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UNITED STATES
DEPARTMENT or AGRICULTURE

DEPARTMENT CIRCULAR 284

Washington, D. C. Issued March, 1926 |

THE STERILIZATION OF AMERICAN
FOULBROOD COMBS

A. P. STURTEVANT

Associate Apiculturist, Bee Culture Laboratory, Bureau of Entomology

CONTENTS
Page Page

BOC HOnet sss 54 styl alee eh eka Fe 1 | Observations—Continued.

“HG TEL NOG IS a Se A a eA se 5 Commercial alcohol-formalin solu-
Diseased material for tests_____. 5 BLE O LG Ys clad ata en US ple a Ne 8
Wullituneseas & bien 2 Bo ee ed 6 Permeability of cappings______-. 14
Polit onsetestedeiws ee ss eee 7 Walcummrtreasbm erie ee 16

MEOSEIRV ALOIS = ee beet hg See ea 8 Perforation of cappings______-—_. aly
SOapESOlUPONS = 2 es ee ee 8 Cappings removed with knife__.. 19
Liquids of low surface tension Wials bine xc om p see ee ee 21

other than ethyl alcohol______. 10 DISCUSSTOME: SEE 9h Se Pa I ES ed i 21
Miscellaneous solutions_________. AEG CONCIUISION Gs FaceUa nk ka reuaws Si Srariea 25
Solutions wath’ diluted alconole=]=— :12-)| Miterature’ cited!= 229 eee 27

INTRODUCTION

A new era in the treatment and control of American foulbrood
has been opened by the use of disinfectants such as an alcohol-
formalin solution for the sterilization of combs infected with this
disease of the brood of bees. Widespread interest has been aroused
throughout the beekeeping industry by the apparent success of this
method of treatment, which eliminates to a great extent the large
losses previously caused by the necessary destruction of all combs
infected with American foulbrood.

Numerous attempts have in the past been made to use formalde-
hyde gas as a disinfectant, but it failed to sterilize infected combs
completely except in a few carefully controlled cases, so that even-
tually the use of this material for disinfecting combs was entirely
abandoned. White (25, 26)1 found that the gas penetrated the
combs slowly, even when used in a tightly sealed jar, and that as
usually applied in the apiary it was insufficient to disinfect the combs
completely. More recently Maassen and Borchert (78) and Bor-
chert (7), experimenting with formaldehyde gas generated from

1 Reference is made by number (italics) to “ Literature cited,” p. 27.
68773°—26——1

) Department Circular 284, U. S. Dept. of Agriculture

solid paraformaldehyde, came to a similar conclusion. The great 4)

difficulty always was found to be the holding of the gas in contact —
with the combs, without leakage to the outside, for a sufficiently
long time to allow for this slow penetration ‘and the complete
sterilization of all the spores of Bacillus larvae.

Late in 1922 the apparently successful use of a new liquid disin-l
fectant for treating infected combs was announced by J. C. Hutzel-
man, of Glendale, Ohio (1/7). The disinfectant is composed prima-
rily of 20 parts of formalin solution to 80 parts of specially denatured

alcohol. The infected combs are placed in a tank and completely —

immersed in this liquid for 48 hours. At the end of that period the
combs are removed and as much as possible of the excess liquid is
removed from them, an extractor being used when available. They

are then allowed to dry, until no odor of formaldehyde remains,

;
é
i
33

before the combs are returned to healthy colonies. The use of ©

alcohol as the carrier for the formaldehyde, which is the active

sterilizing agent in the solution, is based, according to Hutzelman,

upon two properties. Since alcohol is a hquid of low surface tension
the solution is supposed to enter readily and permeate thoroughly
all empty spaces in the combs. Further, it is claimed that this alco-

holic solution, because of its solvent action, penetrates the wax and

propolis, as well as cells filled with pollen, all of which may contain

spores of Bacillus larvae. Hutzelman is of the opinion that the

sterilization of all of these is necessary for the successful disinfection
of infected combs. He has also stated that while all sealed honey
cells must be opened, sealed brood cappings need not be removed if
the combs are treated for 48 hours, as the brood cappings are porous
and are penetrated by the alcoholic liquid. Much credit is due
Doctor Hutzelman for thus being the first to demonstrate the prac-
tical possibility of holding the formaldehyde in more or less direct
contact with the diseased material in combs infected with American
foulbrood for a sufficiently long period to kill the spores of B. larvae.

In June, 1924, Dan H. Jones (7/4), of Ontario, Canada, reported
the results of some cultural experiments with various disinfectants,
including the alcohol-formalin and also water-formalin solutions.
He states that the alcohol-formalin solution

is effective in killing the spores of Bacillus larvae in 24 hours in foulbrood —

affected combs when it comes in contact with the larval scales in open cells.
In case of capped cells, however, it would appear that 48 hours’ immersion is
essential.

Concerning various mixtures of water and formalin he says:

Formalin diluted with water in as high a dilution as 15 per cent formalin to

85 per cent of water is effective in destroying in 24 hours the spores of Bacillus —

larvae as they occur in larval scales in open cells. Although in our experiment
a 48-hour immersion in the formalin and water mixtures gave us a hundred
per cent sterile results in the cultures made from capped cells, we can searcely
expect that such would always be the case, as the porosity of the cell caps
varies considerably.

In a recent article Jones (1/5) gives a further report of investiga- —

tions with water-formalin solution, as indicated by the following
table from his paper:

tacit

The Sterilization of American Foulbrood Combs 3

Combs immersed 24 hours Combs immersed 48 hours

Disinfectant Cultures from Cultures from Cultures from Cultures from

capped cells uncapped cells capped cells uncapped cells

1 2 3 1 2 3 1 2 3
Rormalin: 1002222522222 <. 0 0 0 0 0 0 0 0 0 0 0 0
Formalin 50, water 50__---- 0 0 0 0 0 0 0 0 0 0 0 0
Formalin 25, water 75___--- 0 0 0 0 0 0 0 0 0 0 0 0
Formalin 20, water 80-_----- 0 Oo; + 0 0 0 0 0 0} 0 0 0
Formalin 15, water 85_----- 0 0 + 0 0 0 0 0 0 | 0 0 0
Formalin 10, water 90-_-___-- 0 + + 0 0 0 0 0 0 0 0 0
Formalin 50, alcohol 50___-- 0 0 0 0 0 0 0 0 0 0 0 0
Formalin 20, alcohol 80____- 0 0 0 0 0 0 0 0 0 0 0 0
Hutzelman’s solution --._-. 0 0 + 0 0 0 0 0 0 0 0 0

He further says:

It will be seen that in the case of uncapped cells, after twenty-four hours’
immersion in all dilutions of formalin used, the spores of B. larvae were
killed. In case of the capped cells, however, a few of the spores were not
killed in this length of time either in the water dilutions or in Hutzelman’s
solution.

After forty-eight hours’ immersion, however, all spores were killed in capped
cells as well as in uncapped cells. Thus, in these experiments, the water dilu-
tions of formalin proved to be as effective as the alcohol solutions in destroy-
ing the spores of B. larvae as they occur in the scales of the infected brood
combs.

Although this method has been in use for only about two years,
one of the bee journals has published several reports concerning the
successful use of the alcohol-formalin solution, describing in all the
treatment of several thousand combs. One of the most notable cases
is that of O. E. Barber, of Ohio, reported by G. S. Demuth (8), in
which some 6,000 combs were treated during the summer of 1923,
apparently without recurrence of disease. There have been other
similar cases, all indicating that this solution is satisfactory. On
the other hand, during the latter part of the summer of 1924, J. L.
Byer, of Ontario, Canada, who had treated 400 infected brood ‘combs
and 1,100 super combs (2), found several cases of recurrence of
disease (3) in colonies made from package bees placed on these
treated combs. Some of these combs were sent to Doctor Jones,
but cultures made from them failed to give growth (4). Two simi-
lar cases of recurrence of disease after the use of an alcohol-formalin
solution have also come to the attention of the Bee Culture Labora-
tory, through correspondence. So far as can be learned, all pre-
cautions were taken in these cases, and no adequate explanation can
be given for the recurrence of disease other than that for some rea-
son the disinfection could not have been complete.

The alcohol-formalin treatment of foulbrood combs received con-
siderable discussion at the annual convention of the Ontario Bee-
keepers’ Association in 1925, as reported by Byer (5) in one of the
bee journals. He says:

While reports in the main were favorable, yet some outstanding failures
were reported, and the department at Guelph is not yet prepared to advise the
treatment of badly diseased combs from the brood chamber. They do, how-

ever, recommend treating all super combs from diseased colonies, as every test
made with such combs failed to produce any disease. From the address of

4 Department Circular 284, U. S. Dept. of Agriculture

Professor Jones (15) we learned that in every case water used with the
formalin gave just as good results as when alcohol was used. The assistant
apiarist, Mr. Jarvis, also substantiated these conclusions. - .

In a recent article G. L. Jarvis (12), of the Ontario Agricultural
College, Guelph, Ontario, describes results of experiments in the
apiary in collaboration with Doctor Jones concerning the effective-
ness of water-formalin and alcohol-formalin solutions. Colonies
made with 2-pound packages of bees were used in which to test
combs treated in various ways. He says:

From the results of these experiments and laboratory tests we have arrived
at these conclusions: First, that both the formalin water and alcohol formalin
solution will kill all germs causing American foulbrood in open cells. Sec-
ond, that there is still a doubt as to the effectiveness of both solutions in the
ease of capped cells.

With this in mind, we are ready to advocate the use of the formalin water
solution for sterilizing super combs from diseased colonies after the honey has
been extracted. The strength of the solution must not be less than 15 per
cent formalin to 85 per cent water and the combs immersed for at least 12
hours. The uncapping must be well done; that is, there must be practically
no sealed honey in the combs. The combs should be held, if possible, until the
following spring before being given back to the bees.

Although in our experiments so far we have had no reappearance of
disease in treated brood combs, except in check colonies, some other bee-
keepers who have tried the alcohol-formalin treatment have not been so suc-
cessful. However, the super comb is the doubtful comb, and we believe it is
well worth while to know of a cheap solution, such as the formalin-water,
which will eliminate this doubt. Again, if all combs from a diseased colony
were melted or burned there would be approximately three super combs de-
stroyed to every brood comb, and the super combs would likely average a better
quality than those in the brood chamber.

G. H. Vansell, in California (23), recently has reported promising
experiments in sterilizing foulbrood combs, in which he used various
mixtures of formalin in soapy waters. The results were found
encouraging as to sterility and cost. No details are given concerning
methods or results.

Since the announcement of this new method of treating combs
many beekeepers, as well as the investigators cited above, have been
stimulated to experiment for themselves. In 1924, 33 samples of
treated comb were submitted by beekeepers to the Bee Culture Labo-
ratory for cultural examinations as to the sterilizing efficiency of
the various solutions used. In a majority of cases the disinfectant
used was not described. In about one-third of the cases some varia-
tion of the alcohol-formalin solution as devised by the person
sending the sample was used. Three of these cases, however, were
specifically said to have been treated with the commercial alcohol-
formalin solution. Of the 33 samples, 10 gave cultures showing
growth of Bacillus larvae from both open and sealed cells. Four-
teen of the 33 samples, including 2 samples that had been treated in
the commercial solution, gave cultures showing no growth from open
cells but good vegetative growth of B. larvae from many of the sealed
cells. Nine of the 33 samples, only 1 of which was known to have
been treated with the commercial solution, gave cultures showing no
growth from either open or sealed cells, thereby indicating complete
sterilization.

It would, therefore, seem probable that, although the method of
treating infected brood combs with an alcohol-formalin solution is a
step in advance in the control of American foulbrood, there apparently

ee se ee) ee

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Pee) 2

vy ees)

The Sterilization of American Foulbrood Combs 5

is room for improvement which will eliminate the danger of occasional
cases of failure. Because of the widespread interest in this subject,
preliminary work was started early in 1924 at the Bee Culture Lab-
oratory with the purpose of making an exhaustive bacteriological
study of the efficiency of various disinfectants, including the com-

‘mercial aleohol-formalin solution as well as water-formalin solu-

tions. It was hoped that the results of the investigation by lab-
oratory methods would form a basis for practical work in the apiary.
As the work has developed, numerous difficulties have been encoun-
tered which indicate that the problem of the perfect sterilization of
American foulbrood combs is neither simple nor as yet fully solved.

The results given herein are of a preliminary nature, the data being
in some cases incomplete; but they are given for what they seem
to indicate. Since these investigations were started Doctor Hutzel-
man has taken out a patent (72) on the solution devised by him,
issued October 14, 1924. In the light of the issuance of this patent
it seems advisable to state that the United States Department of
Agriculture can assume no responsibility for the use of any of the
solutions or processes described and discussed in this paper if they in
any way infringe the patent.

METHODS

DISEASED MATERIAL FOR TESTS

In devising methods for testing the efficiency of various disin-
fectant solutions, procedures were adopted corresponding as closely
as possible, on a reduced scale, to the actual practice of beekeepers.
Combs affected with American foulbrood, containing as many scales
as possible both in sealed cells and in open cells, were obtained from
various sources. From the brood areas of these combs test pieces
were cut approximately of the standard size of 114 by 21% inches.
Glass specimen jars of about 160 cubic centimeters capacity, with
fitted glass covers, were used to hold the test solutions and pieces
of comb treated. A standard volume of liquid of i00 cubic centimeters
was used throughout the tests, the proportion of liquid to comb be-
ing approximately that in the the regular 10-frame tanks used by
beekeepers for disinfecting combs. Loss of liquid after each con-
secutive comb had been removed was made up to 100 cubic centimeters
with more solution before a new piece was immersed. Several
pieces of comb were passed through each lot of solution consecu-
tively, in keeping with the actual apiary practice of treating many
combs in the same solution. The piece of comb to be tested was
placed in the empty jar and fastened down with a wire spring to
prevent its floating. The solution was poured into the jar slowly
to permit the liquid more readily to enter the open cells. In accord-
ance with the most approved apiary practice, there was no shaking
of combs to aid in removal of air bubbles. The test combs were
then allowed to soak for 24 or 48 hours, as the case might be. No
immersions of less than 24 hours were tried in this series of experi-
ments.

Upon removal of the pieces of comb from the disinfectant, as
much as possible of the excess liquid remaining in the cells and on
the surface of the comb was removed by vigorous shaking. Each

6 Department Circular 284, U. S. Dept. of Agriculture

piece was then inclosed in a piece of filter paper to protect it from
dust and was allowed to dry at the ordinary room temperature of
the laboratory until no odor of formalin could be detected.

CULTURES

Yeast-extract egg-yolk agar medium, as described in a recent paper
by the writer (21, p. 136) was used in the form of slants for the pur-
pose of making the cultural tests of the scales treated by the various
solutions. In order that there should be a sufficient amount of water
of condensation at the base of the slants, the necessary number of
tubes of fresh medium for each group of combs to be tested were
made up by the addition of sterile egg yolk to tubes of the base
medium. With each new lot of medium control cultures were made
from untreated scales of American foulbrood.

The removal of scales or other diseased remains from the cells of
the treated combs was accomplished by means of a lancet-shaped
dissecting needle which had just been sterilized in a flame and
allowed to cool. The scale (or other remains) was then carefully
placed in the water of condensation of the tube of culture medium,
one scale or material from one cell to a tube of medium. Cappings
from sealed brood were removed with the hot needle, and after
resterilizing the needle the scale contained in one cell was removed
and placed in a culture tube. After the diseased material had been
allowed to soak in the condensation water of the tube for about an
hour, or longer ii necessary, until the dried scale had softened, it
was macerated and spread over the surface of the slant by means of
a stiff platinum loop.

Cultures were at hrst made of material from one open cell and one
sealed cell from each comb treated, on the assumption that for a
given solution the sterilization would be uniform for a definite period
of time, but it soon became evident that there was a variation in the
rapidity with which the various solutions penetrated the brood cap-
pings. At least five cultures were therefore made from open cells
and five from sealed cells in each piece of comb, amounting to ap-
proximately 3 to 5 per cent of all cells in a piece of comb of the size
used in these experiments. The percentage cultured of the scales
actually present is really much higher than this, since a piece of
comb of that size seldom has a scale in every cell.

After incubation for 48 hours at 37° C., cover-glass smears were
made from the material on the surface of the slants. A large loop-
ful of a mixture of material from different parts of the slant in a
drop of distilled water was used in making a good-sized smear on
the cover glass. After drying the cover glass in the air and passing
it quickly through a flame three times the smears were stained for
about 20 seconds with Ziehl-Neelson carbol-fuchsin diluted with an
equal quantity of distilled water. The cover glasses were then care-
fully washed in water, dried, mounted with Canada balsam, and
examined under the microscope. At least 20 to 30 fields were ex-
amined to determine whether there had been any germination of
spores of Bacillus larvae or vegetative growth not visible on the
slant. When no germination of spores was observed, as a rule only
one examination of the culture was made at the end of 48 hours’
incubation, since if there are any spores in a condition to germinate

a ae eee

The Sterilization of American Foulbrood Combs 7

they will do so within that pened _ When a few spores were found
to have germinated, but without evidence of any vegetative growth,
a subculture was generally made, using a generous quantity of mate-

- rial from the surface of the original slant. When good vegetative

growth was observed it was so recorded as positive growth. In
some instances when there was doubt regarding a culture several
more cells of the same kind were cultured from the same piece of
comb.

SOLUTIONS TESTED

In the earlier part of this investigation an attempt was made to
find a substitute for alcohol as a carrier for the disinfectant, because
of the difficulty of purchasing pure grain alcohol and the compara-
tively high cost of the commercial alcohol-formalin solution. Since
certain substances are used in various insecticide sprays to increase
the wetting and spreading powers of the spray solutions (6, 19), it
was suggested that if a liquid could be found which would spread
easily over the surface of the wax comb and diseased remains, forma-
lin added to such a liquid would be carried by it and brought into
contact with those remains; and that it would penetrate sufficiently
to kill the spores, even though the cells, particularly sealed cells,
might not be completely filled with the liquid. This property in
insect sprays is often obtained by the addition of soaps of various

_ kinds to the solutions. The addition of soap also tends to lower the

surface tension of a water solution. Dilute solutions of various types
of soaps, both soft and hard, were used to form the carrier for the
20 per cent of formalin. By experimentation with dry pieces of
comb it was found that only a comparatively small quantity of soap
can be used in a solution, as too much causes the liquid to become
viscous and jelly-like, inhibiting its entrance into the cells. In the
tests with soap solutions varying quantities of half-normal sodium
oleate soap, cottonseed-oil soap, and a 10 per cent solution of one of
the common hard toilet soaps were used in making up the solutions
for the disinfection of diseased combs. From 0.5 to 2 cubic centi-
meters of each of the various soaps was used per 100 cubic centi-
meters of a 20 per cent solution of formalin in water; if much more
than 2 cubic centimeters was used the solution became too viscous.

In a recent article King (76) describes experiments in sterilizing
American foulbrood combs by using a disinfectant solution consist-
ing of the 20 per cent water-formalin solution, with sufficient soap
to form suds (about 1 pound to 5 gallons of solution), the addition
of the soap causing the solution to enter the cells more readily.
After 48 hours’ immersion, followed by extraction and a short drying
period, the combs were given to a healthy colony. Only one colony
was used in the experiment, but King reports that no disease ap-
peared in it when observed carefully at intervals for seven weeks
after it had been given the treated combs.

Among substances similar in physical properties to ethyl alcohol,
a commercial methyl-ethyl ketone solution, acetone, and iso-propyl
alcohol were tried as carriers for formalin, but acetone and iso-propy]
aleohol were too expensive for practical use.

The use of hydrochloric acid in the sterilization of imported
hides, as a preventive of anthrax (20), a disease caused by a spore-

8 Department Circular 284, U. S. Dept. of Agriculture

forming organism similar in nature to Bacillus larvae, suggested ©
its use in the treatment of American foulbrood. Various dilute |
solutions of iodine were suggested, since iodine is successfully used |
in the sterilization of drinking water. <A solution of formalin in~
water, with the addition of acetic acid to increase its penetrating ©
power, is often used for fixating histclogical preparations; for
the treatment of American foulbrood a similar solution was ac-—
cordingly suggested. ui

A number of tests were made to compare the germicidal efficiency —
of the commercial alcohol-formalin solution, now frequently used
by beekeepers, with that of the various other solutions tried, and
in particular with the 20 per cent solution of formalin in water. ~
Kronig and Paul (77) have shown that increasing quantities of ©
either ethyl or methyl alcohol added to a solution of formalin in~
water progressively decreases the germicidal efficiency of the solu-
tion. Tests were therefore made with various dilutions of dena-
tured alcohol as the carrier for the 20 per cent solution of forma-_
lin. Formula No. 1 (22, p. 100) for specially denatured alcohol was
used as a basis for these dilutions. Commercial alcohol-formalin —
solution was diluted in a similar manner by the addition of varying —
quantities of a 20 per cent solution of formalin in water. ‘These-
tests were made to determine whether the presence of a greater pro- —
portion of water mixed with the alcohol changes the germicidal —
efficiency of the solution. %

In this preliminary work no measurements were made of surface ©
tension and other physical properties, but the bacteriological re-
sults were used as a criterion of the relative efficiency of the various —
disinfectant solutions tested.

OBSERVATIONS

SOAP SOLUTIONS

In the use of soap-formalin solutions it was found immaterial —
whether soft or hard soap is used. The greatest difficulty with
the soap solutions, aside from the fact that they do not seem to —
penetrate sealed cells uniformly, which will be discussed later, is —
a rapid change in reaction from alkalinity to marked acidity during
the period of immersion of one piece of comb. The acid reaction
causes the soap gradually to precipitate, so that the solution is ~
soon little better than ordinary water-formalin solution. Even the
addition of sufficient normal sodium-hydroxide solution to bring
the reaction back to: alkaline after the comb is removed fails to
remedy this fault, as the precipitated soap does not return into
solution on the addition of the sodium hydroxide. The prolonged
action of soap and alkali on the wax of the comb during an im-
mersion of 48 hours causes more softening of the cell walls than
is desirable, and makes the combs quite fragile. Since some of the
sealed cells in practically every series of combs tested failed to be
sterilized, as indicated from the cultures made (Table 1), this’
solution was dropped from consideration for use with combs having”
sealed brood. |

In the case of the sodium-oleate-formalin solutions, 59 open and-
59 sealed cells were cultured in 10 series, varying in length (Table

The Sterilization of American Foulbrood Combs 9

1). Cultures from 54 open cells showed no growth of Bacillus
larvae, whereas 5 culture tubes were contaminated. Cultures from
45 sealed cells showed no growth of B. larvae, 2 culture tubes were
contaminated, 11 showed ood growth of B. larvae, and 1 showed
only a few spores germinated, or 12 positive cultures in all. In
brief, of the sealed cells 20.5 per cent were not sterilized.

TABLE 1.—Cultural results of various tests with samples of comb treated 48
hours in water-soap-formalin solution

ge OS a pA
comer males Open cells |= = Sealed cells ae
BE Hes B28
Merete cine n leow|s ! Sas n >| ' cas
D> Sa x) les g — BL SS 3 iaSicy aes Paitin
ala |32 |e (Sete 18 lee Fea RS LAS foots
= Sia |Ssaqle |BSi8 |S [Ses | |ES/E_|S | gsm
ieee 5 |ES|gRS\ viemleals la. | sisalSeie | aa.
om | 6 aaa pes o/s BI »/2O 1 ot Ol} SOs
Tal SSNS) es c of aig Al Lian = aida
=I GIS e455 SSIS 8128 ec e SHlos SMne ROSS
a [aU |Sha Ss eee WS Als oe RIS SES IE olf") S2ER
~ S S reat ephed Sols. |3 oO Ese Sis Sal Os
af 2) B/E sSe/B See ue ewes See |S |neen
3 3 R16 Sog| 5 js wis (3B |e Rwols pools |B |e wo
ole |e IA Ie 4 jOnjO° jo. ie Zz 1010 10 0)
Cre | €3¢.), C. ca, Cc.
0.5} 80] 20] 0 7 ee i ase ln 4/ 0| 4] 0
0.5; 80| 20; 0 4) 4) 05048 feyly| ete sz 4}-01'2| 2
1 | 80] 20] 0 ° p : 6 0 Sh tec 5 1| 4] 0} Third.
ASE) 80i | OGIO) EIT EDIT Sy oO AG ene eee 2). 3} 0 0.
Half-normal sodium |} 1 80} 20 1 Gilg OTRO Galt Onl see 6| 0| 6| 0 |
oleate. | Ae ea Orono Gi ys CaF 20> E71 G6:7F> Othe Stee 6| 0} 6; 0|
1 | 80]} 20} 1 EV Og Be i pee 9\11] 8] 0, Second
oe ge oa ei ne a St) eens BT 2 | 3 |c0 eine
2 80 | 20 Ziad elf | eee ee 2) 7] O| six
a2 80.1 SON 1 6| 6] 0 6) 0 j-------- ae 2 | Ot Rirst:
1p BOM 20H s1b ees 91 8 |p Ot Oto On} 228 2 ae 9} 1] 8] 0 :
Pel. 80) 20. 2 Ne PRC ee De tae es Sa 9/32) 7| 0| Third
Pate soe 20's 1 Dales 2 ne Oy ye. [ee On} teen 2/11] 1] 0| Second
ee SOs) soon ies = 42) 5 44) 20)| ae | ee 41/22/ 1| 1| First.
1 po 20 1 7 a iH fa a al ee gow tum | kaa) Second
: Dit We: <25 l= 0 4 Od ee SE 4{| 3] 1] 0| First
Brea iolit cee he | ek 25] Op a 52) 0) 4) 0) .4| 3| 1| 0] Second.
: 2+) 80) 20) 2 6 Bae O leet 6 120! 26 1.50
2 <0 | 2 Gere Ga Osi Br pe ore t | 561 0| 6] 0
Oras 200 OS 4th Atl OE 4) Olea). 4| 1] 3] 0| First.
1 | 90; 10| 0 A al GS Aw Of ll. 4111] 3] 0| Fourth.
0.5 | 15| 0 42) 4 [ARIE Qt O| First. |-4} 2) 2) 0.| First.

1 Few spores germinated.

? Cottonseed-oil soap.

“One showed few spores germinated.

‘Used entire brood combs in tank.

5 Sealed cells with cappings perforated compared with open cells.

In the case of the solutions of hard toilet soap in 20 per cent
formalin, 39 open and 39 sealed cells were cultured. Cultures from
38 open cells showed no growth, and the culture from 1 was contam-
inated. From the sealed cells, 22 cultures showed no growth, 1
was contaminated, 13 showed good growths of Bacillus larvae, and
in 3 only a few spores had germinated; in all, there were 16 posi-
tive cultures, showing that 41 per cent of the sealed cells were not
sterilized. The variation in the quantity of soap added to the
solution seemed to have little effect on the variable penetration
and sterilization of the scales in sealed cells, but the hard-soap
solutions appeared somewhat iess efficient than the solutions of
soit soap.

Three short series (see bottom of Table 1) were tried, using
smaller quantities of formalin, but these solutions proved inefficient
as germicides in open as well as in sealed cells.

68773 °—26——2

10. Department Circular 284, U. S. Dept. of Agriculture

LIQUIDS OF LOW SURFACE TENSION OTHER THAN ETHYL ALCOHOL

The results of tests with these liquids are presented in Table 2.

Acetone gave indications of being a rather more efficient carrier than
those previously used, both in the proportions of 80 parts acetone
to 20 parts formalin, and even when diluted with water in the pro-
portions of 50 parts acetone to 30 parts water and 20 parts forma-
lin. Two series, one of 4 open and one of 4 sealed consecutive
combs, and one series of 9 sealed combs, showed no cells giving
growth of Bacillus larvae, while each of three series of sealed
combs showed only 1 cell giving growth. Two short series were
tried with the use of smaller proportions of formalin, 15 parts and
10 parts, respectively, per 100 parts. ‘These proportions proved
to be inefficient as germicides in both open and sealed cells. In the
light of later work, it seems possible that if more cells from each
comb had been examined a few more positive cultures from sealed
cells might have been found; but in comparison with other solu-
tions tested under the same procedure of cultural examination, ace-
tone gave about the best results as a carrier. The comparatively
high cost of acetone eliminates it as a substance that can be used
economically in apiary practice.

The ketone solution previously mentioned was mixed with forma-
lin in the proportions of 80 parts ketone to 20 parts formalin. This
solution, as may be seen from the table, sterilized the open celis,
but was unsatisfactory in its action on scales in sealed cells.

TABLE 2.—Cultural results of various tests with samples of comb treated for 48
hours in liquids of low surface tension, other than ethyl alcohol

Composition| 8 8 EE Ba
Merlin aeake E Open cells SE 5 Sealed cells BE
oe Ss. Shs
—_ oO = t = No
a3 3 igsj8 |/8/3 8 fa igsl8 |8| sae
— aS = : rom’ ~ cc
He aff |g |ESl~ | S| Sas |e |Esle |e) S88
Liquid used 2 1a qa LES g het Ragen | rey = g | Ee
BlSesl.S\"RES| |B RM [Sa/*gl/Ssl se} 225
= SHSisk& or} P| 7 ae) Nios; Ss] aae
Lo} Solos 3 s|Q Be qd o28s page a i =——=s3
g wa sae 2 fo) aS g ga.° os Ss iS S 2ees
5 a= ng Bi mS] ct] oe be 5 °d hoe ite Ble) oes
OHWI OD O| OH] 0 2 o |S ooh KY) oa] od
3 | .,/ 8 |acsdia SES £ EYla |§STs 5 Seer
Sa sales Fo 38 Bol8 | Bl ewok lg |BEIS |B) es
&, S/S |SSL5 (sels 15 | #885 \5 Sel5 |B] 88s
1a |e fe |Z Z |O 5 |8|s 42 jO%O0 |O| &
| C.c.| C.c.| Cc. | |
80 | 0| 20 AA 30th 40 eee ee oe 4/ 0} 4| 0
80} 0| 20 LOU LOS (Oe LOu Os =a o ase LOS 9} 0} Tenth
80 |: 0] 20 10) ) A050 RON OieR sa ek ee 105 tra Do.
'}50 | 30 | 20 OU) Ose Salta On eee ee ae 9} OT Sai eb
ee Ce LOBE rege eee eT ae Vd eed ee I MC OR ag ps Reo 219] 81 | sarah
40 | 40 | 20 4 4 0 3 1 eee Sos 4 Col 216 |
1185} O| 15 one owl 2} 0} Second__| 3 1} 2} 0} Second
(90 | 0 | 10 3/3/11]. 2] O| Third.) 31-1] 2 (90s phe
(a0. 86 Kod) Jie 5] 401) Ie iO: eee 5/24} 1| 0| First.
Methyl-ethy] ketone- - ---._-| 80} 0 | 20 Cp) fw a) gee IN) Se ad ar | ae FE 5/33] 1 1 | Second
80 | 0} 20 Be SP Be: Bel Bap tdes Bees 5| 1 | 3.1/2 Do.
| . Lod Led Led l rh
Iso-propyl aleobol. ...-.---- {80 30)20| 7/7] 0| 7| ofc 7] 0] 7] ol
| i
1 Few spores germinated. *Three showed few spores germinated. ‘ Two showed few spores germinated. —

Iso-propyl alcohol, another liquid manufactured on a commer-

cial scale resembling ethyl alcohol in its physical properties, but too

The Sterilization of American Foulbrood Combs 11

expensive for practical use, was also tested. Only four series, each
of seven cells, were tried with this carrier for purposes of com-
parison, two of open and two of sealed cells, the proportions used
being 50 parts of iso-propyl alcohol, 30 parts of water, and 20
parts of formalin. Of all the 28 cells cultured only one sealed cell
was found to contain viable Bacillus larvae. This solution is there-
fore in about the same class as that of acetone.

MISCELLANEOUS SOLUTIONS

The results of experiments with these solutions are presented in
Table 3. The use of iodine solutions proved entirely impracticable
for sterilizing infected combs. In dilutions of 1 to 50,000 in water
and even 1 to 500, with an immersion of 48 hours, no germicidal
action on the spores from diseased material in either open or sealed
cells could be demonstrated. A solution of 1 to 20 iodine killed all
the spores, both in open and sealed cells, but the comb was attacked
to such an extent as to make it too soft for further use. The probable
reason for the lack of germicidal action on the part of iodine is
that this substance-combines readily with the fatty-acid constituents

—

TaBLe 3.—Cultural results of vartous tests with samples of comb treated for 24
hours in various solutions

95 per cent alcohol,80 c. c., forma-
lin, 20 c. ec.

nos |
Sts Open celis a 2 Sealed cells aS
2s aS aE
fy = =
a lef 2 & le || Se. (2 & Ig {e| S88
2 isa |S |B 3| 883 {a |B 3| Sas
glee |* joie ja) 283 |" |G.\e la] aes
Do = ieee = = oO
Composition of solution E : q LS ae Ec . 5 3 ae gs Ed e Sac
= [OBSIos ~ {38 Sad |Ss|BSISe| 8 | sae
S fsn8} Siau/"S1 8) aay | S]aqi"s)/%| oss
BR ja sale dinnia | a H@s [1 olPujin™ oa q°s
2 |Sosl2 |oo/8 © OS Dear oe © eu.
Fe) I & S| 3 o as Q & 3 =} OL
SAS Sele Se | Sseeag |B IE Sas ea
BS |Posis is is 13] 3°) |5 ff is | si Ses
A ia Z 6) ld 2) Fy Zi OD |O oO fy
Hours
Iodine, 1-50,000 water__.__________- 48 Zul enone yoni Ones Oat BETS === |e 2028 Oals OF IESt
Todine, 1-500 water________________- 8 del oul lek O>f Ole dose) cal O10 0
fadine, 1-20 water... -- 2-2. 48 2 21 0 2 Uh sags pe are 2 Os 2) 1 0
Denatured alcohol (50 per cent), 93 24 AAAS | EOE ST AUP OWE eee A) tah -OR30 Do.
¢ Ear ay acid, 5 c. ¢c.,
orm c. Cc.
Denatured alcohol, 90c.c.,hydro-| 24) 4| 4/ 1] 3] 0) Third_| 4| 4{ 0] 0} Do
chloric acid, 5c. c., formalin, 5c. c.
Denatured alcohol (50 per cent), 93 48 Sth yal pee (| Sey eA Oe eee ee S173) Oi 50 Do
e C., paaveblonc acid, 5 ¢c. c.,
ormalin, 2c. c.
Denatured alcohol, 90 c. c., hydro- 48 Salou: Lele 2s hee Opis BTsGetoon| re Sulake ss eal nO Do.
ebloric acid, 5c.c., formalin, 5c. c.
Benito oe te c. c., water, 70 c¢. c., 48 Srleehls OR FOale FO} Ses! es Se Ss ee Fourth.
orm Cc. Cc. |
~ ees 3 ee la ACES) ty [ONMetiiels AGO 8| 3| 5] 0] Do
protic ae a c. c., water, 75 c. c., 24 Tal, pial Olle re | Que es [ti WES al: QO Do.
ormalin, 20 c. c.
eek a8 koste3 d95 lll 3e| wOxlt. see | 31 2] 1] 0| Second.
eeured canon eC formalin, 24 4.) 4 1-0) 4 | Oa PSE ena | 4); 4; Oj; O| First.
c. c., glycerin, 2¢. ¢.
3S eee eee Fe ee a eg Pa ee 5| 1| 4] 0| Third.
1 per cent solution of gelatine, 80 48 Feo eOs| ea) | we Onerane oem 3/12] 1] O} Second.
|) eee aloes Sh-0| 3510 a1 9t | pelea D
ee ee oe ee SO ee war fh ote UO Pe ont YU poo =~ oO.
10 per cent ketone solution added to 48 Chie Any b Sa 4) 21 sey a Wi Ce 1 nd oe pe 7.1 CQ eee 1 Do.

1 One showed few spores germinated. 1 Few spores germinated,

12 Department Circular 284, U. S. Dept. of Agriculture

of the wax, as well as with the fatty residue in the diseased remains
of the brood, thereby nullifying the action of the iodine as a germi- ©
cide.
The use of denatured alcohol in various dilutions containing 5
er cent concentrated hydrochloric acid, and a small quantity of ©
poririilih to prevent deleterious action of the acid (0) on the ©
supporting wires of the frame, gave unsatisfactory results with im-
niersions of 24 and of 48 hours. Each of the sealed cells tested gave ©
a good growth of Bacillus larvae; in two cases growth was obtained
from open cells, although some spores apparently were killed. This ©
type of solution was not tested further. ;
Varying quantities of acetic acid added to a 20 per cent solution
of formalin in water contributed nothing to the germicidal or pene-
trating action of the solution so far as sealed cells were concerned,
as several such cells in each series tested gave each a good growth of
Bacillus larvae. These results are very similar to those obtained
with plain water-formalin solution, which will be considered later.
Several other miscellaneous solutions were tried whose composition
is indicated in the table, all containing 20 per cent of formalin, but
varying in the composition of the carrier. Al proved unsatisfactory
when used with sealed cells, and will not be discussed further.

SOLUTIONS WITH DILUTED ALCOHOL

A few dilutions of denatured alcohol, as well as dilutions of
alcohol-formalin solution, were made, varying the alcohol content
from about 30 per cent to over 60 per cent, as indicated in Table 4,

the 20 per cent formalin, however, being kept constant in the mix- —

tures with denatured alcohol. Tests were made of combs treated ~
for 24 hours and for 48 hours. in every case no growth was ob-
tained from open cells, and, as would be expected, there were fewer
positive cultures from sealed cells in combs treated 48 hours than
from those treated 24 hours, although several sealed cells showed no

growth, even in the 24-hour series. In each of the 48-hour series

there was at least one sealed cell from which was obtained a growth
of Bacillus larvae, but, with the few observations made, no signifi-
cant differences could be found between the few dilutions tested of
alcohol or alcohol-formalin solution. Further work is necessary to
demonstrate whether results comparable with those of Krénig and
Paul can be obtained.

For purposes of comparison, as a preliminary test, three series
of six samples each from diseased combs were treated with a solution
containing no alcohol, composed of formalin 20 parts and water
80 parts (Table 9). No significant difference could be seen between
these results and those obtained with the various alcoholic solutions. —
No open cells treated with this solution gave cultures showing —
growth, but 8 sealed cells, 1 or more in each series, gave cultures
showing growth of Bacillus larvae, the growths in 8 of the 8 cultures -
being noted as “ few spores germinated.”

ithe

The Sterilization of American Foulbrood Combs 13

TABLE 4.—Cultural results of various tests with samples of comb treated for 24
hours and for 48 hours in various dilutions of aicohol-formalin and commer-
cial alcohol-formalin solutions

COMMERCIAL ALCOHOL-FORMALIN SOLUTION

Ten series of combs were treated with the commercial alcohol-
formalin solution at various times. From these combs 61 open
and 61 sealed sells were cultured. It was intended to use these series
as controls for comparison with other solutions, since published
reports of the success obtained with this commercial solution in
apiary practice indicated that negative cultural results would be
obtained. As may be seen in Table 7, however, in the case of
every one of the 10 preliminary 48-hour series 1 or more sealed
cells, 22 in all, or 36 per cent, were found to give a growth of
Bacillus larvae. In 3 cases of the 22 the growths were noted as
“few spores germinated.” No growth was obtained from the 61
open cells cultured. At first it was thought that the solution might
have deteriorated; fresh solution was obtained and tested, with
practically the same results. A chemical analysis of the actual
formaldehyde content of the fresh solution, and a similar analysis.
of solution through which some 500 combs had been passed, showed
an actual imcrease with use in the percentage of formaldehyde.
This increase was without doubt due to the more rapid evaporation
of the alcohol than of the formaldehyde content. These results seem
to indicate that there must be a variation in the permeability of
cappings, the decrease in permeability slowing up or even pre-
venting the penetration of disinfectants to the scales and spores.

14. Department Circular 284, U. S. Dept. of Agriculture

PERMEABILITY OF CAPPINGS

A simple experiment was undertaken in an effort to learn whether
the permeability of the cappings is variable. Such a variation might
account for the fact that sealed cells are not always sterilized, and
for the variation in the number of cultures of Bacillus larvae from
sealed cells in different combs.

If cappings are carefully removed from a piece of comb and
examined under the microscope, it is seen that their structure is
apparently very variable. The cappings are composed of criss-
crossing cocoon fibers, pollen grains, and granules of wax, and conse-
quently vary in structure. Cappings from brood cells of different
ages are found to vary greatly in thickness. Freshly sealed cappings
are much thicker and more opaque than cappings from the cells of
more nearly matured pupe, the latter often being gnawed by adult
worker bees.

A simple piece of apparatus was devised to test the variability in
porosity of cappings. A piece of glass tubing slightly smaller in
outside diameter than the inside diameter of worker cells was drawn
to a fine capillary tube and broken off, making a capillary opening
on the end of a 6-inch tube. Numerous cappings were then removed
from various samples of comb, both diseased and healthy, with a
sharp scalpel, and cut so as to leave on the capping a rim of cell
wall about one-sixteenth to one-eighth-inch wide, care being taken
not to rupture the capping. These cappings were then sealed on the
larger end of the glass tube with liquefied beeswax. ‘The end of
the tube covered by the capping was then submerged 3 centimeters
below the surface of the chosen disinfectant, so as to have a uniform
upward pressure on all the cappings successively tested. The rise
of the liquid in the tube was measured at the end of a five-minute
period unless the liquid was able to pass rapidly through the cap-
ping and to rise to the level of the outer liquid in less than that
time. The capillary opening in the upper end of the tube somewhat
retarded the exit of air and, unless the capping was cracked, or
slightly perforated, the contained liquid would not reach the level
of the outside liquid in five minutes. When the rise was more rapid

the apparent reason was recorded. A considerable number of cap-

pings were tested with fresh and used alcohol-formalin solution, and

with fresh and used water-formalin solution (Table 5). As will be ~
seen, there was not much difference in the results. between the fresh ~

and used solutions. It was clearly indicated, however, that there
was a great variation in the rapidity with which the solutions passed
through the various cappings.

Twenty cappings were tested in the alcohol-formalin solution. :
In 5 cases no solution passed through the cappings within the five- —

minute period; in 6 cases the liquid in the tube rose 1 centimeter or
less; in 2 cases it rose between 1 centimeter and 2 centimeters; in 4

cases there was a rise of between 2 centimeters and 3 centimeters; and —

in 8 cases the liquid rose to the 3 centimeter mark in less than five
minutes. Twenty cappings were likewise tested in the water-for-
malin solution. In 9 cases no solution passed through the cappings

in periods varying from 5 to 10 minutes, and, in 1 case, even 60°

minutes; in 4 cases there was a rise of liquid in the tube of 1 centi-
meter or less, one of these after 10 minutes; there were 8 cases of a rise

a dtd OP. Ge

The Sterilization of American Foulbrood Combs 15

of between 1 centimeter and 2 centimeters, 1 of these being attained
after 5 minutes, 1 after 15, and 1 not until 60 minutes had elapsed;
there were 3 cases of a rise of between 2 centimeters and 3 centi-
meters, 2 of them after 5 minutes, and 1 after 15 minutes had elapsed ;
in 1 case the liquid rose to the 8-centimeter mark in 3 minutes.
When no liquid passed through within the test period, ropy, diseased
material was found smeared on the inside of the capping, or the cap-
ping appeared unusually thick. As was to be pea the alcohol
solutions passed through more readily in most cases than did water
solutions. One capping, not recorded in the table, was submerged in
-water-formalin solution for three days with no perceptible passage
of liquid into the tube.

TABLE 5.—Tests of the permeability of brood cappings to disinfectants‘

Description of cappings Immersion | Rise in tube
Fresh commercial aleohol-formalin solution: Minutes | Centimeters
Dark brown from diseased comb, medium thick________________________----- 5 2.2
Darkebrown from diseased: comb @fairly thick .--.-2 _ 22522 = 5 .9
iDark'brown irom: diseased.comb; quite thin_22-. 5... - se ls 3 3.0
Warkibrowl irom Giseased, COMID LHICK=. 20s AN ee 5 Ay:
Dark brown from diseased comb, slightly cracked____________________-_-__-- 2 3.0
Wauk brows from diseased comb, thick. °° !7=_ i 2.222. kl le. RG Jase | 5 0
Dark brown trom.Giseased comb; medium:_-- 2.222 se oe ee 5 1.1
Ae Drown ArOll GIscaseg*ComMpa.thick oo 2 2 ee 5 0
Manke HLOwal trom: Guseased contb, thin 222122 we sb a ee 5 2.8
Darks brows irom diseased comb; thick 222-2". 2. rd ees oe | 5 0
Used commercial a!cohoi-formalin solution:
Light brown from diseased comb, dried, medium thick___________________._- | 5 2a)
Dark brown, old, from diseased comb, dried, thick___.-_____________________- 5 .9
Brown, old, from diseased comb, medium thin____-.___________-___.___-_--- 5 1.2
Seale SMeAnOd OVeGINSIGG. sticks = =e eet 5 a)
ark prowmlirom, Giscased:comp, thick. = =. a ees Or 5 0
Daric DroOwANiLoM: Giseased comb; thin’). if= oh a eee Se 3 3.0
Dark brown from diseased comb, some scale in capping ----_..-.-.---------- 5 20
Dark brown from diseased comb, dry, medium thick____________.__________- 5 2.8
Light brown from diseased comb, good condition__..______________________--| 5 1.0
Warkebrown:tronicdiseased combs thick=s2- se eee 5 .2
Fresh water-formalin solution:
Dark brown from diseased comb, dried, thick_..____-___._______-.--__------ 5 0.9
Darkabrown irom diseased: comb: thick. -_ 2. 22) 20 eae ee 10 0
Darke brownenrom~-diseased Comb; thine. <2 22229 8 a ee 15 Ibe
Wark brown frony disessed-comb,-normal._- a: 52 ee 5 0
Dark brown from diseased comb, dried, thin_._____________________-_____.-- 10 0
TD Utes Rees Ne 2 Fe Re a pep nl Lee a en 15 2.4
Dark brown from diseased comb, slightly cracked______________-______.-__-- 5 2.8
Dark brown fromdiseased: comb, thick: * 2.252532. A ee 10 0
PDVN ES Se ee Os Se ie ape Oa eS ie ae IS ai eeu cr eee ge eee 10 0
Se pp ae pe ee eee ne Kee en ied MD) Te ee i ee ree 10 0
Used water-formalin solution:
Dark brown from diseased comb, dried, thick_________________________---_-- 10 0. 4
Dark brown from diseased comb, dried, cracked_____..___-__---.-_---------- 5 1.5
Wank DROW: (roim-diseased. cour, thin-----f-- 2. fae 10 0
Wanksbrowiironndiseased comb; very thin _-~ -—_-. 222.2 2 2 5 2.1
Hark brown iron diseased:comb, thin®.-— > So. S se ee tee 60 1B
Dark brown from diseased comb, small hole___..___________________--_------ 3 3.0
Dark brown from diseased comb, dried, thin_______._____________-_-__---_--- 60 0
irk Drow nei Lom,disessed.comb, thin... <2 eee 0 0 :
(OR we, Ss ee re a I a 2 ie ie ie re te A el od a .
Dark brown from diseased comb, cracked__..-_.---_------------------------ 5 8
1 Tests made at a uniform depth of 3 centimeters below surface of solution. 2 Trace.

To observe what actually takes place in a sealed cell when sub-
merged in a disinfectant solution, some artificial cells were made
with pieces of glass tubing of the same diameter as those used in the
experiment just described. Pieces of tube about three-quarters of
an inch long were sealed at one end and sterilized in a hot-air steril-
izer, cotton plugs closing the open ends. Scales containing virulent
spores of Bacillus larvae removed from diseased combs were then

16 Department Circular 284, U. 8. Dept. of Agriculture

with aseptic precautions placed in a number of these glass cells, and
cappings were sealed on the open ends as in the previous experiment.
The sealed glass cells were then submerged for 48 hours in alcohol-
formalin and water-formalin solutions, after which they were al-
lowed to dry for a few days. Observations made at the end of 48
hours showed that no perceptible quantity of liquid had entered the
cells. Enough moisture had been absorbed through the cappings,
however, possibly in the form of vapor or of an indistinguish-
able film, to cause the dried scales to become slimy or almost ropy,
like diseased remains before they have dried down. After drying,
cultures were made in the usual manner (Table 6). Three scales
out of 20 so treated, 1 in alcohol-formalin solution and 2 in water-
formalin solution, apparently were completely sterilized, and from
several, most of which had been treated in the alcohol-formalin solu-
tion, cultures were made which showed only a comparatively few
germinated spores and having a slight growth. This seemed to in-
dicate that not much actual disinfectant gains access to some at
least of the sealed cells.

TasLle 6.—Culiurai results of various tests with spores of Bacillus larvae
inclosed in artificial glass cells, capped, and treated for 48 hours in formalin
solutions

Cells Cells Cells

d showing | showing | contami- Remarks
B. larvae \no growth nated

Ceils

Solution tested culture

Fresh alcohol-formalin_______-_-
Used alcohol-formalin__________

5 carcely perceptible growth. ©
5 :
Fresh water-formalin___-_____- 5 Aye) eae eer ee es OLS
5
2

Mostly good growth.
Do.

Used water-formalin___________ Do.

Control, not treated__________-

1 All showing few spores germinated; one very few.
2Two showing many spores germinated.
3 One showing very few spores germinated.

VACUUM TREATMENT

A method of forcing disinfectant solution into the cells was de-
vised to demonstrate whether alcohol fills all spaces in a submerged -
comb. Pieces of comb of the same size as those used in previous
experiments and containing numerous sealed cells were cut from
infected brood combs and submerged in 180 cubic centimeters of —
disinfectant solution in graduated cylinders. One of these was
allowed to soak for 48 hours. The cylinder containing the sub-
merged comb was then subjected to a vacuum of 28 inches, which
caused the air still remaining in many of the open cells to rush out
in considerable amount and the air in sealed cells to bubble through
and in some cases to burst the cappings. When the pressure was
allowed to become normal the free liquid in the cylinder had de-
creased by from 25 to 30 cubic centimeters in displacing the air in
the cells. Similar results were obtained by applying the vacuum
as soon as the combs were immersed. When combs so treated were
subjected to a vacuum again after 48 hours’ immersion they were
found still to release a few air bubbles from sealed cells. These ex-
periments made it evident that even in open cells of combs immersed
for 48 hours at ordinary atmospheric pressure a considerable por-

The Sterilization of American Foulbrood Combs 17

tion of the cell space is often filled with air. Of course the solution,
particularly an alcoholic solution when used with open cells, forms a
film on the surface of the cell around the bubble, as has been de-
scribed by Demuth (9), and thus comes in contact with the larval
remains. In the case of capped cells, however, under normal atmos-
pheric pressure this bubble can not get out, and only a small quan-
tity of liquid can gain access, in some cases probably not enough to
form this moist film.

It was thought at first that this vacuum treatment might be a
satisfactory method of disinfecting foulbrood combs, but aside from
the cost of apparatus it was found that after this procedure it was
practically impossible to remove the disinfectant from the sealed
cells, even by means of centrifugal force, particularly from those
whose cappings had not been broken, until after removal of the cap-
pings. Liquid in the perfectly capped cells evaporated slowly, and
a deposit of solid paraformaldehyde would probably remain in them.
This residue has been found objectionable if not positively detri-
mental to the bees when combs with such a residue are given to a
eolony. Cultures made from a few combs so treated and allowed to
dry for a long time gave completely satisfactory results, no growth
being obtained from any cells, either open or sealed. For combs
from which all cappings have been completely removed this method
of filling all the cells with liquid, thus assuring actual contact of all
cell surfaces and cell contents with the water disinfectant, shoud be
satisfactory, provided a simple and inexpensive vacuum apparatus
can be devised.

PERFORATION OF CAPPINGS

It seemed evident that because of the greater impermeability of
many of the cappings the diseased material in some of the sealed
cells of the immersed combs was prevented from coming in contact
with sufficient disinfectant to kill the spores of Bacillus larvae in the
infected remains. A preliminary method of perforating brood cap-
pings was tried. By means of a blunt needle holes variable in size
and intended to resemble perforations in cappings made by the
bees, were made in all cappings, through which the solution might
be able to enter the cells more readily. -Series of samples of combs
with cappings perforated in this manner were treated in both alco-
hol-formalin and water-formalin solutions, some for 24 and others
for 48 hours, as well as in two lots of soap solution for 48 hours. In
the 24-hour tests with alcohol-formalin solution, of the 20 scales cul-
tured (Table 8) 5 gave positive growths of B. larvae, whereas in the
ease of the water-formalin solution only 1 scale from a perforated
cell, of 20 cultured (Table 10), showed a few germinated spores.
In the 48-hour tests with alcohol-formalin solution only 1 scale»
showed growth out of 20 cultured from perforated cells (Table 7).
In the 48-hour tests with water-formalin solution 2 scales, of the
20 cultured from perforated cells, showed a few germinated spores
(Table 9). In tests with soap-formalin solution and perforated cap-
pings (Table 1) the contents of all such cells were apparently ster-
ilized as far as this particular experiment was carried. These results
indicate that in the case of both solutions the action was aided by
perforating the cappings, but in a few instances the sterilizing action

18 Department Circular 284, U. 8. Dept. of Agriculture

was apparently still incomplete, probably because a trapped air
bubble had prevented sufficient solution from passing through the
opening.

TABLE 7.—Cultural resulis of various tests with samples of comb treated for 48
hours in commercial alcohol-formalin solution

[>] 1 oot 1 ep
ES Open cells = 23 Sealed cells ane
ges Bes B88
aSimealie 2 Silay ry °
zesla [esle |Sas (@ |esia | S28
Smeal id & 3 | op 8 ae a |B §]} oo 2
“s2i8 |S -j4 | BSS |S |s=|8 | Sq

n . Co
5922/8 aQes aM [Bq/@QFal oo
siigh| S]2E) Sas |s§] SIGE os°
al bee ee cae § aq Plog iy = 8248
Ba Als ey oOo) GOD 3 oO, % bo S225
2$ sa Ss Els o FS/Q2 |5BI8 o Fs
f2cie 2cl8 (pees is Ses) eee
Seals sais | eS8RES\5 Iphld | £885
Z 7 IO |O | me is Ol IDeses
2 Wea tae. lg Ya aa Nol bn Pa 4/14] 0| First.
S| 2 BAN TOR Sa eeeee See ee 3 | 1] 2) Second
Salt Sr Ob|c: gles ee Sie Ee Delieieste
x (iis pac eA ASE ee 7\12| 51] Fifth.
Prelitainary tests) iif Lalo Ge ae uion 3 Ho Sih ilo Bal dt poorest ae
5 5 0 Buss Seen 642 1 4 | Third
Toh geal ROU teed eee 7| 5} 2] Second.
Ce Ts OP Wad | See ee 7| 3| 44 First.
10/10} 0} 10 | eae bes 10; 2] 8 | Ninth.

Open (uncapped) cells compared with sealed cells; |f 10 | 50] 0} 50 |-----___-- 40 110] 30 | Third.

used solution. VO e508): Os tcOF | 23 say ae 40 (318 | 22 Do.

Open (uncapped) cells compared with sealed cells; 10} 505) 10 SON ieee 50 |425 | 25 | First.

fresh solution.

Fresh solution; control for samples washed after 10), 50G)) 10"| 50 ee. |e | ee eee

treatment.

Open cells, washed in water after treatment _-_--_--- 10 | 50 |°23)) 47 | Wirst-222|22cs ae

Cells with perforated cappings compared with { 10 | LOR OU el On eases 10| 0| 10

open cells. 10 | HOE OS Oh 2 eee 10 | 1| 9] Tenth.

1 One showed few spores germinated.
3 Few spores germinated.

3 Three showed few spores germinated.
4 Eight showed few spores germinated.

TABLE 8.—Cultural results of various tests with samples of comb treated for 24
hours in commercial. alechol-formalin solution

|
¢

oud * oot ' wo
= | Open cells a= as Sealed cells as a
SAS|, g28 | Soc
zacia lesig |83s ia [eylg | 34s ©
Oo 'S!2 |B S| oe 3 ye = EB] v0 @ tn
Sole |6S8la | eas 2 |oSia | Bag .
Asia ldute.| 8s. i lesle_| 385 @
Les Peay ee eect faa} Solel ES a
COS as wise 4s Fede. ay ASE OS
Salos| C46) 29° |es] Slee 25 2

Prelimiinar ystests Pek te ie) | ra PN a oes 5) 6 0 P Big STS pet 5| 2) 3} First.

Uncapped cells only; used solution .____-------.---- { in a a Ba Paesvaer PP iow GES.

Uncapped cells only; fresh solution. ______________- 10.) 60. | 1 \ 495), ‘Tenth__.\p) oo oa) o se

Fresh solution; control for samples washed after 10 /|° 50) | 237 "477" Sixth= 2 |ho se sees

treatment; uncapped cells only. ;
Open cells, washed in water after treatment. -_-____- 10), 60: |: 3:4.) 46. | tirst)223|sSeseee ee 2
Cells with perforated cappings compared with | { 10") 102) 2305 |t0s) eee ae 10} 3] 7 | Bighth.
open cells. ‘ 10.) 10.)...0 10st aee Se 10 |22] 8] Fourth.

1 Few spores germinated.

2 One showed few spores germinated,

’ Two showed few spores germinated.

The Sterilization of Amertcan Foulbrood Combs 19

Tasus 9.—Cultural resulis of various tests with samples of comb treated for 48
hours in water-jormalin solution

Lo} ' als) ' ‘ to
= © | Open cells | 2.23 | Sealed cells gas
oAS| 4 EE fo} 8.472 3 worwlo S48
Basie ISs\4 jars [5 |gsi8 | avs
a= bee o )
Saag |e8ia | Eas |8 [BSP | oa.
Leora laelea| 2Se (8 Apg|B.q| 2°
8/90/"7Alosl a 9 jad OP! Aan
Lele e| aSEl Sas is8l SE] see
be on?.e araloa 8 Bc ic8 abla Es Saf
Suhis |ssio™ oon, 1S [ogo GBs
aSarg (BEI | Su52\g (ZEB | sm58
Ss A.eis BES Baas is |lecg | 485
Z BO to ley 7 OOS Es
GaleeGy | rOatulGy |e eee eee 6|14]| 2] Third
Parellningry COStS <a 2 eas ee Ses see | g ¢ 4 iS fg of er : 3 3 4 re
Mesh TSS FLL ifth.
Open (uncapped) cells compared with sealed cells; { ROM 5 Oe te Oly oO.) eee ene | 40 (112 | 28 | Fourth
used solution. i BO SUN On] (OU somes Ser | 40 |210 | 30 Do.
Open (uncapped) cells compared with sealed cells; TOW POON es OF PbO ee ees 50 |315 | 35 | Third
fresh solution.
Fresh solution; control for samples washed after LORE SOLIES OF SOR | SS A Ee ee ee
treatment. ‘
Open cells, washed in water after treatment______- LOM SSO a ONE Ow 22 we 2 Leake ee ee eee tee
Cells with perforated cappings compared with open { LOS) FLO >| eeO ie slO | 2S ses ee /10}12] 8] Fourth
cells. TOY LOM On pel On| ae aan ' 10} 0] 10

1 Two showed few spores germinated.
2 One showed few spores germinated.
3 Seven showed few spores germinated.

TABLE 10.—Cultural results of various tests with samples of combd treated for 24
hours in water-formalin solution

oO We teteye 1
©2 | Open cells Aad Sealed cells Bas
“e sO os
Sel Sin Wes icw lh Sete 3 lw [o 6.48
SAB lasif |S@s [8 jasl8 | 4s
AB "S|° TESla | Suk na |Eeloo | 2
S248 losla aS 18 joslAa BS
Ese jaclea| 284 |S_iU/Eal 2°
“Se ay; Rios) A 4 ay) Qos| Sos
Oe Sl sies (FB aks r= | E 22°
SolCB] 9/79 akc se o|290 a4
Sek s |Sa/S8h Sos, js") Sa/8h Bese
2og 2 (ded | 2, Fol |Selid | © Fs
gee |SS/8 |exct la |SEIB | euoa
= wont} BIS HBSS [3 Shs HAS
Z iO Outs V2 NOs Ss =
: : (SeTOU 50 TFT Os 250) eee bee eee ae epee ae
Open cells only; used solution.____......-.-------- WOH esol CalnnOn ison be Ls Vissi mica ae
Open cells only; fresh solution_________-_--_.-_--_- LOM FOOR | Or aoO: (poe esate S [Acie eee
Fresh solution; control for samples washed after LOS ROM Ou t5O05 (Resse eke = Se 0 oa eed ee
treatment. : : |
Open cells washed in water after treatment_______- AOU SO PSS 47 oe Rarsh See hoe elses See Oe
Cells with perforated cappings compared with open { TOW AOS Ou WO) 2 ee 10 |?21} 9 | Seventh.
cells. OE ETO} es inl Os | eee a eens 2 LORE On Elo

1 Germination of few spores; doubtful in the case of two cultures.
1 Showed few spores germinated.

CAPPINGS REMOVED WITH KNIFE

Considerable experimenting with various methods of opening cap-
pings sufficiently to allow easy access of solutions into the cells dem-
onstrated that the only satisfactory way is to use an uncapping knife
and actually to cut away the cappings, as is done when uncapping
honey, before the combs are placed in the disinfectant solution. If
the knife is sharp and well heated, brood cells may be as easily un-
capped as honey cells, leaving a sharp, complete opening and adding
but little to the labor factor in handling the combs. If the knife is

20 Department Circular 284, U. 8. Dept. of Agriculture

dull, it will tear the comb, closing many of the cells, so that the solu-
tion can not enter. All honey cells that previously have not been ex-
tracted can be uncapped at the same time as the brood cells.’

Several series of combs uncapped in this manner were treated with

both the alcohol-formalin and water-formalin solutions for 24 and
for 48 hours. In some of the 48-hour tests the cappings were cut
from only one side of the combs, so that cultures for comparison

might be made from sealed cells at the same time and to obtain defi-
nite figures for the percentage of sterilization of the sealed cells. To
reduce the probable error arising from the missing of occasional cells
that would give growth, 5 open cells and 5 sealed cells from each
comb, instead of only iL: ‘were cultured. In the 24- hour tests and in
the last 48-hour series only open cells were cultured, since from the

previous experiments it was found that 24 hours and in many cases —

even 48 hours was not invariably sufficient to sterilize all sealed cells.

In tabulating these results the number of combs in each series of

samples and the total number of cells cultured is recorded.

As indicated in Table 7, a total of 281 scales from open cells of
combs treated for 48 hours in alcohol-formalin solution, including
the cells originally open and those opened by uncapping but exclud-
ing those washed after treatment, were cultured, none of which gave
any growth of Bacillus larvae. A total of 191 scales from 191 sealed
cells of the same combs (omitting 20 combs having perforations in
the “sealed” cells and other combs from which cultures were made
from open cells only) gave 115 cultures showing no growth of B.
larvae, 61 showing good growth, and 15 showing a few germinated
spores, or 75 positive cultures; 39.8 per cent of these cells were there-
fore not sterilized. In the 24-hour tests of scales from open cells,
including all considered in Table 8 except those washed after treat-
ment, a total of 225 scales cultured gave 219 tubes showing no growth
of B. larvae, 2 showing good growth, and 4 showing a few germ-
inated spores, or 6 positive cultures, a percentage of 2.7 for open cells
not sterilized. Only one short (preliminary) series of scales from
sealed cells of combs treated for 24 hours was tested, with 2 out of 5
cultures showing growth of B. larvae.

As indicated in Table 9, a total of 238 scales from open cells from
samples of combs treated for 48 hours in water-formalin solution,
including cells originally open and cells uncapped, but no cells
washed after treatment, were cultured, none of cle gave a growth
of Bacillus larvae. A total of 148 scales from sealed cells from the
same combs when cultured gave 103 showing no growth of B. larvae,
382 showing good growth, and 13 showing a few germinated spores,
or 45 positive cultures, a percentage of 30.4 for sealed cells not ster-
ilized. In the 24-hour tests of scales from similar open cells cul-
tured (Table 10), 220 scales were cultured, all the cultures showing
no growth of B. larvae. No 24-hour sealed cells were tested with

2In a recent article Vincens (24), who has charge of the station for apicultural research.

of the Institute of Agricultural Research at Cagnes, France, describes his method for re-
moving cappings and soaking the contents of the cells previous to immersion in a water-
formalin solution. A jet of water reduced to a spray is shot against the surface of the
comb at an adgle with considerable force. This destroys or _berforates the cappings and
fills the cells. After 12 hours the combs are again treated in like manner. Then after
passage through an extractor the combs, which are now thoroughly wet, are immersed
in a water-formalin solution containing only 10 per cent formalin for 24 hours, after
which the solution is removed and the combs allowed to dry. Vincens reports suecess
s0 far with this method used on a small scale.

The Sterilization of American Foulbrood Combs Da

water-formalin solution. A shorter period of immersion than 24
hours was not tried during these investigations.

WASHING COMBS

In one or two instances combs have been treated with alcohol-
formalin solution in cold weather and allowed to dry slowly at a
comparatively low temperature. These, when returned to the bees,

have caused considerable trouble, and even loss of colonies by de-
-sertion of the bees. Because of the retarded rate of evaporation
_at lower temperatures, the formaldehyde, instead of evaporating,

undergoes a transformation to the solid and much less volatile para-
formaldehyde. ‘This is probably more often true of the water-
formalin solution, which evaporates more slowly. When evapora-

tion takes place slowly, a residue sufficient to be poisonous or ob-
' noxious to bees may remain in the cells for a considerable time.
_ Washing combs in water after treatment was tried by Jones (15)
_ to remove the odor of formaldehyde, and has been recommended by
Corkins (7) as a preventive against this difficulty. No observa-

tions in the apiary or results of cultural tests aiter such treatment
have been recorded. Several series of combs were tested, using
_ pieces from which the cappings had been cut, treating some for 24

and some for 48 hours, and comparing the combs allowed to dry
without washing with those washed in water.

As indicated in Table 7, 50 scales were cultured from open cells

of combs washed in water after treatment for 48 hours in alcohol-
_ formalin solution. Of these, 3 cultures showed only a few germi-

nated spores of Bacillus larvae. The control series of combs with
open cells (Tabie 7) not washed in water gave no cultures showing
any growth. In the 24-hour tests 50 scales were cultured from open

cells of combs washed in water after treatment for 24 hours in
_ alcohol-formalin solution (Table 8). Of these, 2 cultures showed

good growth of B..larvae and 2 a few germinated spores. The
control series of combs (Table 8) not washed gave 2 cultures show-
ing good growth, and 1 showing a few germinated spores.

As shown in Table 9, 50 scales were cultured from open cells of
combs washed in water after treatment for 48 hours in water-
formalin solution. None of the cultures gave any growth of Ba-
cillus larvae. ‘The control series of combs (Table 9) that were not
washed gave from open cells no cultures showing any growth. In
the 24-hour tests 50 scales were cultured from open cells of combs
washed in water after treatment for 24 hours in water-formalin
solution (Table 10); of these, 1 culture showed good growth of
B. larvae and 2 showed a few doubtful germinated spores. The con-
trol series of combs with open cells (Table 10) not washed in water
gave no cultures showing any growth of B. larvae.

DISCUSSION

Throughout these preliminary laboratory experiments on the
germicidai efficiency of the various types of disinfectants tested, no
sohition was found which sterilized the contents of all sealed cells
uniformly in ali of the several series of combs immersed in it.

This fact is probably of importance in the sterilization of brood

22 Department Circular 284, U. 8. Dept. of Agriculture

combs infected with American foulbrood by means of various fer-
maldehyde disinfectant solutions as used by beekeepers. It was
found to be true even with solutions of low surface tension and sup-
posedly good penetration, such as alcohol-formalin solution, as
well as with solutions having high wetting and spreading powers
with regard to waxy surfaces, such as soap-formalin solutions. In
- all of the several series of combs treated, even after a treatment of
48 hours, cultures from varying numbers of scales from capped cells
gave growths of Bacillus larvae. A comparison of the percentage
of sealed cells not sterilized indicates that the efficiency of the alco- —
hol-formalin solution is relatively no greater in this regard than
that of water-formalin solution. |
The explanation of this failure of the formaldehyde disinfectants
to sterilize all the sealed brood may be found in the varying per-—
meability of the brood cappings, particularly many of those cover-
ing remains of American foulbrood. In some cases no solution was
able to pass through the cappings, low surface tension of the liquid ©
and solvent action on the wax seeming to be ineffective factors. In i
these cases the glue-like remains of diseased dead larve have often —
become smeared over the inner surface of the cappings during the
routine handling of the combs, drying there and clogging the pores —
of the cappings. As a result, no solution could penetrate them, at —
least within a length of time practicable for treating combs with-—
out subjecting them to action causing undesirable deterioration. -
Even on the same comb there is wide variation in the structure of —
the cappings themselves. As can be noted in the various tables,
there is great irregularity as to which comb of a series shows the —
first sealed cells giving a growth of Bacillus larvae, indicating in-
complete sterilization. The experiments with glass cells indicate the
possibility that the complete sterilization achieved in many of the
sealed cells is brought about by the penetration into the cells of —
formaldehyde gas and water vapor liberated from the disinfectant —
solution, rather than by actual liquid sufficient to cause sterilization.
Experiments with the use of a vacuum in treating combs indicated ©
that relatively little solution enters the majority of sealed cells at
usual atmospheric pressure, but that the method should be effective —
with all uncapped cells. These facts may explain the few cases of re- _
currence of disease so far noted with the method now in use, and serve :

as the basis of a prediction of more cases in the future. Why more —
such recurrences have not been observed is not easy to explain, but
it is to be remembered that this method of disinfecting combs me

been in use for only about two years and much concerning it is yet
to be learned. —

A certain proportion of the cultures of scales from sealed cells
(25 per cent of those treated with alcohol-formalin solution and ~
about 33 per cent of those treated with water-formalin solution)
showed comparatively few germinated spores. In some of these
cases no growth was visible on the surface of the culture medium.
Some few of these cultures on subculturing failed to show further
growth or germination. The question here arises whether contact
with formaldehyde insufficient to kill may lower the vitality of these —
organisms so that they fail to continue growth, or may lower their
virulence, thereby inhibiting infection of healthy colonies from suc
sources. It is also possible that an insufficient number of spor

pnd

The Sterilization of American Foulbrood Combs 23

remain alive to start the disease, as experience in cultivating Bacil-
lus larvae from diseased material on artificial culture media has
demonstrated that a certain quantity of inoculum is necessary to
start growth, whereas growth will not begin when there are only a
few spores. These are among the problems awaiting solution.
Another important fact brought out by the various tests of solu-
tions containing 20 per cent of formalin is that in the case of all
combs that were treated for 48 hours no cultures made from scales
taken from open cells or from cells the cappings of which had been
removed gave any growth of Bacillus larvae. For this length of
treatment complete sterilization was accomplished by the water-
formalin solution as well as by the alcohol-formalin solution. In
the case of the 24-hour tests a few of the scales from open cells of
combs treated with alcohol-formalin solution failed to be completely
sterilized, 6 of the 220 such scales cultured giving growths of
B. larvae. Apparently a 24-hour treatment is somewhat below the
minimum time in which complete sterilization of the scales in open
cells may be expected. On the other hand, none of the cultures from

_ 220 scales from open cells of comb treated 24 hours in water-forma-

lin solution showed any growth of B. larvae. These results indicate

that the water-formalin solution is more efficient as a germicide for

American foulbrood than is the alcohol-formalin solution, provided

ue solutions come in actual contact with the scales for at least 24
ours.

All cappings, those over brood cells as well as honey cappings,
shoufd be carefully removed to insure sterilization of combs infected
with American foulbrood. Even when this is done, apparently a
48-hour treatment is still necessary when an alcohol-formalin solu-
tion is used. From the fact that all cultures were negative which

were made from scales in open cells of combs treated 24 hours in

a water solution containing 20 per cent of formalin, a treatment of
24 hours in such a solution appears to be sufficient. When, after
combs have been soaked in the disinfectant solution, the excess liquid
has been removed in an extractor, and the combs are allowed to dry
without further treatment, there is still some disinfectant left which
continues to act while they are drying, until it is entirely evaporated.
If circumstances, such as the necessity for drying treated coinbs at
a comparatively low temperature, require the washing of the treated
combs in water in order to prevent the formation of an undesirable
residue caused by the retarded evaporation, the combs should be
treated 48 hours, whether in water-formalin or in alcohol-formalin

solution; an additional 12 hours in the case of alcohol-formalin

Reys'

solution would give a greater margin of safety. If this is not done,
the discontinuance of the germicidal action may permit a few scales »
to emerge with spores still capable of causing disease, while a period
of treatment shorter than 48 hours before washing in water would
be entirely insufficient for complete germicidal action.

The uncapping of all brood cells removes the necessity of using
a solution capable of penetrating the wax. With the cappings re-
moved, the dissolving action, or penetration of the wax of the comb,
seems of questionable value. It is noteworthy that not one case has
definitely been recorded of disease resulting ee the use in healthy
colonies of tons of comb foundation that for years has been made
from wax rendered from combs once containing American foulbrood.

24 Department Circular 284, U. S. Dept. of Agriculture

The uncapping of all brood cells eliminates the necessity for a

solution with low surface tension. The much less expensive water- |

formalin solution will be found to enter the open cells sufficiently
to soften, loosen, and sterilize the scales as effectively as an alcoholic
solution, although the entrance may not be quite so rapid. The
slower entrance into the cells is more than counterbalanced by the

greater germicidal efficiency of formalin in the presence of water as —

compared with that of the same disinfectant in an alcoholic solution.
The water-formalin solution readily softens and penetrates the
masses of pollen in cells, since stored pollen is held together by small
amounts of honey, readily soluble in water. Aside from the count-
less spores contained in the diseased remains and those in the infected
honey or pollen, practically the only other spores that might in any
way get to healthy larve and cause disease are not in masses, but are
scattered individually over the surfaces of the comb, having acci-
dentally been carried there by the bees in their work.

White (27, p. 32) has shown that spores suspended in a 20 per
cent formalin solution would be killed inside of a few hours; it
follows that when individual spores on combs come in direct contact
with the disinfectant they must be killed in a comparatively short
time. If, as was found to be the case in the laboratory experiments,
soaking for 24 hours of scales of American foulbrood in open cells
kills all spores embedded in them, probably immersion for only a

few hours is necessary to kill these spores on the comb surfaces,

as would be the case with dry extracting combs that have been
in diseased colonies but have contained no dead brood. The few
spores that might become embedded in propolis are as negligible as
those in the wax. .

The uncapping of all brood cells, as well as of any sealed cells of

honey in diseased brood combs, naturally adds somewhat to the ~

labor cost of treating these combs; but if results in the apiary com-
pare at all with the results in the laboratory, the lowering of the
cost by elimination of the alcohol, using only formalin in water, will
more than offset the slight additional labor cost. However, before
treatment it is almost always necessary to uncap some sealed honey
in combs from diseased colonies; if the brood is uncapped at the
same time, comparatively little extra effort or time will be required.
The washing of combs, when necessary after treatment for 48 hours
in the water-formalin solution, also adds to the labor costs; but in
this case again the extra labor should be repaid by the results.

The data presented indicate that a 20 per cent solution of formalin —

in water is the most efficient as well as the most economical disin-
fectant so far found for the sterilization of combs infected with
American foulbrood, provided the cappings are all completely re-
moved. It is hoped that apiary tests will be made to determine the
practicability of these results. Naturally, even with the most re-
liable process, carelessness in handling the combs, and more particu-
larly carelessness in the treatment of the diseased colonies from which
the combs are taken, will be fatal to success in gaining control
over this disease which has thus far caused such great losses. The
must approved methods for the treatment of American foulbrood
have been discussed elsewhere.*

® Methods of treatment and control of American foulbrood are discussed in United
States Department of Agriculture Farmers’ Bulletin 1084.

The Sterilization of American Foulbrood Combs 25

CONCLUSIONS

A number of soap solutions were found unsatisfactory as carriers
for 20 per cent of their volume of formalin to be used as a disinfect-
ant for treating American foulbrood combs; (1) because of the diff-
culty of controlling the reaction of the solution and of preventing the
precipitation of the soap and (2) because of the failure of the soap
solution to penetrate all cappings of sealed cells sufficiently to kill all
spores of Bacillus larvae contained in the diseased material therein,
and to do this within a period of 48 hours.

Certain liquids of low surface tension other than ethyl alcohol,
such as acetone and iso-propyl alcohol, are somewhat more efficient
as carriers for formalin than most of the solutions tried, including
ethyl alcohol, as indicated by the comparatively few sealed cells
which failed to be completely sterilized by them within a period of
48 hours. On the other hand, these liquids are too expensive for
practical use, even if satisfactory as carriers for the formalin. Less
expensive liquids tried, such as a commercial methyl-ethyl ketone,
are unsatisfactory because of failure to sterilize scales in sealed cells
in 48 hours.

Miscellaneous disinfectants such as dilute hydrochloric acid, var-
ious dilutions of iodine, acetic acid added to water-formalin solu-
tion to increase its penetrating power, and such substances as gela-
tine, added to water-formalin solution to increase the wetting and
spreading powers, are all unsatisfactory as disinfectants for steriliz-
ing American foulbrood combs, since none of them completely steri-
lizes all sealed cells in 48 hours. The hydrochloric acid solutions and
all but quite concentrated iodine solutions even fail to sterilize all
the open cells. Formaldehyde, used as formalin solution, when em-
ployed in sufficient proportions is the most efficient and practical —
germicidal agent thus far used for the purpose of disinfecting
American foulbrood combs. A solution containing less than 20 per
~ cent of formalin is found to be unreliable.

The results obtained with various dilutions of alcohol and of
alcohol-formalin solution as the carrier for 20 per cent of their vol-
ume of formalin are not sufficiently complete to warrant conclusions
as to their relative efficiency. All of these solutions are unsatis-
factory, since they do not completely sterilize all sealed cells in 48
hours. A 20 per cent solution of formalin in water, without al-
cohol, isyshghtly less efficient than the alcoholic solutions in steriliz-
ing in 48 hours the contents of sealed cells, because of its failure to
penetrate many of the cappings; but it sterilizes all open cells in that
period.

The commercial alcohol-formalin solution, like all the other so-
lutions tested, fails to sterilize completely the scales from all sealed
cells with a treatment of 48 hours.

The variation in the permeability of many of the brood-cell cap-
pings accounts for the failure to sterilize many of the sealed cells
within a period of 48 hours. In some cases cappings completely
resist the passage of liquid or vapors into the cells, thereby making
low surface tension and solvent action unavailing within the period
of time practicable for the satisfactory treatment of combs.

The results of experiments with glass observation cells, as well as
of the application of a vacuum to the solutions containing combs

26 Department Circular 284, U. 8S. Depl. of Agriculture

under treatment, indicate that little if any actual liquid enters cells
where the capping is intact and more or less impermeable. It is
probable that when such sealed cells are sterilized the disinfection is
brought about by the passage through the capping of gas and
vapor liberated from the solution.

The perforation of brood cappings adds to the efficients of the dis-
infectant solutions, both alcoholic and water; but, aside from the
difficulty of doing this in a practical way, the sterilization of such
cells is not always uniformly complete, even after a treatment of
48 hours.

To obtain uniformly complete sterilization of all infected cells in
American foulbrood combs, no matter what solution is used, all cap-
pings, covering both brood and honey, should be carefully removed
before the combs are immersed in the solution. This can be easily
done if care is used, with.a hot, sharp, uncapping knife, and adds
little to the labor costs.

The sterilization of combs infected with American foulbrood,
when all the cells are uncapped and alcohol-formalin solution is
used, requires, if complete from the standpoint of the cultural re-
sults obtained, more than a 24-hour treatment. A treatment of 48
hours would give a margin of safety.

The sterilization of combs infected with American foulbrood,
when all the cells are uncapped and a 20 per cent solution of for-
malin in water is used, if complete from the standpoint of the cul-
tural results obtained, requires treatment for 24 hours at least. A
somewhat longer period would give a greater margin of safety.

The washing of combs in water, to prevent the formation of a
residue of paraformaldehyde after treatment with disinfectants con-
taining 20 per cent of formalin, should not be attempted unless the
combs have been treated at least 48 hours in the solution. A some-
what longer period would give a greater margin of safety, particu-
larly with the alcohol- formalin solution.

To reduce the chance of missing any cells that have not been ~

completely sterilized, when testing the efficiency of any disinfectant
solution, a sufficiently large number of cells should be cultured. The
proportion should be approximately from 38 to 5 per cent of the cells
of each class, open and closed, so distributed as to represent fairly all
the cells treated.

In view of the cultural results obtained, a 20 per cent solution of
formalin in water was found the most satisfactory disinfectant for

use in sterilizing combs infected with American foulbrood, with re-

gard to both germicidal action and low cost, provided the proper
precautions are taken. All honey should be extracted, all brood
cappings should be completely removed, and the combs should be
treated at least 24 hours, or 48 hours if it is found desirable to wash
them in water after treatment. Before such a procedure can be rec-
ommended unreservedly exhaustive tests must be carried out under
aplary conditions.

Care must be taken not only with the process of disinfecting combs
infected with American foulbrood, no matter what solution or

method is used, but equal or gr eater care must be exercised in the

treatment of the diseased colonies themselves to eliminate the danger
of recurrence of disease from that source. The successful steriliza-
tion of the combs will otherwise be of little avail.

Pathe i CEA, 2 tube

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(2)

(3)

(4)

(5)

(6)

LITERATURE CITED

BorcHent, A.

1921. Die Formaldehyddesinfection in der Bienenwirtschaft in der
Form des Autanverfahrens sowie experimentelle Untersuchun-
gen tiber die Tiefenwirkung des mit Wasserdampf gesattigten
Formaldehydgases. In Arb. Biol. Reichsanstalt Land- u.
Forstw., vol. 10, Heft 6, pp. 522-557.

ByerJ.. 1:
1924. Alcohol-formalin treatment. In Gleanings Bee Cult., vol. 52, No.
4, pp. 230-231.
1924. Disease in treated combs. In Gleanings Bee Cult., vol. 52, No.
9, pp. 584-585.
1924. In Ontario. [Further report on recurrence of disease with treated
combs.] Jn Gleanings Bee Cult., vol. 52, No. 10, p. 662.
1925. In Ontario. [Note on alcohol-formalin vs. water-formalin treat-
ment.] In Gleanings Bee Cult., vol. 53, No. 1, pp. 38-89.
Cooper, W. F., and NurtTaL1, W. H.

1915. The theory of wetting, and the determination of the wetting
power of dipping and spraying fluids containing a soap basis.
In Jour. Agr. Sci., vol. 7, pt. 2, pp. 219-239, 3 figs.

(7) Corxins, C. L.

(8)
(9)
(10)

(11)

(12)
(13)

(14)

(15)
(16)

(17)

in ;

1924. In Wyoming. [Note on washing of combs with water after treat-
ment with alcohol-formalin solution; to prevent losses from
formation of paraformaldehyde.] In Gleanings Bee Cult., vol.
52, No. 10, p. 659.

DEMUTH, G. S.

1923. American foulbrood ousted. In Gleanings Bee Cult., vol. 51, No.

11, pp. 712-717.

1924. Sterilizing diseased combs. Jn Gleanings Bee Cult., vol. 52, No.
Aap) At.
GRIFFIN, R. C.
1920. The solubility of metals in acids containing formaldehyde. In
Jour. Indus. and Engin. Chem., vol. 12, No. 12, pp. 1159-1160.
HUTZELMAN, J. C.
1922. Can the combs be saved? New treatment for American foul-
brood by immersion in disinfectant solution. In Gleanings
Bee Cult., vol. 50, No. 12, pp. 764-766.

1924. Honeycomb sterilizer. [U. S. Patent No. 1511762. Patented Oct.
i 1o24-)- U.S. Patent Office,. Off. Gaz., vol. 327, p. 387.
JARVIS, G. L.
1925. Apiary experience in treating combs. Jn Beekeeper, vol. 33, No.
os DD boo:
JONES, Dan H.
1924. Control of American foulbrood. Summary of some laboratory
tests with disinfectants in this disease. In Gleanings Bee Cult.,
vol. 52, No. 6, pp. 264-365 and 396.

1925. Chemical treatment of foulbrood. In Beekeeper, vol. 33, No. 1,
pp. +5 and 10.
Kine, GEORGE E.
1925. Disinfecting diseased combs. In Gieanings Bee Cult., vol. 53, No.
7, pp. 438-440.
Kronie, B., and Pau, TH.
1897. Die chemischen Grundlagen der Lehre von der Giftwirkung und
Desinfection. In Ztschr. Hyg. u. Infectionskrank., vol. 25, pp.
1-112,

(18)

(25)

(26)

(27)

Department Circular 284, U. S. Dept. of Agriculture

MAASSEN, A., and BorRcHERT, A.
1920. Uber die Bekimpfung der antsteckenden Bienenkrankheiten und
iiber Entseuchungsversuche mit Formaldehyd in der Form des
Autanverfahrens. Jn Mitt. Biol. Reichsanstalt Land- w.
Forstw., Heft 18, pp. 151-156.
Moore, WILLIAM.
1921. Spreading and adherence of arsenical sprays. Univ. Minn. Agr,
Exp. Sta. Tech. Bul. 2, 50 pp.
RIDEAL, S., and RIpEAL, EE. K.
1921. Chemical disinfection and sterilization. London. 313 pp.
STURTEVANT, A. P.
1924. The development of American foulbrood in relation to the meta-
bolism of its causative organism. In Jour. Agr. Research, vol.
28, No. 2, pp. 129-168.
UNITED STATES TREASURY DEPARTMENT, BUREAU OF INTERNAL REVENUE.
1920. Regulation No. 61. Laws and regulations relative to the produe-
tion, tax payment, ete., of industrial alcohol and to the manu-
facture, sale, and use of denatured alcohol. 107 pp.
VANSELL, G. H.
1925. Promising experiments in sterilizing foulbrood combs. Jn Amer.
Bee Jour., vol. 65, No. 2, p. 55.
VINCENS, F.
1925. Désinfection des rayons contaminées par la “loque pernicieuse”
4 Vaide d’une solution aqueuse de formol. Jn La France Apic.,
vol. 31, No. 8, pp. 174-175.
Wuirte, G. F.
1904. The further investigation of the diseases affecting the apiaries
in the State of New York. In N. Y. State Dept. Agr. Ann. Rpt.
Comr. 1903, pp. 103-114.

1906. The bacteria of the apiary, with special reference to bee diseases.
U. S. Dept. Agr. Bur. Ent., Tech. Ser. 14, 50 pp.

1920. American foulbrood. U. 8S. Dept. Agr. Bul. 809.

~ 4

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‘ ORGANIZATION OF THE
UNITED STATES DEPARTMENT OF AGRICULTURE

January 14, 1926

meereiary 0; Agriculture... = —-=_-—_-__- W. M. JARDINE.
PECL ILLES COL CUI) = ae me ie Ss R. W. DUNLAP.
marecctor of Scientific Workiw 2) 32-_--_—- a,
Director of Regulatory Work______-_-__-- WALTER G. CAMPBELL.
musector of Hxtension Work-——_—----—-~--=-. C. W. WARBURTON.
BEreLOr.-0) INTOrmalionz2=— = oe NELSON ANTRIM CRAWFORD.
Director of Personnel and Business Ad-

MITE SEG LEO: Se en SE Le W. W. STOCKBERGER.
DRG RS SS eS aS ee ee R. W. WILLIAMS.
BCPILOI TR UT COE ene oe ale 22 eT CHARLES F. Marvin, Chief.
Bureau of Agricultural Economics____-~-~- THomAS P. Cooprr, Chief.
eureau of Animal Industry_——_.._—~-==--= JOHN R. MOHLER, Chief.
mureau of Plant Industry____._-~.---_=-. WILLIAM A. Taytor, Chief.
BERECSLENCRUICE soe ote aN has LT gra ee W. B. GREELEY, Chief.
RRCIHE OT CILEMISEFY === 2s ee C. A. Browne, Chief.
DIGCHLIE CO; NOLS S22 x 2 eee) ee EE MILTON WHITNEY, Chief.
mural Of; Entomology. 22> L. O. Howarp, Chief.
Bureau of Biological Survey___-__--------- E. W. NELson, Chief.
mCi OF .Pu0lie Redds222— = soo 2 et THomas H. MacDona.Lp, Chief.
Bureau of Home Hconomics____.-.____---. LouISsE STANLEY, Chief.
RS OF PR OIT GTI Set a es ee C. W. Larson, Chief.
Fized Nitrogen Research Laboratory____- F. G. CortTreiyt, Director.
Office of Experiment Stations_____________ BH. W. ALLEN, Chief.
Office of Cooperative Extension Work___--. C. B. SmitH, Chief.
GIT Tf tek SE a SS Een CLARIBEL R. BARNETT, Librarian.
meeaeral Horticultural Board__________—--- C. L. Maruatt, Chairman.
Insecticide and Fungicide Board__--____-- J. K. Haywoop, Chairman.
Packers and Stockyards Administration__._. JOHN T. CAINE, in Charge.
Grain Futures Administration___________- J. W. T. DUVEL, in Charge.

This circular is a contribution from

mureau of Entomology___________________. L. O. Howarp, Chief.
Bee-Culture Investigations____________ J. I. HAMBLETON, in Charge.

ADDITIONAL COPIES
OF THIS PUBLICATION MAY BE PROCURED FROM
THE SUPERINTENDENT OF DOCUMENTS
GOVERNMENT PRINTING OFFICE
WASHINGTON, D. C.

AT
6 CENTS PER COPY

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