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THE USE OF FORMALDEHYDE A8
A DISQNFECTANT IN THE CONTROL
OF INCUBATOR TRANSMITTED
PULLORUM DISEASE

Thesis for the Degree (if M. 3.
MICHIGAN STATE COLLEGE

Howard C. Zindel
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THE USE OF FORMALDEHYDE AS A DISINFECTANT IN THE CONTROL

OF INCUBATOR TRANSMITTED PULLORUM DISEASE

by

Howard Carl Zindel

“an!!!“

A THESIS
Submitted to the Graduate School of Michigan
State College of Agriculture and Applied
Science in partial fulfilment of the
requirements for the degree of

MASTER OF SCIENCE

Department of Poultry Husbandry

Year 1941

THESIS

CONTENTS

Introduction
Review of Literature
Purpose
Methods and Materials
Effect of Formaldehyde Gas Upon Chick
Amount of Gas Inhaled by Chick During Fumigation
The Lethal Action of Formaldehyde
Effect of Fumigation Upon Chicks in Incubator
Resulting Viability of Fumigated Chicks
Effect of Fumigation Upon Egg Shells, Cheese
Cloth Squares and Cover Glasses
The Incidence of S. Pullorum in Fecal
Material After Fumigation
Discussion
Summary
Conclusion
Recommendations
Literature Cited

References

iSIQIQ

Page

12
15
34
48

54

62
66
68
69
70
71

74

ACKNOWLEDGEMENTS

The author is indebted to Professor C. G. Card, Professor of
Poultry Husbandry; Doctor H. J. Stafseth, Professor of Pathogenic
Bacteriology; and Doctor W. L. Mallmann, Professor of Sanitary Bac-
teriology, Michigan State College. Their constant interest in the
problem and cooperative effort in supplying materials and equipment
are greatly appreciated.

Appreciation is also expressed to Doctor P. J. Schaible,
Research Associate in Chemistry; Doctor E. S. Weisner, Extension
Specialist in Poultry Pathology; Doctor E. W. Henderson, Assistant
Professor of Poultry Husbandry; and J. A. Davidson, Associate Pro-
fessor of Poultry Husbandry, Michigan State College, for helpful

suggestions and cooperation in supplying certain facilities.

INTRODUCTION

Pullorum disease is still a perplexing problem to the poultry
industry. It is difficult to control due to its many modes of trans-
mission. This disease is passed from the infected hen through the
egg to the chick and the chick may transmit the disease to non—in—
fected stock during the hatching period or the first few weeks of
brooding. The spread of the disease and the mortality of chicks is
directly influenced by conditions during the first two weeks of
brooding.

The specific cause of pullorum disease is a bacillus known as

Salmonella pullorum. This organism enters the body through the re—

 

spiratory or alimentary tract through the agency of contaminated air,
feed or water; or it may have been in the egg from which the chick
hatched. The organism is found in the blood, in the unabsorbed yolk,
in the bone marrow, and in the internal organs of the chick following
death from the disease.

The control of pullorum disease depends largely upon two fac—
tors: first, the detection of the infected breeding fowls by means of
agglutination tests and their immediate removal from the breeding flock
and second, the sanitary protection of healthy stock against infection
in incubators, brooder houses and runways.

The value of formaldehyde in the control of pullorum disease

infection in the incubators is the basis for this thesis.

REVIEW OF LITERATURE

Since it was discovered that pullorum disease was Spread via
the incubator, the incubator manufacturers have been looking for
methods of combating this mode of dissemination. T. S. Townsley (l),
in 1930, research director of the Smith Incubator Company was one of
the incubator men to work upon incubator hygiene as a control of
pullorum disease. However, Moore, Upp and Hinshaw (2) in 1926 were
the first to discover that pullorum disease could be disseminated in
the forced draft incubators. The work of Hinshaw and others was con-
firmed by Bunyea and Hall (3).

Many types of disinfectants were tested without apparent suc—
cess until the workers tried formaldehyde (HCHO).

The discovery of formaldehyde is usually attributed to Hoffmann
(4) who in 1867 succeeded in preparing the aldehyde of the methyl
group, by the partial oxidation of methyl alcohol. Almost as soon as
it became commercially available, formaldehyde was hailed as an effec-
tive disinfectant. In 1888, Low (5) recognized the strong antiseptic
properties of formaldehyde. In 1892, Trillat (6) discovered that a
bouillon containing 1 : 50,000 of formaldehyde was not suitable for the
growth of the anthrax germ. E. A. de Schweinitz, (7) Chief of the Bio-
chemistry Division of the United States Department of Agriculture in
1896 states: "Formaldehyde possesses most of the properties of a good
disinfectant". Other early experiments with formaldehyde were carried
out by Miguel, Bandet, and Trillat (8) for the disinfection of rooms.

The summary in the 1896 Yearbook of Agriculture is as follows:

1. "Formalin in concentration of 1 : 10,000 makes growth of
tuberculosis, anthrax, cholera, typhus pus, and diphtheria germs im~
possible.

2. "In gaseous form, a weak dilution is sufficient to check
the growth of the above.

3. "A one per cent solution will kill pathogenic organisms
in an hour.

4. "Spraying with formalin solution and subsequent inclo—
sure of the articles in a closed Space will easily sterilize them.

5. "Feces are deodorized by a one per cent solution and in
13 minutes are germ-free. Buildings are readily disinfected by a one
per cent to one and one-half per cent volume of gas in 6 to 13 hours.

6. "Formaldehyde is useful as an etching material and pre—
servative.

7. "Odor can be readily dissipated by the use of a dilute
solution of ammonia, which readily absorbs the gas."

In the U. S. D. A. Bulletin (9) the following advantages and
disadvantages of formaldehyde are listed.
The advantages summarized:

1. It is a powerful germicide.

2. Its action is not hindered greatly by albuminous

substances or organic matter.

3. It is relatively non-poisonous.

A. It is not injurious to delicate fabrics, to paint,

or to metals.

5. It is the only known gaseous disinfectant which can

be used effectively and safely in the household.

The disadvantages summarized:

1. The gas has a strong tendency to condense in cold
weather and it is not reliable as a disinfec—
tant when the temperature of the air is much
below 65° F.

2. It has a very penetrating odor and the gas is
irritating to eyes and nose.

3. To accomplish disinfection by the gas a long period
of exposure is necessary and considerable work
is required in the proper sealing of rooms
which are to be disinfected.

Many of the workers on this problem agree that the germicidal
efficiency of formaldehyde gas is greatly influenced by the relative
humidity. When the amount of moisture is decreased, the germicidal
efficiency of formaldehyde is decreased.

Two methods of releasing the formaldehyde have been employed,
namely: the cheesecloth method and the potassium permanganate method.
It is with the latter method that we are concerned.

Graham (13) of the Illinois Experimental Station reports that
formaldehyde in the amounts recommended by that station destroyed
S, pullorum in 43 minutes. Graham states, "There is no known incubator
fumigant as efficient as formaldehyde in forced draft incubators for
the suppression of S, pullorum and not—with—standing claims to the con—
trary, formaldehyde possesses a superior efficiency in incubator dis-
infection and costs much less". Doctor Graham (13) reports that chicks

are not injured by exposure to formaldehyde and recommends three dis—

tinct fumigations twelve hours apart.

Some workers have recommended the practice of fumigating the
chicks at the time of hatching so as to destroy organisms that may be
liberated by the chicks at this time. Dakin and Speer (11) recommend
three fumigations during the process of hatching. The first fumiga-
tion was to take place before ten per cent of the chicks are hatched,

a second treatment twelve hours later with the immediate removal of

all dry chicks, and the third treatment at the end of 48 hours. They
also recommended the dosage of 0.2 gm. potassium permanganate and

0.4 cc. formalin per cubic foot of air Space for a period of ten minutes.

Graham and Michael (13) have done considerable work on pullorum
disease and their work shows that "Formaldehyde fumigation of forced
draft incubators by either the cheesecloth method or the potassium
permanganate method is of definite value in the suppression of pullorum
disease". These men recommend fumigation during and before the hatch
comes off.

Bushnell and Payne (14) report: "With a temperature of 99 to
100 degrees and a wet bulb reading of 90 degrees, treatment with 0.35 00.
formalin and 0.175 gm. potassium permanganate per cubic foot of space
kills practically all exposed pullorum organisms within five minutes
after the formaldehyde has been liberated". "Chicks subjected to a
ten minute exposure of formaldehyde liberated from 0.35 cc. formalin
and 0.175 gm. of potassium permanganate per cubic foot of air space
with a wet bulb reading of 9OQF are apparently not injured".

Winter (15) reports, "fumigation between hatches or before
hatches is preferable to fumigation during the hatch. There is no dan-

ger of hurting hatchability if fumigation is used during the seventeenth

or eighteenth day of incubation, while fumigation during the hatch may
injure the chicks". However, Winter gives directions for the fumiga—
tion of chicks during the hatch in this same bulletin, but he says that
fumigation during hatching period is not recommended. "Chicks of low
vitality may be injured by routine fumigation and good chicks are cer-
tainly not improved by it. Chicks more than 48 hours old should not

be subjected to formaldehyde fumigation".

Bushnell and Brandly (16) declare that fumigation during the
hatching period is desirable and recommend three periods of fumigation
eight hours apart. "Neither the very young chicks nor the eggs seem
to be injured if the formaldehyde fumigation is carried out according
to recommended directions". They also recommend 0.35 cc. formalin and
0.175 gm. KMnOA for each cubic foot of air space for ten minutes.

In checking with.the incubator manufacturers, it was found that
two companies, the Buckeye and Smith both recommend fumigation before
the hatch comes off and during the hatch. Smith Incubator Company (17)
recommends that the first fumigation should be used when 15 to 20 per
cent of the chicks are hatched, the second when 50 to 60 per cent are
hatched and the third fumigation when the hatch is about complete.

The Buckeye Company (18) states: "To be effective the wet bulb
reading in the incubator should be at least 85°F and preferably 90°F.
The higher the humidity the more effective the gas and the less danger
of injuring the chicks". The Buckeye Company states: "Fumigation of
eggs is effective in preventing 'Mushy Chicks'". They recommend fumi-
gating the chicks once when about two—thirds are hatched or six to

eight hours before the hatch is completed.

Both companies recommend neutralizing the formaldehyde gas with
26 per cent ammonia hydrate for a ten minute period.

Carus Chemical Company (21) at LaSalle, Illinois, one of the
manufacturers of potassium permanganate crystals, states in one of its
advertising leaflets: "Fumigation during the hatching period, while
the chicks are still damp or not completely dry and fluffed out, will
prevent the spread of this disease from the diseased to the disease
free chicks hatching at the same time. It will not cure the chick that
hatches from the infected egg". They further state: "Do not expose
eggs to fumigation until they have been in the incubator at least for
four days. Fumigation during the first four days of incubation may
prove injurious to the chick embryo in its early stage of development".

Scott (22) in 1928 compared the germicidal powers of phenol and
formaldehyde and found that a l to 200 formaldehyde solution killed
all of the aerobic bacteria both the spore forming and the non-spore
forming in six to twelve hours. He states, "Phenol in a strength up
to five per cent acts very slowly on the anaerobic organisms while for-
maldehyde in dilutions of 0.5 to 0.75 per cent sterilizes anaerobic
cultures rapidly".

According to McCulloch (23) the phenol coefficient of formalin

(40 per cent formaldehyde) is 1.3. I found that with Eberthella typhosa

 

and S. pullorum the phenol coefficient was 1.3 in each case, thus con-
firming his findings.
Tilley and Schaffer (24) in 1928, using 19;. tyjphosa as the test

organism, obtained a phenol coefficient of 1.05 for formaldehyde.

Mallmann (12) and staff did extensive work on this subject but
their results showed that fumigation with formalin with the amounts
recommended and even with greater amounts had very little effect on
the incidence of S, pullorum in the chamber employed (unpublished work).
They were unable to kill all the bacteria upon either the dry or the
wet chicks. "Chicks exposed for ten minutes to the formalin gas
showed marked respiratory disturbances and died 36 hours later".

Weisner (20) states: "Every setting of eggs should be sub—
jected to the fumigation but care should be exercised not to fumigate
after the eggs have begun to hatch. We do not recommend the fumigation
of chicks". However, in his recommendations for fumigation of eggs
the time of exposure to the formaldehyde gas has been raised from ten
minutes to three hours.

Gwatkin (10) demonstrated conclusively that S, pullorum will
live upon pieces of egg shell in an incubator for twelve days and at
room temperature for at least 47 days. The fact that this organism
will persist in an incubator justifies the use of fumigation to rid
the machine of the infection. He recommends the use of 0.5 gm. of
formalin and 0.25 gm. of potassium permanganate per cubic foot of air
Space with an exposure of two hours.

According to Bushnell and Payne (14), the lethal dose of forma-
lin for S. pullorum is between 55 and 60 cc. of formalin per hour for

eight and one—half hours.

PURPOSE

It is the purpose of this study to further the knowledge of
the value of formaldehyde in the control of pullorum disease infec—

tion in the incubator.

EFFECT OF FORMALDEHYDE GAS UPON CHICK

In the preliminary eXperiments, it was found that chicks sub-
jected to formaldehyde gas showed marked reactions. It was then
decided to autOpsy each chick, after subjecting it to definite amounts
of formaldehyde gas for a definite period of time, to learn the action
of the gas upon the flesh, internal organs, and eyes. The following
results were secured after placing day old White Leghorn chicks in an
air tight box and fumigating with potassium permanganate crystals and
40 per cent formalin. The box was built in such a manner that the

actions of the chicks could be clearly seen.

 

Fig. l.

10

 

 

Photograph of Chicks in Fumigation
Chamber Through Top Window.

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AMOUNT OF FORMALDEHYDE GAS INHALED BY
CHICKS DURING FUMIGATION

Due to the fact that formaldehyde is made by the partial oxi-
dation of methyl alcohol, a question is raised as to whether the death
loss, in chicks from fumigation, is due to the effect of the formal—
dehyde or whether it is due to methyl alcohol poisoning. Tests were
conducted to determine just how much formaldehyde gas is actually
taken up by the chick during the fumigation process. A process, by
which the amount of formaldehyde gas that a chick actually inhaled
during the fumigation period, was developed. One school of thought
believes that the loss of chicks, due to mortality, from the day of
hatching until four weeks of age, may be due to fumes of formaldehyde
inhaled during the fumigation period. In the first experiments, an
attempt was made to determine the amount of gas actually taken into
the lungs. It is also thought that death might be due to methyl al-
cohol poisoning. Attempts to kill chicks with methyl alcohol fumes
caused the chicks to stagger as if in a drunk stupor but they did not
die. After a short period of time these chicks seemed to recover
their normal balance. The writer was unable to find any literature
dealing with chick mortality due to methyl alcohol poisoning.

Using a bell jar as an inclosure, a chick was exposed to fumes
of formaldehyde gas liberated by adding formalin to finely ground
crystals of potassium permanganate. The chicks' actions could be

closely observed. Each chick had great difficulty in breathing, and

13

attempted to clear its nose and mouth by shaking its head. As the
chick found it more difficult to breathe, it was noticed that a thick,
sticky, mucous secretion issued from its mouth. This secretion was
tested for presence of formaldehyde. First a color indicator was set
up using a certain percentage of the indicator, Phloroglucinol (Merck's
Reagent), and straight formaldehyde. The indicator, Phloroglucinol
turns red upon coming in contact with formaldehyde. According to the
reference book, the indicator shows a red color in dilutions up to

l : 30,000, but in our tests it would show red only up to l : 10,000.
No formaldehyde could be detected in the mucous secretion. The lungs
were then removed and macerated with fine sand and alcohol and tested
without detecting any trace of formaldehyde using the indicator. I
also macerated the lungs by pressing them between two pieces of filter
paper without finding any traces of formaldehyde. The windpipe was
removed and alcohol was passed through it. This fluid was then tested
after it ran out of the Chick's mouth. This procedure showed faint
signs of formaldehyde. A repetition of this procedure, using distilled
water instead of alcohol, was unsuccessful.

To check under a more practical condition, twenty day—old chicks
were fumigated in the recommended manner and upon removal from the
hatching compartment were tested in all of the above mentioned ways
without any signs of formaldehyde. At the conclusion of this work, it
was decided that it was impossible to determine the amount of formal—
dehyde taken in by the chicks during fumigation period, using the

procedures discussed.

 

 

 

 

 

Fig. 2. Photograph of Experimental Brooder Showing
Front View with Doors in Place.

15

THE LETHAL ACTION OF FORMALDEHYDE

In the next tests, it was decided to determine the lethal
action of formaldehyde upon.S, pullorum artificially infected chicks
in both moist and dry condition.

To be assured that a known type of chick was being used, all
chicks were hatched from a flock of Single Comb White Leghorns.

These birds were blood tested and in some cases retested to be sure
that all adult stock was pullorum-free. In these experiments the same
section of a Jamesway incubator was used for hatching in order to pre-
vent concurrent contamination. The hatching compartment of this
section of the incubator had 7.35 cubic feet of air space with the
following dimensions: 28.75 in. by 27.25 in. by 13.5 in.

One half of these chicks had the web on the right foot between
the first and second toes cut for purpose of identification. These
birds were called controls and were not inoculated with S, pullorum.
The remaining 80 chicks were then inoculated with S. pullorum by
swabbing the mouth and throat of each chick with a swab moistened with
S, pullorum. After inoculation all birds were placed in separate
hatching compartments on the same tray and fumigated. The amounts of
formalin and potassium permanganate, the exposure periods, wet bulb
and dry bulb readings varied in each experiment. After exposure all
birds were placed in the experimental brooder in the following order:

Pen 1 — Twenty infected and twenty clean chicks

Pen 2 - Forty clean chicks
Pen 3 - Twenty infected chicks and twenty clean chicks
Pen 4 - Forty infected chicks

 

16

The brooder temperature for the first two days was set at 95°F.
and then lowered to 850F. for the remaining five days. Each brooder
unit had an individual thermostatic control which regulated the tem-
perature for each compartment. Each pen contained a 32 inch feeding
tray and a water fountain. The floor was covered with about two
inches of wood shavings. Prior to use, the brooders were cleaned and
disinfected with iodine suspensoid.

After remaining in this brooder for seven days each group of
chicks was placed in a battery brooder. These chicks were fed the
Spartan Starter for the first three weeks and then gradually changed
to the Spartan Grower for the remainder of the experiment.

Each chick, upon death, was checked bacteriologically by smear—
ing portions of the heart, liver, and lung upon plates of brilliant
green liver infusion agar and Eosin—methylene—blue (E.M.B.) agar.
After 24 hours of incubation, colonies, appearing to be S, ullorum,
were transferred to agar slants and later were planted in sucrose,
lactose, dextrose, maltose, and mannite broth.

Until the cultures were made, the dead chicks were stored in
a refrigerator. All cultures showing acid and gas in dextrose and
mannite and no change in sucrose, lactose and maltose were reported
as S, pullorum.

The first two trays containing 321 eggs were set in the James-
way incubator on May 28, 1940. The first hatch of chicks was taken
off on June 19. One hundred and sixty good strong chicks were se-

lected for this series of experiments. During the fumigation period,

17

the temperature was set at lOOOF. and the wet bulb reading was 85°F.
giving a relative humidity of 54 per cent. On June 20, all the
chicks in Pen 1 were found dead due to overheating. The thermostat
became stuck allowing the heat to rise to 120%“. It was decided to
carry on the remaining pens as if nothing had happened to the number
one pen.

For brevity, fumigation will be referred to by dosages used.
For example, a single, double, or triple dose of formalin and potas—
sium permanganate was used for a certain time period. In each case

the following is meant:

Single dose — 0.175 gm.* KMnOA and 0.35 cc.* formalin per cubic foot

or 1.35 gm. KMnO4 and 2.7 cc. formalin for 7.74 cubic feet.
Double dose - 2.70 gm. KMnOA and 5.4 cc. formalin for 7.74 cubic feet.

Triple dose - 4.05 gm. KMnOA and 8.1 cc. formalin for 7.74 cubic feet.

*Amounts recommended by Robert Graham of University of Illinois,
Circular No. 403.

18

Table II

The Isolation of S. pullorum at Autopsy of
Chicks Examined

Date of hatch - June 19, 1940
Fumigation - Single dose for 10 minutes
Dry bulb reading — 100°F.)

) Relative humidity 54 per cent
Wet bulb reading — 85°F.)

 

Distribution of clean and infected chicks

 

 

 

 

 

 

: : Pen 1 : Pen 2 : Pen 3 : Pen 4
Age of: Mor- :20 infected :40 uninfectedz20 infected : 40 infected
chick: tal- :20 uninfected: :20 uninfected:

: lty : No. No. in—: No. No. in-: No. No. in-: No. No. in-
jdays): : fected : fected : fected : fected
6 Died 0 0 l l 0 0

10 Died 1 0

Chicks
11 Died all died 1 1

from over—
16 Died heating 2 l
18 Died 2 2
2O Died 1 O l l
21 Died 1 O
22 Died 1 O
27 Killed 39 0 36 0 34 0

Total 40 O 40 2 40 4

 

19

Table III

The Isolation of S. pullorum at Autopsy of
Chicks Examined

Date of batch — July 2, 1940
Fumigation — Single dose for 10 minutes
Dry bulb reading - lOOoF.)

)Relative humidity 68 per cent
Wet bulb reading - 90°F.)

 

Distribution of clean and infected chicks

 

 

 

 

 

 

: : Pen 1 : Pen 2 : Pang} : Pen 4
Age of: Mor— :20 infected :40 uninfected:20 infected : 40 infected
chick: tal- :20 uninfected: :20 uninfected:
: 1ty : No. No. in-: No. No. in-: No. No. in-: No. No. in-
(days): : fected : fected : fected : fected
: : : : :
9 Died 2 2
10 Died 4 4
Chicks
11 Died all died 1 0
from over-
12 Died heating 1 O l l l l
31 Killed 39 0 36 3 35 5
Total 40 0 40 6 40 10

 

Table IV

The Isolation of S, pullorum at Autopsy of
Chicks Examined

Date of hatch — July 10, 1940
Fumigation - Single dose for 10 minutes
Dry bulb reading — 98°F.)

)Relative humidity 99+-per cent
Wet bulb reading - 98°F.)

 

Distribution of clean and infected chicks

 

 

 

: : Pen 1 : Pen 2 : Pen 3 : Pen 4
Age of: Mor— :20 infected :40 uninfected:20 infected : 40 infected
chick: tal- :20 uninfected: :20 uninfected:
: itY' : No. No. in-: No. No. in-: No. No. in-: No. No. in-
(days): : fected : fected : fected : fected
l Died 1 l
2 Died 1 0 l l
3 Died 1 l l l
6 Died 2 l 2 2
9 Died 1 0
29 Killed 38 O 40 0 36 0 36 0

 

 

 

 

Total 40 - 2 40 ,0 40 l 40 4

 

The Isolation of S,

Date of hatch

Fumigation

Table V

pullorum at Autopsy of
Chicks Examined

Dry bulb reading — loo°F.)
)Relative humidity 59 per cent

Wet bulb reading -

87°F.)

 

July 21+: 1940

Single dose for 10 minutes

21

 

Age of: Mor. :20 infected

O
O

 

Distribution of clean and infected chicks
Pen 1 : Pen 2 : Pen 3 Pen 4
:40 uninfectedz20 infected 40 infected

chick :tality:20 uninfected:

:20 uninfected

 

 

 

 

 

.0 O. O. O. .0 O.

 

: : No. No. in-: No. No. in—: No. No. in— No. No. in-
jdays): : fected : fected : fected fected
l :Died : : : l l l 1
5 Died 1 l

6 Died 1 l
8 Died 1 0
10 Died 3 3
l2 Died 1 0 l l 2 2
13 Died 1 0 2 2 l l
14 Died 1 l l 0 l l
21 Died 1 l
22 Died 1 l l 0
26 Died 1 0 l O l 0 l 0
55 Killed 35 0 36 0 32 0 31 0
Total 40 4 40 0 40 7 4O 6

 

Table VI

The Isolation of S. pullorum at Autopsy of
Chicks Examined

Date of hatch - August 1, 1940
Fumigation - Single dose for 20 minutes
Dry bulb reading — loo°F.)

)Relative humidity 83 per cent
Wet bulb reading — 95°F.)

 

Distribution of clean and infected chicks

 

 

 

 

 

 

 

: : Pen 1 : Pen 2 : Peng3» : Pen 4,
Age of: Mor— :20 infected :40 uninfected:20 infected : 40 infected
chick :tality:20 uninfected: :20 uninfected:

: : No. No. in-: No. No. in-: No. No. in—: No. No. in-
(days): : fected : fected : fected : fected
2 :Died : : : 2 2 : 4 3
4 Died 2 2 4 4 3 3
5 Died 1 0 l l

6 Died 1 l
8 Died 1 l
9 Died 1 0 2 1
l4 Died 1 0
20 Died 1 l l O
21 Died 2 l
25 Died 1 0 2 0 3 0
34 Killed 34 l 39 0 30 0 24 0
Total 40 5 40 0 40 7 40 9

 

Table VII

The Isolation of S. pullorum at Autopsy of
Chicks Examined

Date of hatch - September 12, 1940
Fumigation - Double dose for 10 minutes
Dry bulb reading — 99°F.)

)Relative humidity 96 per cent
Wet bulb reading — 97°F.)

 

Distribution of clean and infected chicks

 

 

 

: : Pen 1 : Pen 2 : Peng3 : Pen 4
Age of: Mor- :15 infected :30 uninfected:15 infected : 30 infected
chick :tality:15 uninfected: :15 uninfected:
: : No. No. in—: No. No. in-: No. No. in—: No. No. in-
(days): : fected : fected : fected : fected
3 Died 1 1 l 0
4 Died 1 0 5 5 2 2
5 Died 3 3 4 4
6 Died 1 l ‘ 2 2 l 1
7 Died 1 l
8 Died 2 2 l 0 2 2
l2 Killed 23 3 27 0 23 7 20 4

 

 

 

 

Total 30 10 30 O 3O 14 3O 14

 

24

Table VIII

The Isolation of S. pullorum at Autopsy of
Chicks Examined

Date of hatch - September 19, 1940
Fumigation - Double dose for 20 minutes
Dry bulb reading — loo°F.)

)Relative humidity 93 per cent
Wet bulb reading - 98° .)

 

Distribution of clean and infected chicks

 

 

 

 

 

 

 

: : Pen 1 : Pen 2 : Pen 3 : Pen 4_
Age of: Mor— :15 infected :30 uninfected:15 infected : 30 infected
chick :tality:15 uninfected: :15 uninfected:
: : No. No. in-: No. No. in—: No. No. in—: No. No. in-
jdays): : fected : fected : fected : fected
: : : : :
4 Died 1 1 l l
5 Died 4 4 6 6 4 4
8 Died 1 l 2 0
9 Died 2 l
10 Died 2 2 2 2
l6 Killed 25 0 30 0 l9 0 21 1
Total 30 5 30 0 30 9 30 9

 

Table IX

The Isolation of S, pullorum at Autopsy of
Chicks Examined

Date of hatch - September 26, 1940
Fumigation - Single dose for 20 minutes
Dry bulb reading — 98°F.)

)Relative humidity 73 per cent
Wet bulb reading — 90°F.)

 

Distribution of clean and infected chicks

 

 

 

 

 

 

 

: : Pen 1 : Pen 2 : Pang} : Pen 4
Age of: Mor- :10 infected :20 uninfected:10 infected : 20 infected
chick :tality:10 uninfected: :10 uninfected:

: : No. No. in-: No. No. in—: No. No. in-: No. No. in-
_1gays): : fected : fected : fected : fected
4 Died 1 1 1 l
5 Died 3 3 l l 4 4
6 Died 2 l 2 2
7 Died 1 0 1 0 l l

9 Died 1 1 l 0 l l

10 Died 1 1

ll Died 1 l l 0 l l 1 l

13 Died 1 0

l5 Killed 13 0 l7 0 13 0 11 0
Total 20 6 20 0 20 5 20 9

 

26

To be more certain of the virulence of the culture, six strains
of S, pullorum from autopsied birds were combined into a 12 hour cul-
ture. Ten cc. of this combination were injected into a White Rock
male. Two days later this bird was bled and a pour plate made with
citrated blood to recover the organism. Eight cc. of the recovered
organism was injected intravenously and ten cc. injected subcutane-
ously into a second bird. Four days later this bird died and the
organism was again recovered from the liver and spleen.

To check the virulence of this organism again, 24 day-old White
Leghorn chicks were selected. Various amounts of culture were in-

jected intraperitoneally.

 

 

Fig. 3.

27

 

 

 

Photograph of Experimental Brooder Showing Interior

28

Table X

Virulence of Culture as Determined by
Injection into Day Old Chicks

Date of Experiment - September 5, 1940

Each chick injected intraperitoneally

 

 

 

Age of: Mor— : Group 1 : Group 2 : Group 3 : Group 4
chick :tality: Controls : 0.1 cc. : 0.5 cc. : 0.25 cc.
: : No. No. in-: No. No. in-: No. No. in-: No. No. in-

(Days): : fected : fected : fected : fected

l Died 1 l

2 Died 2 2 3 3 2 2

3 Died 1 1 2 2 2 2

4 Died 1 1 2 2

5 Died 1 1

6 Died 1 l

7 Killed 6 0

 

 

 

 

Total 6 0 6 6 6 6 6 6

 

The Isolation of S. pullorum from Infected Chicks
by Streaking Tissues on Plain Agar and

Table XI

E.M.B. Agar Plates After Fumigation

Date of Experiment - September 17, 1940

Dry bulb reading — loo°F.)
)Relative humidity 62 per cent
Wet bulb reading - 88°F.)

 

 

Group : Medium : Dosage : Time : Results
: used : : minutes :
1 Plain agar . Single . lO . - *
l A Plain agar Single 10 + **
2 E.M.B. Double 10 -
2 A E.M.B. Double 10 +
3 Plain agar Single 20 —
3 A Plain agar Single 20 +
4 E.M.B. Double 20 —
4 A E.M.B. Double 20 +
5 Plain agar Double 30 -
5 A Plain agar Double 30 +
6 E.M.B. Triple 10 —
6 A E.M.B. Triple 10 +

 

* - S, pullorum not isolated.

*% _‘S. pullorum isolated.

A - Controls

30

This experiment, the isolation of S. pullorum from infected

 

chicks by streaking the tissues on plain and E.M.B. agar plates after
fumigation, was repeated with the relative humidity readings of 66
and 68 per cents, in which results identical with those recorded

in the preceding table were obtained.

 

 

Fig, 4, Photograph Showing Special Chamber Used in Fumigation Experiments.

31

Table XII

Germicidal Efficiency of Formaldehyde in Destroying
S, pullorum Streaked on E.M.B. Plates
From Broth and Slant Cultures

Date of Experiment — October 4, 1940
Dry bulb reading — loo°F.)

)Relative humidity 54 per cent
Wet bulb reading — 85°F.)

 

Time : Exposed : Controls
Minutes : Plate Broth Slant : Plate Broth Slant

Group : Dosage

 

1 Single 10 l N * N l G** G
2 G N 2 G G
3 N N 3 G G
4 N N 4 G G
2 Double 10 l N G l G G
2 N N 2 G G
3 N N 3 G G
4 G N 4 G G
3 Single 20 1 N N l G G
2 N N 2 G G
3 N N 3 G G
4 N N 4 G G
4 Double 20 l N N 1 G G
2 N N 2 G G
3 N N 3 G G
4 N N 4 G G
5 Double 30 l N N 1 G G
2 N N 2 G G
3 N N 3 G G
4 N N 4 G G
6 Triple 10 l N N 1 G G
2 N N 2 G G
3 N N 3 G G
4 N N 4 G G

 

* N — no pullorum growth
** G — pullorum growth

32

A fumigation chamber with the following inside dimensions was
made: 24 inches wide, 23% inches long, and 23 3/4 inches deep. This
box was made of one-half inch plywood, fitted carefully and glued so
as to make it air tight. See photograph Figure 4, page 30. The
opening to this chamber was four inches square, fitted so that when
clamped the chamber was air tight. This opening was on the front side
located near the base of the box. The top of this chamber was made
with a glass, four inches square, so that one could look into the box
and observe the reaction of the chicks. This box was also equipped
with an electric light bulb. The air space of this box was 7.74 cubic
feet and it was on this figure that the amounts of formalin and potas—
sium permanganate were calculated.

Beginning June 25, 1940, eggs from Single Comb White Leghorn
hens were placed in the Jamesway incubator each week. The number of
chicks varied each week depending on the hatchability of the eggs. Be—
ginning October 7, 1940, the chicks on livability test were kept in a
battery brooder. The fecal material was removed from the trays daily.
The water troughs were scrubbed daily. In every case, precautions

were taken to avoid the Spread of the S, pullorum from one section of

 

the brooder to another. The temperature, of each section, was kept
constant in order to obtain the best growth possible. Every effort was
made to maintain ideal conditions at all times.

The following experiments are quantitative and were performed
in an attempt to prove that not all the organisms on the chicks are

killed.

33

For brevity, fumigation will be referred by dosages used.
For example, a single, double, or triple dose of formalin and potas-
sium permanganate was used for a certain time period. In each case

the following is meant:

Single dose - 0.175 gm.* KMnO4 and 0.35 cc.* formalin per cubic foot
or 1.35 gm. KMnOA and 2.7 cc. formalin for 7.74 cubic feet
Double dose - 2.70 gm. KMnOA and 5.4 cc. formalin for 7.74 cubic feet

Triple dose - 4.05 gm. KMn04 and 8.1 cc. formalin for 7.74 cubic feet

* Amounts recommended by Robert Graham of University of Illinois,
Circular No. 403.

34

EFFECT OF FUMIGATION UPON CHICKS IN INCUBATOR

This experiment was to determine the number of S, pullorum
found on chicks after subjecting them to formaldehyde. The amounts
of formaldehyde and exposure periods varied.

Eleven chicks were dipped into a suspension of S, pullorum.
Chicks were dried and then fumigated in groups. The chicks were then
dipped in sterile water after fumigation. The rinse waters were
plated in brilliant green agar using one and five cc. amounts. All

plates were incubated at 370 C. for 24 hours.

Table XIII

The Exposure of Dried Chicks to Formaldehyde

 

 

 

Date of Experiment - October 11, 1940
Group : Dosage : Time : Chick ‘: Dilutions

: : minutes : : 1 cc. : 5 cc.

1 Single 10 1 P * P

2 P P
3 P N **

4 P P

2 Double 20 l N P

2 N N

3 P P

4 N N

3 Control 1 P P

 

* P - S, pullorum present.

** N -.S. pullorum not present

35

Twenty chicks were dipped into a 24 hour culture of S, pullorum
and without drying were then fumigated. After fumigation each chick
was dipped into sterile water to wash off any organisms present and
the usual dilutions were made. Plain agar was added to the plates
and incubated at 37°C. for 24 hours.

Figures quoted refer to number of S. pullorum organisms isolated.

Table XIV

Number of Bacteria Removed from Formaldehyde
Treated Chicks. Wet Before Exposure.

Date of Experiment - October 30, 1940

 

No. of bacteria : Average Number
per ml. : of bacteria
of rinse water : per chick

Time : Chick
minutes :

Group Dosage

 

O. .0 .0 O.
O. I. O. O.
O. O. C. O.

0

7,500

37,500
37,500 20,625

1 Single 10

2 Double 10 322,500
37,500
180,000
7,500 136,875
3 Single 20 15,000
7,500
45,000
37,500 26,250
4 Double 20 15,000
0
0
0 3,750
5 Controls 405,000
383,500
457,500
405,000 412,750

PWMH bWMH 1*me {Shawl-J PWNH

 

36

Twenty chicks were dipped, dried, fumigated, and the colonies

on plates were counted at the end of 24 hours incubation.

Table XV

Number of Bacteria Removed from Formaldehyde

Treated Chicks.

Date of Experiment

Dried Before Exposure

November 6, 1940

 

Group

Dosage : Time
: minutes

Chick

No. of bacteria

per ml.

of rinse water

Average Number
of bacteria
per chick

 

O. O. O. .0

Single 10

Single 20

Double 10

Double 20

Controls

‘PWNH {>me waDI—J

bWNi—J waH

12,075,000
10,800,000
14,025,000

5,625,000

9,075,000
8,775,000
11,325,000
1,200,000

4,200,000
9,000,000
2,325,000

300,000

30,750,000

8,130,000
13,275,000
13,950,000

607,500,000
618,975,000

19,050,000

125,550,000

10,633,750

7,593,750

3,956,250

16,526,250

342,768,750

 

37

Ten chicks were dipped in a suspension of S, ullorum, dried,

and fumigated. After fumigation the chicks were rinsed in sterile

water and then put into brooder to check the livability of the two

groups.

 

 

 

l

 

f

Fig. 5. Photograph Showing Method of Dipping and Rinsing Chicks.

38

zmdopss p0 eopsaomw asaoHHSm .m.*

 

 

a amen 00m.e0m.m 00m.80m.m H aHoaasoo a
m aeoao 000.e04 000.moH m

m aooam 00m.m00 m

H asses 000.mam H 0H oHasoo m
m asoam 000.mmw 000.000 m

a asoam 00m.mee N

H asoaa 00m.m0e.H H 0m onaHm m
a aeoHa mam.eaa 000.0es m

a aeoHa 000.mwm N

HH oon 000.eHm H 0H onsam H

Ammmov u guano mod ” amps: omsfla mo u u mopscws « u

Moano mo om< u mflpmposn u .Hs mod n Moflso u mews u ommmoa u macho

aeaHasaas

mo honsss omsao>¢ mwaopomn mo honssz

 

ovma aw honso>oz I psosHamQHm mo open
naoHso espouse maaeHsnoa
mo aeaHaosas are one oasnoaxm oaoaom soaps
mxoflmo oopmope ophsopamshom aohm poboEom mfiaopomm mo nonssz

H>N canoe

39

Fifteen one—day old chicks were fumigated. After first dipping
into a'S. pullorum culture and drying, these chicks were then fumigated

in groups.

fir
. * ' .53 ,

.
0
w. . '
‘ l
I
.
u
.4?" ..
- I
2'- 0. 7‘ l
o- _ V .
a" ' I
\ . .

A

A
\

‘

f 'v /

o
I
w]

\1"

 

Hat )7

Fig. 6. Photograph Showing Section of Jamesway
Incubator Used for Experiments.

40

 

 

 

meOpdm pm ompmaomfl asaoaadm .m.*
m ooHHae 000.040.o 000.m00.H m
m ooHHax 000.mmm.0 m
m eoHHae 000.00m.MH H aHoaesoo m
N aooao 000.0mm.m 000.me0.0 m
m aooao 0 N
a asses 000.mea H 0m eHoaoo a
m aooao 000.m4m.H 00m.eHe.H m
m *ooaa 00m.m0m m
N aooam 000.mm0.m H 0H oHoaoo m
m aeoaa 000.0m<.H 00m.mmm.H m
m aooao 000.mme.H m
m asoaa 00m.mem.H H 0m eHmsam m
m asses 000.0em 000.mmw m
m aeoHo 00m.800 m
H eoHo 00m.eem H 0H onsHm H
Ammsov » Moflmo pod u popes omcflp mo u u mopdsas “ “
Moflno mo ems « swaopomn u .Hs you « Moano u mafia « mwmmom u macho
hpwafinsfl> " mo Aspens mmmao>< u manoeomn Mo nonssz u u u u
owma .mm nonso>oz I psosfiaomxm mo open

so asHHHbsa> one see oasnoaam oaooom eoaan

aonso ooeeose meaeHsaom

mxoflso oopoona oehnooamsaom Bong vo>osom swamposm mo honssz

HH>M oHQmB

Eighteen day—old chicks were dipped in a suspension oqu.

pullorum, dried thoroughly and then fumigated in groups of one to

 

six. An additional group six was added in this experiment whereby
the chicks were exposed to a triple dose of formalin for a time

limit of ten minutes.

i
:

 

 

Fig. 7. Photograph Demonstrating Method of Streaking Chick Organs.

hmepsm cod: oopoaomfl Esaoaasm .w.*

 

 

 

N aoon 00m.N 000.m0H m
N aeoHo 000.m0H N
N aooaa 00m.e H 0H oHaNae o
m eoHHae 000.m00.H 00m.Nma m
m ooHHae 00m.NmN.H N
m eoHHNe 000.0a0.H H nHoaesoo m
N aeoaa mmw.ame 000.e00 m
m Aeoasoaav eoHo 000.04N N
N aeoao 00m.em0.H H 0N oHasoo a
N aeoHa 000.0He.N 000.0Hm.m m
N asses 000.00m.N N
N woman 000.mm0.N H 0H oHosoa m
N asoam 000.mHN.N 000.0ea.H m
N asoaa 000.mam.H N
H aooao 000.0m0.m H 0N aHmaam N
m ooHHae 000.mm0.H 00m.NH¢ m
e aeoam 000.m0m.H N
o aooao 00m.emm.H H 0H onsam H
Ammsov u xoflzo pom " hopes omsfln mo u u mopscfls u «
mowno mo oma ” swampomp u .Hs mod n guano u mafia « omwmom u azopo
hpwaflnma> u mo popes: ommao>< " mahoposn Mo nonssz u u u u
oaaH .eN sonaoeoz esosHaoaam No been

naoHao ooeaoaa NsHaHsnom
mo hpflaflnmfl> on» one mnemoaxm oaomom omens

mxoflso oopmoha ophnopHMENOM Bopm embosom swampomm mo monasz

HHH>N oHQmB

43

Five groups of chicks were again fumigated. However, groups
four and five had only two chicks each as this week's hatch was very

small. Livability was again checked for each group.

 

 

 

 

 

 

 

 

Fig. 8. Photograph Before Battery Brooder Used in Experiments.

hmaopdw cons umpmaomfl ammmwmmm..m *

 

 

 

0H 0¢HHH0 000.HHm.H 000.>H<.H m
e 0mH0 000.m00.H H mHoppqoo m
H *0mH0 000.qu 000.00m m
H *0mH0 000.0sq H om oHpsoa e
m *00H0 000.m¢0 000.0Hm m
a *emHa 00m.me N
m *0mH0 00m.000 H 0H oHnsoa m
m *0on 00m.>ms 000.mm< . m
m *0oH0 00m.>mo.H m
m *0mH0 000.0Hw H om mHmnHm m
m *eme 00m.m>m.H 00m.mqm.H m
m *emHa 00m.~0m.m m
a *0mH0 00m.mmm.H H 0H mequ H
Ammwwv “ Moano Hon u nova: omcfin mo u « mmgdafla u «
Moflno mo mm¢ u wwpmpown “ .HS pom u Mowno » mafia u ommmom n @3090
hpwaflnww> u we Hones: ommpm>¢ « wfihmpomp mo pmnasz u u u u
oqoa «m Amnemomm I pqmaflpomxm “0 open

meHgo empmmpa msHpHsmmm
mo spHHHan> 009 03a masmoaxm opoemm emHnm
mMOflgo condone 00%Smuadanom Scam Um>o€om wflhopowm mo honezz

NH>N oHan

45

Table XX

Number of Bacteria Removed from Formaldehyde
Treated Chicks. Dried Before Exposure.

Summary of Tables XV to XIX inclusive

 

 

: : : : No. of bacteria : Average Number
Group : Dosage : Time : Chicks: per ml. : of bacteria
: : minutes : : of rinse water : per click
1 : Single . 10 : 16 : 55,679,500 : 3,479,968
3 Double 10 16 31,327,500 1,957,968
2 Single 20 16 46,147,000 2,884,187
4 Double 20 12 79,281,500 6,606,791
5 Triple 10 3 277,500 92,500
6 Controls 13 1,401,506,500 107,808,190

 

Table XXI

46

Number of Bacteria Removed from Formaldehyde

Treated Chicks.

Summary of Table XIV

Wet Before Exposure.

 

(
No. of bacteria

Average Number

 

Group : Dosage : Time : Chicks: per m1. : of bacteria
: : minutes : : of rinse water : per chick
I : Single 3 10 3 A 3 82,500 : 20,625
3 Double 10 4 547,500 136,875
2 Single 20 4 105,000 26,250
4 Double 2O 4 15,000 3,750
5 Controls 4 1,651,000 412,750

 

47

Summary

In every instance the treated chicks carried high counts on
both the wet and dry chicks. Data show that although some organisms
are removed, there are still enough organisms left to cause the
disease.

Because only four chicks are used for each part of the ex-
periment, it is very difficult to secure results which.are very
compatible on the quantitative basis. The results obtained from
the difference in time period and dosages were variable but the
trend shows that there is a slight diminution for the longer periods
and stronger dosages as there is a decrease in the number of organisms
over the controls. However, there are still enough organisms left on

the chick to reinfect the chicks as shown by the death loss.

48

SULTING VIABILITY OF FUMIGATED CHICK

This experiment was carried on to determine resulting liva-
bility of chicks after exposing them to certain fumigation periods.
At the beginning of these particular experiments, it was decided to
fumigate these chicks in the specially built fumigation chamber, but
it was finally decided to finish the series by fumigating in the
hatching compartment of the Jamesway incubator. It was thought that
the formaldehyde fumes were not as completely removed from the fumi-
gation chamber after each fumigation period as readily as from the
Jamesway incubator. Furthermore, the experiments run in this manner
were more comparable to the conditions found in the field.

Each group of chicks was fumigated in the hatching compart-
ment and Subjected to various doses of formalin and potassium per-
manganate for various periods of time. All chicks were then put into

the battery brooder and checked for livability.

49

Table XXII
Resulting Viability of Fumigated Chicks
Date of Experiment - October 30, 1940
Fumigation - Single dose for 10 minutes
(Temp. 100°F.
Relative humidity — 77 per cent(
. (wet bulb 93°F.

Number of chicks used - 32

 

 

 

 

Age of chicks : Group 1 : Group 2
(days) : Controls : Fumigated

l . 0 dead 3 A dead

2 0 dead 2 dead

3 0 dead 1 dead

4 1 dead 0 dead

Totals 1 dead . 7 dead
26 15 killed 9 killed

 

The remaining birds were killed November 25. Upon autopsy
the nine birds fumigated showed congested lungs and unabsorbed egg

yolks, while the controls appeared normal.

50

Thirty-two day—old chicks were divided into two groups of

sixteen chicks each.

Table XXIII
Resulting Viability of Fumigated Chicks
Date of Experiment - November 13, 1940
Fumigation — Double dose for 10 minutes
(Temp. lOOOF.
Relative humidity - 54 per cent(
(wet bulb 85°F.

Number of chicks used — 32

 

 

 

 

Age of chicks : Group 1 : Group 2
(days) : Controls : Fumigated
l : 0 dead : 1 dead
7 0 dead 1 dead
11 0 dead 1 dead
12 0 dead 1 dead
12 0 dead 4 dead
13 0 dead 2 dead
14 0 dead 1 dead
15 0 dead 0 dead
Totals 0 dead 11 dead
31 16 killed 5 killed

 

The remaining birds were killed on December 5. Sixteen con-

trols and five fumigated chicks were autopsied. In each case the

fumigated chicks showed congested lungs and unabsorbed egg yolks.

51

Table XXIV
Resulting Viability of Fumigated Chicks
Date of EXperiment — November 20, 1940
Fumigation - Double dose for 20 minutes
(Temp. 100°F.
Relative humidity — 78 per cent(
(wet bulb 90°F.

Number of chicks used - 26

 

 

 

 

Age of chicks : Group 1 : Group 2
(days) : Controls : Fumigated

l 3 0 dead : 0 dead

2 0 dead 0 dead

3 0 dead 1 dead

4 0 dead 1 dead

5 - 20 0 dead 0 dead

Totals 0 dead 2 dead
30 13 killed 11 killed

 

The two chicks which died appeared normal when autopsied.
The remaining chicks were killed and autopsied on December 24 and

they appeared normal.

52

Table XXV
Resulting Viability of Fumigated Chicks
Date of Experiment - November 27, 1940
Fumigation - Triple dose for 10 minutes
(Temp. lOOOF.
Relative humidity - 83 per cent(
(wet bulb 95°F.

Number of chicks used — 3O

 

 

 

 

Age of chicks : Group 1 : Group 2
(days) : Controls : Fumigated

1 ° 0 dead . 0 dead

2 1 dead 2 dead

3 1 dead 0 dead

4 ° 1 dead 3 dead

5 1 dead 1 dead

6 0 dead 1 dead

8 0 dead 1 dead

9 0 dead 1 dead

10 0 dead 2 dead

13 0 dead 1 dead

Totals 4 dead 12 dead
25 11 killed 3 killed

 

Upon autopsy, the three

fumigated chicks showed lung congestion

and unabsorbed egg yolk in each case while the control chicks appeared

normal.

53

Another hatch of 20 chicks was divided into two groups of ten
chicks each. Ten chicks were subjected to a single dose of formalin
for twenty minutes. These chicks were put in the battery brooder to

check comparative behavior.

Table XXVI
Resulting Viability of Fumigated Chicks
Date of Experiment — December 5, 1940
Fumigation — Single dose for 20 minutes
(Temp. 100%".
Relative humidity — 96 per cent(
(Wet bulb 99°F.

Number of chicks used — 2O

 

 

 

 

Age of chicks : Group 1 : Group 2
(days) : Controls : Fumigated

l : ' 0 dead 3 0 dead

2 0 dead 1 dead

3 1 dead . 1 dead

4 0 dead 0 dead

5 0 dead 1 dead

6 0 dead 1 dead

7 0 dead 1 dead

Totals 1 dead 5 dead
26 9 killed 5 killed

 

,The remaining chicks were killed and autopsied on December 30.

All chicks appeared normal.

54

EFFECT OF FUNIGATION UPON EGG SHELLS, CHEESE
CLOTH SQUARES AND COVER GLASSES

This experiment was carried on to study the effect of fumi—
gation upon egg shells, cheese cloth squares and cover glasses.
These experiments were similar to those in which chicks were used
for fumigation trials, except that in this case egg shells, cheese
cloth squares, and cover glasses were impregnated with S, pullorum
after first being sterilized.

In this test, the cheese cloth squares, shells and glasses
were dipped in the culture of S. pullorum and while still wet were
fumigated in groups and then immediately washed in 100 cc. of
sterile water. Appropriate dilutions were made and plain agar was
poured onto the plates after which they were incubated at 37° C.
for twenty-four hours.

All figures quoted refer to number of S, pullorum organisms

isolated.

55

Table XXVII
The Number of Bacteria Removed from Formaldehyde
Treated Egg Shells, Cheese Cloth Squares and
Cover Glasses. Wet Before Exposure.

Date of Experiment - November 11, 1940

 

O. .0 .0

No. of bacteria

 

Group Dosage Time Medium per ml. : Average
minutes of rinse water :

1 Single 10 Cloth 9,300,000

Shells 12,000,000

Glass 17,250,000 12,850,000
2 Double 10 Cloth 23,750,000

Shells 10,575,000

Glass 19,275,000 17,866,666
3 Single 20 Cloth 8,175,000

Shells 2,625,000

Glass 300,000 3,700,000
4 Double 20 Cloth 11,925,000

Shells 15,375,000

Glass 18,075,000 15,125,000
5 Controls Cloth 31,800,000

Shells 65,325,000

Glass 12,900,000 36,675,000

 

56

In this test the egg shells, cheese cloth squares and cover

glasses were dried after dipping into the S, pullorum culture.

Table XXVIII

The Number of Bacteria Removed from Formaldehyde
Treated Egg Shells. Dried Before Exposure.

Date of

Experiment - November 11, 1940

 

Group

A

Dosage

: No. of bacteria
Egg : per ml.
shells : of rinse water

Time
minutes

Average

 

1 Single

2 Single

3 Double

4 Control

10 877,500
75,000

225,000

\UNH

20 1,950,000
1,650,000

900,000
10 8,925,000
7,200,000

1,425,000

P le-J WADE-J

31,575,000

392,500

1,500,000

5,800,000

31,575,000

 

57

Table XXIX
The Number of Bacteria Removed from Formaldehyde
Treated Cheese Cloth Squares.
Dried Before Exposure.

Date of Experiment - November 11, 1940

 

No. of bacteria

 

Group : Dosage : Time : Cloth : per ml. : Average
: : minutes : squares : of rinse water :

1 Single 10 1 O
2 255,000

3 975,000 410,000
2 Single 20 1 23,625,000
2 45,000,000

3 975,000 22,866,666
3 Double 10 1 1,102,500
2 127,000

3 67,500 423,333

4 Control 1 61,425,000 61,425,000

 

58

Table XXX

The Number of Bacteria Removed from Formaldehyde
Treated Cover Glasses. Dried Before Exposure

Date of Experiment - November 11, 1940

 

No. of bacteria

 

Group : Dosage : Time : Cover : per m1. : Average
: : minutes : glasses : of rinse water :
3 Double lO 1 1,417,500
3 1,400,000 2,041,666
4 Control 1 9,157,500 9,157,500

 

Table XXXI

The Number of Bacteria Removed from Formaldehyde
Treated Egg Shells, Cheese Cloth Squares,
and Cover Glasses. Dried Before Exposure

Summary of Tables

XXVIII to XXX inclusive.

59

 

No. of bacteria

 

: z z : :
Group : Dosage : Time : Medium : per m1. : Average
: : minutes : : of rinse water :
Egg
1 Single 10 3 1,177,500 392,500
3 Double 10 3 17,650,000 5,883,333
4 Control 1 31,575,000 31,575,000
Cloth
Sguares
1 Single 10 3 1,230,000 409,999
2 Single 20 3 79,600,000 ‘6,533,333
3 Double 10 3 1,297,000 432,333
4 Control 1 61,425,000 61,425,000
Cover
Classes
3 Double 10 3 6,125,000 2,041,666
4 Control 1 9,157,500 9,157,500

 

Table XXXII
The Number of Bacteria Removed from Formaldehyde
Treated Egg Shells, Cheese Cloth Squares and
Cover Glasses. Wet Before Exposure.

Summary of Table XXVII.

 

No. of bacteria

 

Group : Dosage : Time : Medium : per m1.
: : minutes : : of rinse water
: : : °

Shells
1 Single 10 1 12,000,000
2 Double 10 1 10,575,000
3 Single 20 1 2,625,000
4 Double 2O 1 15,375,000
5 Controls 1 65,325,000

Cloth

Sguares
1 Single 10 1 ' 9,300,000
2 Double 10 1 23,750,000
3 Single 20 1 8,175,000
4 Double 2O 1 11,925,000
5 Controls 1 31,800,000

Cover

Glasses
1 Single 10 1 17,250,000
2 Double 10 1 19,275,000
3 Single 20 1 300,000
4 Double 2O 1 18,075,000

5 Controls 1 12,900,000

 

61

“Sm."w

In this series, egg shells, cheese cloth squares, and glass
cover slips were used instead of baby chicks. These data show
that the fumigation lowers the bacterial count when compared with
the controls. However, enough organisms are left to act as a
possible source of danger to other chicks. The trend shows a

diminution for the longer periods of time and stronger doses.

THE INCIDENCE OF S, PULLORUM IN FECAL MATERIAL
AFTER FUMIGATION

The tetrathionate broth acts as an inhibitor for Escherichia

 

991;, The feces were first weighed and then a suspension of S.
pullorum was thoroughly mixed into the feces. A very thin film of
the feces was Spread over the bottom of Petri dishes and fumigated.
After fumigation, the feces were removed and introduced into ten cc.
of tetrathionate broth and allowed to incubate for eighteen hours
at 370 C. Then a smear was made from the broth and streaked on
freshly prepared MacConkey agar plates and again incubated at 370 C.
for twenty-four hours. The plates were then read.

At the end of the first eighteen hours, the tetrathionate
broth seemed to be very cloudy indicating that there was growth in
every test tube. This broth was then transferred to MacConkey agar
plates and at the end of a twenty—four hour incubation period, the
fumigated plates showed no growth, while all plates had growth at
the end of forty-eight hours. This growth was later demonstrated to

be due to S, pullorum.

63

Table XXXIII

Incidence of S. pullorum in Fecal
Material After Fumigation

Date of EXperiment - December 3, 1940

 

Readings made from

 

tetrathionate : MacConkey
broth : agar
24 hours : 48 hours

Time Tube

minutes

Group Dosage

 

1 Single 10 No growth *
No growth

No growth

‘+«+-+

(0

Single 20 No growth
No growth

No growth

+-+-+-

3 Double 10 No growth
No growth

No growth

-+-+‘+

4 Double 20 No growth
No growth

No growth

5 Controls S, pullorum

S. pullorum
S.pm1mum

mMi-J WMH \DNH bowl-4 bomb-J

4-+-+- +-+ +

 

* This growth later (December 5) was demonstrated to be due to

S. pullorum.

64

Before completing the above series of experiments with the
tetrathionate broth, another experiment was made testing the effect
of formaldehyde gas upon the organism 3°.2211- I concluded that S.
£91i_was so thoroughly dispersed in the fecal material that in order
to kill this organism the fumigant had to be potent. It proved im—
possible to obtain a suspension of S, pullorum which was exactly
comparable to that existing naturally as in the case of S, ggli.

On November 18, fifteen one-gram samples of fresh chicken
fecal material were carefully weighed out. Each sample was carefully
Spread over the bottom of sterile Petri dishes. These plates were
then fumigated. Upon removal from the fumigation chamber, nine cc.
of 0.85 per cent physiological salt solution was poured into the
dish and the fecal material was mixed into the salt solution. From
this mixture, dilutions of l : 10, 1 : 100, 1 : 1000 were made into
a new medium, single strength lauryl sulphate tryptose lactose broth.
This new broth is being used by Doctor Mallmann in checking water
samples. Sb p21; is definitely shown to be present if gas is pro-
duced in this broth.

The tubes were examined at the end of twenty-three houws of

incubation at 370 C.

65

Table XXXIV
Colon Index After Fumigation

Date of Experiment - November 18, 1940

 

Colon Index

Time :
: per cc.

minutes

Group : Dosage Sample

 

1 Single 10 1000
1000

1000

1000
1000
1000

2 Single 20

3 Double 10 1000

10
1000
4 Double 20 1000
1000
1000
5 Control 1000
1000
1000

wNH wNH bowl-4 WNW WNH

 

Only three tubes showed absence of gas production.

66
DISCUSSION

It has been demonstrated in the data submitted that fumigation
with formaldehyde is definitely a marginal disinfection. One must
remember that the dosages used must be exact and the type of material
used must be constant, otherwise, ineffective fumigation would result.
Fumigation as practiced by the hatcherymen is definitely a marginal
disinfection practice.

Inasmuch as the organism (S. pullorum) emanates from the intes-
tinal tract of the chick, discharges, after fumigation, will reinfect
any area traveled by the chick. Thus, fumigation will only give tem-
porary freedom from disease organisms in this area even though the
fumigation process were 100 per cent effective. To effect the elimina-
tion of the organisms throughout the entire hatching period, it would
be necessary to have continuous disinfection which is neither feasible
nor practicable.

Even though some of the organisms are destroyed during the
fumigation period, not all of the organisms are destroyed. Therefore,
the value of fumigation with formaldehyde is limited.

It has been established that a dry formaldehyde gas has no
germicidal action on dry bacteria. To become effective, the gas must
be in solution. The data presented in this paper substantiate this
theory.

Any fecal matter found in the hatching trays is in a dry state

and the S, pullorum exists imbedded in the dry fecal matter. Because

67

formaldehyde has little penetrability, it is impossible to destroy
these organisms by fumigation.

Chicks may be injured by the process of fumigation with for—
maldehyde even though the fumigation is carried out according to
the recommended manner. This damage to the chicks may offset any
benefit gained by destroying a few pullorum organisms. The damage
to the chicks is clearly demonstrated by the mortality suffered

during these experiments.

68

SUMMARY

The following results were secured when fumigation with for-

maldehyde was practiced in doses recommended by various authorities:

1. Fumigation with formaldehyde caused marked injury to the

chicks as demonstrated by the mortality.

2. Fumigation with formaldehyde did not destroy S. pullorum

 

on the infected chicks.

3. Fumigation with formaldehyde did not destroy.S. pullorum
on the infected chicks and allowed the disease to spread to the clean

stock.

4. Fumigation with formaldehyde did not destroy S. pullorum

in fecal matter.

5. Fumigation with formaldehyde did not destroy S, pullorum

on infected egg shells, cheese cloth squares, and glass cover slips.

6. The greatest mortality from S. pullorum occurred about
the sixth day although death may result any time between one and

twenty-eight days.

69

CONCLUSION

1. Pullorum disease in incubators cannot be controlled

merely by fumigation with formaldehyde.

2. If the eggs introduced into the incubator are free from
pullorum disease and the incubator is free of any contamination,

then fumigation with formaldehyde is unnecessary.

7O

RECOHMENDATIONS

1. It is recommended that clean stock be used, that
incubators be kept disease free by exercising care in preventing

the introduction of diseased stock.

2. As a safety factor, terminal disinfection of the

incubator should be practiced.

2.

3.

4.

7.

9.

10.

11.

71

LITERATURE CITED

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Hoffman: As cited by E. C. McCulloch in Disinfection and
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Low: As cited in the 1896 U. S. D. A. Yearbook of Agriculture.

Trillat: As cited in the 1896 U. S. D. A. Yearbook of
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Dorset, M.: Some Common Disinfectants. U. S. D. A. Farmers'
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Gwatkin, R.: Some Experiments on the Disinfection of Eggs
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. The Disinfection of Incubators with Formaldehyde

 

During Hatching. Biol. Absts., 1929, 5, 11221.

Dakan, E. L., and F. Speer: Sanitation in the Hatchery. Ohio

State Univ. Ext. Bull. 90. 1929.

12. Mallmann, W. L., and J. M. Moore: Studies of Pullorum
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: Studies of Pullorum Disease. I. The Influence

 

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13. Graham, R., and W. M. Michael: Studies in Incubator Hygiene.
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: Incubator Hygiene in Control of Pullorum

 

Disease. Univ. Ill. Circ. 403.

 

: Studies on Incubator Hygiene. III. Germi—
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14. Bushnell, L. D., and L. F. Payne: Fumigation of Forced Draft
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: Dissemination of Pullorum Disease in the

 

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22.

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73

Smith Incubator Company. T. S. Townley: Fumigation
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flcCulloch, E. C.: Disinfection and Sterilization.

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1.

2.

.3.

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