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THE INFLUENCE OF CATlflNS ON

AEROBIC SPORE FORMATION
IN A LIQUID MEDIUM

THESIS FOR THE DEGREE OF M. 8.

Claude S. Bryan

1932

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THE INFIUENCE OF CATIONS 0N AEROBIG SPORE FORMATION
IN A LIQUID MEDIUM.

THE INFLUENCE OF CATIONS 0N AEROBIC SPORE FORMATION
IN A LIQUID MEDIUM

A Dissertation submitted to the Graduate Faculty in

candidacy for the Degree of Master of Science.

by

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Claude Syggyan

September, 1932.

:n-nzez’e1

Introduction

History of cultures

Method

Results
'1. -Influence of menovalent salts
2. Influence of pH
3. Influence of di-valent salts
4. Influence of tri-valent salts
5. Influence of tetra-valent salts

Discussion

Conclusions

Literature cited

Acknowledgment . 2

{}4£§?C’

Introduction.

PhysiOIOgical studies as to the nature of spores
have received considerable attention since the organism
Bacillus anthracis was first isolated and demonstrated to
have the ability to form spores. The greatest amount of
research thus far has concerned itself with a study of the
activities and preperties of these spores.

The exact nature of the processes which take place
within the organism.immediately before the formation of
the spore are not very definite at the present time.
Schreiber (17) states that an organism.which has the in-
herent prOperty of forming spores may do so at any time;
in other words the number of rapid transfers without
sporulation does not affect its ability to form spores
later. Thus we can see that this ability is a definite
criterion of the spore forming organisms.-

In general a spore is considered as a condensed,
dehydrated protoplasmic mass consisting chiefly of
nuclear material. Investigators have analyzed the spores
chemically and report that they consist chiefly of
proteins. mellon and.Anderson (16) studied the protein
of the spore and the protein of the vegetative stage
tmmunologically and state that the two proteins are
different. To date the exact nature or process of spore
formation is not explained, but the moving pictures by
Bayne-Jones (1) do give us a visible picture of the
formation of the spore. These pictures indicate that the

-2-

actual process of spore formation is not a long-drawn
out process but a rapid one. It may require some tume
to establish the proper conditions but the physiological
act takes place quickly. It appears that granules are
formed within the bacterium and immediately before the
spore is formed these granules migrate to the Opposite
end from.that in.which the spore is formed.

Mbgoon (15) in some earlier work concludes that
spore formation is a normal process and the spore is a
result of the union of granules which are formed within
the organism. Thus we can readily see that the exact
process of spore formation is vague and not fully ex-
plained.

German investigators have done some work in studying
spore formation in bacteria, especially of §;.anthracis.
The views, as to the reason for spore formation, of these
early investigators are indicated by the two theories
advanced by Buckner (5) (6) and Turro (19). Buckner
states that the stimulus of the organism to form spores
cause when the nutrient material has become deficient
immediately surrounding the organism. Working with the
bacillus of anthrax he states that by the renewal of this
nutrient material, in the local area, before the organism
has reached a certain.point the organism can be held to
countless generations of only vegetative cells.

Turro presents the view that spore formation °£.§a

anthracis is due to the accumulation of products of

 

-3-

metabolic activity.

Schreiber presents a view, partially in accord with
both of the above, in which he gives the impression that
the deficiency of nutrient material can not be a direct
cause but can be considered as the inciting cause of spore
formation. He also demonstrated that oxygen of the air
is a Specific and necessary condition for spore formation
in the case of aerobic spore forming organisms.

The work which follows the above investigations is
limited more to the study of the reaction of the spore to
various physical and chemical factors. Work typical of
the above is on spores of the anaerobic organisms as the
clostridia group, since their importance in the food and
meat canning industry is indeed very great.

In.more recent work Williams (20) (21) shows that
various concentrations of peptone do exert slightly
different influences and he recommends using a one per
cent peptone solution in studying the ratio of spores_to

vegetative cells.

History of Cultures.

The fbur aerobic spore forming organisms used in this
study were Bacillus subtilis, Bacillus cereus, Bacillus
mesentericus and Bacillus megatheriumw The culture of
Bacillus gg£g§,was very kindly furnished by Doctor L. F.

Rettger of Yale University and was isolated by him.from

-4-

a hay infusion. The other three cultures were obtained
from.the stock collection of the Department ovaacteriology
at Michigan State College having been isolated earlier

from soil and hay infusions. Each culture was plated out
(by the loop dilution.method and transfers made from.well
isolated typical colonies. To insure pure cultures the
above transfers were identified according to Bergey's

manual of Determinative Bacteriology (a).

Method.

The study of spore formation necessitated using a
solid medium where the culture could be obtained in a
vegetative stage for subsequent inoculations into the
liquid medium containing various molalities of the salt
under study. It was also essential that a liquid medium
be used which of itself did not stimulate spore formation.
Preliminary work confirmed the "spore cycle" of spore
forming organisms, as determined by.Magoon (15). Thus
twenty-four hours were taken as the thus for these
organisms to go from the spore stage through the vegetative
stage and back into the spore stage again. Eighteen to
twenty hours incubation was taken as the time for the
organisms to go into the spore stage if the inoculum.was
in the vegetative stage. Application was therefore made
of the "spore cycle" in this study.

A variety of slanted mediums were inoculated with a

-5-

culture of each one of the four organisms; these cultures
had been transferred previously at several twelve hour
intervals to insure all vegetative growth. Observations
for spores were made at intervals by making spore stains
of the smears made from the various mediums. Two of the
mediums studied proved interesting and useful; dextrose
agar stflmulated each one of the four organisms to almost
100 per cent spore formation in about twenty hours, while
on the beef liver infusion agar introduced by Stafseth (18)
and further developed by Huddleson (13) the cultures
formed very few spores within the same time limit as the
dextrose agar. The liver infusion agar was therefore
adOpted to maintain the cultures used in this work. Various
liquid mediums were similarly studied including plain broth,
Dolloff's medium (8) Leifsons medium.(14) and Hotchkiss

medium (12) consisting of one per cent Bacto-peptone. Of
1 these the one per cent peptone medium used by Hotchkiss
in her work was adopted as it of itself did not stimulate
spore formation of the cultures studied within eighteen
hours. All mediums were sterilized by autoclaving for
.twenty minutes at fifteen pounds pressure. The solid
mediums were adjusted so final pH after autoclaving was
6.6. The pH of the distilled water used in.making the
one per cent peptone medium.was such that final pH of the
medium.was near 6.6 after sterilization.

All glassware used was of Pyrex type, cleaned by

soaking in cleaning solution overnight, wrinsed in tap

-5-

water followed by distilled water and sterilized in dry
heat at 180'0. for three hours.

The chloride salts, whose effect on spore formation
in the one per cent peptone medium.were studied, can be
classed for convenience into four groups according to
valence. The monovalent salts used were Nacl, K01, NHaCI,LiCI,
sodium lactate; the divalent salts were M3012, MnOlg 43.0,
Baclg, 00012 6H30, PbClg and NiClg; the tri-valent salts
were A1013, CeCla, Feels 6H20 and the tetra-valent salts
3n01.. All of the salts were Baker's analyzed products.

Stock solutions of a definite molality of these salts
were prepared in sterile distilled water and tested for
sterility. The desired amount of the salt solution was
added to five cubic centnmeters of two per cent peptone
medium.and made up to 10 c.c. with sterile distilled
water. Thus each tube contained 10 c.c. of one per cent
peptone containing a definite molality of the salt under
study.

In.making a determination, a very small amount of
the aerobic spore formers used were transferred with a
needle from the liver infusion agar slant into 10 c.c.
of one per cent peptone medium.and incubated for eighteen
to twenty hours at room temperature. One cubic centimeter
of each of these spore free cultures was added to a series
of tubes containing the desired molal concentration of
each salt, also to a tube containing 10 c.c. of one per

cent peptone which served as a control. These tubes were

-7-

then incubated at room.tsmperature for eighteen to

twenty hours after which the number of bacteria per cubic
centimeter and per cent spores present were determined by
using Breed's (5) (4) method for directluicrossopio counts.
Ordinary microscopic slides were marked off into one
square centimeter areas, using a diamond point. From.each
of the tubes containing concentrations of the salt under
study 0.01 c.c. amounts of the uniform suspensions were
placed on the slide and spread evenly over a one square
centimeter area. These were allowed to air-dry and then
stained by.AnJeszky's spore stain method (11) which gives
a red spore and a blue sporangium.

Hydrogen ion determinations were made electrometric-
ally, using a Leeds and Northrup potentismeter in con-
Junction with a saturated calamel cell and a quinhydrone
electrode.

Preliminary determinations were made with each salt
and each one of the four organisms to determine the range
of stimulation, if any, and the point of decrease due to
the toxicity of that molality of salt.

When this range was found "three molalities" were
selected to be used in the final study of the salt under
consideration. The three molalities selected were (a)
the one which gave maximum stimulation, (b) a molality
at the point between the maximum.viability and no growth
due to toxic effect of the salt and (c) a molality at a
point lower than that of the maximum.viability due to

-8-

insufficient stimulation caused by the low salt concentra-
tion. Determinations were made in all cases employing
these "three molalities" and a control. Not less than
four separate determinations were made on each series of
molalities of each salt, before centinuing to the next
salt. Each figure in the tables, therefore, represents
the average of not less than four determinations. The
separate determinations paralleled each other very closely

with no wide variations.

Results.
I. Influence of monovalent cations in combination with
chlorine.

For convenience the salts under study were taken up
in groups according to valence. The monovalent salts were
studied first. The "three molalities" were selected by
preliminary determinations. Four series of molalities
plus controls were prepared and each series inoculated
with one of the four organisms.

Table I gives the influence of NaOl exerted on the
viability and sporulation on each one of the four aerobic
spore-forming organisms under consideration. These data
show that Nacl in.mo1al concentration 0.25 gave maximum
stimulation for viability and also maximum stimulation
for spore formation. The point or molal concentration
below the maximum showed a stimulation over the control
and an influence that definitely marks the molal concentra

tion 0.25 as maximum. The molality above the maximum

-9-

definitely shows a decrease of stimulation due to the
approach of the point of toxicity of the Nacl. The pH
determinations were made electrometrically and come

within the range of 6.5 to 7.46.

2. Influence of pH

To determine the influence exerted by pH in.a medium
otherwise favorable to viability and spore formation the
following experiment was performed. One per cent peptone
medium.aontaining a molality of 0.25 of Nacl was pre-
pared and equal amounts were adjusted to various pH's.
The pH's to which the medium.was adjusted were 5.0, 5.5,
6.0, 6.5, 7.0 and 7.5 and-covered the pH range found in
the study of these salts. The data in Table 2 show that
there is a slight variation in a favorable medium.between
the four organisms studied at the same pH. It also shows
that each one of the organisms has a wide range of pH
before a noticeable effect is produced upon the formation
of spores. On this basis the pH of the medium, when
various molalities of salt were added, was considered
favorable if it came between the range of pH 5.0 and 7.5.

The influence of man is shown by the data in
Table 5. The pH's throughout were lower than in the case
of Nacl but still well in the favorable range for growth
and sporulation. Table 4 demonstrates the influence
exerted by K01. The pH's were slightly higher than with
NHIOl and compare very favorably with those found with

-10-

NaCl. In the case of both N3401 and K01 the maximum
point of spore formation was at a.mo1ality of 0.25 which
coincides with that found for NaOl.

In recent years L101 has found many applications
in.bacteriology and thus makes it_a very interesting salt
to study in this problem. The data in Table 5 show that
it exerted a similar influence as the other salts insofar
as exerting a stimulation of processes is concerned. Its
maximum.point of spore formation was at a.lower molality
(0.125) than was the maximum for the other monovalent
salts (0.25).

The monovalent salts thus far studied were salts of
high dissociation constants. It, therefore, seemed advis-
able to study the influence of a substance like sodium
lactate with a very low dissociztion constant. The
influence of sodium lactate, as shown by the data in
Table 6, was identical to the other monovalent salts which
have the maximum.point of stimulation at molality of 0.25.
The pH determinations also indicate that it was within

the favorable range.

3. Influence of di-valent cations in combination with
chlorine.
Following the study of the above monovalent salts
attention was turned to the di-valent salts as a group.
The data in Table 7 indicates thatngCIn does not exert

a very noticeable stimulating effect on growth and

-11-

reproduction of the organism as is evidenced by the count.
Although the pH's were within the favorable range for
spore formation.Mg01g did not stimulate the production

of spores. .

The influence exerted by Mncle 4320 and Ba012, as
shown by the data in Tables 8 and 9, are identical to
Mgclg. Preliminary work did not indicate any molality,
of these di-valent salts under study, which stimulated
reproduction of the bacteria, and so the molalities of
salt used in the experiments were arbitrarily decided
upon.

The three more toxic di-valent salts chosen for
study were 00012 6320, Pbclg and NiOle. although slight-
ly different molalities of salt were used in each case,
the influence exerted by them can be considered at the
same time. The toxicity of these three salts on the
bacteria was definitely shown by the great decrease in
numbers of organisms in the molalities of the salt as
compared to the numbers of organisms in the control tube.
Upon further studying the results in Tables 10, 11 and
12 we also find that the pH range was favorable and yet
in none of the di-valent salts did we have any stimulation
exerted on the organism.to cause or allow it to form

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-19..

4. Influence of tri-valent cations in combination.with
chlorine.

Tables 13, 14, and 15 give the results of the influence
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Feels GHgO. Here again, as in the case of the di-valent
salts, there was no stimulation of bacteria to reproduce
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favorable.

5. Influence of a tetra-valent cation in combination with
chlorine.

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-22...

Discussion.

The four organisms chosen to be used in this study
are representative of the aerobic spore forming group:
_1§_._ subtilis, 'B_._ cereus, g; mesentericus, and §_._ megatherium.
The influence exerted by the various salts was quite
constant as far as the four organisms are concerned.
Slight individual variations were obtained which are ex-
plained by the fact that we were dealing with three different
species of bacteria that can be expected to exhibit such
slight variations one from.the other.

The most common.method to demonstrate the presence
of spores is the heat reeistence of a culture. The great-
est amount of literature concerning spores deals with the
spores after they are formed and their reactions to
various external factors. The problem.herein presented
deals with the factors which induce the formation of the
spores. As such it seemed very important to know the
correlation between numbers of organisms and per cent of
spores formed. Preliminary experiments showed a very
close correlation between the heat resistance method and
spore staining method of demonstrating the presence of
spores. It is for these reasons that the spore stain was
adopted as the criterion of the presence of spores.

Leifson (14) states that, in his experiments with
certain anaerobic organisms, he found a slightly acid
medium.more favorable for sporulation than an alkaline

one. The results in this paper indicate that for the

-23..

four aerobic spore forming organisms there is a con-
siderable range of pH where sporulation can take place.
This is in part contrary to the work reported by Cook (7)
where he states that growth and spore formation ofggg
subtilis was noted only at a pH between 6.0 and 7.0.

A comparative wide range is herein reported and an
optimum is indicated in an acid medium.

The exact nature, of the influence as exhibited by
the salts included in this study, is not clear. The
cations of the monovalent salts seem.to correlate with
the theory of sporulation as expressed by Buckner. Since
at the Optimum.molality, 0.25 in the case of all mono-
valent salts studied except L101 and at 0.125 in that
case, we do have an increase in number of organisms over
control and thus a "local" exhaustion of food material
may incite the organisms to form.spores.r Nevertheless
in some of the other salts as SnCl. (table 16) we again
have an optimum of bacterial viability at a definite
molality and the pH is within the optimal range, but
here no spores were formed. If the accmmulation of
metabolic products were the essential factors causing
spore formation we would again expect SnCl. to form spores
in molality of 0.00005. The results herein presented
confirm.the results of Magoon (15) where he states that
neither insufficient food, comparable to local exhaustion

of nutrients, nor the accumulation of metabolic products

.24.

cause spore formation.

Fitzgerald (10) having worked with aerogenes bacilli,
reports that NaCl did not exert any appreciable stimulation
of spore formation as compared to the spore formation
obtained in the peptone bouillon be used as a basal medium.
It is a known fact that the bouillon contains some NaCl;
this salt is no doubt responsible for the spore formation
in his controls. The NaCl added when he studied this salt
may have exerted a toxic effect due to presence of too
much salt. Thus the per centage of spores would be almost
the same as when no additional salt was added. In this
study NaCl as all other cations of monovalent chloride
salts gave a definite and consistent stimulation of spore
formation.

From a study of the data presented it is quite
evident that the cations of the salts studied fall into
two groups (a) the cations of the monovalent salts which
stimulate spore formation and (b) the cations of di, tri
and tetravalent salts which as a group do not stimulate
spore formation. The formation of spores was most
abundant at the point of maximum.viability of the organisms.
This fact indicates that spore formation is a normal
physiological process which occurs under the most favor-
able conditions of growth and viability.

Falk (9) made a review of the literature up to 1923,
upon the theories proposed to explain the physiological
activity,exerted by the ions. He presents the view that

to better understand the roles played by electrolytes in
metabolic or physiologic processes of protoplasm there
must be advancement and application of colloidal chemistry.
The idea then presents itself that of the salts studied
the cations may fall into a series where the monovalent
ions are the most active precipitants or coagulants and
thus cause the organisms to form spores. If such is the
case the ions might fall into a series such as the
Hofmeister series but the action of the salts studied
does not definitely follow this or any other series. It
would be interesting indeed to study more ions keeping
in mind a definite series of activity which has been
applied in colloidal chemistry.

A comparison of the molality of the various salts
studied at the point of maximum.stimulation with those
secured by Hotchkiss shows a remarkable agreement. In
her work a non-spore forming, gram negative organism,

Escherichia coli, was used; while in this work four gram

 

positive spore forming organisms were used. The remark-
able correlation, of the molality at which.maximwm
sthmulation occurred, between the two groups of widely
different species would indicate that the stimulating
effect of the various cations upon bacterial viability
was not confined to any one species but was of general

physiological significance to bacteriology.

.26-
Conclusions.
1. Cations of monovalent chloride salts as - NaCl, LiCl,
NH401, KCl and sodium lactate - exerted a distinct
influence of stimulation on aerobic spore formation in

a liquid medium.

2. Cations of divalent chloride salts as MgClz, MDClg 4320,
B8012, 00012 6H20, PbCla and NiClg; of trivalent chloride
salts as A1013, CeCle and FeCle 6H20; and of a tetra-
valent chloride salts as SnClx, had no influence on
stimulation of spore formation in aerobic bacteria in a

liquid “dime

3. Spore formation was most abundant at the point of
maximum.stimulation of viability. This would indicate
that spore formation is not due to a deficiency of
nutrient materials or to an accmmulation of metabolic
products as has been postulated in the past. It would
appear that it is a normal physiological process occurr-

ing under the most favorable conditions.

4. The pH of the medium studied did not materially
affect the formation of spores within a favorable growing
range pH 5.0 to pH 7.5. However, an acid reaction was

slightly more favorable for their production.

6. The determination of spores by staining campares
very favorably with the heat method as a means of determin-

ing the presence or absence of spores.

-27-

6. The molal concentration of the different salts at
which.maximum.stimulation occurred for the four organisms
studied, at cereus, _B_._ subtilis, _B_._ mesentericus, and

g; megatherium, correspond to a remarkable degree with
those for §;.£2l$.33 previously determined by other
investigators. This work, therefore, confirms and
extends our knowledge of the physiological effect of
cations upon bacterial viability.

7. The results of this work would indicate that there

is a relationship between valence and spore formation.

1.

2.

4.

5.

6.

8.

9.

10.

11.

12.

13.

14.

15.

-28-
Bibliography.

Bayne-Jones, S. Phases of growth in size of bacilli
and yeast measured from motion pictures.
Jour. Bacteriology 23(1932)l4.

Bergey's Determinative Bacteriology. Williams and
Wilkins 00., Baltimore, Md. '

Breed, R. S. The determination of the number of
bacteria in.milk by the direct microscopic examination.
Centbl. f. Bakt. Abt. II 30(19111337.

Breed, R. S. and Brew, J. D. Counting bacteria by
means of the microscope. N. Y. Agr. Sta. Cir. 58(1918)

Buckner, H. Ueber die Ursache der Sporenbildung beim
JMilzbrandbacillus. Centbl. f. Bakt. 8(1890)

Buckner, H. Ueber die physiologischen Bedingungen der
Sporenbildung beim.Milzbrandbacillus. Centbl. f.
Bakt. 20(1896)806.

Cook, R. P. Some factors influencing spore formation
in B. subtilis and the metabolism of spores.
Centbl. f. Bakt. Abt. 1(Oct. 1931)329.

Doloff, A. F. A shmple synthetic medium for the
cultivation of bacteria. .Science 64(1926)254.

Talk, I. S. Bibliographic Review - Role of certain
ions in bacterial physiology. Abstracts of Bacteriology
7 1923 87.

Fitzgerald, M. P. The induction of sporulation in
the bacilli belonging to the aerogenes capsulatus
group. Jour. Path. and Bact. 15(19111147.

Giltner's Microbology - Laboratory Manual - AnJesyky'e
Spore Stain, p. 88, John Wiley Sons' Book Co., H.Y.

Hotchkiss, Margaret. The stimulating and inhibitive
effect of certain cations upon bacterial growth.
Jour. Bacteriology 8(1923)l41.

Huddleson, I. 3., Hasley, D. E. and Torrey, J. P.
Further studies on the isolation and cultivation on
bacterium abortus. J. Infect. Dis. 40,2(1927)352.

Leifson, Einar. Bacterial spores. Jour. Bacteriology
21(1931I33l.

imagoon, C. A. Studies on bacterial spores.
Jour. Bacteriology 11(1926)253.

16.

17.

18.

19.

20.

21.

L29-

iMellon, R. R. and Anderéon, L. M. Immuniologic
disparities of spore and vegetative stages of
B. subtilis. J. Immunology 4(19191203.

Schreiber, Oswald. Ueber die physiologischen
Bedingungen der endogenen Sporenbildung'benl
Bacillus anthracis, subtilis und tumescens.
Centbl. f. Bakt. 20(18961429.

Stafseth, H. J. Studies in infectious abortion.
Agr. Exp. Sta. Tech. Bul. 49, pt. 2 (1920)

Tarro, R. Contribucion ad estudio de la esporulacion
e1 bacillus anthracis. Centbl. f. Bakt. 10(1891)91.

Williams, 0. B. Spore formation by B. subtilis
as influenced by peptone concentration.
Jour. Bacteriology 19(1930)ll.

'Williams, 0. B. Bacterial endospore formation in
media of varying biologic value. Proc. Soc. Exp.
B101. and Med. 28, 6(193l)6l5.

-50-

Acknowledgment.
I hereby wish to acknowledge the helpful suggestions
made by Doctor F. W. Fabian and other members of the

Department of Bacteriology and Hygiene.

MICHIGAN STATE UNIVERSITY LIBRARIES

 

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