The bacteria of the apiary

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The bacteria of the apiary
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Table of Contents
    Front Cover
        Page i
        Page ii
        Page iii
        Page iv
    Title Page
        Page 1
    Letter of transmittal
        Page 2
    Preface
        Page 3
        Page 4
    Table of Contents
        Page 5
        Page 6
    Introduction and technique
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
    Part I. Bacteria of the normal apiary
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
    Part II. The diseases of bees
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
    Index
        Page 47
        Page 48
        Page 49
        Page 50
    Back Cover
        Page 51
        Page 52
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TECHNICAL SERIES, No. 14.

U. S. DEPARTMENT OF AGRICULTURE,
/
BUREAIT OF E TrOM1OLO G-.
L. 0. HOWARD, Entomologist and Chief of Biureau.





THE



BACTERIA OF THE APIARY,


WITH SPECIAL REFERENCE TO

BEE DISEASES.


BY

GERSHOM FRANKLIN WHITE, PH. D.,
Expert in Animal Bacteriology, Biochemic Dirisioa, Bu rea u of Animal Induistry.


ISSUED NOVEMBER 6, 1906.


WASHINGTON:
GOVERNMENT PRINTING OFFICE.
1906.














LETTER OF TRANSMITTAL.


U. S. DEPARTMENT OF AGRICULTURE,
BUREAU OF ENTOMOLOGY,
JWashington, D. C., September 24, 1906.
SIR: I have the honor to transmit the manuscript of a paper c
the bacteria of the apiary, with special reference to bee diseases, I
Dr. G. F. White, expert in animal bacteriology in the Biochem
Division of the Bureau of Animal Industry. This paper wias pr
pared by Doctor White.as a thesis in part fulfilment of the requir
ments for the degree of doctor of philosophy, at Cornell Universit
in June, 1905. The Bureau of Entomology considers itself fort
nate in obtaining it for publication, since in this way a wider disti
bution can be made than would be possible were it published in
journal devoted exclusively to bacteriological investigations. It
hoped that the publication of these facts may help to clear up t]
confusion which now exists concerning the causes of the two mo
common diseases of the brood of bees. I recommend that the man,
script be published as Technical Series, No. 14, of this Bureau.
Doctor White wishes to acknowledge his indebtedness to D
Veranus A. Moore, professor of comparative pathology and ba
teriology of Cornell University, under whose direction this woi
was done; to Dr. E. F. Phillips, acting in charge of apicultui
Bureau of Entomology, United States Department of Agricultur
for encouragement and assistance in the preparation of this mani
script; and to Messrs. Mortimer Stevens, Charles Stewart, N. I
West, and W. D. Wright, bee inspectors of the State of New Yor
for their interest in the work.
Respectfully, L. 0. 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 wotld 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 alcei 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





PREFACE.


but since Bacillus alvei is not found in this disease it obviously is n
the same disease as that described by Cheyne. It would be well-nil
impossible, however, to change the name of this disease, and any effo
in that direction would merely result in complicating laws now in for,
which control the infectious diseases of bees and would serve no go(
purpose. This disease is here designated "American foul brood
These names have been chosen only after consultation with some
the leading bee keepers of the United States, and these distinguishii
terms were chosen by the majority of those consulted as indication
the place in which the diseases were first investigated in a thord
scientific manner. Both diseases are found in Europe, as well as.
America, so that the names indicate nothing concerning the ge
graphical distribution of the maladies.
Strangely enough, certain writers for our American apicultur
papers have seen fit to take exception to some of the statements ma(
in this paper without having first found out the reasons for the d
cisions herein published. Apiculture will not be advanced to ai
appreciable extent by such eagerness to rush into print, especial
when there is not a semblance of scientific investigation back of tl
criticism.
E. F. PHILLIPS,
Acting in Charge of Apiculture.













CONTENTS.


Page.
Introduction ------------------------------------------------------- 7
Technique -----------------------------------------------------
Obtaining material for study ---------------------------------
Obtaining cultures ------------------------------------------
Differentiation and identification of bacteria ------------------------9
The 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 11. 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 iii Americ -----------------------33
The present investigation ------------------------------------------"4
Bacillis alrei ----------------------------------------------------36
Inoculation experiments -------------------------------------------37
Distribution of Bacillhs alrei in infected hives 38
Experiments with formaldehyde gas ------------------------------39
American foul brood --------------------------------------------------40
Symptoms -------------------------------------------------------40
The present investigation ------------------------------------------ 41
Bacills lare ---------------------------------------------------- 42
The so-called '"pickle brood "------------------------------------------- 43
The so-called "black brood --------------------------------------------43
Palsy or paralysis ----------------------------------------------------44
Summary to Part II --------------------------------------------------44
Conclusions --------------------------------------------------------- 45
Bibliography to Part II -----------------------------------------------46
Index ----------------------------------------------------------------47
5


































































t












THE BACTERIA 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 cncerning 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 larvve 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





THE BACTERIA OF THE APIARY.


comes in loaded with pollen and honey. After the victim alights
the entrance board, by the aid of a pair of forceps, before she disa]
pears within, one can easily lodge her safely in a petri dish. It i
however, an advantage to study the young adult bees as well as t1
older ones, and if young ones are desired they may be taken fro
the combs or from the front of the hive, near the entrance.

Obtaining Cultures.

(a) From cornbs.-With sterile forceps small pieces of the con
are put directly into gelatin or agar for plates or incubated in boui
Ion for 24 hours and then plated. Growing in bouillon and pla
ing on gelatin is usually preferable.
(b) From polhl;n.-The same technique is used as for combs, bi
the direct inoculation of gelatin tubes for plates is generally pr
ferable.
(c) From honvey.-With sterile loops honey is taken from uncapp(
and capped cells. The caps are removed with sterile forceps and tI
honey is plated (liroctly on glatin or agar. Bouillon tubes are ii
oculated also with varying quantities of the honey.
(d) From lar,;.-The larva is carefully removed to a sterile disl
and with sterile scissors the body is opened and the contents plat(
directly, or bouillon cultures are first made and later plated, if
growth appears.
(c) From parts of the adult bee.IIn studying the adult bee,
small piece of blotting paper wet with chloroform is slipt undi
the cover of the petri dish in which the insects have been placed, an
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 ti
abdomen, the intestine being removed, is placed in bouillon and, aftl
24 hours incubation, plated. preferably on gelatin.
WNhen it is desired to make a study of the bacteria of the intestin
the intestinal tract is removed and studied as follows: The bee
flamed and held in sterile forceps. With another sterile pair of fo
ceps the tip of the abdomen is seized and, by pulling gently, the ti
and the entire intestine are easily removed. This can then be plat(
directly. If gelatin, which is preferable, is used, the-intestine itse
must not be left in the gelatin or the medium will become liquefiE
by the presence of the tissue. If one desires to obtain cultures of t1
ana~robe, which is quite common in the intestine, it is most easi]
obtained in pure culture by the use of the deep glucose agar (Liboriuc
method). Cover glass preparations made direct from the walls (
the intestine or its contents give one some idea of the great number (
bacteria frequently present.





MORPHOLOGY, STAINING PROPERTIES, ETC.


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 experilnenta-
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 niav 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. 1-le 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 "iipartively
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 complete descriptions
which can be found make it impossible to identify nany 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. Froin 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).a

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.






THE BACTERIA OF THE APIARY.


corded dimensions should always be accompanied by facts con
the medium, age, and temperature of incubation. The me
ments recorded in this paper were all taken of organisms in prepa
tions made from a 24-hour agar culture stained with carbol-fuch!
The involution forms are not reckoned in the results.
(b) Spores.-The presence of spores was determined in each C
by staining the various cultures at different ages. A check was m:
on their presence by means of the thermal death point.
(c) FafgcU(a.-Loeffler's method, as modified by Johnson
Mack, was used for staining the flagella (2).
(d) ifoti/ity.-Motility may be present in cultures when first
lated, but after artificial cultivation appear to be entirely lost.
reverse of this also may be noted. No cultures should be record
as nonmotile until cultures on various media at different temperate
and of different ages shall have been studied. Hanging-drop prep
tions were .made from cultures on agar and bouillon, both incuba
and not incubated, and on gelatin.
(e) A'taI/l! proprbc'c.-Basic carbol-fuchsin was the stain u
almost exclusively. In the use of Gram's staining method, carbi
gentian violet (.5 per cent carbolic acid 20 parts, saturated alcoh
solution gential violet 2 parts) was applied to a cover-glass prepa
tion from a 24-hour culture on arI.i for 5 minutes, placed in Lug
solution 2 minutes, and )laced. without insing, ii 95 per cent alco
for 15 niiniutes, removed, washt in water, and allowed to dry.
(f) Oxyge rcq? iremeuts.-I)eteminationswere made by
serving whether a growth took place in the closed or open arm
both, of the fermentation tube containing glucose bouillon.

Media Employed and Suggestions as to the Description of Cultures.

(a) Bouillo.-All bouillon used was made from beef (mea
part, water 2 parts), to which infusion 1 per cent Witte's pepton
siccum and one-half per cent sodium chlorid were added. The
action of the solution was then determined by titrating, and m
+1.5 to phenolphthalein.
In describing a culture growing in bouillon as a medium, th
is usually a more extended description given than in the case
sugar and sugar-free bouillons, since cultures in these media do
differ materially in gross appearance from those observed in
plain bouillon.
(b) Sugar-free bouillon.-This bouillon is made free from sui
by the use of B. coli cornmunis, after which peptone and sodi-
chlorid (NaCI) were added as in bouillon.
(c) Sugar bouillons.-Five different sugars-glucose, lactose, s
charose, levulose, and maltose, as well as mannite-were used in
study. If a 1-per-cent solution of glucose in plain bouillon was f


10






MEDIA EMPLOYED, ETC.'


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 prodctw.n of ra.-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 (11). 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.
(f) 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.
(h) Serum.-The serum used is taken from the horse, sterilized at
550 C. and congealed at 800 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.
(i) 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.





THE BACTERIA OF THE APIARY.


(j) Potato water.-To potatoes sliced very thin is added an equal
amount of -water by weight and the mixture is then boiled. This i
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.
(k) Mi k.-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 plhenolnena appear.
(1) Litmas m;il.The reaction as shown by the litmus and the dis-
charging of the color are the chief points observed.
(n) atiu.-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 apl)roxiliiate.
(n ) Indol.-The cultures are allowed to grow in sugar-free pep-
tonized bouillon for 3 to .5 days, an(1 are tested with potassium nitrite
(KNO.) and sulfuric acid (H2SO4) after the ring method. Too
much stress may be 1)laced upon the ability of an organism to form
indol. This character has been shown to be a somewhat transient
one (3).
(o) Red~wtion 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


12






BACTERIA FROM THE COMBS.


of the hives, combs, honey, pollen, larvw, 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 anyx 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-organisins were being
subjected at the tine, 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, see-s 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 fond on conibs, but inas-
much as they appear most frequently from other sources they are
described there. One species of Saccharolnmces from the comb, also,
is described under the heading Saccharoniyces and fungi."

Bacillus A.

(B. Itescnterics ?)

Occurrence.-Found very frequently on combs, on scrapings from hives, aind
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 4ju in length, and from 0.9A to 1.2g
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 Grain's stain.
Oxygen requirements.-Arobic 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 BACTERIA OF THE APIARY.


heavy white sediment to form in the bend of the tube. The reaction is at I
slightly acid, but subsequently becomes alkaline. No gas is formed.
Lactose.-lReaction alkaline.
sfucch rosc.--Reaction alkaline.
Lcuthiosc.-Reaction acid.
1altosc.-Reaetion acid.
Mantitc.-Reaction alkaline.
Pota to wai tcr.-Reaction alkaline.
Agar slant.-A luxuriant growth takes place on this medium. The gro
gradually increases to a moist, glistening one, being then friable and of a gra,
brown color.
scrun).-A luxuriant, brownish, glistening, friable growth spreads over
entire surface. No liqoefaction is observed.
Potato.-An al)undant fleshy growth of a brown color spreads over the en
! urface. Ti water SUpports a heavy growth. The potato is slightly discolo
Milk.-Precipitation takes l)lace rapidly, followed by a gradual digestion
the casein. the medium clinging from the top downward to a translu4
liquid, becoming alstlast semi-transparent and viscid.
Litmius ni//k.- Lrecipitation of the casein takes place usually within 24 ho
followed by a gradual peptonization. Reduction of the litmus occurs rapi
Ieaving the medi slightly brown later the blue color will return on expo:
the milk to the air by shaking. Reaction alkaline.
Gcl tin.-.\n abundant growth takes place with rapid, infundibuliform 1i,
faction. A heavy, white, friable membrane is formed on the surface of
liquefied medium. A floc-uleit sediment lies at the bottom of the clear li,
fled portion.
lcit aJar. ( rowth takes plae.
lI (I!/ (I.- ('I has been obsIIn-ed.

Nitrate.-Reducti )1 to nitrite is positive.

Bacterium acidiformans. (Sternberg, 1892.)

Occnrrc~t'.-Isulatet from the scraping of propolis and wax from the b
and fr;1ies of healthy colnies.
Gelatin foWonies.-The sul)erficial colonies are friable, convex, opaque,
white with even border: when nmgnified they are finely granular, someti
adliately marked. They are from 1 to 4 milhimeters in diameter. The (
colonies are spherical or oblong and entire.
Morphology.-When taken from an agar slant 24 hours old, the rods
short, with rounded ends., singly and in pairs. Length about 1.6g, thick
0.8. They stain uniformly with carbol-fuchsin. Flagella are apparently
sent.
Motility.-No motility has been observed in any medium.
oSpores.- Spores are apparently absent.
ra 's stain.-Tle bacteria are decolorized by Gram's method.
Oxygen requirements.-Facultatively anaerobic.
Boillo.-The medium becomes slightly clouded with a feeble ring of grc
on the glass at the surface of the liquid. A moderate amount of white fri
sediment is formed. Reaction alkaline.
Glucose.-Uniformly and slightly clouded. No gas is formed. Reach
acid.
Lactose.-Reaction- acid.
Sacch arose.-Reaction alkaline.
Levulose.-Reaction acid.






BACTERIA FROM POLLEN.


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
-iquefaction takes place.
Potato.-A gray growth covers the inoculated surface.
M1ilk.-Heat causes a ready coagulation of the casein. Reaction acid.
Litmus nilk.-Coagulation of casein occurs )ron)tly on boiling a culture 2
weeks old. Reaction acid.
Gelatiti.-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.
Xitrate.-No reduction to nitrite could be observed.
U
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 granulir margin, and clear and sharply
defined border. A peculiar toruloid growth is often observed.
M1orplology.-The organisms are short rods with rounded ends, which stin
uniformly with carbol-fuchsin, and are 1g to 2g in length. Few short involu-
tion forms occur.
Motility.-The bacilli are actively motile in young cultures.
Sporcs.-No spores have been observed.
Grain's stain.-The bacilli are decolorized by Grams stain.
Oxygen req uirew en t.s.-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 -i- 3






THE BACTERIA OF THE API.


Mannite.-Reaction slightly acid, later alkaline.
Agar slant.-A moderate, slightly yellow, nonviscid glistening growth app
along the inoculated surface. This growth gradually spreads and deepen
color to an egg-yellow.
Potato.-A moderate, egg-yellow, nonviscid, glistening growth spreads
the entire surface. The potato is slightly discolored.
Milk.-The milk is covered by a yellow growth of the culture, resemt
cream. Coagulation takes place on boiling.
Litmus nmilk-Reaction alkaline.
Gcatin.-Growth takes place along the line of inoculation. Deep in
medium the colonies are white and spherical; the surface growth is ye]
After a few days liquefaction begins, and at the end of 2 weeks one-half
tube is liquefied. The liquefaction is infundiluliform. Liquefied gelatin is
mounted by a friable, egg-yellow pellicle. The growth in the liquefied poi
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 LARVA.

Comb honey from a laroe number of sources has been exami
and found to be quite uniformly sterile. The healthy larvm likely
are usually sterile.

BACTERIA UPON THE ADULT BEES.

On the external part of the bee we again find only a few differ
species. Bacillus A, described as found upon the combs, is
quently isolated from the bee. Other species which are found
quently are described below.

Bacterium cyaneus (Micrococcus cyaneus).

Occurrencc.-Isolated from the body of a healthy honey bee and from pollE
Gelatin colon ics. -The colonies are lemon-yellow, with entire border, grc
taking place readily on this medium. The superficial colonies, having 1
defined border. are finely granular, and liquefy the medium within 3 to 6 e
Morpliology.-Short oval rods 0.8, to 1.7,u in length, 0.711 to 0.8g in thickr
Short involution forms are present. The rods occur singly, paired, am
clumps. No flagella have been demonstrated.
Motility.-No motion has been demonstrated.
ASporcs.-No spores have been de i'Lonstrated.
Grain's stain.-The bacterium takes Gram's stain.
Oxygen requircments.-A robic.
Bouillon.-At first a slight cloudiness appears, the medium becoming tu
in old cultures. A heavy yellowish-white, slightly viscid ring forms on
tube at the surface of the medium. The sediment, and sometimes the med
show marked viscidity. Reaction alkaline.
Glucose.-The growth of the culture is confined entirely to the open bull
which the medium becomes turbid. No gas is formed. Reaction alkaline.
Lactose.-Reaction alkaline.
Saccharose.-Reaction alkaline.
Levlose,-Reaction alkaline,


16






BACTERIA UPON THE ADULT BEES.


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 produce, a soluble pigment that dif-
fuses thru the agar, giving it a dark-pink color.
Serum.-Luxuriant growth takes phtce, accompanied by liquefaction.
Potato.-A lemon-yellow, fleshy, 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.

Occurrenec.-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 0.8A in diameter, occur in pairs and in small
clusters.
Motility.-Nonmotile.
Spores.-Spores are apparently absent.
Grain's stain.-The coccus takes the Gram's stain.
Oxygen req u irei en ts.-Ailrobic.
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 slaWt.-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.






THE BACTERIA OF THE APIARY.


Litms vilk.-ln this medium coagulation takes place, accompanied
reduction of the litmus. Reaction slightly acid.
(l0ti.After a day or two infundibuliform liquefaction occurs, bei
followed by tratiforin liquefaction; the liquefied gelatin is turbid. Grow
l)clow this portion is in, the form of small splherical coloinies.
Vtcid tiqqr.-A white, fleshy. nonviscid growth is observed.
lndal.-A trace was observed.
Nitratcs.-Reduced to nitrites.

BACTERIA OF THE INTESTINE OF THE HEALTHY HONEY BEE.

A great many investigations have 1een made in recent years on f,
bacteria found present in the intestines of vertebrates (4, 51 0, 7,
). andl striking similarities are notice( in the species found in ma
of them. In this investiation the ilitestinal contents of about I
bees, mostly from on(e al)iarv, have bIeen studied more or less thoro]
Several species which are found to be constant in many of the vert
brakes are found ill the ilntestine of the honey bee. Since the tel
perattire of the )ee al)l)roxilmtes much of the time, especially wh,
in the hive, that of the warnil-blooded ainnuls, many of the -ar
species of bacteria in1habit the intestine of this insect as are foi
thriving in the same localitly in 1mu1n a 111d other animals. A stain,
cover-gla-s 1)rel)aratiii miade directlyy fr-oui a healthy adult field 1
reveals, almost without exception, a iiultituide of bacteria.
In a stldy ()f the i)acterial flora stress has been phced upon t
different species which were found to be-imore or less constant rath
than IIl)Oli the actflzal number of 1)acterila or species in any quanti
of material fro(n a single bee. Froum the observations which h1
been made, it appears that the number of species in any individu
is conl)aratively small. )ut the number of bacteria is in many caE
ey large. Sometimes, however, the latest show very few colonil
while cover-glass lvre1l).ti 11s show a very large number of bacter.
These organisms are prolballv the aiiarobe, which is quite constai
as shown b1y cultures made direct froni the intestine into glucose ag
(Liborius' method).
When a loopful of the material front the intestine was used for t
inociation, the following data give the approximate findings:
Bee No. 1, 300 to 40() yellow colonies, probably ailike.
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 fow fungi.
Bee No. 11, no growth.
Bee No. 12, 300 colonies, slightly yellow.


18







BACTERIA OF THE INTESTINE. 19

Bee No. 13, 2,000 or more gray colonies.
Bee No. 14, yeast cohmies and a few colonies of bacteria showing ground-
glass appearance. ,
Bee No. 15, 2,000 or more colon-like colonies (B. cloact).

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 Sacliaromvees and fnngi."

Bacterium D.

Occurreiec.-Frequent in ie intestine of the healthy honey bee.
Agar colojig.-Deep colonies when niagifiied are coarsely granular, showing a
dark brown center, with a thin and ill-defined border.
Morphiology.-A preplrationi made from a young cult'e taken from a glu-
cose fermentation tube shows rods with rounded ends, occurring siinlv aind in
pairs, staining easily and uniformly with carbol-fuchsin, and ineasuring 0.7)U to
1.5/ in length and 0.5A to 0.71. in thickness.
Motility.--No motility could be observed.
Apores.--No spores could be demonstrate(I in young cultures. In old cultures
their presence is questional e.
Oxygen reqa i'cjnts.-trictly anaroldc.
Boa illo.-In straight tul es 110 grwth1 ec(urs.
Gluco.-.-A lmoderate cloudiness ca-in be seeni in the closed arm. while the
open bull) remains clear. No gas is produced. Reaction about neutral.
GllCo. c agar, (Liborius's method).-Growth is rather slow. After : days a
moderate growth ma ty bIo observed; later, if cultures have recently been iso-
lated from the bee's intestine, the growth imparts to the iiedium a diffused
haziness or cloudiness. After many generations the culture loses this property.
Glitco e geltih (Liborius's niethod ).-Very slow growth occurs in the depth
of the medium. No liquefaction takes place.

Bacillus cloaca.

Occurrnce.-Found in the intestine of a large number of healthy honey bees.
Gelatin colon ies.-Superfitcial colonies are thin ind blue to gray ill color ; deep
colonies, brown. regular. ,ranular, ,nd 1 splieri(al to lenticular.
Agar colon is.--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 lA to 2, and in width from 0'.7,u to o.911. They are usually
found singly or in pairs. Involution forms are not .iol nlion. With carbol-
fuchsin they stain uniformly. This species possesses a few peritrichic flagella.
Motility.-Active motility is observed in young cultures.
Sporcs.-No spores are fornied.
Graia's stain.-The bacillus does not take Grain's stain.
Oxygen rfq irem en ts.-Facultatively anai;robic.
Bouillon .-A uniform cloudiness appears in 24 hours. Growth continues until
the medium becomes heavily clouded, followed by a gradual settling of many of
the organisms, forming a viscid grayish-white sediment. A gray friable mem-
brane, which adheres to the sides of the tube at the surface of the medium, is
sometimes produced. Upon agitation this membrane breaks up and sinks to the







20


THE BACTERIA OF THE A


bottom, leaving a gray ring of the growth adhering to the glass. Reac
alkaline.
Ghcose.- The medium in the bulb becomes turbid, while that in the
arm is uniformly cloudy. A heavy grayish-white sediment is formed .
reaction is at first slightly acid. but in a few days becomes alkaline. A
and rapid gas formation takes place, filling usually from one-half to nint
of the closed arm. The ratio of hydrogen to carbon dioxid is approxi
1 to 2; that is, the ratio of hydrogen to carbon dioxid is less than 1.
Lactosc.-In this medium gas formation takes place more slowly t
glucose. At the end of 8 days one-fourth of the closed arm is filled w I
The ratio of hydrogen to carbon dioxid is greater than 1. Reaction acid.
Sacclaros.-Gas is formed abundantly and rapidly; more than one-ball
the tube is usually filled with gas. The ratio of hydrogen to carbon dilox(
less than 1. Reaction alkaline.
Lcrhosce.-A rapid fermentation takes place: more than one-half of the dc
arm is filled with gas. The ratio of hydrogen to carbon dioxid is approxima.
1 to 5; that is, less than 1. A slight fornmtion of acid takes place at first,
the reaction rapidly becomes alkaline.
]laltwse.-Foration of gas takes place with the result that at the end C
days appIroximately one-hilf of the tube is filled. The ratio of hydrogen
carbon dioxid will approximate that of I to 1. Reaction acid.
Miinnitc.-Gas is fornmed rapidly and abundantly ; at the end of 5 days
closed an "is usually much more thn half filled with the gas. The reaction
at first slightly acid, but soon becomes alkaline. The ratio of hydrogen to i
bon dioxid is approximateIly 1 to 2 ; that is, less than 1.
Potato ratcr.-(as forms rapidly and fills lhlf the closed arm. The rat!(
hydrogen to carbon dioxid is as 1 to 2; that is, less than 1.
Ag/ar wlat. -A moderate, grayisli-white, glistening, friable growth app
along the line of inoculation, which usually spreads to the sides of the tube.
Scrun.- Moderate gray growth appears, which is confined quite closely to
line of inoculation. Liqluefaction takes place slowly after 3 weeks.
Potto.A moderate" amount ()f ray fleshy growth covers the slope.
potato is slightly discolored.
Milk.-Coalq,ilation takes llace after 4 days' growth. Gas is formed.
Litmaus milk.-A marked )roduction of acid, takes place, followed by I
coagulation.
Gclatin.-A heavy white growth takes place along the line of inoculation;
surface growth is flat, bluish-white, and spreads with an uneven margin. S
infundibuliform liquefaction takes place after 2 weeks.
Acid agar.-A growth takes 1hwe.
Imdol.-A trace is sometimes produced.
Nitratcs.-Reduction to nitrites is positive.

B. coli communis.

Oce-rrenee.-Found in the intestine of healthy honey bees.
Gelatin colon ies.-The superficial colonies are blue, lobate-lobulate,
slightly spreading; when magnified they are brownish yellow in the ce
and more transparent toward the margin; the deep colonies are spherica
lenticular and brownish yellow, with well-defined borders.
Morphology.-The short rods with rounded ends measure 1.5/ to 21 in l&
and 0.7g to 0.8/A in thickness. They occur singly or in pairs, stain uniform
and are motile by means of a few peritrichic flagella.






BACTERIA OF THE INTESTINE. 21

Motility.-The bacilli are actively motile from some cultures.
Spores.-No spores are formed.
Gram's stain.-The bacillus is decolorized by Gram's method.
Oxygen requiiremets.-It is a facultative anaerobe., "
Bouillon.-The medium becomes uniformly clouded in 24 hours, with a slight
acid reaction; the medium later becomes alkaline, with a gray and friable
sediment. A feeble pellicle is formed ad a growth of the organism often
adheres to the glass at the surface of the liquid.
Glucose.-Botb branches of the fermentation tube become clouded. The
sugar splits by fermentation into gas and acid, one-half or more of the closed
arm being filled. The ratio of hydrogen to carbon dioxid is 2 to 1.
Lactosc:-Gas fills one-fourth of the closed tube. Reaction acid.
Saccharosc.-Gas fills one-sixth of the closed tube. Reaction acid.
Levulos.-Gas fills one-half of the closed tube. The value of hydrogen to
carbon dioxid is 2 to 1. Reaction acid.
Maltose.-One-sixth of the closed arm is filled with gas. Reaction acid.
Mannitc.-One-half of the closed tube is filled with gas. Reaction acid.
Potato watcr.-Reaction acid.
Agar slant.-A moderate, gray. nonviscid, spreading growth takes place on the
surface of the inclined agar.
Serum.-A gray, glistening, nonspreading growth is observed on the inclined
serum. No liquefaction takes 1)lace.
Potato.-A moderate, fleshy, glistening growth spreads over the inoculated
surface. Potato slightly discolored.
Milk.-Coagulation of the casein takes place in al)out 4 days. A small quan-
tity of gas is produced.
Litmus inilk.-Coagulation occurs. Reaction strongly acid.
Gclatia.-A moderate growth occurs along the line of inoculation; the growth
is spreading with an irregular margin on the surface. No liquefication occurs.
Acid agar.-A moderate grayish growth occurs on surface.
Indol.-A trace was obtained in some cultures.
Nitrates.-Reduced to nitrites.

B. cholerae suis.

Occurred e.-Isolated from the intestine of healthy honey bees.
Gelatin colonics.-Colonies are translucent by transmitted light; bluish to
gray by reflected, the border being uneven and well defined. When the colonies
are magnified they appear brownish and finely granular.
Moi'phology.--The rods are short, with rounded ends, occurring singly and
in pairs, and staining uniformly with carbol-fuchsin, 1 to 2.8A in length, and
0.6ju to 0.8/- in thickness. A few peritrichic flagella are present.
Motility.-Usually only a few are motile at a time in the field, and these
present a rapid whirling motion.
Spores.-No spores are formed.
Gram's stain .-The bacteria are decolorized by Gram's stain.
Oxygen required ents.-Facultatively anaerobic.
Bouillo.-A uniform, moderate cloudiness arises in this medium in 24
hours; later a grayish-white membrane is formed which, upon shaking the
tube, sinks to the bottom, forming a gray sediment. The reaction is at first
slightly acid, but later becomes alkaline.
Glucose.-The medium becomes clouded in both arms of the fermentation
tube, with the production of a small amount of gas. Reaction acid.






22


THE BACTERIA OF THE APIARY.


Lactose.-Growth takes place in both arms of the tube, but the sugar is not
split into either acid or gas.
Saccharose.-Growth occurs in both arms of the tube, neither acid nor gas
being formed.
Levulosc.-Growth takes place in both arms with the production of gas and
acid; one-third of the closed arm is filled. The ratio of hydrogen to carbon
dioxid is about 3 to 1-that is, greater than 1.
Maltosc.-The medium in both arms of the tube becomes clouded. Fermenta-
tion results in the pr(Jductio)l of gas sufficient to fill about one-fifth of the
tube. Only a small portion of the gas is absorbed by sodium hydroxid, leaving
behind an explosive gs.
Mannite.The medium in both branches of the tube becomes clouded; gas
is not formed. Reaction alkaline.
IPotato w(ltcr.-About one-fifth of the closed arm is filled with gas. Reaction
acid.
A gar x lalnt.-A moderate, grayish-white, glistening, nonspreading growth is
forlne(l along the surface iiioculated with the loop.
,$er"uuii. A moderate. gray, glistening. nonspreding growth takes place on
the inclined surface. No liluefaction occurs.
Potato.-A feeble, grayish growth is observed. The )otato becomes slightly
discolored.
Mill.-No coagulatioi occurs. and no gas is pro(luced. Reaction alkaline.
Litbins 11ill-.The medium slowly becomes more and more alkaline.
Gelatin.-A moderate. white growth takes place along the line of inocula-
tion. On the surface it sIpreads with irregular margin. No liquefaction occurs.
ACid agar. A moderate growth appears.
Indlol.Indol is produced.
Nitratcs.-RIeuction to nitrites (?).

Bacillus E.

Occurrenre.-Isolated from the intestine of healthy honey bees.
(Gel~a tin coli is.TIhe (.olonies are lemon-yellow. Surface colonies are con-
vex, smooth, with entire margin ; when magnified they are finely granular.
Deep colonies, when magnified, are lenticular, finely granular, and may appear
darkre. L quefaction takes place slowly.
Mlorphology.-The rods are short, with rounded ends, and usually occur singly.
The bacilli are 1.5/1 to 2,4 in length and 0.7,u in thickness. This species pos-
sesses a fev peritrichic flagella.
Motility.-The bacteria are actively motile.
Spores.-No )spores are )resent.
Gram's N/ii,. They stain with Gram's stain.
Oxygen requi irem cnts. -Airobic.
Boitillon.-The medlium becomes uniformly clouded in 24 hours. Later a
tough, yellowish-white memnbrane is formed, which sinks upon shaking. The
medium is very viscid in old cultures. Reaction alkaline.
Glucose.-Growth is confined to the open bulb. No gas formation occurs.
Reaction slightly acid.
Lactosce.-There is a marked mucous-like appearance in the medium. Reac-
tion alkaline.
Saech arose.-Reaction acid.
Levulose.-Reaction alkaline.
Maltose.-Reaction alkaline.
Mannite.-Reaction slightly acid.






BACTERIA OF THE INTESTINE.


23


Potato water.-Reaction alkaline.
Agar slant.-A moderate, yellowish-gray, nonviscid growth takes place on the
surface.
Serum.-A strong growth takes place and the medium is liquefied.
Potato.-A yellowish-gray, nonviscid growth is observed over the entire
inclined surface.
Milk.-Precipitation of casein takes place with very slight digestion (?).
Litmus milk.-Precipitation of the casein occurs. Reaction alkaline.
Gelatin.-A white growth forms along the line of inoculation, which becomes
slowly liquefied from above.
Acid agar.-A moderate, slightly yellow growth is observed.
Indol.-None demonstrated.
Nitrate.-No reduction to nitrites occurs.

Bacillus subgastricus.

Oceurrence.-Isolated from the intestine of a healthy honey bee.
Gelatin colony.-The colon-like, superficial colonies are thin, blue, spreading,
and lobate-lobulate. When magnified they are finely granular, with brown
center. Deep colonies are spherical and yellow.
Morphology.-Short rods, singly and in pairs, are from 1.5A to 2.5A long and
from 0.6/ to 0.81A thick. They stain uniformly with carbol-fuchsin.
Motility.-M.Narked whirling motion from gelatin cultures.
Spores.-No spores could be demonstrated.
Gram's stain.-The bacilli are decolorized with Gram's stain.
Oxygen requirements.-Facultatively anai~robic.
Bouillon.-This medium becomes clouded in 24 hours. A slight band of
growth is formed on the glass at the surface of the liquid. Later a feeble
pellicle is sometimes formed. Reaction at first slightly acid, later becomes
alkaline.
ii Glucose.-The medium in both branches of the tube becomes clouded. Gas
is readily formed until about one-fourth of the closed branch is filled. The
ratio of hydrogen to carbon dioxid is 2 to 1--that is, greater than 1. Reaction
strongly acid.
Lactose.-Gas formation occurs. About one-sixth of the tube is filled with
gas, part of which is absorbed by sodium hydroxid and another part is explo-
sive. Reaction acid.
Saccharose.-This sugar is fermented to the point of formation of acid, but
no gas is formed.
Levulose.-This sugar splits in the process of fermentation to form acid
and gas, the gas filling about one-sixth of the tube. A portion of the gas is
absorbed by sodium hydroxid, the remainder being explosive.
Maltose.-Fermentation takes place with the formation of acid. No gas is
produced.
Mannite.-One-fifth of the closed arm is filled with gas. A portion of the gas
is absorbed by sodium hydi:oxid and a portion is explosive. Reaction acid.
Potato water.-Reaction alkaline.
Agar slant.-A moderate, translucent, gray, nonviscid and glistening growth
spreads slowly from the surface inoculated with the loop.
Serum.-A moderate, glistening growth appears along the surface inoculated.
No liquefaction occurs.
Potato.-A grayish growth takes place on the sloped surface.
Milk.-Firm coagulation of the milk takes place with the formation of a
small amount of clear serum. A small amount of gas is produced.
9583-No. 14--06 m---4






THE BACTERIA OF THE APIARY.


Litmus milk.-Reaction strongly acid. Coagulation occurs in about six days.
Gelatin.-White, spherical colonies appear along the line of inoculation. The
surface growth is grayish blue and spreading, with irregular margin. Slow
liquefaction takes place, beginning usually in 2 weeks.
Acid agar.-A growth takes place. -
lndol.-None could be demonstrated.
Nitrates.-No reduction to nitrites occurs.

Bacterium mycoides.

Occurrctce.-Isolated from the intestine of a healthy honey bee.
Gelatin colon ics.-A rapid growth of root-like colonies appears in 24 hours.
In macroscopic appearance it somewhat resembles cotton fibers; when magni-
fied these appear thick and somewhat felted in the center, while toward the
margin they are beautifully filamentous. After a day or two the gelatin begins
to liquefy.
Morphology.-The rods are large, scarcely rounded at the ends, and frequently
in chains. They measure from 2.5/ to 5.5u long and 1.51A thick. No flagella
have been demonstrated.
Motility.-No motility could l)e demonstrated.
Spores.-Spores are present.
Gram's stain.-The bacteria are not decolorized by Gram's stain.
-Oxygcn rcqu irem e) ts.--Facultatively analrobic.
Bouillon.-A decided fleecy growth with heavy, cotton-like sediment occurs.
Glucose.-No gas is formed. Reaction acid.
Lactose.-Reaction acid.
Sacc7harose.-Reaction acid.
Levulose.-Reacfion acid.
Maltos.-Reaction acid.
Manmite.-Reaction acid.
Potato water.-Reaction alkaline.
Agar slant.-A luxuriant growth that appears root-like takes place on this
medium. This growth tends to extend into the agar, which causes it to adhere
to the medium.
Serum.-A luxuriant growth is formed, accompanied by liquefaction.
Potato.-A thick, gray, moist growth is found, the potato not being discolored.
Milk.-Coagulation occurs promptly, with formation of a clear serum.
Litmus mil-k.The color is discharged in 48 hours.
Gelatin.-Hair-like outgrowths occur along the line of inoculation. Lique-
,faction begins at the surface and proceeds along the needle tract. In a few days
the entire medium is liquefied.
Indol.-No indol is produced.
Nitrates.-Reduction to nitrites is positive.

Pseudomonas fluorescens liquefaciens.

Occurrence.-Isolated from the intestine of the healthy honey bee.
Gelatin colonies.-Before liquefaction, the superficial colonies, when magni-
fied, are finely granular, with regular margin; deep colonies are spherical,
brown, with regular margin. Liquefaction takes place rapidly. The surface
of liquefied gelatin is covered by a friable membrane. Later the liquefied gela-
tin takes on a green fluorescence.
Morphology.-The bacteria are short rods, varying from 11L to 2/ In length
and from 0.5A to 0.7,A in thickness. They stain uniformly with carbol-fuchsin
and are motile by means of one or more polar flagella.






SACCHAROMYCES AND FUNGI.


Spores.-No spores could be demonstrated.
Gram's stain.-The bacteria do not take Gram's stain.
Oxygen requirements.-Airobic
Temperature requirements.-Culture must be grown at room temperature.
Bouillon.-The medium becomes clouded in 48 hours, forming a moderately
tough p licle. A greenish-yellow fluorescence begins at the surface, which
gradually increases until the entire medium takes on that appearance. Reac-
tion alkaline.
Glucose.-A cloudiness is formed in the open arm, but the closed arm is clear.
Reaction alkaline.
Lactose.-Reaction alkaline.
Saccharose.-Reaction alkaline.
Levulose.-Reaction alkaline.
Maltose.-Reaction alkaline.
Mannite.-Reaction alkaline.
Agar slant.-At first a gray friable growth is formed confined to the surface
inoculated, which later takes on a brown hue. Greenish-yellow fluorescence is
observable in the medium.
Serum.-A slow liquefaction occurs.
Potato.-Very scanty growth occurs with slight discoloration.
Milk.-Rapid liquefaction of the casein takes place.
Litmus milk.-Rapid liquefaction of the casein takes place. Reaction alkaline.
Gelatin.-Infundibuliform liquefaction takes place rapidly.
Acid agar.-No growth occurs.
Indol.-No indol observed.
Nitrates.-No reduction to nitrites occurs.

SACCHAROMYCES AND FUNGI.

The first yeast plant described below is of very frequent occurrence
in the intestine of the normal bee. Scacelharomyces roses can be iso-
lated from the comb. A large number of common fungi were found
in the flora of the intestines and in cultures from the pollen and
combs.
In addition to the above the third Saccharomyces here described
was found in two samples of brood apparently diseased, which could
not be diagnosed as any disease commonly known.

Saccharomyces F.

Occurrence.--Very common in the intestine of healthy honey bees.
Gelatin colonies.-Colonies form slowly; the superficial colonies are white,
glistening, convex, capitate, and about 1 to 2 millimeters in diameter. When
magnified they are finely granular, brownish yellow, with entire margin. Deep
colonies are finely granular, with uniform margin, spherical to lenticular, and
brownish green.
Morphology.-The cells are oval and on agar in 24 hours attain their full
size of 4.5A in length and 3.5,u in thickness. They stain uniformly with carbol
fuchsin.
Motility.-The yeast is not motile.
Gram's stain.-The cells take the Gram's stain.
Oxygen requirements.-Airobic


26






26 THE BACTERIA OF THE APIARY.

Bouillon.-This medium remains clear, with the formation of a friable white
sediment. Reaction neutral.
Glucos.-The closed arm remains clear. No gas is formed. Reaction acid.
Lactose.-Reaction neutral.
Sacch arose.-Reaction neutral.
Le rulose.-Reaction neutral.
Maltose.-Reaction neutral.
Mannite.-Reaction neutral.
Agar.-A white, nonspreading growth occurs.
Serum.-White, moderate, nonviscid, nonspreading growth occurs along the
surface inoculated. No liquefaction takes place.
Potato water.-Reaction neutral.
Potato.-Gray, luxuriant, fleshy growth occurs.
Milk.--No change occurs.
Litm us milk.-- No change occurs.
Gelati.-A moderate growth is formed, accompanied by no liquefaction.
Acid agar.-Moderate growth takes place.
Indol.-Negative.
Nitrates.-Reduced to nitrites.

Saccharomyces roseus.

Occurrence.-Isolated from comb of healthy hive.
Gelatin colotties.-Superficial colonies are pink, convex, capitate, with lobate-
lobulate margin; when magnified, the deep colonies are irregular, brownish-
yellow, and finely granular.
Morplology.-This cell is oval. attaining about 6.5ji in length and 3.5/A in
thickness. The cells stain uniformly.
Motility.-No motility occurs.
Grami's stain.-The cells are not decolorized by Gram's stain.
Oxygen rcquirements.-Arobic.
Bouillon.-This medium remains clear, forming a pink, friable sediment. A
pink band forms at the surface of the medium and adheres to the glass.
Glucose.-The closed arm remains clear. No gas is formed. Reaction acid.
Lactose.-Reaction neutral.
Saccharose.-Reaction neutral.
Levulose.-Reaction slightly acid.
Maltose.-Reaction slightly acid.
Mannite.-Reaction neutral.
Potato water.-Reaction acid.
Glucose agar.-Luxuriant, red growth forms on the surface.
Serum.-A pink, fleshy, nonspreading growth is formed, accompanied by no
liquefaction.
Potato.-A thick, nonspreading, red growth occurs.
Milk.-No apparent change takes place. The milk coagulates on boiling.
Litmus milk.-Reaction alkaline.
Gelatin.-Moderate pink growth is formed, accompanied by no liquefaction.
Acid agar.-Slow growth occurs.
Indol.-Negative.
Nitraes.-Reduction to nitrites is positive.

Saccharomyces G.

Occurrence.-Found in the dead larvm of diseased adult bees.
Morphology.-They appear in hanging-drop preparation in large clusters,






SACCHAROMYCES AND FUNGI.


27


stain uniformly with carbol-fucbsin and are oval, nearly spherical, attaining
the length of 4.5/ and thickness of 3.5gL.
Gram's stain.-The cells are not decolorized by Gram's stain.
Oxygen requirements.-Airobic.
Bouillon.-A slight, friable, white sediment is formed, with a clear medium
above. Reaction slightly acid.
Glucose.-The medium in the closed arm remains practically clear and about
one-fifth of the closed arm is filled with gas. Reaction acid.
Lactose.-Reaction neutral.
Saccharose.-Reaction neutral.
Levulose.-Reaction slightly acid.
Maltose.-Reaction slightly acid.
Mannite.-Reaction neutral.
Potato water.-Reaction acid.
Agar.-A moderate, white growth is formed.
Serum.-Very feeble growth occurs, accompanied by no liquefaction.
Potato.-A luxuriant, moist, white growth occurs.
Milk.-No appreciable change takes place.
Litmus milk.-No appreciable change takes place.
Gelatin.-A moderate, white growth occurs along needle tract and on the
surface. No liquefaction results.
Acid agar.-A feeble white growth occurs.
Indol.-None could be demonstrated.
Nitrates.-No reduction to nitrites occurs.
Glucose agar.-A thick, white, fleshy growth occurs.








TABULATION OF MICRO-ORGANISMS NORMALLY PRESENT IN THE APIARY.


The following table will serve to summarize the
going pages:


descriptions of the micro-organisms considered in the fore-


IBiology. I


Species.


Bacillus A .................
Bact. acidiformans ........
Bacillus B .................
Bact. cyaneus .............
Micrococcus C ............
Bacterium D ..............
B. cloaca .................
B. coli communis......
B. cholera suis ...........
Bacillus E ........ ...
B. subgastricus ...........
Bact. mycoides ........
Ps. fluor. lique ........
Saccharomyces F ......
Saccharomyces roseus ....
Saccharomyces G......


Morphology.


Dimensions in
microns.


3 -4
1.6
1 -2
.8-1.7
.8
.7-1.5
1 -2
1.5-2
1 -2.8
1.5-2
1.5-2.5
2.5-5.5
1 -2
4.5
6.5
4.5


E4-


0.9-1.2+
.8-
......+
.7- .8-
.8-
.5- .7-
.7- .9+
.7- .8
.6- .8+
.7+
.6- .8+
1.5 ?
.5- .7+
3.5-
3.5-
3.5-


0
04

0
+42
0


Physiology.


Cultural features.


6
~J.
~

4.)


0

0


4;

V.0



-1
++








++


+
+

+
+
1+

1+
1+
.+


Biochemic properties.


1+
3 -
3-
1+
2i+




+..
2+


4


+i+


++



++


Gas production.


i+;



+ +


+


Z


Acid production.


+


+
+


Remarks.


B. mesentericus (?).


Indol trace.
SPores (?).
Indol (?).
Indol trace.


. I I


I I I I I





BIBLIOGRAPHY TO PART I.


SUMMARY TO PART I.

The results of the study of the bacteria found normally in the
apiary 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 ab'ei, 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 rose us,
and a Micrococcus referred to here as Micrococcus C, have been iso-
lated and studied.
(5) Honey from a healthy hive is, as a rule, sterile.
(6) The normal larvae are, as a rule, sterile.
(7) There is an anaerobe 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 cloacae, Bacillus coU communis,
Bacillus choler(e suis, Bacillus subgastricus, Bacterium mycoides,
Pseudomonas fliuorescens liquefaciens, and two referred to as Bacillus
E, and Saccharomyces F. Others less frequently present have 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.
1. FULLER, GEO. W., and JOHNSON, GEO. A. On the Differentiation and Distribu-
tion of Water Bacteria. 2. JOHNSON, 0. P., and MACK, W. B. A Modification of Existing Methods for
Staining Flagella. 3. PECKHAM, ADELAIDE W. The influence of environment upon the biological pro-
cesses of the various members of the colon group of bacilli. Medicine, Vol. II, No. 5, p. 549, 1897.
4. FORD, WM. W. The Classification and Distribution of the Intestinal Bacteria in
Man. 1903.
5. KING, W. E. A Study of the Bacterial Flora of the Intestinal Mucosa and Eye
of the Common Fowl. 6. BULLARD, M. J. A Study of the Bacterial Flora of the Intestinal Mucosa of the
Normal Rabbit. 7. DYAR, HARRISON G., and KIETH, SIMON C., jr. Notes on the Normal Intestinal
Bacilli of the Horse and of other Domesticated Animals. Quarterly, Vol. VI, No. 3, 1893.


29





THE BACTERIA OF THE APIARY.


8. MooRE, V. A., and WRIGHT, F. R. Observations of Bacillus coli comunis
from certain species of Domesticated Animals. III, No. 13, p. 504, 1902.
9. LOEBER, E. A Bacteriological Study of the Intestine of the Fish. Vol. VII, No. 4, p. 152, 1904.
10. MATZUSCHITA, T. Bacteriologische Diagnostik, 1902.
11. CHESTER, F. D. A Manual of Determinative Bacteriology, 1901.
PART II.-THE DISEASES OF BEES.
The bee industry in this country, and other countries as well, is
suffering large losses from various diseases among bees. Those which
are most destructive attack the brood and weaken the colony by kill-
ing off large numbers of the young larvae which would otherwise
mature. There are other diseases which attack the adults and so
decrease the strength of the colony in that way.
In order to combat a disease to the best advantage it is clear that
its cause must be known, as well as the means by which the infection
is transmitted and the environmental conditions which are favorable
for the breaking out of an epidemic. The brood diseases among bees
are on the increase. The custom of selling and shipping the honey,
which is now carried on more extensively than formerly, the manner
in which the products of the apiary are handled, and the absence of
a general knowledge by the mass of bee keepers of the nature of the
diseases are conditions which must be met before the spread of these
diseases can be checked. When a colony is diseased, very little or no
profit is realized from it; consequently the wealth and comfort of a
very large number of people are greatly endangered by the existence
of bee diseases. This suggests the importance, from an economic
standpoint, of a thoro knowledge of these disorders.
BRIEF HISTORY.
The attention of investigators has been attracted by these diseased
conditions, not only from the economic interests attached thereto,
but from the scientific point of view as well. The writings of Aris-
totle (12) contain an account of certain disorders which were then
prevalent among bees; at that time it was thought that the blight of
flowers bore a relation to bee diseases. In 1769 Schirach (13) gate
the name foul brood to a diseased condition of the brood of bees;
he attributed the cause to (a) unwholesome food, and (b) the placing
of the larvae with head inward in the cell. Leuckhart (14) thought
the cause to be a fungus, related to the cause (Panhistophyton ova-
turn) of the disease of the silkworm. Muhlfeld (15), in 1868,
thought the trouble to be of two kinds-infectious and noninfec-
tious-and that the cause of the infectious one is the larva of a para-
sitic fly (Ichneumon apium mellificarium) feeding upon the larve of
the bee. In 1868 Preuss (16) exprest the view that the cause of


130






BRIEF HISTORY OF BEE DISEASES.


foul brood is a fermenting fungus belonging to the genus Cryptococ-
cus. Geilen (17), in 1868, thought that when bees alight on the.
remains of animal bodies the putrefying matter thus carried with
them may cause foul brood. The fermentation of bee bread was
thought by Lambrecht (18) to be a sufficient cause of the disease;
while Hallier (19) thought that various fungi could produce the
disorder. On the contrary, Cornallia (20), in 1870, exprest the
opinioii that a fungus ((Cryptococws8 aleari.) is the specific cause of
the trouble. Fischer (21), in 1871, supposed that a predisposing
factor of foul brood is to be found in insufficient nourishment. In
1874 Cohn and Eidem received from Schonfeld samples of foul brood
and, upon examination, they found spores and rods. In 1885 Chesh-
ire and Chevne (22) determined the cause and named the germ
Bacilbis al,ei. Dickel (23) claimed that a number of different
species might be the cause of foul brood. In 1900 Harrison (24)
writes on foul brood and Bacillus al,ei, its cause. Doctor Lambotte
(25), in 1902, made some interesting studies concerning the relation
of Baillvs a(1ei and tUC it 1e,Uevfterlicsi8 0Il(ats.
Since so many conflicting views have been held as to the cause of
foul brood, one might conclude that the term foul brood has been
applied incorrectly to a number of different disorders. In the light
of more recent work this supposition is strengthened.
In June, 1902, the author, tnder the direction of Dr. Veranus A.
Moore, began an investigation of bee diseases, especially as they ex-
isted in New York State. There were recognized at that time by
bee inspectors of that State a number of distinct diseases which
attacked the brood. Those which caused the greatest loss to the
apiarists were known to the bee experts as black brood," foul
brood," and pickle brood." The results of the investigations of
1902 (26). 1903 (27), and 1904 (28) on these disorders; and on palsy
or paralysis, are embodied in the following pages.

THE TERM FOUL BROOD AS HITHERTO APPLIED.

In the discussion of foul brood of bees it must be remembered that
until recent years the name has been applied to what is now known to
be two distinct diseases.
Schirach, in 1769, gave the name foul brood to a diseased condition
in the brood of bees, but it is impossible to know to which of the two
he referred. It may be that both diseases existed then as now and
that he did not observe the. fact that the two were different. We
have reason to think that there are, at the present time in Europe.,
two distinct diseases to which the name foul brood is being applied.
It is definitely knowfi that such is the case in America.
It becomes necessary, then, to have two names to designate these






THE BACTERIA OF THE APIA


two diseased conditions in the brood of bees. For reasons given
Dr. E. F. Phillips, in the preface to this paper, it has been eonsidei
advisable to. retain the name foul brood and to use a qualifying w(
to distinguish the two diseases. "European foul brood a
"American foul brood" are the names by which these two disea4
conditions are to be designated.
In 1885 Cheyne (22) in England (Europe) found present in 1
decayed larva suffering from a diseased condition known as fi
brood" a new bacillus, which he named Bacillus alvei and to wh
ie ascribed the cause of the disease. The diseased condition wh
contains Bacillhos alci is to be called European foul brood," beca
this fact was first observed by an investigator working in Eur(
(England). In 1903 (27) the author observed that there was c,
stantly present in the other (iseased condition known as foul broo
another bacillus which was new, and to which the name Bacil
lara' is given. In view of the fact that Bacillus laroe was c,
stantly found to be present in the larva, suffering from this disor,
in the brood of bees, by investigations carried on in New York St
(America) (27) (28), this (liseased condition is to be called "Am(
can foul brood." From a scientific standpoint this choice of nar
for two distinct diseases might be easily criticized, but from
standpoint of the apiari4 the selection of these names as the comn
ones for these two distinct disorders seemed almost necessary, or
least advisable.

EUROPEAN FOUL BROOD (FOUL BROOD OF CHEYNE).
The first scientific investigation of this disease bacteriologi c
was performed by Cheyne in 1885 (22). At this time he isolated
new bacillus from the (lead larva. It was described by him I
given the name R acilhu al#,ei (literally, hive bacillus). This afford
then, a means for a positive (iagnosis of this diseased condition.
Symptoms.

The symptoms of European foul brood, as given by Dr. E.
Phillips in Circular No. 79, Bureau of Entomology, are as follows
Adult bees in infected colonies are not very active, but do succeed in cleai
out some of the dried scales. This disease attacks larvtp earlier than (
American foul brood, and a comparatively small percentage of the disef
brood is ever capped; the diseased larv, which are capped over have sun
and perforated cappings. The larv, when first attacked show a small ye
spot on the body near the head and move uneasily in the cell; when d(
occurs they turn yellow, then brown, and finally almost black. Decaying la
which have died of this disease do not usually stretch out in a long thi
when a small stick is inserted and slowly removed; occasionally there is a i
slight ropiness," but this is never very marked. The thoroly dried larvM f,
irregular scales which are not strongly adherent to the lower side wall of


32






CONFUSION REGARDING FOUL BROOD IN AMERICA.


cell. There is very little odor from decaying larve which have died. from
this disease, and when an odor is noticeable it is not the glue pot odor of
American foul brood, but more nearly resembles that of soured dead brood.
This disease attacks drone and queen larvw' very soon after the colony is
infected. It is, as a rule, much more infectious than American foul brood and
spreads more rapidly. On the other hand, it sometimes happens that the
disease will disappear of its own accord, a thing which the author never knew
to occur in a genuine case of American foul brood. European foul brood is
most destructive during the spring and early summer, often almost disap-
pearing in late summer and autumn.

Confusion Regarding Foul Brood in America.

Prof. J. J. Mackenzie in 1882 made what seems to have been a
short study of a bee disease as it appeared in Ontario, Canada, which
was known to the apiarists of that Province as foul brood. Ile says
very little of the character of the species of bacteria with which he
was working, but he supposed that they were Bacilu, ,l'e i of
Cheyne. The author has examined samples of brood from Ontario
which have what, in the opinion of bee experts, is the most prevalent
disease, and- has not found Bwilliis (dre; present in any one. The
bacteriological findings and the experience of bee-disease exl)erts
show that American foul brood is the prevalent disease in that, Prov-
ince. As the bee experts see the disease in the light of recent studies,
there is no authentic report of which we are aware that European
foul brood exists in Ontario. We can safely say, then, that BaecIlius
alvei can not be isolated from larva' taken from the, prevalent disease
in the above-named Province. No difficulty is exprest on the part
of Professor Mackenzie in the isolation of lhilltts abel,e ffroni any
sample. The author is inclined to think, therefore, that this investi-
gator was in error as to the identity of his culture, and therefore his
conclusion can have little weight.
The foul brood of bees received some attention also from Prof.
F. C. Harrison, of Ontario. In a paper of some length he gives a
description of a species of bacteria which he identified as Baelais
alvei. The description which he gives and the accomn)anvin, photo-
micrographs (another plate which was given being after Cheyne
and correct for Bacillus alcei) might easily be that of a member of
a group represented by and described as Bacillus "A" in Part I of
this paper. He also says that he has isolated Bacillus alirei from
diseased larve from 13 States of the Union, ranging from New
York to California and from Michigan to Florida. European foul
brood has had a very limited geographical distribution, spreading
only recently from New York to adjoining States. In Professor
Harrison's work, too, there seems to have been no difficulty in iso-
lating Bacillus alvei from diseased brood diagnosed by bee inspectors


33





THE BACTERIA OF THE APIARY.


as foul brood thruout the United States and Canada. In the experi-
ence of the author it has not been possible to obtain Bacillus alei
from diseased brood which the inspectors in most of the States and
in Canada have been calling foul brood. For the above reasons the
author believes that Harrison, too, has made a serious error in'the
identity of his culture and therefore was not working with Bacillus
alicei at all. The author considers himself unfortunate in that he
was unable to obtain a culture of Bacillus alvei for study and identi-
fication from Professor Harrison.
Dr. William R. Howard, of Fort Worth, Tex., also studied foul
brood somewhat, and gave a description of Bacillus alei as he found
it. From his description and from the fact that he, too, worked with
a diseased condition which does not contain Bacilluts alvei, and ex-
prest no difficulty in obtaining his cultures from any samples, the
author believes that this investigator made an error in the identifica-
tion of the culture with which he was working.
Some wi ters-Cowa Bertrand, and others-have attempted the
positive diagnosis of foul brood with the microscope alone from a
prelarationi mitade direct from the dead larvae. If the reader will
remember that with the microscope alone it would be impossible to
distinguish between Balu'il la(ra and Btcilluts alei, the verdict of
these men can have nt weight. As shown later in this paper under
lack brood (pp. 3-44). the Doctor Howard, of Fort Worth, Tex., re-
ferred to above, made an error in supposing that the European foul
brood was a new disease and naming it New York bee disease" or
black brood."
It is very unfortunate for the apiarist that these men should have
fallen into error as to the identity of their culture with Bacillus alvei,
as it has caused great confusion in the names of bee diseases. This
confusion in the identity of cultures may be excused to a certain ex-
tent by the fact that European foul brood did not appear in this
country, or at least did not attract much attention, until after Mac-
kenzie, Harrison, and William R. Howard had done their work on
foul brood.
The Present Investigation.

When the author's investigations were begun in 1902 there were
two especially troublesome diseases in this country, which were then
known to the bee experts as "black brood" and foul brood."
The following summary and table shows the results of the exami-
nation of a number of samples of diseased brood from different
apiaries, sent by the New York State bee inspectors during the sum-
mer nf the year 1902:


34







THE PRESENT INVESTIGATION OF EUROPEAN FOUL BROOD.


Table showing the results of examinations of European fol brood. (The
samples were called "black brood by the apiarists at that time.),


Brood sent by- Date. Bacteriological findings.


W. D. Wright .....................
W. D. Wright ......................
N. D. West .......................
N. D. West .......................
N. D. West .......................
N. D. West .......................
N. D. West .......................
N D. W est .........................
N. D. W est .........................
W. D. Wright ..................


June 4 ..........................
June 12 .........................
June 12 ...................
June 12 .........................
June 12 .........................
June 12....................---
June 12 .........................
June 12 .........................
Aug. 5 ..........................
Oct. 8 ...........................


Bacillus alrei.
Bacillus alvei.
Bacillus alvei.
Bacillus alvei.
Bacillus alvei.
Bacillus ahei.
Bacillus adei.
Bacillus alrci.
Bacillus alvei.
Bacillus alcdi.


It can be seen clearly from the above table that the diseased condi-

tion which the apiarists were calling black brood is really the
disease foul brood" of Cheshire and Cheyne, because of the con-
stant presence of Bacillus albei.

The work upon European foul brood was continued during the

year 1903. The following table gives the results of the examination
of specimens received during that year. The samples were taken

from different apiaries.

Table giving a switiimaryl of the e.ramiation of spechcii8e of turopeait foul brood
(" black brood ").


Brood sent by-


W. D. Wright ..............
W. D. Wright ................
N. D. W est ...................
N. D. W est ....... ............
N. D. West ...................
N. D. W est ...................
N. D. W est ...................
N. D. W est ...................
N. D. West .................
N. D. W est ...................
N. D. W est ...................
N. D. W est ...................
N. D. W est ...................
N. D. W est ...................
N. D. West ..................
N. D.. W est ...................
N. D. W est ...................
N. D. West..............
N. D. West...............
N. D. West ..................
N. D. W est ...................
N. D. W est ...................
N. D. W est ...................
N. D. West .................
N. D. West...............
N. D. West...............


Date.


May
May
June
June
June
June
,June
.July
J uly
!July
July
July
July
July
July
July
July
July
July
July
July
July
July
July
Aug.
Aug.


Sources of brood ii New York.


Columbia County ...................
Albany County ...........................
Schoharie County ........................
Schoharie County ........................
Schoharie Courn y ........................
Schoharie County ........................
Schoharie County ........................
Schoharie County .......................
Schoharie County ........................
Schoharie County ........................
Montgomery County .....................
Schoharie Count ....................
Schoharie County .......................
Schoharie County ....................
Schoharie County. ..................
Montgomery County .................
Schoharie County .......................
Schoharie County ........................
Schoharie County ......................
Schoharie County ........................
Schoharie County ......................
Greene County ...........................
Albany County ...........................
Greene County ...........................
Greene County ...........................
Greene County .........................


Bacteriological
findings.

Bacillus alhei.
Bacillus ahei.
rBacluis (dcei.
Bacillus altei.
Bacillus acei.
Bacillus alhei.
JUillfls alvci.
Bacillus alei.
Jacillu. alei.
Bacillus alvei.
Bacillus ah ci.
Bacillus alvei.
Bacillus alei.
Bacillus alvi.
Bacillus alhce.
Bacillus alci.
Bacillus alei.
Bacillus alhei.
Bacillus ahcei.
Bacillus alwei.
Bacillus adei.
Bacillus ahcei.
Bacillus alei.
Bacillus alhei.
Bacillus alrei.
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 larvar, 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


35






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.
Morpihology.-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 3.9A in length, and from 0.5A to 0.7A 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 1.51 to 2/A in length and from
0.7/ to IA in breadth; they exhibit polar germination. The few flagella are
arranged peritrichic.
Oxygen rcquircmrents.-This bacillus is a facultative anaerobe which grows at
room temperature, but better at 370 C.
Boitillwi.-The medium becomes uniformly clouded in 24 hours; later it
shows a tendency to clear by a settling of the organisms. A somewhat viscid
sediient 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.
Gliwose.-The moium in both branches of the fermentation tube becomes
uniformly clouded. Gas is not formed. Reaction acid.
Lactoe.-The m4liumn 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.
iaeeharwe.-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 plate-s.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
olonies are on the plate, the granular center of the colony is surrounded by
numerous 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 dlait.-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
3 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 increased 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.





INOCULATION EXPERIMENTS WITH BACILLUS ALVET.


Gelatin colonies.-Gelatin is a medium in which it develops slowly. The col-
ony becomes very irregular in outline, owing to threhd-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 inldol reaction is obtained.
Power to resist .disif[cctaiis.--Prelilinary observations give the following
results: The spore form resists drying for a considerable time. Spores which
have been drying for 1 year germiniate 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, I to 1,000.
kills in 1 minute; mercuric chlorid solution. 1 to 2,000, kills in 2 minutes.
Spore form.-Mercuric chlorid, 1 to 1.000, kills in !30 minutes.
Pathogn esis in r'crtebr tcs.-Iinoculati(;hs 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 syn)tols 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
BacillMs alvei. Similar feedings were given to these bees from one
to three times a week until Septeniber 2S, but symptoms of foul
brood did not develop. On August 6 cultures were made from a
few of the hive larvT. 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 BRaillis alr'ei
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 Bacillas abei were freshly isolated from foul-brood
specimens and kept in stock on bee-larva agar (described under
American foul brood, pp. 41-42). All cultures were incubated at 340
C., which temperature is observed to be slightly below that of the
hive. The spore form of Bacillus altei was used. .
Inoculations were made in different ways. A diseased condition


37





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
inixt with sterile sirup. A jelly glass, in the lid of which holes had
been punctured, was filled and inverted on strips of wood inside the
hive. In this way the bees take up the culture with the sirup as
rapidly as it flows out of the glasses. A colony having brood free
from Bacillus alcei was fed in the above manner on August 8, with
repeated feedings on the 9th, 10th, 12th, 13th, 15th, and 17th. On
the 12th Bacillus alvei was found in the living larva- and on the 17th
many larvae were dead under cappings and some were dead which
were not capped; all were soft and of a dull color. Many of the
capped cells containing dead larva, had their capping freshly punc-
tured. BacilllIs aei was usually obtained from these larv in pure
cultures. In no cell examined where the cell capping was punctured
did we find gas-producing organisms; this fact would suggest the
conclusion that these punctures which are found in the capping in
foul brood are made by the bees and not by gas-producing organ-
isms. During this series of inoculations the days were quite cool
and sometimes cloudy and damp. O)n the 20th of August the tem-
perature was much higher, the bees were more active, and much of
the dead brood had been cleaned out by the bees. On the 22d no
dead brood was noticed by casually looking over the brood nest. On
the 24th of the same month a careful search was made by uncapping
all the cells of one brood frame, and 12 decaying larva of a brown
color were found. At this time the larv\ were not viscid. All the
remaining (lead brood had evidently been cleaned out by the bees.
A condition similar to this, where only a few scattered about in the
brood nests contain (lead larva, occurs sometimes in affected apiaries.
Two other colonies which were near by but not inoculated gave no
signs of disease.
Mr. N. D. West reports that the climatic conditions seem to have
something to do with the extent of the ravages of European foul
brood, since the disease is much more destructive in cool, damp
weather. This seems to be a very plausible idea. The larvAl at such
times may receive more infected food than when fresh is being
rapidly gathered; the resistance of the body of the larvae to the
growth of Bacillus al'ei is at such times much lessened; and the
adult bees being less active, the dead larvae are not cleaned out of the
combs so rapidly. The results of the experimental work seem to
confirm this theory.
Distribution of Bacillus alvei in Infected Hives.
In order to combat this disease it is well to know where these patho-
genic bacteria may be found. The following is a summary of the
results of the investigation along this line:


38






EXPERIMENTS WITH FORMALDEHYDE GAS.


1. The greatest number of infecting germs are found in the bodies
of dead larve.
2. The pollen stored in the cells of the foul-brood combs contains
many of these infecting organisms.
3. The honey stored in brood combs infected with this disease has
been found to contain Bacillus alei in small numbers.
4. The surface of the combs, frames, and hives may be contami-
nated.
5. The wings, legs, head, thorax, abdomen, and intestinal contents
of adult bees are found to be contaminated with Bacillus ahiei.
6. Cheshire (29), Mackenzie (30), and others have found Bacillus
alvei in the ovary of the queen. This has suggested a means of in-
fection. From a bacteriological examination of queens from three
badly infected hives we were able to isolate Batl (l/ei in small
numbers in two cases. Since a very large number of this species of
bacteria may be found in the intestinal tract and upon all parts of
the body, it is very probable that such findings are the results of con-
tamination in maki ng cultures and have no special significance.
Experiments with Formaldehyde Gas.
Within the last few years several articles have appeared in the bee
journals entertaining great hopes that a cure for foul brood has been
found in the use of formaldehyde gas. The methods described for its
use have been tested by the apiarists and bee experts in New York
State, with the result that the disease sometimes breaks out anew in
colonies so treated.
In order to test the value of formaldehyde gas as a disinfectant
when used in foul-brood combs a number of experiments were made
in the laboratory. A common frane hive was first used, in which
were placed specimens of foul brood. The hive was charged with
gas by heating formalin in a closed vessel which was in communica-
tion with the hive; 15 c. c. was llsed each time and evaporated to
dryness. The charging of the hive with gas ws repeated in this
way at the end of 2, 4, 6, and 20 hours. Before each charging and
at the end of 24 hours after the first application of gas, cultures were
made. Of all the tubes inoculated 90 per cent showed Baillus aliei
to be present. There was no decrease in the number of tubes in
which Bacillus aiei appeared following the several applications of
formaldehyde gas.
The examination of specimens of foul brood which had been treated
with the gas by an apiarist gave the following results:
Thirty tubes which were inoculated from larve, capped and un-
capped, showed the presence of Bacillus alei in 21.
Thirty tubes which were inoculated with pollen in cells gave
Bacillus alei in 28.


39






THE BACTERIA OF THE APIARY.


Four series of agar plates showed apparently no diminution in
the number of bacteria present.
Further experiments were made by using Novy's anarobic jar,
(a very tight chamber) as a chamber in which to put the diseased
brood combs and cultures. This vessel will retain the gas much more
I)erfectly than the devices made for practical use in the apiary.
Treatment of brood in this jar by recharging with the gas resulted
usually in complete disinfection after 2 days. Agar plates con-
taining spores and cheese cloth on which cultures were spread and
dried were disinfected after a short length of time by the applica-
tion of formaldehyde gas.
From the experiments made the conclusion can be drawn that
formaldehyde gas is a good disinfectant, but that it penetrates very
slowly and that 24 hours' application of the gas to the combs, as
usually applied, is not sufficient to kill all the spores in the decayed
]arva (27).
AMERICAN FOUL BROOD.
The diseasedd con(lition which we shall call American foul brood
and the HiirO-01'"anisi found constantly present in the diseased and
dead larva,, which we shall call BIJ.illas Iart'w. were, for convenience,
referred to, respectively, as Brood and R'"cilbiN "A in a
former report (27). This disease has lbeeii called fo0 brood by
many bee keepers in this country and in other countries as well. It
is the diseased condition with which Mackenzle, Harrison, and
Williaii R. Ihoward were working largely, if not altogether, in their
investigations of foul 1brood. The disorder is, as a rule, dreaded less
than European foul brood by the apiarist. yet in the aggregate the
bee industry suffers enormous lossePs from the trouble. The general
'character of the diseased brood is so much like that of foul brood
that the two may be easily confused by those unfamiliar with the
variety of apipearance which one finds in each disease and a few
characters which are differelntial. Therefore it is not strange that
the mistaken diagnosis should be made from the symptoms mani-
fested by these two diseases. When, however, European foul brood
and American foul brood are subjected to a bacteriological exami-
nation, the diagnosis is easy. Experts when comparing specimens
of the two diseased conditions are able to see a difference in the
gross appearance.
Symptoms.
The symptoms are given by Dr. E. F. Phillips in Circular No. 79,
Bureau of Entomology, as follows:
The adult bees of an infected colony are usually rather inactive and do little
toward cleaning out infected material. When the larvae are first affected they
turn to a light chocolate color, and in the advanced stages of decay they become


40






THE PRESENT INVESTIGATION OF AMERICAN FOUL BROOD.


darker, resembling roasted coffee in color. Usually the larve are attacked at
about the time of capping, and most of the cells containing infected larvu are
capped. As decay proceeds these cappings become sunken and perforated, and,
as the healthy brood emerges, the comb shows the scattered cells containing
larvwp which have died of disease still capped. The most noticeable charac-
teristic of this infection is the fact that when a small stick is inserted in a
larva which has (lied of the disease, aid slowly removed, the broken-down
tissues adhere to it and will often stretch out for several inches before break-
ing. When the larva dries it forlmis a tightly adhering scale of very dark
brown color, which can best be observed when the comb is held so that a bright
light strikes the lower side wall. I)ecaying larvwe which have died of this disease
have a very characteristic odor, which resenibles a 1)oor quality of glue. This
disease seldom attacks drone or queen larva,. It appears to be much more
virulent in the western part of the United States than in the East.
A microscopic preparation from the diseased, but not dead larvw,,
or from larvae recently dead, at first shows a few comparatively long
slender rods; later these increase rapidly in nimnber, and spores also
are seen. In the later stages of decay in the ropy mass and the dried
scales spores only are found; these occur in very large numbers.
When this investigation was begun, in 1902, it was observed (26)
that in the dried dead larva,-t there are very large numbers of s1)ores,
but these, when inoculated into the ine(lia commonly used in the
laboratory, fail to grow. The cultures were sterile, except for an oc-
casional contamination.

The Present Investigation.

The following samples from different sources were examined in
1902:
Results of examination of specni en. of Amcricau foul brood diagnosed by the
experts at that time simply as "foal brood.''

Brood sent by- Date. Source. Bacteriological
findings.

Charls Stewart ............ June 12 ...... New York ........................... No growth.
W. D. Wright .............. Sept. 19 ...... Wisconsin ....................... 2 unidentified ba-
cilli.
W. D. Wright ............... Oct. 19.......Canada ........................ No growth.
W. D. Wright ............... Nov. 11 ......Wisconsin ........................... No growth; 4 sam-
pes.

Inasmuch as Bacillus altei was absent, it is evident that this condi-
tion is not European foul brood (26).
In 1903 the investigations were continued. Several media were
devised in which it was hoped that it would be possible to obtain a
germination of the spores which were observed the year before and
which failed to grow on our ordinary media. The one which proved

successful was prepared as follows: Larvue are picked front the brood
combs of a number of frames of healthy brood and a bouillon (bee-
larvo bouillon) is made from them following the same directions as
when bouillon is made from meat. Our first growth from these


41






THE BACTERIA OF THE APIARY.


spores was secured in an agar (bee-larve agar) made from this special
bouillon when Liborius's method for cultivating anairobes was used.
The technique for making cultures successfully from the diseased
material is not difficult if the following method is used: Place a
loopful of the decayed tissue of the larvw. into a tube of bouillon;
heat to 65' C. for 10 minutes to kill any vegetative forms which might
be present; incubate for 12 hours, and heat again to 650 C. for 10
minutes. This is usually sufficient, but it may be necessary to repeat
the same process. Liquefied bee-larvic agar in a test tube is then in-
oculated and incubated. The successive heating will destroy the veg-
etative stage of any spore-producing species which is common about
the apiary, e. g., members of the group represented by Bacillus A, as
described on p. 13-14 of this paper. Agar slant and bouillon, when
inoculated from this source, remain sterile; but when bee-larvae agar
is used a slow but abundant growth takes place. Under certain con-
ditions the growth appears very near or at the surface when cultures
are made in the above manner. A surface growth can be obtained
after a few generations by reinoculating slant agar of this same
medium.
The above method was used successfully in diagnosing the follow-
]ng samples from different apiaries:
i, cults of cxvamination of spccimiis of American foul brood, formerly called
simply foul brood."
B Bacteriological
Brood sent by- Date. Source. findings.

W. D. Wright ................ Oct. 19, 1902 Canada ............................. Bacillus larval.
W. 1). Wright ................ Nov. 11, 1902 Wisconsin .......................... Bacillus larva.
W. D. Wright ................ Nov. 11, 1902 Wisconsin .......................... Bacillus larvr.
C. II. W. W eber ............. .July 24, 1903 Ohio ................................ Bacillus larvr .
N. I). West................Aug. 3,193 Broome County, N. Y .............. Bacillus larvx.
N. 1). West .............. ... Aug. 3, 1903 Broome County, N. Y ............... Bacillus larvx.
N. D. West ................... Aug. 3,1903 Chenango County, N. Y ............ Bacillus larvx.

The results of these examinations show that Bacillus larve 1was
present in all the specimens examined, which suggests that it very
probably figures as an etiological factor in this disease. Other bac-
teria of different species are occasionally found associated with this
bacillus.
Bacillus larve.
Occurreitce.-Constantly present in diseased brood from colonies affected with
American foul brood.
Gelatin.-There is no growth.
Morph ology.--It is a slender rod, having a tendency to form in chains. This
is especially true when grown in bee-larva- bouillon.
Motility.-The bacillus is rather sluggishly motile.
Spores.-Spore formation takes place. This can be observed best in the dif-
ferent stages of the disease and decay of the larvae.
Oxygen requ iremenits.-When Liborius's method is used, the best growth
usually appears near to but not on the surface. After a few generations a
surface growth may be obtained.


42






THE SO-CALLED BLACK BROOD. 43


Bouillon.-There is no growth.
Glucose bouillon.-There is no growth.
Lactose.-There is no growth.
Saccharose.-There is no growth.
Agar plate.-There is no growth.
Bee-larvw agar.-The inoculations must be made with the medium liquefied.
The growth takes place near tV but rarely on the surface. Cultures must
pass thru a few generations before a satisfactory surface growth can be
specured.
Bee-larvw agar slant.-On the surface of this medium a thin, gray, nonviscid
growth takes place.
Glucose agar.-Slight growth has been observed in the medium. No gas is
produced.
Potato.-There is no growth.
Milk.-There is no growth.
Litmus milk.-There is no growth.
Fermentation.-In bee-larv-e bouillon no gas is produced.
Indol.-There is no growth in sugar-free bouillon.

THE SO-CALLED PICKLE BROOD."

The name pickle brood was given by Dr. William R. Howard. of
Fort Worth, Tex., to a disorder found in the brood of bees. He
stated that the cause of the disease was a specific finigus which he
called Aspergillas pollinis. His results have not been confirmed by
other investigators.
The bee keepers are sustaining a loss from a diseased condition in
their apiaries which they are diagnosing as .. pickle brood." The
larv usually die late in the larval stage. Most of them are found
on end in the cell, the head frequently blackened and the body of a
watery, granular consistency.
The following table gives a summary of the results of an exanina-
tion of specimens received labeled pickle brood:"

Results of exaniination of speciimeins1 of so-called "pickle brood."

Brood sent by- Date. Bacteriological findings.

W. D. Wright ...................... June 17,1902 .................... Two unidentified micrococci.
W. D. Wright ....................... July 31,1902 .................... No growth.
W. D. Wright .................... Aug. 4, 1902 .................... No growth.
M. Stevens ........................ Aug. 20, 1902 .................Unidentified bacilli.
W. D. Wright ...................... Sept. 2, 1902 .................... Unidentified bacilli.
W. D. Wright ...................... June 24, 1903 .................... Unidentified bacilli and yeast.
N. D. West ......... ......... ....No growth.
M. Stevens ........................ Aug. 20, 1903 .................. No growth.

The results of the examinations show that Aspergillus pollinis was
not found.. Further investigations must be made before any conclu-
sion can be drawn as to the real cause of this trouble.

THE SO-CALLED BLACK BROOD."

In 1890 some specimens of diseased brood were sent from New
York State to Dr. William R. Howard, of Fort Worth, Tex., and
unfortunately, after a short and inadequate study of the disease, he


43





THE BACTERIA OF THE APIARY.


reported it to be a new disease and called it "New York bee d
or "black brood." He described as its cause a species of bacteria
which he called Bacillus millii (31).
III our investigations of this diseased condition, which have covered
five years, we have not found an organism corresponding to Bacillus
mllii in any of the specimens that we have received; but we have
found Badibis alcei, the supposed cause of foul brood, to be present
constantly in samples of brood which the bee experts of New York
State say are samples of the same diseased condition as that received
by Howard.
From this we conclude that the diseased brood that has received
the name of New York bee disease" or black brood is really
genuine European foul brood.
PALSY OR PARALYSIS.
The disease known to the apiarists as palsy or paralysis attacks
the adult bees. The name is suggestive of the symptoms manifested
by the diseased bees. A number of bees affected were received from
Messrs. W. 1). Wright and Charles Stewart, taken from apiaries in
New lork State. 1I 1903 bacteriological examinations were made of
a number of bees so affected. Sev-eral species of bacteria were isolated
and sonie experimental inoculations made. but no conclusions could be
drawn frmol the results obtained as to the cause of the disorder.
From a study of the normal flora of the bee it was soon found
that we had here quite a number of species of bacteria present.
This fact stimulated a study of the normal flora, the results of which
are recolded in Part I. From this )oint the work can be carried
on with the hope that, if the disease has a bacterium as an etiological
factor, it may be found. It is believed by some bee keepers that
Bacll7ts qaytobi of Cheshire is the cause of paralysis, but this is not
claimed by Cheshire, and the belief is not grounded on bacteriological
findings.
SUMMARY TO PART II.
Following is a brief summary of the results of the present investi-
gation of bee diseases :
(1) There are a number of diseased conditions which affect the
apiary.
(2) The disease which seems to cause the most rapid loss to the
apiarist is European foul brood, in which is found Bacillus alvei-
first isolated, studied, and named by Cheshire and Cheyne in 1885.
(3) The distribution of Bacillus alvei in the infected hive is as
follows:
(a) The greatest number of infecting germs are found in the
bodies of dead larvae.
(b) The pollen stored in the cells of the foul-brood combs contains
man? of these infecting organisms.


441






CONCLUSIONS.


(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 Bacilb alniei.
(f) Bacillvs al'ei 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 Aniericanl foul brood, and the organism which
has been found constantly present in the disease we have called
Bacilifs larca'. 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 BardluN altci.
(6) It appears that European foil lrood was erroneously called
New York bee disease "or black brood by Dr. Win. R. Howard
in 1900.
(7) There is a diseased condition affecting the brood of bees which
is being called by the bee keepers pickle broo(d. No conclusion can
be drawn from the investigation so far as to the cause of the disease.
(8) A.1sper;rlwt llbib,;, ascribed by Dr. William R. IIoward 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 is 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 insuffi-
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."


45






46


THE BACTERIA OF THE APIARY.


BIBLIOGRAPHY TO PART 31.
12. ARISTOTELES. 13. SCHIRACI, 14. LEuCKHART. 15. MUHLFELD. 16. PREUSS. 17. (iEILEN. 18. LAMBRECHT. 19. HALLIER. 20. CORTNALLIA. 21. FIsCHER. 22. CHESHIRE and CHEYNE. The pathogenic history and history under cultivation
of a new bacillus (B. alvei) the cause of a disease of hive bees hitherto known
as foul brood. 23. DICKEL. 24. HARiSON, F. C. The foul brood of bees. College. Also in Centralblatt ffir Bakteriologie, Parasitenkunde und Infek-
tionskrankheiten, Zweite Abtheilung, VI Band, 1900.
25. LAMBOTTE. Recherches sur le Microbe de la "Loque," maladie des abeilles.
26. MOORE, V. A., and WHITE, G. FR.ANKLIN. A report on the investigation of an
infectious bee disease. 1903.
27. WHITE, G. FRA.NKLN. A report of the further investigation of bee (iseases of
the State affecting the apiaries of the State of New York. department of agriculture, Jan., 1904.
28. WHITE, i6. FRANKLIN. A report of the work on bee diseases for 1904. York State Department of Agriculture, Jan., 1905.
29. CHEShIRE. c Bees and bee keeping. Vol. TI, London. 1885.
30. MACKENZIE. Ontario Agricultural College Report, 1893.
31. IVOWAI{I, XXM. R. New York Bee Disease, or Black Brood. Bee Culture, Feb. 15, 1900.
32. BENTON. 33. SMITH, NA. G. <.British Bee Journal, Vol. XIV, p. 1225, 1886.
34. JONES, S. A. Foul Brood, its management and cure. 35. MCLEAN. 36. WARD, F. F. 37. SChIREuTER. 38. KLAMANN. 39. Reports of the bee keepers' association of the Province of Ontario, 1890.
40. PLANTA. 41. ItOWA RDI, W. R. Foul Brood; Its natural history and rational treatment.
42. McEvoy. Foul Brood, its cause and cure. 43. ROOT, A. I. 44. CoWAN. 45. GOVAN. 46. Formalin as a cure for foul brood. 13, p. 544, 1902.
47. WEBER, C. H. W. Formalin gas as a cure for foul brood. 1903.
48. BURR, R. Bakteriologische Forschungen uber die Faulbrut. Bienen-zeitung, Nos. 10 and 11, 1904.
49. REIDENBACH. Ist das Vernichten der Faulbrautstocke das iicherste Mittel zur
Bekampfung der Faulbrut? 50. NEUMANN. Zur Klarung der Faulbrutfrage.
















INDEX.



Page.
Acknowledgments of author --------------------------------------------- 2
Apiary, diseases --------------------------------------------------------- 30-46
normal, bacteria found ------------------------------------------- 12-30
tabulation of micro-organisms ----------------------------- 28
technique for study of bacteria ----------------------------------- 7-13
Aspergillus pollinis, probably not cause of "pickle brood" ------------------ 43, 45
Bacillus A (B. mesentericus ?), description --------------------------------- 13-14
mistaken for Bacillus alrei ----------------- 3, 29, 33
on combs in normal apiary ------------------- 13, 29
on healthy adult honeybees -------------------16
alvei, confusion with Bacillus A --------------------------------- 29, 33
description ------------------------------------------------ 36-37
-discovery -------------------------------------------------- 31
distribution in infected hives ------------------------ 38-39, 44-45
European foul brood produced experimentally by inoculation.. 37-38
name wrongly given to other bacteria ---------------------33-34
not present in American foul brood ------------------------4, 32
present in European foul brood ----------------------- 3, 32, 35, 44
so-called "New York bee disease" or "black brood" 3
relation with Bacillus ?uesentericus rulgatus -------------------- 31
B, description --------------------------------------------------- 15-16
in pollen and intestine of healthy honeybees ------------------ 15, 29
cholera suis, description ------------------------------------------ 21-22
in intestine of healthy honeybee --------------------- 21, 29
cloac.T, description ----------------------------------------------- 19-20
in intestine of healthy honeybee -------------------------- 19, 29
coli communis, description ---------------------------------------- 20-21
in intestine of healthy honeybee ------------------20, 29
E, description ---------------------------------------------22-23
in intestine of healthy honeybee ------------------------------- 22, 29
gaytoni, considered by some as cause of paralysis of bees ------------ 44
larvx, description ----------------------------------------------- 42-43
formerly termed Bacillus X -------------------------------- 40
present in American foul brood --------------------- 32, 40, 42,45
mesentericus ? (See Bacillus A.)
mesentericus vulgatus, relation with Bacillus alvei -------------------- 31
millii, not found in so-called "black brood" ----------------------- 44
subgastricus, description -------------------------------------23-24
in intestine of healthy honeybee --------------------- 23,29
X=Bacillus larvx --------------------------------------------- 40
47






48 INDEX.


Bacteria, from combs of normal apiary ----------------------------- 13-15
pollen of normal apiary .................................... 15-16
in healthy larve not usual ....................................... 16,29
honey from normal apiary not usual ------------------------ 16,29
intestine of healthy honeybee ................................. 18-2
of apiary, cultures, how obtained ................................. 8
suggestions for description ..................... 10-12
which are described in paper .................. 9
differentiation and identification ........................ 9
material for study how obtained -----------------------7I
media employed for cultures ........................... 10-12
oxygen requirements --------------------------------- 10
staining properties ..................................... 10
technique in study ------------------------------------ 7-13
variations in size ...................................... 9-10
of normal apiary ----------------------------------------------- 12-30
on healthy adult honeybees ------------------------------------- 16-18
Bacterium acidiforians, description ........................................ 14-15
on combs in normal apiary ------------------------- 14
cyIneus (Micrococcus cyancus), description- .------------ -.. 16-17
on combs of normal apiary --------- 29
on healthy adult bees and pollen .... 16
D, description .................................................. 19
in intestine of healthy honeybee .............................. 19-29
mylcoides, description ........................................... 24
in intestine of healthy honeybee ........................ 24,29
Bee bread, fermentation considered cause of foul brood formerly ............. 31
diseases ............................................................. 30-46
history -------- ------------------------------------30-32
modified names necessary ..................................... 34
theories as to cause ............................................ 30-31
Bees, diseased adult, parts of body infected by Bacillus alrei ................. 39,45
healthy adult, bacteria found externally .............................. 16-18
in intestine ............................. 15, 18,25,29
Saccharomyces F in intestine ........................... 25,29
ovary of queen, Bacillus alrei present accidentally ................ 39,45
Bibliography to Part I ...................................... .............. 29-30
II -------------------------------------------------- 46
Black brood" European foul brood ------------------------------------ 44,45
=foul brood of Cheshire and Cheyne ....................... 3
occurrence of Bacillus ahei ................................. 35
origin of term .......................................--. 31,43-44
term may be discarded ----------------------------------- 45
Blight of flowers, supposed by ancients related to bee diseases ---------------- 30
Brood, diseased, occurrence of Saccharomyces G .......................... 26-27,29
Climatic conditions, as affecting European foul brood ----------------------- 38
Combs, of healthy apiary, occurrence of bacteria ------------------------ 13-15
fungi ---------------------------25
Saccharomyces roseus ................ 25,26
diseased apiary, occurrence of Bacillus alvei ...................... 39,45
Oryptococcus alvearis, formerly considered cause of foul brood ---------------- 31
formerly considered cause of foul brood ------------------------ 31
Cultures, of bacteria of apiary, how obtained .................... 8------------ 8






IINDEX. 49

Page.
Cultures, of bacteria of apiary, media employed -------------------------- 10-12
suggestions for description ------------------ 10-12
those described ----------------------------- 9
Differentiation of bacteria of apiary --------------------------------------- 9
Formaldehyde gas, insufficient disinfectant against European foul brood as
ordinarily used -------------------------------------------------------- 39-40
Foul brood, American, application of term -------------------------------- 45
author's investigations ----------------------------- 41-42
Bacillus alrei not present -------------------------- 4, 32
larvx present ------------------------32, 42, 45
confusion with foul brood of Cheshire and Cheyne. 4, 40, 45
symptoms ---------------------------------------- 40-41
confusion regarding diseases ----------------------------------- 33
disease of Cheshire and Cheyne renamed European foul brood. 3, 32, 44
European, application of term -------------------------------- 45
author's investigations ----------------------------- 34-36
Bacillus alvei present ------------------------ 4, 32, 35, 44
foul brood of Cheshire and Cheyne ------------- 3, 32,44
formaldehyde gas insufficient disinfectant as ordinarily
used -------------------------------------------- 39-40
more destructive in cool, damp weather -------------- 38
produced by experimental inoculation with Bacillus
ahvei ------------------------------------------- 38
symptoms ------------------------------------32-33
of Cheyne, named European foul brood ------------------------ 32
term applied to two distinct diseases ------------------------ 31-32,45
use of term in New York State -------------------------------- 31
Frames, in diseased apiaries, occurrence of Bacillus alrei ------------------- 39, 45
Fungi, formerly considered cause of foul brood ----------------------------- 31
in intestines of healthy honeybees --------------------------------- 25
pollen and combs of normal apiaries ----------------------------- 25
Fungus. (See Aspergillus pollinis, Cryptococcus, Cryptococcas alvearis, and Paii-
histophyton ovatum.)
Hives, of diseased apiaries, occurrence of Bacillus alrei --------------------- 39, 45
temperature approximates that of warm-blooded animals ------------- 29
Honey, from foul-brood combs, occurrence of Bacillus dlei ----------------- 39, 45
healthy hives, quite uniformly sterile ----------------------- 16, 29
Ichneumon ap ium mellficarium, formerly supposed cause of infectious bee dis-
ease ------------------------------------------------------------------ 30
Identification of bacteria of apiary ---------------------------------------- 9
Intestine of healthy honeybee, occurrence of bacteria --------------------- 18-25
fungi ------------------------- 25
Saccharomnyces F -------------- 25, 29
Larve of honeybee dead from disease, occurrence of Bacillus alrei ----------39, 44
healthy honeybee, usually sterile ------------------------------ 16, 29
Micrococcus C, description ------------------------------------------------ 17-18
on combs of healthy honeybees ------------------------------ 29
healthy adult honeybees --------------------------------- 17
cyaneus. (See Bacterium cyaneius.)
Micro-organisms normally present in the apiary, tabulation ----------------- 28
Morphology of bacteria of apiary ----------------------------------------- 9-10
'New York bee disease." (See "Black brood.")






50 INDEX.

Page.
Nonpathogenic bacteria of honeybees, necessity for study ---------------
Oxygen requirements, of bacteria of apiary ................................. 10
Palsy. (See Paralysis.)
Pahistophyton oraum, a related fungus, formerly supposed cause of bee disease. 30
Paralysis, of honeybees, cause unknown .................................. 44,45
"Pickle brood," Aspergillus pollinis probably not cause ...................... 43,45
bacteriological findings from author's examinations .........
disease of bees ..................................... 31,43,44,45
Pollen, in foul-brood combs, occurrence of Bacillus alvei ------------------- 39,44
healthy combs, occurrence of bacteria --------------------------- 15, 16
fungi ............................. 25
Propolis. (See Combs.)
Pseudomonaswfluorescen~s liquefciens, description ............................. 24-25
in intestine of healthy adult honeybee -. 24,29
Saccharonmyces F, description ........................ : ...................... 25-26
in intestine of healthy adult honeybee ------------------- 25,29
G, description ............................................. 26-27
in dead larvae of diseased honeybees -------------------- 26,29
in normal apiary ............................................ 25-27
roses, description ........................................... 26
in comb of normal apiary --------------------------- 25,29
Staining properties of bacteria of apiary ----------------------------------- 10
Summary to Part I ....................................................... 29
I -----.......................... -..................... 44-45
Technique in study of bacteria of apiary ---------------------------------- 7-13
Variations in size of bacteria of apiary ------------------------------------ 9-10
Wax. (See Combs.)
"X Brood" = American foul brood ........................................ 40

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