STUDY OF THE TOXIN AND
CELATlN-SPLITTING ENZYME OF
CLOSTRIDIUM TETANI IN
RELATION TO POPULATION

Thesis for the Degree of M. S.
Iva L. Ferguson

1936

THESE

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Tthis

 

 

STUDY OF THE TOXIN AND GELATIN-SPLITTING ENZYME
OF CLOSTRIDIUM TETANI IN RELATION TO POPULATION

STUDY OF THE TOXIN AND GELATIN-SPLITTING ENZYME
OF CLOSTRIDIUM TETANI IN RELATION TO POPULATION

THESIS

Submitted to the faculty of Michigan State College
in partial fulfillment of the requirements for the

degree of Master of Science.

BY
Iva L. Ferguson
7

December 1936

TABLE OF CONTENTS

Introduction ———————— ° ............ 1
Methods:
Anaerobic Method ---------------- 5
Preparation of Seed Culture ---------- 5
Preparation and Inoculation of Broth ----- 4
Sampling and Order of Testing --------- 5
Estimation of Bacterial Population ------ 5
pH Determination --------------- 6
Gelatin-Splitting Enzyme Determination - — - - 6
Estimation of Toxin Potency ---------- 8
Results ---------------------- 9
Comparison of Individual Experiments ----- 10
Comparison of Similar Phenomena ------- 32
Discussion ____________________ 47
Summary and Conclusions -------------- 52
Bibliography -------------------- 54
Appendix --------------------- 56

lfibiJU

ACKNOWLEDGMENT

I wish to express my appreciation to Dr. C. C. Young
of the Michigan Department of Health for making this work
possible, and to Dr. G. D. Cummings for his generous

guidance and criticism of this problem.

INTRODUCTION

A study of the many methods of tetanus toxin production
leads one to believe that these methods were developed
empirically, without a definite aim of discovering the
principles of toxin formation. A comparison of the methods
employed by producers of tetanus toxin reveals a wide variation
in the medium used, in the time of incubation, and in the
bacteriological technique. The potencies obtained by the
different producers are nearly as varied as the methods
employed. Most workers have been content to work toward the
regular production of a toxin of uniform potency, with little
interest in the mechanism of the production (1, 2, 5, 4, 5).

The literature contains little on the theories of toxin
production. It has been customary to accept one of two theories
developed in the early days of bacteriology. These theories
are:

'(l) Toxin is formed in the bacterial cell and liberated
through the cell wall during the life of the cell or at its
death’on autolysis.

(2) Toxin is a substance formed in the medium surround—
ing the bacterial cell, or it reaches its final form in the
surrounding medium.

The most recent publications were by Palitzsch and
Valbum (6) and Walbum and Reymann (7) who favor the theory
that exotoxins are substances formed, or reach final form,

in the medium surrounding the bacterial cell.

This study was initiated as a preliminary effort to
gather fundamental data on the mechanism of toxin production.
The sound approach appeared to be the accurate measurement
of as many as possible of the phenomena occurring during the
growth of Clostridium tetanus in ordinary veal infusion broth.
‘To this end an attempt was made to measure, each day, the pH
of the medium, the potency of the toxin, the activity of the
gelatin-splitting enzyme, and the vegetative and spore cell
population by count.

1.

METHODS
Anaerobic method;

A modified Fildes jar (8) was used to obtain anaerobic
conditions (Fig. l and 2). The technique for the operation
of the jar was as follows: Hydrogen gas was supplied to the
Jar from a hydrochloric acid-zinc generator. A fixed
volume of gas was passed slowly into the jar with the
inlet and outlet valves open. Both valves were then closed.
The jar, and a 50 ohm resistance unit were connected in
series with a 110 volt light circuit for eight minutes.

The circuit was then broken and the inlet valve opened.
Additional hydrogen was passed into the jar to re-establish
atmospheric pressure. The Jar was allowed to cool an
additional five minutes and hydrogen was again added to
atmospheric pressure. The jar was then placed in the
incubator at 57.5°C.

Preparation of the seed culture:

A culture of Cl. tetani (serological Type III,

Pease) which had been maintained in 0.5% veal infusion
semi-solid agar (Appendix I) for six months was used for
the seed culture.

The agar tube was placed in boiling water until the
agar was melted. The contents of the tube were pipetted
into a tube containing 0.5% dextrose veal infusion broth
(Appendix 4) from which the air had been previously
removed by boiling. The broth tube was incubated
aerobically at 57.500. for 24 hours. Five tenths of a
cubic centimeter of this broth culture was pipetted to the

 

 

 

 

 

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bottom of a peptic digest agar slant (Appendix 5) and
incubated anaerobically for 48 hours at 57.500. At the

end of this time growth was visible at the top of the slant.*
A loopful of the growth from the top of the slant was

placed in a second heated and cooled dextrose broth tube.

This broth tube was incubated aerobically at 37.50C. for

24 hours. The toxin broth used in each experiment was
inoculated from a seed tube prepared in this fashion.

The initial seed tube was stored aerobically at
57.50C. between each batch of toxin broth and was used
for the preparation of subsequent seed tubes by the
above technique.

3. Preparation and inoculation of tetanus toxin broth;

Five liters of tetanus toxin broth (Appendix 7) were
prepared for each experiment. The broth was dispensed in
500 cubic centimeter amounts in sixteeneounce French

square bottles, autoclaved for 25 minutes at 121°C., and

cooled rapidly to room temperature in running water.
Fifteen.cubic centimeters of sterile 20% dextrose solution
were added to each bottle of broth following cooling.
Sixteen bottles were inoculated with one cubic centimeter

of the seed culture and incubated aerobically at 37.500.

* All motile strains of Cl. tetanus will grow over the
entire surface of a peptic digest agar slant when incubated
anaerobically and will rapidly outdistance contaminating
organisms which may be present. This characteristic allows

easy purification of any motile strain of Cl. tetanus.

4.

5.

Preliminary experiments indicated that more
accurate results could be obtained by using single
bottles for each day rather than one large bottle for
the entire test period. It was found that tests carried
out on a large volume of broth were inaccurate because
of continuous reduction of volume and because of
frequent contamination.

Sampling and order Qf_testing;

A 50 cubic centimeter sample of broth was withdrawn

 

from one bottle daily for sixteen days for testing. As
each sample was taken the bottle was discarded.

Daily tests were carried out in a constant order
and time following removal of the sample. Samples were
removed at eight o'clock each morning. Bacterial counts
were made immediately after the sample was taken. The
balance of the sample was kept at room temperature for
four hours during which time enzyme tests from the
previous day were completed. The sample was filtered
through a sterile Seitz filter and a colorimetric pH
determination was made. The enzyme test was next
carried out on the filtrate and closely followed by the
toxin potency test.

Estimation of bacterial population;

Daily counts of the vegetative and spore forms

present were made in a Petroff-Hauser counting chamber.

Specimens were diluted with physiological saline

(Appendix 8) according to the turbidity of the sample.
The maximum dilution factor used was 1:10.

Vegetative and spore forms were counted separately
under 011 immersion lens (720 diameters) with bright
field illumination.

The number of organisms in eighty small squares
were enumerated and counts per cubic centimeter were

calculated according to the following formula:

Number of organisms; dilution; 20000 X 1000 __ Organisms
Number of smallest squares counted ‘~ per cubic

centimeter

 

Three daily counts were made and averaged. Bacterial
debris present was recorded qualitatively as not present,
moderate or marked.

6. pgggetermination;

Daily measurements of hydrogen ion concentration

were made colorimetrically with buffer standards.
7. Gelatin-splitting enzyme determination;

Seven rows of sterile 5%)(5/8" culture tubes were

 

placed in a rack in a 37°C. water bath. The number of
tubes in each row varied from five to eight depending
upon the number of dilutions of filtrate measured.

The first row was labeled pH 4, the second pH 5 and so
on until pH 10 was reached. In the first tube of each
row was placed the minimum volume of broth filtrate, in
the second row an increased volume, and so on until the
last row contained the maximum amount. The volumes of
filtrate ranged from 0.01 cubic centimeter to 1.5 cubic

centimeters. One cubic centimeter of gelatin-buffer

mixture (Appendix 9) was added to each tube. Each tube
was made up to 5 cubic centimeters with sterile distilled
water.

Each pH level was controlled by a tube containing
one cubic centimeter of gelatin-buffer mixture plus two
cubic centimeters of sterile distilled water.

All tubes were thoroughly shaken and incubated for
eighteen hours at 57°C. in a constant temperature water
bath.

At the end of the incubation period one drop of
phenol red indicator (Appendix 12) was added to each
tube, including the controls. Tenth normal sodium
hydroxide or tenth normal hydrochloric acid was used to
adjust each tube to pH 7.0 (faint pink to colorless).
Each tube was made up to four cubic centimeters with
sterile distilled water and all tubes were placed in a
lO-lSOC. icebox for thirty minutes. The degree of
liquefaction of gelatin was recorded as follows:

: Liquefaction limited to meniscus of gelatin.

1: Up to 50% liquefaction.

‘F 50-100% liquefaction.

++ Complete liquefaction.

For the purpose of interpreting results the amount of
liquefaction was expressed in units. The number of units
in each tube was expressed as the reciprocal of the

volume of filtrate.

8. Estimation of toxin potency;
The potency of the toxin present in the filtrates

was measured in twenty—gram mice. The number of dilutions
made on each daily filtrate was governed so as to determine
the maximum number of mice minimum lethal doses*Present
per cubic centimeter of filtrate. All dilutions of the
filtrates were made in physiological saline (Appendix 8)
according to a standard scheme of dilutions. One cubic
centimeter portions of each dilution of filtrate were
injected subcutaneously over the abdomen. Dilution
levels for each experiment ranged as follows: undiluted,
1:5, 1:50, 1:100, 1:500, 1:1000, 1:5000, 1:5000, 1:7000,
1:10000, 1:12000, 1:15000, 1:20000, 1:25000, 1:50000,
1:55000, 1:40000, 1:45000, 1:50000, 1:55000, 1:60000.

No less than five dilutions of filtrate were tested on
any one day. Animals were observed daily for symptoms

of tetanus until six days had elapsed, at which time all

living mice were destroyed.

* For the purpose of this study a mouse minimum lethal
dose was defined as the smallest amount of toxin which would
kill a twenty-gram mouse within six days with typical symptoms

of tetanus.

RESULTS

Eight experiments were carried out but the results
from only four were presented in this report. The first
four experiments were performed more to standardize the
technique and to eliminate experimental error than to
yield data for study.

For the sake of clarity, data are presented in two
sections: the first, summarizing the findings in each
experiment; the second, comparing similar phenomena
observed in each experiment.

Early experiments showed that results obtained from
the samples withdrawn each day from a single flask contain-
ing 1800 cubic centimeters of broth were comparable in pH
and toxin potency to samples withdrawn from individual
French square bottles over the test period. In two of the
four experiments reported here, daily pH measurements were
made on a single volume of 1800 cubic centimeters of
medium for the purpose of controlling possible pH variations
in individual French square bottles. In all cases pH values

were comparable.

COMPARISON OF INDIVIDUAL EXPERIMENTS

EXPERIMENT I

Table I

A Comparison of the Vegetative and Spore Population, the

Hydrogen Ion Concentration, the Toxin and the Gelatin,

Splitting Enzyme, and the Bacterial Debris of a Strain of

Cl. Tetani Growing in.Dextrose Veal Infusion Broth at

57.500. for 14 Days.

10

 

 

By Day
Vegetativ pore Cell Minimum Enzyme*
HDay Cell Count Count pH’ Lethal Units . Debris
Millions ‘Millions Doses per c.c.
per c.c. per c.c. of Toxin Measured
per c.c. at pH 7.0
1 108 - 7.4 50 - Not
2 71 15 7.4 1000 5 recorded
5 108 65 7.5 5000 5 "
4 145 60 7.6 6000 6 fl
5 92 40 7.6 11000 20 "
6 61 45 7.4 15000 20 "
7 56 55 7.6 15000 20 "
8 25 22 7.7 18000 5 fl
9 52 55 7.7 20000 20 n
10 18 72 7.7 25000 5 "
11 18 52 7.7 29000 20 Murked
12 6 25 7.7 29000 20 "
15 5 19 7.8 50000 20 n
14 6 12 7.4 50000 20 "

 

 

 

 

 

 

 

 

 

* This table and the tables in following experiments
includes the number of units of gelatin-splitting enzyme meas-
ured at pH 7.0 since this pH reaction closely approximated the
reaction of the medium during incubation. The data on other pH

levels is summarized in Table II.

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The vegetative cell population reached a maximum of 145
million cells per cubic centimeter on the fourth day. There-
after, the count decreased rapidly to reach a constant level
at the twelfth day.

The spore cell population rose to 65 million cells per
cubic centimeter on the third day, dropped gradually to 22
million cells by the eighth day and rose again to 72 million
cells on the tenth day. Thereafter, the spore population
diminished gradually to 12 million cells on the fourteenth
day. The vegetative cell population reached its maximum one
day later than the spore cell population reached its first
peak.

The pH rose from 7.4 to 7.6 in the first four days,
returned to 7.4 on the sixth day and gradually rose to a
maximum of 7.8 on the thirteenth day. The pH reaction on the
fourteenth day was again 7.4. There appeared to be some corre-
lation between the peak of the vegetative cell population
and the rise in pH in the early stages of incubation. After
the sixth day, however, the pH rose as the vegetative cell
population diminished.

The potency of the toxin increased gradually to a
maximum of 50,000 minimum lethal doses on the thirteenth day.
. Exact end points were obtained on the fifth, eighth, eleventh
and twelfth days only, so that the data can only be interpreted
as showing the general trend of toxin production without

reference to peak amounts.

It is apparent, however, that.the probable peak in potency
was reached about nine days after the peak in vegetative cell
population. The high toxin potency appeared to correlate
well with high pH reaction in the late stages of incubation.
The amount of gelatin-splitting enzyme measured at pH
7.0 rose to 20 units by the fifth day, thus coinciding with
the increase in the vegetative and spore cell populations
and the pH reaction. Thereafter, the amount of enzyme varied
between 20 and 5 units and showed no correlation with any of

the other phenomena observed.

14

Table 2
The Influence of Hydrogen Ion Concentration on the Activity
of the Gelatin-Splitting Enzyme of a Strain of Cl. Tetani
Growing in Dextrose Veal Infusion Broth at 57.5°C. for 14 Days.

 

 

By Day
Day » pH 4 pH 5 pH 6 pH 7 pH 8 1 pH 9 pH 10
l — - - - 2 2O 2O
2 - - - 5 2O 20 20
5 - - - 5 2O 20 20
4 l 2 2 6 20 20 20
5 2 5 5 2O 20 20 20
6 2 6 5 20 2O 20 20
7 2O 6 20 2O 20 2O 20
8 l 1 1 5 6 20 20
9 6 20 6 2O 2O 2O 20
10 1 1 . 5 5 20 20 20
ll 20 2O 2O 2O 20 2O 20
12 20 20 20 20 2O 20 20
15 20 20 20 20 2O 20 2O
14 5 20 2O 20 20 20 20
15

 

 

 

 

 

 

 

 

 

 

The influence of the hydrogen ion concentration upon
enzyme activity was marked. The enzyme was completely
inactivated at pH 4, 5, and 6 for the first three days.
Thereafter, the number of units observed varied from 1 to 20
sporadically, reading the highest level of activity in the
late stages of incubation. At pH 7.0 the enzyme was inactivated
only on the first day. Thereafter, a level of 20 units was
reached by the fifth day and, with two exceptions (eighth and

tenth days), was maintained.Enzyme activity was greatest at

pH levels 8, 9, and 10 with 20 units measurable by the
second day of incubation. In general, a high pH reaction
appeared to be much more favorable for enzyme activity

than a low pH reaction.

15

EXPERIMENT 2

Table 5

Chart 2

A comparison of the Vegetative and Spore Population, the

Hydrogen Ion Concentration, the Toxin and the Gelatin-

Splitting Enzyme, and the Bacterial Debris of a Strain of

’Cl. Tetani Growing in Dextrose Veal Infusion Broth at

57.5°C. for 16 Days.

By Day

16

 

 

 

VegetativaSpore Cell . Minimum Enzyme
Day Cell Count Count pH Lethal Units Debris
Millions Millions Doses per c.c.
per c.c. per c.c. of Toxin I Measured
per c.c. at pH 7.0

1 52 1 7.2 1000 50 -

2 151 45 7.7 5000 - -

5 145 40 7.7 10000 2 —
4 102 45 7.5 14000 20 -

5 77 52 7.5 16000 20 Moderate
6 5O 55 7.5 18000 5 fl

7 5O 25 7.2 18000 5 fl

8 51 27 7.1 40000 6 fl

9 15 11 7.5 40000 2 n
10 52 20 7.2 40000 2 Marked
11 17 14 7.7 40000 5 "
l2 5 10 7.7 15000 5 "
l5 5 27 7.7 50000 6 n
14 9 19. 7.5 50000 2 "
15 5 16 7.7 45000 6 "
16 5 29 7.6 50000 2 "

 

 

 

 

 

 

 

 

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The vegetative cell population reached a maximum of 151
million cells per cubic centimeter on the second day. There-
after, the population decreased rapidly to reach a constant
level on the twelfth day. The peak population compared closely
with the peak population in Experiment I.

The spore cell population rose to 45 million cells per
cubic centimeter on the second day and maintained this level
at the fourth day. Thereafter, the population diminished
gradually to 16 million cells on the fifteenth day. The
vegetative and spore cell population reached the maximum level
on the same day.

The pH rose from 7.2 to 7.7 on the second day, dropped
gradually to 7.1 by the eighth day, and rose again to 7.7 on
the tenth day. Thereafter, it fluctuated between 7.7 and 7.5
until the sixteenth day when the pH was 7.6. The peak
vegetative cell population coincided with the first peak in
pH reaction, and the decline in population was paralleled by
a decline in pH reaction.

The potency of the toxin increased gradually to a
maximum of 40000 minimum lethal doses on the eighth day and
remained at this level until the eleventh day. The potency
dropped sharply to 15000 minimum lethal doses on the twelfth
day, then rose to a new level of 45000 minimum lethal doses
on the fifteenth day. The potency on the sixteenth day was
50000 minimum lethal doses. The sudden drop in potency after
the eleventh day coincided with the second peak in pH reaction

19

and the subsequent rise in potency coincided with a drop
in pH reaction.

The amount of gelatin-splitting enzyme measured at pH
7.0 was 50 units per cubic centimeter at the end of the first
24 hours. There was no measurable enzyme on the second day.
Twenty enzyme units per cubic centimeter were present on the
fourth day. Thereafter, the number of units fluctuated
between 2 and 6 until the end of the test period.. There
appeared to be a close correlation between the rapid rise
in the vegetative cell population and the amount of gelatin-
splitting enzyme present in the initial stages of incubation.
The sudden drop in enzyme content on the second day was

accompanied by a sharp rise in pH reaction.

Table 4
The Influence of Hydrogen Ion Concentration on the Activity
of the delatin-Splitting Enzyme of a Strain of Cl. Tetani‘
Growing in Dextrose Veal Infusion Broth at 37.5°C. for 16 Days.

 

 

By Day
ay pH 4 pH 5 pH 6 [ pH 7 pH 8 pH 9 pH 10
1 - - - 50 50 50 100
2 - - - - - 25 100
3 - - - 2 100 100 100
4 100 33 100 20 100 100 100
5 100 25 100 20 100 100 100
6 100 100 2 3 100 100 100
7 - - - 3 100 100 ' 100
8 - - - 6 100 100 100
9 - - — 2 6 100 100
10 v- - - 2 6 50 100
11 - — 2 3 100 100 100
12 - - 3 3 20 100 100
13 - - 3 6 20 100 100
14 - - 2 2 3 6 100
15 — - 3 6 20- 100 100
16 - - 2 2 3 100 100

 

 

 

 

 

 

 

 

 

 

The influence of the hydrogen ion concentration.upon
enzyme activity was marked. The enzyme was completely
inactivated at pH 4.0, 5.0, and 6.0 for the first three days.
On the fourth, fifth and sixth days there was marked enzyme
activity with a maximum of 100 units measurable. No enzyme
was measurable at pH 4.0 and 5.0 from the seventh through
the sixteenth day, while from 2 to 3 units were observed

at pH 6.0 from the eleventh through the sixteenth day.

20

21

At pH 7.0 the enzyme activity was of a low order
ranging from 2 to 50 units over the test period.

Enzyme activity was greatest at pH levels 8.0, 9.0, and
10.0 with 100 units the most frequent amount present at any
measurement.

In general, a high pH reaction appeared to be much

more favorable for enzyme activity than a low pH reaction.

EXPERIMENT 5

Table 5

Chart 3

A Comparison of the Vegetative and Spore Population, the

Hydrogen Ion Concentration, the Toxin and the Gelatin-

Splitting Enzyme, and the Bacterial Debris of a Strain of

‘Cl.lTetan1 Growing in Dextrose Veal Infusion Broth at

37.5°C. for 16 Days.
By Day

22

 

VegetativJSpore Cell

 

. Minimum Enzyme
Day Cell Count Count pH Lethal Units Debris
Millions Millions Doses per c.c.
per c.c. per c.c. of Toxin. Measured
per c.c.' at pH 7.0

l 38 1 7.3 1000 - -

2 56 21 7.3 5000 - -

3 83 28 7.2 7000 0.6 -

4 75 54 7.3 10000 1 -

5 51 16 7.2 20000 1 -

6 30 41 7.3 25000 3 Moderate

7, 19 29 7.3 25000 3 fl

8 21 23 7.2 25000 3 fl

9 13 28 7.3 30000 2 "
10 10 29 7.6 30000 2 "
ll 5 9 7.6 30000 1 Marked
12 ll 17 7.3 25000 1 n
13 5 25 7.4 40000 1 "
14 6 15 7.3 20000 1 "
15 5 20 7.7 25000 1 fl

6 3 15 8.0 25000 2 "

 

 

 

 

 

 

 

 

 

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24

The vegetative cell population reached a maximum of
85 million cells per cubic centimeter on the third day.
Thereafter, the population decreased rapidly to 3 million
cells on the sixteenth day. The cell population in this
experiment was much lower than in the two previous experiments,
but the peak was reached at approximately the same time.

The spore cell population rose to 54 million cells per
cubic centimeter on the fourth day. Thereafter, it decreased
gradually to 15 million cells on the sixteenth day.

The pH reaction rose to 7.6 on the tenth day, dropped
to 7.3 on the twelfth day, and reached a maximum of pH 8.0
on the sixteenth day. The pH remained more or less constant for
the first nine days. Thereafter,there appears to be some
correlation between the rise in pH reaction and decrease in
the vegetative and spore cell populations. The pH reaction
in this experiment and in Experiment 4 was determined
immediately after filtration each day. It was discovered that,
if the filtrate was allowed to stand in a flask exposing a
large surface area of the filtrate, the pH reaction would
rise to about 7.7 within two hours. This fact probably
accounts for the pH variations in Experiments I and 2 in the
early stages of incubation since the pH reactions were
determined after the filtrates had stood in 250 cubic
centimeter flasks for varying periods of time.

The potency of the toxin increased gradually to a maximum
of 40000 minimum lethal doses on the thirteenth day. Exact

end points were obtained each day in this experiment. An

25

increase in potency of the toxin tended to occur from one
to two days after a drop in pH reaction.

The amount of gelatin-splitting enzyme measured at
pH 7.0 rose to a maximum of 3 units by the sixth day, dropped
to one unit on the eleventh day, and increased to 2 units on
the sixteenth day. After the sixth day, there appeared to be
a correlation between decreased enzyme activity, low pH
reaction, vegetative and spore cell populations, and increased

toxin potency.

Table 6
The Influence of Hydrogen Ion Concentration on the Activity
of the GelatinPSplitting enzyme of a Strain of Cl. Tetani

26

Growing in Dextrose Veal Infusion Broth at 37.5°C. for 16 Days.

 

 

 

 

 

 

 

 

By Day
1
Day pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10
1 - - - - 3 3 100
2 - - - - 2 1 100
3 - - 1 0.6 2 2 100
4 - - 0.6 l 2 2 100
5 - 0.6 0.6 l 2 3 100
6 - 0.6 l 3 3 20 100
7 - 1 1 3 3 100 100
8 - 0.6 l 3 3 20 100
9 - 0.6 1 2 3 3 100
10 - 0.6 1 2 3 3 100
11 - 0.6 1 1 2 ‘2 100
12 - 0.6 1 1 1 2 100
13 - 0.6 1 1 1 1 100
14 - 0.6 l 1 2 3 100
15 - 1 1 l 2 3 100
16 0.6 1 2 2 2 3 100

 

 

 

 

No gelatinpsplitting enzyme could be measured at pH 4.0
until the sixteenth day. The enzyme was inactivated at pH
5.0 for the first four days, and at pH 6.0 for the first
two days. One to three units of enzyme per cubic centimeter
were measured at pH 8.0. At pH 9.0, 100 enzyme units were
measurable on the seventh day. Thereafter, the number of
units present dropped to three on the sixteenth day. One
hundred enzyme units were measured every day for sixteen
days at pH 10.0.

In general, a high pH reaction appeared to be more

favorable for enzyme activity than a low pH reaction.

EXPERIMENT 4

Table 7

Chart 4

A Comparison of the Vegetative and Spore Population, the

Hydrogen Ion Concentration, the Toxin and the Gelatin-

Splitting Enzyme, and the Bacterial Debris of a Strain of

.Cl.”Tetani Growing in Dextrose Veal Infusion Broth at

57.5°C. for 16 Days.

27

 

 

 

 

 

 

 

 

 

By Day
Vegetativespore Cell Minimum Enzyme
Day Cell Count Count ‘ pH Lethal Units Debris
Millions Millions Doses per c.c.
per c.c. per c.c. of Toxin Measured
per c.c. at pH 7.0I

1 68 5 7.3 1000 0.6 -

2 105 21 7.1 10000 2 -

3 83 32 7.1 10000 20 -

4 101 33 7.1 15000 20 —

5 40 43 7.3 25000 100 Moderate

6 30 35 7.1 25000 20 n

7 17 20 7.3 50000 20 "

8 20 19 7.1 40000 100 "

9 23 30 7.3 30000 100 V
10 18 21 7.3 30000 20 Marked
-ll 16 17 7.4 45000 20 "
12 10 16 7.4 30000 20 V
13 10 21 7.6 30000 3 "
14 11 15 7.6 30000 100 "
l5 7 18 7.7 30000 20 "
16 8 18 7.7 30000 20 "

 

 

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29

The vegetative cell population reached a maximum of 105
million cells per cubic centimeter on the second day, dropped
to 83 million on the third day, and rose to 101 million cells
per cubic centimeter on the fourth day. Thereafter, the
count decreased rapidly to 7 million cells on the fifteenth
day and 8 million cells on the sixteenth day.

The spore cell population reached a maximum of 43 million
cells per cubic centimeter on the fifth day. The count
gradually diminished to 15 million cells on the fourteenth
day, rising to 18 million cells on the fifteenth and sixteenth
days. The spore cell population reached its peak three days
after the vegetative cell population.

The pH varied between 7.1 and 7.3 for the first ten days,
rising to a maximum of pH 7.7 on the fifteenth day.

The potency of the toxin increased gradually to a maximum
of 50000 minimum lethal doses on the seventh day, decreased
to 30000 minimum lethal doses on the ninth day and reached
another peak of 45000 minimum lethal doses on the eleventh
day. End points were obtained for each day.

The amount of gelatin-splitting enzyme measured at pH 7.0
rose to 100 units on the fifth day. Thereafter, the number of
units varied between 3 and 100. As in Experiment 3, there was

a correlation between low enzyme activity, low pH, low vegetative

cell and spore cell counts, and increased potency after the

first half of the test period.

Table 8
The Influence of Hydrogen Ion.Concentration on the Activity
of the GelatinPSplitting Enzyme of a Strain of Cl. Tetani.
Growing in Dextrose Veal Infusion Broth at 37.5 C. for 16 Days.

 

 

 

 

 

 

 

By Day
*Day pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10
l - - - 0.6 0.6 l 100
2 - - 1 2 3 2 100
3 - 0.6 2 20 3 100 100
4 - 2 3 20 20 100 100
5 l 2 3 100 100 100 100
6 l 2 3 20 20 100 100
7 1 3 20 20 100 100 100
8 l 3 3 100 20 100 100
9 100 20 3 100 20 100 100
10 l l 3 20 20 100 100
11 100 3 3 20 100 100 100
12 l 3 20 2O 3 100 100
13 0.6 1 2 3 3 20 100
14 20 100 20 100 100 100 100
15 100 3 3 20 100 100 100
16 100 3 20 20 100 100 100

 

 

 

 

 

The gelatinrsplitting enzyme was completely inactivated
until the fifth day at pH 4.0, the third day at pH 5.0, and
the second day at pH 6.0. Thereafter, the number of units
varied between 0.6 and 100 units per cubic centimeter. The
increased enzyme activity in.this experiment possibly was
due to the fact that the tubes were neutralized in the water
bath. When mixtures of this kind are kept at a temperature
slightly above room temperature for a short time, the gelatin-
splitting process may be initiated or furthered (7).

Temperature of the room caused the mixtures to solidify

51

partially before neutralization of all the tubes could be
completed. At pH 7.0, 100 units were measured on the fifth
day. Thereafter, the number of units present varied from 3
to 100. Similar enzyme behavior was observed at pH 8.0.
One hundred enzyme units were consistently present each day
at pH 9.0 and 10.0.

In general, a high pH reaction appeared to be more

favorable for enzyme activity than a low pH reaction.

 

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34

The curves showing the trends of vegetative cell
population were nearly identical. Each curve showed a sharp
increase in cell population in the early stages of incubation.
The population peaks were respectively at four, two, three and
two days. At these peaks there were respectively 145 million,
151 million, 85 million and 105 million vegetative cells per
cubic centimeter.

Each curve showed a rapid decline in cell population
for four to five days after the respective peaks were reached,
and each curve indicated a constant level of population after
the seventh to ninth days. At fourteen days, the curves had
flattened'out and the populations were respectively 6 million,
9 million, 6 million and 11 million vegetative cells per
cubic centimeter.

From these curves it is evident that the cell population
rises and diminishes rapidly followed by a gradual diminution

in number until a relatively constant population is reached.

 

 

 

 

 

 

 

 

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37

The spore cell population curves were similar in
These curves were typified by

Experiments 2, 5, and 4.
early peaks at two, four, and five days respectively. At

these peaks there were respectively 45 million, 54 million,
and 45 million cells per cubic centimeter. The curve in

Experiment 1 showed a sharp increase to a peak of 63 million
cells on the thind day, followed by a gradual decrease until

the tenth day when the count rose to 72 million cells per

Thereafter, the count diminished rapidly.

cubic centimeter.
At fourteen days, the spore cell populations had diminished

to 12 million, 19 million, 15 million, and 15 million cells

per cubic centimeter respectively.
It was evident from these curves that the spore cell

population rose rapidly to an early peak and then gradually

declined.

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40

The curves showing the trends in pH varied considerably.
The curves in Experiments 5 and 4 showed the same trend for
the first eight days. Experiment 3 showed sporadic rise and fall
in pH after the eighth day and Experiment 4 a gradual increase
in pH to a maximum of 7.7. The peaks in Experiments I and 2
for the first eight days were probably due to the fact that
the filtrates stood in 250 cubic centimeter flasks for vary-
ing periods of time before measurement.

From these curves it appeared that the pH remained at a
level of 7.1 to 7.3 for the first eight days followed by
sporadic rise and fall in pH during the last half of the
test period. In every experiment but Experiment I, pH values
at the end of the test period were high. In Experiment I,
the pH reaction on the thirteenth day was 7.8, and on the

fourteenth day it was 7.4.

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43

The curves showing the trends of toxin potency for the
first six to eight days were nearly identical. Each curve
showed a gradual increase in potency. Experiment I continued
to show a gradual increase in potency up to 30000 minimum
lethal doses on the eleventh day. This potency persisted for
'the remainder of the test period. Experiments 8, 3, and 4
showed a marked rise and fall in potency in the second half
of the test period, reaching a maximum of 45000 minimum
lethal doses on the fifteenth day in Experiment 2, 40000
minimum lethal doses on the thirteenth day in Experiment
5, and 50000 minimum lethal doses on the seventh day in
Experiment 4. The potencies at fourteen days in the respective
experiments were 30000, 30000, 20000, and 50000 minimum lethal
doses.

It was evident from these curves that potencies increased
gradually for the first six to eight days, then rose and fell
sharply one or two times during the last half of the test
period.

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46

The curves showing the trends in gelatin-splitting
enzyme activity showed no correlation with one another.

Enzyme activity varied sporadically throughout the test

periods.
There appeared to be no correlation between the enzyme

results of one experiment as compared with another.

47

DISCUSSION

The purpose of this study was to observe daily certain
phenomena which occur in tetanus toxin production. It was
hoped that such data might pave the way to an understanding
of the factors which govern toxin production. Certain
assumptions as to the outcome of the various experiments
were unavoidable.

It was thought, for example, that the spore population
would increase in almost direct proportion to the decrease in
vegetative cell population, allowing for a certain amount of
autolysis. This was not the case and the influence of
autolytic phenomena therefore assumed an important position
in theoretical considerations.

It was thought, also, that the pH reaction would show some
constant type of behavior. Instead, each experiment showed
a wide variation in pH values over the test periods. In no
instance did the pH value on the final day of each experiment
give any indication of the amount of variation which had
taken place. In the routine production of toxin it is
customary to measure the pH of the toxin broth at the
beginning of the incubation period and at the time of harvest
‘ten to fourteen days later. If this had been the case in
these experiments, the initial and final pH values would
have been respectively 7.4 and 7.4, 7.2 and 7.6, 7.5 and
8.0, and 7.3 and 7.7. In every case but one these pH values

.appeared to indicate an increase over the period of incubation.

48

An inspection of the daily record, however, showed marked
fluctuation in pH values between the initial and final days
of the test period.

The assumption was that toxin production was a progressive
phenomenon indicated by steady increase in the number of lethal
doses until some constant level was reached. This assumption
was borne out in Experiment I. In the other experiments the
progression was orderly for the first six or seven days.
Thereafter, the number of lethal doses to be expected on any
day was unpredictable. An excellent illustration of this
behavior occurred in Experiment 2. The potency on the eleventh
day was 40000 minimum lethal doses, on the twelfth day it was
15000 minimum lethal doses. By the fifteenth day, the potency
had risen to 45000 minimum lethal doses. The same phenomenon
was encountered in Experiment 4. This type of variation
emphasizes the difficulty in the selection of the proper day
for harvesting toxin. It also illustrates the fallacy of the
assumption that a fixed incubation period can be established
for routine toxin production. The results have indicated
that most of these assumptions were wrong.

Certain phenomena stand out clearly when one considers
the actual data collected. For example, the trend in bacterial
population was definitely constant. Vegetative cell and spore
populations increased and diminished rapidly until a period
of nearly constant population was reached. The accumulation

of debris occurred progressively. Moderate debris was observed

49

by the fifth or sixth day. Marked debris appeared on the
ninth to thirteenth day.

It was discovered at the beginning of Experiment 5 that
exposure of large surfaces of broth filtrate markedly increased
pH values. This was not true if the filtrate was stored in
test tubes. This factor was controlled in Experiments 5 and 4.
pH values varied within narrow limits for the first seven or
eight days. Thereafter, pH values varied erratically with
a tendancy toward the high levels. The first trend toward
high pH levels was accompanied by the appearance of marked
debris.

The production of toxin was progressive and rapid for the
first six or seven days. Thereafter, the toxin values
fluctuated between wide limits. The peak of toxin production
was reached five or six days after the peak of the vegetative
cell population and was preceded by the appearance of moderate
bacterial debris. The peak of toxin production in turn
preceded the rise in pH in three of the four experiments.

The amount of gelatin-splitting enzyme measured at pH 7.0
in the various experiments was not constant and did not
correlate with any of the other phenomena observed. The
enzyme exhibited a much increased activity when measured at
pH 8.0 to 10.0.

The data presented in this report lends support to the
theory that tetanus toxin is directly evolved by C1. tetani.
The rapidity of evolution of toxin appear: to weaken an

enzyme hypothesis. It is admitted, however, that the approach

50

to the enzyme phase of the problem had many shortcomings.

Only one enzyme was selected for measurement with no evidence
that this particular enzyme was at all associated with toxin
production. As Walbum has pointed out, a more accurate
approach might be made through the albumose—splitting enzymes.
The most that can be said is that the gelatin-splitting

enzyme appeared to play no part as a precursor to the formation
of toxin.

A broad consideration of the data presented does indicate,
however, that simple evolution of toxin by the vegetative cells
cannot be considered as the whole story, because it was
obvious that some toxin was being produced long after the cell
population had been markedly diminished.

The chain of events over an incubation period of fourteen
to sixteen days appears to divide into three fairly well
defined phases.

Phase I included the first five to six days of incubation.
Important indications of this phase were the peak in vegetative
and spore cell populations, the relatively constant pH, and
the orderly production of toxin. Phase 2 was marked by the
appearance of moderate debris, the productiOn of more toxin,
and a rise in the general pH level. Phase 3 was marked by
wide fluctuations in the toxin potency, high pH levels, and
marked debris. The gelatin-splitting enzyme did not appear
to be correlated with any of the phases.

Phase I approximates most closely the current theory of

exotoxin production. Phase 2 seems to be the result of an

autolytic phenomenon because of the continued production of

51

toxin in the face of low cell counts and considerable cell
debris. This toxin might be due either to direct liberation
of toxin by remaining bacterial cells or to the action of
some autolytic enzyme on naturally formed bacterial debris.
Phase 3 appears to represent a phase following toxin
production. No appreciable toxin is produced but the toxin
already present fluctuates in potency according to prevailing
conditions. Not the least interesting phenomenon is that
apparent inactivation and reactivation of the toxin. The
factors governing this behavior must be carefully studied
if the maximum amount of toxin is to be harvested in routine

production.

SUMMARY AND CONCLUSIONS
The vegetative and spore cell populations, pH reaction,
potency, activity of the gelatin-splitting enzyme, and the
presence of cell debris were observed over a period of

fourteen to sixteen days aerobic incubation at 57.5°C. The

results led to the following conclusions:
1. Toxin production appeared to be a three-phase phenomenon:

Phase I. A period of simple exotoxin production.

Phase 2. A period of toxin production apparently due
to autolytic phenomenon.

Phase 5. A period following toxin production during
which the toxin present varied in potency
according to prevailing conditions.

2. There was no correlation between vegetative and spore cell
populations, and toxin potency.

5. pH values varied within a narrow limit for the first seven
to eight days. Thereafter, considerable variation took
place with a tendency toward increased pH values as the

cell debris became marked.

eh

. The production of toxin was progressive and rapid for the
first six or seven days. Thereafter, the toxin values
fluctuated between wide limits.

5. The gelatin-splitting enzyme measured at pH 7.0 showed no

constant activity and did not correlate with any of the

other phenomena observed.

53

6. The factors governing the data gathered in these experiments
must be carefully studied in future experiments if maximum

toxin production is to be obtained.

54

BIBLIOGRAPHY
Wilcox, H. L. A Modification of the Hygienic Laboratory
Method for the Production of Tetanus Toxin. Jr. Bacty.
l, 555, 1916. ‘
Wilcox, H. L. The Effect of Peptone Upon the Production
'of Tetanus Toxin. Jr. Bacty. 6, 407, 1921.

Bertholet, A; Ramon, G; and Mlle. Amoureux. 0n the
Conditions of Media Which Influence the Production
of Tetanus Toxin. Comptes Rendus. 96, 52, 1927.

Condrea, P. New Method for the Preparation of a Tetanus
Toxin of High Titer. Comptes Rendus. 105, 1044, 1950.

Legroux, R. and Ramon, G. Production of Tetanus Toxin.
Comptes Rendus. 115, 861, 1955.

Palitzsch, Sven, and Walbum, L. E. On the Optimum
Concentration of Hydrogen Ions for the First Phase
of the Tryptic Decomposition of Gelatin. Comptes
Rendus. 9, 200, 1912.

Walbum, L. E. and Reymann, C. G. Nature of the Gelatin-
Melting Enzymes Formed by the Gas-Gangrene Bacteria.
Jr. Path and Bacty. 59, 669, 1954.

Cummings, G. D. A Modification of the Fildes Anaerobic
Jar. Am. Jr. Pub. Health. 22, 1952.

Clark, W. M. The Determination of Hydrogen Ions. 2nd
edition, p106, 1922.

55

10. Fildes, P. Isolation, Morphology, and Cultural
Reactions of B. Tetani. Brit. Jr. Exper. Path.
6, 65, 1925.

11. Society of American Bacteriologists. Manual of Methods
for Pure Culture Study of Bacteria. Leaflet 9. 4th
edition. June 1950.

56

APPENDIX
1. Veal Infusion Agar 0.5%. Semi-solid. 0.5% dextrose added.
(a) Formula:
Veal infusion broth (Appendix 2) 1000 mils.
Agar, Bacto 5 gms.
(b) Procedure:

Dissolve agar in broth in steam kettle with
constant stirring. When the agar is completely dissolved,
adjust reaction to pH 7.5 and boil for 10 minutes. Hold
for 2 hours at 60-80°C. Filter through cotton-gauze
filter. Add 5 grams of dextrose. Make up to volume
and dispense. Autoclave for 25 mdnutes at 15 lbs.
(121°C) pressure. Check reaction. Do not allow agar
to solidify before sterilization.

2. Veal Infusion Broth
(a) Formula:

Ground veal, fat free (455.6 gms) 1 lb.

Peptone, Bacto 10 gms.
Sodium Chloride C.p. 5 gms.
Distilled water 1000 mils

(b) Procedure:

Grind veal and infuse in coldroom over night. Skim
off fat and boil in steam kettle until meat is thoroughly
cooked. Filter infusion through gauze filter, drain
broth out of meat and cool filtrate in kettle until

57

remaining fat has solidified. Skim off fat and
make up to volume. Dissolve peptone and sodium
chloride in infusion, adjust to pH 7.4 and boil
for 10 minutes. Cool to allow sediment to settle,
filter through hard paper and make up to volume.
Dispense and autoclave 25 minutes at 15 lbs. (121°C)
pressure. Check reaction.
5. Veal Infusion Broth. Sugar-free.
(a) Formula: _
Veal infusion broth (Appendix 2) 1000 mils.
E. coli communior, 24 hour culture 2.5 mils
(b) Procedure:
Inoculate the sterile broth with the 24 hour
culture of B. coli communior (2.5 c.c. broth culture
per liter of broth). Incubate for 24 hours at 37.500.

Autoclave at 15 lbs. (121°C) pressure for 10 minutes
and adjust the reaction to pH 7.5. B011 10 minutes

in steam kettle and then cool to allow sediment to
settle. Filter through hard paper filter, then candle,
make up to volume, dispense and autoclave for 20 minutes

at 15 lbs. (121°C) pressure.

58

4. Veal Infusion Broth plus 0.5% Dextrose.

(a) Formula:

Ground veal, fat free (455.6 gms.) 1 1b.
Peptone, Proteose 10 gms.
Sodium Chloride C.p. 5 gms.
Sterile 20% dextrose solution 25 mils.
Distilled water 1000 mils.

(b) Procedure:
Mix ground veal and water and infuse in coldroom
over night. Infuse for 1 hour at 45°C. Raise to
100°C. and hold for 1 hour. Strain through gauze,
add peptone and sodium chloride and dissolve by‘
stirring. Make up to volume, adjust reaction to pH
7.4 and filter through hard paper, Dispense and
autoclave for 25 minutes at 15 lbs. (121°C) pressure.
Check reaction. Add sterile dextrose solution
aseptically to cooled medium.
5. Peptic Digest Agar (10)
(a) Formula:
Veal infusion agar 2%, pH 7.4 (Appendix 15) 500 mils.
Sterile peptic digest of sheep cells
(Appendix 6) 1-5 mils.
(b) Procedure:
Melt sterile agar and cool to 40°C. Add digest

aseptically and whirl to mix thoroughly. Dispense

59'

with sterile funnel or pour directly into sterile petri
dishes. Incubate 24 hours at 57.5°C. to check sterility.
6. Peptic Digest Solution (10)
(a) Formula:
5 Physiological saline 0.85 (Appendix 8) 150 mils.

Hydrochloric acid 10 normal 6 mils.
Defibrinated sheep cells 50 mils.
Pepsin, granular, Bacto 1 gm.

(b) Procedure:
Introduce ingredients in the order listed into a
250 c.c. Erlenmeyer flask and shake to dissolve. Place
in water bath at 55°C. for 24 hours, shaking occasionally
at the beginning of the time interval. Add 12 c.c. of
5N sodium hydroxide accurately prepared and adjust
reaction to pH 7.6 using more 5N sodium hydroxide if
necessary. To test, the samples are removed from the
bottle with a sterile pipette and diluted with distilled
water until color is light enough to distinguish the
change on the addition of a few drops of indicator.
Adjust mixture to pH 7-7.2 with N/l hydrochloric acid
and add 0.25% chloroform. Paper cap the bottle and use
in 2-5% amounts in media.
7. Tetanus Toxin Broth (1) (Modified)
(a) Formula:
Ground veal, fat free (455.6 gms) 1 1b.

60

Peptone, Witte 10 gms.
Sodium chloride C.p. 5 gms.
Sterile 20% dextrose solution 50 mils
Distilled water 1000 mils

(b) Procedure:
Mix ground veal and water and infuse in coldroom
2 hours. Infuse for 1 hour at 45°C. Raise to 100°C.
and hold for 1 hour. Strain through gauze, and cool
until fat has solidified. Skim off fat. Heat, add
peptone and sodium chloride and dissolve by stirring.
Make up to volume, adjust reaction to pH 7.5 and filter
through hard paper. Dispense and autoclave 25 minutes
at 15 lbs. (121°C) pressure. Check reaction. Add
sterile dextrose solution aseptically to cooled medium.
Inoculate as soon as cool.
8. Physiological Saline
(a) Formula:
Sodium chloride C.p. 0.85 gm.
Distilled water - up to 100 gms.
(b) Procedure:
Dissolve sodium chloride in distilled water and
stir to mix thoroughly. Adjust reaction to pH 7.0
using 0.1% potassium phosphate secondary solution for
titration and 1.0% potassium phosphate secondary solution
for adjustment. Dispense and autoclave for 20 minutes

at 15 lbs. (121°C) pressure. Check reaction.

 

61

9. Gelatin-Buffer Mixtures (7)
(a) Formula:
20% gelatin (Appendix 10) 5 gms.
Buffer solution (Apendix 11) 5 mils plus factor
10. 20% gelatin
(a) Formula:

Gelatin, Bacto 400 gms.
Distilled water 2000 gms. i
Thymol 1 gm.

(b) Procedure:

Heat distilled water and add slowly 400 grams
of gelatin. Make up to near the total volume with
distilled water. Adjust reaction to pH 7.0 with
N/l Sodium hydroxide. Make up to volume. Add
thymol dissolved in a little 95% alcohol.

11. Standard Buffer Mixtures (9)

pH 4 - 50 cc 11/5 KH Phthalate

0.4 cc M/5 NaOH

Dilute to 200 cc with distilled water.
pH 5 - 50 cc M/5 KH Phthalate

25.85 cc M/5 NaOH

Dilute to 200 cc with distilled water.
pH 6 - 50 cc M/5 KH2PO4

5.70 cc M/5 NaOH

Dilute to 200 cc with distilled water.

62

pH 7 - 50 cc M/5 KH2PO4

29.65 cc M/5 NaOH

Dilute to 200 cc with distilled water. *
pH 8 - 50 cc M/5 H5B05, M/5 KCl

5.97 cc M/5 NaOH

Dilute to 200 cc with distilled water.
pH 9 - 50 cc M/5 H5805, M/5 K01

21.50 cc M/5 NaOH

 

Dilute to 200 cc with distilled water
pH 10 - 50 cc M/5 H5805, M/5 KCl
45.90 cc m/s NaOH
Dilute to 200 cc with distilled water. I
These mixtures were overbalanced with N/l sodium
hydroxide or N/1 hydrochloric acid, so that when mixed with
gelatin, broth filtrate, and distilled water, the final pH
reaction would be as given above.
12. Phenol Red Indicator
Phenol red indicator 0.02 mg.
95% alcohol 100 mils
15. Veal Infusion Agar 2%
(a) Formula:
Veal infusion broth (Appendix 2) 1000 mils
Agar, Bacto 20 gms.
(b) Procedure:

Dissolve agar in broth in steam kettle stirring

63

constantly. When agar is completely dissolved, adjust
the reaction to pH 7.4 and boil for 10 minutes. Hold
for 2 hours at 60-80°C. Filter through cotton-gauze
filter. Make up to volume and dispense. Autoclave

25 minutes at 15 lbs. (121°C) pressure. Check reaction.
Do not allow agar to solidify before sterilization.

 

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