Albert Fl. Mann Library Cornell University

Dr. Roger a. Morse

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SUREAU OF ENTOMOWay.

L. O. HowABD, Entomologist and Ohief of Bureau.

C. L. Mablatt, Entomologist and Acting Chief in absence Of Chief.

R. S. Ci.ift6n, Chief Clerk.

F. H. Chittenden, in charge of breeding experiments.^

A. D. Hopkins, in charge of forest, insect investigation^.

W. D. IIuNTEB, in charge of cotton holl weevil investigations.

F. M. Webster, in charge of cereal and forage-plant ijisect investigations.

A. L. QuAiNfANCE, in charge of deciduous-fruit insect investigations.

J)., M. UooERS, in charge of gipsy and hrown-tail moth work.

A. W. MoERiix, engaged in white fly investigations. E. S. G. Titus, in charg^ of gipsy moth laboratory. C. J. GiLTJss, engaged in silk Investigations.

R. P. CuKEiE , assistant in charge of, editorial^ work. Mabel Colcoed, librarian.

Apictjltueal Investigations. .

Feank Benton, in charge (absent).

B. F. Phillips, acting in charge. '

J. M. Rankin, in charge of apicultural station, Chico, Cfit. Jessie E. ^abks, apicultural clerk.

Technical Series, No. 14.

U. S. DEPARTMENT OF AGRICULTURE,

L. 0. HOWARD, Entomologist and Chief of Bureau.

THE

BACTERIA OF THE APIARY,

WITH SPECIAL REFEREI^CE TO BEE DISEASES.

GERSHOM FRANKLIN WHITE, Ph. D.,

Expert in Animal Bacteriology, Biochemic Division, Bureau of Animal Industry.

Issued November 6, 1906.

WASHINGTON:

GOVERNMENT PRINTING OFFICE. 1906.

LEHER OF transmittal;

U. S. Department or Agricultttee,

Bureau of Entomology, Washington, D. G., September 2Ji., 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. ^Vhite, 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 Entomolo^ 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.

EespectfuUy, L. O. Howard,

Entomologist and Chief of Bureau.

Hon. James Wilson,

Secretary of Agriculture.

PREFACE

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 it 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. K. 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.

E. F. Phillips, Acting in Charge of Apiculture.

CONTENTS.

Page.

Introduction 7

Technique 7

Obtaining material for study 7

Obtnining^ cultures 7

Differentiation and identification of bacteria 9

Tbe cultures which are described 9

Morphology, staining properties, and oxygen requirements, with sug- gestions on variations 9

Media employed and suggestions as to the description of cultures 10

PART I. BACTERIA OF THE NORMAL APIARY.

Bacteria from the combs 13

Bacteria from pollen 15

Bacteria in honey and normal larvae 16

Bacteria upon the adult bees 16

Bacteria of the intestine of the healthy honey bee 18

Saccharomyces and fungi 25

Tabulation of micro-organisms normally present in the apiary 28

Summary to Part I 29

Bibliography to Part I 29

PART II. THE DISEASES OF BEES.

Brief history ^ 30

The term "foul brood" as hitherto applied 31

European foul brood (foul brood of Cheyne) 32

Symptoms 32

Confusion regarding foul brood in America 33

The present investigation 34

' Bacillus alvei 36

Inoculation experiments 37

Distribution of Bacillus alvei in infected hives 38

Experiments with formaldehyde gas . 39

American foul brood 40

Symptoms 40

The present investigation 41

Bacillus larval 42

The so-called " picljle brood" 43

The so-called " blacic brood" 43

Palsy or paralysis 44

Summary to Part II 44

Conclusions 45

Bibliography to Part. II 46

Index 47

6

THE BACTERIA OF THE APIARY WITH SPECIAL REFERENCE TO BEE DISEASES.

INTBODTJCTION.

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- plication 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 larvae 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 BACTEEIA OF THE APIAEY.

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.

{h) From pollen. The 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 larvm. 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 hee. In studying the adult bee, a small piece of blotting paper wet with chloroform 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 anaerobe, 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 PKOPEETIES, ETC. 9

Differentiation and rdentification 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-teuths of tbe 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- ience 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 Bequirements, 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-

o 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 BACTEEIA OF THE APIAEY.

corded dimensions should always be accompanied by facts concerning the medium, age, and temperature of incubation. 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.

(5) 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. Loeffler'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 nonmotile 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 froperties. Basic carbol-fuchsin was the stain used 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.

(/) 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 tlie Description of Cultures.

{a) Bouillon. All bouillon used was made from beef (meat 1 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 -j-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.

(6) Sugar-free houillon. 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 bouillons. Five 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 Avas fer-

MEDIA EMPLOYED, ETC. 11

merited 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) Rcaetion 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.

(/) Agar. One per cent agar is used. The description of the growth on this medium is made from the appearance as seen 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.

(A) 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.

(«') 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.

12 THE BACTEKIA OF THE APIAEY.

(j) Potato water.— To potatoes sliced very thin is added an equal amount of water by weight and the mixture is then boiled. This is btrained 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.

(k) Milk.— If a micro-organism breaks up glucose with the forma- tion of gas, a fermentation tube of milk is inoculated with the culture; if not, straight tubes are used. Separator milk is used. The coagulation of the casein with or without liquefaction 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 liquefaction 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.

(w) Indol. The cultures are allowed to grow in sugar-free pep- tonized bouillon for 3 to 5 days, and are tested with potassium nitrite (KNOj) and sulfuric acid (H,S04) 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).

{o) 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

BACTEEIA PROM THE COMBS, 13

of the hives, combs, honey, pollen, larvae, and adult bees was begun, to determine the bacteria normally preseftt. 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 PBOM 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.

Oelatin 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 grauu lar, arranged singly, in pairs, and in chains. The flagella are distributed over the body. The rods measure from Sn to 4/i in length, and from 0.9/4 to L2|U in thickness.

Motility. The bacUli 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. Aerobic and facultatively anaerobic.

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 BACTEEIA OF THE APIARY.

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 alkaline.

Agar slant. A luxuriant growth takes place on this medium. The growth gradually 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 within 24 hours, followed by a gradual peptonization. Reduction of the litmus occurs rapidly, leaving 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 radiately 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 1.6|ti, thickness O.Sfi. They stain uniformly with carbol-fuchsin. Flagella are apparently ab- sent.

Motility. No motility has been observed in any medium.

Spores. Spores are apparently absent.

Gram's stain. The bacteria are decolorized by Gram's method.

Oxygen requirements. Facultatively anaerobic.

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.

BAOTEEIA FROM POLLEN. ' 15

Maltose. Reaction acid.

Mannite. Reaction acid.

Potato water. Reaction acid.

Agar slant. A moderate, gray, glistening growth, confined to tlie 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 taljes place.

Potato. A gray growth covers the inoculated surface.

Milk. Heat causes a ready coagulation of the casein. Reaction acid.

I/itmus 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 PROM 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.

dclatin 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-fuchsln, and are 1/i to 2|H in length. Few short involu- tion forms occur.

Motility. The bacilli are actively motile in young cultures.

Spores. No spores have been observed.

Oram's stam.— .The bacilli are decolorized by Gram's stain.

Oxygen requirements. Facultatively anaerobic.

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

16 THE BACTERIA OF THE APIAKY.

Mannite. Reaction slightly acid, later alkaline.

Agar slant. A moderate, slightly yellow, nonviscid glistening gi:pwth 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.

BACTEEIA IN HONEY AND NORMAL IiAIlV.ai.

Comb honey from a large number of sources has been examined and found to be quite uniformly sterile. The healthy larvae 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 3 to 6 days.

Morphology. Short oval rods 0.8/n to l.T/j, in length, O.Y/i to 0.8|U 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 demonstrated. ' Gram's stain. The bacterium takes Gram's stain.

Oxygen requirements. Aerobic.

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 culture 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.

BACTEEIA UPON THE ADULT BEES. 17

Maltose. Reaction allialino.

Mannitc. Reaction allialine.

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 tleshy, nonviscid, and lemon-yellow. It produces a soluble pigment that dif- fuses thru the agar, giving it a dark-pink color.

Scniiii. Luxuriant growth takes place, lurompanled 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 iiiillc. 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 mediimi a moderate lemon-yellow growth is observed.

Indol. None could be observed.

Xitrates. No reduction of nitrates could be observed.

Micrococcus C.

Occurrence. Isolated from the body of a healthy honey bee.

Gelatin colonics. 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 0.8|U in diameter, occur in pairs and in small clusters.

Motility. Nonmotile.

Spores. Spores are apparently absent.

Grants stain. The coccus takes the Gram's stain.

Oxygen requirements. Aerobic.

Bouillon. ^This medium becomes uniformly clouded in 24 hours after in- oculation, growth increases, and friable sediment forms. The liquid clears somewThat 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 BACTEEIA OF THE APIARY.

Litmus milJc. In this medium coagulation takes place, accompanied bj 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.

y Urates. 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, Y, 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 animals, 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. A stained cover-glass preparation made directly from a healthy adult field bee reveals, almost without exception, a multitude of bacteria.

In a study of the bacterial flora stress has been placed upon the different species which were found to be more or less constant, rather than upon the actual number of bacteria oi- species in any quantity of material from a single bee. From the observations which have been made, it appears that the number of species in any individual is comparatively small, but the number of bacteria is in many cases very large. Sometimes, however, the plates show very few colonies, while cover-glass preparations show a very large number of bacteria. These organisms are probably the anaerobe, which is quite constant, as shown by cultures made direct from the intestine into glucose agar (Liborius's method).

When a loopful of the material from the intestine was used for the inoculation, the following data give the approximate findings :

Bee No. 1, 300 to 400 yellow colonies, probably alilie.

Bee No. 2, a few colonies of fungi only.

Bee No. 3, 500 colonies, mostly yeast.

Bee No. 4, 100 or more colon-like colonies.

Bee No. 5, 2,000 or more, mostly yellow.

Bee No. 6, 20 or more colonies, mostly yeasts.

Bee No. 8, 400 or more yellow colonies.

Bee No. 9, 30 yeasts with a few fungi.

Bee No. 10, 50 yeast colonies with a few fungi.

Bee No. 11, no growth.

Bee No. 12, 300 colonies, slightly yellow.

BACTERIA OP THE INTESTINE. 19

Bee No. 13, 2,000 or more gray colonies.

Bee No. 14, yeast colonies and a few colonies of bacteria showing ground- glass appearance. Bee No. 15, 2,000 or more colon-like colonies {B. cloaca;).

The following are the species which have been found to be most constant. The reader is referred also to the description of the yeast plant found very frequently in the intestine of the normal honey bee, described under " Saccharomyces and fungi."

Bacterium S.

Occurrence. Frequent in the intestine of the healthy honey bee.

Agar colony. Deep colonies when magnified are coarsely granular, showing a dark brown center, with a thin and ill-deflned border.

Morphology. A preparation made from a young culture taken from a glu- cose fermentation tube shows rods with rounded ends, occurring singly and in pairs', staining easily and uniformly with carbol-fuchsin, and measuring 0.7^ to 1.5/» in length and 0'.5/i to 0.7/t in thickness.

Motility. No motility could be observed.

Spores. No spores could be demonstrated in young cultures. In old cultures their presence is questionable.

Oxygen requirements. Strictly anaerobic.

Bouillon. In straight tubes no growth occurs.

Olucose. A moderate cloudiness can be, seen in the closed arm, while the open bulb remains clear. No gas is produced. Reaction about neutral.

Glucose agar (Liborius's method). Growth is rather slow. After 3 days a moderate growth may be observed; later, if cultures have recently been iso- lated from the bee's intestine, the growth imparts to the medium a diffused haziness or cloudiness. After many generations the culture loses this property.

Glucose gelatin (Liborius's method). Very slow growtji occurs in the depth of the mediiuu. No liquefaction takes place. '

Bacillus cloacae.

Occurrence. Found in the intestine of a large number of healthy honey bees.

Oelatin colonies. Superficial colonies are thin and blue to gray In color ; deep colonies, brown, regular, granular, and spherical to lenticular.

Agar colonies. Superficial colonies are partially opaque, brown, finely granu- lar, with well-defined margin ; deep colonies are regular, spherical, or lenticular, with well-defined margin.

Morphology. The rods from 24-hour agar cultures have rounded ends, vary- ing in length from V to 2 it and in width from 0.7/i to 0.9 /i». They are usually found singly or in pairs. Involution forms are not uncommon. With carbol- fuchsin they