me EFFECT or METHYL BROMIDE UPON
SALMONELLAzPULLORUM

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MICHIGAN STATE UNIVERSlTY

Theodore Augustus Maag
1960

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THE EFFECT OF METHYL BROMIDE UPON SALMONELLA PULLORUM

BY

Theodore Augustus Maag

A THESIS

Submitted to the School of Graduate Studies of Michigan
State University of Agriculture and.Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1960

This work is
respectfully dedicated
to

MY FAMILY

ACKNOWLEDGEMENT

The author is grateful to Dr. S. C. Schmittle for his untiring
encouragement, guidance and assistance he has given freely of since the
onset of this project. The work of poultry disease research as under-
taken by Drs. S. C. Schmittle and H. J. Stafseth has been an inspiration
to me. I will be eternally grateful for having had the privilege of
studying under them.

The instruction and guidance of Dr. Mallmann in the field of
sanitary bacteriology has proved to be of invaluable aid in the work
encompassed by this dissertation. I also gratefully acknowledge the
guidance and assistance rendered by Dr. E. D. Devereux and Dr. H. C.
IZindel during the preparation of this manuscript.

The author is grateful to the Michigan Chemical Corporation
of St. Louis, Michigan, for the donation of funds which made it possible

for me to obtain an assistantship.

INTRODUCTION. .

TABLE OF CONTENTS

REVIEW OF LITERATURE

Gaseous Disinfection
Introduction . . . . . .
Formaldehyde . . . . . .
Ethylene Oxide . . . . .
Beta-propiolactone . . .
Methyl Bromide

LA general review .

Action against animal

Technical Data on Methyl Bromide

Commercial Products . . .
Chemical and Physical Characteristic.

Chemistry of its Bactericidal Activity

MATERIALS AND METHODS

Methyl Bromide . . . . . . . .
Gas Measuring Apparatus . . .
Fumigation Chambers . . . . .

Dosage Calculation . . . . . .
Salmonella pullorum . . . . .

 

EXPERIMENTAL PROCEDURES.AND RESULTS

Experiment I -

Experiment II
Experiment III
Experiment IV
Experiment V
Experiment VI
Experiment VII

DISCUSSION . . .

pathogens

Method of.Administering Methyl Bromide .

o
MUTUJN

22
22
24

29
3O
30
32
32
33

Effects of Methyl Bromide on Bacteriological Media 34

Adequacy of Counting Techniques

Dosage and Time . . . . . . .

Temperature Effect
Inactivation Rate
Humidity Effects

Incubator Studies

SUMMARY AND CONCLUSIONS . . . . .

REFERENCES . . .

.APPENDIX .

iii

8

36
37
41
43
48
50

52

58

59

66

FIGURE

Alkylating reaction of methyl bromide with a protein

containing labile carboxyl, amino, sulfhydryl, and

LIST OF FIGURES

hYdr oxy]. groups 0 O 0 O O O O O O I O O

The fumigation apparatus
Non-autoclaved nutrient agar plates fumigated and not
fumigated with methyl bromide at 11 1b/1om ft3 for 48 hours

Dosage of methyl bromide and exposure time required to kill

Percent survival of_§L pullorum.after eXposure to methyl
bromide at 5 lb/lOOO ft
MacConkey agar plates streaked with §L pullorum.fumigated

3
with 3 lb/lOOO ft of methyl bromide for varying time

intervals .

3

at 24 C.

iv

.§L pullorum at room temperature (24 C) .

PAGE

25

31

35

40

46

47

LIST OF TABLES

TABLE PAGE
1 Minimal quantity of methyl bromide in lb/looo ft3

required to kill poultry pathogens under different

conditions . . . . . . . . . . . . . . . . . . . . . . . . . 20
2 Technical data - methyl bromide . . . . . . . . . . . . . . 23
3 Relative activity of certain disinfectants against bacterial

spores and against vegetative cells . . . . . . . . . . . . . 28
4 Variations in numbers of §; pullorum upon standardized filter

paper strips . . . . . . . . . . . . . . . . . . . . . . . . 38
5 Hours required to kill S; pullorum with methyl bromide at

24 C and 100 percent relative humidity . . . . . . . . . . . 39
6 Effects of varying temperature on the death of_§L pullorum

at 5 1b/1000 ft3 . . . . . . . . . . . . . . . . . . . . . . 42

7 Percent survival of Salmonella pullorum after exposure to

 

methyl bromide for varying periods of time in the presence

of a water saturated atmosphere . . . . . . . . . . . . . . 44
8 Effects of different humidity levels on the ability of

methyl bromide at 5 1b/1000 ft3 to inactivate g; pullorum

and Staph. aur eus O O O O O O O O O O O O O O O O I O O O O 49

 

9 Fumigation of embryonated eggs with methyl bromide in

large desiccator jars . . . . . . . . . . . . . . . . . . . . Sl

INTRODUCTION

After an outbreak of an infectious disease in a poultry
house, disinfection of the house and premises is generally recommended.
E3pecially is this needed when young birds are being introduced as re-
placements. During the time interval between the diSposal of the old
birds and the placing of new birds in a house, disinfection is again
advised to prevent the exposure of replacement birds to a heavy micro-
biological infection.

In disinfecting any poultry house the major problems are the
porous Surfaces, the residual organic matter and the dust which remains
even after cleaning. Many poultry houses have dirt, or porous, rough
-concrete floors. Therefore, a desirable disinfectant for poultry houses
should: a. have Superior penetrating activity, b. applied easily,

c. kill micro-organisms in presence of soil and organic matter, d.

be non-toxic to poultry, and e. be non-toxic to man. There are other
important attributes of a disinfectant Such as being non-corrosive to
equipment, lingering or residual effect, dosage level, etc. which could
also be included.

The objective of this investigation was to ascertain the
effectiveness of gaseous methyl bromide as a disinfectant against a

poultry pathogen.

REVIEW OF LITERATURE
Gaseous Disinfection
Introduction

Phillips (1957) briefly reviewed gaseous disinfection and
stated that vapors were used in Biblical times. The burning of incense,
such as frankincense and myrrh, was associated with purification.

During medieval epidemics, the materials on which messages were written
were held in smoke in the belief that this procedure would prevent the
spread of disease. The first authenticated report of scientific dis—
infection with an aerosal was recorded by Lister who Sprayed phenol in
operating rooms with the Specific objective of controlling microbes.

Early use of gaseous disinfectants took two directions. Air
purification was the object in one and the other was terminal or com-
plete sterilization of the air and all objeCtS exposed within an en-
closed space. .Air purification involved substances, such as the glycols,
which were directed toward aerosol sanitation. Harry (1956, 1957) pre-
sented an excellent review of air purification as related to poultry
rearing industry. Terminal sterilization with vapors was first used in
sick rooms to kill infectious disease agents. This practice began in
the late decades of the nineteenth century and has continued into this

century. Some countries made routine fumigation of sick rooms mandatory

whenever an infectious disease occurred. Present knowledge of the

actions and limitations of the various disinfectants used for these fum-
igations makes it doubtful if total disinfection was thus obtained.

The early gaseous disinfectants were sulfur dioxide and
chlorine. These gases were extremely damaging to many objects which was
a definite disadvantage to their use. Formaldehyde was discovered in
1894 as a bactericidal agent and was the first practical gaseous disin-
fectant. It came into general use because of its effectiveness and gen-
eral safety for personnel and equipment. Numerous papers had been
written concerning formaldehyde as a disinfectant. Nordgren (1939)
reviewed the early literature on gaseous disinfectants and Phillips
(1957) and Sykes (1958) have summarized published research of more re-

cent date.

Formaldehyde

Formaldehyde gas is generated either by vaporizing parafor-
madlehyde or by heating a solution of the gas in a water mixture called
formalin. Formaldehyde is stable only at temperatures above 80°C when
in high concentrations. Paraformaldehyde is solid polymer of formalde-
hyde existing as a mixture of polyoxymethylene glycols. When heat is
applied to paraformaldehyde depolymerization occurs with formaldehyde
and water vapor resulting. Formalin is the most common source of for-
maldehyde. It exists in a 35 t percent concentration as a hydrate. Com-
mercial formalin is stabilized with 8 to 15 percent methanol to prevent

formation of the solid polymers. One of the most common methods used

to volatilize formaldehyde gas is by the heat of the oxidative reaction
produced in mixing two parts of formalin and one part potassium perman-
ganate.

When used as a disinfectant, formaldehyde is a surface disin-
fectant. Formaldehyde has little activity in penetrating even the most
porous substances. Cracks, crevices and pores are penetrated with dif-
ficulty. On a smooth surface, microorganisms may be easily protected
by a covering of organic matter. The poor penetrability of formaldehyde
is due to polymerization upon all surfaces with which it comes in con-
tact. These polymers are extremely obnoxious in that they may linger
for several days and slowly break down to formaldehyde gas. Formalde-
hyde, because of its instability, must be used at a temperature above
20°C with the relative humidity 60 to 80 percent. Only relatively low
concentration can be maintained in the atmosphere. At room temperature
only about 3 ounces can be maintained per 1000 ft3 of Space. Sykes
(1958) reviewed the activity of formaldehyde against bacterial cells
and observed that the reaction was reversible. The inactivated cells
are apparently in a state of suspended animation; but that certain
“neutralizers” plus a normal nutrient medium will revive these bacteria.
Perhaps this phenomenon will explain the conflicting evidence concerning
the effectiveness of formaldehyde on organisms that have been exposed
to the gas.

In conclusion, formaldehyde disinfection is only effective
if several varied conditions are accomplished. There are also adverse

side effects present in many types of applications.

Ethylene Oxide

Ethylene oxide was used first as an insect fumigant by Cotton
and Roark (1928). Schrader and Bossert (1936) applied for patents in
Germany during 1928 and in the United States in 1929. In the patent
applications it was stated that the alkylene oxides were capable of
killing pests and germs of all kinds. Later, ethylene oxide was widely
advocated as a microbicidal agent in the sterilization of spices, food-
stuffs and other common organic products by Griffith and Hall (1938,
1940a, 1940b, 1940c, 1943), Kirby, Atkins, and Frey (1936), Yesair and
Cameron (1938), and Hall (1938). Phillips and Kaye (1949), Phillips
(1949), Kaye (1949), and Kaye and Phillips (1949) introduced the prac-
tical field of gaseous sterilization as it is used today. These four
publications by Phillips and Kaye reviewed the earlier published data on
ethylene oxide as a bactericidal gas and presented extensive original
data substantiating the earlier work.

Phillips (1957), Phillips and Warshowsky (1958), Sykes (1958),
and Phillips (1958) reviewed extensively the various uses of ethylene
oxide as a sterilizing agent. Textiles, plastics, paper, wood, oil
paintings, medical and biological preparations, plaster of Paris band-
ages, medical instruments, arteries, animal feeds, eggs, bacteriologi-
cal media, soil, airplanes, trucks, delicate electronic instruments,
and analytical balances are some of the various materials sterilized.
All species of bacteria, fungi and viruses are affected by the lethal
action of ethylene oxide.

The boiling point of ethylene oxide is 10.8 C. It has an

ether-like smell, is highly eXplosive, generally safe to use on most

types of objects and is relatively non-toxic to humans. This gas has
extremely good penetrating power and does not require Special conditions
in order to be reactive. To suppress the explosiveness of ethylene
oxide a mixture of 10 percent ethylene oxide and 90 percent carbon diox-
ide is commonly used. This dilution lowers the concentration of the gas
and increases its cost considerably.

Kaye and Phillips (1949) found that only extremes in humidity
were important factors in the sterilizing activity of ethylene oxide.
At 28 percent relative humidity ethylene oxide is most active, while
at 65 percent it is one-fourth as active and at 100 percent but one-
tenth as active. At complete dryness of a product such as would occur
in lyophilization, the action of ethylene oxide is inhibited. They
advanced two theories as to the role of humidity in the sterilizing
action of ethylene oxide. First there must be a mechanical role in that
some moisture must be present within the cell to dissolve the ethylene
oxide and permit its contact with the cellular protein. Too much moisture
causes a dilution effect upon ethylene oxide before it comes into contact
with the organism. {A second theory postulates that ethylene oxide reacts
with and adds onto groups within the protein molecules, such as hydroxyl,
sulfhydryl, amino, or carboxyl groups which contain a labile hydrogen.
To do this the labile hydrogen would have to ionize before the ethylene
oxide could react with them.

Hawk and Mickelsen (1955), Bakerman, Romine, Schricker,
Takahashi, and Mickelsen (1956), Windmueller (1956), Windmueller and

Engel (1956), and Windmueller, ACkerman, and Engel (1956) reported

research concerning the adverse effect of ethylene oxide on the diets
of experimental animals.

The availability of thiamine, riboflavine, pyridoxine,
niacin, and folic acid is adversely affected. Pantothenic acid,
biotin and vitamin B12 remained unaffected. Ethylene oxide fumi-
gated casein, as a protein source, for rats gave no growth but this
condition was rectified by addition of supplemental methionine and
histidine in the diets. It appears that some chemical reaction in-
activates various nutrients in the diets without imparting toxicity.

Several workers have used ethylene oxide to sterilize media
for the cultivation of microorganisms and insects without adverse
effects on the subsequent growth of several different species (Hansen
and Snyder 1947, Wilson and Bruno 1950, Judge and Pelozar 1955, and
Barlow and House 1956). Sykes (1958), however, reported that he has
found that some lots of serum sterilized with ethylene oxide were

less capable of Supporting the growth of some sensitive bacteria.

Beta-propiolactone

Hoffman and Warshowsky (1958) were the first to report the
bactericidal activity of beta-propiolactone vapor. Their report
shows that beta-propiolactone vapor is 50,000 times more effective
than methyl bromide and 40,000 times more effective than ethylene
oxide and 25 times more effective than formaldehyde vapor for a 90
percent kill of bacteria.

Phillips (1958) and Phillips and Warshowsky (1958) briefly

reviewed beta-propiolactone as a gaseous sterilizing agent. They make

this statement:

"The present authors feel that the compound possesses
almost all of the advantages of formaldehyde, and
avoids two of its disadvantages, slow action and ex-
treme persistency as a vapor, following use. It is
believed that beta-propiolactone should in the future
replace formaldehyde in many applications where it is
now used, and Should find additional applications where
the disadvantages of formaldehyde have limited its use."
Beta-propiolactone has a relatively high boiling point
(155 C) hence low vapor pressure at ordinary temperatures. This gas
is similar to ethylene oxide, methyl bromide, and formaldehyde in that
it is also bactericidal by virtue of its alkylating ability. It is
active at a low concentration so it is easy to obtain sufficient vapor
concentration in an atmosphere to be bactericidal. This gas, like
formaldehyde, requires a high humidity (70 percent or above) and an
effective concentration can be maintained in an area without it being
hermetically sealed. Beta-propiolactone does not penetrate porous
materials easily but is rapid in its inactivating action, two hours
of exposure being adequate compared to formaldehyde which needs ten
or more hours in order to be effective. unlike formaldehyde, beta-
propiolactone does not polymerize on surfaces and fumigated areas are
readily aired free of gas. This gas is lachrymatory but less irritating
than formaldehyde.
Phillips in a personal communication (1959) states:
"...we (workers in the field of gaseous sterilization
at Fort Detrick, Frederick, Maryland) would believe
from our own work, that beta-propiolactone would be

more effective than methyl bromide in the control of
poultry diseases."

Methyl Bromide

.é general review - Le Goupils (1932) demonstrated the toxicity of

 

gaseous methyl bromide for insects. It quickly came into extensive
usage by the plant, grain, and food industries as an effective agent
against insects and rodents. The advantages of methyl bromide over
other fumigants then in use was its high penetrability and low toxicity
to most plants at a low concentration, dormant stock and seeds, low
residual toxicity, freedom of damaging chemical action on equipment and
materials, and wide range of effectiveness at different temperatures.
Richardson and Johnson (1935), Mackie and Carter (1937), Phillips and
Easter (1943), Steinweden (1945), Latta, Richardson and Kindler (1946),
Phillips (1955), Dow Chemical Corporation manual and Michigan Chemical
Corporation manual have reviewed methyl bromide, the methodology of its
uses and effects. These are but a few of the many publications which
have dealt with methyl bromide and its uses in the plant agricultural
field and insect control. Through its use in the fumigation of green-
houses, nurseries, and buildings to control insects and rodents, methyl
bromide was also found to be highly lethal to many other organisms.
These included weeds and other plants, insect larvae and other phyto-
pathogenic and free-living invertebrates, as well as fungi, bacteria,
and viruses which are pathogenic to plants.

The New Jersey Experiment Station in 1939 reported that 2-1/2
lb of methyl bromide per 1000 ft3 of soil for 2-1/2 hours did not

affect the root knot nematode Heterodera marioni. Hawkins (1939)

 

10

eliminated the white fringe beetle Pontomorus leucloma from potting

 

soil by a treatment with 40 ml of liquid methyl bromide per yd3 of

soil. Taylor and McBeth (1940 and 1941) presented the first evidence
of methyl bromide's wide range of activity in soil when the gas concen-
tration was maintained through the use of gas—tight cover over the soil.

These workers used methyl bromide to kill Heterodera marioni and innum-

 

erable Species of free living nematodes in the soil. They also reported
that bacteria and fungi were killed in the treated soils. Taylor and
McBeth noticed that less damping off occurred with plants grown upon
the treated beds. These workers used 80 ml/M3 of soil in vault fumi-

gations and 1-1/2 1b/200 it2

for fumigation under a gas-tight cover.
Livingstone (1940) used methyl bromide to control the larvae of the
white fringed beetle in the soil. An anonymous article in Florist
Exchange and Horticulture Trade World in 1940 reported the use of
methyl bromide to control grubs of the Asiatic beetle in azalea beds.
Mark (1941) used methyl bromide and ethylene oxide in con-
trolling spoilage organisms of dates. Salle and Korzenovsky (1942)
reported that methyl bromide in two hours exposure at an unstated
concentration did not kill bacterial spores. Godfrey and Young (1943)
reviewed soil fumigation for plant disease control through part of

1942. They used methyl bromide at 2-1/2 m1 of the liquid per ft3

of
soil in gas—tight containers and killed nematodes and fungi pathogenic.

for plants. In other trials using a gas—tight cover, methyl bromide

killed Sclerotinia sclerotiorum buried eight inches in wet potting

 

soil at 53 F with a dose of 2 1bs/yd3. When the soil was dry,

11

Sclerotinia was not killed but the damping off organism was killed.

 

Baerwald (1945) patented a method of sterilizing foodstuffs such as
sausage with methyl bromide and ethylene oxide. Baerwald stated that
l, 2, or 3 g of a methyl bromide snow (50 percent methyl bromide, 50
percent water mixed and frozen then ground) would completely sterilize
a heavily contaminated sausage wrapped in pliofilm or cellophane. .After
sterilizing the package the methyl bromide would diffuse out of the
wrapper leaving no toxic residues. Johnson (1945) inactivated the
virus of wheat mosaic disease using gaseous methyl bromide.

Whelton, Phaff, Mrak, and Fisher (1946) fumigating freshly
inoculated slants for 24 hours at room temperature, found the following
concentration of methyl bromide necessary to effectively kill: bacteria

(Bacillus, Escherichia, and Pseudomonas) 5 m1 of the liquid per 1 of

 

air, fungi (Aspergillus and Penicillium) and yeasts (Endomycopsin,

 

 

‘gygosaccharomyces, Saccharomyces, and Hanseniospora) l ml/l. Ethylene

 

oxide mixture (20 percent ethylene oxide in ethylene dichloride) was
approximately five times more effective than the methyl bromide in
this trial.

Newhall (1946) reviewed methyl bromide as a fungicide and
nematocide. Using gallon crocks with no vapor seal, Stark and Lear
(1947) obtained control of the damping off organisms with methyl
bromide. Christie (1947), using methyl bromide without adequate means
to confine the gas, found that nematodes but not fungi were killed in

their trials. Newhall and Lear (1948) recommended methyl bromide for

12

soil fumigation as a result of experimentation in which they showed
that methyl bromide killed fungi and was easily administered with good
penetration through the soil with great effectiveness. Phillips (1949)
working with ethylene oxide and methyl bromide found the latter bacter-
icidal but less so than ethylene oxide.

Lear and Mai (1950) killed golden nematode cysts on used bags
tied in bundles of fifty or in soil, 10 lb per paper sack, when fumi-
gated with methyl bromide. The gas was confined by means of either gas
proof flexible plastic tarpaulin, a copper lined fumigation chamber, or
a 55 gallon metal drum with a removable cover. Lear (1951) in a review
presents some new data on the effectiveness of methyl bromide as a soil
fumigant.

Swank (1951) reported that methyl bromide killed damping off

and root rot organisms in the Rhizoctonia, Pythium, and Fusarium genera.

 

Lear (1952) advocated methyl bromide fumigation of bags and machinery
on the farm as a method of controlling the spread of the golden nema-
tode of potatoes. .A lethal exposure consisted of 23 lb for 16 hours

or 46 lb of methyl bromide per 1000 ft3

for two hourS. The tempera-
ture needed to be, at least, 60 F. According to a report by Stover
and Koch (1952) muck soil fumigated with 2 lb of methyl bromide per
100 ft3 was free of all tobacco pathogens. a dosage of l 1b/100 ft3
killed the damping off organism and all weeds. The work of Wensbey
(1953) states that methyl bromide is more effective against fungi and

bacteria than against actinomycetes. This work was done utilizing a

13

L. D. 90 (90 percent of the organisms killed) as the endpoint. Soil
moisture did not affect methyl bromide’s activity. High organic content
soils slowed up the action by combination with methyl bromide and slowed
penetration by the gas.

Trickel (1952) used methyl bromide in controlling molds on
cheese. He found that ethylene oxide required 16 hours and methyl
bromide 8 hours for complete kill of all Penicillium roqueforti, using

5 1b of the gas per 1000 ft3 at 74 F. Methyl bromide did not kill_§L

 

roqueforti in 20 hours with a 10 1b dose at 40 F. .At 60 F dosages of
3

 

2, 3,4, 5, and 10 lb/lOOO ft of fumigated volume killed completely

.2; roqueforti in 20, 12, 8, 6, and 4 hours respectively. Trickel

 

studied various factors as they might affect the activity of methyl
bromide to kill molds. The pH of the media upon which the molds were
exposed, moisture content of the atmosphere, negative pressure, for-
mation of inhibitory substances in the media upon fumigation, and var-
ied Susceptibility of the spores and mycelium were not found to be
factors which altered the effectiveness of methyl bromide. .Also sur-
veyed in this paper was the activity of methyl bromide to penetrate
various plastic films. Trickel also studied the disinfectant proper-
ties of methyl bromide on various organisms. Methyl bromide killed
Saccharomyces cerevisiae, Candida krusei, Aspergillus niger, Geotrichum

3

candidum, and Cladosporium herbarum at 10 lb/1000 ft for four hours

 

of 5 1b after eight hours of exposure, but Streptococcus lactis,

 

Escherichia coli, Serratia marcescens, and Mycoderma Sp. were not

 

inactivated during these same exposure conditions.

14

Using pathogenic plant fungi Munnecke and Ferguson (1953)
effectively fumigated these fungi in soil within flats stacked under
a plastic cover. The lethal dosage necessary was found to be 4 lb/
1000 ft3. Mac Lachlen, Monro, Radicot, and King (1953) found that
neither 50, 100, nor 150 mg/l of methyl bromide when used for eight

or sixteen hours killed Corynebacterium.sepedonicum. McKeen (1954)

 

found that methyl bromide was more effective against fungi than against
bacteria and actinomycetes.

The bacteria had a greater range of variability than fungi
in their susceptibility to methyl bromide. Some fungi were killed at
3/5 lb of methyl bromide per 100 ft3 of soil. Other fungi had a mini-
mum lethal dose of 2 lb/100 ft3 of soil except for some Sporeformers

which survived at dosages as high as 4 lb/lOO ft3

of soil. To com-
pletely inactivate non-Sporeforming soil bacteria dosages from 10 to
20 1b of methyl bromide per 100 ft3 were needed.

McKeen fumigated soil for a 24 hour period at temperatures
between 70 and 80 F. The moisture content of the soil in these exper-
iments did not influence the number of organisms killed.

Munnecke and Lindgren (1954) fumigated 225 flats of soil

3 and

under a plastic cover with 4 1b of methyl bromide per 100 ft
studied the concentration and the fungicidal activity of the gas
throughout the pile. Methyl bromide rapidly penetrated the soil
throughout all flats. The gas rapidly diffused out or was absorbed

in the soil in the first few hours until about 1-1/2 1b of methyl

bromide per 100 ft3 remained in the air. Upon fumigating 24 hours

15

Verticillium alboatrum frequently survived but other fungi did not.
Newhall (1955) gave a review of soil disinfection using

methyl bromide.
Phillips (1959) in a personal communication concerning work
that his group had done with methyl bromide and ethylene oxide states:

"We did not publish on this (methyl bromide) compound
because it was not particularly promising to us. It
is only about 1/10 as effective as ethylene oxide for
example. We did measure its activity at various tem-
peratures against B; subtilis (g; globigii) Staph.
aureus, E; coli and the T-2 phage of_E; coli. It
killed all of these organisms, but Slowly and as with
other alkalating agents there was not the very marked
difference in rate of kill between Spores and vege-

tative agents as is found with other types of disin-
fectants.“

 

Action against animal pathogens - Swanson and Taylor (1943) first used
methyl bromide against nematodes parasitic for mamma1S. They fumigated
sandy loam plots of 63 ft2 each with 1 lb of methyl bromide. These
plots were covered with glue coated kraft paper for 22 hours after
fumigation. The soil normally contained free living nematodes of
several genera and was artificially contaminated with several other
free living forms. The plots were also contaminated with the larvae

of several parasitic Species cultured from calf feces: Haemonchus,

 

Ostertagia, Trichostrongylus, Oesophaggstomum, Cooperia, Bunostomum,
and Nematodirus. All the represented genera were inactivated in the
fumigated plots but remained active in the unfumigated plots. In addi-
tion it was noticed that moving protozoa were present in the control
soil but none was present in the fumigated soil. These workers only

checked the soil to a depth of 12 in.

16

Andrews, Taylor, and Swanson (1943), using similar conditions

and dosage as above, killed Ascaris lumbricoides embryos within the

 

eggs, the infective larvae of Strongyloides and Oesophagostomum, im-

 

 

mature larvae within kidney worm eggs, coccidia, and the earthworm as
the intermediate host of the swine lung worm. They suggest methyl
bromide as a means of cleaning up animal runs or other Small lots used
in extensive growing operations. Cram and Hicks (1944) fumigated
sewage, a mixture of digestion sludge and sand, at a temperature of 61
to 68 F at the same dosage as used in the preceeding work. Ascaris

lumbricoides eggs with only slight development were killed, but eggs

 

with more mature embryos were not inactivated.
Boney (1948) considered methyl bromide to have possible
economic value as a fumigant in the control of cecal coccidiosis of

chickens. He found that methyl bromide inactivated Eimeria tenella

 

under practical conditions in soil plots or in plots covered with cane
pulp litter. These plots were enclosed under wet Sisal Kraft paper and
fumigated for 13 hours at a temperature of 65 F. Fifteen-hundredths to
0.3 m1 of liquid methyl bromide per ft2 (approximately 1 lb/lOOO ftz)
was found to be lethal t°.§; tenella. In practical type of fumigations
involving small brooder houses, E; tenella was inactivated. The dosage

used was 2 lb/lOOO ft3

for a house sealed completely within a gas-tight
cover or 3 1b/1000 ft3 in a house sealed with masking tape and wet news-
papers only. The fumigation period was 13 hours at 65 F. Clapham (1950)
fumigated plots ten yards Square for 24 hours with 25 g of methyl bromide

using a covering of rubberized ballon fabric. She effectively controlled

l7

Heterakis gallinae, Ascaridia lineata (galli), Colinus virginianus,

 

 

Trichostrongylus tenuis, Syngamus trachea, Capillaria and Histomonas

 

meleagridis. In addition to these parasites all other life in the

 

soil of the fumigated plots were killed, even at the 9 inch depth which
was as deep as soil samples were taken. These plots were not plowed
and had been part of a pasture for several years. They were rough
with plant growth typical of a mediocre pasture. Birds reared upon
these fumigated plots were kept essentially free of parasites for a
period of a year. The source of recontamination seemed to be from
bordering unfumigated plots and wild birds. Clapham advocated methyl
bromide as having excellent possibilities for fumigation of bird runs
once a year as a control measure of many poultry parasites.

Kolb and Schneiter (1950) and Kolb, Schneiter, Floyd, and
Byers (1952) showed that gaseous methyl bromide is germicidal and

sporicidal for Bacillus anthracis upon exposure for 24 hours in the

 

presence of moisture at a concentration of 3.4 to 3.9 g/l. This is
equal to a concentration of 211 to 248 1b/1000 ft3 of methyl bromide.
Kolb and Schneiter used six different strains of virulent organisms
from different animal species. When the organisms were placed upon
filter paper Strips and exposed to methyl bromide in presence of
moisture for one hour spores survived in 17 of the trials. However,
no survival was evident at 24 hours of exposure. In the absence of
moisture, some spores survived in 13 of their tests regardless of the

length of exposure time. Liquid methyl bromide was ineffective in

18

killing Spores of E; anthracis. In another series of experiments using
excessively dehydrated Spores, the action of methyl bromide seemingly
was not impaired. Saiki (1952) found that methyl bromide would kill

Vibrio cholerae, Bacillus dysenteriae, and Bacillus typhosa on Endo

 

agar plates, dry and wet gauze, and in peptone water when these were
fumigated in a sealed room. Schmittle (1955) reported that methyl
bromide inactivated Newcastle disease virus (NDV) on feed sacks at a
concentration of 5 lb/1000 ft3 at 25 C for 21 hours, but was ineffec-
tive at 4 C. Using large desiccator jars as fumigation chambers, NDV
was killed in 21 hours at 4 C with 8 1b dose, at 25 C with a 2 lb dose,
and at 30 C with a 1 lb dose per 1000 ft3.

Edgar and King (1955) Stated that methyl bromide was effec-

tive in killinggspergillus fumigatus on agar plates, and Ascaridia

 

galli and EL tenella in a small amount of water within a Petri plate.
These organisms were fumigated with 1 m1 of methyl bromide per a one
qt fruit jar used as a fumigation chamber. This dosage was approxi-

mately equivalent to 105 1b methyl bromide per 1000 ft3

of fumigated
area. In a field type experiment, a methyl bromide impermeable plastic
cover was placed over used litter, and in the litter were buried Petri
dishes containing the above mentioned organisms. A dose of l lb/lOO
ft2 inactivated all the organisms. Used litter was fumigated in the

1 qt jars at the rate of 1 ml liquid methyl bromide in each. This

litter contained viable coccidial oocysts, two Species of nematode

eggs, mites, fungi and several species of bacterial contaminants. All

19

these organisms were killed upon fumigation. In summary, Edgar and
King stated that methyl bromide may prove practical in freeing heavily
contaminated premises of undesirable disease agents.

Whelton fig 31. (1956) reported that approximately 540 lb/lOOO

ft3 were necessary to inactivate Bacillus, Escherichia and Pseudomonas

 

 

species. No explanation can be given as to why this high concentration
was needed for inactivation unless the fumigation was done in such a way
as to allow the gas to escape.

Manns and Schmittle (1956) patented the use of methyl bromide

for inactivation of Salmonella_pullorum, coccidia, and the virus of

 

Newcastle disease in used feed bags. The minimum stated amount needed

3 for 24 hours

for this purpose is 5 lb of methyl bromide per 1000 ft
at 70 F. Table l is a summarization of their results which Show dosages
needed of methyl bromide per 1000 ft3 at various temperatures and ex—
posure times with the three different organisms.
Schmittle (1959) stated that methyl bromide at 50 1b/1000
ft3 will not penetrate tightly packed bales of bags and enact sterili-
zation. If the bags are fumigated in an unbaled or loose bale form
with proper distribution of methyl bromide, sterilization will result.
Schmittle and Holderried (1959) reported results from the
use of methyl bromide in field type experiments. They used a poultry
house of concrete block construction with composition roof and con-
crete floor. The house in an unclean condition with litter not removed

nor with any other cleaning, was fumigated. In these experiments the

gas concentration in various parts of the building was determined along

20

TABLE 1

MINIMAL QUANTITY OF METHYL BROMIDE IN LB/lOOO FT3
REQUIRED To KILL POULTRY PATHOGENS
UNDER DIFFERENT CONDITIONS*
Time of Sterilization

24 hours 36 hours

Temp. of Sterilization Temp. of Sterilization

 

4C 200 30G 4C 20C 300
Newcastle
Disease Virus 8 lb 2 1b 1 1b
Salmonella
pullorum 9 4.75 4.5 5 lb 3 lb 3 lb
Eimeria
tenella 2 1 0.25

* From.MannS and Schmittle (1956)

21

with the effect of methyl bromide upon agar plate cultures of Aspergillus

 

fumigatus and g; pullorum. The agar plates were placed on top of and
beneath the litter and at ceiling level at various locations within the
building.

The results of Schmittle and Holderried showed that a building
of such a construction, even with all the windows and vents sealed with
gas proof plastic, would not maintain a sufficient concentration of
methyl bromide to sanitize. Completely enveloping the building with
gas proof plastic rectified this problem. A 5 lb dose per 1000 ft3 did
not give adequate control in these experiments but a 7-1/2 1b dose was
adequate. This was mainly due to leakage of the gas. The following were
found to be important factors which influence the effectiveness of
methyl bromide in any test: (1) outside wind velocity as it affects gas
leakage, (2) temperature within the building, (3) air movement within
the building as it affects layering of the gas, (4) the evaporation
rate of methyl bromide after introduction into the building. In cal-
culating the expense of fumigation a cost of 80 cents per lb of methyl
bromide was used. The dosage rate was 7-1/2.lb/1000 ft3. Using these
figures, 3 building with an 8 ft ceiling could be disinfected for 4.8
cents per ft2 and sealed in plastic for approximately 2.7 cents addi-
tiOnal per ftz, including labor and materials. The total calculated

2

cost of disinfection was approximately 7.5 cents per ft of floor area.

22
Technical Data on Methyl Bromide

Commercial products

Methyl bromide is available as a liquid packaged under pres-
sure in steel cylinders of capacities of 10, 40, 50, 100, 150, and 400
lb or in small 1 1b "tin" cans. The steel cylinders have air added to
bring the total pressure within the cylinder up to 60 lb/in2 at 40 F.
This added pressure assists in forcing the methyl bromide from the
cylinder. The cylinders dispense methyl bromide as a liquid by means
of a tube from the valve to the bottom of the cylinder. This method
preserves the air charge used to force the methyl bromide from the
cylinder.

Commercially, methyl bromide is available as a 99 percent pure
product or with 2 percent chloropicrin (tear-gas) added to serve as a
warning agent of a contaminated atmosphere. Several preparations are
also manufactured consisting of methyl bromide dissolved in various

solvents which lower its vaporization pressure.

Chemical and Physical Characteristics

The general pr0perties of methyl bromide are listed in Table
2. Methyl bromide is a gas which becomes a liquid at 3.6 C and a
solid at -93.7 C. This gas is heavier than air, nonflammable, colorless,
practically odorless, and is highly diffusible and penetrating. Methyl
bromide is generally non-reactive with most inanimate objects. Upon
exposure of gaseous methyl bromide to a source of intense heat such

as a flame or a glowing metal surface, decomposition of the gas takes

23
TABLE 2

TECHNICAL DATA - METHYL BROMIDE

Synonyms . . . . . . . . Bromomethane, Monobromomethane
FOI’IIIUla o o o o o o o 0 CH3 Br
Molecular weight. . . . 94.95

Density, liquid . . . . 1.732 at 0 C and 0 pressure
vapor . . . . 3.974 g/l at 20 C and 760 mm mercury pressure

Melting point. . . . . . -93.7 C
Boiling point. . . . . . 3.56 C

Specific heat, solid at -96.6 C . . . . . . . . . 0.165 cal/gm
liquid at -13.0 C . . . . . . . . 0.197 cal/gm
vapor at 25 C . . . . . . . . . . 0.107 cal/gm

Heat of fusion at -93.7 C . . . . . . . . . . . 15.05 cal/gm
Heat of vaporization at 3.6 C . . . . . . . . . 60.20 cal/gm
Heat of transition at —99.3 C . . . . . . . . . 1.20 cal/gm

Vapor pressure . . . . . 1420 mm Hg at 20 C
2 atmospheres at 23.3 C
5 atmOSpheres at 54.8 C
10 atmospheres at 84.0 C

Solubility . . . . . . . water - at 20 C and with a total pressure
of 748 mm Hg 3 1.75 gm per 100 gm of
solution
soluble in all portions in alcohol, ether
and carbon tetrachloride

Flash point . . . . . . nonflammable over wide range of concen-
tration in air with intense source of
ignition.

Description . . . . . . Colorless, transparent, volatile liquid
or gas with a burning taste and slight
chloroform-like odor

Uses . . . . . . . . . . Fumigant, fire extinguisher, reagent in
manufacture of dyes, and the pharmaceutical
industry

Data from: Handbook of Chemistry and Physics, Michigan Chemical Company,
and Encyclopedia of Chemical Technology

24

place. The products evolved are hydrogen bromide, bromide, carbon
oxybromide, carbon dioxide and carbon monoxide. These decomposition
products are corrosive and highly irritating.

Methyl bromide is relatively insoluble in water but readily
soluble in most organic solvents. Liquid methyl bromide is a vesicant,
if skin is in prolonged contact with it. The gas is toxic to almost
all forms of life. Primarily, the commercial use of methyl bromide is
fumigation for rodents and insects within grain mills, warehouses or
food plants. Methyl bromide also has extensive usage as a soil fumigant
in the control of insects, weeds and microorganisms for tobacco seed-
beds, nursery plots, and greenhouse soils. The gas is also used in fire
extinguishers, and in organic synthesis by the dye and pharmaceutical

industries.

Chemistry of its Bactericidal Activity

The lethal action of methyl bromide as proposed by Phillips
(1952 and 1958) is a result of its action as an alkylating agent.
Alkylation consists of the replacement of an active hydrogen atom in
an organic compound with an alkyl group. The lethal alkylation re-
action takes place with the reactive groups which are parts of essen-
tial complex enzymes or proteins of the cell. Phillips presents
alkylation as the means of action of formaldehyde, ethylene oxide,
beta-propiolactone and methyl bromide. Figure 1 presents diagramati-
cally the chemical action involved with methyl bromide acting upon a
protein containing free carboxyl, amino, sulfhydryl, and hydroxyl

groups. These data were presented by Phillips (1952). The methyl

25

 

 

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26

group of methyl bromide acts as the alkyl group with the bromine com-
bining with the labile hydrogen atom forming hydrobromic acid. Lewis
(1948) presented data that methyl bromide is capable of combining with
sulfhydrylic groups and produced a progressive and irreversible inhibi—
tion of the enzymes urease, succinic dehydrogenase, papain, and yeast
reSpiration. In this reaction Lewis found that hydrobromic acid was
formed which then formed bromides. The alkylation theory appears to
be supported by the findings of Kolb, Schneiter, Floyd, and Byers (1952)
in that neither hydrobromic acid nor methanol killed anthrax Spores in
the presence of moisture in such concentrations as were effective in
the case of methyl bromide.

A review was presented by von Oettinger (1955) of the action
and toxicity of methyl bromide upon higher forms of life, especially
man and laboratory animals. Dixon and Needham (1946) studied the vesi-
cant effect of methyl bromide. They found that hexokinase was inhibited
by methyl bromide. Church, Halvorson, Ramsey, and Hartman (1956) demon-

strated heterogeneity in the resistance of spores of Bacillus Sp. to

 

ethylene oxide. The percent of resistant organisms could not be'
increased by selection of resistant Spores. These workers also demon-
strated that Susceptibility to ethylene oxide varies with the fat con-
tent of the organism. Removal of the fat with a solvent decreases the
resistance.

Phillips (1952) presented some interesting data on the

activity of the alkylating agents and other disinfectants in killing

27

Spore and non-spore forming bacteria. Table 3 presents data as given
by Phillips. This table lists the relative ratio of resistance of a

spore forming bacterium Bacillus globigii as compared to non-Spore

 

formers Micrococcus pyogenes and Escherichia coli for various disin-

 

fectants. The spore forming bacteria are from 0.5 to 15 times as re-
sistant to the alkylating agents as are the non-spore forming bacteria.
Comparing these results with those of the other common disinfectants
it is noted that the Spore forming bacteria are exceedingly resistant
to the lethal action of those disinfectants. Spore forming bacteria
are ten thousand to a hundred thousand times more resistant than are
the non-spore forming bacteria to the action of the common disinfec-
tants.

Phillips explains the increased susceptibility of the Spore
state to the alkylating agent in that the reaction can take place with
any labile hydrogen. The other disinfectants, however, primarily react
only with the sulfhydryl groups. The sulfhydryl group is protected
through some means in the process of spore formation. The action of
the common disinfectants is thus blocked with the Spore protection of
the sulfhydryl group but the alkylating agents have many other groups

which are Still in a reactive Stage.

28

TABLE 3

RELATIVE ACTIVITY OF CERTAIN DISINFECTANTS AGAINST
BACTERIAL SPORES AND AGAINST VEGETATIVE CELLS

Disinfectant Ratio of
Resistance

Alkylating agents

Ethylene oxide Between 2 and 6
Ethylene imine Between 0.5 and 10
Methyl bromide Between 2 and 5
Formaldehyde Between 2 and 15
Chlorine
Sodium hypochlorite About 104
Silver
Movidyn About 104
Phenol 3
Trichlorophenol About 10
Quaternaries
4-Cetyl-4-methyl- About 104
morpholinium

methyl sulfate

29
MATERIALS AND METHODS
Methyl Bromide

The methyl bromide used in this study was provided by the
Michigan Chemical Corporation, St. Louis, Michigan. The gas was the
commercially available product marketed under the trade name
"Pestmaster". This product was the commercially pure product, 99 per-
cent CH3Br. It was packaged in one pound metal containers in which the
methyl bromide was maintained as a liquid under pressure.

.A small, clean, steel cylinder of the Size and design come
monly called a lecture bottle was used as the container for methyl
bromide. This cylinder was equipped with a micro valve (Hoke valve)
in order to diSpense the gas at finely controlled flow rates.

The air was removed as completely as possible from this
cylinder with a vacuum pump. The cylinder and a 1 lb container of
methyl bromide were chilled overnight in a walk-in freezer. Working
within the freezer the metal can of methyl bromide was then opened
(methyl bromide is a liquid at atmospheric pressure at this tempera-
ture) and transferred into the cylinder by suction utilizing the nega-
tive preSSure within the cylinder. Care was taken not to admit any
air into the cylinder. Upon returning the cylinder to room temperature
the methyl bromide vaporized giving a pure, easily controlled supply

of gaseous methyl bromide.

30

Gas Measuring Apparatus

Figure 2 illustrates the gas measuring apparatus. The fol-
lowing description describes the apparatus in this figure.

The methyl bromide-filled, lecture cylinder lA' together
with micro-valve 'B' was attached thru means of pyrex tubing to
three-way stopcock 'C'. Ball and socket joint 'F' served as a means
of disconnecting the cylinder for replenishing purposes. Gas burette
'D' graduated in 0.1 ml with 100 ml capacity was connected with the
three—way stopcock 'C' at one end and leveling bulb 'E' at the other
end by means of flexible 'Tygon' tubing. Flexible tubings of rubber
or plastic were unsatisfactory in that they were easily penetrated by
gaseous methyl bromide. Additional pyrex tubing connected the three-
way stopcock 'C' with the ball part of ball and socket joint 'F'. In
order to maintain the rigidity necessary for this dispensing apparatus
it was mounted securely on an upright piece of plywood. ,All stopcocks
and ball and socket joints were sealed by Dow-Corning Silicone stopcock

grease.

Fumigation Chambers

The fumigation chambers consisted of 250 mm Size vacuum
desiccators of pyrex glass. These are Similar to those described
under catalog number 8-631 in the Fisher Scientific Company catalog
number 59. These desiccators had a total volume of 11,075 m1. Attached

to the desiccator was a three-way stopcock 'H' and the pyrex ball and

 

 

 

 

 

 

 

 

 

FIGURE 2

THE FUMIGATION APPARATUS

32

socket joint 'F2'. The jars and stopcocks were similarly sealed with

Silicone stopcock grease.
Method of Administering Methyl Bromide

The desiccator was attached by means of the ball and socket
joint 'Fz'. A slight quantity of air was then removed from the
desiccator through the three-way stopcock 'H'. Methyl bromide was
then admitted from cylinder 'A} into gas burette 'D' by means of valve
'B' and stopcock 'C'. The gas displaced the distilled water in the
gas burette into the leveling bulb 'E'. By changing the direction of
flow at stopcock 'C' and adjusting stopcock 'H' air could be flushed
out of the system by purging with methyl bromide. Methyl bromide
could be admitted to the chamber '6' in a measured amount of pure
gas from the burette 'D' by readjusting stopcock 'H' and changing the
height of leveling bulb 'E'. .After admission of the desired amount
of gas, stopcock 'H' was then adjusted in such a position that air
from.the room atmOSphere could be admitted to equilibrate pressure

within the desiccator with that of the external atmosphere.
Dosage Calculation

Referring to Table l the density of methyl bromide vapor is
listed as 3.974 g/l at 20 C and at an atmospheric pressure of 760 mm
of mercury. From the Handbook of Chemistry and Physics, 35gb edition
the following infonmation was obtained: 1 lb Avoirdupois 3 453.5924 g

and 1 ft3 : 28316 m1. Using appropriate mathematical calculations,

33

0.0040309 ml of gaseous methyl bromide will give a concentration in
1 ml of fumigated volume equal to 1 lb of methyl bromide per 1000 ft3.

Dosage was calculated for all this work using this formula:

Dosage in pounds per 1000 ft3 X volume in ml of fumigation chamber X
0.0040309 = m1 of gaseous methyl bromide

If dosage was expressed as oz/lOOO ft3, it would be

analogous to mg/l.

Salmonella pullorum

 

The culture of Salmonella pullorum which had just recently

 

been isolated was obtained in the Michigan State University poultry
diagnostic laboratory.

After receiving the culture, several nutrient agar (No. Bl
Difco*) slants were inoculated and after 24 hours of incubation these

were refrigerated and used as Stock cultures.

 

*Difco Manual, 9gb eidtion: Difco Laboratories, Inc.,

Detroit 1, Michigan, 1953.

34
EXPERIMENTAL PROCEDURES AND RESULTS

Effects of Methyl Bromide on Bacteriological
Media - Experiment I

Sterile nutrient agar and MacConkey agar (No. B 75 Difco)
were placed in 20 ml amounts into sterile pyrex Petri dishes 100 x
15 mm. Also prepared were the same media but dispensed without auto-
claving into unsterile Petri dishes. One plate of each medium and
of each preparation (sterile and non-sterile) was then fumigated.
Replicate unfumigated plates were used as controls.

The fumigated plates were exposed for 48 hours at a concen-
tration equal to 11 1b of methyl bromide per 1000 ft3 of fumigated
area. The control plates were held under identical conditions minus
the exposure to methyl bromide. .After fumigation the plates were
aired in room atmosphere for four hours. At this time the sterile,
fumigated and control plates were streaked with_§L pullorum obtained
from the stock culture using an inoculating loop. The inoculation was
heavy and was done so as to give good coverage of the plate. Figure 6
illustrates the general procedure of plate streaking. All the plates
were then incubated for 48 hours at 37 C and the results recorded.

After fumigation, the autoclaved and non-autoclaved agar
plates were identical in appearance to the unfumigated, autoclaved
plates. The non-fumigated non-autoclaved agar plates showed con-

siderable growth of many different bacteria and fungi. Figure 3

FIGURE 3

NON-AUTOCLAVED NUTRIENT AGAR PLATES FUMIGATED AND NOT FUMIGATED
WITH METHYL BROMIDE AT 11 LB/lCOOFT3 FOR 48 HOURS

 

36

illustrates the growth present on the non-autoclaved, non-fumigated
plate and the absence of growth on the fumigated»‘non-autoclaved plate.

The growths of g; pullorum on the fumigated and on the auto-
claved non-fumigated plates after incubation were essentially identical
in amount and appearance.

Adequacy of Counting Techniques -
Experiment II

An experimental procedure was developed whereby a known
number of organisms could be exposed to methyl bromide for a period
of time. After exposure the number of survivals of this population
could be determined and the percent of survival calculated. For this
work bacteria absorbed onto filter paper strips of a standard grade
and size were used. This method offered uniform exposure of the
organisms to the gas and provided means of experimentally varying the
moisture present in the surroundings of the organisms.

The filter paper strips were sterilized by autoclaving within
Petri dishes. After autoclaving the dishes and strips were dried over-
night in a 120 C oven. A culture tube of a 24 hour nutrient broth
(No. B 3 Difco) culture of_§; pullorum was aseptically poured into
the Petri dish over the filter paper strips. After allowing five
minutes for adsorption, each strip was removed asepticallthith sterile
forceps to a sterile Petri dish.

To obtain the experimental variation present, the organisms
on twelve strips were counted. Each strip was aseptically removed

from its Petri dish and placed into a water dilution blank containing

37

100 ml of saline (0.85 percent NaCl). The bacteria were washed into
suSpension by vigorous shaking of the bottle and plated.

Table 4 gives the counts obtained from each filter paper
strip. The highest count obtained was 8.4 million and the lowest,
3.1 million. The mean was 5.9 million with the variation in count

being 44 percent.

Dosage and Time - Experiment III

Using streaked nutrient agar plates, a series of trials
was carried out to obtain the time and dosage relationship necessary

for complete kill of S. pullorum. The procedure was essentially the

 

same as outlined in Experiment I. Sterile nutrient agar plates were
heavily streaked with §L pullorum from the stock culture. The
streaking was done with an inoculating loop and in such a manner as
to assure heavy growth over the total plate. Duplicate plates were
made with one being an unexposed control and one being fumigated.

The plate for fumigation was placed into the desiccator
jar which had 100 ml of distilled water in the bottom to maintain
proper humidity. The covers of the Petri dishes were not removed.
The gas was then introduced according to prior stated procedures.
The fumigation was carried out at room temperature which averaged
24 C. The control plates were held under identical conditions in
another desiccator jar except that no methyl bromide was added. After
fumigation the plates were removed from the jars and incubated for 48
hours at 37 C and the results recorded.

Table 5 and figure 4 constitute a synopsis of the results of

42 trials. The concentration of methyl bromide in lb per 1000 ft3 is

5,500,000
5,400,000
4,500,000
6,100,000
6,200,000

7,800,000

TABLE 4

VARIATIONS IN NUMBERS OP_§; PULLORUM UPON
STANDARDIZED FILTER PAPER STRIPS

3,100,000
4,900,000
8,400,000
3,800,000

7,700,000

5,800,000

38

39

TABLE 5

HOURS REQUIRED TO KILL.§L PULLORUM WITH METHYL BROMIDE
AT 24 C AND 100 PERCENT RELATIVE HUMIDITY

Dosage Exposure for
Methyl Bromide Complete Kill
per 1000 £t3

2 lb 42 to 47 hours
3 42 to 44
4 30 to 35

5 18 to 23

Hours of exposure

40

FIGURE 4

DOSAGE OF METHYL BROMIDE AND EXPOSURE TIME REQUIRED TO

60

50

40

30

20

10

 

KILL_§; PULLORUM.AT ROOM TEMPERATURE (24 C)

X - Viable

O - Killed

 

 

q
d

V

2 3 4 5
3

4

Pounds methyl bromide per 1000 ft

41

listed in the first column. In the right-hand column are listed the
shortest and longest eXposure periods in hours found necessary to
kill é; pullorum at the respective dosages in a series of several

trials.

3 it required from

42 to 47 hours to completely kill g; pullorum. At 3 lb/lOOO ft3, 42

With 2 lb of methyl bromide per 1000 ft

to 44 hours were necessary, whereas at a 4 lb dose 30 to 35 hours
proved sufficient. Eighteen to 23 hours of exposure were required
to kill g; pullorum at a concentration of methyl bromide equal to

5 1b/1000 ft3.

Temperature Effects - Experiment IV

Using the same methods as outlined in experiment III the
effect of temperature was studied on the activity of methyl bromide
in killing g; pullorum. To vary the temperature of fumigation the
jars were placed in a walk-in refrigerator having an average tempera-
ture of 5 C and in an incubator with an average temperature of 32 C.
In all cases the fumigation chambers were allowed to reach temperature
equilibrium prior to administering the gas. The plates were removed
from the desiccator jars at the end of the exposure period and in-

. cubated for 48 hours and the results read. The control plates were
maintained under similar conditions except for the exposure to methyl
bromide.

Temperature caused a major change in the rate of killing of

.§; pullorum by methyl bromide. Table 6 is a listing of the results

42

TABLE 6

EFFECTS OF VARYING TEMPERATURE ON THE DEATH OF
g, PULLORUM AT 5 LB/lOOO ET3

Temperature Exposure for
Complete Kill
5 C 50 to 111 hours
24 18 to 23

32 10 to 11

43

of this experiment compiled from data obtained in eight trials using

5 1b of methyl bromide per 1000 ft3. The variation listed in hours
was the shortest and longest period of time needed for killing of g;
pullorum in the various trials at that temperature. .At 5 C the lethal
and point varied from 50 to 111 hours. Ten to eleven hours were
necessary for killing at 32 C. Room temperature (24 C) data were ob-
tained from experiment III. At 24 C the time required varied from

18 to 23 hours.
Inactivation Rate - Experiment V

Using filter paper strips impregnated with a broth culture
of §; pullorum the death rate was studied. This method using strips
of filter paper was essentially the same as outlined in experiment II.
The Petri dishes containing the inoculated strips were fumigated for
varying time intervals. Bacterial counts were then made as was done in
experiment II. These fumigations were carried out at room temperature
with each desiccator jar containing 100 m1 of distilled water. Methyl
bromide was administered at the rate of 5 lb/lOOO ft3. The results
are reported as percent kill with unexposed strips serving as con-
trols. Thirty-four trials were carried out at the following time
intervals: 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 15, 18, and 21 hours of
exposure to methyl bromide.

The average percent survival of_§; Eullorum after exposure
to methyl bromide at 5 lb/lOOO ft3 for varying time intervals is

listed in Table 7. The percent Survival figures used are the average

TABLE 7

PERCENT SURVIVAL OF SALMONELLA PULLORUM AFTER EXPOSURE TO
METHYL BROMIDE FOR VARYING PERIODS OF TIME IN THE
PRESENCE OF A.WATER SATURATURED ATMOSPHERE

 

Exposure period3 Average
to 5 1b/1000 ft survival
1 hour 74.2%
2 49.7
3 45.0
4 42.3
5 33.3
6 41.8
7 1.40
8 8.92
9 .165
12 .003
15 .002
18 .001
21 .000

24 .000

45

of all the data collected. Figure 5 is a graphic representation of
these results.

At one hour of exposure approximately 25 percent kill
occurred with an additional hour of exposure only about 50 percent
of the organisms were viable.’ At the 3, 4, 5, and 6 hour intervals
the percent survival did not decrease appreciably. During the next
three hours of exposure,_§L pullorum was killed at a rapid rate. By
the end of the ninth hour of exposure less than one percent survived.

Twelve additional hours of exposure were necessary however to
kill the remaining few viable organisms. Figure 6 illustrates results
obtained when MacConkey agar plates streaked with g; pullorum were
fumigated with a concentration of methyl bromide equal to 3 1b/1000 ft3
.for varying time intervals. These plates were fumigated as a part of
experiment III but are presented here to illustrate the rate of death
as seen visually be examination of the agar plates. The unfumigated
control plate in the center illustrates the heavy streaking of each
plate with g; pullorum. After 24 hours of exposure to methyl bromide
a clearly distinguishable reduction in numbers of the viable bacteria
occurred. At.thirty hours a very small percentage of organisms re-
mained viable compared with the hundreds present in the original
inoculum. Only one organism survived the eXposure to methyl bromide
for 36 hours. The plate showed no growth being present after 42 hours

of fumigation.

Percent survival

100

90

80

70

60

50

40

30

20

10

PERCENT SURVIVAL OF S.

 

FIGURE 5

PULLORUM.AFTER EXPOSURE TO METHYL

BRmIDE AT—S LB/ 1000 PT3 AT 24 c

 

 

H4

NA

La"

b‘

U1.-

0“

mi

12 15

Hours of exposure

46

:h—

18

21

LWWW/N) l GH7E-0

CONf/‘T 01

FIGURE 5

MAC COITREI' ATARI, PLATES STREAKED WITH _§_._ PULLORLJ’L’I FUMIGATED WITH 3 LB/IOOO
.. S 3 Oz? METHYL BROMIDE FOR VARYING TIME INTERVALS

 

48
Humidity Effects - Experiment VI

Again using the filter paper strip method, the effect of
atmospheric moisture was studied as to its influence on the power
of methyl bromide to kill g; pullorum. Difficulties were encountered
in maintaining viable controls at low humidities with_§L pullorum.

Therefore Staphylococcus aureus was used in conjunction with_§L

 

pullorum in this study as it was more resistant to desiccation.
Trials were made in duplicate using a dose of 5 1b of methyl bromide
per 1000 ft3 for a 24 hour exposure period. Humidity was controlled
by using distilled water and calcium chloride. For a dry atmosphere
pure calcium chloride was used in the bottom of the desiccator jar.
A saturated atmosphere was obtained using 100 m1 of distilled water
with the 50 percent relative humidity level obtained by a mixture of
35 percent calcium chloride and 65 percent water on a weight basis.
Results are recorded as the number of organisms present upon a nutrient
agar plate inoculated with 1 ml of the saline used to wash the filter
paper strip.

Table 8 is a summary of 24 trials in which §L pullorum and

Staph. aureus were exposed to a concentration of methyl bromide of

 

5 lb/1000 ft3 for 24 hours at 0, 50, and 100 percent relative humidity.
At 100 percent relative humidity the results were the same as those

in previous experiments. All the fumigated bacteria were killed. The
unfumigated controls at fifty percent relative humidity and in a dry
atmOSphere had difficulty surviving. No fumigated organisms survived

the exposure regardless of the atmOSpheric moisture.

TABLE 8

EFFECTS OF DIFFERENT HUMIDITY LEVEL ON THE ABILITY 0F
METHYL ERmIDE AT 5 LB/1000 FT TO INACTIVATE
_§_._ PULLORUM AND STAPH. AUREUS

Surviving Organisms

 

.SL pullorum Staph. aureus
Relative
Humidity Fumigated Control Fumigated Control
100% 0 T.N.T.C.* 0 10,000
50 0 10 0 1,000
0 0 3 0 l

* Too numerous to count

50

Incubator Studies - Experiment VII

To learn of the possible use of methyl bromide to control
.§; pullorum contamination in chick incubators, embryonated eggs were
fumigated. Twenty eggs each of 5, 9, 13, 17 and 18 day-old embryos
were fumigated in the desiccator fumigation chambers. Table 9 gives
the dosage and time of fumigation used. Controls were placed in
desiccators for the same length of time but without methyl bromide.

All fumigated embryos were killed and the control, unfum-
igated embryos involving a long period of exposure to the limited
atmosphere of a desiccator jar also died. The control embryos kept

in the jars for six hours or less remained viable.

51

TABLE 9

FUMIGATION OF EMBRYONATED EGGS WITH METHYL BROMIDE
IN LARGE DESICCATOR JARS

Age of Fumigation Survival
Embryos
Period Dose Fumigated Controls
Days Hours Lb/lOOO ft3 Percent Percent
5 45 3 0 0
9 45 3 0 0
l3 l7 3 0 0
17 6 5 0 95

18 3 5 0 100

52
DISCUSSION

The results obtained in experiment I show that under the
experimental conditions methyl bromide was bactericidal for micro-
organisms contaminating nutrient agar and MacConkey agar media poured
into unsterile plates. .As evidenced by growth 0f.§; pullorum, no
loss of essential nutrients resulted nor did a residual toxicity re-
main on the fumigated plates. These results agree with the work men-
tioned in the description of the experiments in which ethylene oxide
was used to sterilize bacteriological and mycological media. The
results do not adequately evaluate methyl bromide as an agent to ster-
ilize bacteriological media. This evaluation should be made using an
organism of fastidious nutritional requirements and different media.
Evidence to support this need is found in the work of Sykes (1958)
who found that ethylene oxide fumigated serum was inferior to unfum-
igated serum in the culture of certain fastidious bacteria.

Experiments III and IV demonstrated that methyl bromide
did not adversely affect the capacity of the media to support growth
of S; pullorum. .Agar plates inoculated with S; pullorum, when exposed
to sublethal dosages of methyl bromide, showed no visible growth at
the end of the fumigation period. The control plates, however, showed
good growth during the fumigation period prior to the actual incubation
of the plate. This demonstrates that methyl bromide is bacteriostatic
in sublethal dosages. Two pounds of methyl bromide per 1000 ft3 was

the lowest dosage used and it proved bacteriostatic.

53

The data presented by Trickel (1952), Mac Lachlen_g£_§l.
(1953), Phillips (1957 and 1959) and McKeen (1954) on the minimal
lethal dose of methyl bromide for microbial life agrees closely with
the results reported here. Their experiments had been carefully con-
trolled with adequate precautions taken to prevent the methyl bromide
from escaping from the fumigation chamber. These workers also used
lengthy exposure periods. Other workers, who have reported that
methyl bromide is only effective in extremely high dosages, have either
failed to maintain adequate methyl bromide concentration for a period
of several hours or the temperature was sub-optimal.

Experiment II showed that the paper strip method had the
disadvantage of considerable variance in the counts. This method
was used, however, in lieu of any other because of its advantage in
that the bacteria were exposed to the varying conditions of the at-
mosphere. In this humidity experiment, two lethal factors were in-
volved, methyl bromide and environment, probably desiccation.

Results of experiment VI in which atmospheric moisture has
little or no effect on the activity of methyl bromide to inactivate
.S; pullorum is in agreement with the work of several other workers.
They found that in soil fumigation excess moisture interfered with
the activity of methyl bromide to penetrate the soil. None of these
workers reported that moisture was necessary for the effectiveness
of methyl bromide. The interference with the diffusability of methyl

bromide by water is probably a result of its slight solubility in

54

water. The moisture present in the agar that was sterilized by methyl
bromide did not seem to interfere appreciably. Trickel (1952) did
not find that moisture was necessary for the inactivation of micro—
organisms. Kolb and Schneiter (1950) also presented substantiating
evidence in that they found that methyl bromide killed excessively
dehydrated Spores of Bacillus anthracis. However, in another trial,
unless moisture was added to the fumigation chamber the organism was
able to survive. Saiki (1952) was able to inactivate members of the
Enterobacteriaceae on wet or dry gauze. Edgar and King (1955), as
well as Schmittle (1955), did not find that supplementary moisture was
necessary.

Kaye and Phillips using ethylene oxide found that it was
not as active in excessively high concentrations of moisture nor on
organisms that had been lyophilized. Their theory as to the role
of moisture in the alkylation reaction is elaborated upon in the re-
view of literature. If these theories can be applied to the action
of methyl bromide then the effect of ethylene oxide being readily
soluble in water should be considered. Methyl bromide is only slightly
soluble in water, therefore, excessive moisture might be detrimental
to its action. The difference in the solubilities of methyl bromide
and ethylene oxide may be the factor that renders ethylene oxide a
more rapid acting bactericidal agent. Because of the lower concen-
tration of methyl bromide ions in a solution, a longer time interval
would be necessary for these ions to seek out and react with the labile

groups of the organisms.

55

Methyl bromide fumigation of chicken embryos is not to be
recommended, the embryos being more susceptible to this gas than is
.§; pullorum.

In experiment V the reaction rate of methyl bromide and_§L
pullorum as evidenced by bacterial mortality, is comparable to that
ascribed to most bactericidal chemicals. 0f significance here is that
99 percent 0f.§; pullorum are killed in less than 9 hours, however,
the remaining one percent of the bacterial population requires an
additional 12 exposure hours to enact complete kill. The practical
application of this fact is that field disinfection would be considered
effective in disease prevention with a 99 percent kill of contaminate
pathogens. Combining this fact with the adverse environmental factors
acting upon S; pullorum, such as desiccation, adequate field dis-
infection using methyl bromide should be easily accomplished.

In all conditions where microorganisms or other forms of
’life were Subjected to methyl bromide, death occurred if the gas
concentration and exposure period was adequate. Relative to its use
by the poultry industry, methyl bromide destroys all parasites even
the most resistant, the intermediate hosts of parasites, bacteria,
viruses, insects and rodents. The combination of this wide killing
spectrum of methyl bromide with its high activity in the presence of
organic matter and penetrability make it an ideal disinfectant for use

by the poultry industry.

56

Methyl bromide has the disadvantage of requiring a tight
almost hermetically sealed space to contain the gas during fumigation.
The gas is less active at lower temperature so that fumigation of ex-
posed areas in the northern states during winter will be limited. The
gas is rather costly for large scale fumigations if used at 5 lb/1000
ft3, the cost being approximately 85 cents to one dollar per pound.
Methyl bromide is toxic to man and animals but its popularity and
widespread use by other agricultural industries demonstrates that if
a few basic precautions are followed this factor is not a serious dis-
advantage. The toxicity for humans is actually of a much lower order
than many other common insecticidal fumigants and sprays in use.

The general non—reactiveness of methyl bromide is a big
advantage. This gives a great deal of safety to the fumigation process,
thus accidents due to explosion are eliminated. AAlso methyl bromide
does not harm most articles ordinarly fumigated.

The explosiveness of ethylene oxide necessitates mixing it
with carbon dioxide which reSults in increased expense. This mixture
then approximates methyl bromide in activity, methyl bromide being
l/10 as active as ethylene oxide.

Ethylene oxide has a slightly higher boiling point, therefore,
it is not as active at low temperatures as is methyl bromide.‘ The pene-
trative activity and general effectiveness of ethylene oxide seems to
equal that of methyl bromide. Although ethylene oxide has been shown
to affect the nutrition of certain animals fed an ethylene oxide fumi-

gated diet, this fact has not been stated as being true of methyl bromide

57

(see appendix on toxicity).

Beta—propiolactone has the same basic disadvantage as
formaldehyde. It has little penetrating power and needs heat and
moisture. Both products require a special procedure for vaporization
of the liquid. Beta-propiolactone is also toxic to humans but less
than methyl bromide. The advantage beta—propiolactone offers over
formaldehyde is its rapid action with resulting easy removal of the
gas after fumigation. Neither formaldehyde nor beta-propiolactone are
effective disinfectants because they are active only on organisms that
are directly eXposed. In application on poultry houses, chick boxes,
litter, poultry yards, and poultry equipment disinfection by beta-

propiolactone and formaldehyde seem unlikely.

58
SUMMARY AND CONCLUSIONS

Eleven pounds of methyl bromide per 1000 ft3 sterilized
nutrient agar and MacConkey agar plates as evidenced by absence of
growth of microbes after 48 hours of incubation. Methyl bromide fumi-
gation did not impart to the media any residual toxicity which inhib-
ited the growth of Salmonella pullorum.

Salmonella pullorum upon agar plates, at 24 C and 100 per-
cent relative humidity, was killed by methyl bromide at 2, 3, 4, and
5 lb/lOOO ft3 with 47, 44, 35, and 23 hours of exposure respectively.
A relative humidity of 0, 50, or 100 percent within the fumigation
chambers did not alter the activity of methyl bromide at 5 lb/1000

ft3 in killing Salmonella pullorum and Staphylococcus aureus.

 

The inactivation rate of Salmonella pullorum by methyl

 

bromide, when percent survival was plotted against time, gave a curve

essentially of an exponential character. Exposure of Salmonella

 

pullorum to 5 lb/1000 ft3 of methyl bromide at 5, 24, and 32 C
necessitated fumigation periods of 111, 23, and 11 hours respectively
for complete kill.

Methyl bromide at 5 lb/lOOO ft3 was lethal for 17 and 18
day—old chick embryos upon eXposure for six or three hours reSpec-

tively.

59

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66

APPENDIX

The following references are listed as being beyong the main
scope of this dissertation. They are however directly pertaining to
the topic of gaseous disinfection with methyl bromide. Their inclusion

here is so that this thesis could serve as a complete reference.
Toxicity

Brooks, V. J. and Jacobs, M. B. 1958 Poisons, Properties, Chemical
Identification, Symptoms and Emergency Treatment. 2nd Ed.
D. Van Nostrand Co., Inc., New York.

Dudley, H. C. 1939 Bromide content of fruits and vegetables follow-
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Dudley, H. C-.E£ a1. 1940 Studies on foodstuffs fumigated with
methyl bromide. U. S. Public Health Reports, 22, 2251-2275.

Irish, D. D. 23 a1. 1941 Chemical changes of methyl bromide in the
animal body in relation to its physiological effects. J.
Ind. Hyg., 21, 408-411.

Lewis, S. E. and Eccleston, K. 1946 Residues in wheat flour fumi-
gated with methyl bromide. J. Soc. Chem. Ind., 92, 149-152.

Miller, C. D. 1945 Thiamine content of cereals before and after
treatment with carbon bisulfide and methyl bromide to destroy
insects. J. Am. Diet..Assoc.,‘gl, 516-517.

Reohm, L. S., Shrader, S..A. and Stenger, V..A. 1942 Bromide residues
in cereals fumigated with methyl bromide. Cer. Chem., 12,
235-242.

Reohm, L. S., Stenger, V. A. and Shrader S..A. 1943 The fumigation
of dairy products with methyl bromide. J. Dry. Sci.,_2§,
205-211.

Spencer, H. C. 23 al. 1944 ReSponse of rats maintained on diets
fumigated with methyl bromide. Food Res., 2, 11-18.

67

von Oettinger, H. F. 1958 Poisoning, A Guide to Clinical Diagnosis and
Treatment. 2nd Ed. W. B. Saunders Co., Philadelphia.

Wintsringham, F. P., Hellyer, G. C. and McKay, M. A. 1958 Effects of
methyl bromide on phOSphorus metabolism in the adult house—
fly, Musca domestica LL Biochem. J. .62, 640.

 

Application methods
Anonymous 1939 Methods of use of methyl bromide on plants. Bur.
Ent. Plant Quar. Bul. 499.

Anonymous 1953 Methyl bromide bulletin, Michigan Chemical Co.,
St. Louis, Mich.

Anonymous 1955 Methyl bromide dooms Khapra beetles. Pest Control,
22(4), 22"26.

Hassler, Karl 1956 Some facts about fumigation tarps. Pest Control,
“24(2), 25-26.

Latta, R. 1941 Methyl bromide fumigation of imported perishable food
products. Bur. Ent. Plant Quar., Cir. E-521.

Parker, M. E. 1948 Food-Plant Sanitation. McGraw-Hill Book Co., Inc.
New York.

PhilliPS, G. L. 1955 Grain fumigation I. Agr. Chem. 19(1), 55-56,
117-121.

Phillips, G. L. 1955 Grain fumigation II. .Agr. Chem._19(2), 41-43,
133, 135.

Steinweden, J. B. 1945 Methyl bromide fumigation. Calif. State
Dept. Agr. Bul.,‘gfl, 4-14.

Young, T. R., Jr. 1955 Inexpensive heat exchanger for methyl bromide
dry wood termite fumigations. Pest Control, g§(3), 45-46.

68
ANALYSIS

Anonymous 1952 Directions for methyl bromide analysis. Michigan
Chemical Co., St. Louis, Mich.

Anonymous 1953 Instruction manual for Cow-Mac gas master. Cow-Mac
Instrument Co., 100 Kings Road, Madison, N. J.

Frear, D. E. H. 1942 Chemistry of Insecticides and Fungicides.
D. Van Nostrand Co., Inc., New York.

Monro, H. A. H., Buck Land, C. T., and King, J. E. 1953 Preliminary
observations on the use of the thermal conductivity method
for the measurement of methyl bromide concentrations in
ship fumigation. Ann. Rept. Ent. Soc. Ontario, §4, 71-76.

Phillips, G. L. and Bulger, J. W. 1953 Thermal conductivity method
for measuring methyl bromide. Bur. Ent. Plant Quar., E-851.

Shrader, S. A., Beshgetoor, A. W. and Stenger, V. A. 1942 Determina-
tion of total and inorganic bromide in foods fumigated with
methyl bromide. Ind. Eng. Chem., Anal. Ed., 14, 1-4.

Stenger, V. A., Shrader, S..A., and Beshgetoor, A. W. 1939 Analytical
methods for methyl bromide. Ind. Eng. Chem., Anal. Ed.,_ll,
121-124.

General
Allen, M. W. and Raski, D. J. 1950 The effect of soil type on the

dispension of soil fumigants. Phyt0path., 49, 1043-1053.

Anonymous 1951 Encyclopedia of Chemical Technology. Interscience
Encyclopedia, Inc., New York.

Bailey, S. F. and Smith, L. M. 1951 Handbook of Agricultural Pest
Control. Industry Publication, Inc., New York.

De Ong, E. R. 1948 Chemistry and Uses Insecticides. Reinhold
Publishing Corp., New York.

Metcalf, R. L. 1957 .Advances in Pest Control Research. Vol. 1.
Interscience Publishers, Inc., New York..

Sampson, R. E. and Ludwig, R. A. 1956 Laboratory studies on the
evaluation and activity of anti-fungal fumigants. Can.
J. 3012., 21:, 37-43.

 

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