Historic, archived document Do not assume content reflects current scientific knowledge, policies, or practices. ts mo c sg pues ‘ ‘ are a Es: ‘ : ee ee TECHNICAL SERIES, No. 14. 3 SEES ere Jf - = see! at yw, oF U: S. DEPARTMENT. OF. AGRICULT | aioe BUREAU .OF ENTOMOLOGY. L. O. HOWARD, Entomologist and Chief of Bureau. E>, aS pms Ts, PT - 2 sees Spee Ee MOR FN ms _ . ase Fs Bs Aig oy ~ Ss ; 2 z Hi 5 - tJ e% 5 > “ye “Ss , ae : i 5 > : s 4 —_," . we 2 ‘_ , € , ae : ki - ‘ § f if : : ; 4 9 es > wy sa eater ae t a - eared a h> r* > - Dies "then Sage BACTERIA OF THE APIARY, WITH SPECIAL REFERENCE TO BEE DISEASES. BY | GERSHOM FRANKLIN WHITE, Px. D., Expert in Animal Bacteriology, Biochemic Division, Bureau of Animal Industry. x ISSUED NOVEMBER 6, 1906. Ip Dinh inllbeiines Waa " We. pa Pha eel Lal ne i i e ¢ . * ) te = x: \) i Jy Hl bf sat! I LA (SS el | J Mn 94 Sill als iy WASHINGTON: GOVERNMENT PRINTING OFFICE. 1906. or a b EK. ) ¢ a i: % E. C. BUREAU OF ENTOMOLOGY. A Re O. “Howarp, Entomologist and” Chief. OF. puceate Ri C. L. MARLATT, Entomologist and Acting Chief in \qosenge ae Chie ae i AS RS BS: oes Chief. Clerk. epg PF. H. bein) in charge of breeding experiments. me - ‘ es A. D. Hopxins, in charge of forest insect investigations. Ate 8 ou ie aS W. D. HunNTER, in charge of cotton boll weevil investigations. Mee on Ua ae M. WEBSTER, in Charge of cereal and forage-plant insect ‘investigations. — Ae Li. QUAINTANCE, in charge of deciduous- fruit insect investigations. ey M. RoceErs, in charge of gipsy and brown- tail moth ae fe, Ww. MorRILL, engaged in white fy investigations. — ‘SS. G. Titus, in charge of gipsy moth laboratory. _ | J. Gituiss, engaged in silk investigations. sf Bice R. P. Currie, assistant in charge of editorial work. ee “Maser Corcorp, librarian. | Be as ah aise or Py, | AprovrruraL INVESTIGATIONS. A She FRANK BENTON, in charge (absent). si Tahal ene My een E. F. Puitiies, acting in charge. AL AGRE NYS ee aa wen J. M. RANKIN, in charge of apicultural station, ‘Chica. Cal. En JESSIE E. MaRKSs, apicultural clerk. PEG) aie Oi ane j Ae ~ iy ike ery | phe pa & sf i v» RS 6 3 kg " “h Ar ‘ . pet Be TECHNICAL SERIES, No. 14. USS DEPARTMENT OF AGRICULTURE, BU EAU [OM HN TOMOLOGY. L. O. HOWARD, Entomologist and Chief of Bureau. THE BACTERIA OF THE APIARY, WITH SPECIAL REFERENCE TO BEE DISEASES. BY GERSHOM FRANKLIN WHITE, Pa. D., Expert in Animal Bacteriology, Biochemie Division, Bureau of Animal Industry. ISSUED NOVEMBER 6, 1906. PETOIOSS5 a ZERY wl rile ines nt wt | oii nial i hall Be > li i ane ry > | is i i 8 mT it be a ii y= i, Sie r J WEA WZ . —— — we. WASHINGTON: GOVERNMENT PRINTING OFFICE. L206. LETTER OF TRANSMITTAL. U. S. DeparTMENT OF AGRICULTURE, Bureau or ENTOMOLOGY, Washington, D. C., September 24, 1906. Sir: I have the honor to transmit the manuscript of a paper on the bacteria of the apiary, with special reference to bee diseases, by Dr. G. F. White, expert in animal bacteriology in the Biochemic Division of the Bureau of Animal Industry. This paper was pre- pared by Doctor White as a thesis in part fulfilment of the require- ments for the degree of doctor of philosophy, at Cornell University, in June, 1905. The Bureau of Entomology considers itself fortu- nate in obtaining it for publication, since in this way a wider distri- bution can be made than would be possible were it published in a journal devoted exclusively to bacteriological investigations. It is” hoped that the publication of these facts may help to clear up the confusion which now exists concerning the causes of the two most common diseases of the brood of bees. I recommend that the manu- script be published as Technical Series, No. 14, of this Bureau. Doctor White wishes to acknowledge his indebtedness to Dr. Veranus A. Moore, professor of comparative pathology and _bac- teriology of Cornell University, under whose direction this work was done; to Dr. E. F. Phillips, acting in charge of apiculture, Bureau of Entomology, United States Department of Agriculture, for encouragement and assistance in the preparation of this manu- script; and to Messrs. Mortimer Stevens, Charles Stewart, N. D. West, and W. D. Wright, bee inspectors of the State of New York, for their interest in the work. Respectfully, L. O. Howarp, Entomologist and Chief of Bureau. Hon. JaAmrs Wiison, Secretary of Agriculture. 2 BRAG Eo The spread of diseases of the brood of bees is to-day a great menace to the bee-keeping industry of the United States. It is therefore of great importance that all phases of these diseases should be investi- gated as thoroly as possible, and this paper, it is believed, will help in clearing up some disputed points in regard to the cause of the two most serious brood diseases. Dr. G. F. White has offered this paper for publication as a bulletin in the Bureau of Entomology because in that way the statements herein contained may become more widely known than would be the case were it published in some journal devoted exclusively to bacteri- ological investigations. Obviously there are many points still un- settled, and it is hoped that some of these may be taken up for in- vestigation in the near future, but the results so far obtained should by all means be made known to the persons practically engaged in bee keeping. | The necessity for the study of nonpathogenic bacteria found in the apiary may not be at first evident to the ordinary reader. When it is seen, however, that some of the investigators of bee diseases have apparently mistaken Bacillus A or some closely allied species for Bacillus alvei 1t will be evident that a study of nonpathogenic germs is necessary to a thoro investigation of the cause of these diseases and a full understanding of the confusion which has existed. The names which should be used for the diseased conditions of brood was a matter which arose after this paper was offered for pub- lication. It was desired that out of the chaos of names in use cer- tain ones be chosen which would be distinctive and still clear to the bee keepers who are interested in work of this nature. Unfortu- nately, after a short investigation, Dr. W. R. Howard, of Fort Worth, Tex., gave the name “ New York bee disease,” or “ black brood,” to a disease which Cheshire and Cheyne described in 1885 as “foul brood.” Since this is the disease in which Bacillus alvei is present, we can not drop the name “foul brood,’ and the word ‘“ European ” is used to distinguish it from the other disease. The bee keepers of the United States have been taught that the type of brood disease characterized by ropiness of the dead brood is true foul brood, 3 4 PREFACE. but since Bacillus alvei is not found in this disease it obviously is not the same disease as that described by Cheyne. It would be well-nigh impossible, however, to change the name of this disease, and any effort in that direction would merely result in complicating laws now in force which control the infectious diseases of bees and would serve no good _ purpose. This disease is here designated “American foul brood.” These names have been chosen only after consultation with some of the leading bee keepers of the United States, and these distinguishing terms were chosen by the majority of those consulted as indicating the place in which the diseases were first investigated in a thoroly scientific manner. Both diseases are found in Europe, as well as in America, so that the names indicate nothing concerning the geo- graphical distribution of the maladies: Strangely enough, certain writers for our American apicultural - papers have seen fit to take exception to some of the statements made in this paper without having first found out the reasons for the de- cisions herein published. Apiculture will not be advanced to any appreciable extent by such eagerness to rush into print, especially when there is not a semblance of scientific investigation back of the criticism. EK. F. PHrures, Acting in Charge of Apiculture. Introduction CONTENDS: = WS EERMOTOG. acct Sar age SS eS aire eae ea i ee acme take Tien Ol ili yee iter ee ete eS ED CREY PUD COL EET eS SI er lg vse al ge FeO Differentiation and identification of bacteria ___-__.._________. athe cuties wich are G@eseripeGg. 6. ie Se Morphology, staining properties, and oxygen requirements, with sug- PCS SEOM SMAI AII GU Ge= tie ere ST NS a) ree Media employed and suggestions as to the description of cultures____ PART I. BACTERIA OF THE NORMAL APIARY. ee aeceeatet 1 renters COMMS ae! ers ee Paree te AmeT aT EPONA pies a os Gee yr re Ses) Pe ek ot Boeenemienoneyoand Tlorimal lary 2. 2 ee feet ee med eran t. Pees = <2. ee ee Bacteria of the intestine of the healthy honey bee___________=___________ Sar eeepeneeA NAD INONUACI Ce aNA CATA OE es we es een es Se oS eh ee Tabulation of micro-organisms normally present in the apiary____________ Summary to Part ie Sa aa 8 Ae ae Sa ee ee DS50 DLO SEEE EY Dy CRY AG ra] BAS Ba (ise wa ce en hate sete wt See eo Sele ee en ann se DESTPLELE LSE CVI ele St ae PS ae eo Oa ny ee nee ee A eee gi Mee A ees eae erin tou) brood.’ -as-hitherto applied]. 2. 2 =e Baurepein: toul. brood. (foul hroed: of Cheyne). 2 SOT ET EDUC — se ee as a PE ed A BS A peat in Sc ieee i Crt A ee Se Commision recardin® foul brood ins America. => 2 Ree PRR CBR CCEOiE TR VECO ATION = 25 cee san 2 Se ga ee ee ee ee ee CES DUCE ITED 208 Mae Boe es sR cea Sp Oe RMN ee ail aT, eRe ge A Seale OTe XCEIMNCI IG. = oo Soe oe td SS ae a ie Disimipuion of Bacillus alvei in. infected hives__=— 2 = se HxpCLNnentsow iti. Lormaldehy de: Gas =" ss ee ee aS TDE PUGET: SCTE NOE et ae eo ae EN we Saree ye eS ee RCS LAE © SUPE CDYECV TS PSS cS SE eM eee ea CI eh eee a eS Mh agate Siti cltd Wie Sid Ad 1G 1h a Se ee ee ee ee ee CETTE Se oF If Dee ip le i ee a oe RLS See ee | Whee calleiien BIGdeapra0d = =< 45. 7s 2a Soa, ee eS eee Wieee ee ede aCe LOORS | as De ete fe ee Ae eaten TN Vachs; 2 ce ek Se gh EE SN Se i ee ee STREPDREN Ere 7 Eat (Saag ean ee Ean pee ore ae aide: 5 oe CODD ETS TES Co SE ae a ie Nee nd a ios NCES TS Ree hs 8 lel macnraettine tee ais bie eet eh tS a a ee eee eee aed ie tne ene) 8 A ee eee ee eee PART II. THE DISEASES OF BEES. OO 1 ] 1 QQ? 10 Tie DACIERIA OF THE APIARY WITH SPECIAL REFERENCE TO BEE DISEASES. INTRODUCTION. Since bacteriology is one of the youngest of the sciences, it is only natural that there should be many problems concerning which there is much confusion, and many others concerning which nothing is known. In a study of the saprophytic bacteria this is especially true; the exploration of this jungle of micro-organisms is scarcely begun. Comparatively few species have been studied and named, and a much less number can be identified. From studies that have been made one is led to believe that the species which might be classed under bacteria outnumber by far all the macroscopic plants known. Comparatively little is as yet known concerning the dis- tribution of these minute organisms in nature, their needs for multi- pleation and growth, their power of endurance, their relations the one to the other, their relations to man and. industries, and their relation to pathogenic species. Both from the standpoint of scien- tific interest and from the standpoint of practical economy these problems call for further investigation. By far the greatest amount of work which has been done in the science of bacteriology has been prompted by the direct or indirect economic importance of the question. This is largely true of the present investigation, since honey bees suffer from a number of diseases, some of which are considered in Part II. . TECHNIQUE. Obtaining Material for Study. If necessary, bees may be conveniently shipped alive by mail in cages constructed for that purpose. Combs also may be sent by mail in small boxes. If combs, honey, pollen, or larve are desired, the hive must be entered. In case older adult bees are wanted it is not difficult to supply the needs from the entrance to the hive. To capture them one may stand at the entrance and catch the unwary toiler as she 7 9583—No. 14—06 M 2 8 THE BACTERIA OF THE APIARY. comes in loaded with pollen and honey. After the victim alights on the entrance board, by the aid of a pair of forceps, before she disap- pears within, one can easily lodge her safely in a petri dish. It is, however, an advantage to study the young adult bees as well as the older ones, and if young ones are desired they may be taken from the combs or from the front of the hive, near the entrance. Obtaining Cultures. (a) From combs.—With sterile forceps small pieces of the comb are put directly into gelatin or agar for plates or incubated in bouil- lon for 24 hours and then plated. Growing in bouillon and plat- ing on gelatin is usually preferable. (6) From pollen—rThe same technique is used as for combs, but the direct inoculation of gelatin tubes for plates is generally pre- ferable. (c) From honey.—With sterile loops honey is taken from uncapped and capped cells. The caps are removed with sterile forceps and the honey is plated directly on gelatin or agar. Bouillon tubes are in- oculated also with varying quantities of the honey. (d) From larve.—The larva is carefully removed to a sterile dish, and with sterile scissors the body is opened and the contents plated directly, or bouillon cultures are first made and later plated, if a growth appears. (e) From parts of the adult bee——In studying the adult bee, a small piece of blotting paper wet with chloreform is slipt under the cover of the petri dish in which the insects have been placed, and in a short time the bees are under the influence of the anesthetic. ‘Then with sterile scissors a leg, a wing, the head, the thorax, or the abdomen, the intestine being removed, is placed in bouillon and, after 24 hours incubation, plated, preferably on gelatin. When it is desired to make a study of the bacteria of the intestine, the intestinal tract is removed and studied as follows: The bee is flamed and held in sterile forceps. With another sterile pair of for- ceps the tip of the abdomen is seized and, by pulling gently, the tip and the entire intestine are easily removed. This can then be plated directly. If gelatin, which is preferable, is used, the intestine itself must not be left in the gelatin or the medium will become liquefied by the presence of the tissue. If one desires to obtain cultures of the anaérobe, which is quite common in the intestine, it is most easily obtained in pure culture by the use of the deep glucose agar (Liborius’s method). Cover glass preparations made direct from the walls of the intestine or its contents give one some idea of the great number of bacteria frequently present. MORPHOLOGY, STAINING PROPERTIES, ETC. 9 Differentiation and Identification of Bacteria. These very low forms of plant life show a marked susceptibility to environmental conditions and those desirous of speculating on prob- lems in evolution may find here food for thought and experimenta- tion. On account of this susceptibility, various cultures which belong to the same species may possess slight variations in some one or more specific characters. Consequently one can not say that a species must possess certain definite characters and no others. It is convenient, then, to think of a species as more or less of a group of individuals whose characters approximate each other very closely. In this paper are described a number of species each of which, in fact, represents a group, the individual cultures of which approxi- mate each other so closely in character that the differences may be easily attributed to environmental conditions which are more or less recent. Concerning the identification of species, the conditions have been well summed up by Chester. He says: Probably nine-tenths of the forms of bacteria already described might as well be forgotten or be given a respectful burial. This will then leave comparatively few well-defined species to form the nuclei of groups in one or another of which we shall be able to place all new sufficiently described forms. The variations which occur and the very incomplete descriptions which can be found make it impossible to identify many species even to a more or less restricted group. For these reasons some of the cultures are not identified or named, but letters are used for conven- lence in this paper to represent the specific part. Migula’s classifica- tion has been used. The Cultures Which are Described. Plate cultures were observed for some weeks, the different kinds of colonies which appeared being especially noted. Subcultures were then made in bouillon, and after 24 hours the subculture was re- plated. Subculturing and replating were then repeated. From this last plate the pure culture was made on agar for study. These were not studied culturally, as a rule, for some weeks, thus allowing time for the organism to eliminate any character due to recent environ- mental conditions (1).¢ Morphology, Staining Properties, and Oxygen Requirements, with Sug- gestions on Variations. (a) Size—The length and thickness of a micro-organism often varies so much with its environmental conditions that certain re- a Numbers in parentheses refer to papers in the bibliography at the end of Part I or that at the end of Part II. 10 THE BACTERIA OF THE APIARY. corded dimensions should always be accompanied by facts concerning the medium, age, and temperature of incubatien. The measure- ments recorded in this paper were all taken of organisms in prepara- tions made from a 24-hour agar culture stained with carbol-fuchsin. The involution forms are not reckoned in the results. (6b) Spores.—The presence of spores was determined in each case by staining the various cultures at different ages. A check was made -on their presence by means of the thermal death point. (c) Flagella.—Loefiler’s method, as modified by Johnson and Mack, was used for staining the flagella (2). (d) Motility.—Motility may be present in cultures when first iso- lated, but after artificial cultivation appear to be entirely lost. The reverse of this also may be noted. No cultures should be recorded as nonmotie until cultures on various media at different temperatures and of different ages shall have been studied. Hanging-drop prepar- tions were made from cultures on agar and bouillon, both incubated and not incubated, and on gelatin. (e) Staining properties.—Basic carbol-fuchsin was the stain psed almost exclusively. In the use of Gram/’s staining method, carbolic gentian violet (5 per cent carbolic acid 20 parts, saturated alcoholic solution gential violet 2 parts) was applied to a cover-glass prepara- tion from a 24-hour culture on agar for 5 minutes, placed in Lugol’s solution 2 minutes, and placed, without rinsing, in 95 per cent alcohol - for 15 minutes, removed, washt in water, and allowed to dry. (7) Oxygen requirements.—Determinations were made by ob- serving whether a growth took place in the closed or open arm or both, of the fermentation tube containing glucose bouillon. Media Employed and Suggestions as to the Description of Cultures. (a2) Bouillon—All bouillon used was made from beef (meat J part, water 2 parts), to which infusion 1 per cent Witte’s peptonum siccum and one-half per cent sodium chlorid were added. The re- action of the solution was then determined by titrating, and made +1.5 to phenolphthalein. In describing a culture growing in bouillon as a medium, there is usually a more extended description given than in the case of sugar and sugar-free bouillons, since cultures in these media do not differ materially in gross appearance from those observed in the plain bouillon. (b) Sugar-free bouillon.—This bouillon is made free from sugar by the use of B. coli communis, after which peptone and sodium chlorid (NaCl) were added as in bouillon. (c) Sugar bowillons—F ive different sugars—glucose, lactose, sac- charose, levulose, and maltose, as well as mannite—were used in the study. If a 1-per-cent solution of glucose in plain bouillon was fer- MEDIA EMPLOYED, ETC. EF mented with the production of gas, fermentation tubes were used for all the sugars and mannite. If no gas was formed in the glucose, the straight tubes were inoculated. The sugars and mannite were used in a 1-per-cent solution in sugar-free bouillon. (d) Reaction of media—The reaction of cultures is determined as it appears on the fifth day in the different media, unless otherwise stated. The medium in the open arm is used to determine the re- action in the fermentation tube. Beginning with a reaction of +1.5 to phenolphthalein, or slightly alkaline to litmus, the detection of an increase in acidity is not difficult. But inasmuch as the production of an alkali is very frequently small in degree, cultures are often in this paper recorded alkaline in reaction when probably the reaction has not changed. (e) Fermentation with the production of gas.—Gas may be formed in such small quantities as not to be observed as such, but to be en- tirely absorbed by the medium. Whenever gas formation is men- tioned as a character, visible gas is meant. The analysis of the gas was made in the usual manner by absorbing a portion with potassium hydrate (KOH) and testing the remainder with the flame. The amount absorbed by potassium hydrate (KOH) is referred to as carbon dioxid (CO,) and the remainder, if an explosion is obtained, as hydrogen (H). This is, naturally, only approximately correct. Since the gas formula may vary from day to day, too much value must not be given to the exact proportion. It is well to observe whether the proportion of hydrogen to carbon dioxid is greater or less than 1. ! . (7) Agar.—One per cent agar is used. The description of the growth on this medium is made from the appearance as séen on the surface of an agar slant. The description is usually very brief, since it has, as a rule, little differential value. (g) Acid agar—This medium is made acid by titrating to +3 to phenolphthalein. The absence or presence, as well as the degree of growth, is noted. (h) Serum.—The serum used is taken from the horse, sterilized at 55° C. and congealed at 80° C. Deep inoculations are made, and the surface of slanted serum is also inoculated. The degree of growth is usually noted. Cultures are observed for 6 weeks to 2 months. The presence or absence of liquefaction is the chief character sought for. Since room temperature varies so greatly, the time at which liquefac- tion begins varies, and little differential value, therefore, can be given to the exact time of this phenomenon. (2) Potato—The composition of potato varies so markedly that a description of a culture on this medium may differ materially from that which is observed on another tube of the same medium. It is the aim to omit for the most part the observed variations due to the composition of the different potatoes. 13 THE BACTERIA OF THE APIARY. (7) Potato water.—To potatoes sliced very thin is added an equal amount of water by weight and the mixture is then boiled. This is strained and distributed in straight and fermentation tubes. The reaction of the solution was made +1.5 to phenolphthalein. If any of the micro-organisms ferment glucose with the production of gas, fermentation tubes are inoculated to test the fermentation of starch; if not, straight tubes are inoculated. (4) Milk.—lf a micro-organism breaks up glucose with the forma- tion of gas, a fermentation tube of milk is moculated with the culture; if not, straight tubes are used. Separator milk is used. The coagulation of the casein with or without lquefaction is the chief character noted. Very little stress is laid upon the time ele- ment in the coagulation of the casein and the other phenomena which are to be observed in milk. Different samples of milk and the different environmental conditions are factors which vary the length of time at which the different phenomena appear. (1) Litmus milk.—The reaction as shown by the litmus and the dis- charging of the color are the chief points observed. (m) Gelatin—The color, degree of growth, the presence or absence of liquefaction, and the form of lquefaction are the chief points observed. The cultures are kept under observation 2 months or longer and, as in serum, the time given at which liquefaction takes place is only approximate. (n) Indol.—The cultures are allowed to grow in sugar-free pep- tonized bouillon for 3 to 5 days, and are tested with potassium nitrite (KNO,) and sulfuric acid (H,SO,) after the ring method. Too much stress may be placed upon the ability of an organism to form indol. This character has been shown to be a somewhat transient one (3). (0) Reduction of nitrates to nitrites —Cultures are cultivated 7 days in a solution of 1 gram of Witte’s peptonum siccum and one- fifth gram of sodium nitrate in 1,000 c. c. of tap water. To such a culture and to a control tube are added a mixture of naphthylamine and sulfanilic acid (napthylamine, 1 part; distilled water, 1,000 parts: sulfanilic acid, one-half gram, dissolved in dilute acetic acid in the proportion of 1 part of acid to 16 parts of water). If nitrate is reduced to nitrite, a pink color develops. The control tube should remain clear, or slightly pink—owing to the absorption of a trace of nitrite from the atmosphere. PART I. BACTERIA OF THE NORMAL APIARY. Before studying the cause of a disease it is necessary that we know what bacteria are normally present, so that later, in studying diseased conditions, a consideration of these nonpathogenic species may be eliminated. In view of this necessity a bacteriological study BACTERIA FROM THE COMBS. 13 of the hives, combs, honey, pollen, larvee, and adult bees was begun, to determine the bacteria normally present. It was not hoped that all the species isolated could be easily identified, or that all would merit a careful description, but it was hoped that those species which seemed to be localized in any part of the apiary, or upon or within the bees, might be studied and described with sufficient care to guarantee their identification upon being isolated again. The chance of varia- tion in morphology, pathogenesis, and cultural characters due to environmental conditions to which these micro-organisms were being subjected at the time, or to which they had been subjected before isolation or study, has been carefully borne in mind. BACTERIA FROM THE COMBS. One might naturally suppose that very many species of bacteria would be present on combs, since these are exposed more or less to the contaminating influence of the air. The reverse, however, seems to be true. The number of different species isolated is comparatively small. Those which appear most often are described below. Some other species mentioned in this paper are found on combs, but inas- much as they appear most frequently from other sources they are described there. One species of Saccharomyces from the comb, also, is described under the heading “ Saccharomyces and fungi.” Bacillus A. (B. mesentericus?) Occurrence.—Found very frequently on combs, on scrapings from hives, and on the bodies of bees, both diseased and healthy. . Gelatin colonies.—Very young colonies show irregular edges, but very soon liquefaction takes place and the colony gives rise to a circular liquefied area, covered with a gray membrane, which later turns brown. Agar colonies.—Superficial colonies present a very irregular margin consist- ing of outgrowths taking place in curves. Deep colonies show a filamentous growth having a moss-like appearance. Morphology.—In the living condition the bacilli appear clear and often granu- lar, arranged singly, in pairs, and in chains. The flagella are distributed over the body. The rods measure from 3 to 4y in length, and from 0.94 to 1.2u in thickness. . Motility.—The bacilli are only moderately motile. Spores.—Spores are formed in the middle of the rod. Gram’s stain.—The bacilli take Gram’s stain. Oxygen requirements.—Aérobic and facultatively anaérobic. Bouillon—Luxuriant growth in 24 hours, with cloudiness of medium; a gray flocculent membrane is present. Later, the membrane sinks and the medium clears, leaving a heavy, white, flocculent sediment, with a growth of the organ- isms adhering to the glass at the surface of the medium. Reaction alkaline. Glucose.—Luxuriant growth takes place in the bulb, with a moderate, floccu- lent growth in closed arm. The gradual settling of the organisms causes a 14 THE BACTERIA OF THE APTARY. heavy white sediment to form in the bend of the tube. The reaction is at first slightly acid, but subsequently becomes alkaline. No gas is formed. Lactose.—Reaction alkaline. Saccharose.—Reaction alkaline. Levulose.—Reaction acid. Maltose.—Reaction acid. Mannite.—Reaction alkaline. Potato water.—Reaction aikaline. Agar slant.—A luxuriant growth takes place on this medium. The growth eradually increases to a moist, glistening one, being then friable and of a grayish brown color. Serum.—A luxuriant, brownish, glistening, friable growth spreads over the entire surface. No liquefaction is observed. Potato.—An abundant fleshy growth of a brown color spreads over the entire surface. The water supports a heavy growth. The potato is slightly discolored. Milk.—Precipitation takes place rapidly, followed by a gradual digestion of the casein, the medium changing from the top downward to a translucent liquid, becoming at last semi-transparent and viscid. Litmus milk.—Precipitation of the casein takes place usually waehatl 24 hours, followed by a gradual peptonization. Reduction of the litmus occurs rapidly, teaving the medium slightly brown; later the blue color will return on exposing the milk to the air by shaking. Reaction alkaline. Gelatin.—An abundant growth takes place with rapid, infundibuliform lique- faction. A heavy, white, friable membrane is formed on the surface of the liquefied medium. A flocculent sediment lies at the bottom of the clear lique- fied portion. Acid agar.—Growth takes place. Indol.—None has been observed. Nitrate.—Reduction to nitrite is positive. Bacterium acidiformans. (Sternberg, 1892.) Occurrence.—Isolated from the scraping of propolis and wax from the hives and frames of healthy colonies. Gelatin colonies.—The superficial colonies are friable, convex, opaque, and white with even border; when magnified they are finely granular, sometimes radiateiy marked. They are from 1 to 4 millimeters in diameter. The deep colonies are spherical or oblong and entire. Morphology.—When taken from an agar slant 24 hours old, the rods are short, with rounded ends, singly and in pairs. Length about 1.6u, thickness 0.8u. They stain uniformly with carbol-fuchsin. Flagella are apparently ab- sent. Motility—No motility has been observed in any EL A Spores.—Spores are apparently absent. Gran’s stain.—The bacteria are decolorized by Gram’s method. Oxygen requirements.—Facultatively anaérobic. Bouillon.—The medium becomes slightly clouded with a feeble ring of growth on the glass at the surface of the liquid. A moderate amount of white friable sediment is formed. Reaction alkaline. Glucose.—Uniformly and slightly clouded. No gas is formed. Reaction acid. ' Lactose.—Reaction acid. Saccharose.—Reaction alkaline. Levulose.—Reaction acid. BACTERIA FROM POLLEN. 15 Maltose.—Reaction acid. Mannite-—Reaction acid. Potato water.—Reaction acid. Agar slant.—A moderate, gray, glistening growth, confined to the area inocu- lated with the loop, is formed on the inclined surface. Serum.—A feeble gray growth is formed only on the inoculated surface. No liquefaction takes place. Potato.—A gray growth covers the inoculated surface. Milk.—Heat causes a ready coagulation of the casein. Reaction acid. Litmus milk.—Coagulation of casein occurs promptly -on boiling a culture 2 weeks old. Reaction acid. Gelatin.—Growth of spherical colonies appears along the line of inocula- tion, the surface growth being grayish and spreading slowly. No liquefaction takes place. Acid agar.—Growth takes place. Indol.—A trace was observed. Nitrate.—No reduction to nitrite could be observed. BACTERIA FROM POLLEN. As in the case of the examination of the combs, the number of spe- cies of bacteria found in pollen is comparatively small. The follow- ing are often found to be present. Other species have been isolated, but their distribution in the pollen is not at all constant. Bacillus B. Occurrence.—Found frequently in pollen and in the intestine of healthy honey bees. : Gelatin colonies.—The colonies are egg-yellow with even border. Liquefac- tion takes place slowly. Surface colonies are about 1.5 millimeters in diameter, have coarsely granular center, finely granular margin, and clear and sharply defined border. A peculiar toruloid growth is often observed. Morphology.—The organisms are short rods with rounded ends, which stain uniformly with carbol-fuchsin, and are lu to 24 in length. Few short involu- tion forms occur. Motility.—The bacilli are actively motile in young cultures. Spores.—No spores have been observed. Gram’s stain.—The bacilli are decolorized by Gram’s stain. Oxrygen requirements.—Facultatively anaérobic. Bouillon.—This medium becomes uniformly clouded, frequently with a scanty, friable membrane. Sometimes the organisms settle, clearing the medium and forming a viscid sediment. A growth of the culture adheres to the glass at the surface of the liquid. This, together with the membrane, is of a light egg-yellow color, which deepens somewhat with age. Reaction alkaline. Glucose.—At first both arms of the fermentation tube are clouded slightly, and the cloudiness later increases. Sometimes a stronger growth occurs in the closed arm than in the open one. Reaction is at first acid, but slowly changes to alkaline. Lactose.—Reaction alkaline. Saccharose.—Reaction alkaline. Levulose.—Reaction alkaline. Maltose.—Reaction slightly acid. 9583—No. 14—06 M 3) o 16 THE BACTERIA OF THE “APTARY. Mannite.—Reaction slightly acid, later alkaline. Agar slant.—A moderate, slightly yellow, nonviscid glistening growth appears along the inoculated surface. This growth gradually spreads and deepens in color to an egg-yellow. : Potato. A moderate, egg-yellow, nonviscid, glistening growth spreads over the entire surface. The potato is slightly discolored. . Milk.—The milk is covered by a yellow growth of the culture, resembling cream. Coagulation takes place on boiling. Litmus milk.—Reaction alkaline. Gelatin.—Growth takes place along the line of inoculation. Deep in the medium the colonies are white and spherical; the surface growth is yellow. After a few days liquefaction begins, and at the end of 2 weeks one-half the tube is liquefied. The liquefaction is infundibuliform. Liquefied gelatin is sur- mounted by a friable, egg-yellow pellicle. The growth in the liquefied portion is flocculent, which, on settling, forms a yellow sediment at the apex. Indol.—None could be observed. Nitrates.—No reduction to nitrites occurs. BACTERIA IN HONEY AND NORMAL LARVZ. Comb honey from a large number of sources has been examined and found to be quite uniformly sterile. The healthy larve likewise are usually sterile. BACTERIA UPON THE ADULT BEES. On the external part of the bee we again find only a few different species. Bacillus A, described as found upon the combs, is fre- quently isolated from the bee. Other species which are found fre- quently are described below. Bacterium cyaneus (Micrococcus cyaneus). Occurrence. —Isolated from the body of a healthy honey bee and from pollen. Gelatin colonies.—The colonies are lemon-yellow, with entire border, growth taking place readily on this medium. The superficial colonies, having well- defined border, are finely granular, and liquefy the medium within 8 to 6 days. Morphology.—Short oval rods 0.84 to 1.74 in length, 0.74 to O0.8u in thickness. Short involution forms are present. The rods occur singly, paired, and in clumps. No flagella have been demonstrated. Motility.—No motion has been demonstrated. Spores.—No spores have been deimenstrated. Gram’s stain.—The bacterium takes Gram’s stain. Oxygen requirements.—Aérobic. Bouillon.—At first a slight cloudiness appears, the medium becoming turbid in old cultures. A heavy yellowish-white, slightly viscid ring forms on the tube at the surface of the medium. The sediment, and sometimes the medium, show marked viscidity. Reaction alkaline. Glucose.—The growth of the cuiture is confined entirely to the open bulb, in which the medium becomes turbid. No gas is formed. Reaction alkaline, Lactose.—Reaction alkaline. Saccharose.—Reaction alkaline. Levulose.—Reaction alkaline. BACTERIA UPON THE ADULT BEES. - 17 Maltose.—Reaction alkaline. Mannite.—Reaction alkaline. Potato water.—Reaction alkaline. Agar slant.—On the surface of the agar there takes place an abundant growth, which is confined to the surface inoculated with the loop. The culture is fleshy, nonviscid, and lemon-yellow. It produces a soluble pigment that dif- fuses thru the agar, giving it a dark-pink color. Serum.—Luxuriant growth takes place, accompanied by liquefaction. Potato.—A lemon-yellow, fieshy, glistening growth spreads over the inclined surface of the potato. Milk.—Precipitation followed by slow liquefaction of the casein occurs: later the medium becomes alkaline and very viscid. Litmus milk.—The litmus is discharged and the casein is liquefied. Reaction alkaline. Gelatin.—Infundibuliform liquefaction soon begins, which is followed by stratiform liquefaction. The liquefied gelatin is turbid and viscid. Acid agar.—On this medium a moderate lemon-yellow growth is observed. Indol.—None could be observed. Nitrates.—No reduction of nitrates could be observed. Micrococcus C. Occurrence.—Isolated from the body of a healthy honey bee. Gelatin colonies—The surface colonies are round and _ slightly yellow. Liquefaction begins in from 2 to 4 days. The magnified colonies are finely granular, with sharply defined, entire border. Morphology.—Cocci, about O0.8u in diameter, occur in pairs and in small clusters. Motility.—Nonmotile. Spores.—Spores are apparently absent. Gram’s stain.—The coccus takes the Gram’s stain. Oxygen requirements.—Aérobic. Bouillon.—This medium becomes uniformly clouded in 24 hours after in- oculation, growth increases, and friable sediment forms. The liquid clears somewhat on standing. Reaction at first slightly acid; later returns to neutral. Glucose.—The medium in the bulb becomes cloudy, while that in the closed arm remains clear. White friable sediment forms in bend of tube. Reaction acid. No gas is formed. Lactose.—Reaction slowly becomes acid. Saccharose.—Reaction acid. Levulose.—Reaction acid. Maltose.—Reaction acid. Mannite.—Reaction acid. Potato water.—Reaction acid. Agar slant.—A grayish white, fleshy, nonviscid, glistening growth takes place along the inoculated surface. It does not spread, and retains a dis- tinct boundary. Serum.—A spreading growth takes place, accompanied by liquefaction. Potato.—A gray, fleshy, glistening, nonviscid growth forms over the entire cut surface of the potato. The potato is slightly discolored. Milk.—This medium becomes firmly coagulated and later the casein liquifies with the formation of a milky serum. 18 THE BACTERIA OF THE APIARY. Litmus milk.—In this medium coagulation takes place, accompanied by reduction of the litmus. Reaction slightly acid. Gelatin.—After a day or two infundibuliform liquefaction occurs, being followed by stratiform liquefaction; the liquefied gelatin is turbid. Growth below this portion is in the form of small spherical colonies. Acid agar.—-A white, fleshy, nonviscid growth is observed. Indol.—A trace was observed. Nitrates.—Reduced to nitrites. BACTERIA OF THE INTESTINE OF THE HEALTHY HONEY BEE. A great many investigations have been made in recent years on the bacteria found present in the intestines of vertebrates (4, 5, 6, 7, 8, 9), and striking similarities are noticed in the species found in many of them. In this investigation the intestinal contents of about 150 bees, mostly from one apiary, have been studied more or less thoroly. Several species which are found to be constant in many of the verte- brates are found in the intestine of the honey bee. Since the tem- perature of the bee approximates much of the time, especially when in the hive, that of the warm-blooded vnimals, many of the same species of bacteria inhabit the intestine of this insect as are found thriving in the same locality in man and other animals. snesod sooATIOIBY DIRE —|+|—|—|--|--|—| —|—} —] 4 oofeefe rf] —|— || —}—|—|—|— I tye ig fle} i— yey JAI eI Aloo] |9's oR aren hak Re J SOOATLOIBY DIRE =-|—|+|— —|—|— SN I a Fl a el te fel dO el a nl ef FL ce tl fl el oe ct rae“ vce! ||) 9 hoe eh enbiy ‘ony “sd al a oa wefrefoe|—eetest ttf ype Peete ie pb bib ioe PIAA i+] 3 eT O° GAGr Oe rine s soplookul “youg fe eee eh ll le lee ie feelers ele l |i i8* 9" a SNOLYSBSQNS * SP IE PE Sa FT MSH basta ea ON halted eye eats eal Go xed nce ed eds Poul redhead thf a ens eae fel fl Fon hl ON Me cea a 3 ota os of SN[[LloRg ST Ee ee ee a ee ee en en a oe Ut ree Gel ee Cl el Ul el ol Ol Gl ol Dl (Ul rl Dl GU: E20 Co SINS BIO[OYD “gq -90B1} Topuy |i I/II IIE IETF IF IAI IIRL IIE] Jai limi eid i@ [eee yee |e pe) i |i 48° 2° ri fel Uae Pe a ey SIUNULULOD I[09 “g "(3) JopuL |—-|AI— |i}! |] III IPI III EI 4lJH pl fm ime ie EPI JG PEPE E ye [yep] |e me" Gos eles owaee a VIVO HED V EE MOL Ee tet eT ee Ne Se lho lite (ha al be dfe SS | INTE PO MICH BL Se MM ae ae leat A ard ad aa fet dee eal eel) ADD ce Cg IL he a EN a q wnweyoRg ‘OOBIL TOPUT tefl eet elt cpp patel ei +l] | | +12 HIT IG [ell Pele 18 a Oe A fits eee RS ) SLODVOVOIOTIT —|—|+|—|—|—|—|-|-|-|- Bea ee ee oer re fe ted rel eet Ua (ant fale Cancer fue me fa far fae PS asl fst fn cles [need ci“) enV] Gets Pues Se a as snouvisd “oRg aha e een oe Ape fo — Ill l—ie [IIT Io JIRA erie Elim ie a q sny[loeg “elle=clferelPtteettaed ttm faelfere hard hay ne Sf Se eee teres pot qa keen lle ls et 9°T ase Rens SUBULIOJIPIO®B “JOVg *(g) snotroquosoun ‘gq |—|+ i i—|—|=- | PE ja ity ieee peice [Aid 8 Eyre) [pepe Fie E60 | fA SPC a V sny[loRg oe el ol le Mea ES 5 eI AS fe SAS lela Gar Cll a cede Markee een Vee ZERIT ECP RSHEISSSZ RCo cE SE sageueecroreiagezgss 2 | F Sg FIRIE|E/Sc/S/SS/FSISlsaiSis/SS(S/Si5 [R/S walZlSisiRieiGls/B ee /Bioa/z/25/e)a2| 2 mR Lea ag im = | Alo Sle |” yO SH [al fl =a fa ‘ u | ct | 6'| ~ = as *) e888) 2/2 mE 8/2 /8 19/8 "|" 2 “|2)s| BIR) 9)% S)B)P "SB/BR Se Bia B = S| ©, ; O19 . lA Si=ig a 5 leet Sel elo | hl te ie Salo| a w ® als . @ |S | ola BIOle ‘ = OQ |= | 2 elalo|/Si sie Fy | iq?) ich | 016] By 5 ct ey 1a e\Q\Bi 2! |. Ml a “5 =| Salen. (| MOP GcIK CIRC a earls le se lleos | de Q| 2 ® ™M Seca 8 | Sa alba) ce, Vel el alts “SY IVULOY et CH o| ro) el ee lo KS a ‘satoodg ‘ ’ ° r ~ eh oO jo) || hae Se n uoTonpoid ploy uoronpord sey ® Z Q 5 : g S 5 5 ; Caton Bye a S$, a UT SUOTSUDTHAL(T Eas eel aan 9 ‘sorjrodoid oTruletoorg, ‘Soin BOT [BINI[TNO ‘ASOLOISAY A “ASoloy dso] *ASO[LOTA | -soord SuLOs -910F OY} UL pasoptsuoo SUISTUVG.10-O.10TUL Of} JO SUOTIATAIOSOP OY} OZLIVUTUINS 0} DATOS TTA 9]qQ"I SUTMOT[OF OUT, oe) NA ‘AUVICV AHL NI LNASAUd ATIVWUON SWSINVOUO-OMOIN HO NOILVTIOEVIL BIBLIOGRAPHY TO PART I, 29 SUMMARY TO PART I. The results of the study of the bacteria found normally in the aplary may be briefly summarized as follows: (1) The temperature of the hive approximates that of warm- blooded animals. (2) Upon adult bees and upon the comb there occurs quite con- stantly a species of bacteria which we refer to in this paper as Bacillus A, and which, it is believed, is the organism that some workers have confused with Bacillus al vei, the cause of European foul brood (p. 33). (3) There occurs very constantly in the pollen and intestine of adult bees a species here referred to as Bacillus B. (4) From the combs Bacterium cyaneus, Saccharomyces roseus, and a Micrococcus referred to here as J/icrococcus C, have been iso- lated and studied. (5) Honey from a healthy hive is, as a rule, sterile. (6) The normal larve are, as a rule, sterile. (7) There is an anaérobe found quite constantly in the intestine of the healthy honey bee. It is referred to in this paper as Bacterium D. (8) From the intestine there have been isolated and studied the following micro-organisms: Bacillus cloace, Bacillus coli communis, Bacillus cholere suis, Bacillus subgastricus, Bacterium mycoides, Pseudomonas fluorescens liquefaciens, and two referred to as Bacillus E, and Saccharomyces F. Others less frequently present ae been isolated, but not studied. (9) In two samples of brood with unknown disease there was found a species of yeast plant here referred to as Saccharomyces G. BIBLIOGRAPHY TO PART I. ti . FULLER, Gro. W., and JoHnson, Geo. A. On the Differentiation and Distribu- tion of Water Bacteria. | Schoharie COmMbiyrss se 7 = ok eee Bacillus alvei. IN Pea eSi a hom SEs cre eune: 292s Schoharie COMliay 2 4 se ta eee oes Bacillus alvei. NEM ANV GSES ee eee Pune -2 9; eoehoharie COUMLY se ee. LS Sess eck Bacillus alvei. NEWER SVCESts oe cee toe ea ee June. 297 Schoharie’ Coumbly:) sssce ses 8s eee Bacillus alvet. NSA VVESES ak tae eae ily oo schoharre Conmty cos 22 oo © eee be ee Bacillus alvei. NZD SWiest=25. = Ie we eS Ae lyes ioe SCH OMATMIeC OUMGYS 2 Hel ou ee Soe Se Bacillus alvet. We, DE NVeS tie cate Sos sane 40 eT yea] el O MATCH COUN LY een on ee ere Bacillus alvet. NED SNUESU oo eset, 2 SSeS: Jaly 7100); Monteomery County 222 = 222 252 8 Se Bacillus alvet. Na DE Wests once cree July 40s: Schoharie: County Ae se) 2a eseee teres | Bacillus alvei. INCREAS ee ce Ce Fe Se July -10.|: Schoharie’ County 2:22.02. 5 --ek se. ee | Bacillus alvei. NE DAW est: ic ea Pe Sots July 104 Schoharie Wounty +. 2ess2s- kee ae | Bacillus alvei. Nea WES wae se acto eC es Le July -t5<|LSehonarie!Coumtysss.02 22 ees oe oe eee | Bacillus alvet. NESS WiESt et ons str eons Julyed5s =—Montzomeny County. s06s oases. eee | Bacillus alvet. OAV CS he ehais Soa a es Julye22q GsevohameCoumtyes > oea as eens | Bacillus alvet. NE NVICS ES = so ees ase Ply 22 =| Schohnrie Couimtyen coe eee es aces ee | Bacillus alvet. Nee CSts2e - =h 2c loos een e ce July 22 4: Sehonarie-Counmty--n-0 see tees eee ee Bacillus alvet. NENW esis eee ee July a0. | ssehOhare County. cass sk ees --Se- te ee Bacillus alvet. BE Wes ee ao en. ce POL ya SOM tC ONATIC COMMU a= ste Geet sate eee Bacillus alvet. NEG VieR ies ese | SULLY, 10" |: Greene? COUMLY. . 642) see eae oe eee oe Bacillus alvei. ee OWVERE SS ees tan ae Se f duly 80%) Al pany ACOUMUGY 2.76) Sota cee eee os | Bacillus alvei. IS WES po 2s oer a og ily 50" | Greene; COUunby so heee eka ae oe ee Bacillus alvet. Ty UR DAN es es eee eee lar Aue. 200 Green ee Oung ye sso oes ee eee ae Cerone Bacillus alvei. Nie ONVEST AAA Sse oe nk oot Se (eATIS-& 20: | Greene COURLY sacses te Poe 402 cee eee | Bacillus alvei. The above table shows that Bacillus alvei was present in each speci- men of European foul brood received. Frequently pure cultures of this species were obtained from dead larve, but with it sometimes were associated other rod-shaped bacteria of different species. In 1904 the work upon bee diseases was confined principally to the diagnosis of the diseased brood sent in and a further study of the organisms found. Bacillus alvei was found in a large number of 36 THE BACTERIA OF THE APIARY. samples received from New York State and in some received from Pennsylvania. | Bacillus alvei. Occurrence.—This bacillus was found in all samples of European foul brood examined. Morphology.—The bacillus is a motile, rod-shaped organism, occurring singly and in pairs, and varying when taken from the surface of agar from 1.2u to 3.9u in length, and from 0.54 to 0.7u in width. Involution forms are some- times present. Spores are produced and occupy an intermediate position in the organism. They are oval and vary from 1.54 to 2u in length and from 0.74 to lu in breadth; they exhibit polar germination. The few flagella are arranged peritrichic. Oxygen requirements.—This bacillus is a facultative anaérobe which grows at room temperature, but better at 37° C. Bouillon—The medium becomes uniformly clouded in 24 hours; later it shows a tendency to clear by a settling of the organisms. A somewhat viscid sediment is thus formed in the bottom of the tube. In older cultures a slightly gray band of growth adheres to the glass at the surface of the me- dium. The acidity is at first slightly increased, and a pellicle is sometimes formed. Glucose.—The medium in both branches of the fermentation tube becomes uniformly clouded. Gas is not formed. Reaction acid. Lactose.—The medium becomes uniformly clouded in both branches of the fermentation tube, but the cloudiness is not so marked as when glucose is used. The acidity is slightly increased, as shown by phenolphthalein. No gas is formed. Saccharose.—The bouillon in this case also becomes clouded in both arms. A heavier growth is observed than when lactose is used, but less than when glucose is used. Acidity is slightly increased. Gas is not formed. Agar plates.—Small, grayish, circular colonies form in 24 hours. When many are on the plate, they do not exceed 2 millimeters in diameter. Under low magnification they appear granular, with no definite margin. When fewer colonies are on the plate, the granular center of the colony is surrounded by pumerous smaller but similar growths. The organism has a tendency to grow into the medium rather than upon the surface. Sometimes, however, when there are but a few colonies on the plate a thin, transparent growth spreads rapidly over the surface. Later it takes on a brown tint. Agar slant.—A gray layer spreads over the surface in 24 hours, which later takes on a slightly brown color. A strong, slightly viscid growth occurs in the condensation water. Acid agar.—Growth takes place with the reactions varying from neutral to 3.5 to phenolphthalein. Serum.—A slightly raised growth which is confined quite closely to the line of inoculation appears on the surface of solidified serum. Potato.—On this medium the bacillus grows rather slowly at first, but after 5 or 4 days a milky growth is observed, which increases until a luxuriant growth is formed, which varies from a lemon-yellow to a gray color, and which later becomes tinted with brown. . . Milk.—Acidity is inereased after inoculation. Coagulation usually takes place after the third day. Litmus milk.—Much of the blue color is discharged, leaving the coagulated milk of a light brown. “I INOCULATION EXPERIMENTS WITH BACILLUS ALVEI. 3 Gelatin colonies.—Gelatin is a medium in which it develops slowly. The col- ony becomes very irregular in outline, owing to thread-like outgrowths which take place in curves from its border. Growth is better when 5 per cent glycerin is added. From the small, white, spherical colonies which form along the line of puncture gray, thread-like growths shoot out thru the medium. In about 2 months the gelatin is changed to a thick liquid, holding gray flocculent masses of organisms which gradually settle, forming a strong, slightly viscid sediment. Indol.—In old cultures a decided indol reaction is obtained. Power to resist disinfectants.—Preliminary observations give the following results: The spore form resists drying for a considerable time. Spores which have been drying for 1 year germinate promptly when introduced into bouillon. The vegetative form: One per cent carbolic acid kills in 10 minutes; 3 per cent carbolic acid kills in 2 minutes; mercuric chlorid solution, 1 to 1,000. kills in 1 minute; mercuric chlorid solution, 1 to 2,000, kills in 2 minutes. Spore form.—Mercurie chlorid, 1 to 1,000, kills in 30 minutes. Pathogenesis in vertebrates.—Inoculations into guinea pigs and frogs have not proven this organism to be pathogenic to these animals. Inoculation Experiments. That part of the investigation which involves the producing of the disease experimentally by inoculating with pure cultures of the organism is usually the most difficult one. Very rarely indeed is one able to produce the disease with symptoms closely simulating those found in nature. The experimental production of a disease involves many variable factors, such as attenuation of the organism, methods of inoculation, resistance of the host, and the immediate environment. On August 4, 1902, we inoculated a hive containing nothing but healthy brood, free from bacteria, by feeding with sirup (sugar and water in equal parts) to which was added the growth from the sur- face of the plate cultures containing spores and bouillon cultures of Bacillus alvei. Similar feedings were given to these bees from one to three times a week until September 28, but symptoms of foul brood did not develop. On August 6 cultures were made from a few of the hive larve. They were found to contain the bacilli. Inoculation experiments were again made in 1903. Because of a failure to produce a diseased condition with cultures of Bacillus alvei in the experiment of 1902, the variable factors above mentioned were carefully considered in the experiment of this year. The inocula- tions were made when climatic conditions were such as seemed to favor the ravages of the disease in the apiaries; namely, low tem- perature, dampness, and cloudiness. A colony of black bees was used, as they were almost universally considered more susceptible. Cultures of Bacillus alvei were freshly isolated from foul-brood specimens and kept in stock on bee-larve agar (described under American foul brood, pp. 41-42). All cultures were incubated at 84° C., which temperature is observed to be slightly below that of the hive. The spore form of Bacillus alvei was used. Inoculations were made in different ways. A diseased condition 38 THE BACTERIA OF THE APIARY., appeared in the hive when the following method was used: The agar from plates on which the culture was grown was finely crusht and mixt with sterile sirup. ) The pollen stored in the cells of the foul-brood combs contains many of these infecting organisms. CONCLUSIONS. 45 (c) The honey stored in brood combs infected with this disease has been found to contain a few bacilli of this species. (d) The surface of combs, frames, and hives may be contaminated. (e) The wings, head, legs, thorax, abdomen, and intestinal con- tents of adult bees were found to be contaminated. with Bacillus alvei. (7) Bacillus alvet may appear in cultures made from the ovary of queens from European foul-brood colonies, but the presence of this species suggests contamination from the body of the queen while the cultures are being made and has no special significance. (4) The disease which seems to be most widespread in the United States we have called American foul brood, and the organism which has been found constantly present in the disease we have called Bacillus larve. This disorder was thought by many in this country and other countries as well to be the foul brood described by Cheshire and Cheyne, but such is not the case. (5) From the nature of American foul brood it is thought that the organism has a similar distribution to that of Bacillus alvei. (6) It appears that European foul brood was erroneously called “New York bee disease ” or “ black brood” by Dr. Wm. R. Howard in 1900. (7) There is a diseased condition affecting the brood of bees which is being called by the bee keepers * pickle brood.” No conclusion can be drawn from the investigation so far as to the cause of the disease. (8) Aspergillus pollinis, ascribed by Dr. William R. Howard as the cause of pickle brood, has not been found in this investigation and is not believed by the author to have any etiological relation to the so-called “ pickle brood.” (9) Palsy or paralysis 1s a diseased condition of the adult bees. No conclusion can yet be drawn as to its cause. (10) Formaldehyde gas as ordinarily used in the apiaries is insufli- cient to insure complete disinfection. CONCLUSIONS. In a paragraph the author wishes, if possible, to present the status of the bee diseases in this country. It should be remembered, firstly, that “black brood” can now be dropt from our vocabulary, and probably does not exist; secondly, that the term “ foul brood” was being applied to two distinct diseases. One of these diseases we now refer to as European foul brood, because it first received a scientific study from a European investigator. We refer to the other disease as American foul brood, because it was first studied scientifically in America. There is one more disorder in the brood of bees which has attracted considerable attention—the so-called “pickle brood.” There are, then, these three principal diseases: European foul brood, American foul brood, and the so-called “ pickle brood.” 12. 26. 21. 28. 29. 50. ol. o2. 30. 4. or oo. 36. ol. 38. oo. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. THE BACTERIA OF THE APIARY. BIBLIOGRAPHY TO PART II. ARISTOTELES. < Historia Animalium, Book IX, Ch. 27. ScoiracH.