A QUANTE?A?€VE COMPAMSON 0F
GRQWTH QF CERTASN M‘EMBERS OF THE

Camus; {QEQSEREBMM 3N comomv
EMWGYED mm: mm CQMPAMYEVE
mm w “FREE Gaawm EN 50m
NEW MEDIA

Thais for H1. Degree a»! M. S.
MICHiGAN STATE COLLEGE
Robert B. Whoaton
1954

THESI!

This is to certify that the

thesis entitled

A Quantitative Comparison of Growth of Certain Members of
the Genus Clostridium in Commonly Employed Media with

Comparative Data on their Growth in Some New Media.
presented by

Robert B. Whe aton

has been accepted towards fulfillment
of the requirements for

MS. degree in Bacteriology

flQW

Major professlr
Date J2m—

0-169

 

 

 

A QUANTITATIVE COMPARISON OF GROWTH OF CERTAIN MEMBERS
OF THE GENUS CLOSTHIDIUM IN COMMONLY EMPLOYED MEDIA
WITH COMPARATIVE DATA ON THEIR GROWTH
IN SOME NEW MEDIA

By
ROBERT E. WEEATON

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science

in partial fulfillment of the requirements
for the degree of

l

FASTER OF SCIENCE

Department of Bacteriology

1954

THESIS

INTRODUCTION . .

HISTORICAL REVIEW

METHODS AND MATERIALS

RESULTS .
DISCUSSION
SUMMARY .

REFERENCES

\

TABLE

OF CONTENTS

‘7‘ 6‘1)“§~S~:J
‘_.:'~'_:" ‘3‘: i 'v a

Page

15
54
60
66

67

T
0 My Dad

ACKNOWLEDGMENTS

The author wishes to thank Dr. Jack J. Stockton,

Assistant Professor of Bacteriology, for his untiring guidance
of this experiment, and Catherine A. Hamilton of Grand Rapids,
Michigan and Esther Narie Barrett of Kalamazoo, kichigan for
typing this Thesis. Thanks are also due Nr. Richard E.
Fuhrman of Kalamazoo, Michigan, for his invaluable aid in

photographically reproducing the Thesis.

INTRODUCTION

Since the discovery of the clostridia in 1861, many
types of media have been devised for their cultivation.
Some of these are relatively simple, and some are rather
complex. Many contain solid particles such as ground muscle
or ground brain tissue. Often large amounts of unidentified
proteins, such as blood serum, are added and in still other
media only pure amino acids and other purified nutrients and
salts are combined to produce completely synthetic media.

Regardless of the various formularies of each of these
individual media they are all intended to accomplish one
primary goal, the propagation of anaerobic bacteria. In its
own way each accomplishes this to a greater or lesser degree.
Most of these media have certain good points and most of the
media have at least one bad point. The purely synthetic
media are expensive often to the point of being prohibitive
for routine and large volume work, particularly by academic
institutions. Those containing tissue particles do not
produce a clear broth and in addition little or no toxin is
obtained from them. Some that contain reducing agents, such
as sodium thioglycollate, produce excellent anaerobic condi-
tions (Reed and Orr, 1943), but they are somewhat toxic to the
bacteria (Malin and Flinn, 1951). The primary problem is,
however, that the anaerobic pathogens are fastidious, diffi-

cult to culture and, with few exceptions, multiply rather

slowly. Whereas the nonpathogenic contaminants, usually
present in gas gangrene wounds, grow rapidly even in the
poorer media. As a consequence, in commercial media now
available, the contaminants are apt to overgrow the pathogens
when one tries to isolate gas gangrene bacilli from morbid
tissue. Also the propagation of cells for vaccine and
bacterin production often results in rather sparse growth.
With all of these facts in mind an experiment was
designed to investigate the realm of new and better media
and to estimate quantitatively the relative value of the
existing ones. The operations and results of that experiment

manifest themselves as the following thesis.

HISTORICAL REVIEW

Bacteria dealt with in this thesis are members of the

genus Clostridium. Members of this genus are comparatively

 

large, gram-positive, spore bearing rods that will not grow
in the presence of oxygen; that is, they are anaerobic. All
the species used in this experiment are pathogenic, and
belong to that division of the genus lestridium designated
the Gas Gangrene Group. Members of this group produce a
clinical entity designated gas gangrene.

The first recorded case of gas gangrene was reported
by Fabricus Hildanus in 1607, Kellett (1939). The case

history reads as follows:

. . .the wheel of the cart tore up the whole of the

inner part of the leg. The periosteum was ripped from
the tibia for a palm's length. . .having washed the wound
with red wine and tepid water. . .I enveloped the leg in
a bandage soaked in vinegar. . .He had a restless night.
On the 4th day. . .I undid the dressings and found. . .
the whole of the outer part of the leg and the foot

itself mortified and covered with large black vesicles
containing water similar to that in which meat had been
washed. . .at a certain place I could make out a sound as
if there were some sort of empty space underneath.
Inferring, therefore, that the disease was present under-
neath in that place I began to explain. . .that amputa-
tion was clearly going to prove fruitless. . .that
evening a vesicle the size of an egg rose in his groin. .
.and within the next two hours the scrotum became swollen
the size of a head and gangrenous. At about the third
hour after midnight he became covered with sweat, at
first hot then cold, and he died peacefully, in the
middle of a sentence, four days, eleven hours after his

illness commenced.

In spite of this accurate description of a typical gas

gangrene case history in 1607, it remained for Pasteur and
Joubert in 1877 to isolate and describe the first pathogenic
anaerobic bacillus. The isolation was made from putrid

sheep blood and the organism named Vibrion septique.

 

Later Koch isolated what he considered to be Pasteur's
Vibrion septigue, but he renamed it the Bacillus of Malignant
Edema. These are now considered to be identical with the
species now designated Clostridium ggpticum. However,

Koch's culture was probably contaminated with Clostridium

§porogenes, or a similar species, since Koch attributed

 

proteolytic properties to the organism.

The first adequate description of Clostridium feseri

 

was recorded by Arloing, Cornevin, and Thomas in 1879.
Welch and Nuttall (1892) first completely described

a pathogenic Clostridium which they named Bacillus aerogenes

 

capsulatus. This organism is now designated Clostridigm

 

 

pgrfringens. It is the principal cause of typical gas

 

gangrene, particularly in humans.

Except for these and other rather crude isolations
the field lay dormant until the World War of 1914 to 1918.
During that war the incidence of gas gangrene following
bullet wounds skyrocketed, and the disease became a very
important military problem. This fostered considerable
research in the technology of the production of anaerobic

systems. The first to be successful in this were McIntosh

5
and Fildes (1916) who reported on their classic anaerobe jar.
This was a heavy glass jar of about two liters capacity,
with a metal cover. A tube protruded through the cover and
was fitted with a hose through which hydrogen was introduced
into the jar under slight pressure. An electric heating
filament was hung from the cover inside the jar. This was
surrounded with a platinum catalyst which in the presence
of the heat from the filament caused the oxygen in the jar
to combine with the introduced hydrogen to form water.

This, of course, removed the oxygen in the jar and produced
an anaerobic environment.

Fildes and McIntosh (1921) improved their anaerobe
Jar by making it more substantial and thus increased the life
expectancy of the filament and palladium asbestos assembly.
However, the jar still had one great disadvantage. Occasion-
ally the hydrogen and oxygen would ignite so violently that
an explosion resulted. This made the use of the jar rather
hazardous to laboratory personnel. This type of anaerobe
jar, with certain modifications, has continued to be used and
today is the most commonly used method for producing
anaerobiosis.

A modification in the McIntosh-Fildes anaerobe jar
came when Brown and Brewer (1938) proposed the use of
illuminating gas instead of hydrogen. This removed the ex-

plosion hazard of hydrogen and made this piece of equipment

6
usable by virtually every laboratory. However, one further
modification was necessary. The illuminating gas as usually
carried in gas lines was at too low a pressure to fill the
jar. Consequently, it was necessary to evacuate the jar to
about 50 cm of mercury pressure with a water aspirator in
order to introduce sufficient gas into the jar to result in
combustion. The jar was kept attached to the gas jet during
the heating process, which lasted from 30 to 45 minutes.

Bridges, Pepper and Chandler (1952) found that
anaerobe jars, similar to the McIntosh and Fildes type,
furnished excellent anaerobic conditions when evacuated to
about 20 mm of mercury pressure and the volume was replaced
with helium bled from commercial cylinders. This eliminated
the necessity for the heating filament and platinum asbestos
that had been employed when the jars were filled with hydro-
gen or illuminating gas.

Spray (1930) employed an oxygen absorbing system of
pyrogallic acid and sodium hydroxide. The two chemicals
were poured into separate depressions in the bottom of a
deep bottomed Petri dish. The tOp of the dish, containing
the agar, was then sealed to the bottom. The whole assembly
was then tilted to allow the two chemicals to run together,
the resulting chemical reaction absorbed all the oxygen in
the air contained by the dish. In this way an agar plate

was sealed in an anaerobic atmosphere.

7

Another means of obtaining anaerobiosis by a chemical
method was advanced by Rosenthal (1937). In this technique,
the cultures to be incubated were placed in a jar and five
grams of powdered chromium metal was added to the jar for
each liter of volume of the Jar. Then 100 ml of 15 per cent
sulfuric acid was added to the jar for every liter of jar
volume, and the jar was quickly sealed. At this point the
chromium reacted with the acid to yield chromous sulfate
and hydrogen. The chromous salt thus formed took up the
oxygen in the jar and became chromic sulfate. This resulted
(in an oxygen free or anaerobic environment. A.mercury trap
was included in the jar so that the hydrogen could escape
without developing excessive pressures.

The chromium-sulfuric acid method of Rosenthal was
modified by Mueller and Miller (1941). They found that
Rosenthal used a great excess of both chromium and acid.
They recommended smaller quantities of these materials and
the addition of a small amount of sodium carbonate to the
jar so that some carbon dioxide would be produced. They
found that the presence of carbon dioxide resulted in better
growth at least in the case of Clostridium tetani.

An ingenious device for the anaerobic incubation of
Petri dish cultures was devised by Marshall and Nordby (1942).
This device consisted of two Petri dishes, one a standard

100 by 15 mm and the other 75 by 10 mm. A layer of agar

medium.was poured into each dish. The larger dish was
streaked with the anaerobic culture to be incubated. The
smaller dish was streaked with a culture of Serratia
marcescens and this dish inverted over the larger dish so
that its edge pressed into the agar of the larger dish
forming an airtight seal. This assembly was then placed in
the incubator where the rapid growth of the aerobic

.§° marcescens quickly removed the oxygen contained in the
space between the two Petri dishes.

McLeod and Gordon (1923) hypothesized that it was the
presence of hydrogen peroxide, rather than oxygen, in the
medium that prevented anaerobes from growing. The theory was
advanced that since the clostridia, unlike most other bacteria,
do not produce catalase or peroxidase there is an accumulation
of hydrogen peroxide when the cells grow in the presence of
oxygen. This accumulating, bacterially produced hydrogen
peroxide then turns upon its creator, so to speak, and des—
troys it. In the absence of oxygen the hydrogen peroxide
does not accumulate in quantities which are toxic or is not
produced at all.

All of the anaerobic techniques develOped did not
involve the use of chemical oxygen absorbing systems. Spray
(1936) supported the use of a medium.made semisolid by the
addition of 0.1 to 0.2 per cent agar. The medium was steamed

just before use to drive off dissolved gases. The semisolid

9
nature of the medium prevented gases from the air from mixing
with the medium except for the surface layer.

The use of semisolid agar did not originate with
Spray, however. It was first demonstrated to be useful in
the cultivation of anaerobes by Pringsheim in 1910. Before
this time only solid agar was used for deep tube cultivation
of anaerobes.

Quastell and Stephenson (1926) found that when organic
reducing agents such as cysteine, glutathione, and thio-
glycollic acid were added to the media, better growth
resulted and the anaerobic atmOSphere surrounding the medium
need not be so exacting.

Brewer (1940) took up the work on organic reducing
agents and found that sodium thioglycollate produced ex-
cellent anaerobiosis. Unlike the thioglycollic acid that
Quastell and Stephenson had used earlier, the sodium salt
was heat stable and could be sterilized by autoclaving.

He also found that if he added 0.05 per cent agar to the
medium.the mixing of air with the medium could be greatly
minimized. This allowed for the incubation of a tubed fluid
medium in the open air. His results showed that the fluid
medium stayed anaerobic for over a month even when not
inoculated.

McClung (1940) showed that Brewer's thioglycollate

medium.compared favorably with commonly used and

10
commercially available media.

McClung (1943b) also observed that semisolid media
containing sodium thioglycollate would initiate growth of
pathogenic clostridia even when the inoculum contained very
few cells. He observed that the growth produced compares
very favorably with that in dried beef heart medium.

Reed and Orr (1943) observed that the initial Eh of
semisolid media with or without sodium thiOglycollate was
approximately equal, but with successive hours of incubation
the media containing the thioglycollate maintained a lower
or more negative Eh than that which did not contain the
thioglycollate.

A new and simple method for the anaerobic incubation
of Petri dish cultures was introduced by Brewer (1942).

This consisted of a standard 100 by 15 mm Petri dish bottom
and a much heavier top. The top was constructed with a rim
that rested on the periphery of the agar layer and enclosed

a very narrow air space above the agar. In order to remove
the oxygen from the enclosed air space Brewer's thioglycollate
agar was employed; that is, the sodium thioglycollate in the
agar took up the excess oxygen.

Malin and Flinn (1951) found that the use of an anion
exchange resin such as "duolite 8-10" in broth.media, used
for the cultivation of clostridia, produced excellent growth.

The resin had a very high oxygen absorbing capacity and when

11
combined with copper ions formed a reduced copper resin
complex that reacted very readily with dissolved oxygen.
They also stated that sodium thioglycollate was inhibitory
to some clostridia in the presence of carbohydrates.

Nagler (1939) observed that when a broth culture

 

filtrate containing the alpha toxin of £1. perfringens was
incubated with normal human serum, an opalescence developed
in the tube. When this material was centrifuged, a fatty
layer separated out on the surface. Nagler hypothesized

that the alpha toxin of 91. perfringens was a lecithinase

 

and it acted on a protein-lecithin complex in the serum to
produce a fatty material. Thus, this was a test for
bacterially produced lecithinase, and could be used to
identify lecithinase producing bacteria.

MacFarlane, Oakley, and Anderson (1941) observed that
the lecithovitellin from chicken egg yolk would give the
same Opalescence in the presence of the alpha toxin of

91. perfringens as was observed earlier by Nagler in blood

 

serum.

Hayward (1941, 1943) develOped an agar plating medium
containing human blood serum to demonstrate the Nagler
reaction in Petri dish cultures.

McClung (1946) investigated the Nagler plate culture
and recommended a new base agar to which egg yolk could be

added. This medium consisted of four per cent proteose

12
peptone #2, 0.5 per cent disodium phOSphate, 0.1 per cent
monopotassium phosphate, 0.2 per cent sodium chloride,

0.01 per cent magnesium sulfate, 0.2 per cent glucose, and
2.5 per cent agar. This was adjusted to a pH of 7.6.
Continuing his work, McClung (1947), demonstrated that the
Nagler plate reaction showed sufficient specificity to
identify many of the Species of gas gangrene pathogens. He
pointed out, however, that this test should be used as a
presumptive identification rather than final proof.

Robertson (1916) reported on a cooked meat medium
that is still popular. The original directions for its
preparation called for eight ounces of bullock's heart
minced and finely ground. To this, eight ounces of tap
water was added. The mixture was heated slowly and allowed
to cook for approximately one hour. The material was then
dispensed into test tubes and sterilized by autoclaving.
This medium produced good growth and had an added value in
being of diagnostic significance. That is, saccharolytic
organisms, which are the pathogenic members of the genus
cause a reddening of the medium. Whereas, proteolytic
clostridia which are generally considered nonpathogenic,
render the cooked meat black and foul smelling due to
digestion of the meat.

McClung (1943) recommended the use of dried beef

heart tissue in the preparation of media for the cultivation

13
of the clostridia and outlined a procedure for the prepara-
tion of the dried tissue.

With the increasing need and desire for gas gangrene
toxoid, free of proteins and peptones, Bernheimer (1944)
perfected a protein and peptone free broth medium that
initiated the production of large amounts of toxin by 91.
septicum. This originally was an entirely synthetic medium.
However, he found that a medium consisting basically of
enzymatically hydrolyzed casein fortified with vitamins
and some salts, most notable of which was ferric chloride,
produced toxin titers equal to those of the synthetic medium.
He also found that the highest toxin titers were produced
concurrently with the highest cell count.

Jones and Clifton (1953) in their work on the
nutrition of 91. feseri found that this organism produced
a mixed type of fermentation, the principal end products of
which were ammonia, carbon dioxide, hydrogen, formic acid,
acetic acid, butyric acid, lactic acid, and succinic acid.
Ethanol and small quantities of acetone were also produced.
They also found that the presence of phOSphate in glucose
broth produced better growth over a wider pH range than did
glucose alone. They found that this species would readily
ferment glucose, pyruvic acid, and serine. Good growth
occurred in a medium composed of an enzymatic digest of

casein with glucose, biotin, nicotinic acid, pantothenic

acid, and pyridoxamine added. However, when an acid

hydrolyzate of casein was used no growth occurred.

14

METHODS AND MATERIALS

All stock cultures were maintained in Difco brain
heart infusion broth made semisolid by the addition of
0.2 per cent agar.

The standard inoculum for the tests was prepared by
inoculating three tubes of brain heart infusion broth with
each of the test cultures. These were placed in a Brewer
jar and the jar evacuated to a pressure of approximately
40 mm of mercury using a Welch Duo Seal vacuum pump.
Illuminating gas, from a laboratory outlet, was then admitted
to the jar until pressure equilibrium was attained. The jar
was thus evacuated and filled five times. At the fifth
addition of illuminating gas only a small amount was admitted
to the jar. Rubber tubing on the jar inlet was sealed with a
screw clamp and the jar placed in the incubator. An electric
connection in the incubator, equipped with a time switch, was
plugged into the electric connection of the Brewer jar and the
heater in the jar was allowed to run for two hours, and thus
removed any trace of oxygen.

After incubation for 24 hours the broth cultures were
removed from the jar and the cells were concentrated by
centrifuging in an International Equipment Company size 2
centrifuge for 30 minutes at a R.C.F. of 1,300 X G. The
supernatant broth was decanted from the packed cells and the

cells were resuSpended in sterile 0.85 per cent sodium chloride.

16
This suspension was again centrifuged and decanted as above
to wash the cells. Sterile saline was again added and the
tubes were shaken to suspend the cells completely. This
cell suspension was then dispensed in one m1 amounts into
sterile 15 by 150 mm pyrex test tubes. The tubes were then
plugged with nonabsorbent cotton and the excess cotton
burned off down to the rims of the tubes. The tubes were
sealed with parafilm. The contents of these tubes were then
shell frozen by immersing the tubes in a freezing mixture of
dry ice and ethanol and rotating the tubes about their long
axis until freezing was complete. The tubes were transferred
to the freezing compartment of the laboratory refrigerator
for storage. The temperature of this freezing compartment
was maintained at —10 C.

The final inoculum was prepared by first determining
the highest dilution of the above cell suspension that
initiated growth of the organisms in brain heart infusion
broth within 24 hours, except for El. feseri for which Difco
AC broth was used. This was accomplished by thawing a tube
of the cell suSpension of each culture at room temperature
and adding nine ml of sterile 0.85 per cent saline to each
tube. This brought about a 1:10 dilution of the original
suspension. Next, a series of seven test tubes, each
containing nine ml of sterile 0.85 per cent saline, was set

up for each cell suspension. One ml of the 1:10 dilution

17
of the suSpension was transferred to the next tube and
thoroughly mixed, thus producing a 1:100 dilution of the
original frozen cell suSpension. This procedure was repeated
for the remaining six tubes of saline. The highest dilution
thus produced was 1:100,000,000. One tenth ml from each
dilution was pipetted into three tubes of brain heart infusion
broth. The inoculated tubes were then incubated anaerobically
in the Brewer jar by the method indicated above. After 24
hours incubation the tubes were removed from the jar and
examined grossly for growth. The highest dilution that showed
growth at this time was selected as the proper dilution for
the inoculum prepared from that particular series of frozen
portions of cell suspension for the culture being tested.

This procedure was carried out for all of the cultures.

Normal sheep livers with all grossly visible fat
removed were cut into small pieces and ground in a power meat
grinder. The ground liver was then weighed and mixed with an
equal weight of distilled water. This mixture was stored in
the refrigerator for six hours and then was placed in an
Arnold sterilizer and steamed for four hours. Next the mix-
ture was removed from the steamer and the solids were fil-
tered off through several thicknesses of cheese cloth. The
resulting liquid was poured into three liter flasks and auto-
claved at 121 C for 15 minutes. The proteins precipitated by

autoclaving and the remaining meat solids were filtered off

18
through grade #541 Whatman fluted filter paper. The remaining
liquid was the liver infusion used in the experiments. This
infusion was used to prepare base media to which carbo-
hydrates and other nutrients were later added to form varia-
tions of the test media. Two principal bases were prepared,
differing only in the peptone employed. One of these contained
Difco proteose peptone #3 and the other contained Difco
tryptose. A third base containing bacto peptone was also
prepared and compared with the other bases but it was not
used as a base for the media variations. For the composition

of the base broths see Table l.

19
TABLE 1
COMPOSITION OF BASE MEDIA
Proteose Peptone Base

Sheep Liver infusion. . . . . . . . . . . . . . . . 1 liter
Distilled water . . . . . . . . . . . . . . . . . . 1 liter
Bacto Proteose peptone #3 . . . . . . . . . . . . . 20 grams
Sodium chloride . . . . . . . . . . . . . . . . . . 10 grams

Disodium hydrogen phosphate . . . . . . . . . . . . 5 grams

Tryptose Base
Sheep Liver infusion. . . . . . . . . . . . . . . . 1 liter
Distilled water . . . . . . . . . . . . . . . . . . 1 liter
Bacto Tryptose . . . . . . . . . . . . . . . . . . 20 grams
Sodium chloride . . . . . . . . . . . . . . . . . . 10 grams

Disodium hydrogen phosphate . . . . . . . . . . . . 5 grams

Peptone Base

Sheep Liver infusion. . . . . . . . . . . . . . . 200 m1
Distilled water . . . . . . . . . . . . . . . . . 200 ml
Bacto Peptone . . . . . . . . . . . . . . . . . . . 4 grams
Sodium chloride . . . . . . . . . . . . . . . . . . 2 grams
Disodium hydrogen phosphate . . . . . . . . . . . . 1 gram

Test media were prepared by adding definite quantities
of certain nutrients to the bases. Composition of test media

employed is given in Table 2.

20

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 2
COMPOSITION OF EXPERIMENTAL MEDIA
Medium 2:01:02: Tryp‘I'ose .‘LBado Isaeledw. Calm... Calcium
Code 52,. Base GIucose Manes. GquatIuo asanhe Casem I‘lydrou'deGIucamfc
A IOO ml _ 0.59.. _ __ __ _ __ _
TA —— IOOml 0.59m __ __ _ __ _ _
B [00ml -- —— — — — L09». ._ ._
B, [00ml _. _ ._ _ (.09m _ __ __
TB, — 100ml __ _ _ Log... _ _ __
C IOOmI —_ _ —— 20.0.,... __ __ _ __
TC — IOOrnI — — ZOO-n3 — _ _ __
D 100ml __ 0.59m _ _. _ 1.09m __ _
D, l00mI — 0.59». —- — I.Ogm ._ ._ _
TD, __ IOOmI 0.59m _ __ 1,09... _ _ _
E IOOmI — 0.59m —— I200... — __ _ __
4 TE —- IDOmI 0.59m -— 20.0., __ _. _ __
F :00 ml —— — — 20.0.... 1.09... _ __ __
d I00 ml —— 0.59m -— —— I.09 m —— LOQm —
TK —-— loo...) _ 059., _ _ __ __ __
TL — [00ml -— —- — -— -— -— 0.59m
TBCK —— 100ml __ 0.59». 20.0m,./.09m __ __ _

 

 

 

 

21

In addition to the test media listed in Table 2, the
sheep liver infusion, sheep liver infusion plus 0.5 per cent
sodium chloride and 0.25 per cent disodium phosphate, and the
three bases were also tested. The commercial media tested
were Difco AC broth, Difco thioglycollate broth, and Difco
brain-heart infusion broth.

All the media used were tubed in 15 x 150 mm pyrex
test tubes in exactly ten ml amounts.

The amount of growth in the test media was determined
turbidimetrically by use of a Beckman model B spectrophoto-
meter.

The incubated broth was centrifuged in an Interna-
tional Equipment Company size 2 centrifuge for 45 minutes
at a R.C.F. of 1,300 x G. The supernatant broth was then
decanted and saved. The packed cells were suspended in.
sterile 0.85 per cent saline solution to which 0.3 per cent
formalin had been added. This suSpension was centrifuged
again as before to wash the cells. The supernatant was
decanted and discarded. Following this, exactly ten ml of
sterile 0.3 per cent formalinized saline were added to each
tube and the cells were thoroughly and evenly suspended in
the saline. One ml of this suSpension was added to nine m1
of sterile 0.3 per cent formalinized saline resulting in a
1:10 dilution. In some instances, dilutions other than 1:10

were made so that the turbidity of the resulting suspension

22
would not vary greatly from the others being tested, thus
insuring greater accuracy.

The Spectrophotometer was turned on and allowed to
warm about one hour. Cuvettes were chosen that gave the same
optical density when filled with distilled water and compared.
These matching cuvettes were used in all cell count deter-
minations.

A standard was prepared by filling one of the cuvettes
with the 0.3 per cent formalinized saline suspending medium.
The spectrophotometer was fitted with a red sensitive barrier
cell. A wave length of 650 mu was used for turbidimetric
determination (Bernheimer, 1944). With the standard in place
optical density was adjusted to zero with the slit width
vernier. The standard was replaced by a sample cell suSpen-
sion and the Optical density of the cell suspension was read
from the meter, and the data thus obtained were recorded.
This procedure was carried out for all samples tested.

In order to convert optical density of the cell
suSpensions to total cell counts a graph was prepared. Four
cell suspensions of each species were prepared and their
optical densities determined by means of the spectrophoto-
meter. A portion of these cell suSpensions were diluted to
give a 1:20 dilution. The number of cells in this dilution
were then counted by use of the Petroff-Hauser counting

chamber employing a phase contrast microscOpe. From this

23

data the total cell count of the original suspensions was
calculated. Dilutions were made of each of the original
suspensions so that final concentrations were 90, 75, 66.7,
50, 33.3, 25, and 10 per cent of the original. Optical
density of each dilution was determined as before. Total
cell count for each dilution was calculated from the cell
count of the original suspensions. These data were analyzed
statistically and a graph of their correlation line was
plotted for each species. See Table 3 and Figures 1, 2, and
3.

Test cultures used in this experiment were as follows:

Clostridium.perfringens, which had been isolated from the

 

abdominal muscle of a cow dead from gas gangrene. The isola-
tion was made in the laboratories of the Department of
Bacteriology and Public Health, Michigan State College. The
organism fermented glucose, lactose, maltose, and sucrose
with the production of acid and gas. Salicin was not fer-
mented. Rapid and violent stormy fermentation in iron milk
medium and liquefaction of gelatin occurred in 24 hourS.

The organism was nitrite positive and indole negative and
produced a wide zone of precipitation on egg yolk medium.

One half ml of a 24 hour broth culture injected intramuscularly
into guinea pigs killed them in 24 to 30 hours.

Clostridium septicum 830 was obtained from Dr. L.S.

 

McClung of the University of Indiana. This organism

24
fermented dextrose, lactose, maltose, and salicin with the
production of acid and gas. Sucrose was not fermented.
Gelatin was liquefied and hydrogen sulfide was produced. The
organism was indole negative and nitrite positive and pro—
duced no zone of precipitation on egg yolk medium. One half
ml of a 24 hour broth culture injected intramuscularly into
guinea pigs killed them in 20 to 26 hours. Autopsy revealed
considerable swelling and bloody edema near the sight of
injection, the abdomen appeared green and the abdominal hair
slipped easily. Microscopic examination of material from
the liver surface showed bacilli in characteristic long

filamentous strands.

 

Clostridium feseri was obtained from the Corn States
Serum Company, Omaha, Nebraska, courtesy of Dr. Earl M.
Baldwin. The organism fermented dextrose, lactose, maltose,
and sucrose but did not ferment salicin. There was no
reaction in iron milk, and on egg yolk medium the colonies
were pearlescent with little or no zone of precipitation
extending beyond the colony. The organism was indole negative
and nitrite positive. Gelatin was slowly liquefied and
hydrogen sulfide was produced. Injection of 0.5 m1 of a
24 hour broth culture intramuscularly into guinea pigs

killed in 30 to 50 hours.

 

TABLE 3

COMPARISON OF OPTICAL DENSITY TO CELL COUNT

CLOSTRIDIUM PERFRINGENS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Medium Percent Concentration of Cell Suspension
100% 90% 75% 66.7% 50% 33.3% 25% 10%
g Optical 0.390 0.370 0.315 0.278 0.200 0.142 0.105 0.048
Density
Cell 98.0 88.2 73.5 65.2 49.0 32.6 24.5 9.8
Count *
g Optical 0.330 0.300 0.250 0.222 0.170 0.115 0.080 0.035
Density
Cell 83.0 74.7 62.1 55.2 41.5 27.6 20.7 8.3
Count *
9 Optical 0.420 0.370 0.310 0.273 0.220 0.145 0.115 0.052
Density
Cell 108.0 98.1 81.0 72.0 54.0 36.0 27.0 10.8
Count *
D Optical 0.575 0.520 0.426 0.380 0.290 0.193 0.147 0.060
- Density
Cell 142.0 127.8 106.5 94.6 71.0 47.3 35.5 14.2
Count 4
Medium 5: proteose peptone base plus 0.5 percent glucose and
one percent Bacto Casitone, incubated 24 hours at
37 0.
Medium 8: tryptose base plus 0.5 percent glucose, incubated
24 hours at 37 0.
Medium 0: proteose peptone base plus one percent Bacto
— Casitone, incubated 48 hours at 37 0.
Medium 2: Difco Brain Heart Infusion broth, incubated 48
hours at 37 C.
* Cell count in millions.

 

26

Figure 1. Correlation graph to convert optical density of

El. perfringens cell suspensions to total cell

 

count. Dots represent observed optical densities

for suspensions of known cell count. Statistically

calculated values for plotting the correlation

line were:

 

 

 

 

Cell Count Optical Density
20,000,000/m1 0.088
40,000,000/m1 0.166
60,000,000/m1 0.244

100,000,000/m1 0.400

 

 

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TABLE 4

COMPARISON OF OPTICAL DENSITY TO CELL COUNT

CLOSTRIDIUM SEPTICUM

 

28

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Medium Percent Concentration of Cell SuSpension
100% 90% 75% 66.7% 50% 33.3% 25% 10%

A Optical 0.310 0.280 0.231 0.203 0.148 0.105 0.075 0.032
Density
Cell 188.0 169.0 141.0 124.0 94.0 62.0 47.0 18.8
Count 4

g Optical 0.370 0.335 0.280 0.250 0.190 0.127 0.097 0.038
Density
Cell 236.0 212.0 177.0 157.0 118.0 78.7 59.0 23.6
Count *

9 Optical 0.240 0.220 0.185 0.163 0.120 0.083 0.057 0.025
Density
Cell 146.0 131.0 109.0 97.4 73.0 48.8 36.5 14.6
Count *

2, Optical 0.410 0.377 0.305 0.280 0.210 0.144 0.108 0.045
Density 1
Cell 252.0 227.0 189.0 168.0 126.0 84.2 63.0 25.2
Count *

Medium 5; Difco AC broth, incubated 24 hours at 37 C.

Medium g: proteose peptone base plus 0.02 percent gluta-

thione, incubated 24 hours at 37 0.
Medium 2: proteose peptone base, incubated 48 hours at 37 C.
Medium 2; tryptose base plus 0.5 percent maltose, incubated
48 hours at 37 C.
4 Cell counts in millions.

29

Figure 2. Correlation graph to convert optical density of

Cl. septicum 830 cell suspensions to total cell

count per m1. Dots represent observed optical

densities for suspensions of known cell count.

Statistically calculated values for plotting the

correlation line were:

 

 

 

 

Cell Count Optical Density
50,000,000/m1 0.084
100,000,000/m1 0.164
150,000,000/m1 0.244
200,000,000/m1 0.323

 

 

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CONPARISCN OF

TABLE 5

CLCSTRIDIUM FESERI

 

OPTICAL DENSITY TO CELL COUNT

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Medium Percent Concentration of Cell Suspension
100% 90% 75% 66.7% 50% 33.3% 25% 10%

A Optical 0.295 -—- ——- -—— 0.155 ——- 0.080 0.035
Density
Cell 300.0 —— ——— ——— 150.0 -—- 75.0 30.0
Count *

2 Optical r 0.325 0.300 0.250 0.223 0.178 0.116 0.090 0.030
Density
Cell 350.0 315.0 262.0 233.0 175.0 117.0 87.0 35.0
Count 4

g Optical 0.280 0.265 0.225 0.200 0.155 0.106 0.078 0.035
Density
Cell 300.0 270.0 225.0 200.0 150.0 100.0 75.0 30.0
Count 4

2 Optical 0.475 0.425 0.363 0.323 0.250 0.172 0.135 0.055
Density
Cell 540.0 486.0 450.0 360.0 270.0 180.0 135.0 54.0
Count 4

Medium A: proteose peptone base plus one percent Bacto

Casitone, incubated 24 hours at 37 C.
Medium B: tryptose base, incubated 24 hours at 37 C.
Medium 9: proteose peptone base plus 0.5 percent glucose

Medium.2:

* Cell

and 0.02 percent glutathione, incubated 48 hours

at 37 C.

Difco AC broth, incubated 48 hours at 37 0.

count in millions.

 

32

Figure 3. Correlation graph to convert optical density of

EA. feseri #6 cell suspensions to total cell count

per ml. Dots represent observed optical densities

for suspensions of known cell count. Statistically

calculated values for plotting the correlation

line were:

 

 

 

 

Cell Count Optical Density
75,000,000 0.080
175,000,000 0.169
275,000,000 0.254
0.370

400,000,000

 

 

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34
RESULTS

Results of this experiment were essentially numerical
and thus best presented by the tables and graphs that follow.
However, it should be stated here that in making up media
it was found that those media that contained isoelectric
casein, developed a heavy, flocculent, white precipitate
after incubation with bacteria. This precipitate obviously
made these media useless for turbidimetric cell counts. As
a result, Bacto Casitone was substituted for casein and
further trouble from this source was allayed. However,
precipitation from other sources did occur. Addition of
calcium hydroxide resulted in the formation of a dense pale
yellow precipitate. A similar precipitate formed when
calcium hydroxide was added to sheep liver infusion alone.
The presence of calcium gluconate was coincidental with the
formation of a pale yellowish precipitate above a pH of 6.2

or after incubation.

35

 

 

 

 

 

 

 

 

 

 

 

TABLE 6

EXPERIMENTAL DATA FROM 24 HOUR CULTURES OF 9A. PERFRINGENS
Medium Dilu- Optical Cell Count/m1

tion‘e Density
Liver Infusion 1:10 0.190 450,000,000
1:10 0.172 410,000,000
1:10 0.155 370,000,000
Liver Infusion plus 0.5 1:10 0.265 645,000,000
percent NaCl, and 0.25 1:10 0.245 600,000,000
percent Na2HPO4 1:10 0.230 575,000,000
Proteose Peptone Base 1:10 0.410 1,020,000,000
1:10 0.390 970,000,000
1:10 0.385 960,000,000
Tryptose Base 1:10 0.450 1,120,000,000
1:10 0.430 1,070,000,000
1:10 0.420 1,050,000,000
Peptone Base 1:10 0.430 1,070,000,000
1:10 0.420 1,050,000,000
1:10 0.400 1,000,000,000
Proteose Peptone Base 1:10 0.415 1,040,000,000
plus 0.5 percent glucose 1:10 0.415 1,040,000,000
1:10 0.402 1,010,000,000
Tryptose Base plus 0.5 1:10 0.375 925,000,000
percent glucose 1:10 0.360 900,000,000
1:10 0.350 870,000,000i

 

 

 

 

* For adjustment of turbidity (See Page 20.)

TABLE 6 (Continued)

36

 

 

 

 

 

 

 

 

 

Medium ' Dilu- Optical Cell Count/m1
tion Density
Proteose Peptone Base 1:10 0.435 1,095,000,000
plus 1.0 percent Bacto 1:10 0.425 1,060,000,000
Casitone 1:10 0.402 1,010,000,000
Tryptose Base plus 1.0 1:10 0.362 910,000,000
percent Bacto Casitone 1:10 0.360 900,000,000
1:10 0.340 850,000,000
Protoose Peptone Base 1:10 0.380 950,000,000
plus 0.02 percent 1:10 0.355 890,000,000
glutathione 1:10 0.320 800,000,000
Tryptose Base plus 0.02 1:10 0.350 870,000,000
percent glutathione 1:10 0.320 800,000,000
1:10 0.312 780,000,000
Proteose Peptone Base 1:10 0.420 1,050,000,000
plus 1.0 percent Casitone 1:10 0.400 1,000,000,000
and 0.5 percent glucose 1:10 0.395 990,000,000
Tryptose Base plus 1.0 1:10 0.405 1,015,000,000
percent Bacto Casitone 1:10 0.390 970,000,000
and 0.5 percent glucose 1:10 0.380 955,000,000
Proteose Peptone Base 1:10 0.470 1,180,000,000
plus 0.5 percent glucose 1:10 0.425 1,060,000,000
and 0.02 percent
glutathione 1:10 0.420 1,050,000,000

 

 

 

 

 

TABLE

6 (Continued)

37

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical Cell Count/m1
tion Density
Tryptose Base plus 0.5 1:10 0.455 1,145,000,000
percent glucose and 0.02 1:10 0.430 1,070,000,000
percent glutathione 1:10 0.425 1,060,000,000
Proteose Peptone Base 1:10 0.425 1,060,000,000
plus 1.0 percent Bacto
Casitone and 0.02 1:10 0.400 1,000,000,000
percent glutathione
1:10 0.395 990,000,000
Tryptose Base plus 0.5 1:10 0.440 1,110,000,000
percent maltose 1:10 0.430 1,070,000,000
1:10 0.412 1,030,000,000
Tryptose Base plus 1.0 1:10 0.490 1,225,000,000
percent Bacto Casitone,
0.5 percent maltose and 1:10 0.470 1,180,000,000
0.02 percent glutathione
1:10 0.425 1,145,000,000
Difco AC broth 1:10 0.360 900,000,000
1:10 0.350 870,000,000
1:10 0.350 870,000,000
Difco Brain Heart 1:10 0.155 370,000,000
Infusion broth 1:10 0.132 315,000,000
1:10 0.096 220,000,000
Difco Thioglycollate 1:10 0.310 770,000,000
broth 1:10 0.300 740,000,000
1:10 0.293 726,000,000

 

 

 

 

 

38

 

 

 

 

 

 

 

 

 

 

 

TABLE 7

EXPERIMENTAL DATA FROM 48 HOUR CULTURES OF 9A. PERFRINGENS
Medium. Dilu- Optical 1 Cell Count/ml

tion Density
Liver Infusion I:10 0.155 370,000,000
1:10 0.150 360,000,000
1:10 0.150 360,000,000
Liver Infusion plus 0.5 1:10 0.210 510,000,000
percent NaCl and 0.25 1:10 0.210 510,000,000
percent NaZHPO4 1:10 0.200 483,000,000
Proteose Peptone Base 1:10 0.340 850,000,000
1:10 0.320 800,000,000
1:10 0.303 750,000,000
Tryptose Base 1:10 0.390 970,000,000
1:10 0.340 850,000,000
1:10 0.330 830,000,000
Peptone Base 1:10 0.375 940,000,000
1:10 0.360 900,000,000
1:10 0.345 860,000,000
Proteose Peptone Base 1:10 0.360 900,000,000
plus 0.5 percent glucose 1:10 0.340 850,000,000
1:10 0.330 830,000,000
Tryptose Base plus 0.5 1:10 0.320 800,000,000
percent glucose 1:10 0.320 800,000,000
1:10 0.310 770,000,000

 

 

 

 

 

39

 

 

 

 

 

 

 

 

 

TABLE 7 (Continued)
Medium Dilu- Optical Cell Count/ml

tion Density
Proteose Peptone Base 1:10 0.430 1,070,000,000
plus 1.0 percent Bacto 1:10 0.420 1,050,000,000
Casitone 1:10 0.415 1,040,000,000
Tryptose Base plus 1.0 1:10 0.325 810,000,000
percent Bacto Casitone 1:10 0.320 800,000,000

1:10 0.310 770,000,000
Proteose Peptone Base 1:10 0.375 925,000,000
plus 0.02 percent 1:10 0.360 900,000,000
glutathione 1:10 0.320 800,000,000
Tryptose Base plus 0.02 1:10 0.340 850,000,000
percent glutathione 1:10 0.325 810,000,000

1:10 0.315 790,000,000
Proteose Peptone Base 1:10 0.390 970,000,000'
plus 1.0 percent Casitone 1:10 0.360 900,000,000
and 0.5 percent glucose 1:10 0.300 740,000,000
Tryptose Base plus 1.0 1:10 0.380 955,000,000
percent Bacto Casitone, 1:10 0.370 920,000,000
and 0.5 percent glucose 1:10 0.355 880,000,000
Proteose Peptone Base 1:10 0.375 925,000,000
plus 0.5 percent glucose
and 0.02 percent 1:10 0.360 900,000,000
glutathione

1:10 0.355 890,000,000

 

 

 

 

 

TABLE 7 (Continued)

40

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical 0611 Count/m1
tion Density
Tryptose Base plus 0.5 1:10 0.360 900,000,000
percent glucose, and 0.02 1:10 0.340 850,000,000
percent glutathione 1:10 0.336 840,000,000
Proteose Peptone Base 1:10 0.390 970,000,000
plus 1.0 percent Bacto
Casitone, and 0.02 1:10 0.380 955,000,000
percent glutathione
1:10 0.375 925,000,000
Tryptose Base plus 0.5 1:10 0.380 955,000,000
percent maltose 1:10 0.360 900,000,000
1:10 0.350 870,000,000
Tryptose Base plus 1.0 1:10 0.445 1,115,000,000
percent Bacto Casitone,
0.5 percent maltose, and 1:10 0.440 1,110,000,000
0.02 percent glutathione
1:10 0.415 1,040,000,000
Difco AC broth 1:10 0.395 990,000,000
1:10 0.350 870,000,000
1:10 0.347 865,000,000
Difco Brain Heart 1:10 0.148 356,000,000
Infusion broth 1:10 0.115 260,000,000
1:10 0.082 183,000,000
Difco Thioglycollate 1:10 0.320 800,000,000
broth 1:10 0.310 770,000,000
1:10 0.310 770,000,000

 

 

 

 

 

41

 

 

 

 

 

 

 

 

 

TABLE 8
EXPERIMENTAL DATA FROM 24 HOUR CULTURES OF EA. SEPTICUM 830
Medium Dilu- ) Optical ' Cell Count/ml
tion Density

Liver Infusion 1:5 0.205 630,000,000
1:5 0.180 550,000,000

1:5 0.170 520,000,000

Liver Infusion plus 0.5 1:10 0.147 900,000,000
percent NaCl, and 0.25 1310 0.130 800,000,000
percent NaQHPO4 1:10 0.125 760,000,000
Proteose Peptone Base 1:10 0.295 1,820,000,000
1:10 0.290 1,800,000,000

1:10 0.275 1,690,000,000

Tryptose Base 1:10 0.297 1,840,000,000
1:10 0.265 1,630,000,000

1:10 0.244 1,500,000,000

Peptone Base 1:10 0.296 1,830,000,000
1:10 0.275 1,690,000,000

1:10 0.260 1,610,000,000

Proteose Peptone Base 1:10 0.200 1,230,000,000
plus 0.5 percent glucose 1:10 0.175 1,080,000,000
1:10 0.165 1,010,000,000

Tryptose Base plus 0.5 1:10 0.175 1,080,000,000
percent glucose 1:10 0.155 930,000,000
1:10 0.144 860,000,000

 

 

 

 

 

TABLE 8 (Continued)

42

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical 0611 Count/m1
tion Density
Proteose Peptone Base 1:10 0.263 1,630,000,000
plus one percent Bacto 1:10 0.247 1,530,000,000
Casitone 1:10 0.240 1,480,000,000
Tryptose Base plus one 1:10 0.258 1,600,000,000
percent Bacto Casitone 1:10 0.250 1,540,000,000
1:10 0.225 1,400,000,000
Proteose Peptone Base 1:10 0.147 900,000,000
plus 0.02 percent 1:10 0.145 890,000,000
glutathione 1:10 0.145 890,000,000
Tryptose Base plus 0.02 1:10 0.144 860,000,000
percent glutathione 1:10 0.132 810,000,000
1:10 0.130 800,000,000
Proteose Peptone Base 1:10 0.254 1,560,000,000
plus one percent Casitone 1:10 0.245 1,520,000,000
and 0.5 percent glucose 1:10 0.240 1,480,000,000
Tryptose Base plus one 1:10 0.244 1,500,000,000
percent Bacto Casitone 1:10 0.240 1,480,000,000
and 0.5 percent glucose 1:10 0.223 1,380,000,000
Proteose Peptone Base 1:5 0.230 715,000,000
plus 0.5 percent glucose
and 0.02 percent 1:5 0.225 700,000,000
glutathione
1:5 0.210 640,000,000

 

 

 

 

 

TABLE 8 (Continued)

43

 

 

 

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical Cell Count/ml
tion Density
Tryptose Base plus 0.5 1:10 0.112 680,000,000
percent glucose and 0.02 1:10 0.105 630,000,000
percent glutathione 1:10 0.092 560,000,000
Proteose Peptone Base 1:10 0.215 1,320,000,000
plus one percent Bacto
Casitone and 0.02 1:10 0.200 1,230,000,000
percent glutathione
1:10 0.195 1,200,000,000
Tryptose Base plus 0.5 1:10 0.290 1,800,000,000
percent maltose 1:10 0.285 1,740,000,000
1:10 0.248 1,530,000,000
Tryptose Base plus one 1:10 0.275 1,680,000,000
percent Bacto Casitone,
0.5 percent maltose and 1:10 0.270 1,650,000,000
0.02 percent glutathione
1:10 0.254 1,560,000,000
Difco AC broth 1:10 0.173 1,060,000,000
1:10 0.160 970,000,000
1:10 0.155 930,000,000
Difco Brain Heart 1:10 0.084 510,000,000
Infusion broth 1:10 0.080 490,000,000
1:10 0.065 400,000,000
Difco Thioglycollate 1:10 0.147 900,000,000
broth 1:10 0.145 870,000,000
1:10 0.140 850,000,000

 

 

44
TABLE 9

EXPERIMENTAL DATA FROM 48 HOUR CULTURES OF EL. SEPTICUM 830

 

Dilu-

 

 

 

 

 

 

 

 

Medium Optical Cell Count/m1

tion Density
Liver Infusion 1:5 0.065 200,000,000
1:5 0.050 145,000,000
1:5 0.044 130,000,000
Liver Infusion plus 0.5 1:5 0.092 280,000,000
percent NaCl, and 0.25 1:5 0.083 250,000,000
percent Na2HP04 1:5 0.065 200,000,000
Proteose Peptone Base 1:10 0.245 1,520,000,000
1:10 0.230 1,430,000,000
1:10 0.223 1,380,000,000
Tryptose Base 1:10 0.250 1,540,000,000
1:10 0.235 1,440,000,000
1:10 0.220 1,340,000,000
Peptone Base 1:10 0.244 1,500,000,000
1:10 0.225 1,400,000,000
1:10 0.222 1,360,000,000
Proteose Peptone Base 1:10 0.253 1,560,000,000
plus 0.5 percent glucose 1:10 0.250 1,540,000,000
1:10 0.225 1,400,000,000
Tryptose Base plus 0.5 1:10 0.240 1,480,000,000
percent glucose 1:10 0.230 1,430,000,000
1:10 0.220 1,340,000,000

 

 

 

 

 

45

 

 

 

 

 

 

 

 

TABLE 9 (Continued)
Medium Dilu— Optical 0611 Count/m1

tion Density
Proteose Peptone Base 1:10 0.250 1,540,000,000
plus one percent Bacto 1:10 0.247 1,530,000,000
Casitone 1:10 0.245 1,520,000,000
Tryptose Base plus one 1:10 0.240 1,480,000,000
percent Bacto Casitone 1:10 0.230 1,430,000,000

1:10 0.223 1,380,000,000
Proteose Peptone Base 1:10 0.245 1,520,000,000
plus 0.02 percent 1:10 0.240 1,480,000,000
glutathione 1:10 0.230 1,430,000,000
Tryptose Base plus 0.02 1:10 0.237 1,460,000,000
percent glutathione 1:10 0.225 1,400,000,000

1:10 0.215 1,320,000,000
Proteose Peptone Base 1:10 0.270 1,650,000,000
plus one percent Casitone 1:10 0.245 1,520,000,000
and 0.5 percent glucose 1:10 0.240 1,480,000,000
Tryptose Base plus one 1:10 0.262 1,620,000,000
percent Bacto Casitone, 1:10 0.250 1,540,000,000
and 0.5 percent glucose 1:10 0.237 1,460,000,000
Proteose Peptone Base 1:10 0.200 1,230,000,000
plus 0.5 percent glucose
and 0.02 percent 1:10 0.190 1,160,000,000
glutathione

1:10 0.178 1,100,000,000

 

 

 

 

 

 

46

 

 

 

 

 

 

 

 

TABLE 9 (Continued)
Medium Dilu— Optical Cell Count/m1
tion Density
Tryptose Base plus 0.5 1:10 0.195 1,200,000,000
percent glucose and 0.02 1:10 0.187 1,140,000,000‘
percent glutathione 1:10 0.175 1,080,000,000
Proteose Peptone Base 1:10 0.256 1,580,000,000
plus one percent Bacto
Casitone, and 0.02 1:10 0.250 1,540,000,000
percent glutathione
1:10 0.244 1,500,000,000
Tryptose Base plus 0.5 1:10 0.320 1,950,000,000
percent maltose 1:10 0.305 1,860,000,000
1:10 0.275 1,690,000,000
Tryptose Base plus one 1:10 0.325 1,980,000,000
percent Bacto Casitone,
0.5 percent maltose, and 1:10 0.315 1,940,000,000
0.02 percent glutathione
1:10 0.285 1,760,000,000
Difco AC broth 1:10 0.198 1,220,000,000
1:10 0.160 970,000,000
1:10 0.155 930,000,000
Difco Brain Heart 1:10 0.055 330,000,000
Infusion broth 1:10 0.053 310,000,000
1:10 0.053 310,000,000
Difco Thioglycollate 1:10 0.128 790,000,000
broth 1:10 0.115 690,000,000
1:10 0.104 630,000,000

 

 

 

 

 

 

EXPERIMENTAL DATA FROM 24

TABLE 10

47

HOUR CULTURES 0F CE. FESERI #6

 

0.11 Count/m1

 

 

 

 

 

 

 

 

Medium Dilu- Optical
tion Density
Liver Infusion nene 0.080 75,000,000
none 0.077 73,000,000
none 0.057 50,000,000
Liver Infusion plus 0.5 none 0.212 225,000,000
percent NaCl, and 0.25 none 0.195 205,000,000
percent Na2HPO4 none 0.164 170,000,000
Proteose Peptone Base 1:5 0.155 790,000,000
1:5 0.140 715,000,000
1:5 0.132 680,000,000
Tryptose Base 1:5 0.178 875,000,000‘
1:5 0.150 770,000,000
1:5 0.120 600,000,000
Peptone Base 1:5 0.133 685,000,000
1:5 0.132 680,000,000
1:5 0.132 680,000,000
Proteose Peptone Base 1:2 0.190 400,000,000
plus 0.5 percent glucose 1:2 0.167 344,000,000
1:2 0.160 325,000,000
Tryptose Base plus 0.5 none 0.297 320,000,000
percent glucose none 0.285 306,000,000
none 0.280 300,000,000

 

 

 

 

 

TABLE 10 (Continued)

48

 

 

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical Cell Count/m1
tion Density
Proteose Peptone Base 1:2 0.295 634,000,000
plus one percent Bacto 1:2 0.282 606,000,000
Casitone 1:2 0.265 570,000,000
Tryptose Base plus one 1:2 0.309 780,000,000
percent Bacto Casitone 1:2 0.260 558,000,000
1:2 0.257 550,000,000
Proteose Peptone Base 1:5 0.245 1,310,000,000
plus 0.02 percent 1:5 0.230 1,230,000,000
glutathione 1:5 0.225 1,190,000,000
Tryptose Base plus 0.02 1:5 0.230 1,230,000,000
percent glutathione 1:5 0.224 1,180,000,000
1:5 0.200 1,060,000,000
Proteose Peptone Base none 0.395 430,000,000
plus one percent Casitone none 0.335 362,000,000
and 0.5 percent glucose none 0.294 315,000,000
Tryptose Base plus one 1:2 0.217 460,000,000
percent Bacto Casitone, 1:2 0.185 388,000,000
and 0.5 percent glucose 1:2 0.155 320,000,000
Proteose Peptone Base 1:2 0.190 400,000,000
plus 0.5 percent glucose
and 0.02 percent 1:2 0.173 360,000,000
glutathione
1:2 0.155 320,000,000

 

 

 

 

 

TABLE 10 (Continued)

49

 

 

 

 

 

 

 

 

 

Medium Dilu- Optical r Cell Count/ml
tion Density
Tryptose Base plus 0.5 1:2 0.190 400,000,000
percent glucose, and 0.02 1:2 0.165 344,000,000
percent glutathione 1:2 0.150 310,000,000
Proteose Peptone Base 1:5 0.225 1,200,000,000
plus one percent Bacto
Casitone, and 0.02 1:5 0.210 1,110,000,000
percent glutathione
1:5 0.205 1,085,000,000
Tryptose Base plus 0.5 1:5 0.145 755,000,000
percent maltose 1:5 0.125 715,000,000
1:5 0.118 598,000,000
Tryptose Base plus one 1:5 0.188 985,000,000
percent Bacto Casitone,
0.5 percent maltose, and 1:5 0.174 910,000,000
0.02 percent glutathione .
1:5 0.160 825,000,000
Difco AC broth 1:5 0.112 550,000,000
1:5 0.110 540,000,000
1:5 0.108 530,000,000
Difco Brain Heart none 0.190 200,000,000
Infusion broth none 0.145 149,000,000
none 0.125 125,000,000
Difco Thioglycollate 1:5 0.136 690,000,000
broth 1:5 0.135 685,000,000
1:5 0.135 685,000,000

 

 

 

 

 

TABLE 11

50

EXPERIMENTAL DATA FROM 48 HOUR CULTURES 0F 99. FESERI #6

 

r D1111-

 

 

 

 

 

 

 

 

Medium. r Optical Cell Count/m1

tion Density
Liver Infusion none 0.110 110,000,000
none 0.095 92,000,000
none 0.093 90,000,000
Liver Infusion plus 0.5 none 0.190 200,000,000
percent NaCl, and 0.25 none 0.175 183,000,000
percent NagHPO4 none 0.167 175,000,000
Proteose Peptone Base 1:5 0.160 820,000,000
1:5 0.155 790,000,000
1:5 0.125 630,000,000
Tryptose Base 1:2 0.340 736,000,000
1:2 0.335 725,000,000
1:2 0.330 715,000,000
Peptone Base 1:5 0.190 1,000,000,000
1:5 0.187 990,000,000
1:5 0.185 975,000,000
Proteose Peptone Base none 0.297 320,000,000
plus 0.5 percent glucose none 0.245 261,000,000
none 0.230 245,000,000
Tryptose Base plus 0.5 a... 0.175 180,000,000
percent glucose none 0.163 170,000,000
none 0.145 150,000,000

 

 

 

 

 

51

 

 

 

 

 

 

 

 

 

 

TABLE 11 (Continued)
Medium Dilu— Optical Cell Count/m1

tion Density
Proteose Peptone Base 1:10 0.205 2,170,000,000
plus one percent Bacto 1:10 0.197 2,070,000,000
Casitone 1:10 0.184 1,920,000,000
Tryptose Base plus one 1:5 0.270 1,450,000,000
percent Bacto Casitone 1:5 0.260 1,360,000,000

1:5 0.247 1,325,000,000
Proteose Peptone Base 1:5 0.302 1,625,000,000
plus 0.02 percent 1:5 0.275 1,475,000,000
glutathione 1:5 0.250 1,340,000,000‘
Tryptose Base plus 0.02 1:5 0.253 1,345,000,000
percent glutathione 1:5 0.230 1,220,000,000

1:5 0.225 1,200,000,000
Proteose Peptone Base 1:5 0.172 900,000,000
plus one percent Casitone 1:5 0.153 785,000,000
and 0.5 percent glucose 1:5 0.142 720,000,000
Tryptose Base plus one 1:5 0.155 790,000,000
percent Bacto Casitone, 1:5 0.140 715,000,000
and 0.5 percent glucose 1:5 0.133 680,000,000
Proteose Peptone Base none 0.280 300,000,000
plus 0.5 percent glucose
and 0.02 percent none 0.277 298,000,000
glutathione

none 0.275

 

 

 

295,000,00

 

TABLE 11 (Continued)

52

 

. Dilu-

 

 

 

 

 

 

 

 

Medium 1 Optical 4 Cell Count/m1
tion Density
Tryptose Base plus 0.5 none 0.230 245,000,000
percent glucose and 0.02 none 0.220 233,000,000
percent glutathione none 0.190 200,000,000
Proteose Peptone Base 1:10 0.208 2,200,000,000
plus one percent Bacto
Casitone, and 0.02 1:10 0.190 2,000,000,000
percent glutathione
. 1:10 0.187 1,945,000,000
Tryptose Base plus 0.5 1:5 0.142 725,000,000
percent maltose 1:5 0.135 680,000,000
1:5 0.132 660,000,000
Tryptose Base plus one 1:5 0.260 1,360,000,000
percent Bacto Casitone,
0.5 percent maltose, and 1:5 0.252 1,340,000,000
0.02 percent glutathione
1:5 0.222 1,180,000,000
Difco AC broth 1:2 0.265 570,000,000
1:2 0.235 500,000,000
1:2 0.200 420,000,000
Difco Brain Heart none 0.230 245,000,000
Infusion broth none 0.180 188,000,000
none 0.172 180,000,000
Difco Thioglycollate 1:5 0.142 725,000,000
broth 1:5 0.138 700,000,000
1:5 0.133 670,000,000

 

 

 

 

 

53

Figure 4. Graph showing cell count per m1 of broth for 24 hour

cultures of 9A. perfringens in each of the experi-

 

mental media. Composition of media is obtained by
noting check marks in media constituent columns on
the left side of graph. Figure 5 is similarily
constructed and indicates cell counts of 48 hour

cultures of 9A. perfringens. Values for El.

 

septicum 830 and EA. feseri #6 are indicated in

Figures 6, 7, 8, and 9.

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60
DISCUSSION

Results obtained from the addition of isoelectric
casein to the experimental media were rather interesting.
In all probability, acid produced by the growing organisms
lowered the pH of the medium sufficiently to precipitate
casein. A strong support for this hypothesis lies in the
fact that addition of small amounts of hydrochloric acid
to media containing casein brought about a similar precipita-
tion. The precipitate that formed When calcium hydroxide
was added to counteract the acid appeared different and was
formed even in the absence of casein. Calcium gluconate
when added to media with a pH above 6.2 produced a
precipitate with an appearance identical with that produced
by calcium hydroxide. Apparently large amounts of casein
were incompatible with the sheep liver infusion.

Differences of optical density and cell count
between species inferred that the various species either
have different light absorption maxima or different cellular
optical densities. Cell size might also have played a part

in this. El. perfringens had larger cells than the other two

 

Species.
The above data on cell count studies showed that the
rate of growth in different media varied considerably with

the Species. It was noticed that all media employed for the

cultivation of 9A. pgrfringens produced about the same

 

number of cells in both 24 and 48 hours. The only exceptions
to this were liver infusion with and without the salts added,
and Difco Brain—heart Infusion.

It was further noticed that generally there were

fewer cells of 9;. perfringens and EA. septicum 830 in the

 

48 hour cultures than in the 24 hour cultures. This
phenomenon can be explained by the possibility that the
cultures had passed their peak of growth by 48 hours and
some cells were beginning to autolyse. Of course, the form-
ation of spores and their subsequent release might account
for this, however, virtually no spores were ever observed
microscopically from these cultures in the relatively short
incubation time employed in this experiment.

The sheep liver infusion without additives, supported
growth of all three species, although, cell counts were low.
With the addition of 0.5 per cent sodium chloride and 0.25
per cent disodium hydrogen phosphate the amount of growth was
increased. This was in keeping with the findings of Jones
and Clifton (1953) in that the liver undoubtedly contained
some tissue glucose, and this was more readily fermented in
the presence of phOSphate ions.

The three base media employed, supported nearly
identical amounts of growth. The base media produced an

amount of growth equal to, or in most cases superior to, the

62
commercially prepared media that were tested. This indicated
that the base media alone were quite satisfactory for the
propagation of Clostridia species. When glucose was added to
the base, no particular change in cell count resulted in the

case of 9A._p§rfringens or in the 48 hour cultures of

 

EA. septicum 830, but considerably less growth occurred in
cultures of 9A. feseri #6 and in the 24 hour incubation of
9A. epticum 830 cultures. When 0.5 per cent of maltose was
substituted for the glucose, growth was equal to that pro-
duced by the base alone except in 48 hour cultures of

CA. septicum.830 where an increased cell count was noted.

As was the case with maltose, the addition of one per cent
Bacto Casitone to the bases did not particularily alter the
number of cells produced except in one instance, but that
was a singularily dramatic one. The 48 hour cultures of

9A. feseri #6 grown in base containing one per cent Bacto
Casitone produced about three times as many cells as the
base alone did. Addition of 0.02 per cent glutathione to the

bases gave uniformly poorer growth in El. perfringens

 

cultures and in 24 hour cultures of El. septicum 830. The
48 hour cultures of El. septicum 830 gave cell counts about
equal to base alone, but encouragingly increased cell counts
were noted in all 9;. feseri #6 cultures. The greatest
increase came during the 24 hour incubation, indicating that

this compound brings about more rapid growth of this rather

63
slow growing and difficult to isolate pathogen. The presence
of a small amount of glutathione in isolation media may well
be the Spark necessary to transform some of the doubtfully
positive "black leg" cases to known 9;. feseri infections.

When both glucose and Bacto Casitone were added, the
growth was no better than that in base alone; and in 48 hour
cultures of 9A. feseri #6 growth was considerably poorer.

The combination of glutathione with glucose produced about
the same number of cells as the addition of glucose alone
did, that is, rather poor growth resulted. This tended to
indicate that the presence of glucose in the range of about
0.5 per cent has some inhibitory effect on these three
organisms.

Addition of Bacto Casitone and glutathione together
gave about the same results as Casitone alone, except in the
case of 24 hour cultures of EA. feseri #6 where growth
paralleled that in glutathione alone. The combination of
these two materials seemed to embody the good qualities of
each. Addition of 0.5 per cent maltose together with Casitone
and glutathione resulted in the best growth of Cl. perfringens
and 9A. epticum 830 of any medium tested. Maltose did not
seem to exhibit the inhibitory tendencies of glucose. This
medium produced good growth of 9A. feseri #6, but not the
excellent growth obtained when glutathione alone and Bacto

Casitone plus glutathione were used.

64

 

Difco AC broth produced more growth of El. perfringens
and 2A. septicum 830 than any other commercially prepared
medium tested. Growth 0f.2l° perfringens in Difco AC broth

 

was about equal in amount to the base media, but somewhat
less growth of 9A, septicum 830 and CA. feseri #6 was pro-
duced than in the base media. Difco thioglycollate broth
produced growth of 9A. feseri #6 in an amount about equal to
that in the base media and thus it was the best of the
commercially prepared media for that Species. However, the
Difco thioglycollate broth produced less growth of the other
two Species than did Difco AC broth. Difco Brain-heart
Infusion broth produced poor results of all three Species.

The range of differences in cell counts for a given
Species in one medium was also observed by Reed and Orr
(1943). They accounted for this variation as the manifesta-
tion of mixing a small amount of air with the medium during
inoculation, thus the oxidation-reduction potential of diff-
erent tubes varied slightly.

From these data it appears that when bacteria are to
be isolated from gangrenous tissue, a greater chance of re-
covering the pathogen would prevail if a sheep liver infusion
base containing 0.5 per cent maltose, one per cent Bacto
Casitone and 0.02 per cent glutathione was employed. In
cases where the presence of 9A. feseri is suspected, a medium

of sheep liver infusion base containing 0.02 per cent

65

glutathione should be used in addition to the above medium.

66
SUMMARY

It was found that the substitution of maltose for glu—
cose in media used for the cultivation of the three anaerobic

pathogens employed in this experiment, 9;. pgrfringens, 9A.

 

septicum 830, and El. feseri #6, resulted in greater cell
production. Addition of a small amount of glutathione to
sheep liver infusion base produced rapid and heavy growth of
El. feseri. Two new media of growth promoting ability
superior to certain commercially prepared media are des-
cribed. They were tested, and are recommended for the
propagation and isolation of gas gangrene producing

clostridia.

67
REFERENCES

Bernheimer, A. W. 1944. Nutritional requirements and fac-
tors affecting the production of toxin of Clostridium
septicum. J. Exp. Med., 80, 321-331.

Brewer, J. H. 1940. A clear liquid medium for "aerobic"
cultivation of anaerobes. J. of Bact., 39, 10.

Brewer, J. H. 1942. A new Petri dish cover and technique
for use in the cultivation of anaerobes and micro-
aerophiles. Science, 95, 587-589.

Bridges, A. E., Pepper, R. E., and Chandler, V. L. 1952.
Apparatus for large scale anaerobic cultures in an
atmOSphere of helium. J. of Bact., 64, 137-138.

Brown, J. H., and Brewer, J. H. 1938. A method for utilizing
illuminating gas in the Brown, Fildes and McIntosh or
other anaerobe jars of the Laidlaw principle. J. Lab.
and Clin. Med., 23, 870-874.

Fildes, P. A., and McIntosh, J. 1921. An improved form of
McIntosh and Fildes anaerobic jar. Brit. J. Exp.
Path., 2, 153-155.

Hayward, N. 1941. Rapid identification of Clostridium
welchii by the Nagler reaction. Brit. Med. J., 1,
EIIZEII

Hayward, N. 1943. The rapid identification of Clostridium
welchii by Nagler test in plate cultures. J. Path.
BfiCt. ’ 55, 285-2930

Jones, L. W., and Clifton, C. C. 1953. Metabolism and
nutrition of Clostridium feseri. J. of Bact., 65,
560' 564 e

Kellet, C. E. 1939. The early history of gas gangrene.
Ann. Med. Hist., 1, 452. This quotation cited from:
Dubos, R. J., et al. 1952. Bacterial and mycotic
infections of man. J. B. Lippincott 00., Philadelphia.

Macfarlane, R. G., Oakley, C. L., and Anderson, C. G. 1941.
Hemolysis and the production of opalescence in serum
and lecithovitellin by the toxin of Clostridium
welchii. J. Path. Bact., 52, 99-103.

68

Malin, B., and Flinn, R. K. 1951. The use of a synthetic
resin in anaerobic media. J. of Bact., 62, 349.

Marshall, M. S., and Nordby, H. P. 1942. Anaerobic plates.
JO Of B80t., 44’ 619-6210

McClung, L. S. 1940. The use of dehydrated thioglycollate
medium in the enrichment of spore-forming anaerobic
bacteria. J. of Bact., 40, 645-648.

McClung, L. S. 1943. Use of dried tissue in beef heart
medium for anaerobic bacteria. J. of Bact., 46, 215-
216.

McClung, L. S. 1943b. Thioglycollate media for the culti-
vation of pathogenic clostridia. J. of Bact., 45, 58.

McClung, L. S., Heidenreich, P., and Toabe, R. 1946. A
medium for the Nagler plate reactions for the identi-
fication of certain clostridia. J. of Bact., 51, 751-
752.

McClung, L. S., and Toabe, R. 1947. The egg yolk plate
reaction for the presumptive diagnosis of Clostridium
gporogenes and certain Species of the gangrene and
botulinum groups. J. of Bact., 53, 139-147.

 

 

McIntosh, J., and Fildes, P. A. 1916. New apparatus for the
isolation and cultivation of anaerobic micro-organisms.
Lancet, 1, 768.

McLeod, J. W., and Gordon, J. 1923. The problem of intoler-
ance of oxygen by anaerobic bacteria. J. Path. Bact.,
26, 332-343.

Mueller, J. H., and Miller, P. A. 1941. A modification of
Rosenthal's chromium—sulfuric method for anaerobic
cultures. J. of Bact., 41, 301-303.

Nagler, F. P. 0. 1939. Observations on a reaction between
the lethal toxin of Clostridium welchii (Type A) and
human serum. Brit. J. Exp. Path., 20, 473-485.

Quastell, J. H., and Stephenson, M. 1926. Experiments on
"strict" anaerobes: I, The relationship of B. Sporo-
genes to oxygen. Biochem. J., 20, 1125-1137.

69

Reed, G. B., and Orr, J. H. 1943. Cultivation of anaerobes
and oxidation-reduction potentials. J. of Bact., 46,
475-4800

Robertson, M. 1916. Notes upon certain anaerobes isolated
from wounds. J. Path. Bact., 20, 327-348.

Rosenthal, L. 1937. "Chromium-sulfuric acid" method for
anaerobic cultures. J. of Bact., 34, 317-320.

Spray, R. S. 1930. An improved anaerobic culture dish. J.
Lab. and Clin. M8d., 16, 205-2060

Spray, R. S. 1936. Semisolid media for cultivation and
identification of Sporulating anaerobes. J. of Bact.,
32, 135-155.

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