. It was not helpful when injected into rat lepro- mata (Cowdry, E. V. and Ruangsiri, C, Arch. Path., 1941, 32, 632-640). Hermann's Fluid. 2% osmic acid, 4 cc; 1% platinum chloride, 15 cc; glacial aceticjflkacid. 1 cc. This resembles HERRING BODIES 156 HOFMANN'S VIOLET Flemming's fluid and is a good cyto- logical fixative. Herring Bodies, see Gushing, H., Proc. Soc. Exp. Biol. & Med., 1932-33, 30, 1424- 1425. Hertzberg's Victoria Blue stain for elemen- tary bodies is described by Seiffert, G., Virus Diseases in Man, Animal and Plant. New York: Philosophical Li- rary, Inc., 1944, 332 pp. It is rather like Gutstein's technique (see Ele- mentary Bodies) except that Hertzberg does not make up the stain with po- tassium hydrate and Gutstein does not " destain in 1% citric acid. Herxheimer's solution for staining fat : scarlet red (scharlach R, sudan IV), 1 gm. ; 70% alcohol, 50 cc. ; acetone C.P., 50 cc. See Sudan IV. Heterophile, see Staining. Hexamethyl violet, see Crystal violet. Hexuronic Acid as antiscorbutic factor (Harris, L. J., and Ray, S. N., Biochem. J. 1933, 27, 58-589). Hickson Purple, a disazo dye, giving in aq. sol. a purple color to leucocytes and a red color to erythrocytes introduced by H. G. Cannan (J. Roy. Micr. Soc, 1941, 61,88-94). Higgins' Ink. This was apparently first used as a vital stain by George Wislocki, see Foot (McClung, p. 114). Dilute with equal volume sterile aq. dest. Warm and inject into marginal vein of rabbit's ear 5 cc. daily for 3-4 days, then every 3 days as long as desired. Since the carbon is relatively insoluble it is a simple matter to fix, imbed, section and counterstain. Smaller amounts are to be used for smaller animals, see Vital Stains. Hirudinea, see Parasites. Hischler's Fluid, see Golgi Apparatus. Hiss's Method for capsule staining, see Capsule. Histamine, improved colorimetric method for estimating (Rosenthal, S. M. and Tabor, H., J. Pharmacol. & Exp. Therap., 1948, 92, 425). For use in de- termination of the histamine content of experimental tumors, see Rosenthal, S. M., J. Nat. Cancer Inst., 1949, 10, 89-91. Histiocyte, a term without value as it simply indicates a "tissue cell," often applied to phagocytic cells of connec- tive tissue. Histones, see discussion of Saint-Hilaire's method under Purines. Histoplasma Capsulatum. The halos about this organism and Sporotrichum schencku as seen in H & E stained tissue sections suggest that they are encapsu- lated. Technique for search for such capsules and its failure to reveal them is described by Kligman, A. M. and Baldridge, G. D., A. M. A. Arch. Path., 1951, 51, 567-574. The Hotchkiss- McManus stain, as employed by Klig- man, A. M. and Mescon, H., J. Bact., 1950, 60, 415 is considered by Kligman and Baldridge to be the best suited one for the demonstration of capsules. Historadiography is the x-ray photography of tissues. By a special technique Larmaque, P., Bull. d'Hist. Appl., 1937, 14, 1-16) rays emitted at a tension of 50-100 KV having a length of 0, 12-0,2 A° are directed upon a section closely applied to a particularly finely grained emulsion. The absorption of the rays by the section depends upon the density of its parts. Total opacity of the tissue to the rays is marked on the photo- graphic negative by white, permeability by black, and there are usually all grades between the two. Subsequent magnifi- cation of about 500 times is possible, but is not advisable. Sections, not more than 4 microns thick, of formalin fixed tissues, are recommended. An illus- trated description of the appearance of epidermis, cartilage, artery wall, thjToid and other tissues is provided by Tur- chini (J. Bull. d'Hist. Appl., 1937, 14, 17-28). Historadiography may have many uses in the measurement of densities in different physiological states and in study of the distribution of sub- stances opaque to x-rays experimentally introduced. In some cases great den- sity may accompany high Viscosity. Histospectrography. This is a very valu- able survey method for minerals in tissues. See Policard, A., Protoplasma, 1933, 19, 602-629; Scott, G. H. and Williams, P. S., Anat. Rec, 1935, 64, 107-127; Cowdry, E. V., Heimburger, L. F., and Williams, P. S., Am. J. Path., 1936, 12, 13-29. Optic lens and cata- racts have been analysed particularly for iron, copper and zinc (Busnel, R. G., Pillet, P. and Tillie, H., Bull. d'Hist. Appl., 1938, 15, 99-109). MacCardle, R. C., Engman, M. F., Jr. & Sr., Arch. Dermat. and Syph., 1941, 44, 429-440 have employed histospectrography to advantage in determination of skin magnesium. See Absorption Spectra. Hodgkin's Disease, see Reed-Sternberg Cells. Hofmann's Violet (CI, 679)— dahlia, iodine violet, primula R water soluble, red violet, violet R, RR or 4RN — Conn (p. 120) says above names are applied rather indiscriminately to stains varying in shade from methyl violet to basic fuchsin which are mixtures of methyl- ated and ethylated rosanilins and pararosanilins having less than 5 methyl or ethyl groups. He further remarks that a mixture of basic fuchsin and HOLLANDE'S FIXATIVE 157 HYALURONIC ACID methyl violet of the color desired may perhaps be made by the worker himself as a substitute for Hofmann's violet which is in fact the composition of some samples sold as Dahlia and Hofmann's violet. Hollande's Fixative. Picric acid, 4.0 gm.; copper acetate, 2.5 gm.; formol, 10 cc; glacial acetic acid, 1.5 cc; aq. dest. 100 cc. Recommended for flagellate protozoa (McClung, 1950, p. 445). Holmium, see Atomic Weights. Holtfreter's Solution, for use in examina- tion of fresh tissues; NacCl, 0.35 gm.; KCl, 0.005 gm.; CaClj, 0.01 gm., NaHCOa-HsO, 0.02 gm., aq. dest. 100 cc. (Holtfreter, J., Arch. Entio.-Mech., 1931, 124). Hookworms. To eliminate opacity in mounts of, see Tahmisian, T. N., Stain Techn., 1945, 20, 26. Hormones. Consult volume entitled New and Nonofficial Remedies published each year by the American Medical Association. See Testosterone, Chro- maffin Reaction, Vulpian Reaction, Os- mic Acid. Hotchkiss'Method, see Polysaccharides. Howell-Jolly Bodies, see Jolly Bodies. Huber's Toluidin Blue stain for Nissl bodies (Addison in McClung, p. 150). This much used method is suggested for autopsy material. Fix in 95% alcohol, 100 cc; trichloracetic acid (Mallinck- rodt), 1.5 gm.; mercuric chloride (Mal- linckrodt), 3 gm. 2-10 days depending upon size of piece of tissue. Change fixative every 2 days for larger speci- mens. Pour off fluid and store in 95% alcohol until used. Do not take out mercury with iodine. Stain paraffin sections in toluidin blue 15-18 hrs. (Make up solution by adding 1 gm. to 500 cc. aq. dest. Heat gently and when it is dissolved add 500 cc. aq. dest.). Pour off stain. Wash in aq. dest. Leave 2 hrs. in lithium carbonate. (Make this by adding 5 gm. to 1000 cc. aq. dest. Boil several minutes. Cool. Filter. To 100 cc. filtrate add 900 cc. aq. dest.) . Differentiate in 70% alcohol 5-30 min. Leave flat in 95% alcohol, 5-15 min. Dehydrate in absolute, clear in xylol and mount in balsam. Humus, see soli. Huntoon's Hormone Medium, see Bacteria, Media. Hyalin. This is usually easily recognizable in sections stained with Hematoxylin and Eosin or by Phloxin and Methylene Blue, by its affinity for eosin or phloxin. Phosphotungstic Acid Hematoxylin colors it deep blue. A hematoxylin- phloxin method is also recommended by Mallory (p. 207). Fix in alcohol or 10% formalin and imbed in paraffin or celloidin. Stain in alum hematoxylin, 1-5 min. or more. Wash in tap water and stain with 0.5% phloxin in 20% alcohol, 10-30 min. or longer. Wash in tap water and treat for |-1 min. with 0.1% aq. lithium carbonate. Wash in tap water, dehydrate, clear and mount. In case of celloidin sections, clear in terpineol or origanum oil from 95% ale Nuclei, blue; fresh hyalin, in- tensely red; older hyalin, pink to colorless. A simple thionin stain is also given by Mallory. It is to stain similar sections for 5-10 min. in 0.5% thionin in 20% ale Differentiate and dehy- drate in 80% alcohol. Then 95% alco- hol, terpineol and terpineol balsam. Nuclei and old hyalin, blue. Hyaluronic Acid. — Written by A. R. Gopal- Ayengar, Barnard Free Skin & Cancer Hospital, St. Louis. (Now at Tata Memorial Hospital, Bombay.) This is a polymer of acetyl glucosamine and glucuronic acid. It occurs in a poly- disperse form in a variety of tissues such as umbilical cord, synovial fluid, vitreous humor, skin, tumors due to virus of leucosis and sarcoma of fowls, and in pleural fluid associated with human mesothelioma. (For an exten- sive treatment of the subject of acid polysaccharides and a comprehensive bibliography, refer to Karl Meyer's re- views on, "Mucolytic enzymes" in Currents in Biochemical Research, In- terscience Publishers, N. Y., 1946; "Mucoids and Glycoproteins" in Ad- vances in Protein Chemistry, Academic Press, N. Y. 1945; "The Chemistry and Biology of Mucopolysaccharides and Glycoproteins" in Cold Spring Harbor Symposia on Quant. Biol., 6, 1938, 91- 102.) The enzyme, hyaluronidase, depolymerizes and hydrolyses hyal- uronic acid. It is a Spreading Factor and has been ably presented, along with other spreading factors, by Duran- Reynals, F., Bact. Rev., 1942, 6, 197- 252; Meyer, K. and Chaffee, E., Proc. Soc Exp. Biol. & Med., 1940, 43, 487- 489; Meyer, K. et al., Proc Soc Exp. Biol. & Med., 1940, 44, 294-296, and others. A histochemical method for the dem- onstration of acid polj'saocharides like hyaluronic acid is described bv Hale, C. W., Nature, 1946, 157, 802. the use of metachromatic stains such as tolui- dine blue while satisfactory for sul- phated polysaccharides like chondroitin sulphate is valueless for hyaluronic acid and for related acid polysaccharides which do not stain metachromatically. Fixation of material is an important factor in the retention of hj'aluronic acid for subsequent staining. The or- HYALURONIDASE 158 HYDROGEN ION INDICATORS dinary aqueous fixatives containing formalin, while eminently suitable for fixing protein components, tend to dis- solve the hyaluronic acid. To preserve intact hyaluronic acid it is therefore imperative to employ dehydrating fix- ing agents like Carnoy. The material after fixation, dehydration and embed- ding is sectioned in the usual manner and treated with an acid solution of ferric hydroxide. The iron combines with hyaluronic acid but not with the neutral polysaccharides or proteins. The combined iron is then characterized as Prussian blue by treatment with hydrochloric acid and potassium ferro- cyanide. A counter stain like fuchsin is recommended in order to bring out sharply the blue stained acid polysac- charides against a background of red stained cells. The detailed outline of the Hale technique is as follows: Fix small pieces of tissue in Carnoy (Abs. alcohol, 6 pts. -H chloroform, 3 pts. + glacial acetic acid, 1 pt., for j hr. Dehydrate in abs. alcohol, clear, embed in paraffin and section in the usual manner. Mount sections on clean slides without albu- men. Bring sections rapidly to water and fiood with a mixture of dialysed iron, 1 vol. and acetic acid (2M), 1 vol., 10 min. (Dialyzed iron may be pre- pared by adding ammonia water to a concentrated solution of ferric chloride and dialysing the resulting solution un- til free or nearly free of ammonium salts. It is a dark red liquid easily miscible with water and contains ap- proximately 3.5 per cent Fe, or 5% Fe20,. M = Molecular Solution, which see.) Wash well with aq. dest. Flood with a solution containing potassium ferrocyanide (0.02M) and hydrochloric acid (0.14M)— 10 min. Wash with wa- ter and counterstain with appropriate contrasting dye. Dehydrate rapidly, clear in xylol and mount in Canada balsam. In order to distinguish hyaluronic acid from other blue staining structures Hale recommends interpolation of another step during the staining proc- ess. The procedure suggested involves use of the specific enzyme-hyaluroni- dase — soon after fixation. The enzyme hydrolyses the hyaluronic acid and prevents the combination of the pol- ysaccharide with iron. Since hyal- uronidase is specific, it has no similar action on other polysaccharides. Hyaluronidase is the spreading factor which increases the permeability of connec- tive tissue by reduction in viscosity and by hydrolysis of Hyaluronic Acid. Commercial preparations of hyal- uronidase from bull testes are available from the Schering Corp., Bloomfield, N. J. Enzyme prepared from certain bacteria apparently have hydrolytic powers different from those of the testicular preparations. Hydrax is a synthetic resin used as a mount- ing medium (Hanna, D., J. Roy. Micr. Soc.,1930, 50, 424-426). Hydrogen Acceptors. These are substances like p-amidophenol, p-phenylenedia- mine and resorcin, recommended to strengthen supravital staining of nerve fibers with methylene blue, see Auer- bach's Plexus. Hydrogen Ion Indicators — Written by L. F. Wicks, Veterans Administration Hos- pital, Jefferson Barracks, Missouri. February 1, 1951. — These are also called acid-base indicators and pH indicators. They are dye compounds which are themselves weak acids or weak bases, more usually the latter, and have defi- nite ionization constants. According to the old theory of Ostwald, the color of the indicator in solution depends upon the degree of dissociation and the relative ratio of dissociated and un- dissociated forms. This ratio, and the corresponding shift in color, varies with the concentration of hydrogen ions present, the effect being a composite one. The color change interval will span a certain range, usually of two pH units or less, and it does not require a shift which crosses neutrality. Of the very many dyes and plant coloring matters which alter color with pH, only a few change sufficiently sharply to be of analytical value. Acid-base indicators may, of course, be employed for adjusting the pH of a solution. If direct addition is not de- sired, small portions of both liquids may be transferred to a spot plate, or the indicator may be applied in the form of test papers. (Some indicators such as litmus are now rarely employed otherwise. This is partly true also for Congo red and nitrazine.) Indicators may also be used to esti- mate the reaction of a solution by the application of a series with different pH ranges. Once roughly determined, there is a procedure ("Gillespie's drop- ratio method") by which a fairly ac- curate pH measurement may be easily made with a single indicator of proper range. Perhaps the commonest use for hy- drogen ion indicators by the analytical chemist (who usually prefers the glass electrode for pH measurement and ad- justment), is as an end point device in acid-base titrations. When titrating a weak acid or a weak base, the choice of HYDROGEN ION INDICATORS 159 HYDROGEN ION INDICATORS indicator is very important. That chosen should be one which has its sharpest color transition near the true equivalence point. For example, when titrating the weak base ammonium hydroxide with hydrochloric acid, the resulting ammonium chloride is an acidic salt, and the indicator selected should be one that shifts below neu- trality, methyl red for example. Again, when titrating a weak acid such as lactic acid with sodium hydroxide, the salt formed is alkaline, and the indicator of choice should shift above neutrality, bromthymol blue, for ex- ample. In titrating strong acids and strong bases against each other, the selection of indicator is not critical, as here the pH change near the end point is very great for only a small increment of the added reagent. One should never titrate weak acids and weak bases together, for the results cannot be accurate. There are several possible sources of error in the use of hydrogen ion indi- cators. Some are present at all times and others especially so in biological fluids. An occasional indicator, with two groups sensitive to acid or alkali, has two ranges of color transition at perhaps widely separately pH values. For example, th3^mol blue shifts from red to yellow at a low range (1.2-2.8) and from yellow to blue at a much higher one (8.0-9.6) . A very few indicators ex- hibit "dichroism" (or "dichromatism") in which the color varies with the depth and concentration of the solution. Bromcresol purple and bromphenol blue are examples. In attempting to determine the pH of a very dilute solution, a false result may be obtained by the use of indicators as they are themselves acids or bases. (Recall, for example, that water in con- tact with the carbon dioxide of the normal atmosphere has a pH of about 5.7.) For such cases, a very small amount of buffer should be present to offset this effect. Indicators are intended for aqueous sj^stems, and the presence of other solvents such as alcohol decreases the dissociation constant. Acidic indi- cators then become more sensitive to hydrogen ions, and basic ones less sensi- tive. A control solution of the same solvent composition may be used for comparison, however. Temperature errors are slight over the usual ranges. Indicators may be altered or de- stroyed by the presence of oxidizing and reducing agents, and thej^ niay unite with heavy metal ions. Fortunately, these are negligible considerations in biological fluids, but greater potential errors exist. Proteins and their hydrolysis prod- ucts are usually amphoteric and may combine with the indicator. Congo red, for example, is almost worthless in protein solutions. The presence of much neutral salt will affect the color of indicator solu- tions, partly by influencing the light absorption and partly by shifting the ratio between dissociated and non- dissociated forms of the indicator. The use of mixed indicators deserves greater attention than it has yet re- ceived. As employed for titrations, they are of two general types. One sort consists of two acid-base indicators which have color transitions in op- posite directions, resulting in a very sharp change at a narrow pH zone. The other kind utilizes for contrast color a dye which itself is not influenced by hydrogen ion concentration. The composite color change resulting is usually much sharper than that of the indicator alone. In recent years, there have appeared "universal indicators" consisting of a mixture of half a dozen compounds with a "spectrum" of colors which may vary over the entire pH range. Such indicators are not very accurate and should be used only as a first rough test on an unknown solu- tion. These mixtures are more com- mon as test papers. It is not surprising that hj^drogen ion indicators have been employed as a sort of vital stain to determine the re- action of various living components. In 1893 Ehrlich injected neutral red in an attempt to determine the reaction about phagocj'tosed granules. Since then, other workers have applied other dyes, striving to estimate the approxi- mate pH of tissues, of the fluids bathing them, and even of individual cells. Alizarin red and litmus have been much used, the later especially with lower organisms. Thus, Steiglitz applied all three dyes mentioned above to estimate the reaction of living kidney (E. J., Arch. Int. Med., 1924, 33, 483-496) and confi.rmed the contention that alkaline urine can be formed by an acidic cortex. Harvey and Benslev (B. C. H. and R. R., Biol. Bull., 1912, 23, 225-249) used pH indicators to indicate that gastric fluid does not arise directly within the cells of the mucosa. Margaria (R., J. Physiol., 1934, 82, 496-497) injected bromcresol purple and bromphenol blue, and claimed to have measured pH changes upon stretching a muscle. Orr (J. W., J. Path. & Bact., 1937, 44, HYDROGExX ION INDICATORS 160 HYDROGEN ION INDICATORS 19-27) employed phenol red to estimate alterations in pH in the skin of tarred mice during carcinogenesis. Chambers and his colleagues have added pH indi- cators to tissue cultures (R., Proc. Roy. Soc, B, 1932, 110, 120-124) and have injected them directly into individual living cells (McClung, pp. 62-109). The most enthusiastic investigator tx) employ the phthalein and sulphon- phthalein indicators is Rous (P., Sci- ence, 1924, 60, 363: J.A.M.A., 1925, 85, 33-35, and many articles in J. Exp. Med., 1925 to 1927). The literature is extensive but scattered. There are brief reviews by Rous (P., J. Exp. Med., 1925, 41, 379-411) and von Mollendorf (W., Ergebn. Physiol., 1920, 18, 141- 306). See W. M. Clark in Simmons and Centzkow 161-171. It is well to question the dependa- bility of data upon pH of living material as apparently indicated by vital staining methods. Consider the ideal require- ments for such a vita,l stain. It should exhibit a sharp and pronounced color change in the proper pH range. It should be fairly soluble, readily dif- f usable, strongly colored, of low toxicity and stable in the organism (not readily oxidized or reduced or precipitated by tissue electrolytes). Of the many indi- cators employed in analytical chemistry, only a few meet these requirements. Certain errors are to be guarded against in their use. The "salt error" and "protein error" are unavoidably pres- ent. In the application of these vital stains changes may take place that will themselves cause a pH change. Among them anesthesia, trauma, loss of carbon dioxide from exposed tissues, interfer- ence with blood supply, and postmortem change deserve special mention. How- ever crude though the methods may be, these dye indicators are of value in pre- liminary experiments or where no better procedure is applicable. The indicator dyes of most promise are certain of the phthalein and sulphon- phthalein compounds. They are gen- erally quite soluble, highly diffusable, show marked color shifts and are fairly constant in composition. The dye solutions diffuse quickly when injected, and quickly appear in the urine and stools. For these reasons, fairly large doses given intraperitoneally are more suitable than subcutaneous injections. But it is doubtful, according to Cham- bers (personal communication), whether the more soluble dyes actually penetrate the walls of most cells. The following selection of indicators is based upon the reports of Rous and others, and upon experiments with mice carried out at The Barnard Free Skin and Cancer Hospital. Their chemical names can be found in The Merck Index or in any good textbook of chemistry. Some are to be used in 1% aq. solutions, others in sat. solutions in physiological saline, litmus in either aqueous or agar solution (Rous, P., J. Exp. Med., 1925, 41, 379), while the remainder, which are acidic (the sulphonphthaleins and methyl red), require to be converted to their corresponding sodium salts be- cause the latter are more soluble in water. Consequently the proper equiv- alent of sodium hydroxide must be reacted with each compound. Rub up 0.1 gm. of the dry dye in a mortar (agate, preferably) with the volume of N/20 sodium hydroxide solution given in cc. below the dj'^e in the table. Filter, wash out the mortar with several small portions of saline (0.9% NaCl) and make all to a volume of 10 cc. For a mouse, 0.5-2.0 cc. of the dye solution should be injected intraperitoneally. It should be emphasized that wher- ever possible, the glass electrode should be employed for direct measurement of pH in biological fluids rather than indi- cators. Micro electrodes, including injection types, have been developed. Indicator Bromphenol blue* 3.0 N/20 NaOH Sodium alizarin sulphonate (Alizarin red) 1% aq. or sat. in aaline Bromcresol green* 2.9 N/20 NaOH Methyl red* 7.4 N/20 NaOH HYDROGEN ION INDICATORS pH Range and Colors Value as Vital Stain yeUow*— 3.0 — 4.6— » blue Very strong stain, too far on acid side. yellow «- 3.8 — 5.0 -► pink yellow <— 4.0 — 5.6 — ► blue red «- 4.2 — 6.3 -♦ yellow Very toxic, weak stain. Strong stain, persistent, well tolerated. Unstable in organism, weak stain, fixes on lipoids. HYDROKOLLAG 161 ICTERUS INDEX HYDROGEN ION INDICATORS— Conitnued Indicator Chlorphenol red* 4.7 N/20 NaOH Bromcresol purple* 3.7 N/20 NaOH Bromphenol red 3.9 N/20 NaOH Methyl violet 1% aq. or sat. in ealine Bromthymol blue* 3.2 N/20 NaOH Phenol red 6.7 N/20 NaOH Litmus, purified (Azolitmin) 1% aq. or in agar sol. Neutral red (Toluylene red) 1-2% aq. or sat. in saline Cresol red 5.3 N/20 NaOH Metacresol purple 5.3 N/20 NaOH Thymol blue* 4.3 N/20 NaOH pH Range and Colors yellow ♦- 4.8 — red — 6.8 -» purple yellow «— 5.4 — 6.6 -♦ purple yellow <— 6.4 — red — 7.0 — ► purple blue- violet <- 6.0 — 7.0 -► violet yellow «- 6.0 — 7.4 -* blue yellow ♦- 6.6 — 7.8 -» red (6.8 — 8.4) (approx.) red ♦- 6.0 — 8.0 -« blue (approx.) red «— 6.8 — 8.0 — » yellow yellow *- 7.2 — 8.4 —> purple-red yellow ♦-7.4 — 9.0 —» purple yellow ♦- 8.2 — 9.4 -» blue Value as Vital Stain Powerful stain, well tolerated. Strong stain but rapidly excreted, is toxic and exhibits dichromatism. Very strong stain, well tolerated. Weak stain, toxic. Weak stain, very toxic to mice, but not for insecte. Rapid, intense stain, very well tolerated . Slow stain, diffuses poorly, usually de- posits in granules. Very weak stain, precipitates out readily in vivo, not toxic if pure. Somewhat toxic, not a strong stain. Very weak stain, not very soluble. Toxic, range too alkaline. The indicators starred are perhaps of of widest ordinary laboratory use. To these may also be added cresol purple (yellow <— 7.4-9.4 — >• purple) and phenol- phthalein (colorless <— 8.0-9.8 — »• red- violet). The latter is usually made up in 70-90% alcohol. It should not be used to titrate ammonia. Hydrokollag, a particulate material em- ployed for injection of Lymphatic Ves- sels which see. Hydrotropes, see Sudan Stains. Hydroxy Tri-Phenyl Methanes. These are the rosolic acids. Amino groups of tri- amino tri-phenyl methanes are replaced by hydroxyls making them aciuic in- stead of basic. Examples : aurin (or rosolic acid); red corallin. Hydroxybenzene Compounds as cytoplasmic fixatives. Details of use ofpyrogallol and resorcinol in neutralized formal- dehyde solutions are given. The sim- plicity and rapidity of the procedures and the ease of thereafter cutting sec- tions 1-2^1 in thickness are cited as the advantages special attention having been paid to mitochondria and secretion granules (Huseby, R. A., Proc. Soc. Exp. Biol. & Mod., 1946, 61. 122-125). Hydroxyquinoline test for iron, see Iron. Hypophysis, see Pituitary. Hypoxanthine, see Murexide test under Purines. Ice-crystal Artefacts in normal and chroma- tolj'tic anterior horn cells (Gersh, I., and Bodian, D., Biological Symposia, 1943, 10, 163-184). Icterus Index is a simple measure of the degree of yellow color of blood plasma, or serum, in comparison with standard potassium bichromate solutions. Make up in tubes of same thickness and bore as hematocrit tubes a series of unit dilutions of the bichromate solution Unit 1 = 1 gm. potassium bichromate in 10,000 cc. aq. dest.. Unit 3 = 3 gm. in 10,000 cc. Unit 5 = 5 gm. in 10,000 cc. etc. The plasma of centrifuged blood in hematocrit is compared with these. If it has a color corresponding to, say, Unit 5 of the bichromate solu- tion the icterus index is considered to be 5. The normal value of the icterus index is usually given as 4-7 units. The measure being that of color, and, since increase in color can bo caused by substances other than bilirubin, the in- dex is not a specific measure of bili- rubinemia. Lipochromes can increase the index. If the blood is unusually IDIOCHROMATIN 162 INDIGO concentrated the index is higher al- though the total amount of bilirubin in the circulation may not be elevated. See much more adequate description by Wintrobe, M. M., Clinical Hematol- ogy. Philadelphia: Lea & Febiger, 1942, 703 pp. Idiochromatin (G. idios, one's own, pe- culiar). The chromatin concerned par- ticularly with reproductive functions such as chromosome formation con- trasted with nutritive trophochromatin (G. trophe, food, nourishment). There is no special technique for it. Illumination. For microscopic work the lighting is of great importance. Direct visible light can best be obtained from various electric microscopic lamps on the market. Only when the light is more intense than that required for routine purposes can it be properly employed for dark field examination or for polari- zation. Therefore an intense source should be available. The intensity can be reduced to optimum by using an iris diaphragm. When it is desired to deliver light into the body to a position behind living tissues or organs for transillumination the Quartz Rod tech- nique is suggested. Even to make the light equivalent in quality to that from the white cloud on a bright day, that microscopists used to search for, is quite unnecessary. If the light is too much screened by "day- light" or other glass its intensity will be impaired. Green light was recom- mended quite enthusiastically about 20 years ago. But it is difficult to secure green light of the necessary intensity and it is unpleasant to work with. Ultraviolet light, which permits higher resolution and is selectively absorbed especially by nucleoproteins, is used occasionally for Ultraviolet Photomicro- graphy. The objects, however, can of course not be seen directly so that to photograph them is a hit and often miss experience, though it is possible to focus on a fluorescent screen. The principal use of ultraviolet light is in the Fluorescence Microscope by which the structures giving off fluorescence can be viewed in a dark background at high magnification. Imbedding, see Celloidin, ParafiBn, Glycol- Stearate, Rubber Paraffin, Ceresin, Double and Gelatin for imbedding preparatory to sectioning. The Mount- ing of sections and whole tissues is a kind of imbedding. Immersion Oils. A special grade of cedar oil is usually emploj'ed for oil immersion objectives. Mineral oil is also sold for this purpose. If a refractometer is not available Lillie (p. 6) recommends ad- justment of the index of refraction of the mineral oil by the addition to it of alphabromonaphthalene gradually to the point where a white glass slide im- mersed in the oil can no longer be seen through the oil. It should be N A 1.515 to 1.520. For darkfield work he uses a mixture of 4 parts of "heavy" mineral oil with one part of alphabromonaph- thalene. Cedar oil as supplied for this purpose may easily get too thick by evaporation. It should never be left to harden on the objective but should always be brushed oft" with lens paper immediately after use. If it does harden on the objective, condenser or slide wet the lens paper with xylol which will dissolve the oil when it is gently applied. See Lens Paper. Immunization of monocytes against foreign erythrocytes with phagocytosis of the latter (Bloom, W., Arch. Path, and Lab. Med., 1927, 3, 608-628). Impedence, see Electrical Resistance. Imperial Red, see Eosin B or bluish. Imperial Yellow, see Aurantia. Impression Preparations, see Smears. Inanition, see Fasting. Inclusion Bodies are any substances in- cluded in a cell, tissue or organ. There is the implication that the substance is included from without, that is to say, it is of extraneous origin. But the designation is so loosely used as to be almost meaningless. It is applied to droplets of fat, ingested pigments, remnants of phagocytosed materials, bodies developed in cells as a result of virus action and so forth. The virolo- gists have taken over the designation from normal cytology in which it is used less and less. In certain virus diseases inclusions form in the nucleus, in the cytoplasm or in both (Cowdry, E. V. in Rivers' book on Virus Diseases, Baltimore, Williams & Wilkins, 1928, pp. 113-154). Since the nucleus is shielded from the environment by the cytoplasm its reac- tivity is restricted and the materials available for the formation of nuclear inclusions are also limited as compared with those in the cytoplasm. Conse- quently the composition of nuclear in- clusions in virus diseases is more uni- form than that of cytoplasmic inclusions. See Nuclear and Cytoplasmic Inclusions Indamin Dyes. Methylated amino deriva- tives of indamin. Bindschedler's green and toluylene blue. India Ink, see Higgins'. Indian Blue 3 RD, see Naphthol Blue. Indicators, see Hydrogen Ion and Oxidation Reduction Potential indicators. Indigo, a fine blue dye produced from the leaves of Indigofera tinctoria, employed INDIGO-CARMINE 163 INSECTS as a stain and a cosmetic for more than 4000 years, and early adopted officially for the uniforms of American and British sailors, its history reads like a romance. (iSee, Leggett, W. F., An- cient and Medieval Dyes. Brooklyn: Chemical Publishing Co., Inc., 1944, 95 pp.) Indigo (CI. 1177) is now produced artificially as well as from plants. Indigo-Carmine (CI, 1180) — indigotine la — This sodium salt of indigosulfonic acid is blue with acid characteristics so that it is a good counterstain for carmine. It has been employed with fuchsin by Shumway, W., Stain Techn., 1926, 1, 37-38. See renal excretion of (Kemp- ton, R. T., Bott, P. A. and Richards, A. N., Am. J. Anat., 1937, 61, 505-521). It was used as a vital stain by Heiden- hain who employed 35-60 cc. of 0.4% suspension for rabbits and 150-1500 cc. for dogs (see Foot, McClung, p. 113). The Bensleys (p. 151) advise intra- venous injection of 4 cc. sat. filtered aq. indigo-carmine per kilogram of body weight. Fix by vascular perfusion with formalin alcohol (neutral formalin, 10 cc; absolute alcohol, 90 cc.) or by im- mersion in it. Counterstain frozen sec- tions with Mayer's Acid Carmine or with 1% acridine red. Another way is to imbed (in paraffin), section, clear and examine with or without this counter- staining. Indigotine la, see Indigo-Carmine. Indin Blue 2rd, see Naphthol Blue R. Indium, see Atomic Weights. Indo Reaction for phenols. Formation by oxidation of an aromatic paradiamine in presence of tissue phenol of a blue or green indamine. A difficult reaction (Lison, p. 142). See Lison's study of the venom gland of toads (Lison, L., C. Rend. Soc. de Biol., 1932, 111, 657-^58). Indol Compounds, see Nitro Reaction, Nitrosamino Reaction. Indophenol Blue (CI, 821). This is formed by oxidation of a mixture p-amino- dimethylaniline and a naphthol. Conn (p. 73) says that this is probably the dye employed for staining fat byHerx- heimer, G., Deut. Med. Wochenschr., 1901,27,607-609. Indophenol 1. See Oxidation-Reduction. Indophenol Oxidase, see Nadi Reagent, Cytochrome, Oxidase. Indophenols. Dyes closely related to inda- mines. Example: indophenol blue. Indulin. 1. Spirit soluble (CI,860)— spirit indulin and spirit nigrosin R. 2. Water soluble (CI, 861)— fast blue B, OB, R, etc., soluble indulin 3B — An infrequently used acid azin dye. Lynch, J. E., Zeit. f. wis. mikr., 1930, 46, 465-469; Cumley, R. W., Stain Techn., 1935, 10, 53-56. Indulin Black, see Nigrosin, water soluble. Infra Red photography shows split appear- ance of chromosomes (Ganesan, D., J. Roy. Micr. Soc, 1939, 59, 75-78) and gives better definition of epiphyseal layers of normal and rachitic bone (Siegel, L., Allen, R. M., McGuire, G. and Falk, K. G., Am. J. Path., 1939, 15, 273-277). Guardabassi, M., C. rend. Soc. de Biol., 1935, 118, 559-561 has used this technique for alcohol fixed sections of brain of rabid dog sensitized with rubrocyanine to demonstrate struc- ture of Negri bodies. Transmission of infra red light through the skin facili- tates photography of superficial veins in the living state. Resolution with this light of relatively long wave length is inferior to that with visible light. Injection, see Microinjection. Perfusion of blood vessels and Neutral Red method of staining pancreas by vascular injection. Innervation, determination by dissection (Wharton, L. R., Anat. Rec, 1937, 67, 467-475). Place tissue sheets or thin organs on writing paper. Allow to adhere 5-10 min. Place in 1 part gly- cerol, 1 part glacial acetic acid and 6 parts 1% aq. chloroal hydrate, 18 hrs. Glycerol, 1 part ; Ehrlich's hematoxylin, 1 part; and 1% aq. chloral hydrate, 6 parts, 24 hrs. or more. If overstained decolorize in first solution or in 1% hydrochloric acid in 70% alcohol. Transfer to glycerol 10 days. Dissect under binocular microscope in fresh glycerol. To make permanent prepara- tions, pass up to 95% alcohol, then through bergamot oil, 2 parts; cedar oil, 1 part; and pure carbolic acid liq- uefied by heat, 1 part, to xylol. Mount in balsam. See Nerve Endings. Inoculation is to introduce materials into the body usually disease producing or antigenic. They are in reality injected and we speak of injecting a host of different substances, see in this connec- tion Microinjection, Perfusion and Transplantation. Insects. For whole mounts of large insects Stapp, P. and Cumley, R. W., Stain Techn., 1936, 11, 105-106, specify abs. ale, 5-15 days; 95, 85, 70, and 50% each 15 min. Ale 35%, 30 min. Equal parts H2O and H2O2 -f trace NH4OH, 12-24 hrs. Ale. 35, 50, 85, and 95%, 15 min. each. Abs. ale. 2-3 changes, 3 days or more. Toluol, 10-21 days. Pass from thin to thick dammar and mount. Perhaps the simplest method for small insects (fleas, etc.) is simply to drop them in creosote, U.S. P. and after 24 hrs. to mount them directly in balsam INTERFERENCE COLORS 164 IODINE NUMBER (Fox, 1., Science, 1942, 96, 478). Sec- tioning is facilitated by methods de- signed to soften Chitin, see also Fleas, Ticks. Use of fluorescence microscopy in entomology (Metcalf, R. L. and Pat- ton, R. L., Stain Techn., 1944, 19, 11- 27). In making preparations of insect tissues one must of course be on the lookout for infecting organisms. A well illustrated volume, giving many technical details, is that of Paillot, A. L'Infection Chez Les Insectes. Im- primerie de Tr^voux, G. Patissier, 1933, 535 pp. Interference Colors for daylight, Newton's scale, see McClung's Microscopical Technique, 1950, p. 633. Intermitotic Cells, see Cell Classification. Intestinal Protozoa. 1. Johnson's rapid iron hematoxylin method (Johnson, C. M., Am. J. Trop. Med., 1935, 15, 551). Fix thin smears 10 min. in Schaudinn's fixative containing 5-10% glacial acetic acid (37°-45°C). Treat for 5 min. with iodine in 95% alcohol (port wine color). After placing in 70% alcohol for 5 min. rinse in tap water 1-3 min. Mordant in 4% aq. iron alum (purple crystals) for 15 min. Rinse in tap water 1-2 min. and stain for 10 min. in 0.5% aq. hematoxylin (10 cc. 5% hematoxylin in 95% ale. plus 90 cc. aq. dest.). Differ- entiate in 0.25% aq. iron alum 6-10 min. for flagellates and 12 min. for amoebae. After washing in running water for 3- 30 min., dehydrate in ale, clear in xylol and mount. 2. Long method of Heidenhain (Q.M. Geiman in Simmons and Gentzkow, p. 616). Recommended for Balanti- dium coll and for permanent mounts. This is practically the same except for longer mordanting and staining. See Iron Hematoxylin and Iron Hematoxylin Single Stain. Intestine. Difference in appearance of wall when contracted and normally distended (Johnson, F. P., Am. J. Anat., 1912-13, 14, 235-250). Alterations in human mucosa from absorption of fat and from fasting (Cowdry's Histology, pp. 302- 305). Effect of different dehydration and clearing agents on intestine (Ralph, P., Stain Techn., 1938, 13, 9-15). Ros- enberg, L. E., Stain Techn., 1940, 15, 53-56 has given an interesting account of postmortem autodigestion. Mingaz- zini phenomenon (Macklin, C. C. and M. T.,J. Anat.,1926, 61, 144-150). See Large and Small Intestines. Intracellular Phase, see Chloride. Intranuclear crystals. Hepatic cells of dogs. Determination of properties (Weatherford, H. L., and Trimble, H. C, Anat. Rec, 1940, 77, 487-502). Intranuclear Inclusions, see Nuclear In- clusions. Intravitam Staining, the same as Vital Staining but different from Supravital Staining, which is upon or after, the death of the animal whose tissues are stained. lodeosin B, see Erythrosin, bluish. Iodides. Histochemical localization not feasible (Gersh, I. and Stielglitz, E. J., Anat. Rec, 1933, 56, 185-193). As ex- plained by Glick, p. 34, a precipitating agent to fix iodide will also precipitate protein and thus prevents its own pene- tration into the tissue. Iodine, detection of: 1. Ionized iodine in the form of iodides. Stieglitz (E., J. Pharm. and Exp. Therap., 1924, 22, 89-98) injects 20 cc. 5% aq. lead nitrate intravenously into an animal to be killed and fixes the tissue in formalin. In the sections, iodine is found in the form of yellow crystals of lead iodide. Methods have been reviewed by Gersh and Stie- glitz (I. and E. J., Anat. Rec, 1933, 56, 185-193). 2. Methods for iodine in organic com- bination appear to be unsatisfactory. The whole subject of iodine has been critically considered by Lison (p. 111- 113). See Gram's and Lugol's solu- tions. Iodine, as a stain is one of the stains used for Glycogen and Starch Grains. It is also advised in the form of Lugol's solu- tion to bring out in frozen sections of nervous tissue certain extremely minute bodies in the cytoplasm and along the processes of nerve cells bv Adamstone, F. B. and Taylor, A.B., Science, 1946, 104, 111. See Gram-Pappenheim stain and Gram Stain for bacteria. lodine-Eosin stain of Donaldson, R., Lan- cet, 1917, 1, 571 is highly recommended by Craig, p. 45 for intestinal amebae and flagellates. Saturate one volume of 5% aq. potassium iodide with iodine crystals and mix with equal volume of sat. aq. eosin (yellow aqueous eosin). Mix small drop with a little feces on slide, cover and examine. Cysts of amebae and flagellates, yellow to green- ish yellow in red background; glycogen bodies within cysts, brown. Iodine Green (CI, 686), closely related to methyl green, only used occasionally. Iodine-Iodide Solution. This term is em- ployed for almost any solution contain- ing iodine and iodide as Lugol's and Grams. Iodine Number of lipids, see Schmidt- Nielsen, K., C. rend. trav. lab. Carls- berg, S^r. Chim., 1944, 22, 87-96 and Kretchmer, N., Holman, R. T. and Burr, G. O., Arch. Biochem., 1946, 10, 101-105. IODINE VIOLET 165 IRON Iodine Violet, see Hofmann's Violet. Iris Blue, see Resorcin Blue. Iris Violet, see Amethyst Violet. Iron occurs in tissues "masked" in organic compounds which are not ionisable and free in inorganic compounds which are ionisable into ferric and ferrous salts. 1. Macallum's hematoxylin method depends upon the formation of a blue black iron hematoxylinate. The tissue is fixed in 95% alcohol 24-48 hours, dehydrated, cleared, imbedded in paraf- fin and the sections are passed down to distilled water. Contact with iron is reduced to a minimum. The microtome knife must be free of rust. Treat sec- tions with a freshly prepared straw yellow 0.5% aqueous solution of hema- toxylin which must be of the highest purity. Inorganic iron produces the blue-black compound which is rela- tively insoluble. Dehydrate, clear and mount in balsam in the usual way. The technique for organic iron is more difficult because it must be un- masked before it will react in this way. The best account is Nicholson, F. M., J. Comp. Neurol., 1923, 36, 37-87. In studying the cytoplasmic iron contain- ing proteins of nerve cells of the medulla of rats, he fixed in 95% alcohol 48 hours ; dehydrated in absolute alcohol 2-5 hours; cleared in cedarwood oil until transparent; imbedded in paraffin (2 changes) and cut sections 7/1. After being deparaffinized, the sections were passed through alcohols to 4% pure sulphuric acid in 95% alcohol held at 60 °C. for 5-60 minutes. This liberated the iron. The sections were washed in 95% alcohol ; passed down through graded alcohols to aq. dest., and placed in freshly prepared 0.5% aqueous hematoxylin, 1-5 minutes in which the blue-black hematoxylinate forms . Then wash in aq. dest. (not tap water). Counterstain in dilute alcohol erythro- sin and mount as usual. As a check the nuclear chromatin of sections not treated with the acid alcohol should not be colored black by this hematoxylin solution . Difficulty may be experienced because the color of the unmasked iron is faint. The reaction is a chemical one of great delicacy and requires practice. Pancreatic acinous cells also afford favorable material. Look for cyto- plasmic iron in the poles distant from the lumen where thechromidial material, which resembles the Nissl bodies, is most concentrated. 2. Prussian blue reaction. Prepare sections in the same way, deparaffinize and test as described in Lee (p. 291). For ferric salts of inorganic iron wash in aq. dest., 2% aqueous potassium ferro- cyanide, 3-15 minutes; Prussian blue is formed, wash, dehydrate, clear and mount. For ferrous salts substitute ferricyanide for ferrocyanide in the test. For both use equal parts of ferrocyanide and ferricyanide. When the iron is organic it is unmasked by treating the sections with 3% pure nitric acid in 95% alcohol for 24-36 hours at room tem- perature or at 35 °C. if necessary. Wash in pure 90% alcohol and in aq. dest. Place in equal parts freshly made of 1.5% aqueous potassium ferrocyanide and 0.5% aqueous hydrochloric acid for not more than 5 minutes. Wash well in aq. dest., colored with eosin or safranin, dehydrate, clear and mount. Hemosiderin gives Prussian blue reaction for inorganic iron. The iron in hemoglobin is not unmasked by these acid alcohols. Brown, W. H., J. Exper. Med., 1911, 13, 477-485, devised special methods for its demonstration. Test- ing for iron in association with calcium particularly in bone is critically de- scribed by Cameron, G. R., J. Path, and Bact., 1930, 33, 929-955. He em- phasizes the fact that exposure of tissues and fluids to dust in a city like London is an important source of error. 3. Microincineration yields a mineral residue that contains iron originally both organic and inorganic. Color of the iron oxides, viewed in the dark field, varies according to Policard (C. rend. Acad. d. sc, 1923, 176, 1187) from yellow to deep red. He suggests that perhaps the yellow to brown ash is of organic iron and the red ash is of free iron. See also Marza, V. D., Marza, E., and Chiosa, L. Bull, d'hist. Appliq., 1932, 9, 213. Scott (McClung, p. 758) warns against confusion with carbon. 4. Hydroxyquinoline test (Thomas, J. A. and Lavollay, J., Bull. d'Hist. Appl., 1935, 12, 400^402). Fix in alco- hol, trichloracetic acid or neutral forma- lin. Avoid formol with alkaline water and fixatives containing chromium. Make up reagent by dissolving 2.5 gra. 8-hydroxyquinoline in 4 cc. pure acetic acid warming gently. Add quickly aq. dest. to make 100 cc. Filter. Wash sections (or smears or cultures) well in neutral aq. dest. Then add few drops of reagent 5-15 min. Pour off reagent. Add to preparation 1 drop 25% aq. ammonia which produces a ppt. Wash in a stream of neutral aq. dest. If large crystals remain wash more ener- getically. Stain nuclei with lithium carmine. Examine in neutral aq. dest. or dehydrate in terpinol and mount in vaseline oil. Iron, green black; nuclei, red. Recommended for localization of iron in granules of vitellus, in red blood IRON HEMATOXYLIN 166 ISAMINE BLUE cells, and in connection with micro- incineration. Said to be better than Prussian Blue reaction for iron. 5. Dinitrosoresorcinol (Humphrey, H. A., Arch. Path., 1935, 20, 256-258). Treat paraffin sections of formalin fixed tissue with 30% aq. ammonium sulphide, 1 min. Rinse in water and immerse in sat. aq. dinitrosoresorcinol (Eastman) 6-20 hrs. A counterstain can be em- ployed. Humphrey does not say which. 1% eosin in 50% alcohol should be satis- factory because the iron containing com- pounds such as hemosiderin are colored green. Wash, dehydrate, clear and mount. Intravenous injections of colloidal solutions of iron in rabbits are described by Duhamel, B. G., C. rend. Soc. de Biol., 1919, 82, 724-726. 6. A clinical demonstration of iron in the skin in hemochromatosis involves intradermal injection of equal parts of sterile 0 5% aq. potassium ferrocyanide and 1/100 N hydrochloric acid. This produces a wheal which turns dark blue in 5 min. A positive reaction can even be obtained after death. (Fishback, H. R., J. Lab. & Clin. Med. 1939-40, 25, 98-99). In special cases, as in the analysis of small amounts of epidermis, resort may be had to a quantitative polarographic determination of iron, see Carruthers, C. and SuntzefT, V., J. Nat. Cancer Inst., 1942,3, 217-220. Kirk, P. L. and Bentley, J. T., Mikrochemie, 1936, 21, 250^259 advo- cate a titrimetric method for iron. The difficulty of interference by small amounts of copper has been overcome by Ramsav, W. N. M., Biochem. J., 1944, 38, 467-469. Click (p. 277) is of the opinion that such methods may be adapted for use with the quantities of material employed in histochemical work. Iron Hematoxylin of Heidenhain is one of the standard stains. It will give excel- lent results after almost any good fixa- tion. Zenker's fluid and formalin- Zenker are suggested. Bring paraffin sections down to aq. dest. Mordant in 5% aq. iron ammonium sulphate (iron alum, light violet colored crystals, dis- card the brownish material accompany- ing them) 12-24 hrs. Rinse quickly in aq. dest. Transfer to 1% aq. hema- toxylin (made up by diluting 1 cc. sat. sol. hematoxylin in abs. ale. with 99 cc. aq. dest.) for 12-24 hrs. Differentiate under microscope in 1% aq. iron alum. Wash thoroughly in tap water. Many counterstains can then be used such as 1% aq. Bordeaux red, orange G., acid fuchsin, acridine red, or Mucicarmine. Dehydrate, clear and mount. Nuclei dense blue-black in background of color selected. See Centrosomes, Nuclei, Regaud's Method for mitochondria. 1. Koneff, A. A., Anat. Rec, 1936, 66, 173-179 advises use with anilin blue. Mordant sections 5-10 min. in 5% aq. iron ammonium sulphate. Rinse quickly in aq. dest., stain 3-15 min. in Harris' hematoxylin. Rinse again in aq. dest. and stain in: anilin blue (Griibler) 0.1 gm. ; oxalic acid, 2 gm. ; phosphomolybdic acid, 15 gm. and aq. dest. 300 cc. Wash in aq. dest., differ- entiate in alcohol, dehydrate (2 changes of absolute), clear in xylol and mount in balsam. If euperal is used for mounting omit the xylol. Nuclei, violet-brown; cytoplasm, light brown; erythrocytes, dark violet; myelin and muscle brown; elastic fibers, reddish brown to red. 2. Lillie, R. D. and Earle, W. R., Am. J. Path., 1939, 15, 765-770 recom- mend employment of a hematoxylin containing ferric and ferrous iron: (A). Ferric ammonium sulphate, violet crys- tals, 15 gm.; ferrous sulphate, 15 gm.; aq. dest., 100 cc. (B). Hematoxylin, 1 gm.; 95% alcohol, 50 cc, glycerin, C.P., 50 cc. Mix A and B in equal quantities before using. For best general discussion of iron hematoxylin, see Lillie, p. 58. Iron Hematoxylin Single Stain — Written by Morris Goldman, Dept. of Parasitology, School of Hygiene and Public Health, Johns Hopkins University, Baltimore. January 29, 1951 — This stain is useful for the purpose of diagnosing intestinal protozoa occurring in fecal smears. It is not intended to replace the longer and more precise iron hematoxylin methods. The stain is prepared from the following relatively stable solutions: Solution A: 1% hematoxylin in 95% ale. (best pre- pared from a stock 10% ale. solution of hematoxylin). Solution B: NH^Fe- (804)2- I2H2O, 4.0 gms., glacial acetic acid, 1.0 ml., concentrated H2SO4, 0.12 ml., and aq. dest. to make 100 ml. Mix equal parts of Solution A and B, filter after several hours and use. Staining time varies from 30 sec. when stain is fresh to 3 min. after 24 hrs. Discard stain after 3 days. Fecal smears are fixed in Schaudinn's, passed through iodine alcohol to 50% alcohol, stained, washed in running tap water 5 minutes, dehydrated and mounted. The stock solutions used in this technique may also be used in the Heidenhain iron hematoxylin procedure. Iron Pigments, see Berlin and Turnbull blue reactions. Iron, Radioactive. See Erythrocytes. Isamine Blue is described by Conn (p. 137) ISLETS OF LANGERHANS 167 JALOWY as a sulfonated naphthyl-rosanilin or naphthyl-pararosanilin. He questions the synonym (alkali blue XG) given in the Colour Index. This acid has been much used as a Vital Stain in European laboratories. It is not made in the United States. Islets of Langerhans of the pancreas . There are many techniques for the study of these cellular masses. 1. To study in the living state the method employed by O'Leary, J. L., Anat. Rec, 1930, 45, 27-58 is recom- mended. It consists essentially of partly withdrawing the pancreas from a mouse and of mounting it in such a way that a thin film of tissue can be closely examined with circulation still active. The islet cells can be studied with oil immersion lenses and the changes in them on the injection of insulin noted. 2. To obtain an idea of the distribu- tion, number and size of the islets supravital staining with Neutral Red or Janus Green is indicated, which see. 3. To stain the cell types specifically Neutral Gentian and other stains advised by Lane, Bensley and their followers are available. The Azan Stain suggested by Bloom, W., Anat. Rec, 1931, 49, 363-371 (see his beautifully colored plate), has been further investi- gated by Gomori, G., Anat. Rec, 1939, 74, 439-459 whose technique abbreviated is as follows : Fix thin slices of pancreas in Bouin's fluid 8-10 hrs. Wash in aq. dest. Imbed in paraffin and cut ifi sec- tions. Stain 45-60 min. at 56°C. in azocarmine. (To make dissolve 0.1% azocarmine in aq. dest. Boil about 5 min. Cool and add 1.0 cc glacial acetic acid to each 50 cc. solution. Before use filter at 60 °C. Stain will keep for months.) Rinse quickly inaq. dest. and blot. Destain in 90% alcohol containing 1% aniline oil until acinous tissue is al- most wholly decolorized and B cells show red against pink background of A cells. Rinse briefly and treat with 5% aq. iron alum for 5 min. or more. Rinse again and stain 2-20 min. in the usual mixture (anilin blue, 0.5 gm.; orange G, 2.0 gm.; + aq. dest. to make 100 cc.) diluted with 2-3 times its volume of aq. dest. until under the microscope colla- genic tissue becomes deep blue. Rinse and blot. Differentiate and dehydrate in absolute alcohol, clear in xylol and mount in balsam. Cytoplasm of A cells rich orange yellow, of B cells fiery red and of D cells sky blue. The author states that by first staining with Bens- ley's neutral gentian, decolorizing and restaining by above Azan method it can be seen that there is no gradation be- tween A and B cells. Isoelectric Points of cellular structures. Methods for their determination at con- trolled pll's by intensity of staining have been critically evaluated by Levine, N. D., Stain Techn., 1940, 15, 91-112. His conclusion is that no true isoelectric points have yet been established for nucleus, cytoplasm or other tissue ele- ments by tliese techniques. See re- ticulo-endothelial cells (Fautrez, J., Bull. d'Hist. Appl., 1936, 13, 202-200). Isohematein, as a biological stain (Cole, E. C, Stain Techn., 1931, 6, 93-96). Greater tinctorial power than hematox- ylin but less selective. Isomerase with Aldolase are known as Zymohexase. Isopentane recommended by Hoerr, N. L., Anat. Rec, 1936, 65, 293 for freezing in the Altmann-Gersh technique. Isopropanal in combination as a new fixative for animal tissues which also dehydrates (Cleverdon, M. A., Science, 1943, 97, 168). Isopropanal, 55 cc; picric acid, 5 gms., acetone, 30 cc; glacial acetic acid, 55 cc; formalin (40% formalde- hyde CP), 5 cc Fix 2 hrs.— 4 days de- pending on size. Store in 70% iso- propanal or imbed in paraffin after first washing in 2 changes nearly absolute isopropanal. Remove picric acid from mounted sections just before staining with 1.5% ammonium hydroxide in 95% alcohol. Isopropyl Alcohol. Has been recommended as a substitute for ethyl alcohol since it mixes with water and xylol. It is said to be less hardening than ethyl alcohol (Bradbury, O. C, Science, 1931,74,225) but it is more expensive. See Herman, C. M., J. Lab. & Clin. Med., 1941,26, 1788. Isorubin, see New Fuchsin, Iso-Safrol is obviously an isomer of safrol which is given as 3,4-methylene-dioxy- allylbenzene in the Merck Index. Iso- saf role is listed among Eastman's organic chemicals. It is sometimes recom- mended as a partly dehydrating and clearing agent (Silver Citrate injection of blood vessels, etc) but in all likeli- hood other clearing agents can be used as substitutes. Isospora, see Coccidia. Jacobson's Organ, innervation, Bellairs, A., J. Anat., 1942, 76, 167-177. Jalowy modification of Hortega method for the skin (Jalowy, B., Zeit. f. Zellf. u. Mikr. Anat., 1937, 27, 667-690). To make reagent wash ppt., formed by adding 20 drops 40% aq. NaOH to 20 cc. 10% aq. silver nitrate, 10 times with aq. dest. Suspend ppt. in 20 cc. aq. dest. Add ammonia drop by drop till it dis- JANSSEX'S IRON HEMATOXYLIN 168 JOHNSON'S NEUTRAL RED solves. Add 100 cc. aq. dest. and store in dark. Deparaffinize sections of tissue fixed 1-2 days in neutral formalin. Treat with above reagent 5-30 miu. at 30°C. Rinse in aq. dest. and in ammonia water. After treating with 1 part neu- tral formalin to 4 of aq. dest. wash in running water, dehydrate, clear and mount in balsam. Collagen, yellow to brownish yellow; reticular fibers, black. Janssen's Iron Hematoxylin recommended in place of Weigert's acid iron chloride, hemato.xylin (Lillie, R.D. and Earle W.R. Stain Technol., 1939, 14, 53-54). Janus Blue can be used in exactly the same ways as Janus green and with equal success. Janus Dyes. Named after the God, Janus with two faces since they often exhibit two colors. Their chemistry and use in histology is described by Cowdry, E. V. Contrib. to Embryol., Carnegie Inst. Washington, 1918, No. 25, pp. 39-148. Janus green (formerly made by Grub- ler) is safraninazodimethylanilinchlo- ride. This is useless for staining mitochondria. Janus green C (Hoechst) is dimethyl safraninazodimethyl anilinchloride. This likewise is useless for mitochondria. Janus green B (Hoechst) is diethyl- safraninazodimethylanilinchloride. This is the most specific stain for mito- chondria and is now supplied by many companies both as Janus Green B and simply as Janus Green. Janus blue G and R (Hoechst) is diethylsafranin-B-naphthol and stains mitochondria as well as Janus Green B. The marks G and R indicate differences in method of manufacture not different dyes. Janus black D, I, II and 0 (Hoechst), of these Janus Black I is a mixture of two substances Janus green B and a brown dye. It colors mitochondria by virtue of the former. Janus gray B, BB (Hoechst) are also safranin derivatives but useless for mitochondria. Janus yellow G, R, (Hoechst) likewise safranin derivatives and no good for mitochondria. Diethylsafranin is a reduction product of Janus green B. It is a red dye which colors mitochondria specifically but not very strongly. Janus Green B (Diazingriin) is diethyl safraninazodimethylanilinchloride. Ja- nus green now sold without the qualifica- tion B is usually the same substance because it has become well known that the dye required must have the composi- tion indicated. Owing to its toxicity Janus green cannot be injected into living animals like trypan blue and other "vital" stains. It is employed as a supravital stain by simply immersing tissues in it or better by its injection into the vessels of a freshly killed animal the individual cells of which remain for some time alive. Janus green is the best supravital stain for mitochondria. Janus green is also very useful for stain- ing the islets of Langerhans of the pancreas and the renal glomeruli of the kidney when injected intravascularly, see Neutral Red. Both islets and glomeruli are colored deep bluish green against a background at first colorless, or faintly green, and changing to pink by reduction of the dye to diethyl- safranin. This permits the counting of islets and glomeruli in pieces of tissue mounted in salt solution and observed at low magnification. When the oxygen is further consumed by the cells the dye is reduced to a second colorless leucobase. It is therefore an oxidation -reduction indicator as well as a specific stain for mitochondria. See Neutral Red-Janus Green stain. Janus Red B (CI, 266), a basic disazo dye of light fastness 4. Action on paren- chyma described (Emig, p. 36). Jaws, see Teeth and Jenner-Giemsa method of Pappenheim (see May-Giemsa). Jenner's Stain for Leishmania as described by Craig, p. 146: To make, mix equal parts 1.2% water soluble eosin (Grub- ler or NAC) in acid free aq. dest. and 1% aq. medicinal methylene blue in a flask. Shake thoroughly and let stand at room temperature 24 hrs. Collect ppt. on small filter paper and wash with aq. dest. till filtrate is almost colorless. Dry ppt. and store in dark at room temperature. Dissolve 0.5 gm. ppt. in 100 cc. pure methyl alcohol (Merck's Reagent). Cover smears with this 1-2 min. Then add aq. dest. drop by drop till metallic sheen forms on surface. Leave 5-15 min. longer as desired for intensity. Method said by Craig to be less reliable than Giemsa, Leishman or Wright techniques. Johnson's Neutral Red stain for Nissl bodies (Addison in McClung, p. 450). Ripen 1% aq. neutral red 1-4 years. Dilute to 0.25-0.5% before using. Differ- entiate and dehydrate in the usual way. Clear in 1 part xylol + 2-3 parts castor oil. Gives good results in thick sections (50m) and can be employed after silver methods on tissues fixed in alcohol or formalin. Kirkman, I. J., Anat. Rec, 1932, 51, 323-326 used the following unripened stain after Bouin and formalin fixatives : neutral red (Coleman & Bell), 1 gm. ; aq. dest., 500 cc, 1% aq. glacial acetic acid, JOINTS 169 KERATOHYALIN GRANULES 2 cc. 10-20 min. is sufficient for counter- staining Weigert-Pal preparations. Then rinse in aq. dest., differentiate in 95% alcohol, dehydrate in absolute, clear and mount. Joints. Meniscus (Raszela, F., Bull. d'Hist. Appl., 1938, 15, 186-210). Jolly Bodies. These structures are nuclear remnants in erythrocytes that are strongly colored by Giemsa's Stain. Jores' Solution, see under Color Preserva- tion of gross specimens. Kabunylin, a dye extracted from beetroot. Said to be good for use with picrofuchsin (Fuse and Hino, Arb. Anat. Inst, zu Sendai, 1937,20, 111-113). Kaiserling's Solution, see under Color Preservation of gross specimens. Kaliichrom, a combination of cresyl violet and auramin recommended for both plant and animal tissues (Kisser, J., Mikr. f. Naturfreunde, 1931, 9, 95). Kardos-Pappenheim modification of Giem- sa's stain (Kardos, E., Folia haematol., Archiv., 1911, 12, 39). To make the methyl green-orange stain mix 2% aq. orange G. with concentrated aq. methyl green. Filter, dry the ppt. and dis- solve in methyl alcohol. Shake well together 5 drops methyl green-orange, 10 drops of Giemsa's stock solution and 15 cc. aq. dest. The fluid under the foam is used for staining. First fix and stain the blood smear with May-Griinwald mixture 3 minutes ; add equal volume aq. dest., 1 minute; pour off and add the methyl green-orange 15 minutes; wash quickly in water and blot dry. Karo, white corn syrup (Corn Products Co.) is a useful medium for mounting whole insects because they can be transferred to it directly from water or weak alcohol and clearing is unnecessary (Patrick, R., Science, 1936, 33, 85-86). Karotin, see Carotin. Karyosome (G. Karyon nut, nucleus + soma, body). A basic staining or chro- matin-nucleolus, in contrast to a plasmo- some, generally more numerous, smaller and of less regular shape often called a net-knot. Kerasin is a Cerebroside. Keratin, a scleroprotein contained in hair, nails, horns, epidermis, etc. There are apparently two sorts. Their chemistry is discussed by Giroud, A., Bulliard, H. and Lebond, C. P., Bull. d'Hist. Appl., 1934, 11, 365-373. See Orange II, Oral Mucosa. Keratohyalin Granules, separation and analysis — Written by Donald L. Opdyke, Dept. of Anatomy, Washing- ton University Medical School, St. Louis, Mo. November 8, 1951 — Highly refractile cytoplasmic granules of vary- ing size found in the cells of the stratum granulosum of stratified squamous epithelia. They were first described by Langerhans in 1882 and were called "keratohyalin" by Waldeyer who as- cribed to them a constant relation to the process of cornification. The tinctorial properties of these granules may be summarized as follows: They stain beautifully with picro- carmine, have an intense affinity for all of the hematoxylin stains and stain as well with unoxidized hematoxylin as with the aged stain. They stain meta- chromatically with toluidine blue, and after staining with crystal violet and iodine they resist destaining by acetone. They do not stain by the Feulgen pro- cedure but give beautifully intense reactions to the Bauer, Iodine, and Best's Carmine technics for glycogen. The reactions with the P.A.S. method of McManus give varying results. The granules are not tannophil; do not osmicate or stain with the Sudan dyes. They are argentophil. Microincineration studies reveal large concentrations of mineral in the granules as demonstrated by quantities of the white ash typical of calcium and/or magnesium. These ashed sec- tions, as well as unashed sections show intense reactions of the granules to the gallamine blue staining method of Stock, A., J. Roy. Micr. Soc, Series III, 1949, 69, 20-24. Enzyme studies have resulted in dis- agreement in the literature with respect to ribonuclease digestibility, but the granules are definitely not totally digest- ible in ribonuclease, hyaluronidase, crystalline trypsin or salivary enzymes. They are readily dissolved by elastase. The most striking features of these granules, both tinctorially and histo- chemically, are their similarities to elastic fibers: These common features include their tinctorial reactions to Verhoeff's procedure, congo red, and orcein; their affinity for mineralization with calcium salts; their solubility in the elastolytic enzyme of Bal6 and Banga (See Elastic Fibers). References for the above data are: Lansing, A. I., and Opdvke, D. L., Anat. Rec, 1950, 107, 379 398; Smith, C, and Parkhurst, H. T., Anat. Rec, 1949, 103, 649-674; and unpublished work of D. L. Opdyke. The origin of these granules is much debated. Stam, Von F. C, Acta Dermato-Venereol., 1951, 31, 407-411 believes them to be extruded nucleoli. Favre of the Bacteriological Institute of Lyon is of the opinion that they are formed by the spiral shaped mito- chondria of the basal layer of the epi- KERMES 170 KIDNEY dermis (Favre, M. Ann. de Dermat. et Syph., 1950, 10 (3) , 241-262) . The litera- ture 1886 to date includes reports of the origin of keratohyalin granules from nuclear substance, intercellular fibrils, and cytoplasm. The particulate nature of the granules has been demonstrated recently by their isolation from epidermis (D. L. Opdyke). Epidermis is separated by the heat method of Baumberger, J. P., Suntzeff, v., and Cowdry, E. V., J. Nat. Cancer Inst., 1942, 2, 413-423. The sole of the foot offers an ideal place to obtain keratohyalin granules. After separating the epidermis from the dermal layers, the proximal side of the epidermal sheet is scraped with a knife, removing all of the epidermis in fine shavings down to the highly trans- parent, thick stratum corneum. The shavings are then homogenized in a Waring Blendor until no cells remain intact. This homogenate is made in a 0.85% NaCl solution of pH 7.3 to which a crystal of thymol is added to minimize bacterial contamination. All proce- dures are carried out in a cold room. The process of cell fractionation in the micro cup of the Waring Blendor re- quires about 45 min. If 2% citric acid is employed as the medium, this time may be reduced to 15 min. The components of the suspension are then separated by differential centrif- ugation. Nuclei and whole cells are found to sediment at 2.5 to 3 Kg/g. The keratohyalin granules sediment at 25.2 Kg/g. after 12 min. of centrifuga- tion. These can be resuspended in fresh saline, washed, and reprecipi- tated. They can be distinguished from mitochondria by their tinctorial properties. Kermes. This scarlet dye was known in Egypt and farther East at a very early date. Kermes is the Armenian term for a "little worm", variously identi- fied as Coccus arborum and Coccus ilicis. Moses referred to it as "Fola" and "Fola shami". Remember the promise of Jehovah: "Though your sins be as scarlet (Fola) they shall be as white as snow; though they be red as crimson (Fola shami), they shall be as wool". So valuable was Kermes that after the subjugation of Spain by the Romans the people were made to pay half of the tribute in Kermes. At about 1640 a Dutch chemist discovered the similarity of this dye to cochineal. Its history affords interesting reading (Leggett, W. F. Ancient and Medieval Dyes. Brooklyn: Chemical Publishing Co. Inc., 1944, 95 pp.). Kidney — Written by Jean Oliver, Dept. of Pathology, State University of New York, Brooklyn 2, N. Y., September 4, 1951 — Techniques for the general demonstration of the elements of the renal tissue, epithelial cells, sustaining tissues, blood vessels and nerves are essentially the same as those used for other organs. Masson's Trichrome stain has the advantage of affording a particularly colorful differentiation of the various elements in a single section. The individual renal organs that make up the kindey, the nephrons, can be isolated in their entirety by maceration and teasing as described by Huber, G. C, Cowdry's Special Cytology, 1932,2, 935-977. Partly wash out blood by in- jecting physiological saline into the renal artery. Then follow with hydro- chloric acid (cone. HCl, 3 parts and aq. dest. 1 part) using care to protect the eyes. Remove and immerse the organ in the same fluid. After a suitable time, determined by excising pieces, wash a block of tissue with aq. dest. and stain in Hemalum. Wash in very dilute aq. sodium hydrate. Isolate individual tubules by teasing with fine needles. Wash, and mount in glycerin. With small mammals Ruber's results were excellent but he was not satisfied with his human preparations. The method has however been well adjusted to the human kidney by Oliver, J., Architec- ture of the Kidney in Chronic Bright's Disease, New York: Paul B. Hoeber, 1939, by a simpler procedure and dis- sected nephrons may be mounted, stained and photographed. A method of montage then affords a demonstra- tion in their natural continuity of the cellular elements of the nephron at high magnification (J. Clin. Invest. 1951, in press). A clear distinction between glomeruli and the renal tubules is important. It is a simple matter to color the former with 1:5000 Janus blue (which is more satisfactory for this purpose than Janus green) in 0.85% aq. sodium chloride by vascular Perfusion and to determine their number, size and distribution against a background of unstained or faintly rose tinged tubules in slices of fresh kidney (Cowdry, E. V., Contrib. to Embryo! . Carnegie Inst., Washing- ton, 1918, 8, 39-160). Perhaps in no other organ is it pos- sible to correlate morphological struc- ture with functional activity so closely and by so many methods as in the kid- ney. See Oliver, J., Am. J. Med., 1950, 9, 88 for a general statement of the problem with various examples of such procedures. As an example, the technique for the microscopic study in KIDNEY 171 KIDNEY vivo of the surface of the guinea pig'a kidney, and for the marking of single tubules with India ink for their later isolation by maceration, is given by Walker, A. M. and Oliver, J., Am. J. Physiol., 1941, 134, 562-595. The micro collection of fluid from single tubules is as the authors state a direct continu- ation of the researches of A. N. Richards. See Oliver, J., Harvey Lec- tures, 1944^5, 40, 102-155. Vital staining of kidney tubules is a valuable method for "seeing" func- tional activity in morphological form. It is usually carried out by techniques not requiring special adaptation. See Vital Staining. The procedure em- ployed by Oliver, J., Bloom, F. and MacDowell, M., J. Exp. Med., 1941, 73, 141-160 deserves mention because it gives a clear demonstration that the cells of abnormal proximal convoluted tubules can be marked by their inability to concentrate trypan blue which con- sequently stains the tubule wall diffusely. This is illustrated in colors. Microscopic observations, having a close relation to function, are easily made on the kidneys of lower forms. See the account of contractility of the ciliated necks of renal tubules in Nec- turus by Lucas, A. M. and White, H. L., Anat. Rec, 1933, 57, 7-11. The functional handling of fluorescent dj^es by the amphibian kidney may be ob- served microscopically in vivo by means of ultraviolet light (Ellinger, P., Quart. J. Exp. Physiol., 1940, 30, 255, and Singer, E., Anat. Rec, 1936, 66, 343). The amphibian kidney also lends itself to the application of the "extra-vital" method where the functional activities (secretion of dyes) of the isolated per- fused kidney are correlated with the structural aspect of the process by subsequent histological examination (Oliver, J. and Smith, P., J. Exp. Med., 1931, 53, 785; Oliver, J. and Lund, E. A., Ibid, 1933, 57, 435). The study of the renal tubules present in tissue cultures is useful in the study of function. Thus Chambers, R. and Cameron, G., Radiology, 1941, 37, 186-193 have found that susceptibility to x-rays is increased when a secretory stimulant is added but that in cultures it is distinctly less than in vivo. See references accompanying this paper. A method has been devised by Crab- tree, C. E., Endocrinology, 1941, 29, 197-203 of measuring by a differential count the number of Bowman's capsules made of cuboidal as contrasted with squamous cells. The count appears to provide an index of age and sex varia- tions in normal mice and of the in- fluence of testosterone propionate on castrated mice. The glomeruli may also be counted in aliquot portions of macerated renal cortex and decreases in their number correlated with the abnormal conditions of disease or senility (Aratabi, M., Am. J. Anat., 1926, 36, 399). Methods for estimating the distribu- tion of enzymes in the tissue components of the rabbit's kidney are given by Weil, L., and Jennings, R. K., J. Biol. Chem., 1941, 139, 421-432. They de- pend on topographic correlation be- tween distribution of cell types in 15 ft frozen sections and decomposition of substrates. The techniques are ca- pable of demonstrating catheptic, ami- nopolypeptidase and esterase activities in all of the epithelial components and of showing that the cells of the proximal and distal convoluted tubules are about twice as active enzymatically as those of the ascending and descending loops of Henle and about 4 times as active as the cells of the collecting tubules. Amylase and dipeptidase activities can also be localized and expressed quanti- tatively in relative terms. For the application to proximal con- voluted tubules in phlorizin glycuresis of the Kabat and Furth procedure for alkaline phosphatase see Kritzler, R. A. and Gutman, A. B., Am. J. Physiol., 1941, 134, 94-101. See Phosphatase. Lipase may also be shown by the pro- cedure of Gomori, G., Arch. Path., 1946, 41, 121. Techniques capable of revealing very interesting data on the shape of cells of the proximal tubule have been devised and employed by Foote, J. J., and Grafflin, A. L., Am. J. Anat., 1942, 70, 1-20. They can probably be employed to advantage in different functional states and to other than renal cells. Methods have been elaborated for measurement of the renal filtration sur- face and data have been supplied for the albino rat (Kirkman, H. and Stowell, R. E., Anat. Rec, 1942, 82, 373-389). The original paper should be consulted. See also the measure- ments of glomerular number and size in various species by Rytand, D., Am. J. Anat., 1938, 62, 507. A valuable collec- tion of measurements of renal struc- tures will be found in v. Mollendorf- Handbuch d. mikr. Anat., 1930, VII-1, 20-34. pH determinations can be made as described by Emmel, V. M., Anat. Rec, 1940, 78, 361-377 by means of a capillary glass electrode (Voegtlin, C. and Kahler, H., Science, 1932, 75, 362) and a vacuum tube potentiometer (Hill, KING'S CARBOL-THIONIN 172 KURLOFF BODIES S. E., Science, 1931, 73, 529). It is significant that increase in activity of the renal cortex immediately follows ligation of the renal artery and that the mitochondria respond by enspherula- tion and fragmentation within 6 min- utes. The kidney is an organ in which mitochondria must be examined with the utmost promptness. A delay in fixation of 15 minutes at room tempera- ture is sufficient to cause disturbances in the mitochondrial rodlets of the proximal convolution. Material from human autopsies is therefore of ques- tionable value. Fuller, R. H., Arch. Path., 1941, 32, 556-568 could find no relation in a rather large number of cases studied between age, hours post- mortem and cause of death (except renal disease) and quantity and distribution of stainable lipoid. The interstitial framework of the kidney in both normal and pathological conditions is well shown by the silver methods that impregnate "reticular" fibers (q.v.). The "polysaccharide" content of the interstitial tissue may be examined by the use of periodic acid (McManus, J. F. A., Amer. J. Path., 1948, 24, 643. See also, Ritter, H. B. and Oleson, J. J., Amer. J. Path., 1950, 26, 639. The blood vessels of kidney may be injected with Neoprene (q.v.) and also with radio-opaque suspensions for a radiographic demonstration of micro- arteriography (Barclay, A. J., Amer. J. Roent., 1948, 60, 1). For a demonstra- tion of the functional status of the renal circulation, the fluorescent dye Vasoflavine may be injected into the living animal and the kidney removed and viewed with ultraviolet light (Moses, J. B., Emery, A. J., and Schlegel, J. V., Proc. Soc. E.xp. Biol, and Med., 1951, 77, 233). The cytoplasmic particulates of the renal epithelium, (mitochondria, micro- somes and droplets of absorbed protein) can be isolated in sucrose suspensions and examined by standard biochemical procedures. Cf. Oliver, J., J. Mt. Sinai Hospital, 1948, 15, 175. King's Carbol-Thionin stain for Nissl bodies (Addison in McClung, p. 450). Stain paraffin or celloidin sections, 2-3 min., in sat. thionin in 1% aq. carbolic acid. Then wash quickly in aq. dest., differen- tiate in 95% alcohol. Pass through equal parts absolute alcohol and chloro- form to xylol and mount in balsam. Kinney's Method for staining reticulum (Kinney, E. M., Arch. Path., 1928, 5, 283). Fix 18 hrs. in 1 gm. sodium sul- phantimonate dissolved in 100 cc. 4% formalin immediately before using. Imbed in paraffin, but more than 1 or 2 hrs. in xylol or cedar oil will remove the dark brown stain from the reticulum. Hematoxylin is contraindicated as coun- terstain because it obscures the color of the reticulum. Other ordinary counter- stains can be used. This method works well even with autopsy material. It is recommended particularly for kidney and pancreas. Results are sometimes patchy in the spleen. Kleinenberg's fixative. Saturated picric acid in 2% aq. sulphuric acid. Embryos and marine organisms. Knee-Joint, method for investigation therein of radioactive gold (Ekholm, R., Acta Anat., 1951, Suppl. 15, 11, 75 pp.). Knisely, see Quartz Rod Technique. Kolatchew Fluid, see Golgi Apparatus. Korfif's Fibers of dentin, see Teeth, De- veloping. Kossa, see his test for Calcium. Krajian's Congo Stain. Elastic fibers (Kra- jian, A. A., Arch. Path., 1934, 18, 378- 380). Fix in 10% formalin, 24 hrs. or more. Cut frozen sections. Wash them in tap water. Place in 2% aq. aluminum chloride 5-10 min. Wash and stain 10 min. in 8 cc. 4% Congo red in 5% aq. sodium citrate -|- 2 cc. glycerin C.P. After washing in tap water trans- fer to 1% aq. KI for 10 sec. agitate. After again washing in tap water, stain 5-10 min. in:anilin blue, 1.5 gm. ; orange G, 2.5 gm.; resorcinol, 3 gm.; phospho- molybdic acid, 1 gm.; aq. dest., 100. Wash carefully in tap water. Blot sec- tions on slides. Dehydrate in absolute alcohol 2 min.; clear in origanum oil; pass through xylol to balsam. Elastic fibers bright red, fibrin dark blue. Krause's End-Bulbs. Methylene blue dem- onstration of in skin of forearm (Wed- dell, G., J. Anat., 1940-41, 75, 346-367). See Skin. Krause's Membrane. Special technique for, see Dahlgren (McClung, p. 427). Kronig's Cement is recommended by Bens- leys (p. 41) for ringing preparations mounted in glycerin jelly or glycerin : 7-9 parts colophonium (resin) melted and stirred with 2 parts beeswax. Kuff, see Nucleic Acid Dye Interactions. Kurloff Bodies are cytoplasmic inclusions which frequently occur in the non-gran- ular leucocytes of guinea pigs. They show particularly well in smears of the spleen, may attain a size equal to that of the nucleus and can be brilliantly colored supravitally by 1:2000 brilliant cresyl blue in physiological salt solution (Cowdry, E. V. chapter in Rivers 'book on Viruses, Baltimore, Williams & Wil- kins, 1928, p. 141). KULTSCHITZKY'S HEMATOXYLIN 173 LEAD Kultschitzky's Hematoxylin is 1 gm. hema- toxylin dissolved in a little alcohol made up to 100 cc. with 2% aq. acetic acid (Lee, p. 526). Lac, a crimson dye obtained from resinous incrustation caused by the insect, Coc- cus lacca, of Siam, Indo-China and Southern India. This dye, introduced into England about 1790 A.D., became an important article of commerce in competition with cochineal of Mexican origin, but before long proved inferior to cochineal and was no longer im- ported. The crimson dyes, Kermes, cochineal and lac have played im- portant parts in the history of civiliza- tion (Leggett, W. F., Ancient and Medieval Dyes. Brooklyn: Chemical Publishing Co., Inc., 1944, 95 pp.) Lacmoid, an indicator similar to Resorcin Blue. Lacteals, see Lymphatic Vessels. Lactoflavin, see Vitamin Bj. Lactophenol, a fixative for Bilharzial Cer- cariae. See Lactophenol-cotton blue technique under Fungi. Laidlaw's Methods. 1. For inclusion bodies (quoted from Pappenheimer, A. W. and Hawthorne, J. J., Am. J. Path., 1936, 12, 625-633, see colored figure, who used it for cytoplasmic inclusions in liver cells). Fix in sat. aq. corrosive sub- limate 100 cc. -f 5% glacial acetic acid or in Zenker's fluid without acetic. Imbed in paraffin, cut sections 3m. Re- move paraffin and pass down to water. Weigert's iron hematoxylin (2%) 5min. Differentiate in 0.5% acid alcohol. Rinse in tap water, then aq. dest. 1% aq. acid fuchsin 5-15 min. Rinse in aq. dest. Mordant in 1% phospho- molybdic acid 30 sec. Rinse in aq. dest. Differentiate in 0.25% orange G in 70% ale. Dehydrate, clear ana mount in balsam. 2. For silver staining of skin and tu- mors (Laidlaw, G. F., Am. J. Path., 1929, 5, 239-247). Fix in Bouin's fluid or in 10% neutral formalin for 3 days. (To make the Bouin's fluid he uses, add 100 cc. commercial formalin and 20 cc. glacial acetic acid to 300 cc. tap water and satu- rate with picric acid). Fix paraffin sections to slides by Masson's Gelatin Glue. Wash Bouin sections for 20 min. in running water, and formalin ones for 5 min. 1% ale. iodine, 3min., rinse in tap water. 5% aq. hypo (sodium thiosul- phate), 3 min., rinse in tap water. ^% aq. potassium permanganate 3 min., rinse in tap water, 5% oxalic acid, 5 min. Wash in running water, 10 min. Aq. dest. 3 clianges in 5-10 min. to clean before adding silver. Heat stock Lith- ium Silver solution to 50°C. and stain in oven for 5 min. Pour aq. dest. over both sides of slides. Flood sections fre- quently for 3 min. with 1% formalin in tap water. Again rinse both sides of slides with aq. dest. 1:500 yellow gold chloride in aq. dest. in Coplin jar at room temperature, 10 min. Rinse both sides with aq. dest. Pour on 5% oxalic acid 10 min. Rinse in aq. dest. Pour on 5% hypo changing as often as it be- comes turbid, 10 min. Wash in running water. Counterstain if desired. De- hydrate, clear and mount in usual way. Reticulum, black threads; collagen red- dish purple. Lake Ponceau, see Ponceau 2R. Lampblack. A colloidal suspension of lamp- black is an excellent substance to inject intravenously to demonstrate phago- cytosis, especially by monocytes. Mc- Junkin, F. A., Arch. Int. Med., 1918, 21, 59-64, advised adding 0.4 gm. of carefully pulverized lampblack to 100 cc. 2% gelatin in aq. dest. Inject intra- venously with 5-9 cc. 10% aq. sodium citrate, as in the case of Higgins'Ink. The method has been slightly modified by Simpson, M. J., J. Med. Res. ,1922, 43, 77-144; Wislocki, G. B., Am. J. Anat., 1924,32, 423-445; and Lang, F.J., Arch. Path., 1926, 1, 41-63. Lanacyl Blue BB (CI, 210), an acid monoazo dye which colors cell walls and paren- chymatous cells light blue but less well than other blue acid dyes (Emig, p. 35). Lanacyl Violet B (CI, 207), an acid monoazo dye of light fastness 3. Directions for staining plant tissue and fungous my- celia (Emig, p. 35). Langerhans, see Islets of. Lard, reactions in tissue to fat stains after various fixations (Black, C. E., J. Lab. & Clin. Med., 1937-38, 23, 1027-1036). Large Intestine. The conditions that in- fluence the appearance of sections are easier to guard against than in the Small Intestine because of the absence of villi and greater uniformity of contents. The pronounced influence of degree of distention is described and well illus- trated by Johnson (F. P., Am. J. Anat., 1912-13, 14,235-250). Lansing, see Collagen, Elastin. Latex-Cast Techniques for study of the circulation have been applied to the spleen by Gall, D. and Maegraith, M. G., Ann. Trop. Med. & Parasit., 1950, 44, 331-338, who give good illus- trations of results and references to previous work. Lauth's Violet, see Thionin. Lead, histological demonstration. 1. Mallory and Parker's method (Mal- lory, F. B. and Parker, F. J., Am. J. Path., 1939, 15, 517-522) : Fix tissues in 95 or abs. alcohol (not formalin). Stain celloidin sections at 54°C. in: 5-lOgm. LEATHER BROWN 174 LEISHMANIA. MEDIA hematoxylin dissolved in few drops abs. or 95% alcohol + 10 cc. freshly filtered 2% aq. K2HPO4 for 2-3 hrs. Wash changing tap water 10-60 min., dehy- drate in 95% ale, clear in terpineol and mount in terpineol balsam. Lead light to grayish blue, nuclei deep blue. Another method applicable to paraffin sections of Zenker fixed material is to stain in 0.1% methylene blue in 20% ale. 10-20 min. Differentiate 10-20 min. in 95% ale, dehydrate, clear and mount. Phloxine is recommended as a contrast stain before the methylene blue. 2. Chromate method (Frankenberger, Cretin). By simply fixing in Regaud's Fluid lead is precipitated as insoluble yellow lead chromate easily identifiable microscopically. This method is strongly advised by Lison (p. 101). It has been used by True (E., Bull. d'Hist. Appl., 1929, 6, 393-399). See Sieber (E., Arch. f. exper. path. u. pharmak., 1936, 181, 273-280) for demon- stration of lead in bones. 3. Attempts have been made to identify lead after microincineration by exposure to hydrogen sulphide, because lead sulphide is black, but Gordon H. Scott emphasizes difficulty in dis- tinguishing it from other sulphides and from carbon in imperfectly incinerated specimens (McClung, p. 660). 4. The method of Sieber, E., Arch. f. exper. Path. u. Pharmak., 1939, 181, 273 depending on production of acid resistant brown-black lead sulfide when tissue is treated with acidulated H2S solution is said to be satisfactory by Gomori, G., J. Mt. Sinai Hosp., 1944- 45, 11, 317-326 when presence of other heavy metals is ruled out. Methods for chemical determination of lead in biological materials are important as checks on above. Consult Smith, F. L. 2nd., Rathmell, T. K. and Williams, T. L., Am. J. Clin. Path., 1941, 11, Suppl. 5, 653-668. For a convenient method of giving colloidal lead intravenously to rabbits see Crawford, B. L., Stewart, H.L., Willoughby, C. E. and Smith, F. L., Am. J. Cancer, 1938, 33, 401^22. The authors describe techniques for direct analysis of lead in the tissues. Leather Brown, see Bismark Brown Y. Leather Yellow, see Phosphine. Leblond, see Radioantographic Technique. Lebowich's soap-wax technique eliminates use of alcohol , xylol and overnight drying of paraffin sections. Takes only 6-8 hrs. (Moritz, C. E., Stain Techn., 1939, 14, 17-20). L. E. Cells. The discovery of these cells in acute disseminated lupus erythe- matosus by Hargraves, M. M., Rich- mond, M. and Morton, R., Proc. Staff Meet., Mayo Clin., 1948, 23, 25-28, was a definite advance in diagnostic pro- cedure. The simplest test for L. E. cells is that of Lee, Stanley L., Am. J. Clin. Path., 1951, 21, 492-496. With- draw 1-2 cc. venous blood into a clear dry test tube. Let clot and remain at room temperature for 2 hrs.; with wooden applicator "fish out" the clot. Lecithin, a compound of phosphoric acid, glycerol, choline and 2 fatty acid mole- cules. It is a phosphatide soluble in alcohol, chloroform, ether and benzene, see Lipoids. Lee-Brown. Modification of Mallory's ani- line blue connective tissue stain (Lee- Brown, R. K., and Laidley, J. W. S., J. Urol., 1929, 21, 259-274). Mallory (p. 155) states that the following tech- nique is particularly valuable for the kidney. Treat paraffin sections of Zen- ker fixed material with iodine to remove mercury. Wash. 1% aq. phosphomolyb- dic acid, 30 sec. Wash in aq. dest. 1-2 min. Stain in: aniline blue, 0.5 gm.; orange G., 2 gm. ; phosphomolybdic acid, 2 gm.; aq. dest., 100 cc. for 30 min. at 55 °C. Wash in an. dest 2-5 min. 1% aq. phosphomolybdic acid, 30 sec. 95% ale . , abs . ale . , xylol , balsam . Glomerular basement membrane and collagen, deep blue; nuclei, orange. Leishmania Donovani, a search for stains that will color more rapidly than Giemsa revealed Astra violet F. F. Extra, Himmelblau, Magenta Lermont and Navy blue shade, each to be used in fresh 10% aq. solution (Takasaki, S., Lues, Tokyo, 1938, 16, 127). Leishmania. Media. Direct microscopic examination of peripheral blood may be negative while detection in culture is feasible. Q. M. Geiman (Simmons and Gentzkow) recommends addition of 10 cc. blood to sodium citrate in physiological saline, centrifuge and in- oculate few drops buffey coat into tubes of NNN medium, incubate 22-28°C. and examine microscopically 10-20th day for motile forms. The following media are abbreviated from Geiman 's ac- count. 1. Blood agar or NNN (Novy, Mac- Neal and Nicolle, 1908). Agar, 14 gm., sodium chloride, 6 gm., aq. dest. 1000 cc. Add 5 vol. sterile defibrinated rabbit's blood cooled to 45°-50°C. Mix, tube long slant. After agar sets, cap with sterile rubber stoppers. Prove sterility by incubation 37°C., 24 hrs. Inoculate material to be cultivated on slant and in water of condensation. Incubate 20°-25°C. Transfer every 20- 30 days to maintain. LENGTH 175 LEUCOCYTES 2. Leptospira (Noguchi, 1924). 0.9% aq. sodium chloride, 800 parts; fresh rabbit serum, 100 parts; 2% nutrient agar pH 7.2, 100 parts, rabbit hemo- globin solution 10-20 parts. (To make this hemo'^^lobin solution take 1 part defibrinated rab!)it's blood and 3 parts aq. dest., centrifuge and use clear super- natant fluid.) Tube, prove sterility by incubation before using. Subculture every 30 days. An increase in hemo- globin solution improves growth of Leishmania. 3. Adler's modification of above. Agar, 1 part; Locke's solution contain- ing 0.2% dextrose, 8 parts; fresh rabbit serum, 1 part. For species of Leish- mania and Trypanosoma cruzi. 4. Modified, Salle and Schmidt (Cleveland and Collier, 1930). Veal infusion (50 gm. Bacto-veal, Difco -t- 1000 cc. aq. dest.), 250 cc; proteose, peptone (Difco), 10 gm.; sodium chlo- ride, 5 gm.; aq. dest., 550 cc. Dissolve make pH 7.4 and autoclave. Add 20 cc. 50% aq. glucose (sterilized by filtration or in autoclave 10 lbs., 10 min.) and 60 cc. horse red cells laked with 2 parts aq. dest. Pour in medium flasks or tubes. Vigorous long lived cultures. Length measurements : Millimeters to inches X 0.0394. Inches tomm. X25.4. SeeMicron. Lens Paper, a specially prepared soft paper indispensible for cleaning immersion oil from objectives. Leprosy Bacilli. Stain by carbol-fuchsin in smears. See Concentration method for collecting bacilli from lesions. For study in sections, see Acid Fast Bacilli. Leptospira Medium, Noguchi's, see Leish- mania. Leptospiras, method for isolation from water (Bauer, J. H., Am. J. Trop. Med., 1927, 7, 177-179. See Spirochetes. Leuco Basic Fuchsin. To make add to 200 cc. aq. sol. fuchsin, 2 gm. potassium metabisulphite and 10 cc. N hydro- chloric acid. After bleaching 24 hrs. add 0.5 gm. Novit, shake 1 min. and filter through coarse paper. Resulting clear solution works nicely in Feulgen tech- nique (Coleman, L. C, Stain Techn., 1938,13, 123-124). Leuco-Dyes as vital stains. Make 001% aq. solutions of methylene blue,azurA, thionin toluidine blue and brilliant cresyl blue. Add to 100 cc. 1-2.5 cc. N/10 NajSaO, and 1-4 cc. N/10 HCl. Mix and store at room temperature in dark. To stain, add 1-2 drops of leucobase to the protozoa, blood cells, etc. in physio- logical saline. Said to give good contrast staining of nucleus and cytoplasm and to be useful in oxidation-reduction determinations (Roskin, G., Arch. Russ. Anat. Hist. Embr., 1937, 16,107-109). Leuco-Patent Blue V, see Lillie, p. 285. Leucocytes. In the broad sense they in- clude all white blood cells but the term is generally restricted to the "granular" leucocytes as compared with the "non- granular" ones (Lymphocytes and Mon- ocytes). In a still narrower sense the leucocytes include only polymorphonu- clear neutrophiles, eosinophiles and basophiles which are easily found in circulating blood as contrasted with less differentiated leucocytes called Myelo- cytes and Myeloblasts generally con- fined to the bone marrow. For mitochondria within leucocytes supravital staining with Janus green is indicated. In smears Giemsa's stain has a little advantage over Wright's in the fact that it better demonstrates any bacteria that may be present. The May-Giemsa technique is most used in Europe. It is, in effect, a double staining because the air dried smears are first treated with the May-Grunwald com- bined fixative and stain and are later colored by Giemsa's stain. It gives satisfying deep colors. TheKardos- Pappenheim modification is suggested when a particularly intense coloration of neutrophilic granules is desired. Ehr- lich's triacid stain may likewise be use- ful because it is said to stain the neutro- philic granules leaving the azur granules untouched. Leucocytes give strong Peroxidase and Oxidase reactions, which are, how- ever, not specific for them. The Golgi Apparatus (reticular material) can be demonstrated by long treatment with osmic acid or by the Cajal uranium ni- trate and silver method (Cowdry, E. V., J. Exper. Med., 1921, 33, 1-11). The demonstration of degenerative leucocytic changes associated with ageing is de- scribed by Lowell (A. L., J. Lab. & Clin. Med., 1937-38, 23, 791-796), of variability in relation to alterations in meteorologic conditions by Berg (M., J. Lab. & Clin. Med., 1937-38, 23, 797-803) and of lipoid components by Bacsich (P., J. Anat., 1935-36, 70, 267-272). Chemotaclic re- sponse and motility can be measured both in tissue cultures (Comau, D. R. Arch. Path., 1940, 30, 896-901) and directly by observing the behavior of leucocytes with relation to bacteria and in temporary mounts (Mallery, O. T. and McCutcheon, M., Am. J. Med. Sci., 1940, 200, 394-399 ) . By the latter method differences in behavior of neutrophiles from seriously ill and normal persons have been reported . Motion pictures are of great assistance in making a thorough analysis of the movements and behavior of leucocytes. Some excellent ones, LEUCOCYTE COUNTS 176 LEUCOCYTES taken by Dr. W. H. Lewis, are available for distribution by the Wistar Institute of Anatomy in Philadelphia. To in- vestigate their behavior after they have left the blood vessels and entered the surrounding tissues is immensely more difficult. The only method that gives promise of important results is to employ for this purpose special chambers in- serted in the ears of rabbits (Clark, E.R. and E. L., Am. J. Anat., 1936, 59, 123- 173) . See Neutrophile, Eosinophile and Basophile Leucocytes. Leucocyte Counts, 1. Total number white blood cells per c. mm. Over 12,000 a leucocytosis, less than 5000, a leucopenia. Average about 7,500. 2. Differential. Smears colored by Giemsa's or Wright's stains are more satisfactory than supravi tally stained preparations because the latter are more difficult to handle and the cells are slowly dying and showing more and more deviations from normal. Relative num- ber of different white cells is expressed in percentages, i.e. neutrophiles 55-75, eosinophiles, 2-4, basophiles, 0-1, lym- phocytes 21-31, and monocytes 4-5. Both total and differential counts should be correlated to avoid misconceptions. 60% neutrophiles in total count of 8,000 amounts to 4,800 neutrophiles per c. mm. 80% neutrophiles in total count of 4,800 is the same, namely 4,800 neutrophiles per c. mm. although a relative neutro- philic leucocytosis exists. 60% neutro- philes in a total count of 16,000 makes on the other hand 9,600 neutrophiles per c. mm. which is an actual neutro- philic leucocytosis. 20% lymphocytes of 9,000 is the same number per c. mm. as 60% of 3,000 ; while 30% of 11,000 is an actual lymphocytosis. 3. Age. Since young neutrophiles have fewer nuclear lobes than older ones counts of the number with from 1-5 lobes were made by Arneth. Today simpler methods are used. The Schilling is the usual one. It is both a total, a differential and an age count combined. The normal is given Total 5,000 to 10,000 £ d-l E 2-4 M 0 J 0-1 Leucocytes St 3-5 S 51-67 L 21-35 Mon. 4-6 above. B = basophile. E = eosino- phile. M = myelocyte (Nucleus large, occupying about half cytoplasmic area. spherical to oval or kidney-shaped, pale staining, chromatin reticulated, nu- cleoli present. Cytoplasm faintly basophilic with few specific granules which are small, often difficult to stain and irregularly distributed). J = juvenile (A little larger than mature neutrophiles. Nucleus saucer to bean shaped. Stains poorly. Circum- scribed basophilic nucleoli). St = stab nuclear (Slightly smaller than juveniles. Nucleus T V or U shaped but not divided into segments by fila- ments and without nucleoli). S = seg- ment nuclear (Fully differentiated neu- trophiles having 2-5 or more segments often joined only by filaments. Nuclei stain intensely.) L = lymphocyte. Mon = monocyte. When the numbers of M. J. St. are increased relative to S., it is called a "shift to the left", meaning that im- mature leucocytes are called into the circulation , which is an unfavorable sign. When the relative number of S is in- creased over the others, it is termed a "shift to the right", meaning that only mature leucocytes are called out, which is a favorable sign if it follows a previous shift to the left. Details are given by Wintrobe, M. M., Clinical Hematology, Philadelphia, Lea & Febiger, 1942, 792 pp. For blood containing gum acacia, see Monke, J. V., J. Lab. & Clin. Med., 1940-41, 26, 1664-1667 and for inter- ference by decreased fragility of eryth- rocytes see Bohrod, M. G., J. Lab. & Clin. Med., 1940-41, 26, 1953-1955. A better method, unfortunately not widely employed, is the filament-non- filament count. Filaments are neutro- philes in which the nuclear segments are connected by delicate strands apparently made up of nuclear membrane only and nonfilaments are those in which the connections are so wide that they can be resolved into nuclear membrane plus nuclear contents. In 100 neutro- philes there are normally 8-16 nonfila- ment cells. A greater per cent is a shift to the left. For counts see Krusen, F. H., Am. J. Med. Sci., 1937, 193. 470- 474. Leucocytes. Developmental series. The technique employed apparently makes a great deal of difference in the conclu- sions reached. See Cowdry's His- tology, p. 99. 1. Maximow and Bloom employing mainly permanent preparations list: Hemocytoblasts: "... large (up to 15) ameboid, non-granular basophil cells of lymphoid nature." Occur extra vascu- larly. Promyelocytes: "The oval or kidney- shaped, clear nucleus contains a loose LEUCOCYTIC INDEX 177 LIEBERMANN-BURCIIARDT chromatin network and several nucleoli. At the indentation of the nucleus there is a distinct cytocentrum . The ame- boid protoplasm is slightly basophil, although it often shows acidophil areas." Specific granules "are scarce and usual- ly confined to the periphery of the cyto- centrum and to the acidophil spots in the cell body." Azurophil granules are present but later disappear. They often show mitosis. Myelocytes: "The protoplasm becomes diffusely acidophil while the specific granules increase in number and fill the whole cell body, except for the cyto- centrum. The nucleus keeps its com- pact form while its previously loose chromatin network becomes coarser and stains darker. The nucleoli are indis- tinct. Mitoses are common." Metamyelocytes: After an unknown number of mitoses a generation appears. The nucleus "as soon as it is recon- structed after the last mitosis, shows a beginning polymorphism and has the shape of a horse-shoe." The mature leucocyte is formed from these cells by individual maturation without division. 2. Sabin and associates relying chiefly on supravital stains list : Reticular cells: They "are small, their cytoplasm is faintly basophilic, as seen in fixed films, and in supravital prepa- rations they show no differentiation of specific substances." Reticular cells "lack the striking rod-shaped mito- chondria which characterize the lympho- cytic strain. . . . The nuclei have less sharp contours and less chromatin than those of lymphocytes." Myeloblasts: These differ "through the elaboration of a marked basophilia and of great numbers of small mito- chondria. ... In supravital technique, the myeloblast has usually no stainable substance except mitochondria . . ."but occasionally a few vacuoles reacting to neutral red are present as well as some which are not colored by it. Myelocytes A: The earliest stage with the specific granules up to 10 "reacts with a single blue granule in the oxydase test." Myelocytes B: "May be conveniently divided into those with less than half and those with more than half the full quota of granules." Myelocytes C: These cells contain the full quota. Metamyelocytes: They "show the earliest signs of the nuclear changes toward polymorphism and the first sign of the transformation of the cytoplasm to a phase sufficiently fluid to allow the flowing of granules which is essential for ameboid movement. In passing through these stages, there is a gradual decrease of basophilia of the cytoplasm and in the numbers of mito- chondria. The basophilia disappears entirely in the early leucocytes, while the mitochondria persist in small num- bers until the stage of senility in the leucocytes." Leucocytic Index, ratio of number of pol- ymorphs to number of lymphocytes, considered by Turley, L. A. and Mc- Clellan, J. T., Am. J. Clin. Path., 1943, 7, 87-95 to be valuable indicator of condition of the patient, a high or rising index being a bad sign and a low or fall- ing one, a favorable sign. Leucocytic Infiltrations. A convenient way to produce an intense local neutrophilic infiltration is to inject starch as de- scribed by Chambers, R. and Grand, C. G., Am. J. Cancer, 1937, 29, 111-115. Cowdry, E. V. and Ruangsiri, C, Arch. Path., 1941, 32, 632-640 made repeated injections of 1% corn starch suspensions in physiological saline in amounts of 0.1-0.2 cc. into leprous nodules of rats. Leucocytozoa, Protozoa, belonging to the Hepatozoidae, which inhabit the mono- cytes of dogs, rats, and other animals particularly in the tropics. See, Wen- yon, C. M., Protozoology. New York: William Wood & Co., 1926, 2,1053-1563. Leucosin, a stored reserve in lower plants (Taylor in McClung, p. 221). Levitation Method, see Floatation Method. Levulose Syrup for fluid mounts. Mallory (p. 99) specifies 30 gms. levulose dis- solved in 20 cc. water by warming at 37 °C. for 24 hrs. Lewis-Locke solution, see Locke-Lewis. Leydig Cells are usually known as inter- stitial cells of the Testis which see. Liang uses Schiff reagent for nerve endings (Liang, H. M., Anat. Rec, 1947, 97, 419). Lieberkiihn's Glands, data on size, surface area, number of cells etc. in human large intestine (Policard, A., Bull. d'Hist. Appl., 1939, 16, 261-262). Liebermann-Burchardt reaction for choles- terol and its esters {cholesterides) . 1. Modification of A. Schultz. Ex- pose frozen sections of formalin fixed tissue at least 4 days (more in winter) to strong light, if possible sunlight. Mount. Dry carefully with blotting paper. Cover with few drops equal parts acetic and sulphuric acids. Drain and examine in the reagent. Cholesterol and its esters dark blue or red purple becoming green. 2. Modification of Romieu (M., C. rend. Acad. d. Sci., 1927, 184, 1206-1208) Mount frozen sections of formol or Bouin (less acetic) fixed tissues and dry. Cover with 1 drop cone, sulphuric acid, 3-15 sec. Stop reaction by adding 2-3 LIGHT BLUE 178 LINDERSTR0M-LANG, ET AL drops acetic anhydride. Wash with several drops of same. Cover and examine immediately. Cholesterol and its esters violet lilac or red purple, be- coming green. The above two methods abbreviated from Lison (p. 210) are in his excellent judgment specific for cholesterol and its esters if positive. A negative reaction does not definitely prove their absence. See Swyer, G. I. M., Cancer Research, 1942, 2, 372-375 for quantitative measurement of the color. Light Blue, see Spirit Blue. Light Green, see Methyl Green. Light Green N, see Malachite Green. Light Green SF yellowish (CI, 670) S— acid green, fast acid green N — Commission Certified. This acid di-amino tri- phenyl methane dye is a sulfonated derivative of brilliant green and a valuable counterstain for safranin. It is used by Twort, F. W., Brit. J. Exp. Path., 1924, 5, 350-351 as a double stain with neutral red for animal parasites and microorganisms in tissues. Un- fortunately light green fades quickly. Conn (p. 110) recommends fast green FCF as a substitute. Lighting, see Illumination. Lignin Pink, a monazo acid dye (British Drug Houses Ltd.). Advised 0.5% aq. solution as a chitin stain and a contrast stain with chlorazol Black E (Cannan, H. G., J. Roy. Micr. Soc, 1941, 61, 88-94). Lilienfeld-Monti test for phosphorus is not a satisfactory microchemical method. See Bensley's method (R. R., Biol. Bull., 1906, 10, 49-65) and criticism by Lison (p. 118). Lillie, see Azure or Toluidin Blue Eosin. Lillie's chrom-osmic-acetic fixative. J% aq. chromic acid, 15 cc; 2% aq. osmic acid, 3.5 cc. ; glacial acetic acid, 3 drops. Used by him for echinoderm eggs. Lime, see Calcium. Linderstr0m-Lang, Kaj. U., and Holier, Heinz, Histochetnical Advances — Written by David Glick, Dept. of Physiological Chemistry, University of Minnesota, Minneapolis 14-, Minn. Octo- ber 17, 1951 — Prof. Linderstr0m-Lang, since 1938 the head of the Department of Chemistry of the Carlsberg Labora- tory, Copenhagen, Denmark, and Dr. Heinz Holter, his associate and head of the Cytochemical Department, are responsible for some of the most sig- nificant advances in the field of quanti- tative histochemistry as well as of protein and proteolytic enzyme chemis- try. Their histochemical work began in 1930 when Dr. Holter came to the Carlsberg Laboratory. Their first ef- forts were directed to the development of micro titration techniques that would enable a thousandfold refinement of common macro methods of biochemi- cal analysis without loss of precision. This degree of refinement constituted a temporary compromise since it was their goal to bring reliable quantitative chemical techniques to bear on the study of the single cell, and a hundred thousandfold refinement would be re- quired to achieve this end in the case of mammalian cells. However the one thousandfold refinement did permit work on single microtome sections, protozoa, and ova of certain marine invertebrates. The first investigation of the series (Linderstr0m-Lang, K. and Holter, H., Compt. rend. trav. lab. Carlsberg, Ser. Chim., 1931, 19, No. 4) described the micro titration apparatus that was de- vised, (the burettes used were graduated in 0.0002 ml. divisions and readings were taken to 0.00002 ml.) and subsequently Linderstr0m-Lang and Holter adapted the equipment to the measurement of proteolytic enzyme activity. They em- ployed this method for a study of the distribution of peptidase along the roots and sprouts of barley (Linderstr0m- Lang, K. and Holter, H., Ibid., 1932, 19, No. 6). When one has developed a new tool it is natural that he employ it for diverse purposes to test its range of usefulness. And accordingly, in the next application of their technique, Holter and Linderstr0m-Lang (Zeit. f. Physiol. Chem., 1932, 219, 223-240) in- vestigated the proteinases of Drosera Rotundi folia, an insectivorous plant, and then Linderstr0m-Lang (Ibid, 1933, 215, 167-178) made a study of the pepti- dase content of single eggs of two marine invertebrates, the California sand dollar and Urechis caupo, before and after fer- tilization. These eggs have a wet weight of only about 5 X 10~' g. Linderstr0m-Lang and Holter next extended their technique to a method for the iodometric determination of reducing sugars with a precision corre- sponding to 25 X 10~* g. of glucose (Compt. rend. trav. lab. Carlsberg, 1933, 19, No. 14), and this was followed by the development of a diffusion method for ammonia sensitive to 14 X 10~' g. of nitrogen (Compt. rend. trav. lab. Carlsberg, Ser. Chim., 1933, 19, No. 20.) The elegance and general applica- bility to diverse problems of the tech- niques and methods being developed began to attract research workers to the Carlsberg Laboratory from other countries, and the series of publications that had evolved under the heading of LINDERSTR0M-LANG ET AL 179 LINDERSTR0M-LANG ET AL Studies in Enzymatic Histochemistry soon had contributions from T. Philipson, who investigated the peptidase activity of single centri- fugally separated parts of eggs of Psammechinus miliaris {Ibid, 1934, 20, No. 4), and D. Glick, who elaborated an acidimetric method for lipolytic en- zymes {Ibid, No. 5) and applied it to a study of the distribution of esterase in the gastric and duodenal mucosa of the hog (Glick, D. 76id, No. 11). The latter was undertaken since Lin- derstr0m-Lang and Holter had already underway similar investigations of the distribution of pepsin {Ibid, 1935, 20, No. 11), acid {Ibid, 20, No. 11), and peptidase {Ibid, 20, No. 11). A. S0e- borg-Ohlsen joined in a collaborative study of the enzj'me distribution in the hog stomach as a function of its histo- logical structure {Ibid, 20, No. 11) and the enzyme pattern in the mucosa was revealed to a significant degree. The need of methods for the chemical determination of additional cellular constituents diverted the Carlsberg Laboratory workers and visiting scien- tists to the task of elaborating them, and for the visitors this served the ad- ditional purpose of providing excellent first hand training and experience in the use of the techniques. With Palmer, A. H. {Ibid, 1935, 21, No. 1), an electrometric chloride titration was developed with a precision of about 14 X 10-' g. of chloride; Levy, M. {Ibid, 1936, 21, 101-110) refined the Kjeldahl nitrogen analysis to 3 X 10""® g. of nitrogen, Linderstr0m-Lang (Compt. rend. trav. lab. Carlsberg, Ser. Chim., 1936, 21, 111-122) worked out an electro- metric titration method for sodium plus potassium sensitive to 1 X 10"^ m. equiv., Norberg, B. {Ibid, 1937, 21, 233-241) evolved a titrimetric estima- tion of potassium isolated as the iodoplatinate that was accurate to 1-2 X 10— ' m. equiv., and with Weil, L. {Ibid, 1935, 21, 7-14) micro methods for arginase and urease were established. Linderstr0m-Lang, K. and Engel, C. {Ibid, 1938, 21, 243-258) employed the method that had been previously de- veloped for reducing sugars for the measurement of amylase activity, the distribution of which they studied in barley, and Holter and Doyle, W. L. {Ibid, 1938, 22, 219-225) subsequently modified the technique to gain greater precision. Meanwhile micro methods for other enzymes were being developed. Holter and Doyle (J. Cell Compt. Physiol., 1935, 12, 295-308) adapted the iodometric method of Stern to the esti- mation of catalase with a precision equivalent to the decomposition of 2 X 10"* g. of hydrogen peroxide, and Glick, D. (Compt. rend. trav. lab. Carlsberg, Ser. Chim., 1938, 21, 2G3-268) extended his esterase method to include the meas- urement of cholinesteraso with a sensi- tivity equivalent to the hydrolysis of 1 X 10~* mole of ester. As the new methods were made avail- able their application to specific prob- lems followed at once. Thus, Linder- str0m-Lang, K., and Duspiva, F., (Ibid, 1936, 21, 53-84) studied the digestion of keratin by larvae of the clothes moth, and Duspiva followed this work by in- vestigations of various proteolytic en- zymes of clothes- and wax-moth larvae {Ibid, 1936, 21, 177-202), as well as of pH of the intestinal juice of these organ- isms for which a micro glass electrode was used {Ibid, 21, 167-176). Holter (J. CellComp. Physiol., 1936, 8, 179-200) studied peptidase localization in marine ova, with Kopac {Ibid, 1937, 10, 423- 437) the localization of this enzyme in amoeba, and with Doyle, W. L. (Compt. rend. trav. lab. Carlsberg, Ser. Chim., 1938, 22, 219-225) the amylase localization in amoeba. Doyle {Ibid, 1938, 21, 291-299) also investigated the catalase and peptidase activity in ma- rine ova. The activation of leucylpep- tidase of single Tubifcx eggs by magnes- ium salts was reported by Holter, H., Lehmann-Bern, F. E. and Linderstr0m- Lang (Compt. rend. trav. lab. Carls- berg, Ser. Chim., 1938, 21, 259-262). Other applications in this period in- clude a study of the distribution of urease in the dog stomach by Linder- str0m-Lang and S0eborg-Ohlsen, A. (Enzymologia, 1936, 1, 92-95), and an investigation of the cholinesterase dis- tribution in the hog stomach before and after administration of drugs that affect gastric secretion by Glick {Ibid, 1938, 21, 269-281). Using a titrimetric dye method previously worked out (J. Biol. Chem., 1935, 109, 433-436) Glick fol- lowed the changes in ascorbic acid in different parts of the developing barley embryo up to the ten-day sprout stage {Ibid, 1937, 21, 203-209). Titrimetric procedures alone had been exploited up to this time. But in the attempt to extend the applicability of this approach to quantitative histo- chemistry, other analytical principles were tested. Colorimetry, if conducted on the required micro scale, would ob- viously open a vast new region for exploitation, but the limiting factor in the middle nineteen thirties was the availability of the necessary equipment. At that time, the Zeiss Pulfrich step photometer, that employed cuvettes re- LINDERSTR0M-LANG ET AL 180 LINDERSTR0M-LANG ET AL quiring 0.2 ml. of liquid, was used by Lundsteen, E. and Vermehren, E. (Ibid, 1936, 21, 147-166) at the Carlsberg Laboratory for the determination of phosphorus and phosphatase in 0.05 ml. blood samples. But for many histo- chemical purposes the liquid volume needed for the colorimetric measure- ment was still too great. This problem was largely solved subsequently as will be mentioned later. In 1937 two new analytical techniques were ingeniously developed by Linder- str0m-Lang for micro-biochemical re- search. One of these was based on the dilatometric principle that in certain reactions a volume change of the total solution occurs which results in a change in specific gravity great enough for measurement. As applied by Lin- derstr0m-Lang (Nature, 1937, 139, 713- 714), a density gradient is set up by half -filling a glass tube with a kerosene- bromobenzene mixture of appropriate specific gravity, and then filling the re- mainder of the tube with a similar mix- ture having a greater proportion of kerosene to give a lower specific gravity. If the liquid in the middle of the tube is stirred, and the tube mounted in a thermostat, a stable vertical density gradient is produced. If into this gradient an aqueous drop of a reaction mixture is placed, it will fall until it reaches the level at which its specific gravity equals that of the surrounding medium. As the reaction within the drop proceeds, the resultant volume change of the drop will alter its specific gravity and the drop will then move to a new level. In some cases the distance the drop will move has been found to be directly proportional to the extent that the chemical reaction has occurred. By employing a cathetometer the move- ment of the drop can be followed to 0.01 mm. and finer movements can be ob- served with an ocular micrometer in the telescope of the instrument. After cali- bration of the gradient with drops of salt solution of known specific gravity, measurements of the excursion of the drop can be used to calculate the amount of reaction products formed in certain instances. The dilatometric method was em- ployed by Linderstr0m-Lang and H. Lanz, Jr. (Ibid, 1938, 21, 315-338), for peptidase measurements, and a study was made by Holter, Lanz, and Linder- str0m-Lang {Ibid, 1938, 23, 1^9) of this enzyme during the first cell divisions in the egg of Psammechinis miliaris. Linderstr0m-Lang, Jacobsen, O. and Johansen, G. {Ibid, 17-25) also em- ployed the density gradient tube for the measurement of the deuterium con- tent of heavy water mixtures. The other new technique that was evolved at this time was based on the principle of the Cartesian diver (Lind- erstr0m-Lang, K. Nature, 1937, 140, 108-108). The diver is a small vessel, open at one end, that will rise or fall, while submerged in a salt solution, as a result of a change in its gas volume. The volume of the first divers used was about 0.01 ml., and a reaction mixture of about 0.001 ml. placed in the bottom of the diver was employed for gaso- metric measurements of enzyme activity or respiration. The measurement was carried out by applying a known posi- tive or negative pressure to the air over the tube containing the submerged diver to bring it to a marked level in the tube. At this level the gas volume in the diver has a fi.xed value, and by determining the external pressure necessary to bring the gas volume back to this value after expansion (in case of a reaction evolving gas) or contraction (in case of reaction using up gas), the actual amount of gas evolved or con- sumed can be calculated. In other words the apparatus is a constant volume, variable pressure, unit similar in this respect to the ordinary Warburg apparatus but capable of measuring gas changes of a magnitude a thousand times smaller. Further refinements were developed later and will be mentioned. The first applications of the Cartesian diver method were to the gasometric measurement of cholinesterase activity in ganglia, Linderstr0m-Lang and Glick (Compt. rend. trav. lab. Carlsberg, 1938, 22, 300-306), the determination of thiamine and cocarboxylase in yeast, Westenbrink, H. G. l-256). Observa- tion of lung through thoracic window in vivo (Terry, R. J., Science, 1939, 90, 43-44), See Alveolar Epithelium, Alveo- lar Fluid, Alveolar Foam Cells, Alveolar Pores, Alveolus, Ammoniacal Silver, Bronchiolar Epithelium, Carmine Dust- ing, Celluloid Corrosion, Dust Cells, Gash Irrigation, Heart Failure Cells, Pneumonocytes, Silver Lineation, Tis- sue Phagocytes, Vacuoloids, Wash-out Recovery Method. Lungs, Uncollapsed, Fixation — Written by LUTECIUM 186 LYMPHATIC VESSELS C. C. Macklin, Dept. of Histological Research, The University of Western Ontario, London, Canada. November 28, 1951— This may be done (1) by prompt immersion of the fresh flayed intact thorax (IIT) in any good fixative which is adaptable to small animals, such as mice; (2) by perfusion of the blood vessels of the intact thorax of the recently exsanguinated animal (PIT); (3) by injection of the fixative into the trachea with the thorax unopened (BF). This is known as bronchial filling. For technique of methods Nos. 1 and 2 see Dust Cells. For method No. 3 a cannula is tied into the trachea and fixing fluid injected in amount equal to one-third to one-half of the volume of the lung in full expiration. Presence of air does not prevent spread. Fix- ation is rapid. After tying the trachea the preparation is allowed to stand one to twenty-four hours. This method may be combined with No. 1. Methods 1 and 3 may be used without previous exsanguination. In methods 1 and 2 the capillaries of the alveolar walls are fully opened, but in 3 they are only partly so. Methods 1 and 2 possess the advantage of not having loose particles washed into the lower part of the air tract. Other advantages are con- sidered under "Dust Cells". Methods 1 and 2 demonstrate the close and ex- tensive relation of the capillaries of the alveolar wall to the pneumonocytes (Macklin, C. C, Trans. Roy. Soc. of Can., Sect. V, 1946, 40, 93-111). Lutecium, see Atomic Weights. Lutein, see Lillie, p. 129. Luyet, see Revival after Ultra Rapid Cooling. Lymphatic Vessels. There are many ways of demonstrating lymphatic vessels. The most convenient is to sit in an easy chair and view the splendid moving picture prepared by Dr. Richard L. Webb of the Department of Anatomy of the University of Illinois College of Medicine entitled: "Mesenteric lym- phatics, their conduct and the behavior of their valves in the living rat". Another easy method is to watch absorption of cream in a cat. A fasting animal is fed | pint of cream and the abdominal cavity is opened under ether anesthesia a few minutes later. At first sight it may be difficult or impossible to see any lyroplmtics in the mesentery although a few bean shaped lymph nodes are visible near its base and can be easily felt. Keep the abdominal con- tents moist with saline. Close the opening. In a little while, when again examined, the lymphatic vessels will be clearly marked in white by the milk fat which has been absorbed by the lacteals and is being transported in them. A simple method to visualize the pathways of lymphatic drainage from the nasal mucous membrane has been described by Yoffey, J. M., Lancet, 1941, 1, 529-530. Anesthetize a cat. Drop into each nostril 1 cc. 5% trypan blue (T. 1824) in physiological saline (0.85% aq. NaCl). T. 1824 is specified because it is a trypan blue isomer which is deeply colored even in high dilutions but any good trypan blue will do. Dis- sect away the side of the neck. Lymphatic vessels, deeply stained, will be seen from the nose and pharynx converging to the deep cervical node and from the posterior border of this node a single deep cervical vessel takes origin and proceeds downward in the neck. The technique delineates a func- tioning system of vessels actually at work. Lymphatic vessels and capillaries constitute a drainage system provided in largest measure beneath the external surface of the body and the invagina- tions of this surface into it in the respira- tory, alimentary and urinogenital systems. They are absent in the brain and bone marrow and rare or absent in skeletal muscle. See detailed information concerning the organ or tissue, in which it is desired to demon- strate them, to be found in Drinker, C. K. and Yoffey, J. M., Lymphatics, Lymph and LymphoidTissue. Harvard Univ. Press, 1941, 406 pp. Methods for the injection of lympha- tics involve forcing fluid containing particulate matter into areas where there are many lymphatic capillaries. A technique for the observation in vivo of the superficial lymphatics of human eyelids is described by Burch, G. E., Anat. Rec, 1939, 73, 443-446. 0.02 cc. of a dilute solution of patent blue V is injected intradermally 5-10 mm. beyond the middle of the lid margin. The lymphatics are apparent in about 5 min. and may be observed as long as 75 min. Consult earlier experiments with this dye by McMaster, P. D., J. Exp. Med., 1937, 65, 347-372. A good way is to utilize the trans- parent ears of white mice to inject the lymphatics with hydrokollag by means of a microdissection apparatus (Pul- linger, B. D. and Florey, W. H., Brit. J. Exp. Path., 1935, 16, 49-61). But the best available technique is closely to examine over long periods of time living non-injected lymphatics in Sandison cliambers in the ears of rabbits (Clark, E. R. and E. L., Am. J. Anat., 1937, 62, 59-92. See India ink method for LYOGLYCOGEN 187 MACERATION renal lymphatics (Pierce, C. E. 2nd., Anat. Rec, 1944, 90, 315-329). Lyoglycogen, see Glycogen. Lyons Blue, see Spirit Blue. Lymphocytes. There is no specific stain for lymphocytes, but identification is usually easy at least for small lympho- cytes. To observe motility, mount fresh blood and ring with vaseline to prevent evaporation. Movements usually begin after the neutrophiles have become active. Examination in the darkfield may be helpful. Mito- chondria can be demonstrated easier in lymphocytes by supravital staining with Janus Green than in polymorpho- nuclear leucocytes because they are not obscured by the specific granulations. In the study of smears the characteristic cytoplasmic basophilia of lymphocytes can be brought out by most of the usual stains (Giemsa's, Wright's). The Peroxidase Reaction of Ijonphocytes is negative, or very strictly limited. Methods demonstrating Cathepsin, Nu- clease, Amylase, Lipase, Lysozyme and Adenosinase in lymphocytes are de- scribed by Barnes, J. M., Brit. J. Exp. Path., 1940, 21, 264-275. To determine the age of lymphocytes is extraordinarily difficult. Perhaps the nearest approach to this goal is the work of Wiseman, B. K., J. Exper. Med., 1931, 54, 270-294. Lysis. In histology this term means the solution of a cell resulting from injury to the cell membrane. A choice may be made of several agents productive of this change. As classified by Danielli (Bourne, pp. 74-75) antibodies and px)lyhydroxylic phenols probably act almost wholly on the protein component of the membrane; lipoid solvents, lecithinase, digitonin, sodium or potas- sium salts of fatty acids and paraffin sulphonates mainly on the lipoid part ; and the heavy metals probably on both. He suggests the probable modes of action. It is therefore possible that these lytic agents may in their action provide clues as to the nature of the plasma membrane. See Cell Mem- branes. Lysozyme a heat and acid resistant enzyme produced from egg white and isolated as a basic protein of small molecular weight by Abraham, E. P., Biochem. J., 1939, 33, 622-630. It is present in many animal and plant tissues. A method for its determination in lympho- cytes and polymorphonuclear leuco- cytes (neutrophiles) is given by Barnes, J. M., Brit. J. Exp. Path., 1940, 21, 264- 275). The use of lysozyme as a cy to- logical agent in bacteriology is de- scribed by Dubos, R. J., The Bacterial Cell. Harvard Univ. Press, 1945, 460 pp. Observation that a bacterium is sus- ceptible to lysozyme is an indication that it contains as an essential part of its structure a substrate for this en- zyme, probably an acetyl amino pol- j'saccharide. Lyssa Bodies are small Negri bodies which look optically hyaline, sec Negri Bodies. Maceration (L. macerare, to soak) is a very important technique by which tissues are soaked for considerable periods of time in various fluids which loosen the connections between the cells and allow them to be easily separated for micro- scopic study. This is a method em- ployed by the great masters in histology which is unfortunately not sufficiently used now-a-days. For nervous tissue Addison (McClung, p. 439) recommends Gage's dissociator which is 2 cc. formalin in 1000 cc. physiological salt solution for 2 or 3 days. After this treatment large ven- tral horn nerve cells can easily be dis- sected out with the aid of a binocular microscope, stained with carmine, picro- carmine or a dilute anilin dye and viewed as units with parts of their processes attached. Smooth muscle of the bladder is well dissociated bv 10-20% nitric acid (Dahlgren, in McClung, p. 423). The resulting fibers are suitable for class use. Thyroid follicles are freed from the surrounding tissue and can be examined individually after maceration in cone, hydrochloric acid 3 parts and aq. dest. 1 part for about 24 hrs. and thorough washing in at least 10 changes of tap water (Jackson, J. L., Anat. Rec, 1931, 48, 219-239). Epidermis can be separated from dermis by maceration in 1% acetic acid, see epidermis. Kidney tubules. Pieces of kidney fixed in 10% formalin or in Kaiserling's solution are placed in cone, hydrochloric acid at room temperature until they become sufficiently softened after 2-7 days. The time depends upon size of piece, degree of fibrosis and other factors. There is no advantage in using fresh tissue. When adequately macerated the almost diffluent tissue is washed in repeated changes of aq. dest. in which it may be kept for several days. Dis- sect out individual tubules with the aid of a binocular microscope (Oliver, J. and Luey, A. S., Arch. Path., 1934, 18, 777-816). Seminiferous tubules. Whole human testicles are fixed in formalin. They are then cut into segments 1 cm. thick parallel to direction of the lobules. The tunica vaginalis is not removed but is slit through in one or two places with a MACNEAL'S TETRACHROME 188 MAGDALA RED razor. Each segment is placed in cone, hydrochloric acid, 75 cc, aq. dest. 25 cc. 1-7 days. Heat just below boiling 20-30 min. Tissue shrinks, turns dark brown and softens. A sediment collects in the dish. Part of acid is drawn off with a pipette, boiled water is added and the process is repeated until practi- cally all of the acid is removed. The water is boiled to prevent formation of air bubbles along the tubules. It turns the tubules a yellowish white color in which condition they should be isolated by careful teasing. When the tubules cannot be easily lifted away from one another, the maceration is insufficient. When, on the other hand, they break it is a sign of over maceration (Johnson, F. P., Anat. Rec, 1934, 59, 187-199). A similar method was used by Johnson in 1916 to separate the lobules of the pig's liver. Bone cells and lamellae. Treat a thin bone section with cone, nitric acid as long as 24 hrs. Mount on a slide and squeeze out bone cells by pres- sure on cover glass. The lamellae can be pealed off easily from a piece of decalcified bone which has been gently boiled in water (Shipley, in McClung, p. 348). Enamel rods. A piece of dental ena- mel is dissociated with 5-10% hydro- chloric acid for 24 hrs. When it has become soft, remove a little with a needle to a slide and tease out. Mount in physiological salt solution under a cover glass. Draw through a little carmine stain with a blotter and wash it out with 10% acetic acid. The specimen can be ringed with paraffin (Churchill, and Appleton, in McClung, p. 372). Nerve cells. Pieces of gray matter of ventral horn are soaked for 2 or 3 days in 0.02 formalin. The tissue softens, the cells are dissected out and stained with carmine or picro-carmine (Addison, in McClung, p. 439). MacNeal's Tetrachrome is a blood stain containing eosin, methylene azure A, methylene blue and methylene violet. It is employed like Wright's stain. For details see MacNeal, W. J., J. A. M. A., 1922, 78, 1122, and Conn, H. J., Stain Technology, 1927, 2, 31. Macrophages. These are the free cells of the reticulo-endothelial system. Al- most any method of exposure to rela- tively non-toxic, finely particulate matter is sufficient to bring them out. The simplest way is to inject mice with trypan blue as described under Vital Staining and to look for the macro- phages in spreads of Loose Connective Tissue. Another method, used by Maximow, is to give rabbits intra- venous injections of saccharated iron oxide or India ink and to examine blood from right ventricle in smears (see Cowdry's Histology, p. 69). Lines of division between macrophages and monocytes, if they exist, are difficult to establish. Supravital staining with Neutral Red and Janus Green is useful to demonstrate neutral red granules and mitochondria respectively. Madder Staining of bone. Madder is a red dye, prepared from the plant Rubia Tinctorum which has been used for thousands of years. It is perhaps the first dye to be used in camouflage in war. With its help Alexander defeated the Persians by staining the clothing of his Greek soldiers red, each garment in a different part so that the Persian leaders at once concluded that all they had to cope with was an already well damaged army. (Leggett, W. F., Ancient and Medieval Dyes. Brooklyn: Chemical Publishing Co., Inc. 944, 95 pp.) Alizarin and purpurin, formed from madder, are now made syntheticall3\ Madder should be employed for the vital staining of growing bone as de- scribed by Macklin (C. C, Anat. Rec, 1917, 12, 403-405; J. Med. Res., 1917, 36, 493-507). Young rats are suggested as material. Each should eat 1-5 gms. of madder, thoroughly mixed with its food, daily. The calcium deposited in the growing bone while madder is thus made available in the circulation is colored red. Staining is noticeable after 1 day but the feeding should be continued for a week or more. The ventral ends of the ribs and the epiphyseal lines of long bones are most intensely colored. The bones selected are fixed in 10% neutral formalin, washed and cleaned in water, dehy- drated thoroughly in alcohol, placed in benzene for 24 hrs., cleared in oil of wintergreen by the method of Spalteholz and examined with binocular microscope as whole objects. Chemistry of madder staining is dis- cussed by Dr. Richter (Biochem. J., 1937, 31, 591-595). The substance giv- ing the intense carmine red color is apparently purpurin carboxylic acid. Madder is one of the most classical of stains. Its history extends back through the centuries and has been well reviewed by F. T. Lewis (Anat. Rec, 1942, 83, 229-253). See Line Test. Magdala Red (CI, 857) — naphthalene pink, naphthalene red, naphthylamine pink, Sudan red — According to Conn (p. 102) this basic naphtho-safranin differs from commercial magdala red which is an acid dye belonging to an entirely dif- MAGENTA 189 MALARIAL PIGMENT ferent group. He calls attention to its use by Kultschitzky, N., Arch. f. Mikr. Anat., 1895, 46, 673-695) in stain- ing elastic tissue of tlie spleen. Used as a fluorochrome for Lipids. Magenta, see Basic Fuchsin. Magenta II is triamino ditolyl-phenyl- methane chloride probably present in most samples of Basic Fuchsin. See Pararosanilin (Magenta O), Rosanilin (Magenta I) and New Fuchsin (Magenta III). Magnafiux is a useful instrument employed in the FBI Laboratory to detect the occurrence of small cracks and defects in the surface of metallic objects. When, for example, a magnetizable object is placed in a magnetic field, created by the magnafiux, the field is distributed throughout the metal if it is sound. Otherwise, magnetizable pig- ments become oriented around the breaks in the surface indicating their location (Hoover, J. E., Scientific Monthly, 1945, 60, 18-24). Obviously metallic laboratory equipment can be tested in this way. Magnesium, Titan yellow method for de- termination of small amounts in body fluids (Haury, V. G., J. Lab. & Clin. Med., 1938, 23, 1079-1084). Methods for detection in plant cells (Broda, B., Mikrokosmos, 1939, 32, 184). (1) Triturate 1 part quinalizarin with 5 parts sodium acetate crystals. Make to fresh 0.5% solution in 5% aq. NaOH. Addition of 1-2 drops to paraf- fin section, then 1-2 drops 10% NaOH results after some hours in blue stain. (2) Add to paraffin section 1-2 drops 0.2% aq. Titan yellow, then 1-2 drops 10% NaOH gives rise to brick red stain of magnesium. (3) Add to paraffin sec- tion 0.1% aq. azo blue. Gives, without the NaOH, a violet stain of magnesium. An attempt should be made to adjust these techniques to human tissues in which a magnesium salt has been injected. By means of a specially constructed electron microscope Scott and Packer (G. H. and D. M., Anat. Rec, 1939 74, 17-45) have accurately localizea magnesium and/or calcium in muscle. The method can be extended to other tissues and perhaps to other minerals. Histospectrography gives data on the amount of magnesium relative to the other minerals in the skin of normal and neurodermatitis patients. In the latter there is a magnesium deficiency (Mac- Cardle, R. C., Engman, M. F., Jr. and Sr., Arch. Dermat. and Syph., 1941, 44, 429^40). If it is desired to supplement micro- scopic and spectrographic detection of magnesium by quantitative analysis of very small amounts of tissue a tech- nique of microdermination with the polarograph devised by Carruthers, C., Indust. and Engin. Chem., 1943, 15, 412-414 will be useful. It has been employed for analysis of pure epidermis by Carruthers, C, and Suntzeff, V., Cancer Research, 1943, 3, 744-748. See Calcium 5. The Spectrophotometric determina- tion of magnesium in human serum has been advanced by Craig, P., Zak, B., Iseri, L. T., Boyle, A. J. and Myers, G. B. (Am. J. Clin. Path., 1951, 21, 394-398) through the making of several innova- tions: (1) destruction of protein by nitric acid digestion, (2) use for stabi- lizer of a sodium lauryl sulfate-poly- vinyl alcohol reagent, (3) choice of a 550 m/x wave length for quantitation and (4) the preparation of calibration curves from standards containing urea and electrolytes normally occurring in plasma. It would seem feasible by different calibration and other adjust- ments to utilize this technique for the estimation of magnesium in fluids other than serum and perhaps in tissue homogenates. Magnetic Particle Technique to investigate the phj'sical properties of the cytoplasm of living cells as determined by move- ments of phagocytized particles of various sorts in a magnetic field (Crick, F. H. C. and Hughes, A. F. W., Exp. Cell Res., 1950, 1, 37-80). This appears to be a very promising method. Malachite, a mineral mined by the Egyp- tians, and applied as a powder gave a green pigmentation about the eyes. It is said to be the oldest coloring mat- ter known to them (Leggett, W. F., Ancient and Medieval Dyes. Brook- lyn: Chemical Publishing Co. Inc., 1944, 99 pp.). Malachite Green (CI, 657) — diamond green B, BX or P extra, light green N, new Victoria green extra, O, I or II, solid green O, Victoria green B or WB — Commission Certified. A feebly basic di-amino tri-phenyl methane dye quite extensively employed as a counterstain for safranin or carmine. Malachite Green G, see Brilliant Green. Malarial Pigment. Produced in erythro- cytes by action of the parasites, black and distinguishable from carbon by its solubility in concentrated sulphuric acid. Among distinguishing character- istics given by Lison (p. 254) are solubility in dilute alkalis, argentaffine reaction negative, specific stains for lipids negative, likewise reactions for iron. But Morrison and Anderson (D. B. and W. A. D., Public Health Rep., MALARIA PLASMODIA 190 MALLORY-HEIDENHAIN STAIN 1942, 57, 90-94) find that when the pigment within the parasites (Plas- modium Knowlesi) is extracted in such a way as not to influence the spectra of hemoglobin it can be identified spectrophotometrically as ferrihemic acid, or hematin, which does contain iron. Malaria Plasmodia. Technique of examina- tion of process of "exflagellation" (Anderson, Ch. W. and Cowdry, E. V., Arch, de I'lnst. Pasteur de Tunis, 1928, 17, 46-72), of quantitative determina- tions of gametocytes (Cowdry, E. V. and Covell, W. P., Ibid., 147-456) and of demonstrating neutral red granules and Golgi apparatus (Cowdry, E. V. and Scott, G. H., Ibid., 233-252). For staining the plasmodia in smears, see Giemsa, Jenner, Marino, Nocht, Plehn, Wilson and Wright's stains. A simple method for staining plasmodia in paraffin sections is described with numerous illustrations by Tomlinson, W. J. and Grocott, R. G., Am. J. Clin. Path., 1944, 14, 316-326. The Barber Eomp thick film method is strongly recommended for surveys. Serlin, N. J. and Lissa, J. R., Am. J. Clin. Path., 1942, 6, 8 advise the follow- ing method when diagnosis depends on finding gametocytes, or malarial pig- ment, in peripheral blood. Completely evaporate 1 cc. 1% aq. potassium oxa- late in a 15 cc. centrifuge tube. Add 10 cc. venipuncture blood. Mix carefully and centrifuge 30 min. at 2,500 R.P.M. Pipette oil all but about J in. of super- natant plasma. Smear on 2 slides by wiping buffer layer with stick applicator having non-absorbent cotton tip. Stain by Wright's method. Study of Giemsa stained smears by dark field is suggested (Goosmann, C., J. Lab. & Clin. Med., 1935-36, 21, 421-424). See Protozoa. Taylor, D. J., Greenberg, J. and Josephson, E. S. (J. Lab. Dis., 1951, 88, 158-162) describe a useful method for the maintenance of intraerythrocytic forms of Plasmodium gallinaceum in a whole medium on vitro. Mallory's Connective Tissue Stain. This is name usually given to his anilin blue-acid fuchsin-orange G stain. See also his Phosphomolybdic and Phospho- tungstic Acid Hematoxylin Stains. (Mallory, p. 155). Fix in Zenker's fluid. Imbed in paraffin or celloidin. Remove mercury from sections with iodine or 0.5% sodium hyposulphite. Stain in 0.5% aq. acid fuchsin, 1-5 min. Drain off stain and put in : anilin blue, water soluble, 0.5 gm. ; orange G, 2 gm. ; 1% aq. phosphotungstic acid, 100 cc, 20 min. or longer. Rinse in 95% ale. 2 or 3 changes until no more stain is removed. Dehydrate in abs. ale, clear in xylol, mount in neutral balsam. For celloidin sections, reduce staining time and pass from 95% ale. to terpineol and mount in balsam. This is one of the most beautiful of all stains and is very widely used. Collagenic fibrils blue, fibroglia, neuroglia and myoglia fibrils red, elastic fibrils pink or yellow. In McClung, p. 405, Mallory and Parker advise 0.25% aq. acid fuchsin and staining in the anilin blue mixture for 1-24 hrs. or for 1 hr. in paraffin oven at 60 °C. The modifications of this stain are almost endless. Adaptation to formalin fixed material is often desirable. Kernohan (J. W., J. Tech. Meth., 1934, 13, 82-84) has outlined the following method of doing this by mordanting. Wash formalin fixed tissue in running water or in ammonia water for short time. Place in Weigert's primary mordant — potas- sium bichromate, 5 gm.; chromium fluoride, 2 gm. and aq. dest. 100 cc. — for 4 days and in his secondary mordant — copper acetate, 5 gm.; chromium fluoride, 2.5 gm.; acetic acid (36%), 5 cc; aq. dest., 100 cc. and formol, 10 cc. — for 2 days. Imbed in paraflin in the usual way. Rexed, B., and Wohlfart, G., Zeit. wiss. Mikr., 1939, 56, 212-215 suggest control of pH of the acid fuchsin. It is stated that fresh 0.1% acid fuchsin has pH 4.49 and that increase in alkalinity makes it defective. To prepare one at pH 3.29 ± 0.01, which is recommended, take acid fuchsin 1 gm.; N/10 HCl, 60 cc. ; aq. dest. 900 cc. ; Storensen's citrate (citric acid crystals, 21 gm.; N/1 NaOH, 200 cc; + aq. dest. to make 1000 cc), 40 cc. Most tissues stain in range pH 3-4, red blood cells alone at pH5-7. In 1936, Mallory considered (Stain Tech., 11, 101-102) the most important modifications of his stain to be Heiden- hain's Azocarmine (Azan), the Lee- Brown and Masson Trichrome methods. See Grossman's modification and Pitui- tary for special adaptations. Mallory-Heidenhain Rapid One-Step Stain for Connective Tissue — Written by Jane E. Cason, Dept. of Pathology, Medical College of Alabama, Birming- ham, Ala. January 27, 1951— Although innumerable modifications of Mallory's aniline blue collagen stain for tissue sections prevail, this procedure appears to be an improvement over the original and subsequent modifications. The necessity for adjusting the in- tensity of the aniline blue and the acid fuchsin in routine staining suggested MAMMARY GLANDS 191 MANGANESE DIOXIDE the possibility of combining the two original solutions at a modified ratio. The results proved quite successful, and three definite advantages of this pro- cedure were apparent: 1) Only one step is involved. 2) sufficient and desirable coloration is accomplished in five min. 3) The intensity of the color is con- sistent. The coloration appears the same as that listed by Mallory: Collagenous fibrils stain intense blue; ground sub- stances of cartilage and bone, mucus, amyloid and certain other hyaline sub- stances stain varying shades of blue; nuclei, fibroglia, myoglia and neuroglia fibrils, axis cylinders and fibrin stain red; nucleoli, red blood corpuscles and myelin stain yellow; elastic fibrils stain pale pink or yellow. This technique is suggested for routine pathologic stain- ing because it is simple to follow, rapid and consistent. To make stain add to 200 cc. aq. dest. Phosphotungstic acid crystals (Merck). . . 1 gm. Orange G (C. I. No. 27) 2 gm. Aniline blue, W. S. (C. I. No. 707) 1 gm. Acid fuchsin (C. I. No. 692 3 gm. Formalin-Zenker is the preferred fixa- tive, but Bouin's, formalin, and alcohol have been used with success. Embed tissue in paraffin, and cut sections at 6^. 1. Deparaffinize in xylene, pass through graded alcohols to tap water. (If formalin-Zenker is the fixative, treat with iodine and sodium thiosulfate.) 2. Stain sections 5 min. in staining solution. 3. Wash in running tap water 3 to 5 sec. 4. Dehydrate rapidly through 95% and 100% ale, clear in xylene, and mount in balsam. Mammary Glands. These can be studied in sections by methods intended to reveal the particular data sought. For general purposes Hematoxylin and Eosin, Mallory's Connective Tissue Stain, or Phloxine-Methylene Blue is recommended after Zenker fixation. For fat use Sudan Black and Oil Red O on frozen sections after fixation in 10% formalin or examine in paraffin sections after fixation in Flemming's fluid or some other osmic acid containing mix- ture. In the case of the small glands of mice, rats, rabbits and other mammals the method of making whole mounts is invaluable in investigations of the responses of mammary glands to endo- crine stimulation. The following is essentially the same technique as that originally described by Turner, C. W. ana Gardner, W. U., Agri. Exp. Res. Stat. Bull., Univ. of Mo., 1931, 158, 1-57 : Remove skin and mammary gland. Stretch out and fasten on a cork block with the external surface of the skin down. Fix in Bouin's fluid 24 hrs. Wash in tap water. Dissect away all tissue over the gland which has been tinged light yellow by the picric acid in the fixative. Remove the gland from the skin. Stain in Mayer's Hemalum. Wash in l%aq. potassium alum and then in water. Differentiate in 70% ale. + 2% of hydrochloric acid until the color has been removed from the connective tissue and the acini and ducts of the glands show up in sharp contrast in a light background. Wash in tap water. Dehydrate in alcohol, clear in xylol, mount in balsam between glass plates and close the edges with sealing wax. Much can be made out when magnified only 2-5 times. Small pieces can be mounted on slides, with edges of cover glasses supported as may be necessary, for examination at higher magnifications. There are many excellent pictures in the paper cited. For examination of fetal mice, see Turner, C. W. and Gomez, E. T., ibid, 1933, 182, 1-43. Valuable data are given in Turner's chapter on mammary glands in Allen's Sex and Internal Secretions, Baltimore: Williams &Wil- kins, 1939, 1346 pp. For techniques to reveal secretory phenomena in mam- mary glands, see Weatherford, H. L., Am. J. Anat., 1929, 44, 199-281 ; Jeffers, K. R., Am. J. Anat., 1935, 56, 257-277, 279-303. Technique for localizing site of fat formation in mammary glands is given by Kelly and Petersen, J. Dairy Sci., 1939, 22, 7. The differential stain- ing of sections of unpreserved bovine udder tissue is to be found in U. S. Dept. of Agri. Circular No. 514, under authorship of W. T. Miller and H. W. Johnson. A method for obtaining serial slices of whole human breasts is described by Ingleby, H. and Holly, C, J. Tech. Meth., 1939, 19, 93-96. Manchester Blue (British Drug Houses Ltd), a dis-azo dye of the benzidine series. In either alcoholic or aqueous solution it gives a sharp deep blue effect (H. G. Cannan, J. Roy. Micr. Soc, 1941,61,88-94). Manchester Brown, see Bismark Brown Y. Manchester Yellow, see Martins Yellow. Mandarin G, see Orange II. Manganese. Histochemical detection un- certain (Lison, p. 98). Manganese Dioxide. Drinker, C. K. and Shaw, L. H., J. Exper. Med., 1921, 33, 77-98 employed a suspension of fine particles in acacia water to investigate phagocytic power of endothelium be- MANN'S FIXATIVE 192 MASSON'S TRICHROME STAIN cause the particles can be seen within the cells and the amounts of manganese in the tissues can be determined by chemical analysis. Mann's Fixative is equal parts 1% aq. osmic acid and sat. corrosive sublimate in phys- iological salt solution (0.85% NaCl). It is a good way to apply osmic acid for the blackening of fat. Mann's Methyl Blue-Eosin Stain. This is used for protozoa and for inclusions caused by viruses. Sections are de- parafhnized, stained 12 hrs. in 1% aq. methyl blue 35 cc, 1% aq. eosin 45 cc. and aq. dest. 100 cc. They are then rinsed in 95% ale, dehydrated cleared and mounted. See Alzheimer's Modi- fication of Mann's method. Manometer for capillary blood pressure, see Landis, E. M., Am. J. Physiol., 1926, 75, 548. Marchi Method. For degenerating nerve fibers. Modification by Swank, R. L. and Davenport, H. A., Stain Techn., 1935, 10, 87-90. Details provided by Dr. J. L. O'Leary. Degeneration time of approximately 14 to 20 days. Kill animal by overdose of nembutal or some other barbiturate given intraperi- toneally. Open left ventricle, insert cannula into aorta and perfuse with 2.5-5% anhydrous (10% crystalline) magnesium sulfate solution containing 2-3% potassium bichromate. Imme- diately afterwards remove the brain and spinal cord and put into 10% formalin for 48 hrs. Place slices 3 mm. thick directly, without washing, in : 1% aq. potassium chlorate, 60 cc. ; 1% aq. osmic acid, 20 cc. ; glacial acetic acid, 1 cc. ; 37% formaldehyde (Merck's reagent), 12 cc. Use about 15 volumes of this fluid to 1 of tissue. Agitate and turn over daily. After staining for 7-10 days, wash in running water, 12-24 hrs., dehydrate in 70% and 95% and absolute alcohol and imbed in low viscosity nitro- cellulose as described by Davenport, H. A. and Swank, R. L., Stain Tech., 1934, 9, 134-139. See Celloidin Im- bedding. Cut 40m sections serially, mount on slides, dehydrate to toluol, placing chloroform in absolute alcohol since low viscosity nitrocellulose is soluble in absolute alcohol. Clear in toluol. Mount in clarite X dissolved in toluol. See these authors (Stain Techn., 1935, 10, 45-52) for artifacts and effects of perfusion in Marchi technique. Rasmussen, G. L., Anat. Rec, 1944, 89, 331-338 has elaborated a very useful cellophane strip method for preparation and study of Marchi serial sections. Marchi's Fluid. Miiller's Fluid, 2 parts; 1% osmic acid, 1 part. Fix 5-8 days; wash in running water. Employed to blacken degenerated nerve fibers. See Nerve Fibers. Method, underlying mechanisms in- volved (Swank, R. L. and Davenport, H. A, Stain Techn., 1934, 9, 11-19; 1935, 12, 45-52). Marine Blue V, see Anilin Blue. Marino's Stain for malaria plasmodia is de- scribed in detail by Craig, p. 286 who states that it gives excellent results; but, owing to its complexity, is little used for routine blood examinations. Marrow, see Bone Marrow. Marshall Red (British Drug Houses Ltd), a disazo dye. Stain sections in sat. aq. solution 20 min. Rinse in aq. dest. Stain in sat. Victoria Green G in 70% alcohol 30 min. Rinse in 95% alcohol, dehydrate, clear and mount in usual way. Myofibrils sage green, nuclei crimson. Advised also for retina (H. G. Cannan, J. Roy. Micr. Soc, 1941, 61,88-94). Martius Yellow (CI, 9) — Manchester yellow, naphthol yellow — An acid nitro dye employed by Pianese (G., Beitr. z. Path. Anat. u. AUg. Path., 1896, Suppl. I, 193 pp.) for investigating cancer tissue in association with acid fuchsin. Conn (p. 44) reports good results in staining of plant tissue with CC product. Masson's Gelatin Glue. Method for mak- ing sections stick to slides (Masson, P., Am. J. Path., 1928, 4, 181-212). Dissolve 0.05 gm. sheet gelatin in 20 cc. aq. dest., warming gentlj'. Filter a large drop on each slide on warm plate. Float paraffin sections on drops. When drops spread place slides upright to drain but do not permit drying. Blot and transfer to dish containing formalin (so arranged that vapor only will act on slides) in oven 45-50 °C. For sub- sequent staining 20 minutes in hot vapor is enough. For silver treatment over- night is suggested. Masson's Trichrome Stain — Written by Pierre Masson, Dept. of Pathology, University of Montreal, Montreal, Canada. October 24, 1951— The prin- ciple established by F. B. Mallory in his famous method of employing acid fuchsin, phosphomolybdic acid, anilin blue, orange G. can be advantageously applied to other acid dyes yielding a more specific staining of the chromatin. It then gives a very instructive com- bination of tints. Due to the intensity of the staining, the sections must be thin; 5^ is opti- mum. In order to prevent swelling of the collagen and its deformation during the desiccation, the sections must not be left too long (20-30 sec.) on the warmed water, or gelatin, especially MASSON'S TRICHROME STAIN 193 MASSON'S TRICHROME STAIN after fixation by picro-formol (Bouin) or formalin. So altered, the collagen does not properly absorb the dyes and differentiates badly. Trichrome stains with iron haematoxylin. All trichromic methods are based upon a common first step: the staining of nuclei by iron alum haematoxylin, followed by differen- tiation with picric alcohol. Three solutions are required: A. Iron alum, violet crystals, 5 gm., aq. dest. 100 cc. B. Regaud's hematoxylin solu- tiin made up by dissolving 1 gm. hema- toxylin in 60 cc. hot aq. dest. Cool and add 10 cc. glycerin and 10 cc. of 95% alcohol. This stain is ready for use at once. C. Differentiating mixture con- sisting of 2 parts sat. picric acid in 95% alcohol and 1 part of 95% alcohol. Step 1. Place iron alum solution and the hematoxylin solution in staining jars in a water bath heated to 40-45°C. Mordant deparaflined sections in heated iron alum, 15 min. Rinse with aq. dest. Stain in heated hematoxylin, 15 min. or more. Rinse the uniformly black sections with 95% alcohol and immerse in picric alcohol. Control the progress of differentiation under the microscope. As soon as the nuclei alone remain colored, wash with running water for 15 min. If after washing, the background of the preparation, particularly the col- lagen remains gray, rinse with alcohol and complete picric differentiation. The chromatin is electively black and opaque. Various authors have proposed to "simplify" this method by staining the nuclei with Weigert's iron perchloride hematoxylin or with alum hematoxylin. I must say that I have long ago tried such modifications before adopting the above method and that I have aban- doned them entirely. The red of the ponceau-fuchsin solution superposes itself on the gray or blue color imparted by hematoxylin so that chromatin takes a dull color lacking in specificity. Rather than to use such modifications it would be better to omit all nuclear staining and start with the next step (ponceau-fuchsin and so forth) : the results are thus comparable to those ob- tained by Mallory's original method. Step 2. Stain in a mixture of acid fuchsin and Ponceau ofxylidin BS (J. R. Geigy, S. A. Basel). No other Ponceaux I have used, French, German or Amer- ican give results comparable to that of Geigy's. Three solutions are required: A. Ponceau, BS (Geigy), 1 gm., aq. dest. 100 cc, glacial acetic acid 1 cc. B. Acid fuchsin (National Anilin Co., New York), 1 gm., aq. dest. 100 cc, glacial acetic acid 1 cc. 1 part of A with 2 parts of B. C. Aq. dest. 100 cc, glacial acetic acid 1 cc. D. Aq. dest. 100 cc, phosphomolybdic acid 1 gm. Place sections stained with the iron hematoxylin in the A B mixture, 5 min. Rinse them with C. Transfer them to Z> in a Copeland or Borrel jar at 40°C. 5 min. or more. The collagen should remain perfectly colorless. Step 3. Stain the collagen with anilin blue or Fast green. To make the former dissolve 2 gms. anilin blue (Na- tional Anilin Co., New York) in 100 cc. warmed aq. dest. Cool and add 1 cc. glacial acetic acid. To make the latter dissolve 1 gm. fast green (National Anilin Co., New York) in 100 cc. aq. dest. and add 1 cc. glacial acetic acid. After the phosphomolybdic differenti- ation (Step 2 jD) rinse the sections in acetic water C Pour on them 8 to 10 drops of the anilin blue or fast green solutions 2 to 3 min. Wash in acetic water C 2 to 3 min. Dehydrate with absolute alcohol, clear in toluol or xylol, mount in balsam or permount. Total length of this technique is approxi- mately 60 minutes. Chromatin is black, cytoplasms stain in various shades of red, granulations of eosino- philes and mast cells stain ruby red, erythrocytes are black, elastic fibers stain red, collagenic fibers and mucus stain dark blue (with anilin blue) or green (if Fast green is used). To this relatively fast technique, I prefer a slow one, based on the use of diluted solutions of Ponceau-fuchsin, anilin blue or fast green. After nuclear staining with iron alum hematoxylin (Step 1) wash and immerse the slides in 1 part A B mixture and 9 parts 1% acetic acid, 30 min. Rinse in acetic water C, 1 min. Transfer the sections into D at 40°C. 5 min. or more. Rinse in acetic water C. Stain Anilin blue solution 1 part {or Fast green solu- tion 1 part) with acetic water C 9 parts, 15 to 30 minutes. Rinse in acetic water. Dehydrate with absolute alcohol, clear in toluol or xylol, mount in balsam or permount. The results are grossly the same as after the rapid method, but more deli- cate and precise. Moreover, the stain- ing of the collagen is slow and progres- sive and can be stopped at the most favorable step; in many circumstances an excessive staining of collagen masks some fine details, for example the fine prolongations of the connective tissue cells. N.B. After staining of collagen with Fast green, it is preferable to mount in MAST CELLS 194 MCILVAINE BUFFERS salicylic balsam or salicylic permount. Spread a drop of saturated solution of salicylic acid in toluol upon a cover slip. Dry. The glass is then covered with a thin crystalline layer of salicylic acid. Put on it a drop of balsam or permount and cover the section taken out of the toluol (or xylol). Salicylic acid instantaneously redissolves in the mounting medium. This addition of salicylic acid is not advantageous after staining with anilin blue. Mast Cells, see Basophile Leucocytes and Tissue Basophiles. Mastigophora, Techniques for given by Mc- Clung, Microscopical Technique, 1950, p. 469. Mastoid Process. Use methods for Bone. Technique for measurements of size of air cell system is given by Diamant, M., Acta Radiol., 1940, 31, 543-548. Mauveine ((T, 846), a basic dj^e of light fast- ness 3, the first dye made from aniline in 1856. Gives stain of plant tissues like Methyl violet (Emi_g, p. 57). Maximow (see Azure II Eosin Hematoxylin method). He has advised as a fixative 90 cc. Zenker's fluid less acetic acid + 10 cc. formalin. This is essentially Formalin Zenker. See Buzaglo's con- nective tissue stain. May-Giemsa stain of Pappenheim (Folia Haematol., Arch., 1917, 22, 15). This is the same as Jenner-Giemsa. Fix and stain air dried blood smears about 3 min. in May-Griinwald mixture (sat. sol. methylene blue eosinate in methyl alcohol). Add same amount aq. dest. and leave 1 min. Pour off (but do not wash) and add diluted Giemsa's solu- tion. Stain in this 15-30 min. Rinse aq. dest. 1 min. or until desired color is reached. Blot dry. This is a good modification of the ordinary Giemsa's stain because it gives slightly more intense colors. May-Griinwald combined fixative and stain is a sat. sol. of methylene blue eosinate in methyl alcohol (Grtibler or Holl- born). If methylene blue eosinate is not available make it as originally de- scribed by Jenner (Lancet, 1899, No. 6, 370). Mix equal parts 1.25% water sol. eosin and 1% methylene blue; after 24 hrs. filter; wash ppt. on filter_ with water; dry and dissolve powder in 200 cc. pure methyl alcohol. It is employed in the May-Giemsa and Kardos-Pap- penheim methods for staining blood smears. May-Griinwald-Giemsa stain in one solu- tion. Strumia (M. M., J. Lab. & Clin. Med., 1935-36, 21, 930-934) gives di- rections for combining the stains and for use and notes that a standardized product is prepared by Coleman and Bell Co. Intensity of coloration is enhanced by the combination. Mayer's Acid Alum Hematoxylin. The following formula is given by Mallory (p. 73). Dissolve 1 gm. hematoxylin in 1000 cc. aq. dest. with a little heat if required. Add 0.2 gm. sodium iodate and 50 gm. ammonium or potassium alum. When latter is dissolved add 1 gm. citric acid and 50 gm. chloral hy- drate. Color turns reddish violet. Does not easily over-ripen. Mayer's Acid Carmine. The Bensleys (p. 131) advise its preparation as follows. Add 4 gm. carmine to 15 cc. aq. dest. + 30 drops hydrochloric acid. Boil until it is dissolved. Add 95 cc. 85% ethyl alcohol. Neutralize with ammonia until the carmine begins to precipitate as seen in a graduate against white paper background. Add 4 more drops ammonia after first precipitation. If this acid carmine stains too quicklj^ slow it down by dilution with 80-90% alcohol. This gives a fine red nuclear counterstain for tissues vitally stained with Indigo-Carmine, Trypan Blue and similar dyes. Mayer's Albumen, see Albumen Glycerin. Mcllvaine Buffers after Stitt from Lillie, R. D., Stain Techn., 1941, 16, 1-6 who employed them to improve Romanowsky staining after various fixatives. See Toluidine Blue Phloxinate Method, (see Molecular Solution) To make M/15 citric acid required dissolve 14.01 gm. mono-hydrateu crystalline citric acid in 500 cc. aq. dest. and add enough neutral methyl alcohol C.P. to make total volume 1,000 cc. after careful mixing. To make M/15 Na2HP04 dis- solve 9.47 gm. anhydrous Na2HP04 in 500 cc. aq. dest. and make up to 1,000 cc. with methyl alcohol. These, in following proportions listed in cc, give pH values indicated. cc. Citric Acid cc. NajHPOi pH 1.3 0.7 3.9 (3.873) 1.25 0.75 4.0 (4.034) 1.2 0.8 4.2 (4.205) 1.15 0.85 4.4 (4.44) 1.1 0.9 4.6 (4.653) 1.05 0.95 4.8 (4.80) 1.0 1.0 5.0 (5.042) 0.95 1.05 5.2 (5.201) 0.9 1.1 5.4 (5.428) 0.85 1.15 5.7 (5.696) 0.8 1.2 5.85 (5.838) 0.75 1.25 6.05 (6.036) 0.7 1.3 6.3 (6.29) 0.65 1.35 6.5 (6.444) 0.6 1.4 6.5 (6.522) 0.55 1.45 6.6 (6.60) Since it is difficult to measure out these MCJUNKIN-HADEN BUFFER 195 MELANINS small volumes accurately at least ten times the volume in each case should be taken and the amount not required simply be discarded. For ordinary purposes employ aq. dest. in place of methyl alcohol. McJunkin-Haden Buffer has pH 6.4 and is useful in place of aq. dest. for diluting Giemsa, Wright and other blood stains. Monobasic potassium-phosphate, 6.63 gm.; anhydrous dibasic sodium phos- phate, 2.56 gm.; aq. dest^ 1000 cc. (Haden, R. L., J. Lab. & Clin. Med., 1923, 9, 64-65). Meat Extract Broth and other media con- taining meat, see Bacteria Media. Mechanical Stages, see Lillie p. 287. Meckel's Diverticulum. Literature on (Curd, H. H., Arch. Surg., 1936, 32, 506-523). Media, see Bacteria, Leishmania, Protozoa, Trypanosomes. Megakaryocytes. These can, like blood cells, be examined in fresh and stained smears of bone marrow. For a deter- mination of their role in platelet forma- tion it is essential to clearly show the granules typical of both. This can best be done in sections of bone marrow prepared by : 1. Wright's method (Wright, J. H., J. Morph., 1910, 21, 263-277). After fixation in sat. mercuric chloride in 0.9% aq. NaCl, dehydrate in alcohol, follow with acetone, clear first in thick cedar oil and then in xylol, embed in paraffin. Sections deparaffinized are covered with equal parts stain (poly- chrome methylene blue solution 3 parts and 0.2% eosin yellowish in methyl alcohol 10 parts) 10 min. A metallic looking scum forms but the stain should not be allowed to precipitate. Stop staining when cytoplasm looks bright red and reticular fibers light red. Wash in water, dehydrate in acetone, clear in turpentine and mount in thick colophonium in pure turpentine oil. See Wright's colored plates. In place of the fixative suggested, Downey (Folia haematol., Archiv, 1913, 15, 25) uses commercial formalin 10 cc. and sat. mercuric chloride in 0.9% aq. NaCI 90 cc. 2. Kingsley's method (Kingsley, D. M., Folia Haemat., 1937, 57, 87-98). Fix in Downey's fluid (given above) 4 parts, saturated picric acid 1 part, 24 hrs. Wash in running water, 18-24 hrs. Dehydrate through alcohols up to70%,i-lhr.each. 80% ale. + iodine, overnight. 95% ale, 45 min. Repeat with fresh ale. A^ butyl alcohol (techni- cal), 1 hr. Repeat with fresh. Paraf- fin (58°C.), ^ hr., then 3 more changes, each 5 hr. Imbed. Prepare stock solutions A : methylene blue (U.S. P. med. 88%), 0.065 gm.; methylene azure A (80%), 0.01 gm.; glycerin, C.P., 5 cc; CH3OH (C.P.), 5 cc; aq. dest., 25 cc; buffer (pH, 6.9), 15 cc B: methylene violet (Beruthsen 85%), 0.013 gm.; eosin, yel. (92%), 0.45 gm.; glycerine, 5 cc; CH3OH, 10 cc ; acetone, C.P., 35 cc. The buffer is 40 cc. of A = 9.078 gm. KHjPOi per liter + 60 cc. of B = 11.876 gm. Na2HPO«-2H20 per liter of aq. dest. Immediately before use mix equal parts of stock stains A and B. After washing deparaffinized sections in aq. dest. stain 8-10 min. Wash off in current of aq. dest. Wash in aq. dest. 100 cc. + 1% acetic acid, 0.8 cc. Wash again in aq. dest. to re- move acid. Blot. Rinse in acetone, 100 cc + 0.001 gm. eosin -f 4 cc. 1% acetic acid. Rinse in n butyl ale -\- a little eosin. Neutral xylol several changes. Mount in neutral xylol dam- mar. See Kingsley's plate for colors. Granules dark red. It is important to fix the bone marrow promptly after death or to obtain it by biopsy. Megaloblasts, see Erythrocytes, develop- mental series. Meibomian Glands. Whole mounts of the glands stained with Sudan IV in a trans- parent background by a method de- scribed for Sebaceous Glands. Meissner's Corpuscles. To investigate by supravital staining with methylene blue in skin of amputated fingers, see Weddell, G., J. Anat., 1940-41, 75, 441-446. Skin from general body sur- face will not do because of rarety of the corpuscles. Meissner's Plexus, see Auerbach's. Melanins. Lison (p. 248) gives many dif- ferential microchemical properties from which the following are selected. Ex- treme resistance to most chemicals, not modified by concentrated acids but soluble in concentrated alkalis. They are depigmented by oxydants. Thus, Schultze treats them with diaphanol (chlordioxyacetic acid) for 24 hrs. in hermetically sealed container in dark- ness ; and Alfiere treats sections with 0.1% potassium permanganate 2-24 hrs. ; washes with much water, treats with 0.3% oxalic acid and again washes. Their power of reducing ammoniacal silver nitrate, Lison regards as very characteristic. Melanins occur nor- mally' in epidermis, hair, choroid of eyes. Greatly increased in Addison's disease. Contain no iron or fat. Difficulties in histological identification (Jacobsen, V. C. and Klinck, G. H., Arch. Path., 1943, 17, 141-151). Use of Bodian method (Dublin, W. B., Am. J. Clin. Path., Techn. Suppl., 1943, 7, 127-128). MELANOBLASTS 196 MESONEPHRIC TUBULES A method for the collection of melanin for analysis by differential Centrifuga- tion is described by Claude, A., Trans. New York Acad. Sci., 1942, II, 4, 79-83. A very complete account of the melanins has been presented by Gordon, H. (Organizing Chairman) : The Bi- ology of Melanomas — Special Publica- tions, New York Acad. Sci., 1948, 466 pp. Lillie (p. 131) cites Alfieri from Romeis as advising for the bleaching of melanin in sections treatment with 0.05% aq. potassium permanganate until thoroughly brown followed by de- colorization in 0.33 aq. oxalic acid and repeating the process if necessary. Obviously stronger solutions could be employed. This removal of melanin might be advantageous in some cases to reveal more sharply other properties of the cells like mitochondrial content. Treatment with 10% hydrogen peroxide, as suggested by Lillie, is perhaps a better method. See Dopa Reaction for melanogen in melanoblasts. Melanoblasts, see Dopa Reaction. Meldola's Blue, see Naphthol Blue R. Mercuric Chloride (corrosive sublimate) in various combinations is an excellent fixative. It can be used in saturated aq. sol. plus 5% acetic acid or in satu- rated ale. sol. with the same amount of acetic acid. See (1) with formalin, glacial acetic and physiological saline for Centrosomes, (2) sat. in 0.9% aq. sodium chloride for Megakaryocytes, (3) sat. in 70% alcohol + 5% acetic for Mitosis, (4) sat. aq. + equal parts 2.5% aq. potassium bichromate for Neutral Gentian, (5) sat. aq. with equal parts abs. alcohol for Thymonucleic Acid, and (6) with nitric acid for Urea. The mercuric chloride is removed from the sections by Lugol's iodine solution. See also fixatives of Zenker, Gilson, Rabl and Petrunkewitsch. Zinc chlo- ride is suggested as substitute for mercuric chloride in Zenker's fluid (Russell, W. O., J. Techn. Methods & Bull. Int. Asso. Med. Museums, 1941, 21,47). Mercurochrome 220. Trade name for di- brora-oxy-mercuri-fluorescein. Can be used as substitute for eosin (Baldwin, W. M., Anat. Rec, 1928, 39, 229) but it has little to commend it. Mercury, microchemical tests for. 1. Method of Almkvist-Christeller. Fix tissues 2 days in sat. aq. picric acid, 100 cc. ; 25% nitric acid 1 cc, saturated with HjS gas, filtered after 1 day. After fixation wash in running water for 24 hrs. Imbed in paraffin. Mercury ap- pears as black ppt. of sulphide. Lison (p. 102) explains that it is necessary to make parallel tests for iron because this method changes iron into the black sul- phide which could be mistaken for the sulphide of mercury. Simonet (M., Arch. d'Anat. Micr., 1929, 25, 372-381) uses instead fixation for 10 hrs. in equal parts alcohol and chloroform, 100 cc, + nitric acid, 2 cc. the mixture satu- rated with HjS by bubbling. 2. Method of Brandino (G., Studi Sassari, 1927, 5, 85). Fix in formalin or in alcohol. Treatment of sections with 1% sol. of diphenylcarbazide which forms with mercury a violet ppt. Gives results with organs of persons killed by mercury poisoning kept in formalin 17 years (Lison, p. 102). 3. Method of Hand et al., J. Lab. & clin. Med., 1943, 28, 1835-1841. Re- agents: (A) Mercurous. 1 cc. thioglycol- lic acid +9 cc. glycerol. (B) Mercuric. Heat until clear 100 cc. glycerol., +5 gm. tartaric acid, +5 gm. stannous chloride. Add few gms. metallic tin and store in glass stoppered bottle. (C) Iodine. Dissolve 50 gm. potassium iodide in 50 cc. aq. dest., add 70 gm. iodine and 95% ale. to make 1,000 cc. (D) Chloroauric acid 1% aq. (E) Con- trol. Let 5 gm. tartaric acid stand over night in 100 cc. glycerol. Cut 15 m frozen sections of tissue. Place on slides and dry. To section on each of 4 slides add 1 drop of one reagent. Add cover glasses. Remove with filter paper excess of reagents. Seal edges of cover glass with commercial gold size, an adhesion intended to hold gold foil on glass. Melted paraffin is less satis- factory but will serve for a short time. After 10 min. examine slides. Metallic mercury visible as small black spherules. These are soluble in C and form gold amalgam losing glossy surface when treated with D. Reagent A gives typi- cal yellow crystals with mercurous mercury in addition to globules of mercury when mercuric mercury also is present. Sections treated with B and E are unchanged (adapted from Click p. 25). Intravenous injections of colloidal solutions of mercury in rabbits are described by Duhamel, B. G., C. rend. Soc. de Biol.. 1919, 82, 724-726. Mesentery spreads, sections and cultures. Maximow, A., Arch. f. exper. Zellf., 1927, 4, 1-42 (nice colored plates). For microinjection of small vessels of the mesentery see Florey, H., Proc. Roy. Soc. B, 1926 100, 269. Mesonephric Tubules, cultivation in vitro and method for collection of fluid there- from (Keosian, J., J. Cell & Comp. Physiol., 1938, 12, 23). METACHROMASIA 197 METHYL VIOLET Metachromasia, see Metachromatism. Metachromatism (metachromasia) is the property of certain dyes to stain (G., meta, beyond) the usual color (G. chroma). The action of some impure methylene blues is sometimes cited as an example. Thus polychrome (many colored) methylene blue stains some objects blue and certain granules red- dish. This methylene blue is however a mixture of methylene blue and methyl- ene red. The latter dye accounts for the staining beyond. Orcein colors nuclei blue and cytoplasm pink. Safra- nin stains nuclei in its ordinary solution color (red) and the ground substance of cartilage that of its free color base (orange). Michaelis (Lee, p. 136) thinks that the appearance of the color base is not occasioned by the alkalinity of the objects stained. The red stain of mucin by thionin can be altered to blue by alcohol and be shifted back to red by water. For colored plates show- ing metachromatic staining of mast cells, see Maximow, A., Arch. f. mikr. Anat., 1913, 83 (1), 247-289. Meta- chromasia of acid dyes is increased by adding strychnine, quinine or clupein and of basic dyes by gum arable or other negatively charged colloid (Bank, O. and Hungenberg de Jona, H. G., Proto- plasma, 1939, 32, 489-516). The dis- tinction between "true" and "false" metachromatic staining with toluidine blue is fully described by Sylvan, B., Acta Radiol., 1945, Suppl. 59, 100 pp. Metacresol Purple. See Hydrogen Ion In- dicators. Metallurgic Microscope. Since the mate- rials routinely studied are opaque the light is reflected vertically down upon them through the objective. This in- strument is of little use in biology and medicine. Metamyelocytes, see Leucocytes, develop- mental series. Metanil Yellow (CI, 138)— acid yellow R, orange MNO or MN, soluble yellow OL, tropaeolin G, yellow M — An acid mono- azo dye employed in the Masson tech- nique, see Foot, N. C., Stain Techn., 1933, 8, 101-110. Methacrylate. Plastic for mounting ali- zarin-red-S preparations. (Holcomb, R. C. and Apterman, P. M., J. Tech. Methods, 1944, 24, 21-24). Methyl Alcohol, see Elementary Bodies. It is much used in many techniques. Methyl Benzoate. Refractive index close to that of cedar wood oil. It can be used in place of immersion oil. In addition it is a substitute for absolute alcohol and an excellent clearing agent but it is expensive. See Ceresin imbedding. Methyl Blue (CI, 706)— cotton blue, Hel- vetia blue — Widely used. Recom- mended for connective tissue by Lillie, R. D., J. Tech. Methods, 1945, No. 25, 47 pp. See Mann's Methyl-Blue Eosin and staining of Elementary Bodies. Methyl Blue-Eosin, see Mann's. Methyl Eosin (CI, 769). The methyl ester of eosin Y, see Eosins, choice of. Methyl Green (CI, 684) — double green, light green — This basic triphenyl meth- ane dye is crystal violet (he.xa-methyl pararosanilin) into which a seventh methyl group has been incorporated. Conn (p. 130) points out that this is loosely bound so that some methyl or crystal violet is always present with the methyl green to which circumstance the metachromatic properties of the dye are partly due. Methyl green is not as stable as most dyes and cannot therefore be kept too long in the powdered state. It is very similar to Ethyl Green. Methyl Green-Pyronin (Pappenheim). Sec- tions of formalin-Zenker fixed tissues are stained about 6 min. in : methyl green 0.5 gm.; pyronin Y, 0.5 gm.; ale. 2.5 cc; glycerin 20 cc; aq. dest. 0.5% carbolized 100 cc. Rinse in aq. dest.; dehydrate in acetone; clear in cedar oil followed by xylol and mount. Opti- mum time of staining must be deter- mined experimentally. A brilliant stain particularly for lymphocytes and plasma cells. Very useful for spleen and lymph nodes. (Slider and Downey in McClung's Microscopical Technique, p. 342). Modification of Scudder (Stain Techn., 1944, 19, 39-44) gives good results on tissue sections and bacteria and has been used for identification of 2 types of nucleic acids (Taft, E. G., Exper. Cell Research, 1951, in press). Dehydration of stained material with tertiary hiilyl alcohol gives better results than with ethyl alcohol. Methyl Orange (CI, 142)— gold orange MP, helianthin, orange III, tropaeolin D — A slightly acid mono-azo dye widely employed as an Indicator. Methyl Red (CI, 211). A slightly acid mono-azo dye widely used as an Indica- tor. See also Carter, J. S., J. Exp. Zool., 1933, 65, 159-179 for vital staining of rabdites of Stenostomum with methyl red. Methyl Salicylate (oil of Wintergreen) is employed in Spalteholz Method of clearing. Methyl Violet (CI, 680)— dahlia B, gentian violet, Paris violet, pyoktaninum coeru- leum — Exists in various shades 2R, R, B, 2B, 3B, etc., depending upon propor- tions of the mixture of tetra-, penta- and he.xa-methyl rosanilins. R indi- METHYLENE AZURE 198 MICRODISSECTION cates reddish and B bluish. 2B is the one which Conn (p. 123) regards as most satisfactory whenever methyl violet, or one of the redder types of gentian violet, is requested. (It is Commission Certified.) The pure hexamethyl com- pound is called crystal violet — a dye much in demand. See Hydrogen Ion Indicators. Methylene Azure (CI, 923) . A basic thiazin dye long recognized as a component of Polychrome Methylene Blue. Conn (p. 76) says that the term, methylene azure, should be discarded because it is composed of three components Azure A, B, and C which see. Methylene Blue (CI, 922)— Swiss blue- Conn (p. 80) says that this basic thiazin dye is theoretically tetra-methyl thio- nin but the homologues of lower methylation are practically always present ; he lists the following grades : methylene blue BX, B, BG, BB, and methylene blue chloride. The last named is Commission Certified and least toxic. Methylene blue Med. U.S.P. is required to be zinc free and is also satisfactory. New methylene blue N (methylene blue NN) is a basic dye of the same type but of a slightly greener shade. Conn (McClung, p. 595) states that it was apparently in certain lots of prewar methylene blue. Methylene blue O is the same as toluidin blue O which resembles azure A, a component of methylene azure produced by poly- chromizing methylene blue. Another of the series is methylene blue GG but it has no particular advantage. Prob- ably no dye, other than hematoxylin and eosin, is more widely used. The oxida- tion products of methylene blue are described by Holmes, W. C, Stain Techn., 1926, 1, 17-26 and the influence of pH on staining of plasma cells and lymphocytes by Kindred, J. E., Stain Techn., 1935, 10, 7-20. Its cytological action has been fully studied by Lud- ford, R. J., Arch. f". exp. Zellf., 1935, 17, 339-359. It is an excellent counter- stain for Acid Fast Bacilli. See Poly- chrome Methylene Blue, Loeffler's Alkaline Methylene Blue, Nerve End- ings, Phloxine Methylene Blue, Mac- Neal's Tetrachrome, Pancreas, Pro- tozoa, etc. For use of methylene blue as a supravital stain fixed with ammo- nium molybdate, see Lillie, p. 245. Methylene Blue NN, see New Methylene Blue N. Methylene Blue T 50 or T Extra, see Toluidin Blue O. Methylene Blue Eosinate, see May-Griin- wald fixative and stain. Methylene Green (CI, 924). This basic thiazin dye is mono-nitro methylene blue. Conn (p. 86) says that it is oc- casionally employed as a substitute for methyl green and gives good results as counters tain for eosin. Methylene Violet. Commission Certified. This feebly basic thiazin dye is, as Conn (p. 86) explains, formed whenever methylene blue is heated with a fixed alkali or alkali carbonate. It may be purified by recrystallization but little is to be gained. The dye is not much used. Metrial Gland. This is a transitory struc- ture of unknown function in the mouse appearing at approximately the 8th d.n}' of pregnancy. Failure of its cells to take up trypan blue seems to eliminate the hypothesis that it is active in phago- cvtosis (Lobo, B. A., and Atkinson, W. B., Anat. Rec, 1946, 94, 77). Micelle (dim. of L. Mica a crumb, micella, micellae). Term introduced by Nageli in 1884 for then hypothetical structural units of the cell. Michiavello Stain. See Rickettsia. Microchemical Reactions. For the microscopic identification of particular elements or substances some micro- chemical reactions are available but it is difficult to sharply distinguish them from other techniques not usually styled microchemical. An attempt is made to list them under the objects demon- strated : Lead, Iron, Vitamin C, Peroxi- dase, etc. Many are generally known under personal names. See for exam- ple: Axenfeld (proteins), Burchardt (gold) , Carr-Price (vitamin A) , Feulgen (thymonucleic acid), Gmelin (bile pig- ments), Lilienfeld-Monti (phosphorus), Millon (tyrosin), Romieu (proteins), Schiff (aldehydes), Vulpian (epineph- rine), etc. Microdissection. In the selection of this method for use in any particular problem it is well to bear in mind several con- siderations. It is of particular value in the direct examination of large cells easily isolated, like sea urchin eggs, and of tissues that exist in thin sheets, like highly vascularized membranes which can be easily approached in the living state without serious injury. The data to be secured relate chiefly to the responses of the cells to the mechanical stimulus of the microneedle, to the character of the connections be- tween fibers, cells and parts of cells as determined by their resistance to at- tempts to separate them and to the physical consistency of cellular and nuclear membranes and of cytoplasm and nucleoplasm. Moreover individual cells can be isolated by microdissection just as Barber was able to isolate single bacteria by the pipette which he intro- MICROELECTRODES 199 MICROINCINERATION duced and which was in fact the inspira- tion of G. L. Kite's first microdissection apparatus. Today this has been very greatly improved chiefly by Chambers and Peterfi. See detailed account by Chambers under Micromanipulation. Microelectrodes, see full discussion, Mc- Clung, Microscopical Technique, 1950, p. 532. Microglia. Method for impregnating with silver in pyroxylin (celloidin) sections (Weil, H. and Davenport, 11. A., Trans. Chicago Path. Soc, 1933, 14, 95-96). Wash 15yu sections in aq. dest. Treat for 15-20 sec. with silver solution (made by adding 10% aq. silver nitrate drop by drop from a burette to 2 cc. cone, am- monia (28%) shaking to prevent ppt. formation until about 18 cc. have been added and the solution has become slightlj' opalescent). Transfer to 15% formalin, moving section rapidly until coffee-brown in color. Pass through 3 changes aq. dest. Dehydrate in alcohol, clear in xylol and mount in balsam. Microglia and Oligodendroglia. In frozen sections 20-40m of formalin fixed mate- rial. Immediately place them in aq. dest. + 20 drops ammonia per lOO cc. Thence pass directly to 5% aq. am- monium bromide 4O^50°C. 10-15 min. Equal parts ammonia, pyridine and aq. dest. 2 min. Then 3-5% aq. sodium sulfite, 2-3 min. Pass through and shake in 3 changes 1 min. each of follow- ing: 8 parts 5% aq. sodium carbonate, 2 parts, 10% aq. silver nitrate + am- monia till ppt. Reduce in 1% formalin less than 1 min. Wash, dehydrate clear and mount (King, L. S., Arch. Neurol, and Psychiat., 1937, 38, 362-364). Microincineration — Written by Gordon H. Scott, July 26, 1946 and revised by him January 16, 1951 — This method is one which has been used by plant and ani- mal histologists intermittently for over a hundred years. In concept it is simple in that it consists primarily of ashing tissue sections carefully so as to retain the minerals in their position in the fixed tissue. The ashing can be done on glass or quartz slides by a vari- ety of heating processes. Most tissues in the body can be treated by the ash- ing process with some success. Those which contain large quantities of phos- pholipids ordinarily do not give as good results as tissues lacking them. The method is one which requires some care and the observance of cer- tain very definite precautions if good results are to be had. Fixation: There are two methods of fixation which can be used. These are the chemical and the frozen-dehydra- tion. If the cryostat or other suitable devices for frozen-dehydration are not available, fixation by absolute alcohol plus 10 per cent formalin yields reason- ably good pictures. This particular fixative is one of the few chemical mix- tures which dissolves the minimum of mineral from fresh tissue and adds none to it. Tissues are passed through re- peated changes of absolute alcohol to dry them and are then infiltrated wdth paraffin in the usual manner. The alternative method, that of frozen-dehydration, is the most suitable for preparation of tissues for micro- incineration. (See Altmann-Gersh and Cryostat.) This technique yields tissues which, except for the ice crystal forma- tion, have not been altered, to any perceptible degree, either ph3^sically or chemicallJ^ Dehydration at suffi- ciently low temperatures maintains an ice-salt equilibrium and no shifting of minerals in the cell results. If the paraffin infiltration is done with care, shrinkage and consequent cellular dis- tortion is avoided. Methods of examination of the in- cinerated preparations are several. One of the simplest and best for study and for photograph}' is the dark-field. Of the several types of dark-field, the cardioid condenser probably gives the most uniform results. Illumination from above with the incident light fall- ing on the slide at an angle of 30° is advised by Policard. This has some advantages over the dark-field but makes the use of higher magnifications difficult if not actually impossible. Cel- lular details are, therefore, to be ob- served best by using the cardioid dark- field. Identification of minerals. Some good results can be achieved by the use of ultraviolet light and with the sub- sequent fluorescence of minerals. Stu- dents should consult reference and text- books on mineralogy for details of identification. Calcium and magnesium are charac- terized in the dark-field by their dense white ash residues. Iron is oxidized during the incineration process and appears as varying tints of red. The amount of this element present can be MICROINJECTION 200 MICROMANIPULATION correlated with the color intensity. Silicon is definitely crystalline in char- acter and is recognizable by its property of double refraction in polarized light. This may at times be confusing since all minerals blend to some extent with the glass. Lead and other elements which yield black sulfides can be de- tected by treating the section with gaseous hydrogen sulfide. Uranium in pathological tissues fluoresces with a unique color under ultraviolet radia- tion. Attempts have been made to quanti- tate the ash residue by photographic means and by the use of a photoelectric cell whose output current is properly amplified. Both methods leave much to be desired both in accuracy and because of the utter relativity of the results obtained. The most useful finding obtained from microincinera- tion, therefore, is the appreciation of the distribution of the total minerals in the cell. Experimental alterations in them can be detected by the technique. See account by Scott in McClung's book and Electron Microscope, Histo- spectrography and Ultraviolet Photo- micrography. Microinjection. This is an important exten- sion of microdissection whereby various fluids are injected directly into the cytoplasm or nuclei of living cells. It is capable of yielding information on Permeability, Hydrogen Ion Concen- tration, Oxidation-Reduction Poten- tial which cannot be secured in any other way, but in reaching conclusions due allowance must be made for the fact that cells thus treated are of necessity severely injured. Microinjection with glass pipettes but without an expensive micromanipulator can yield worthwhile results as described by Knower (Mc- Clung, pp. 51-61) but for direct work on cells the micromanipulator is essential. Microliter Burettes are essential in some histochemical techniques. They are of two sorts. In the first a capillary glass tube is calibrated so that volume is indicated by the level of the meniscus. In the second the tube is not calibrated but instead a screw determining the level is provided as a micrometer. The best micrometer burette was designed by Scholander and has been improved by Scholander, P. F., Edwards, G. A. and Irving, L., J. Biol. Chem., 1943, 148, 495-500. In selecting a microliter burette consult Click, pp. 255-264. Micromanipulation — Revised by Robert Chambers, Dept. of Biology, Washing- ton Square College of New York University, New York. May, 1950 — Broadly speaking, this term covers two types of operations: delicate free-hand operations in which the only accessory may be a dissecting microscope, and, second, operations conducted by means of micrurgical instruments under high magnifications. For freehand operations considerable training is required in using a com- pound microscope because of the in- version of the image. This, however, can be corrected by using the so-called erecting ocular. A decided help to relieve fatigue from too long holding of, a dissecting needle, for instance, is to have the shaft of the needle held in the apex of a pyramid of plastic clay, the base of which has been pressed down on the stage to one side of the microscope. The operator's hand en- circles the mound of clay which bends as his fingers guide the needle. The tiring fingers can be released at any time while the needle tip remains in position. Descriptions of excellent methods for injecting minute vessels, such as the marginal vein of chick embryos or lymphatic vessels of frog tadpoles, are as follows: H. McE. Knower, Chapter in McClung's Hand- book Microscopical Technique, 3rd ed., New York: Hoeber, 1950; A. L. Brown, Anat. Rec, 1922, 24, 295. Micromanipulation in its more re- stricted sense applies to the use of mechanical devices for controlling the movements of the tips of microneedles and micropipettes in the field of high powers of the compound microscope. A full account is given in McClung's Handbook. Several instruments are now being built. The ones in most general use in this country are those of Chambers, P6terfi and Emerson. The micro- needles or pipettes extend into a moist chamber on the stage of the microscope so that their tips can be inserted into hanging drops of fluid suspended from the undersurface of a coverslip which roofs the chamber. The essential con- dition of an instrument is that the movements be sufficiently smooth and controllable under the highest magni- fications of the compound microscope. P^terfi's instrument was manufac- tured by Carl Zeiss Co. and is now difficult to procure. Chambers' was manufactured by E. Leitz and has MICROMAN IPULATION 201 MICROMANIPULATION undergone many modifications as to its accessory parts. It is now being manufactured by the Gamma Instru- ment Company, Great Neck, New York. Both instruments are supplied with two main holders each independent of the other for carrying a microneedle or a micropipette. The advantage of Chambers' is that the two holders are so adjusted as to permit the needles and pipettes to extend parallel to one another on the microscope stage. This permits the insertion of the needles into the moist chamber through one opening, thus increasing the chances of main- taining moisture conditions in the chamber. More recently an instrument devised by de Fonbrune of Paris is being manu- factured by A. S. Aloe Co., St. Louis, Mo. An early form of it is described in L'lllustration, February 15 and 22, 1941. It depends for its fine move- ments on hydraulic pressure conveyed through flexible tubing from a hand operated lever. Circus movements in the horizontal plane are performed by rotating the lever, the vertical move- ment being accomplished by a thrusting action of a plunger in the shaft of the lever. Two such instruments mounted on opposite sides of the microscope per- mit the use of two microneedles. The instrument has great possibilities but its specific usefulness for the perform- ance of circus movements is fully covered by the Emerson instrument which, incidentally, is sturdily con- structed. The Emerson is a first class machine with a mechanically controlled lever using circus movements in a horizontal plane, also a separate fine adjustment device for the vertical ones. Emerson has two models, the lower priced one having a lever control for both hori- zontal and vertical fine adjustments. These are being supplied by the J. H. Emerson Company, Cambridge, Massa- chusetts. The Chambers instrument is the only one supplied with a microinjection ap- paratus a description of which is given in the latter part of this article. Micrurgical instruments lend them- selves to several tj^pes of operations : (1) Microdissection and injection of animal or plant cells and tis.sues for studies in cell anatomy and physiology, also cj'to-chemistry in which chemical reactions can be obtained by applying chemical agents not only to individual cells but to localized regions within a given cell. (2) Chemical reactions in micro-drops. A very useful method is to deposit the droplets with a micro- pipette in a hanging drop of an inert oil. This prevents evaporation and the sphericity of the droplets in the oil permits quantitative determina- tions. Application of the technique to certain phases of microchemistry are given by Benedetti-Pichler in his book Introduction to the Microtechnique of Inorganic Analysis, New York: Wiley, 1942. (3) Isolation studies for obtain- ing pure line cultures (of bacteria, pro- tozoa, etc., breaking of asci and isola- tion of the liberated spores, etc.). A good isolation technique is given by Reyniers, J. A., J. Bact., 1933, 26, 251. The movements of the instruments can be controlled in any of three dimen- sions; the horizontal permits circus movements in one plane. Circus move- ments in the horizontal plane are best managed with the de Fonbrune and Emerson instruments. The vertical movement is operated by a separate controlling screw. Micro operations also can be performed under relatively low powers of the microscope. How- ever, the operator should realize that the compound microscope, even though it be binocular, is monobjective. This means that the position of an object in the vertical plane can be deduced only by observing whether the object is in or out of focus. The lower the magnifying power of the objective the greater is the depth of its focus. Hence, there may be occasions when the tip of the microneedle and the ob- ject to be operated upon are at different levels although both are in focus to the eye. The mechanical stage of the micro- scope is a useful adjunct for micrurgy. Particularly for injections, the most satisfactory way is to keep the tip of the micropipette in the center of the field and to perform the operation by raising the tip into the object to be injected after having brought the ob- ject into position by means of the mechanical stage. The manufactured instruments are supplied with instructions as to their use. Emerson supplies two types, one for coarser movements although it is possible to use this model for remark- ably fine operations. The only way to select an instrument is to know what is wanted and then to decide after having the instrument demonstrated to him. All require the use of a good mechanical stage to move the moist chamber which carries the drops containing the tissue to be operated on. All in all, micro- manipulation requires not only ability but mechanical aptitude on the part of the would-be operator. It is one thing MICROMANIPULATION 202 MICROMANIPULATION to have an instrument and a good micro- scope. It is another matter to build the many accessories, with cement, out of wood, glass or plastic, which the operator may need for his special pur- poses. Any gadget built may well mean a new discovery. Tissues and cells to be operated on often require special means for holding them in place. Actively moving pro- tozoa can be kept quiet by immersing them in egg albumen or a solution of hemi-cellulose. Strips of the epidermis of onion or tulip bulbs, immersed in varying concentrations of cane sugar, offer good objects for operation on pro- toplasts under different degrees of plasmolysis, likewise stamen hairs of Tradescantia which show mitotic figures. Similar studies may also be made on the epidermis of the tails of tadpoles. For these, the operator should use frogs' Ringer solution to maintain the proper balance of elec- trolytes in the medium. Muscle fibers stripped from the semitendinosus of the frog are good material. Urodeles fur- nish excellent material. An effective means of obtaining red cells undergoing mitosis is to bleed a Necturus or other member of the same order and take a sample of blood after a week or so. The microneedles and micropipettes are usually made from glass capillary rods or tubes. Serviceable sizes with an outside diameter of 1-2 mm. can be drawn out in a bunsen flame. The needle tips are made over a microfiame by heating and pulling the shaft of a capillary held at both ends with the two hands. A serviceable gas micro- burner for this purpose is a hypodermic needle. When successful, the drawn- out tips taper to a point rapidly enough so that the invisibly, fine tip is sup- ported on a relatively rigid shaft. The shaft about 2 mm. from the tip, is bent in the microfiame to about a right angle. The other end of the capillary is then inserted into a specially constructed needle-holder and mounted in a micro- manipulator so that the tip extends over the microscope stage into a moist chamber. The bent-up tip is adjusted with the screws of the instrument until the tip lies in a hanging drop of fluid suspended from a glass cover-slip serv- ing as the roof of the moist chamber and in the field of the microscope. Mechanical contrivances for drawing out the end of a glass capillary into a tapering tip are available. The most elaborate one is that of de Fonbrune, the microforge. This is a highly am- plified mechanism with a built in micro- scope and coarse adjustment parts for holding, one, a glass capillary and the other a platinum loop. The microscope is adjusted to view the loop of an elec- trically incandescing fine platinum wire to which one end of the glass capillary is approached and then withdrawn as the glass begins to melt. Then prop- erly done the tip of the capillary is drawn out to a tapering tip. The principle of the microforge is based on a much simpler device long ago devised by S. L. Schouten and de- scribed by him in 1934. This can be readily built in any laboratory equipped with an ordinary microscope and micro- manipulator. A fairly good mechanical device for drawing microtips is being supplied by the Gamma Instrument Company. It depends upon springs to which the two ends of a glass capillary (1-2 mm. diam.) are fastened while the middle of the capillary passes through several coils of a fine platinum or nichrome wire. When the wire is caused to incandesce electrically the glass softens and the taut springs pull the capillary in two, each part with a tapering tip. A more precise needle puller based on the same principle is the Livingston Micro Pi- pette Puller made by Otto K. Hebel, Swarthmore College, Pennsylvania. There are several possible ways by means of which a person with some ingenuity should be able to devise from the usual laboratory equipment a simple mechanical device for drawing needle tips. If the glass capillary is of tubing the microtips can be used for micro- capillaries. Injections are performed bj^ breaking the tip of a micropipette against the undersurface of the coverslip while the tip is in view under the microscope. Capillarity draws fluid into the shaft of the pipette when the open tip is in- serted into a hanging drop of fluid, be it oil or any given solution. For micro- injection, the pipette holder, mounted on the instrument, is attached to a looped, capillary brass tube of which the other end is attached to the nozzle of a syringe. Before mounting the micropipette, the syringe is filled with water previously boiled to be air-free and, by means of the plunger, the water is driven into the brass tubing and the pipette holder after which the micro- pipette is inserted. Thus, we have a water-filled system extending from the syringe to the base of the micropipette the shaft of which may contain air. Micro-amounts of any given solution are then drawn into or ejected from the tip of the micropipette by a delicate handling of the plunger of the syringe. MICROMETRY 203 MICROSCOPES Considerable deviations are possible in the matter of the microinjection technique. For example, if it is deemed desirable to have no air in the system, the shafts of the capillary tubing, on which the microtips are drawn, may previously be filled with either oil or water. By using some ingenuity the entire microinjection apparatus can be built in the laboratory, the parts re- quired being a Luer syringe, hypodermic needles the shafts of which can be cut off, a strip of flexible brass or copper tubing, glass tubing and of course the operator's constant companion: a stick of deKhotinsky cement or an analo- gous superior sealing wax. The instruments are generally supplied in pairs, one part carrying a micro- needle for holding the tissue to be injected, the other carrying the micro- pipette. For microdissection, the in- strument carries two needles, each of which can be operated independently. Wilhelm Pfeffer, to whom we owe the term "plasma membrane" for the limit- ing boundary of protoplasm, stated, in one of his papers in 1887, that an instru- ment with which one could operate delicate needles and pipettes in the field of a compound microscope would go far toward the elucidation of the nature of living cells. Pfeffer's dream has been realized in the development of the special field of science called today Micromanipulation or Micrurgy. Of general interest, and also for many details not described elsewhere, are the following: Barber, M. A., Philippine J. Science, B, 1914, 9, 307; Chambers, R., Anat. Rec, 1922, 24, 1; P^terfi, T., in methodik der wissensch. Biologic, 1928, 1 (4), 5; and Schouten, S. L., Zeit. f. wiss. Mikr., 1934, 51, 421. An excellent book which covers a broad range of the field of Micrurgy is that edited by J. A. Reyniers on Micrurgical and Germ-Free Techniques, C. C. Thomas, 1943, an article on micromanipulation by Chambers, R. and C. G. Grand, Encyclopaedia Britannica, 1948. Micrometry is the measurement of an object observed microscopically. This can be done either by using an ocular microm- eter in which there are lines which can be accurately moved the length of the structure to be measured or by inserting a ruled disc in an ordinary ocular with which it can be compared. Both must be standardized in relation to a microm- eter slide generally ruled with lines 10m apart. See Cell Measurements. Micromicron (nn) = 1/1 ,000,000th part of a micron = 1/1, 000 ,000 ,000th part of a mm. = 10- » mm. = 0.000,001 micron = 10~^A. Unfortunately often used syn- onymously with o millimicron (m/i) = 0.001 micron = lOA. Micron (Gr. Mikros, small) expressed by Gr. letter n = approximately 1/25,000 inch = 1/1000 part of a mm. = 0.001 mm. = 10-3 mm. = 10,000 A (see Millimicron and Micromicron). Microphotometer, see Photoelectric. Microradiographic examination. This con- sists of magnification of a Roentgen ray image after it has been registered pho- tographically. The essential point is to use film of very fine grain emulsions. Thus the Gevaert Lipmann emulsion permits enlargement 300 times without much loss of detail. In some cases it is helpful before microradiographic examination to increase the absorption of Roentgen rays by "absorption stain- ing" through adding radio-opaque mate- rials such as barium sulp'aate and thoro- trast. The application of this technique in the study of biologic materials is described and illustrated by Clark, G. L. and Bick, E. J., in Glasser's Medi- cal Physics, 730-733. The importance of extremely soft Roentgen rays and the properties of fine-grained emulsions are described by Engstrom, A. and Lindstrom, B., Acta Radiol., 1951, 35, 33-44. See also their illustrations of striated muscle fibers and of bone by microradiography. Microrespirometer to indicate production of carbon dioxide by bacteriophages, viruses and bacteria (Bronfenbrenner, J., Proc. Soc. Exp. Biol. & Med., 1924, 22, 81-82. See Capillary Respirometry. Microscopes (From Cowdry's Histology, 1950). While excellence in histological technique is important, knowledge of microscopes and of how to get the best service out of them is also important. There are several kinds from which a choice must be made of the one capable of yielding the information required. The following account of what these instruments are, what their particular use is, what their limitations are, sup- plemented by leading references to literature on the subject is offered for guidance. It was written for Cowdry's Histology by Dr. T. B. Rosenthal. The ordinary compound microscope is a precision optical instrument designed to give magnified images of 50 to 1200X. Daylight, or strong artificial light, is used to illuminate by transmission a more or less thin, flat, transparent object. The image appears in its nat- ural form and color, but inverted and reversed in position, at a distance of about 10 inches from the eye. Height and depth are not reversed, however, so it is possible to judge or measure MICROSCOPES 204 MICROSCOPES the elevations on an object by differen- tial focussing. Three lens systems, condenser, ob- jective, and ocular cooperate to form the final image. The purpose of the condenser is two-fold: to gather light for illumination, and to focus this light on the object in a cone of the proper dimensions so that the full resolving power of the objective lens can be gained. By means of the objective lens a real, magnified, inverted, re- versed image of the object is formed at the upper end of the tube. The ocular further magnifies this primary image to yield a virtual, inverted, reversed sec- ondary image. Total magnification is calculated by multiplying the magnify- ing powers of the objective and the ocular. Inspection of the markings on a set of objectives will show their magnifying powers and another optical property called the numerical aperture (N. A.). Usually the 10 X (also called 16 mm) objective is marked with the number "0.25;" the 44X (4 mm, "high dry"), "0.66;" and the oil immersion, 95X (1.8 or 2.0 mm), "1.25." Numerical aperture, calculated from a geometrical property of the front lens element, is proportional to the theoretical resolving power of the objective and is related to the maximum power of the ocular that can be profitably used with that objective. In order to understand this relation between magnification, resolution, and N. A. it is necessary to consider certain fundamentals. As successively higher powers of mag- nification are brought to bear on a tis- sue section more and more fine detail becomes visible, which is, after all, the only purpose of microscopy. But to fineness of detail a limit is set, not by magnification, but by the nature of light and by the optical properties of lenses. As long as the object to be seen is large compared to the wave length of the light which illuminates it the microscopic image will be sharp. If structural details are so small that their size nearly approaches the wave length of light, the image becomes fuzzy. Although magnification may be secondarily raised by employing stronger eyepieces no further resolu- tion of detail takes place; the image remains fuzzy, and we have what is called "empty magnification." The formula R = X/2 N. A. gives a general relationship, where X = wave length of light used, N. A. is a given constant of the objective lens, and R is the size of minimum resolvable detail (given in /i if X is given in fx). Taking an average value for X in white light to be 0.5 fi and substituting the N. A. values mentioned above, we have for the lOX objective, R = 1.0 m; for the 44X, R = 0.4 m; and for oil immersion, R = 0.2 /x. Thus, with the best lenses, details lying less than 0.2 m apart are not discerned. Another general rule in microscopy states that the maximum total magnifi- cation should not be pushed higher than about one thousand times the N. A.; otherwise empty magnification results. With the lOX objective, 1000 X 0.25 = 250. Therefore a 25X ocular is the maximum. For the oil immersion, a 13X ocular gives the limiting useful magnification. It is thus evident that the objective lens is the heart of the microscope. The ocular brings out only the details which already have been resolved by the objective. The role of the condenser may be reviewed in the light of these interpre- tations. Historically the condenser received its name from its original pur- pose: it was simply a lens used to con- centrate light on the subject. E. Abbe (1840-1905), who contributed a great deal to theoretical and practical microscopy, including the concept of numerical aperture, came to the con- clusion that an objective lens could not work at its maximum N. A. unless it were matched with a condenser lens system of equal N. A. Hence the modern condenser is also rated in terms of N. A. and is provided with a variable iris diaphragm to alter its N. A. in order to match with that of the objective. Since the standard microscope pro- vides all the convenient magnifications and even more resolution than is usually required in routine histology, it is not necessary to take extraordinary pains to coUimate (parallel) the microscope with the light source or to worry over the fine points of diaphragm control. However, good photomicrography de- mands such attention to details, be- cause the plate is much more sensitive to inequalities of lighting than the ob- server's eye. On the other hand the eye is subject to fatigue unless the proper illumination is employed. In student classes one commonly finds that the lighting with monocular micro- scopes is too bright and with binoculars too dim. To minimize visual fatigue the brightness of the microscopic field of view should about equal the bright- ness of the table-top and be in keeping with the general level of illumination in the room. The following suggestions for setting MICROSOMES 205 MICROSOMES up the microscope apply to the oil immersion lens. When dry lenses are to be used omit step 2. 1. Clean the slide with alcohol. Put a drop of xj-lol on the oil immersion lens and polish it with lens paper. Do the same for the eye piece. 2. Raise the condenser so that its upper surface is on a level with the stage, or slightly below it. Put a drop of immersion oil on the condenser and lay the slide down. Put a drop of oil on the slide, lower the lens, and focus as usual. 3. Open the diaphragm wide, and removing the eye piece, look down the tube. Adjust the position of the light source and mirror so that the aperture appears sj^mmetrically illuminated. 4. At this point the aperture should look like a bright disk surrounded by a rim of dim illumination. Now, close down the diaphragm until about nine- tenths of the area of bright central field remains visible. Replace the eye piece. The N. A. of condenser and objective are now approximately equal. 5. Modify the brightness of the image for visual comfort by altering the in- tensity of illumination; not by altering the diaphragm. If the image is too bright put tissue paper over the lamp or pull the lamp away and readjust by repetition of Steps 3 and 4. If the image is not bright enough move the lamp in, and if necessary dispense with the substage mirror. As the final step, try a slight change in the position of the condenser, but avoid breaking the oil pool between the condenser and the slide. Resolving power may be improved somewhat for striated, or periodic, structures by using oblique illumina- tion. Light is sent diagonally from below so that only part of it enters the objective, with the striations (as in muscle) lying across the direction of the light. If the condenser is not laterally movable it should be masked below so that light enters only from one side. By trial and error a favorable orientation of condenser, slide and mask can be found. The limitations imposed on the re- solving power of the conventional microscope cause no inconviences in general histology. It is with the finer details of cytology that need is felt for greater resolution. As is obvious from the formula R = X/2 N. A., either an increase of N. A. or a decrease of X will reduce the value of R. The best available oil immersion objectives and condensers are rated at 1.40 N. A. A special lens working at 1.60 has been designed, but the improvement in re- solving power over one at 1.40 is not important. Fortunately we can ob- tain the shorter values of X by em- ploying ultra-violet light and photog- raphy (see ultraviolet-microscope). A word should be said in favor of water-immersion objectives, items for- merly found in every laboratory of microscopy but now no longer listed in the catalogues of American manufac- turers. These are made in a series of magnifications, and in resolving power are intermediate between air and oil- immersion objectives. For study of living aquatic organisms and tissue cul- tures the lOX and 44X objectives are very useful since they are made to be lowered directly into the medium. For histological slide material the high- power water-immersion objective is probably not inferior to the oil-immer- sion, besides being more convenient to use. It is commonly believed that a bin- ocular, i. e., two eyepiece, microscope is superior to the monocular because it affords stereoscopic vision. Actually, it cannot be so since a single objective forms only one primary image regard- less of whether it be viewed by one or two eyes. Nevertheless our habit of seeing with two eyes probably creates an illusion of stereoscopic vision with the binocular microscope. On the other hand a binocular microscope em- ploying two objectives on a pair of converging tubes does provide stereo- scopic views. The dissecting micro- scope is built on this pattern, having in addition a set of reversing and erect- ing prisms so that the final image is normally orientated. Magnifications above 30X become rather useless for dissection purposes because the depth of focus is greatly reduced. Manipula- tion is usually by hand. See Centrifuge, Color Translation, Darkfield, Electron, Phase Contrast, Metallurgical, Polarizing, Reflecting and Ultraviolet Microscopes. Many excellent books on microscopes and photomicrography are available. The following are suggested: Gage, S. H., The Microscope. Ithaca: Cornstock, 1941, 616 pp. Shillaber, C. P., Pho- tomicrography in Theory and Prac- tice. New York: John Wiley and Sons, 1944, 773 pp. Wredden, J. H., The Microscope, Its Theory and Applica- tion. New York: Grune and Stratton, 1948, 296 pp. Microsomes (G. mikros, small, soma, body). Term introduced by Hanstein in 1880 originally to indicate tiny granules — as MICROSPECTROPHOTOMETRY 206 MICROSPECTROPHOTOMETRY compared with ground substance. Claude, A. Biological Symposia, 1943, 10, 111-129 estimates their size to be 50-300 ran and therefore beyond limits of ordinary microscopic visibility. These microsomes of Claude are ob- viously not the ones which Hanstein had in mind. According to Claude they are essentially ribose nucleopro- teins and phospholipins in definite proportions. Microspectrophotometry — Barry Commo- ner, The Henry Shaw School of Botany, Washington University, St. Louis 5. November 28, 1951 — Microspectropho- tometry is a technique for examination of cells and cell structures designed to yield data on the chemical composition of these objects. The method is based on the fact that given molecular configurations absorb specific wave- lengths of ultraviolet, visible or infra- red radiation. Under ideal conditions, as in dilute solutions, the molecular group and therefore the substance in which it occurs may be identified from the shape of the absorption spectrum, and its concentration in the sample determined from the amount of absorp- tion at a characteristic wavelength. Unfortunately, cytological objects never offer such simple, readily analyz- able situations. Consequently, special steps must be taken to evaluate the absorption spectra of cytological objects, and in most instances, data com- parable with those obtained from solu- tions are not yet attainable. Never- theless, the technique has thus far proved to be a valuable source of in- formation on cell composition, and if used with care can be advantageously applied to a number of biological prob- lems. The essential measurement in micro- spectrophotometry is determination of the reduction in intensity of a light beam after passing through a cytologi- cal object. This measurement, made at a series of specific wavelengths, gives the absorption spectrum of the object. The measurements may be made by passing a monochromatic beam through the object; or by passing a heterogene- ous beam through the object and then dispersing it into a spectrum. Equip- ment employing the second of these methods has been described by (Mellors, R. C, Science, 1951, 112, 381-389). Since most microspectrophotometry has employed the first principle of operation, details for this type of ap- paratus are given below. 1. General. The fundamental parts of a microspectrophotometer consist of a suitable light source, a monochrom- ator, microscope optics, a photoelec- tric tube which receives the projected image of the object, and appropriate means of measuring the phototube response. 2. Light sources. Ordinary automo- bile headlight tungsten lamps operated on a storage battery are suitable for work in the visible spectrum. For work in the ultraviolet ranges mercury dis- charge tubes provide adequate light intensity. High pressure mercury lamps (such as General Electric AH6) emit a continuous spectrum in the vis- ible and ultraviolet superimposed on a number of bright and dark lines. In the infra-red ranges Nernst glowers are suitable. 3. The monochromator. The light beam from source is directed into the entrance slit of a monochromator, thus giving an emergent beam of a determined mean wavelength. The monochromator should supply the con- denser of the microscope with a beam sufficient to fill the aperture of the latter. 4. The microscope. The optical ar- rangement of the microscope must be such as to give a true light image of the object at the plane of the photocell. The requirements for this condition have been presented by Caspersson, T. (Cell growth and cell function, New York: Norton, 1949). For visible work any good apochromatic system of suffi- cient numerical aperture is adequate. In the ultraviolet range quartz lenses or reflecting objectives must be used. The latter have the advantage of being achromatic in the ultraviolet spectrum. 5. The light detector. The most suitable detector for this type of work is the photomultiplier tube. The tube is supplied with a suitable power source and its output amplified and read off a microammeter. Less conveniently the direct output may be detected with a galvanometer. The construction of microspectro- photometric equipment has been de- scribed in the following papers: Norris, K.P. and Wilkins,M.H.F., Discussions of the Faraday Society, 1950, No. 9, 360-363, Barer, R., Discussions of the Faraday Society, 1950, No. 9, 369- 378, Mellors, R. C., Discussions of the Faraday Society, 1950, No. 9, 398-406, Thorell, B., Discussions of the Faraday Society, 1950, No. 9, 432-436, Walker, P. M. B. and Davies, H. G., Discus- sions of the Faraday Society, 1950, No. 9, 461-470, Commoner, B., Ann. Mo. Bot. Card., 1948, 35, 239-254, Burch, C. R., Proc. Physic. Soc, 1947, 59, 41-46, Grey, D. S., J. Opt. Soc. MICROSPECTROPHOTOMETRY 207 MICROSPECTROPHOTOMETRY Amer., 1950, 40, 283-290, Mellors, R. C, Science, 1951, 112, 381-389. The prob- lem of instrumental accuracy is dis- cussed by Click, D., Engstrom, A. and Malmstrom, B. G. (Science, 1951, 114, 253-258) and Caspersson, T. (Cell growth and cell function, New York: Norton, 1949). The light absorption due to the ob- ject may be determined in the following ways. 1. Direct method. The microscope is focused on an area of the object slide adjacent to the object itself. The light impinging on the photo-cell is then adjusted to give a fixed response in the detector circuit. Without alter- ing the optical conditions the slide is then moved so that the object is now centered in the field and a reading of the detector response is taken. This measurement compared with the first one yields the per cent of the incident light which is transmitted by the ob- ject. 2. Split-beam method. In this method the light beam is split before entering the microscope and a small fraction directed toward an accessory phototube and detector circuit. It is then possible to evaluate the intensity of the light reaching the main photo- tube in terms of the intensity of the beam directed toward the seconday phototube. This method has the ad- vantage of being independent of random fluctuations in the intensity of the light source. As in the direct method the absorption of the object is determined by comparison with a blank area in the slide. Interpretation of data: The derivation of valid conclusions from intracellular absorption data is a considerably more difficult task than the experimental work itself; in fact, it is frequently the case that data are obtained for which there are as yet no valid interpretations. The difficulties in interpretation arise from the fact that little is known con- cerning the physical state of intracel- lular objects and its effect on their optical properties. Since the rules for analysis of absorption data are based exclusively on the optical properties of homogeneous systems such as gases and dilute solutions, entirely new methods need to be developed for the hetero- geneous structures of the cell. For homogeneous solutions of ab- sorbing materials, the following rela- tions (the Beer-Lambert Laws) are found, within limits, to hold: D = kcd where c is the concentration of absorb- ing material, d is the length of the opti- cal path through the absorbing layer, and k is the extinction coefficient of the substance in question. D, the optical density, is defined by the expression D = logio y, where lo is the intensity of the incident beam and I the intensity of the transmitted beam. The Beer- Lambert relationship holds only where all light absorption is due to the trap- ping of photons by the substance in question, and where each molecule in the light path contributes equally to the absorption. In microspectrophotometric work, the value of D for a given optical sec- tion through the object is determined experimentally at various wavelengths. This gives the absorption spectrum of the entire object. Since the aim of the measurement is usually the identifica- tion and estimation of a specific sub- stance present in the object, the initial spectrum needs to be evaluated in the light of the above relationships. In order to identify a specific sub- stance from the absorption spectrum of an object believed to contain this substance, the following conditions must be met. 1) It must be known that the object contains no absorbing material other than the substance in question; or the relative contents and absorption spectra of extraneous sub- stances must be determined. Without this information one cannot conclude that a given absorption maximum in the spectrum of the object is due to a spe- cific substance ; such a maximum may re- sult from the superposition of the spectra of two or more different sub- stances. 2) It must be known that the absorption of light at any given wave- length is due only to the trapping of photons by the substance in question and is not a consequence of scattering and similar non-specific processes. In general, light scattering rises with in- crease in frequency but since sharp changes in refractive index occur near the absorption maximum of a sub- stance, scattering effects may some- times pass through a maximum with variation in wavelength. If in addition to identification an estimation of the relative arnount of the substance present in the intracel- lular object is to be made, the follow- ing further conditions must be fulfilled. 3) The length of the optical path through the object must be known. This is not necessarily identical with the thickness of the object for in hetero- geneous systems internal reflections may cause a significant fraction of the entering beam to be reflected through MICROSPECTROPHOTOMETRY 208 MICROSPECTROPHOTOMETRY a total path length considerably longer than the dimensions of the object. This effect will enhance the absorption of the object and may be variable with wavelength. 4) The relationship be- tween the optical density of an object and the concentration of the specific substance must be demonstrated since it cannot be assumed that the linearity predicted by Beer's Law will hold. 5) The effect of inhomogeneous distri- bution of the absorbing material in the planes perpendicular to the optical axis must be estimated since relatively small departures from homogeneity will lead to significant alterations in the absorption due to a given amount of material. If the absolute quantity of a given substance in an intracellular object is to be determined, two further condi- tions must be met. 6) The specific extinction of the substance in question, that is, the absorption per mole per liter must be known. 7) The influences of the conditions noted above on the extinction must be determined quan- titatively. If accepted analytical prac- tices are to be followed, this would mean that the absorption due to a known amount of the specific substance when added to the intracellular object be determined or that the amount of the substance found in the object by inde- pendent analytical means be correlated with its absorption characteristics. Unfortunately, presently available techniques can meet the above condi- tions only in part. The procedures thus far worked out or suggested are noted below. / . The problem of complex composition of the object. If it can be shown that the composition of the object is uniform throughout its optical depth, then the relative contribution of individual sub- stances to the overall absorption spec- trum may be worked out by comparison of the latter with individually deter- mined spectra of the separate compo- nents. However, large differences in the height of the maxima frequently make this procedure a difficult one. To demonstrate that this optical uni- formity exists, it must be shown that the absorption spectrum of the object is constant for all thickness (obtained, for example, by sectioning). Where this is not possible as in the case of structures enclosed within intact liv- ing cells an optical method may be used (Commoner, B., Discussions of the Faraday Society, 1950, No. 9, 449-460). This procedure may be applied to ob- jects in which one component is dis- tributed in an invariant layer in a cell which varies considerably in thickness. In this case it is possible to determine the absorption due to the separate com- ponents in situ by measuring the ab- sorption spectra of two regions in the cell which differ in thickness by a known amount. In favorable instances this method may be used to determine the absorption spectrum of the nucleus con- tained within a living cell. Thus far the procedure has been applied only to certain types of plant cells. Failure to conform with this condition casts doubt on the meaning ascribed to the absorption spectra of structures such as the nucleolus which have been ob- tained without detailed analysis of the contribution made by over-lying and under-lying material. 2. Non-specific light losses. It has been frequently assumed (Caspersson, T., Cell growth and cell function. New York: Norton, 1949) that light lost due to scattering is related to wave- length according to the Rayleigh equa- tion. Using this assumption and the further assumption that at some spe- cific wavelength range such as 300-350 mM no specific absorption occurs, the scattering losses are calculated by ex- trapolation from the readings obtained in this limited range. The usefulness of this method is considerably weakened by the fact that the first assumption has never been demonstrated to be true for intracellular objects and that the second assumption can be true only very rarely. An experimental determina- tion of losses due to scattering may be made by an apparatus described by Caspersson, T. (Cell growth and cell function, New York: Norton, 1949) which measures the light emerging from the object at various angles from the optical axis. Scattering losses may vary considerably with the physi- cal state of the object and are, there- fore, very sensitive to fixation proce- dure, etc. 3. The length of the optical path. Thus far no method for determining this value has been proposed and in prac- tice the only approach to the value of this dimension is the observed thickness of the object. Thus in making this assumption, calculations are exposed to an error of unknown magnitude. 4- The validity of Beer's law. This essential determination has thus far been carried out in only one instance (Commoner, B., Discussions of the Faraday Society, 1950, No. 9, 449-460) in which the cellular material occurred in solution in the vacuole of a mature plant cell. In this case it was possible to alter the concentration of the dis- MICROTOME 209 MILK solved material by plasmolysing the cell to various degrees and thereby al- tering the water content of the solution. This method permits comparison of the optical density of the vacuole at various volumes and provides a direct test of Beer's law. In the case of solid struc- tures such as the nucleus the osmotic method is probably invalidated by the fact that the absorbing material is not freely dissolved throughout the struc- ture. When the cytological object contains oriented absorbing material in signifi- cant amounts, considerable departure from the Beer-Lambert relationship is to be expected (Commoner, B., Science, 1949, 110, 31-40). It may be possible to avoid this difficulty by the use of a polarized incident light beam. 6. The effect of lateral inhomogeneity. This problem has been discussed from a theoretical point of view (Glick, D., Engstrom, A. and Malmstrom, B. G., Science, 1951, 114, 253-258; Danielli, J. F., Cold Spring Harbor Symp., 1949, 14, 32-39). It is apparent from these discussions that large errors may arise from this effect but at present there is no experimental way of determining their magnitude. In general it would seem essential that objects studied be homogeneous at least with respect to their microscopic appearance. 6. The determination of absolute quan- tities. In some instances (Caspersson, T. and Schultz, J., Proc. Nat. Acad. Sci., 1940, 26, 507-515) investigators have calculated the absolute content of, say, nucleic acid in a nucleus from meas- urements of the optical density of the nucleus and the specific extinction of nucleic acid solutions. Such a proce- dure has never been validated by the customary tests of the analyst. 7. The effect of ultraviolet light on the optical properties of the cell. One of the most serious difficulties encountered in the microspectrophotometric work is the fact that exposure to ultraviolet light is damaging and frequently lethal to the object. At the same time irra- diation seriously alters the absorption properties of the cell. It was first shown by Brumberg, E. M. and Larionov, L. P. (Nature, 1946, 158, 663-664) that in living cells undamaged by ultraviolet the optical density of the nucleus does not exceed that of the cytoplasm. When the cell has been killed by ultraviolet radiation the opti- cal density of the nucleus rises sharply whereas the absorption of the cytoplasm drops. Similar changes have now been observed by a number of other workers (Bradfield, J. R. G., Discussions of the Faraday Society, 1950, No. 9, 481-490; Walker, P. M. B. and Davies, H. G., Discussions of the Faraday Society, 1950, No. 9, 461-470). These observa- tions show that 1) absorption measure- ments of living cells must be made on cells which actually survive the ex- perimental procedure, 2) fixation pro- cedures may seriously alter the apparent distribution of ultraviolet absorbing material and 3) the structural ar- rangement peculiar to the living nu- cleus probably has a significant effect on its absorption properties. Microtome. The freezing, rotatory and sliding microtomes are well known and advertised. The high speed micro- tome required for cutting especially thin sections for Electron Microscopy is essential for research. See account by G. H. Scott in McClung's Micro- scopical Technique, 1950, p. 720; also FuUam, E. F. and Gessler, A. E., Rev. Sci. Inst., 1946, 17, 23 and Gessler, A. E. and Fullam, E. F., Am. J. Anat., 1946, 78, 245. Geren, B. B. and McCuUoch, D. (Exp. Cell Research, 1951, 2, 97-102) have made adjustments in the Minot rotary microtome by which sections of tissues, prepared by the methacrylate embedding technique of Newman, S. B., Borysko, E. and Swerdlow, M. (Science, 1949, 110, 66) can be cut 1/20 n in thickness. The edges of some sec- tions are much thinner. Glass knives with water trough (Latta, H. and Hart- mann, J. F., Proc. Soc, Exp. Biol, and Med., 1950, 74, 436) are a great improve- ment over steel ones. Microtome Knife, sharpening. There is no easy method. Care and long practice are essential. (See Bensleys, p. 57.) For the usual oil and water stones a ground glass is now sometimes substi- tuted (Uber, F. M., Stain Techn., 1936, 11,93-98). Micrurgical Technique (Gr. micros, small + ergon, work) is referred to under the heading of microdissection. Mikado Yellow G (CI, 622)— Stilbene Yel- low— a direct dye of light fastness 3. Similar to Sun Yellow but lighter in color (Emig, p. 46). Milk, bacteria in, a modification of Newman technic (Broadhurst, J. and Paley, C, J. Am. Vet. Med. Assoc, 1939, 94, 525-526). To prepare stain add 0.4 cc. cone. H2SO4 to 54 cc. 95% alcohol. Mix with 40 cc. technical tetrachlor- ethane in flask and heat to 55 °C. but no higher. Add about 1.0-1.2 gm. methy- lene blue while mixture is still hot. Shake until dye goes into solution. Then add 8.0 cc. 1% basic fuchsin in 95% alcohol. Mix, cool, filter and put up in glass stoppered bottle. Spread MILLIMICRON 210 MITOCHONDRIA 0.01 cc. milk over area of 1-2 sq. cm. on slide. Dry on flat warm surface 5 min. Flood with stain 15 sec. Drain off ex- cess and dry while flat with gentle heat. Wash in cold water till all blue is re- moved and a faint pink color appears. Dry and examine. Technique for the rapid detection of Mycobacterium tuberculosis in cows, milk is described by Maitland, M. L. C, J. Hyg., 1950, 48, 397-401. Millimicron (m^) = 1/lOOOth part of a micron = 1/1, 000,000th part of a mm. = 10~' mm. = 0.001 micron = 10 A (see Micromicron). Millon's Reaction. For microchemical pur- poses it is necessary, as Bensley and Gersh (R. R., and I., Anat. Rec, 1933, 57, 217-233) point out, for the reagent to act without the aid of heat, to give almost immediately with tyrosin in vitro an intense red color yielding red ppt. not changing to yellow within 24 hrs. They give the following directions. Add 600 cc. aq. dest. to 400 cc. cone, nitric acid (sp. gr. 1.42) making 40% by volume. After 48 hrs. add 1 part to 9 parts aq. dest. Saturate with mercuric nitrate crystals frequently shaking sev- eral days. To make the reagent take 400 cc. filtrate, add 3 cc. original 40% solution plus 1.4 gm. sodium nitrite. Mount sections (preferably after freez- ing and drying technique) to slides without using water. Immerse in rea- gent in cold. Maximum reaction should be within 3 hrs. when sections show noticeable rose color. However use several slides, remove them from reagent in a Coplin jar at intervals, dip imme- diately in 1% aq. nitric acid, dehydrate quickly in absolute alcohol, clear in xylol and mount in balsam. Bensley and Gersh found that mitochondria are positive to Million's reagent. Mineral Oil, reactions in tissue to fat stains after various fixations (Black, C. E., J. Lab. & Clin. Med., 1937-38, 23, 1027-1036). See Immersion Oils. Mingazzini Phenomenon in intestinal villi interpreted as an agonal or early post- mortem change (by Macklin, C. C. and M. T., J. Anat., 1926, 61, 144-150). Mites. The techniques given for Ticks and Insects are applicable for making whole mounts. The simple creosote method (see Insects) is recommended. Mitochondria — Written by Geoffrey Bourne, London Hospital Medical College, Lon- don, England. November 5, 1951 — Granules, rods and filaments existing in the cj'toplasm of practically all liv- ing cells of plants and animals. They can be studied in living cells unstained and after supravital staining, and in fixed tissues. They can be seen in living cells even with direct illumination if it is critical. In mammals they probably are best seen unstained in very small pieces of pancreas mounted in normal saline and flattened out by the pressure of the cover glass. The distal poles of the acinous cells, facing the glandular lumen may be identified by densely packed, highly refractile zymogen gran- ules. The proximal poles are nearer the surrounding blood vessels and com- paratively free from zymogen granules. With direct illumination a careful search with an oil immersion objective will distinguish the mitochondria as delicate, slightly refractile filaments oriented in general with their long axes parallel with the length of the cell. Wernicke illumination (phase contrast) will permit immediate recognition of these bodies which are shown up clearly by this method. Phase contrast en- ables mitochondria to be studied with ease in a variety of living unstained cells (H. U. Zollinger, Rev. d'Hematol., 1950,5,696). The clarity with which mitochondria may be observed with phase contrast in living unstained cells has rendered their supravital staining less necessary. However, on occasions, this may be desirable. When very dilute methylene blue is added to tissue culture cells the mitochondria become stained a bril- liant blue (Ludford, R. J., Arch. f. Exp. Zellf., 1935, 17, 339-359). Other vital dyes are diethylsafranin, Janus blue, Janus black 1, Pinacyanol, Rhodamin B. and Janus green B. (diethylsafra- ninazo-dimethyl-analin chloride) . The latter is used, as an example, as follows: Place a small drop of 1:10,000 Janus Green B in 0.85% aq. sodium chloride, dj^e should be added from a 1% stock The solution in aq. dest. because the power does not dissolve easily in salt so- lution. In this drop a variety of small pieces of tissue, may be teased, and then covered with a cover glass, and mito- chondria may be demonstrated very beautifully in blood leucocytes by add- ing a drop of freshly drawn blood to a drop of 1 in 10,000 solution of Janus Green B and examining after adding a coverslip. Mitochondria become col- ored a dark bluish-green after 5-10 min. They first appear in the lymphocytes and then between the granules of the granular cells. R. R. Bensley (Am. J. Anat., 1911, 12, 297-388) has described a method of intravascular staining of mitochondria using Janus Green B. Janus Green preparations of mito- chondria are not permanent — they MITOCHONDRIA 211 MITOCHONDRIA bleach in from 30-60 min. to a colorless leucobase. Mitochondria show up bril- liantly in living, particularly tissue culture, cells using dark-ground il- lumination (see Strangeways, T. S. P. and Canti, R. G., Quart. J. Micr. Sci., 1927, 71, 1-14). They may be also photographed at high magnification with the electron microscope (R. Claude and E. F. Fullam, J. Exp. Med., 1945, 81, 51-62; H. U. Zollinger, E.x- perientia, 1950, 6, 16-17). Mitochondria can be isolated from tissues by the process of differential centrifugation (R. R. Bensley and N. Hoerr, Anat. Rec, 1934, 50, 251, 499). This technique has been developed by Porter, K. and his colleagues (J. Exp. Med., 1945, 81, 233-246); Claude, A, (Science, 1943, 97, 451-456; J. Exp. Med., 1944, 80, 19); Hogeboom, G. H. and his colleagues (J. Biol. Chem., 1946, 165, 615-630). These authors not only developed the technique of isola- tion of mitochondria by differential centrifugation and were able to obtain mitochondria which were morphologi- cally identical with those in the living cell, but by a series of chemical studies were able to show that such isolated mitochondria contain the greater part of the respiratory enzymes of the cell, a fact which suggests, not only that the mitochondria may be the main respira- tory centers of the cell but that they can function as synthetic centers as well. The conception of the respira- tor}^ function of mitochondria was sug- gested as long ago as 1912 by Kingsbury A. (Anat. Rec, 1912, 6, 39). Isolation of Mitochondria. Hoge- boom, G. H., Schneider, W. C. and Pallade, G. E. (Proc. Soc. Exp. Biol. Med., 1947, 65, 320-321) have described a method of obtaining morphologically intact mitochondria from rat liver. Rat liver is homogenized by the method of Potter, V. R. and Elvehjem, C. A. (J. Biol. Chem., 1936, 114, 495-504) in 0.88 M. sucrose. The homogenate is centrifuged 3 times at 600 g. for 10 min. This removes nuclei and intact cells. The supernatant is then centrifuged at 24,000 g. for 20 min. This brings down the mitochondria together with a few microsomes. Mitochondria obtained in this way remain stable in form for several days at 4°C. There are a variety of methods for making perma- nent preparations of mitochondria in tissue sections. 1. Altmann's method. See Carleton, H. M. and Leach, E. H., Histological Technique, Oxford, 1949. Fix in Champy's fluid or in Flemming without acetic. Postchrome for 3 days (trans- fer tissue direct to 2§-3% aq. potassium dichromate). Wash 12-24 hrs. in run- ning water, dehydrate, embed and sec- tion. Bring sections to water, then: (1) flood slide with aniline fuchsin (aniline water, made by adding 10 cc. of aniline to one half or one litre of hot aq. dest. in a flask and ashing, cooling and filtering, 100 cc, acid fuchsin 12 gm.). Warm slide with bunsen flame till the stain steams (but does not boil). Leave for 5 min. (2) rinse rapidly in aq. dest. (3) differen- tiate in slide jar of picric acid, sat. sol. in ab. ale. 20 cc, 30% ale 80 cc. Dif- ferentiation should be stopped when the red dye has diffused out of the nuclei and cytoplasm and the mitochondria are bright red. (4) rinse in aq. dest. (5) dehydrate rapidly, mount in bal- sam. 2. Heidenhain's iron hematoxylin method. Tissues may be fixed in formaldehyde, Helly's fluid, Zenker for- maldehyde or Flemming-without-ace- tic, for 24 hrs. Sections are mor- danted in a 5% aq. iron alum for 5 to 24 hrs. according to the nature of the tissue. Rinse in water. Stain in 0.5% aq. hematoxylin for a period of time equal to that of the iron alum treat- ment. Rinse in water, differentiate in iron alum solution and control dif- ferentiation with microscope until only nuclei and mitochondria are black. Counterstain if necessary, dehydrate, mount in balsam. 3. Cain's method (Quart. J. Micr. Sci., 1948, 89, 229-231). Fix in Helly's fluid, 6 hrs. postchrome 48 hrs. at 37°C. in sat. aq. potassium dichromate, wash overnight in running water, embed in parafRn wax and cut sections about 3 n. Bring sections to water treating with iodine (i in 70% ale) and then 5% aq. sodium thiosulphate on the way. Dry slide, except sections, flood with aniline fuchsin, (see Altmann's method) and heat until steaming as for Alt- mann's technique. Wash off acid fuch- sin with aq. dest. Irrigate with alka- line solution (one drop of aq. sodium carbonate in 10° cc. of aq. dest.). Dif- ferentiate 30 sec. to I5 min. To stop differentiation dip slide into 1% HCl. Wash in aq. dest., counterstain 1% aq. water-soluble methjd blue. Wash aq. dest., dip into 1% acid 3 sec. only. Wash aq. dest., dehydrate and mount in balsam. The Bensley-Cowdry acid fuchsin and methyl green method (Cowdry, E. V., Contrib. Carnegie Inst., Wash., VIII, 1918) gives beautiful results. In it the methyl green is used both as differen- tiator and counterstain. MITOCHONDRIA AND BACTERIA 212 MITOSIS Schridde's method (Ergn. Anat. u. Entw., Bonnet, XX, 1911) can also be recommended as giving beautiful prep- arations of mitochondria. 4. Pritchard's silver method (J. Anat., 1951 , 85, in press) . This method, which is dependent upon reduction of silver on the slide, gives very beautiful and precise preparations of mitochondria which show up black. Small pieces of tissue should be fixed in Regaud's or Helly's fluid for 3 da3's and post- chromed for 4 days in 3% aq. potassium bichromate. Prepare paraffin sections in the usual way, avoiding excessive heat in flattening and drying. Remove paraffin and proceed to aq. dest. Slides placed for 20 sec. with agitation into a dilute solution of silver diamino hy- droxide (Wilder's Solution diluted with an equal volume of aq. dest. to which is added 2 drops of 8% NH4OH per 50 cc). Drain quickly and without rins- ing immerse in a large volume (e.g. 200 cc.) of very dilute formalin (1/1000 commercial formalin in aq. dest.) agi- tating for 10-20 sec, not more. Fresh formalin solution for each section is preferable. Wash in aq. dest. Dif- ferentiate carefully under the micro- scope with 1-2% aq. potassium ferri- cyanide until mitochondria show up black against a clear background. Wash in aq. dest., counterstain with 1% Safranin or Ponceau fuchsin. Regaud's fixative gives the best results but after Helly's fluid the Golgi element is often sharply impregnated as well as the mito- chondria. Mitochondria and Bacteria. Demonstration in the same cells. See Cowdry, E. V. and Olitsky, P. K., J. Exper. Med., 1922, 36, 521-533, Cowdry, E. V., Am. J. Anat., 1923, 31, 339-343. Stain as for mitochondria with Anilin Fuchsin and Methyl Green. Mitochondria are col- ored crimson. When the bacilli are acid fast as in leprosy they are colored a dark reddish purple; but when they are not acid resistant they are stained bluish green. Mitogenic Radiations. It is questionable whether these rays, said to generate mitosis, really exist. A critical and well balanced statement is afforded by Glasser, O., in Glasser's Medical Phy- sics, 760-763. Mitosis (G. Mitos, thread). Indirect nu- clear division in which the chromatin forms a thread which breaks up into chromosomes. Material should be freshly fixed, less than half hour after removal. But mito- sis can be seen in some tissues 24 hrs. or longer after death, especially if the body is kept at a low temperature but the number is less and the details not so clear as after quick fixation (Mallory, p. 108). Sat. mercuric chloride in 70% ale. plus 5% acetic acid, Zenker's fluid, formalin-Zenker , Bouin's fluid and Flem- ming's strong fluid are satisfactory fixatives but the last named penetrates very badly. The most beautiful stain for mitotic figures is safranin light green but the mitoses can be more clearly distin- guished without the green counterstain. Simply deparaffinise and stain sections in anilin-safranin (Babes), wash quickly in tap water, differentiate in acid alcohol until the resting nuclei are less intensely colored than the dividing ones, wash in 95%, dehydrate in abs. clear in xylol and mount in balsam. Another excellent method is to apply the Feulgen reaction for Thymonucleic Acid to sections of tissues preferably fixed in Carnoy's fluid or acetic subli- mate. This demonstrates thymonucleic acid in the chromatin, and the dividing nuclei, as with safranin, are more deeply stained than the others. This method is displacing the older safranin tech- nique. To demonstrate mitosis in whole mounts of epidermis place freshly ex- cised skin (circumcision specimen pre- ferred) in 0.1% aq. acetic acid in the icebox over night. Wash quickly in aq. dest. Strip off the epidermis with needles, stain it like a section with anilin-safranin or with Harris' hema- toxylin and mount with the outer sur- face uppermost. This technique could probably be adapted to relatively flat epithelia of the respiratory digestive, urinary and genital systems. In order to reveal the maximum num- ber of mitotic figures it is important to study the mitotic rhythm of the par- ticular tissue or organ and take tissues at the peak which in the case of the human foreskin is probably between 9 p.m. and midnight (Cooper, Z. K. and Schiff, A., Proc. Soc. Exp. Biol. & Med., 1938, 39, 323-324). The relation of alimentation and nutrition to cyclic variations in mitotic activity is pre- sented by Blumenthal, H. T., Growth, 1950, 14, 231-250. To experimentally increase the num- ber of mitosis use colchicine which ar- rests the process chiefly in the meta- phase by causing failure of the mitotic spindle to form and function (Ludford, R. J., Arch. f. exper. Zellf., 1936, 18, 411-441). Consequently as long as the cells are under the influence of colchi- cine— a matter of a few hours only — mitosis begins as usual; but, since it is not completed, the proportion of mitotic iMOIST CHAMBERS 213 MOROSOW'S METHOD figures to resting nuclei is temporarily greatly increased. Sodium cacodylate, auramine and other substances listed by Ludford likewise influence mitosis. For checks on the method of estimating growth by counting arrested mitoses, see Paletta and Cowdry (F. X. and E. v., Am. J. Path., 1942, 18, 291-311). Aisenberg (E. J., Bull. d'Hist. Appl., 1935, 12, 100-122) has found that mitosis of epidermal cells is arrested in the metaphase simply by passing a ligature around a frog's leg and keeping the foot in distilled water. The mitoses ac- cumulate in large numbers but continue when released from the hypotonic environment. Aisenberg {ibid. 1936, 13, 265-286) also discovered low concen- tration of ethyl alcohol to stimulate mitosis, 0.4-0.8 M to arrest in meta- phase, 1.2-1.5 M. to cause gelatinization of mitosis and higher concentrations to kill the cells. Comprehensive data on the influence of Colchicine are supplied by Levine, M., Ann. N. Y. Acad. Sci., 1951, 51, 1365-1408. Moist Chambers, Small for stage of micro- scope (Chambers, R. and Kopac, M. J. in McClung's Microscopic Technique, 1950). Large in which hands can be used (Holter, H., C. rend. trav. lab. Carlsberg, S6r. Chim., 1945, 25, 15fr- 167). See diagrams for air-condition- ing (Click, p. 182). Molecular Film Technique, see Taylor, H. S., Lawrence, E. O., and Langmuir, I., Molecular Films, the Cyclotron and the New Biology, Rutger's University Press, 1942, 95 pp. Molecular Solution is the molecular weight of the substance in grams made up to 1 liter with aq. dest. Thus M oxalic acid (COOH)2-2H20 is 126 gms. with aq. dest. added to 1 liter; but A-^ oxalic acid is half of this concentration. See Normal Solutions. The molecular weight expressed in grams is called the gram-molecular weight or mole. Millimole is 1/1000 of a mole. Milligram equivalent (milliequiva- lent). The equivalent weight, the gram-equivalent, or the equivalent of a substance is the weight in grams which in its reaction corresponds to a gram atom of hydrogen, or of hydroxyl, or half a gram atom of oxygen, or gram atom of a univalent ion. Milliequiva- lent is 1/1000 of the equivalent weight, i.e., the equivalent weight of sodium carbonate is j the molecular weight, or 53.0. Therefore, the milliequivalent (m.e.) or the weight in 1 ml. of normal solution is 0.0530 gm. Molybdenum, see Atomic Weights. Mono-Azo Dyes. Amarnth, azo fuchsin, benzene-azo-a-naphthylamine, bordeaux red, brilliant yellow S, chromotrope 2R, chrysoidin Y, fast yellow, janus green B, metanil yellow, methyl orange, methyl red, narcein, nitrazine, oil red O, orange G, orange I, orange II, orange IV, ponceau 2R, sudan R, thiazine red R. Monocytes. When "typical" these are easily recognized in stained blood smears and in supravital preparations but there is no technique by which they may always be distinguished from all Lym- phocytes and Macrophages. That is, they possess no single feature, like the eosinophile granules of eosinophile leuco- cytes, for their certain identification (see Cowdry, p. 66-71). They ingest particulate matter including Trypan Blue and similar vital stains and are therefore to be considered as com- ponents of the Reticulo-Endothelial System. Many of their properties can to great advantage be investigated in Tissue Cultures. The best way to demonstrate the remarkably close rela- tion that paay exist between monocytes and contained bacilli is to stain leprous tissue_ for acid fast bacilli (see Leprosy Bacilli). See Bacterium Monocyto- genes. Monolayer technique is a physico-chemical line of investigation that gives valuable data on the structure of protein and lipoprotein films and consequently on the plasma membrane of cells. See Schulmann (Bourne, pp. 51-67). Moore, see Fungi. Mordant (L. mordere, to bite), a substance, like alum, employed to make a dye bite into the tissue and hold on. The dye combines with the mordant which is itself in high concentration in the structures to be stained. In the Iron Hematoxylin technique the sections are mordanted with iron alum. They are briefly washed in aa. dest. to remove some of the excess mordant. Then they are stained with a dilute aqueous solu- tion of hematoxylin and differentiated in the mordant which draws out most of the hematoxylin until it remains only in the structures which took up the mordant most energetically in the first place and which therefore alone remain colored. Copper salts are also good mordants. See Weigert's mordants. Morosow's Method for elementary bodies as modified by Fonta and Triboudeau and given by Seiffert, G., Virus Diseases of Man, Animal and Plant. New York: Philosophical Library, Inc., 1944, 332 pp. Dry thin smear in air. Place vertically in aq. dest,, 10-15 min. and MOSKOWITZ 214 MOUNTING MEDIA dry again. Cover with mixture of acetic acid, 1 cc; 40% formalin (com- mercial formaldehyde), 2 cc; aq. dest., 100 cc. Rinse well in aq. dest. and heat till steam rises in mixture of carbolic acid, 1 cc; tannin, 50 gm.; aq. dest., 100 cc Rinse in aq. dest. j min. and heat slightly 1-2 min. till smear becomes brown or slightly black in silver solu- tion made up as follows: To 20 cc. aq. dest. add "platinum loop" of 25% am- monia and then drop by drop from pipette of 10% aq. silver nitrate until an opalescent ppt. appears. About 0.5 cc. of silver solution will be needed. After silvering smear rinse well in aq. dest., mount and seal edges with paraffin. Moskowitz, see Protein Silver for Staining Protozoa. Mosquito larvae, technique of raising anoph- eline (Bates, M., Am. J. Trop. Med., 1941, 21, 103-122). Bodian technique for mosquito nervous system (Rogoff, W. M., Stain Techn., 1946, 21, 59-61). Motion Pictures. The technique of making motion pictures of living cells and or- gans has proved its worth. The movies can be projected again and again and the sequence of events made very clear. It is important to remember that mo- tile cells do not run around at the speed indicated, because the actual distance travelled is far less than on the screen and the time much greater. The Wis- tar Institute of Anatomy in Philadel- phia is distributor of a comprehensive series of motion picture films on either a purchase or rental basis. Motor End Plates. The particular morpho- logical type of nerve ending in muscle does not concern us here ; but reference can be made if desired to the classifica- tion by Hines, M., Am. J. Anat., 1931, 47, 1-55. The methods advocated for histological demonstration are legion. Reference is made to 2 gold techniques (Craven's and Carey's) and to 1 silver method (Chor's). The former can be ultimately traced back to Ranvier and the latter to Cajal. See also techniques described under Nerve Endings. Mounting Media. The refractive index of the medium is important and a table giving the indices for many substances used is supplied by Lee (p. 218). As pointed out, the greatest transparency is secured when the refractive indices of media and tissues are equal and media of lower index than the tissues give some- what greater visibility of tissue com- ponents, while those of higher index provide less visibility. There are nmny media to choose from, the refractive indices of which are more or less satis- factory. The selection will depend more upon whether the medium can be employed for the particular tissue and its relative permanence. For frozen sections and tissues to be mounted from water and aqueous solu- tions various glycerin mixtures are popular : Lactophenol, Glychrogel, Brandt's and Kaiser's glycerin jellies. Having taken the easiest one to prepare, for their merits are about equal, the tissue is mounted and covered and it is necessary to seal the edges. In the case of temporary mounts a little paraffin applied with a heated scalpel, or wire, will suffice. Lee (p. 230) advocates Peter Gray's sealing medium made up by melting together 4 parts anhydrous lanolin, 1 part Canada balsam (dry) and 8 parts resin which becomes solid on cooling. Apply to edges in the same manner as the paraffin. Eronig's ce- ment is employed in Bensley's labora- tory. Duco cement is very worthwhile because it is insoluble in xylol, alcohol and other chemicals used to clean micro- scopic preparations. Mallory (p. 99) dilutes it with an equal volume of ace- tone. See Karo. For sections and tissues which are first dehydrated and cleared the investiga- tor must choose the mounting medium best adapted to his purpose from a con- siderable number proposed of which the following are given elsewhere in this book : Balsam, Cedar Oil, Clarite, Colophonium, Damar, Diaphane, Eupe- ral, Nevillite, Sandarac, Terpineol Balsam. The chief desiderata are a medium which will harden fairly quickly, which will not become acid and bring about the fading of anilin dyes and which will not crack or develop granules. Clarite is competing for first place with balsam* because, to make the balsam neutral ana keep it so, is a troublesome job. Direc- tions for its preparation are given by the Bensleys (p. 39). But the balsam ordi- narily purchased is satisfactory for hematoxylin and eosin and iron hema- toxylin preparations except when the latter are counterstained with an anilin dye. The writer used to employ cedar oil (for immersion objectives), in mount- ing sections stained by Giemsa's method, which is superior to balsam, but it drys slowly and is not better than clarite. Damar has been recommended for stains likely to fade and colophonium for thick sections of the nervous system for which covers are not used; both however appear to be less valuable than clarite. Museum specimens require an aque- ous mounting medium which preserves MUCICARMINE 215 MUCUS colors. See Color Preservation. See Plastics for museum work. Mucicarmine for mucus, Mayer's (Mallory and Parker in McClung, p. 417). To make up stain, mix carmine, 1 gm.; aluminum chloride, 0.5 gm. ; and aq. dest., 2 cc. Heat over flame for 2 min. Color of solution darkens. Add grad- ually 100 cc. 50% ale. stirring constantly until dissolved. After 24 hrs. filter. Filtrate keeps well. Stain parafTin sec- tions of absolute alcohol fixed tissue in carmine sol. 5-10 min. Wash in water, dehydrate, clear and mount. Mucus is red. When nuclei also are colored red, add few drops 1% aq. sodium bicarbon- ate to the stain. It is customary to stain cells and nuclei before hand with alum hematoxylin. Bensley (Cowdry's Special Cytology, 1932, p. 203) uses alcoholic chrome sublimate (sat. mer- curic chloride and potassium bichromate in 95% ale.) and increases the content of carmine 5 times. Mucigen, intracellular antecedent of Mucin. Mucihematein for mucus, Mayer's, Mal- lory and Parker in McClung, p. 416). Alake up : (A) hematein, 0.2 gm. ; alumi- num chloride, 0.1 gm.; glycerin, 40 cc; aq. dest., 60 cc. and (B) hematein, 0.2 gm.; aluminum chloride, 0.1 gm.; 70% alcohol, 70 cc; nitric acid, 1-2 drops. A is advised except when the mucus swells much in which case use B. Stain paraffin sections of absolute alco- hol fixed tissue 5-10 min. Wash in water. Dehydrate in 95% ale. and in abs. Clear in xylol and mount in bal- sam. Mucus stains blue. The other materials are colorless. _ Preliminary coloration with carmine is suggested. Bensley (Cowdry's Special Cytology, 1932, p. 203) used alcoholic chrome sub- limate fixation (sat. mercuric chloride and potassium bichromate in 95% ale.) and increased the content of hematein five times. Mucin, one of several glycoproteins found in mucus. See Mucus and Mucicarmine, Mucihematein and Mucisudan stains, also Polysaccharides. Mucinase. Enzyme (s) which hydrolyze mucus or mucoproteins. They are not very well understood, and specific preparations are not yet available. Purified enzymes, capable of differenti- ating the various mucopolysaccharides one from another would be very valu- able. Mucisudan is a dye of undetermined compo- sition made by hydrolysis of sudan black B with acetic acid and recom- mended as a new stain for mucin (Leach, E. H., J. Path, and Bact., 1938, 47, 637-639). Mucoproteins. A method for histological distinction between the chondroitin sulphuric acid protein of connective tissue mucus and the mucotin sulphuric acid protein of epithelial ti-ssues has been worked out by L. H. Hempelmaiin, Jr., Anat. Rec, 1940, 78, 197-206. Briefly stated toluidin blue in 1:280,000 will stain the former vividly and the latter not at all. Click, p. 46, notes that con- firmation of Hempelmann's work is still lacking. Metachromatic staining with toluid in blue is specific for mucoid compounds containing polysaccharide esters of sulfuric acid provided that Lison's tech- nique is followed closely in the opin- ion of Sylvan, B., Acta Radiol., 1945, suppl. 59, 100 pp. Fix tissue (12-24 hrs.) in equal parts 8% aq. basic lead acetate and 14-16% formalin. Stain some paraffin sections (30 min.) in 0.1% toluidin blue in 1% ale. and others in 0.1% toluidin blue in 30% ale (first allowing these stains to age for several days). Wash in ale briefly, mount in natural cedar oil. See Michaelis, L. and Granick, S., J. Am. Chem. Soc, 1945, 67, 1212-1219 and Hess, M. and Hollander, F., J. Lab. & Clin. Med., 1947, 32, 905-909 for data about meta- chromasia. Leach, E. H., Stain Techn., 1947, 22, 73-76 has advocated Bismark brown as a stain for muco- proteins. Mucus means slime. It is a viscid, stringy material which ordinarily stains with basic dyes and is found in many parts of the body. The chemical composition of mucus is not uniform. It may consist of one of several glycoproteins, called mucins, which are by contrast definite chemical substances. The term mu- cous is an adjective describing a cell or tissue which produces or contains mucus. Mucigen is the intracellular antecedent of a mucin. Since there are several mucins there are several corre- sponding mucigens. Pathologists sometimes divide mucins into two categories, epithelial and con- nective. The connective tissue type is found in the ground substance of bone, synovial fluid and in other locations. It increases in amount in the myxedema (G. myxa, mucus -|- oidema, swelling) of certain thyroid deficiencies as well as in arteriosclerosis and various tumors. The ubiquitous fibroblast is said to be a great former of mucins. Epithelial mucins are produced by epithelial secretory cells. The goblet cells are easily recognized by the fact that the material to be discharged is held in a goblet like expansion of the cell. Other mucous cells can be distinguished from MUELENGRATH TEST 216 MYELOBLASTS serous or zymogenic cella by several criteria : 1 . The nuclei instead of being roughly spherical are often, but not always, pressed against the cell membrane re- mote from the lumen. 2. The mitochondria are usually of smaller diameter and shorter than in zymogenic cells. 3. The secretion antecedents (Muci- gens) of mucous cells are more difficult to see in the fresh state, more labile, and in fixed tissues are metachromatic and can be stained almost specifically with mucicarmine and mucihematein. See Mucicarmine and Mucihematein of Mayer. A simple method for mucus has been described by Lillie (R. D., J. Tech. Methods, 1929, 12, 120-121). Sections of tissue fixed in formalin or in Zenker- formol (Helly) are passed to water. In the case of the latter remove mercury with iodine and sodium thiosulphate as usual. Stain 1 min. in 0.2% aq. toluidin blue. Wash in water. Dehydrate in pure acetone, clear in xylol and mount in balsam. Mucus, reddish violet; nuclei, blue ; red cells, yellow or greenish yellow. In the case of old formalin material rinse in 95% alcohol before the acetone. McManus, J. F. A., Nature, 1946, 158, 202, recommends the use of Schiff's Reagent followed by periodic acid. Material fixed in Zenker-formal is de- hydrated and embedded in the usual manner and the sections transferred to water after treatment with iodine and hypo and placed in a 0.5% aq. periodic acid 2 min. The slides are washed in tap water and aq. dest. and kept in Schiff's reagent for 15 minutes; rinsed in Sulphurous Acid, dehydrated and cleared in the alcohol and xylol series respectively and mounted in balsam. According to McManus, the mucus of the goblet cells of the human intestine and bronchus, mucus salivary glands, certain pituitary cells, the colloid of the pituitary stalk and thyroid, granules in some nerve cells in the medulla of the rat and in the human intestine, the basement membranes of the tubular epithelium and of the glomerulus in the kidney were tested by this method and an intense coloration detected in all instances. Hotchkiss, R. D. deserves credit for reporting in a personal communication to Glick (p. 43) in 1946 the independent discovery of a similar technique to that of McManus employing periodic acid. This is given in detail by Glick. Muelengrath Test, see Icterus Index. Miiller's Fluid. Potassium bichromate, 2- 2.5 gm.; sodium sulphate, 1 gm.; aq. dest., 1 gm. This was formerly much used for long fixation and mordanting of nervous tissue. See Chromaffin Reac- tion, Decalcification, O'Leary's Bra- zilian Method, Weigert Method. It is now largely replaced by Orth's Fluid which is really formalin -Miiller. Mumps. Refractile, eosinophilic bodies in red blood cells are very small first 5-6 days. Increase in size and elongate 7-14 days. (Parsons, H. H., Military Surgeon, 1938, 83, 541-543). Murexide Test, see Purines. Muscle, to distinguish in sections from con- nective tissue, Dahlgren (McClung, p. 306) suggests Retterer's and Van Gieson's stains, picronigrosine and Unna's orcein to which may be added Mallory's stain. Demonstration of chloride in muscle fibers (Heilbrunn, L. V. and Hamilton, P. G., Physiol. Zool., 1942, 15, 363-374). For contrac- tion bands and wave mechanics, see Carey, E. J., Arch. Path., 1940, 30, 881-892, 1041-1072. A technique for separating nuclei from cytoplasm for analysis is given under Nuclei. If microdissection is contemplated the pioneer paper by Kite, G. L., Am. J. Physiol., 1913, 32, 146-164 should be consulted. The experimental produc- tion of myocardial segmentation is described by Saphir, O. and Karsner, H. T., J. Med. Res., 1923-24, 44, 539- 556. Methods of Maceration are often useful in the isolation of single fibers. Mitoses can only be induced in excep- tional cases (Allen, E., Smith, G. M. and Gardner, W. U., Am. J. Anat., 1937, 61, 321). An electron microscopic technique for localization of magnesium and calcium is described by Scott, G. H. and Packer, D. M., Anat. Rec, 1939, 74, 31-45. Muscle gives beautiful fluorescent colors in ultraviolet light with many fluorochromes (Metcalf, R. L. and Patton, R. L., Stain Techn., 1944, 19, 11-27). See Myosin and Fur- kinje cells and fibers. Museum Specimens, see Color Preservation. Mycelia, see Lillie, p. 289. Mycobacteria, see Acid Fast Bacteria. Mycological Techniques, see Fungi. Myelin, see various methods for demonstra- tion of Nerve Fibers. Myeloblasts. The recognition of these cells is a fine art ; because, by definition, they are so little dififerentiated that the granules characteristic of the 3 types of leucocytes are absent. For contrasting views, dependent largely on whether supravital staining or fixed and stained preparations are used, see Cowdry's Histology, p. 100, also Leucocytes, de- velopmental series. MYELOCYTES 217 NAPHTHOL GREEN B Myelocytes, see Leucocytes, developmental series. Myeloidin is the term applied to the mate- rial of certain spheroidal or cuboidal bodies of wax-like luster present in the bases of retinal pigment cells of monkeys and some other animals but reported as absent in man and said to resemble myelin. For literature see Arey, L. B. in Cowdry's Special Cytology, 1932, 3, 1218. Myocardium. Method for separation of fiber bundles (Mall, F. P., Am. J. Anat. 11,211-266). Myofibrils. The best method is to fix in Zenker's fluid or strong Flemming's mixture and to stain with iron hema- toxylin (see Dahlgren in McClung p. 425). Microincineration is useful for the demonstration of minerals. Myoglia is a fine network of fibers associated wuth muscle cells well demonstrated by Mallory's Connective Tissue Stain. Myonemes of trypanosomes, gold chloride method for (Ogawa, M., Arch. f. Protistenk., 1913, 29, 248). See Wen- rich, D. H. and Diller, W. F. in Mc- Clung's Microscopical Technique, 1950, p. 464. Myosin is a protein, present in muscle, the molecules of which are needle-shaped. Cross striations of muscle are thought to depend on their arrangement. In the isotropic (non-birefringent) bands the myosin molecules are believed to be disposed at random and in the aniso- tropic (birefringent) bands parallel to the length of the fiber (see Bourne, p. 30). Myriapoda, see Parasites. Nadi Reagent is dimethyl-paraphenylene- diamin -|- a naphthol. Indophenol oxidase catalyses oxidation of nadi to indophenol blue and that of parapheny- lene diamin to diamin. Nails. These very interesting structures are seldom examined microscopically despite the fact that changes in them may provide significant clues to the con- dition of other tissues. They are chiefly made up of stratum lucidum thickened by much eleidin. It is a simple matter to macerate cut finger or toe nails in 40% aq. potassium hydroxide or in con- centrated sulphuric acid for a few days and then to isolate the individual cells some of which are nucleated. Mac- Leod, J. M. H., Practical Handbook of the Pathology of the Skin. London: H. K. Lewis, 1903, 408 pp. gives Heller's method which involves fi.xation of un- gual phalanx for a few days in Muller's fluid, prolonged washing^ decalcification for 4-6 days in 1 part nitric acid and 3 parts of water followed by thorough imbedding in celloidin. The sections can then be stained with hematoxylin. gentian violet, safranin or any other of a number of dyes. Naphthalene Pink, see Magdala Red. Naphthalene Red, see Magdala Red. Naphthamine Blue 3BX, see Trypan Blue. Naphthamine Brilliant Blue 2R, see Dianil Blue 2r. Naphthol Blue Black (CI, 246). Lillie, R. D., J. Tech. Methods, 1945, No. 25, 47 pp. has reported that this dye (NAC- 7080 and DuFont L 6401) gives excellent staining in combination: Stain with Weigert's iron hematoxylin, 6 min. Wash in water and counterstain 5 niin. in 3 parts 1% brilliant purpurin R (CI, 454) in 1% aq. acetic acid and 2 parts 1% azofuchsin (CI, 153) likewise in 1% aq. acetic acid. Rinse in 1% aq. acetic acid and stain 5 min. in 1% naphthol blue black (CI, 246) in sat. aq. picric acid. Rinse in 1% aq. acetic acid, 2 min. Dehydrate and clear in alcohol, alcohol and xylol, xylol and mount in clarite. Collagen, reticulum and base- ment membranes, dark green; smooth muscle, brown; nuclei brownish-black. Naphthol Blue R (CI, 909)— fast blue 3R, Indian blue 2RD, Meldola's blue, new blue R, phenylene blue — An oxazin dye used by Harvey, B. C. H., and Bensley, R. R., Biol. Bull., 1912, 23, 225-249 as a supravital stain for gastric mucosa. The Bensleys' report that this dye has proved useful in the localization of un- suspected parathyroid and thyroid tis- sue in experimental animals. After vascular perfusion in a concentration of 1 : 40 ,000 of 0 .85% aq . sodi um chloride the thyroid, parathyroid and lymph nodes become colored intensely blue ; whereas other tissues, muscles, salivary glands etc., are colored pale greenish blue. Naphthol Green, see Naphthol Green B. Naphthol Green B (CI, 5) — acid green O, green PL, naphthol green — An acid nitroso dyefor whicha probable formula is given by Conn (p. 42) and which he thinks was the naphthol green used by v. Volkmann, R. and Strauss, F., Zeit. f. Wis. Mikr., 1934, 51, 244-249, and by MoUier, G., Zeit. f. Wis. Mikr., 1938, 55, 472-473. Lillie, R. D., J. Techn. Methods, 1945, No. 25, 47 pp. recommends naphthol green B for connective tissue. Stain sections 6 min. in Weigert's or other iron hematoxylin. Wash thoroughly in water and stain 3 min. in 1% aq. eosin Y (CI, 768 . Rinse in water and mordant 4 min. in 10% dilution of U.S. P. ferric chloride solution. Rinse in water and stain 5 min. in 1% naph- thol green B. Differentiate 2 min. in 1% aq. acetic acid. Dehydrate in aceton, clear in acetone-xylene and in .xylene and mount in clarite xylene or NAPIITHOL ORANGE 218 NASAL CELL SMEARS in salicylic acid balsam. Connective tissue, green; muscle and cytoplasm, pink. Y (CI, 2) — fast printing green, Gam- bine — An acid nitroso dye apparently not used in histology. Naphthol Orange, see Orange L Naphthol Red S, C or O, see Amaranth. Naphthol Yellow, see Martins Yellow. Naphthyl Red (CI, 856), a basic dye of light fastness 5. Only nuclei of mature plant cells colored fugitive red (Emig, p. 57). Naphthylamine Brown (CI, 170), an acid monoazo dye which stains plant tissues darker in presence of potassium bi- chromate (Emig, p. 34). Naphthylamine Pink, see Magdala Red. Naples Yellow, an exogenous pigment — Pb3(Sb04)-.— Lillie, p. 134. Narcein (CI, 152). An acid mono-azo dye. Was used by Ehrlich in combination with pyronin and methylene blue or methyl green to produce a neutral dye (Conn, p. 54). No longer available. Nasal Cell Smears — Written by Marian Pfingsten Bryan, Dept. of Otolaryngol- ogy, Washington University, St. Louis 10, Mo.— September 14, 1951— Since 1927 the study of the cytology of nasal secretions has been recognized as an important aid in the clinical diagnosis and differentiation of certain nasal and sinus conditions. Eyerman, C. H., one of the early investigators (Ann. Otol., Rhinol. and Laryngol., 1927, 36, 808-815), identified the main tj^pes of cells found in nasal secretion. Tillot- son, R. S. (Arch. Otolaryng., 1929, 10, 262-265) reported on the value of cyto- logic studies in the diagnosis of sphen- oiditis. Sewall, E. C. and Hunnicut, L. (Arch. Otolaryng., 1929, 10, 1-15) dis- cussed cytologic examination of the antrum. Dean, L. W. (J. A. M. A., 1932, 99, 543-546) emphasized the sig- nificance of the nasal smear in helping to diagnose allergy of the nose and si- nuses. Hansel, F. K. (J. Allergy, 1934, 5, 357) contributed many observations on the cytology of these secretions, es- pecially in regard to allergy of the nose and paranasal sinuses. There is a good review of the numerous articles in the literature dealing with nasal cytology by Hansel, F. K., Allergy of the Nose and Paranasal Sinuses, St. Louis: C. V. Mosby Co., 1936, p. 383. The particular emphasis has been on the number of eosinophilic cells in rela- tion to neutrophiles; but, as knowledge has progressed in nasal cytology, the value of the study of smears has been extended. The objectives of nasal and sinus cytology include the following: First, the demonstration of eosinophiles in the secretions in relation to neutro- philes may be helpful in distinguishing allergic conditions from infections, or in establishing the coexistence of both. In the second place the method makes possible detailed observations of the ciliated columnar epithelial cells, ex- foliated in the early stages of acute upper respiratory infections. These studies may aid further in differentiat- ing the common cold from allergic rhinitis. Thirdly the smear technique proves to be a reliable help, along with biopsy, in the diagnosis of carcinoma, other tumors and lesions of the upper respiratory tract. Fourth, for the microscopic study of radiation changes produced in cells after treatment with x-ray and radiation, it is invaluable. Fifth, it is a useful tool for working out unknown cellular patterns which may support clinical diagnoses of other nasal and sinus conditions. For cytological studies, nasal secre- tion is collected, after blowing the nose, on wax paper or on a cellophane hand- kerchief. Specimens are preferably taken separately from each side of the nose. The material may also be blown directly on to a clean glass slide, then gently smeared with a probe (not a dry cotton applicator). Thin smears are more suitable for microscopic study than thick ones. In order to obtain a sampling from a normal nose, the naso- pharynx is wiped with a small cotton applicator, slightly moistened with saline. For abnormal dry noses, the raping technique may also be used be- fore application of any drugs. A saline tampon, inserted into the nostril, may stimulate the flow of secretion in order to obtain a specimen. Secretions from the sinuses may be collected by aspira- tion of the nose, displacement or irri- gation of sinuses, or be taken directly from the area during an operation. These secretions, direct from the re- spective paranasal sinuses, are often of value in locating the source of the in- fection and in determining the type of cellular response. They thus help to complete the diagnosis. The cyto- logical picture presented in the nose may be somewhat different from the cellular pattern observed when the ma- terial is obtained directly from a par- ticular sinus. Both should be studied and correlated whenever possible. In the differential diagnosis of acute and chronic infections from allergic ones, or in demonstrating the joint ex- istence of both, the numerical propor- tions of eosinophilic to neutrophilic cells in the nasal smear should be eval- uated. The ratio is usually an expres- sion of the nature and stage of the nasal NASAL CELL SMEARS 219 NASAL CELL SMEARS condition. Hansel, F. K., St. Louis: C. V. Mosby Co., 1936, gives a useful scheme on page 380 for recording the cytology of nasal secretions similar to that used in the recording of skin tests. In view of the irregularitj- of distribu- tion of the cells and the amount and type of secretions, it is difficult to de- termine the cells in percentages. Long personal experience in the observation of smears and in the correlation of them with clinical symptoms is of the utmost value. The smears should be repeated in the same patient in order to obtain a complete picture of the sequence of events. In estimating the numbers of neutrophiles in the secretion, it must be realized that when acute or chronic in- fections complicate allergic responses, the neutrophiles outnumber the eosino- philes in the secretion about 10 to 1. In seasonal hay fever, however, un- complicated by infection, there may be a few neutrophiles along with squamous epithelial cells, but the major cellular response will be eosinophilic. Some cases of hay fever show a response of mononuclears with coarse blue granules filling the cell and nearly obscuring the nucleus. Their actual significance is still unknown. A criticism of Hansel's scheme is that it does not take into con- sideration the other cellular elements besides eosinophiles and neutrophiles. The type, color, quantity and consist- ency of the secretion should be included and correlated with clinical symptoms, but the macroscopic appearance cannot be relied upon as an indication of the microscopic cellular contents of the se- cretion. The smears must also be ex- amined for the presence of microorgan- isms and the tj'pes of epithelial cells should be carefully studied. Smears from normal noses may or may not show a few scattered neutro- philes. A small amount of thin mucus is usualljr present with some squamous epithelial cells. Ciliated cells are seen if they have been wiped from the nose with the applicator. In the normal nose they are rarely exfoliated. In cases of nasal polyps, eosinophiles usually are predominant; but repeated studies of the secretions are necessary to determine whether or not complicat- ing acute or chronic infection is present. If a marked predominance of neutro- philes persists, a chronic infection is present. The cytology of nasal polypi is fully discussed by Walsh, T. E., and Lindsay, J. R. (Arch. Otolaryng., 1934, 20, 649). Stagnation and secondary infection are frequently present inthese cases, since polypi cause obstruction in the nose and a resultant neutrophilic response. A single failure to find eosin- ophiles in a smear does not rule out the existence of an allergic condition, but repeated failures to demonstrate eosin- ophiles usually means that an active nasal allergy is not present. The differentiation of an allergic rhinitis from a common cold may at times be difficult clinicall3^ In these cases microscopic examination of the secretions is helpful; but nevertheless, even with cases of acute upper respira- tory infections in non-allergic patients, the eosinophilic response is so marked that it may closely approach the num- ber found in allergic individuals during an infection. In the non-allergic per- sons the eosinophiles may disappear before the neutrophiles but not in many of them. In allergic persons, the eosin- ophiles often increase as the neutro- philes disappear, but these differences are uncertain. Acute allergic exacer- bations may so closely resemble com- mon colds that in numerous intances, even microscopically, it is difficult to distinguish between them unless re- peated consecutive smears are examined throughout the course of the condition. Hilding, A. C. (Tran. Amer. Laryngol. Assoc, 1934, 253-271) carefully studied fresh unstained nasal secretions from common colds in adults and noted that ciliated columnar cells were shed in the exudates during the first few days of the infection. More recently Bryan, W. T. K. and Brvan, M. P. (The Larvngo- scope, 1950, 60, 523-531) applied the staining technique of Papanicolaou to nasal secretions. In a series of upper respiratory infections, in which the secretions were examined at timed intervals during the course of the in- fection, it was possible to chart a de- generative pattern of change exhibited by the ciliated columnar epithelial cells. These changes suggest virus etiology. In seasonal allergic hay fever the ex- foliation of the ciliated columnar cells and their degenerative patterns of change have thus far not been observed. Smears from these cases reveal that large numbers of clumped or grouped eosinophiles are significant when they are not complicated bj^ infection. Ciliated epithelial cells are of rare occurrence, but some basal and squa- mous epithelial cells are frequently encountered. Consequently exfoliation of the ciliated columnar epithelial cells in acute upper respiratory infections may have diagnostic value. Within the last few years cytological studies of nasal diseases have been ex- tended by use of the smear technique to include diagnostic information in regard NASAL CELL SMEARS 220 NASAL CELL SMEARS to malignancies, other tumors and le- sions occurring in this area. Morrison, L. F., Hopp, E. S. and Wu, R. (Ann. Otol., Rhinol. and Laryngol., 1949, 58, 18-31) have employed the smear tech- nique as an adjunct in the diagnosis of exfoliating neoplasms of the naso- pharynx. This proved so reliable that a positive smear demanded discovery of the source of the malignant cells. Sooy, F. A. (The Laryngoscope, 1950, 60, 964-992) in a study of primary tu- mors of the nasal septum, has also used the smear technique to advantage. Early diagnosis of carcinoma of the maxillary sinus in a series of cases re- ported by Fitz-Hugh, G. S., Moon, C. N. Jr. and Luptom, C. H. Jr. (The Laryngoscope, 1950, 60, 376-387) was thus facilitated. This cell smear method is not only an aid in diagnosis, but also is a convenient means for studying the microscopic course of a lesion during and after treat- ment. Biopsies are not always possi- ble, but smears are easily obtained with- out discomfort to the patients. The effects on the cells of x-ray or radium therapy comprise a whole new field of cytological research. New knowl- edge thus gained is of great importance when closely correlated with clinical symptoms. Satisfactory techniques for staining nasal secretions include the following: 1. Wright's Stain. Slides are dried in air (avoid flaming). Many direc- tions advise staining as a blood film; but two points will improve this tech- nique for nasal work, namely the use of a buffer solution for the diluent and the shortening of the time recommended for blood smears to only 15 or 20 sec. of staining. After marking off the ends of the slides with a wax pencil, they are flooded with the dye for 15 sec. The buffer diluent is added to the stain and allowed to mix well for 15 sec. more. The slides are then washed in buffer solution and placed on end on a blotter to drain and thus to dry more rapidly. This light rapid stain- ing shows cellular detail better in the nasal smear than the usual technique. The slides can be kept and stored for many years without coverslips and thereafter show no signs of deteriora- tion. The cellular details are even better if the slides are stained within 30 min. after being made and dried. The stain is especially good for eosino- philes, neutrophiles and mononuclears. It is not very suitable for differentiating epithelial cells. 2. HanseVs Stain. Color slide 30 sec. with his dye which is an eosin- methylene blue combination and can be obtained directly from him (Dr F K. Hansel, 634 N. Grand Blvd., St. Louis 3, Mo.). Then add alkaline water which is made by adding one drop of 1% potassium carbonate to 60 cc. aq. dest., for 30 sec. Wash in alkaline water followed by washing in acid water which is made by adding one drop of 1% hydrochloric acid in 60 cc. aq. dest. Wash again in alkaline water and finally rinse in 95% ethyl alcohol. This stain is especially good for eosinophiles. The granules are very brilliant and re- fractile. Other cellular detail may be somewhat dark and indefinite. 5. Gtemsa Stain. The preparation from Gradwohl is very satisfactory. The dilution is one drop of stain to 1 cc. ac[. dest. The slide is flooded with diluted stain for one minute then washed with aq. dest. If overstaining occurs, this may be decolorized with ethyl alcohol. The restulting colora- tion is excellent for eosinophiles but is not especially recommended for the other cells. 4. Supra-Vital Staining. This tech- nique is particularly useful for study- ing secretions in the fresh condition when one wishes to observe motility of eosinophiles and neutrophiles, the phagocytic activity of neutrophiles and of mononuclears, as well as the ciliary activity of the exfoliated columnar epithelial cells. One drop of exudate may be mi.xed with a drop of 1:15,000 aqueous Neutral Red or Janus Green, or of both in combination. The tech- nique is fully described by Sabin, F. R. (Bull. Johns Hopkins Hosp., 1923, 34, 277-288) who used it to study living human blood cells. The stock solution of Neutral Red contains 100 mg. of dye to 10 cc. of absolute alcohol. The dilute solution contains 0.4 cc. of stock Neutral Red in 10 cc. of absolute al- cohol. An even dye film is obtained by flaming the slide, then flooding it with the dilute Neutral Red solution or with a mixture of Neutral Red and Janus Green, which is 2 cc. of dilute Neutral Red to 3 drops of saturated solution of Janus Green in absolute al- cohol. The slide is quickly drained and placed upright to dry. Fresh exudate IS mounted on the slide with coverslip and ringed wth vaseline. The cells may last for 2 to 3 hrs. if examined un- der a warm stage. The mitochondria of the cells may be studied when Janus Green is used. This dye is more toxic to the cells than Neutral Red. The ciliated epithelial cells are of particular interest and the motility of the cilia is not impaired. Nuclear staining of the NASAL PASSAGES 221 NECROBIOSIS cells with either of the dyea is indica- tive of cell death. The supra-vital staining technique was applied to the study of nasal secretion by Pfiugsten, M. G., in 1933 and reported to the E. N. T. clinical conference, Barnes Hospital (unpublished). It is possible to distinguish cells which are stimu- lated to activity from degenerating cells with this technique. A good azure stain may be obtained by using 10-12 drops of Wright's stain in absolute al- cohol, but this is more toxic than Neu- tral Red. 5. Papanicolaou Stain. This stain, as developed by Papanicolaou, G. N. (Science, 1942, 95, 438-439) is the first satisfactory stain and fixation found which permits accurate identification of the different types of epithelial cells found in nasal exudates. Ciliated columnar cells are well preserved and can be readily identified from the squamous and basal types. The nu- clear detail of all cells in this stain is very sharp and clear. The eosinophilic granules, however, are not as distinct as in the other stains. Slight modifica- tions can be made to bring out certain details in nasal smears. The chromatin masses may be more distinct when the time in hematoxylin is shortened to 2 to 3 min. The granules of the eosin- ophilic cells and other acidophilic bod- ies are brighter red if the time in eosin is increased to 3 min., or, if a trace of phlo.xine is added (0.5% solution in 95% alcohol). All cellular details disclosed by the Papanicolaou stain are revealed by Wright's stain, even the patterns of epithelial degeneration, but they are noted with greater difficulty. It follows that one stain is not suffi- cient to see all cellular details to the best advantage. It seems important to use several staining methods on the same material to obtain more complete knowledge of cellular responses. The smears, with their cellular patterns, may be considered to be an approximate index of the pathological processes oc- curring in the tissues. Nasal Passages. The fluid, when present in unusual amounts can obviously be studied in Smears. Nasal clearance depends upon the movement by the cilia toward the pharynx of a mucous sheet (to which foreign materials be- come attached) over a layer of fluid in which the cilia act as can be demon- strated by the techniques of Lucas, A. M. and Douglas, L. C, Arch. Oto- laryng.. 1934, 20, 518-641 and others. Methods for Mucus and Cilia are given under their respective headings. The wall of the nasal passages exhibits marked regional diversity (Hilding, A., Arch. Otolaryng., 1932, 16, 9-18). The nasal mucous membrane covering the septum can be removed in toto by the dilute acetic acid method (see Epider- mis) and examined as a whole mount which gives valuable data impossible to secure from the study of sections. Those interested in wound healing would do well to consult a paper by Boling, L. R., Arch. Otolaryng., 1935, 22, 689- 724. An easy and graphic method for visualization of lymphatic drainage is described under Lymphatic Vessels. For numerous suggestions as to tech- nique see Proetz, A. Applied Physi- ology of the Nose. St. Louis: Annals Publishing Co., 1941, 395 pp. Nasal Sinuses. The mechanism of clear- ance is similar. To make sections of the nasal sinuses, especially the smaller ones, fixation in Formalin Zenker is suggested followed by Decalcification and Celloidin Imbedding. The sec- tions can be stained by the method best adapted to the purpose in mind. Nasmyth's Membrane, see Enamel cuticle. n-Butyl Alcohol (prophylcarbinol). Rec- ommended by Stiles (K. A., Stain Techn., 1934, 9, 97-100) to replace higher concentrations of alcohol in histo- logical technique especially for lightly chitinized insects but also as a routine for vertebrates. After fixation in Gil- son's Fluid pass the tissues through 35% (ethyl) alcohol ^1 hr.; 90 cc. 45% ale. + 10 cc. butyl, 2 hrs.; 80 cc. 62% ale. -f 20 cc. butyl, 2 hrs.; 65 cc. 77% ale. + 35 cc. butyl, 4 hrs.; 45 cc. 90% ale. + 55 cc. butyl, 6 hrs. to days; 25 cc. abs. ale. + 75 cc. butyl, 6 hrs. to over night; butyl 2 changes several hrs. (or store in butyl if desired). To imbed transfer to mixture of butyl and paraffin and to paraffin, n Butyl alcohol is helpful in making permanent prepara- tions of tissues freshly stained with Methylene Blue, which see. It should not be confused with Tertiary Butyl Alcohol. Necrobiosis was for Minot (C. S., The Problem of Age, Growth and Death. New York, G. P. Putnam's Sons, 1908, 280 pp.) a condition in which the cells continue to live but change their chemi- cal organization so that their substance passes from a living to a dead state. "Here (he says) life and death play together and go hand in hand." The term is current but is of little use be- cause it has no advantage over the word Necrosis for the disorganization of death seldom if ever takes place simul- taneously throughout the substance of any living thing. See Dead Cells. NECROSIS 222 NEODYMIUM Necrosis (G. ne/crosis, a killing). The term is usually appliea to indicate the local death of a cell or of group of cells, not that of the body as a whole. Death is defined by Webster and others as the "cessation of life" which merely poses the question of what life is. Perhaps the most fundamental vital phenomenon is the oxygen consumption involved in respiration. This may persist in eryth- rocytes even after the loss of their nuclei (Harrop, G. A., Arch. Int. Med., 1919, 23, 745-752). But cells frozen by special techniques do not respire while frozen. They endure in a state of suspended animation (called vitrifica- tion) indefinitely. They are not dead since they retain the structural organi- zation, which, when unlocked by in- crease in temperature, confers renewed vitality (see Luyet, B., C. rend. Soc. de biol., 1938, 127, 788-789 and many others). Death can therefore be better defined as the disorganization of living matter which makes permanently im- possible all vital phenomena. Since the organization of different sorts of living cells is fundamentally different the loss of organization in them is likely also to be different. See various forms of Degeneration. In general necrosis of tissue is often evidenced by a break- ing up of the nucleus known as caryor- rhexis (G. Karyon, nucleus, -f- rhcxis, rupture) or by its solution, caryolysis (G. lysis, solution). Consequently any good nuclear strain such as hematoxylin or methylene blue is satisfactory. See techniques for Dead Cells, Necrobiosis. Neelsen, see Carbol-Fuchsin. Negative Stains are used to show the back- ground in which bacteria and other organisms are present in smears and by contrast thus to reveal them unstained, that is in a negative way. The tech- nique is very simple. Simply mix the fluid containing the organisms with the "stain", smear on a slide, dry and examine. Higgins' India Ink is usually employed; but congo red (Cumley, R. W., Stain Techn., 1935, 10, 53-56) and azo blue (Butt, E. M., Boynge, C. W. and Joyce, R. L., J. Inf. Dis., 1936, 58, 5-9) are among many other materials used. See Azo Blue. Negri Bodies. 1. Rapid section method (Schleif stein, J., Am. J. Pub. Health, 1937, 27, 1283-1285). Fix in Zenker's fluid, wash, dehydrate in dioxan, embed in paraffin, cut at 4 microns, mount, deparaffinize. Flood slides with 1 drop 1:40,000 aq. KOH in 2 cc. stock solution of stain (Rosanilin of Grubler 1.8 gm., methylene blue, Nat. Col., 1 gm., gly- cerol 100 cc. and methyl alcohol 100 cc). Steamgentlv5min. Rinse in tap water. Decolorize by gently moving in 90% ethyl alcohol until color is faintly violet. Pass quickly through 95% alcohol, absolute, xylol and mount in balsam. Negri bodies deep magenta with dark blue inclusions. 2. Rapid smear method (Dawson, J. R., J. Lab. & Clin. Med., 1934-35, 20, 659-663). Remove brain to be examined as quickly as possible. Cut several small segments (3-4 mm. thick) from Ammon's horn perpendicular to its long axis and place in Petri dish. Cut away adjacent tissue leaving only the horn. Place a segment, cut surface down, on small end of a new 1 in. cork. With wooden applicator, or match, gently wipe peripheral tissue outward and downward. The segment is thus more firmly attached to the cork and the gray matter containing the pyra- midal cells bulges upward. Press this gently against a slide (clean and entirely free from grease) held at one end be- tween thumb and forefinger. Repeat 3 or 4 times, starting at end away from fingers, quickly so tissue does not dry. Immediately immerse in abs. methyl alcohol 5 min. or more. Rinse in run- ning water and stain in 2% aq. phloxine 2-5 min. Wash off excess stain in run- ning water and color in Loeffler's alka- line methylene blue, 10-20 sec. De- colorize in 80% ethyl ale, dehydrate in 95% and 2 changes of absolute, clear in xylol and mount in balsam. Handle slides with forceps and avoid danger from contact with tissue throughout process. Pyramidal cells blue, Negri bodies bright red to reddish brown. Time including examination 25 min. Stovall, W. D. and Black, C. E., Am. J. Clin. Path., Tech. Suppl., 1940, 4, 8 recommend control of pH in staining with eosin methylene blue (see Buffers) . Stain with 1% eosin in 95% alcohol at pH 6.0 or more alkaline. ISIegri bodies pale red. The red is much more intense if the pH is 3.0. Loeffler's methylene blue is best as counterstain at pH 5.3. At pH 6.0 it removes eosin. Azur B is advised for staining of Negri bodies by Jordan, J. H., and Heather, H. H., Stain Techn., 1929, 4, 121-126; see also Carbol-Anilin Fuchsin methyl- ene blue. Neisserian Infection. A differential stain favorable for diagnosis (Scudder, S. A., StainTechn., 1931, 6, 99-105). Neisser's Stain for Diphtheria Bacilli, which see. Nemathelminthes is the phylum of round worms. See Parasites. Nematodes. See Glychrogel for mounting. See Parasites. Neodymium, see Atomic Weights. NEON 223 NERVE FIBER DEGENERATION Neon, see Atomic Weights. Neoprene, injection of blood vessels (Lieb, E., J. Tech. Methods, 1940, 20, 50-51). Neoprene is a colloidal, finely divided suspension of synthetic chloroprene in an alkaline aqueous medium. Instruc- tions for the human kidney. Cannulate renal artery and wash with tap water at slow but constant rate. Ligate grossly leaking vessels. Continue 8-18 hrs. until organ is pale gray. Cover and keep in ice box 6-7 hrs. or until the next day. Keep specimen at room temperature about one hour before in- jection. If it feels cold warrn it with tap water. Connect cannula with bottle containing neoprene. A special appara- tus for maintenance of 150^160 mm. Hg. is advised by Lieb but it is probably sufficient to provide gravity pressure by raising the bottle 5 ft. or more. Close vessels ejecting the neoprene with hemostats and tie them when ves- sels are completely filled. Rinse in warm water. If a corrosion specimen is wanted leave kidney in cone, com- mercial HCl in tightly covered vessel at 56 °C. over night. Next morning pour off acid and allow stream of water to flow over the cast itself in the bottom of the container. When all debris is removed examine under water with dissecting microscope. Store in 0.3% Dowicide sol. (American Anode Inc., 60 Cherry St., Akron) to avoid mold. Lieb gives more details and describes combined corrosion, histological and roentgenological methods. Technique should be adapted to other organs. (Revised by Ethel Lieb, May 16, 1946). Lieb's method has been modified in several respects by Duff, G. L. and More, R. H., J. Tech. Methods, 1944, 24, 1-11. The technique for mounting separately for detailed microscopic examination small sprigs of the renal cortical arteries greatly increases its usefulness. Neoprene Latex. Emplo3^ed for injection of coronary arterial sj'stem, well illus- trated and with a list of earlier papers (Smith, J. R. and Henry, M. J., J. Lab. & Clin. Med., 1945, 30, 462-466). Nerve Endings. These may be demon- strated in many ways. Nothing will adequately take the place of their study in vivo (Speidel, C. C., J. Comp. Neur., 1942, 76, 57-73) ; but no method should be used with expectation of satisfactory results the first time. Experimentation is required. Most of the silver methods for neurofibrils show nerve endings. The writer has obtained good results by Bodian's Method applied to paraffin sections of experimental tumors. Cra- ven's Gold Chloride method may be tried. For silver impregnation of intra- cellular nerve endings in pars inter- media of pituitary, see Tello, F., Trab. d. Lab. Rech. Biol. Univ. Madrid, 1912, 10, 145-183. Methylene blue is, since the time of Ehrlich, a very popular stain for nerve endings. Addison (McClung, pp. 477-480) has given a full account of the technique. Commission Certified zinc-free methylene blue is suggested. Dye can be applied locally or by vascular perfusion. 1. Local application. Place tissue in shallow dish on thin layer of glass-wool moistened with 0.1-0.05% methylene blue in physiological salt solution. Add enough stain every few minutes to keep tissue moist and covered by film of stain. Beginning after 15 min. examine frequently at low magnification until nerves are colored blue. Fix stain by immersion in cold 8% ammonium molyb- date in physiological salt solution or Ringer's (^ hr.). Wash in cold water. Dehydrate in alcohols in refrigerator a little above 32 °C. Either clear in xylol and mount in balsam or imbed in paraffin and section. Cole (E. C, J. Comp. Neurol., 1925, 38, 375-387) proceeded much in this way. He immersed whole alimentary tract of frog in 1:10,000 methylene blue solution for 1 hr. and cut it in pieces. 2. Vascular perfusion. Insert can- nula in main artery leading to the tissue. Inject 1:10,000 methylene blue in physiological saline until tissue becomes light blue. Leave 15 min. Remove thin pieces or slices. Place in dish and moisten with methylene blue solu- tion. Examine uncovered at low magni- fication at intervals until nerve fibers and endings are stained. It is essential as in local application not to exclude air from tissue by covering with too much fluid. Fix in ammonium molybdate and continue as described above. For large fetuses use Langworthy's method (O. R., J. Comp. Neurol., 1924, 36, 273-297), for the lungs of rabbits that of Larsell (O., J. Comp. Neurol., 1921, 33, 105-131), for arteriovenous anasto- moses Brown's (M. E., Anat. Rec, 1937, 69, 287-295), and for skin Weddell's (G., J. Anat., 1940-41, 75, 441-416). Staining may perhaps be accentuated by hydrogen acceptors, see Auerbach's Plexus. See Pacinian Corpuscles, Meissner's Corpuscles, Krause's End Bulbs, Motor End Plates, Boutons Terminaux and Synapses. Nerve Fiber Degeneration. The standard techniques are the Marchi Method by which the lipids produced by degenera- tion are blackened with osmic acid and the staining of lipoids by Sudan III. NERVE FIBERS 224 NERVOUS SYSTEM In addition 3 other much quicker methods are recommended: 1. To stain vitally with neutral red (Covell, W. P. and O'Leary, J. L., J. Tech. Meth., 1934, 13, 92-93). In- tensity of staining of degenerating myelin depends upon amount and con- centration of the dye. It can be applied in 3 ways: (1) Inject 4 cc. 4% neutral red in physiological salt solution into marginal ear vein of a rabbit over period of 1 hr.; (2) Perfuse through aorta with large volume of 1:1,000 solution; (3) Immerse finely teased piece of degenerated nerve in 1:10,000 solution for about 12 min. Vital stain- ing permits immediate determination of extent and degree of degeneration. See the author's excellent colored figures. 2. To examine by polarized light (Weaver, H. M., J. Lab. & Clin. Med., 1940-41, 26, 1295-1304). Lay excised nerves without stretching on piece of wooden tongue depressor and fix 24 hrs. or more in 10% neutral formalin. Cut longitudinal frozen sections 10 microns thick. Float them onto slides from water, mount in neutral glycerin and examine. Weaver gives diagrams to aid in interpretation of findings. See also Pritchett, C. O. and Stevens, C, Am. J. Path., 1939, 15, 241-250; Rad- hakrishana, BLao, M. V., Ind. J. Med. Res., 1938, 26, 103-106. 3. To demonstrate early changes in the axis cylinders (cores of the fibers) Alzheimer's modification of IVIann's eosin-methyl blue method is strongly recommended by Mallory as showing normal axis cylinders deep blue and degenerated ones, red. Nerve Fibers. Many excellent methods present themselves : the continuous direct observation of the growth of individual fibers in living tissues of lower animals (Speidel, C. S., Biol. Bull., 1935, 68, 140-161); the micro- dissection of living fibers (De Renyi, G. S., Cowdry's Special Cytology, 1932, 3, 1370-1402); x-ray diffraction studies of the sheaths (Schmitt, F. O., Bear, R. S. and Palmer, K. J., J. Cell, ana Comp. Physiol., 1941, 18, 31-42) and microincineration (Scott, G. H., Proc. Soc. Exp. Biol. & Med., 1940, 44, 397- 398). For their demonstration in fixed tissues consult methods of Bodian, Davenport, Golgi, O'Leary, Osmic Acid, Weigert and WeiL The methylene blue technique of staining nerve fibers is given under Auerbach's Plexus. See Nerve Endings, Motor End Plates, Bouton Terminaux. Use of quartz io