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ACTIVATION 0F BACILLUS STEAROTHERMOPHILUS
SPORES

Thesis for the Degree of MS.
MICHIGAN STATE UNIVERSITY
YUSEF ESSANUSI ET - MABSOUT
1977

 

LIBRARY

Michigan State
University

 

 

ABSTRACT

 

 

ACTIVATION OF BACILLUS STEAROTHERMOPHTLUS SPORES

By

Yusef Essanusi El-Mabsout

The objective of this study was to investigate the

activation of Bacillus stearothermophilus spores after mild

 

heat treatments at Tow pH. The spores were produced in
32-02. bottles containing a layer of Nutrient Agar +
0.03% MnSO4 adjusted to pH 6.8 (NAM). The bottles were
surface inoculated and incubated at 55 C for 2 days. The
spores were harvested, washed 3 times with sterile distilled
water, treated with 0.l mg/ml lysozyme for 2 hr at 37 C,

and washed 3 times with distilled water. The cleaned spore
suspension was stored at 4 C.

Initially, >80% of the spores were dormant as deter-
mined by comparison of direct microscopic counts and the
numbers of spores which would form colonies on NAM at 55 C.
Spore suspensions in distilled water were adjusted to pH
1.1-4.0 with HCl and heated at 40-70 c for 30-l20 minutes.
The effects of various treatments on spore activation were
determined by plating on NAM at 55 C. The rate of spore
activation was accelerated during treatments at~pH l.l-2.0
and 60-70 C.

Activation of spores was paralleled by transformation

of spores to a more heat-sensitive form. Normal or

Yusef Essanusi El-Mabsout
heat-resistant spores were not significantly affected at
pH 7.0 by heating below 100 C (086 > l000 minutes). How-
ever, at pH 7.0, the heat-sensitive spores were destroyed
during heating at 86 C (086 l 9 minutes). When activation
of spores was >90% as measured by colony formation on NAM,
heating at 86 C and pH 7.0 could be used to measure the
fraction of spores which were heat-resistant, and thus,
presumably not activated. By following the destruction of
spores during heating at 86 C it was apparent from the
initial counts that the heat-sensitive forms were viable,
activated spores.

Treatment at pH l.l-2.0 and 60 C for 60 minutes was
considered to provide the best conditions for activation in
this investigation. While treatment at 70 C resulted in
faster activation, results indicate that some spores may be
inactivated during treatment at that temperature. Heating
the spores for 90 or l20 minutes at 60 C also resulted in
less than maximal plate counts. The results of this inves-

tigation indicate that activation of 8; stearothermophilus

 

spores at low pH may be used under certain conditions as a

substitute for a severe heat shock.

ACTIVATION OF BACILLUS STEAROTHERMOPHILUS SPORES

 

By

Yusef Essanusi El-Mabsout

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1977

ACKNOWLEDGMENTS

The author gratefully acknowledges the assistance and
cooperation given him throughout this study by his major
professor, Dr. K.E. Stevenson. Thanks are also extended to
Dr. L.G. Harmon and Dr. D. Heldman who reviewed this manu-
script. Sincere appreciation is also due to M.K. El-Khoja
whose companionship made the writing of this manuscript
possible and Dr. J.C. Canada, Gerber Products Company, for

his cooperation.

ii

TABLE OF CONTENTS

Introduction.
Review of Literature.
Activation of bacterial spores.
Heat activation
Methods of activation other than heat
Low pH.
Ca-DPA.

Reducing agents and other chemical
compounds

Chemical manipulation of the heat resistance
of Bacillu§_stearothermophilus spores

Materials and Methods
Organism.
Preparation of heat sensitive spores.
Preparation of heat resistant spores.
Activation of spores.
Heat treatment at 86 C.
Experimental Results.
- Growth and sporulation. . . . . . . . . .“.

Measurement of activation by plating.

Heat resistance of H-form and Ca-form spores.

Heat resistance of activated spores

Page

d

xooooo-pww

IO

II
T4
14
15
15
16
I7
18
I8
18
20
20

Activation of spores at pH l.l
Effect of temperature.
Rate of activation at 50 C
Effect of pH on activation of spores

Activation of thermophilic flat sour spores in
flour samples. .

Discussion
Conclusions.

Bibliography

iv

Page
22
22
24
24

24
28
37
38

LIST OF TABLES

Activation of spores at pH 2.0 and 50 C as
determined on NAM agar after incubation for
48 hr at 55 C .

Activation of spores at pH 2.0 and 60 C as
determined on NAM agar after incubation for
48 hr at 55 C

Survival of activated (treated at pH 2.0 and
60 C for 60 minutes) spores at 86 C as
determined on NAM agar after incubation

for 48 hr at 55 C

Enumeration of thermophilic flat sour spores
in oat flour.

Page

I9

T9

20

27

LIST OF FIGURES

Page
Survivor curves of H-form and Ca-form spores
heated at 86 and TDD C. . . . . . . . . . . . . . 2]
Survivor<nnwes of spores heated at 86 C.
Spore were incubated at different temperatures
for 60 minutes at pH l. l before heating at 23
86 C. . . . . . . . . . . . .

Survivorcurvesof spores heated at 86 C.
Spores were incubated for different periods .
at pH l.l and 50 C before heating at 86 C . . . . 25

Survivor curves<yf spores heated at 86 C.

Spores were incubated for 60 minutes at

60 C at different pH values before heating

at 86 C . . . . . . . . . . 26

vi

 

INTRODUCTION

The study of activation of Bacillus stearothermophilus

 

spores is of great importance to food microbiologists due to
the unique heat resistance and high degree of dormancy of

the mature spores. Because of their extreme heat resistance,
they are often used in the food industry as an indicator of
the adequacy of the thermal process used for commercial
sterilization. Also, large annual economical losses in the
canning industry are partially attributed to flat sour
spoilage of low acid foods, such as peas, beans, whole kernel

corn and cream-style corn, caused by B; stearothermophilus.

 

Accordingly, the food industry is quite concerned with the
methodology involved in determining spore counts in raw
materials and the survival of spores in finished products.

The concern over activation of B; stearothermophilus spores

 

is due to the fact that when a food sample is evaluated for
the preSence of these organisms, only a small fraction of
the spores will form colonies on appropriate media while the
majority of the spores remain dormant. Thus resulting in a
lower count than the actual number of thermophilic spores
present in the sample.

Presently, methods used in determining flat sour orga-

nisms utilize severe heat treatments for activation of

spores. This may result in under-estimating the spore
content of a given food sample due to a variety of reasons,
such as over-heating or killing some of the spores, heat-
induced dormancy, or inadequate conditions for activation.
The objective of this study was to investigate the

activation of B; stearothermophilus spores by mild heat

 

treatments at low pH.

REVIEW OF LITERATURE

Activation of Bacterial Spores

 

As stated by Murrell (l96l): "Viable spores failing to
germinate in apparently favorable conditions are said to be
dormant". In most cases fresh spores are dormant and will
not germinate, even under favorable conditions unless they
are preheated or otherwise treated. The process by which
dormant spores are converted into spores which have the
ability to germinate is called activation. In some cases,
slow germination may take place even without previous treat-
ment, therefore, activation may be defined as: the condi-
tioning treatment or the process by which spores are rendered
less dormant and germination of spores is accelerated.

Historically, mold spore activation was discovered
before activation of bacterial spores. Shear and Dodge
(T927) were the first to apply heat to mold spores for acti-
vation and subsequent germination. 0n the other hand
Weizmann(1) (T919) was the first to apply heat activation to
bacterial spores. Although he did not realize its direct
effect on spores, he used a heat treatment of 90-l00 C for

one to two minutes to activate Clostridium acetobutylicum

 

spbres for use in the fermentation of acetone. In his

 

(l) Gould and Hurst, T969

 

4

opinion, he heated the spore suspension to eliminate con-
tamination with vegetative cells which might have interfered
with normal fermentation by inhibiting spore germination

or competition for nutrients. However, Curran and Evans
(T945) were the first to note and understand the direct
activation effect of heat on bacterial spores. They observed
that in a given spore suspension, a greater number of spores
germinated if the suspension was given a mild heat treatment
before plating (Desrosier and Heiligman, T956). Curran and
Evans (T945) studied both mesophilic, as well as thermo-
philic strains, including Bacillus stearothermophilus strain I

NCA 1518.

 

 

Although heat treatment is not the only method of
activation, the majority of the reports on activation have
concerned use of heat. Many workers studied the effects of
heat on different spore suspensions and different types and
strains of spores. Other methods have also shown results
similar to heat activation. In this review, heat activation

as well as activation by other methods will be discussed.

Heat Activation

 

Heat activation is defined by some workers as a sub-
lethal heat treatment (Fields, T970) and it is the simplest
method of activation (Keynan and Evanchik, l969)J Spores of
different species vary in their temperature requirements for
activation. These variations also exist among different
strains of the same species and among different batches of

spores produced from the.same strain. Brachfeld (l955) and

Titus (T957) showed that spores of B; stearothermgphilus

 

could be activated at temperatures above T00 C. In another '
study, Finely and Fields (T962) found that maximal activa-
tion of B; stearothermophilus spores occurred only at tem-
peratures above TOO C, namely l00-ll5 C depending upon the
strain and the spore suspension. They also noted heat-
induced dormancy when the spores were heated in distilled ffifi
water at 80, 90 and l00 C. Similar studies on heat activa-
tion showed that only a few minutes at 60 C were required

for optimal activation of spores of some strains of Bacillus

 

1"“ n..-

megaterium whereas spores of B; stearothermophilus and other

 

thermophilic and thermotolerant bacteria required from l05-
TT5 C for optimal activation. It has also been shown that
Bacillus coagulans required 5 minutes of heating at 85 C

(Desrosier and Heiligman, T956) and Bacillus cereus T

 

required 45 minutes of heating at 65 C (Keynan 33 al., l964)
for optimal activation.

In addition to species and strain differences in heat
requirements for activation, which are endogenous to the
spore, other factors also affect heat activation. One factor
is the composition of the medium on which growth and sporu-
Tation occurs. As an example, Keynan t al. (T961) found

that spores grown on media containing different amounts of
Ca++ and phenyl alanine required different periods at 65 C
to obtain maximum activation rates and the times necessary
for activation correlated well with the Ca-dipicolonic acid

(DPA) content of the spores.

The extent of heat activation is also dependent upon
other factors. These include: composition of the heating
medium; the presence of salts and heavy metals; and the pH
of the medium.

Beers (T958) observed that it was impossible to acti-
vate dry lyophilized spores and spores suspended in a high
concentration of glycerol. Earlier Powell and Hunger (T966) .r_.
and Maeda ££._l- (T975) reported that heat activation must

be carried out in the presence of water. Finely and Fields 1

(T962) and Fields and Finely (T963) studied the effects of

 

phosphates and carbohydrates in phosphate buffer. They I
found that regardless of strain and suspension source, phos-
phate buffer (0.0083 M) influenced the germination of B;
stearothermophilus.) In another experiment, Fields and
Finely (T963) found that different spore germination respon-
ses occurred when spores were heated in the presence of
monosaccharides, disaccharides and polysaccharides in

0.0083 M phosphate buffer (pH 7.l). These differences were
observed among various strains, and among spore suspensions
from the same strain. In decreasing order of efficiency
glucose, lactose, peptone, skim milk, glucoSe-nutrient agar,
beef extract, glucose-nutrient broth, distilled water and
sodium chloride were found to affect heat activation (Curran
and Evans, T945). 0n the contrary, Busta and Ordal (l964)
noted no effect on heat activation for various suspending
media containing glucose, xylose, robose, NaCT, or sodium

phosphate, nor was there a marked effect due to change in

pH from 5.0 to 8.0 when heat activation of Bacillus subtilis

 

strain 5230 spores was carried out at 75 C. Other investi-
gators (Halmann and Keynan, T962; Heynan gt al., T965)
showed that salts interfere with germination if present
during heat activation. However, if the spores were washed
after the heat treatment, germination was not affected.
This suggested that salts did not affect activation, but 'F“
interfered only with germination.

Various reports also show that heat activation of bac- 7

terial endospores is pH dependent. Keynan gt al. (l964)

 

showed that §;_cereus T activation was inhibited at pH e
values above 8.5. At pH 7.0, activation was optimal when

spores were heated at 60 C for one hour. In comparison,

T0 minutes at 60 C gave similar activation when the pH was

below 2.0. It has also been demonstrated that some spores

respond differently to pH variation during heat activation.

Gibbs (T966) found that activation of Clostridium bifer-

 

mentans spores was optimal at high pH values and inhibited
at low pH.

The effect of carbohydrates on heat activation of B;
stearbthermophilus was studied by Fields and Finely (T964).
Their results show that with increased carbohydrate concen-
trations (using 0.00T, 0.0T and 0.l M glucose, fructose,
sucrose, maltose, dextrins and starch), the dominant trend
was reduced activation. Fields (l964) found that heat
activation of B; stearothermophilus spores at lTO C in 20%

sucrose solution caused the rough variant to decrease in a

8
mixed population of rough and smooth variants of strain M.
The smooth variant, however, increased. Similarly, the rough

variant of the strain NCA T5T8 of B; stearothermophilus

 

decreased when heated in a mixed population at Tl0 C in the

presence of 20% sucrose solution. Other studies have also

shown that age of the spores and the presence of lysozyme

have a marked effect on heat activation (Fields and Jenne, 'r-fi

1962).

Methods of Activation Other than Hegt

Although heat application is the predominant method

 

for activation of bacterial spores, other methods have also
been described. These methods involve the use of low pH,
Ca-DPA, reducing and surface active agents and other chemi-

cals and ionizing radiation.

Low pH

Normally, spores are heat activated and germinated at
normal pH. However, heat activation as described earlier is
pH dependent and the Tower the pH, the lower the temperature
required for activation. Moreover, low pH replaces heat
activation under certain conditions. In a study of dormancy
and activation of bacterial spores, Lewis t al. (T965)

showed that the rate of germination of §t_stearothermophilus

 

spores increased from T8 to 80% when they were subjected to
pH l.5 for 80 minutes at 25 C. They also showed that dor-
mancy was restored if the spores were exposed to 0.02 M Ca++

ions (pH 9.7). Keynan gt gt. (l964) reported that spores

of fit cereus T germinated spontaneously (without heat acti-
vation) when incubated for a long time in a buffer solution
at low pH. Brown gt gt. (T968) in studying activation of

§t_stearothermophilus spores found that spores subjected to

 

0.5 N HCT at 25 C gave increased colony counts which were

equal to the total counts of spores.

Ca-DPA

"Since the demonstration by Powell (T953) that bac-
terial spores contained dipicolinic acid (DPA), there has
been much interest in its biological role", (Lee and 0rdal,
T963). Riemann and Ordal (T96T) showed that spores of 8t
subtilis and E; cereus T. germinated in solutions of equi-
molar concentrations of Ca++ and DPA without heat activation.

Later, Lee and 0rdal (T963), showed that 8t megaterium

 

spores were activated and germinated spontaneously in dis-
tiTTed water when dormant spores were placed in a solution
of 40 mM Ca-DPA (pH 7.0) for 70 minutes at 7 and TO C.
However, germination of activated spores was inhibited by a
chelating agent, o-phenanthroline, and by various cations
such as Cu++, Fe++, Ag+ and Hg++. The activation effect

was reversed by treatment with acid. In another study,
Ca-DPA incorporated into tyrptone-glucose yeast extract agar

was used to germinate spores of gt subtilis, Bg_stearother-

 

mophilus, gt megaterium and 8t coagulans for their enumer-

 

 

ation on media without heat activation (Busta and Ordal,

l964). However, Freese and Cashel (T965), reported that

 

10

spores of B; subtilis T68 did not germinate when inoculated
in Ca-DPA for T60 minutes but were only activated for L-
alanine germination and 98% of the spores remained refrac-

tile after the Ca-DPA treatment.

Reducing agents and other chemical compounds

Reducing agents which reduce disulphide bonds such as
thioglycollate and mercaptoethanol were found to replace
heat treatment as a means of activation. Keynan eth T.

(T964) found that when spores of Bacillus cereus strain T

 

were incubated in 0.2 M thioglycollate or 0.2 M mercapto-
ethanol at 28 C, partial germination occurred after washing
and addition of L-alanine and adenosine. However, a long
time was required (T2 hours) to observe the activation
effect of these reducing agents. In another study, (Gibbs,

T966) thioglycollate failed to activate gt bifermentans

 

spores which suggests that spores of different species
respond differently to treatment with reducing agents.
Antibiotics such as D-cycloserine and 0-carbamyl-D-serine,
and polar solvents like dimethylformamide also were used
as replacements of heat for spore activation (Gould, T966).
Recently, ionizing radiation has been used to activate
spores of g; cereus PX (Gould and Ordal, T968) and E; gggg;
terium (Levinson and Hyatt, T960).

Generally speaking, activation of bacterial endospores
is reversivle. However, there are a few exceptions where

activation has been shown to be irreversible (Busta and

11

Ordal, T964; Keynan et al., 1965 and Gibbs, T966). Refrac-
tibility and heat resistance of spores are not affected by
the activation process. I i

The mechanism through which activation occurs is still
controversial, and gg single hypothesis has yet been sug-
gested that could account for all of the known activation
treatments and conditions. However, many of these hypothe- 'f‘fi
ses deal with a particular factor which may trigger activa- .

tion such as a change in spore permeability (Lee and Ordal,

"‘7'! I. '

T963), deactivation of an inhibitor or toxic factor (Mef-

9.‘ ' Li'J

 

ferd and Campbell, 1961), reorientation of spore components

and denaturation of a spore protein (Keynan gt 1., 1964).

However, more information is required before the real nature

of activation is known.

Chemical Manipulation
of the Heat Resistance of
Bacillus stearothermophilus Spores

The refractile dormant spores of gt stearothermophilus

 

are known to be extremely heat resistant. Among the factors
that are known to influence their heat resistance are age of
the spores, the chemical composition of the medium on which
spores were produced, the growth and sporulation tempera-
ture, pH and composition of the heating medium (Stumbo,
T973), and the chemical state of the spore (Alderton and
Snell, T969)._ However, only those pertaining to chemical
manipulation of the heat resistance of spores will be dis-

cussed below.

 

 

12

Certain divalent cations such as Ca++ and Mn++ play
an important role in determining the thermal stability of
mature spores. Amaha and 0rdal (1957) showed that heat
resistance of bacterial spores was reduced when the concen-
tration of Ca++ and Mn++ was reduced below certain levels
in the sporulation medium. 0n the contrary, a high phos-
phate level in the medium markedly decreased the heat resis- .egm.
tance (ET-Bisi and Ordal, 1956). The chemical composition
of mature spores also affects their heat resistance. The

results obtained by Murrell and Narth (1965) indicated a

significant relationship between heat resistance and Ca++ ,

 

content of the spores. Their results also showed that the
lowest heat resistance occurred in spores having the highest
Mg:Ca ratio; in heat resistant spores, Ca content was high,
Mg:Ca ratio was fairly low, and CazDPA ratio was high.
However, in mature heat resistant spores, Ca++ and DPA
occurred in nearly equimolar amounts (El-Bisi _t gt., T962)
and these compounds were released together with spore pep-
tides during germination (Powell and Strange, T953).
Further studies on the physico—chemical properties of
spores have shown that mature spores under pH-controlled
conditions were capable of undergoing cation exchange
(Alderton and Snell, T963). Divalent cations such as Ca++
and Mg++ (presumably responsible for the increased heat
resistance of mature spores) are replaced by hydrogen ions

or vice versa, thus allowing formation of hydrogen-form

(H-form) and calcium-form (Ca-form) spores. As stated by

13

Alderton and Snell (1963), the spore cation exchange system
resembles that of a resinous, weak cation exchange system,
such as methacrylic acid polymers, rather than the strong
sulfonic acid type of cation exchange system. They also
suggested that carboxyl, phosphorus-containing and chelating
groups which are pH controlled are possible functional
groups for base binding in the spore cation exchange system.
The H-form spores have a markedly lower heat resistance
than Ca-form spores. This difference in heat resistance is
of a large magnitude. Alderton and Snell (l969b) stated
that the temperature at which equal survivor rates of fit
stearothermophilus spores are obtained changes about 28 C
between the heat-sensitive (H-form) and the heat-resistant
(Ca-form). The chemical states of spores can be prepared
by treating mature spores with an acid (e.g. HCT) or buf-
fered metal cations (e.g. Ca(0H)2 in calcium acetate buffer).
The change in heat resistance of spores which is introduced
by such treatments persists even after the spores are
thoroughly washed and the rate of change can be manipulated
by changing the pH and the temperature as well as the dura-

tion of the treatment (Alderton and Snell, l969b).

 

MATERIALS AND METHODS

Organism

The organism used in this study was 9; stearothermo-

 

Ehilus E-2 obtained from the culture collection of the
Department of Food Science and Human Nutrition at Michigan
State University. It was maintained in a frozen form in

nutrient broth (Difco) and stored at -20 C.

 

The frozen culture was thawed and incubated at 55 C
for 48 hr., and nutrient agar (Difco) slants (NA) were ino-
culated and incubated at 55 C for 48 hours. One-ml portions
of the 48-hr NB were used to inoculate double strength
nutrient agar + 0.03% MnSO4 (NAM) plates, and the plates
were incubated for 3 days at 55 C. The cultures were har-
vested by adding 4 ml of sterile deionized water to each
plate and rubbing the growth off with a sterile glass rod.
The harvested cells were centrifuged at 4080 x g for 20
minutes in a SorVal Superspeed RC-2 automatic refrigerated
centrifuge, suspended in sterile water and cleaned 3 times
by centrifugation. The spores and cells were suspended in
water and stored at 5 C. This spore suspension was used in
preliminary investigations.

For preparing larger amounts of spores, 32-02. pre-

scription bottles containing 200 ml of NAM were sterilized

14

15

at 121 C for 15 minutes. After solidification of the agar,
bottles were left on their wide side for 2 days over the
surface of the agar. The bottles were incubated for 2 days
at 55 C. Spores were harvested in 10 ml sterile, distilled
water, centrifuged, cleaned 3 times with sterile, distilled
water, treated with 0.1 mg/ml lysozyme, 3 x crystalline
(Nutritional Biochem. Corp., Cleveland, Ohio), at 37 C for
2 hr, and cleaned 3 times with deionized sterile water by
centrifugation. The cleaned spore slurry was suspended in

water and stored at 4 C.

 

Preparation of Heat Sensitive Spores E ~~
Heat sensitive spores (H-form) were prepared by mixing

normal spores in a solution described by Alderton and Snell

(1969a) containing 1% tryptone, 0.5% glucose and 0.1% solu-

ble starch. The pH of the solution was adjusted to 1.1

with 3N HCT. The spore suspension was incubated overnight

at 20 C, centrifuged, washed 3 times and suspended in

sterile, deionized water. Heat sensitive spores were also

formed by treatment with some of the methods described below

in the section on Activation of Spores.

Preparation of Heat Resistant Spores

 

Heat resistant spores (Ca-form) were prepared by
mixing normal spores in a solution described by Alderton and
Snell (l969a) containing 0.02 M calcium acetate, adjusted to
pH 9.7 with aqueous Ca(0H)2. The suspension was incubated

overnight at 20 C and heated in a steam cabinet for 15

16

minutes. The suspension was centrifuged, washed 3 times

and suspended in deionized sterile water.

Activation of Spores

 

Temperature, time and pH were the main factors consi-
dered in this investigation. The general approach was to
change one of these factors at a time, thus allowing for the
evaluation of its effects on activation of spores.

In one set of experiments, 5-10 ml samples of spore
suspension were pipetted into screw-capped test tubes (Pyrex
no. 9825) each containing 10 ml of 0.2N HCl giving a final
pH of 1.1. These sample suspensions were stored at 50 C
for 0, 30, 60, 90 or 120 minutes.

For determining the effect of temperature, 4-10 ml
samples of normal spore suspensions were added to screw-
capped test tubes containing 10 m1 of 0.2N HCT. These sam-
ple suspensions were incubated at 40, 50, 60 or 70 C for one
hour.

For determining the effect of pH, 4-10 ml aliquots of
acid-buffer solutions (pH adjusted to 1.1, 2.0, 3.0 or 4.0)
were added to screw-capped test tubes. Each contained 10 ml
of cleaned normal spore suspension. These sample suspen-
sions were incubated for 1 hr in a water bath adjusted to
60 C. c

Other methods used in this study included the standard
method for the determination of thermophilic flat sour

spores described by the American Public Health Association

 

17

(APHA, T966) and the method described by Dr. J.C. Canada of
Gerber Products Company (Personal communication) which is
used for examining raw materials for heat resistant aerobic
thermophiles and flat-sour spores. A description of this
method is given below.

A sample was weighed and dispersed in H20 by blending
or shaking at 1:10. Twenty milliliters of the suspension
were pipetted into a 250-ml Erlenmeyer flask containing
100 ml sterile dextrose-tryptone agar with brom cresol
purple. The flask was cotton plugged and swirled to mix

contents and autoclaved at 108 C (5 psi) for 10 minutes.

 

The autoclave was previously standardized to give a heat

treatment equivalent to FL?

= 0.74 when heating was com-
pleted with the autoclave set for slow exhaust. The flask
was immediately removed and the contents were cooled to 43-
45 C in running water. The contents were poured into 4-5
petri dishes and the plates were inverted and incubated at

50-55 C for 48 hrs.

Heat Treatment at 86 C

 

One milliliter of each treated sample was added to 9
ml of phosphate buffer, pH 7.6, in a screw-capped test tube.
The sample suspensions were heated in a water bath at 86 C
and consecutive dilutions were made from suspensions which
had been heated for various time intervals. Appropriate
dilutions were surface-plated on NAM plates, and the plates

were inverted and incubated at 55 C for 2 days.

EXPERIMENTAL RESULTS

Growth and Sporulation

Most workers indicate that fit stearothermophilus spor-

 

ulates readily on nutrient agar, a nutritious medium which
contains 0.3% beef extract, 0.5% peptone and 1.5% agar.
However, preliminary investigation showed that §t_stero-

thermophilus strain E-2 grew poorly and failed to sporulate

 

on several media such as dextrose tryptone brom cresol
purple agar (DTBA), plate count agar (PCA), and nutrient
agar (NA).

It is mentioned in the literature that MnSO4 can be
used to supplement nutrient agar in order to enhance the
production of B; stearothermophilus spores (Alderton and
Snell, 1969; Titus, 1957). When 0.03% MnSO4 was incorpo-
rated into NA in this investigation, better growth and spor-
ulation was observed. Luxuriant growth and approximately

80% sporulation was observed when 8t stearothermophilus

 

strain E-2 was cultured on NAM for 2 days at 55 C. Thus,

NAM was used in subsequent experiments.

Measurement of Activation by Plating

 

Table 1 shows average plate counts of spores which
have been activated at pH 2.0 and 50 c for different periods

vs. nonactivated spores. The total count of spores, as

18

 

19

determined by direct microscopic count, was m9xlO7 spores/

ml. Colony counts were obtained from plates containing a
6

 

 

 

TO- dilution of the original spore suspension.
Table 1. Activation of spores at pH 2.0 and 50 C
as determined on NAM agar after incubation
for 48 hr at 55 C.
__Time of activation treatment in minutes
0 30 60 90 120 "'1
Colonies/platea 9 19 74 55 ' 51

 

aNumbers represent the average count on duplicate plates at
a 10-6 dilution.

 

These results indicate that activation has taken place L
since the plate counts increased from 9x106 at 0 Time (un-

treated control) to 7.4x107

at 60 minutes, which represents
the optimal activation time under these conditions. The
counts decreased slightly during extended treatment.

When activation was carried out at a higher tempera-
ture (60 C), the same trend was observed. However, higher
activation was obtained with the 60 C treatment (Table 2).
After 60 minutes, plate counts increased more than 8-fold,

Table 2. Activation of spores at pH 2.0 and 60 C

as determined on NAM agar after incubation
for 48 hr at 55 C.

 

 

Time of activation treatment in minutes
0 3o 50 90 " 120

Colonies/platea 1o 31 83 57 59

 

 

aNumbers represent the average count on duplicate plates at
a 10-6 dilution.

20

from 1.0x107 to 8.3x107 spores/ml. The data also show that

the numbers of activated spores increased to at least 90% of
the total count of spores in the original spore suspension

(9.0x1o7 spores/ml).

Heat Resistance of H-form and Ca-form Spores

Data presented in Figure 1 show survivor curves for
heat sensitive (H-form) and heat resistant (Ca-form) spores
when heated at 86 and 100 C. These data indicate that the
Ca-form spores were extremely heat resistant and the total
number of spores remained virtually unchanged when heated
for 30 minutes at 86 or 100 C. 0n the other hand, 99.95%
of the H-form spores were killed after the first 5 minutes
of heating at 100 C. In addition, when the H-form spores
were heated at 86 C, over 99.99% of the total count of

spores were destroyed after 30 minutes.

Heat Resistance of Activated Spores

 

Spores subjected to an activation treatment of 60
minutes at pH 2.0 and 60 C show a marked decrease in their
heat resistance. Table 3 shows the number of treated
spores which survived heating at 86 C.

Table 3. Survival of activated (treated at pH 2.0
and 60 C for 60 minutes) spores at 86 C
as determined on NAM agar after incuba-

tion for 48 hr at 55 C.

Heating time in minutes at 86 C
0 5 TO 15 20 30

Colonies/platea 300 249 183 53 38 5

 

 

 

aNumbers represent the average count on duplicate plates at
a 10‘5 dilution.

IL
No

Survivor Log

 

 

 

 

 

 

 

 

0 IR; - - A 1;;
N\ 9*; 3—' 5* 3.555
Ca-form
-1 _.
-2 __ \.
..3 —‘
-4 —-
100 c H-form 86 C
5 ? I L I I T I
' O 5 TO 15 20 25 30
Time in minutes
Figure 1. Survivor curvescrf H-form and Ca-form

spores heated at 86 and 100 C.

F'”

22

These data indicated that over 90% of the spores were heat
sensitive. This is comparable to the percentage of spores
activated during treatment at pH 2.0 and 60 C for 60 minutes
(Table 2). Thus, during activation under these conditions
(60 C and pH 2.0) spores were transformed to a heat-sensi-
tive state. Data obtained on the heat resistance of

untreated spores at 86 C indicate that Bt_stearothermophilus

 

spores are very heat resistant. This confirms the previous
conclusion that activation of spores was accompanied by
their sensitization to heat.

In measuring activation, the use of plate counts
(before and after activation) is limited to m80-90% of the
tOtal count. However, at higher levels, activation can be
approximated by measuring the proportion of heat sensitive
spores. Thus, heating at 86 C was used in the following
experiments to monitor the proportion of spores which were

activated, i.e. heat-sensitive.

Activation of Spores at pH 1.1

 

Effect of temperature

Figure 2 shows survivor curves for spores which were
treated at pH 1.1 for 60 minutes at temperatures of 40, 50,
60 or 70 C, and then heated at 86 C. Results indicate that
at least 90% of the spores were activated (heat sensitive)
during treatment at 40 and 50 C. Activation increased to
approximately 99.9% and 99.99% for treatment at 60 and 70 C,

respectively.

-2
o
22
05
O
...l
S.
o
>
0t-
>
S.
3

V’ -3

-4

-5

23

 

 

 

 

 

60C
70:
I I I I I I
0 5 10 15 20 25 30
Time in minutes
Figure 2. Survivor curves of spores heated at 86 C.

Spore were incubated at different tem-
peratures for 60 minutes at pH 1.1 before
heating at 86 C.

 

24

Rate of activation at 50 C

Figure 3 shows a survivor curve during heating at 86 C
for spores treated at 50 C and pH 1.1 for various periods of
time. The data show that 55% of the total spores were heat
sensitive (activated) when treated for 30 minutes under the
aforementioned conditions. Similarly over 90% of the spores
were activated by treatment for 60 and 90 minutes. The

120-minute treatment gave approximately 99.9% activation.

Effect of pH on Activation of Spores

For spores activated for 60 minutes at 60 C, maximum
activation occurred at pH 1.1-2.0. At higher pH values
(3.0 and 4.0), no significant activation was observed.
Figure 4 shows survivor curves for spores heated at 86 C
after treatment for 60 minutes at 60 C and pH values of 1.1,
2.0, 3.0 or 4.0. These data indicate that over 99.9% of
the spores were activated at pH 1.1 and 98% at pH 2.0. At
pH values of 3.0 and 4.0 activation was minimal.

The effect of pH was also shown by the fact that over
99.99% activation was obtained when the spores were treated
at pH 1.1 and 20 C overnight (Figure T). On the contrary,
an overnight treatment of spores at pH 9.7 and 20 C resulted

in essentially no activation.

Activation of Thermophilic Flat Sour Spores in Flour Samples

 

Data obtained (Table 4) indicated that few thermo-
Philic flat sour spores were activated by the low pH-mild

heat (pH 1.1 and 60 C) treatment. Higher plate counts were

25

 

 

 

 

 

 

 

0 ~—-—. A A k - '1
c f V ' fig '1 '1
‘1 , ..
~ 30 111:1
-1 __ ‘ '
a 60 Min
\\\\“fl 90min
1!
o
22

at v
o
_1
s. '2 _
o
.3
>
s.
3
m

-3 .-

9 120 min
-4 1 1 1 1 1 I
0 5 10 15 20 25 30

Time in minutes

Figure 3. Survivor curves of spores heated at 86 C.
Spores were incubated for different

periods at pH 1.1 and 50‘C before heating
at 86 C. .

25

 

 

 

 

 

 

 

O _‘. - - Li -.
c v v v V'J (TITO
‘1 -
" 30 11 1
-1 __ ‘ .
v . a” 50 min
\ 90 min
i‘
O
ziz
U1 ‘
o
_1
s. '2 '—
O
>
'>'
s.
3
m
-3 .—
° 120m1n
_4 1 1 1 1 1
O 5 TO 15 20 25 30

Time in minutes

Figure 3. Survivor curves of spores heated at 86 C.
Spores were incubated for different

periods at pH 1.1 and 50‘C before heating
at 86 C. I _

O

-T
To
22
on
o

_. -2
s-
o
>
'2
S.
3
m

-3

-4

 

 

 

 

 

 

 

3h .1
pH. 3.

C)C.)

pH 2.0

pH 1.1

 

1 1 I 1 1 1
O 5 TO 15 20 21 22
Time in minutes
Figure 4. Survivor curves of spores heated at 86 C.

Spores were incubated for 60 minutes at

60 C at different pH values before
heating at 86 C.

 

27

were obtained when the recommended method for enumeration

of thermophilic flat sour spores (APHA, 1966) was followed.

Table 4. Enumeration of thermophilic flat sour
spores in oat flour.

 

 

Method Spore count per 109 sample
Recommended method 170
Low pH-mild temperature 35

Gerber method 30

 

DISCUSSION
Preliminary investigations suggested that Mn++ was
necessary at a certain level in the plating medium for

improved growth and sporulation of gt gtearothermophilus

 

Strain E-2. Similar situations were encountered by other
workers. Rowe gt _t. (1975) found that manganese was
required at relatively high concentrations (1.0 uM) for the
growth of §t_stearothermophilus 1503 in a defined liquid
medium. The addition of 15 to 30 ppm of manganese (Mn++)
to a defined liquid medium containing tryptone was found to
stimulate sporulation of g; stearothermophilus (Thompson

4..

and Thames, 1967). Addition of Mn+ to nutrient agar for

increased sporulation of gt stearothermoghilus has been

 

utilized by various investigators (Schmidt, 1950; Kim and
Naylor, 1966; Titus, 1957).

The 086-value (decimal reduction time, or the time
required to destroy 90% of the spores at 86 C) obtained when

gt stearothermophilus strain E-2 spores were heated at

 

pH 7.0 at 86 C indicated significant differences between
activated and normal spores. These differences were depen-
dent on the conditions (pH, time and temperature) under which
the spores were activated. The minimum 086-value (086 =

m7 minutes) was observed when the spores were activated at

28

29

pH 1.1 for 60 minutes at 70 C. When the temperature of
activation was decreased to 60 C and 50 C, the 086 value
increased to N9 and 20 minutes, respectively. However,
similar D-values were shown for the spores which have been
treated overnight at pH 1.1 and 20 C and the spores which

have been treated at pH 1.1 at 60 C for 1 hr. Thus, the
higher Dgs-value for spores treated at 50 C probably

represent partial activation or a partial change to the
heat-sensitive state.

On the other hand, the Ca-form spores have shown ex-
treme heat resistance when heated below 100 C at le7.0,
indicating a 086-value of over 1000 minutes, which is
similar to the D-value obtained with natural (untreated)
spores heated under the same conditions.

Difficulties were encountered when attempts were made
to compare these findings to those of other workers. That
is, the conditions under which their experiments were carried
out were different from those which were employed in this
study. However, the D-values obtained with natural spores
are comparable to those obtained by other workers. When 8;
stearothermophilus NCIB 8919 spores were heated in water at
100 C, a Dloo-value of 3000 minutes was obtained (Briggs,
1966). At a higher heating temperature (115 C) a lower 0-

value (0115 = 22.6 minutes) was reported by the same worker.

 

For spores of'gt stearothermophilus NCIB 8919 a DHS value
of 18.3 minutes was obtained (Cook and Gilbert, 1968). For

spores of Bt stearothermgphilug ATCC 7953 two D-values

 

30

(0100 = 459 and 714 minutes) were reported (Murrell and
Warth, 1965). The difference between the two values was
attributed to differences in CazDPA ratios of the spores.
A comparison between these findings and the data obtained
with §t_gtearothermophilus E-2 native spores would indicate
a fairly good agreement, and the discrepancies are quite
explainable in light of the factors which affect heat resis-
tance described earlier in the review of literature:

The effect of acid wash (Slepecky and Foster, 1959) on
spore heat resistance was investigated by Murrell and Warth
(1965). They reported little effect for such treatment (at

5 C) on the heat resistance of four Bacillus species. The

 

Dloo-values for these species as measured before and after

the treatment were 0.99 and 1.0 minute for Bacillus strain

 

668, 35.2 and 34.6 minutes for Bacillus strain 636, 270

 

and 232 minutes for Bt_ggagulans; and 2.38 and 2.17 minutes

 

for Bacillus strain 645. However, when spores of gt stearo-
thermophilgg strain NCA 1518 were treated in a liquid cul-
ture medium, previously adjusted to pH 3.0 with HCT, for 60
minutes at 70 C, the heat resistance was reduced greatly as
compared to the untreated spores.

The time needed to reach a 100,000-fold reduction in
the untreated spores was 11 times greater than for heat sen-
sitive spores when both spore suspensions were heated at
115.6 C and pH 5.95 (Alderton and Snell, 1969b). Alderton
and Snell (1969b) stated that the change in heat resistance

amounted to several hundred-fold between the heat sensitive

31

and heat resistant forms. Thus the data obtained with H—

form and natural spores of Bt stearothermophilus E-2 are

 

comparable and the general trends in heat resistance of
treated and untreated spores are quite similar to those
obtained by other workers.

Various studies have shown that activation of Bacillus

 

stearothermophilus spores will take place primarily at tem-

 

peratures above 100 C. With spores of Bt stearothermo-

 

philus NCA 1518, Brachfeld (1955) found that maximum acti-
vation was obtained when the spores were heated for 5 minutes
at 105 C. He also found that heating at lower temperature
(55-85 C) resulted in heat-induced dormancy and lower plate
counts. Furthermore, his data have shown that even after
heating the spores at 105 C for 5 minutes, complete activa-
tion was not observed and a portion of the spores were
unable to form colonies.

Lewis gt gt. (1965) have shown that activation of gt

stearothermophilus spores is possible at lower temperatures

 

(25 C) and under conditions of low pH (pH = 1.5). Thus the
effect of pH in conjunction with temperature and length of

duration on activation of Bt stearothermophilus spore was

 

investigated.

The results of this study indicated that it stearo-
thermgghilus E-2 spores were activated by a relatively mild
heat treatment (50-70 C) under controlled pH conditions
(l.l-2.0). At higher pH value (pH 3 3.0) and lower tempera-

ture (40 C) little activation was observed. The optimal

32

time for activation under these conditions (pH 1.1-2.0 and
50-70 C) was found to be approximately 60 minutes. However,
other treatment times (30, 90 or 120 minutes) at pH 2.0 and
50 or 60 C resulted in less activation. .

Activation of dormant spores was measured by two
methods. These methods are: the regular plating method
(plating before and after treatment) and by measuring the
heat sensitive spores. The assumption was that if the spores
were sensitized to heat by the acid-Tow heat treatment, even
if the acid was removed and heating was carried out at pH
7.0 then the amount of activated spores should be comparable
to the amount of heat sensitized ones. The data presented
earlier (Tables 2 and 3) indicated that this relationship
existed in this investigation. At higher levels of activa-
tion (>90% of the total count of spores) the plate count
method becomes less sensitive in measuring activation.
However, under these circumstances the amount of activation
can be more accurately measured by determining the proportion
of heat sensitive spores.

When the results of this study were compared to the
finding of other workers, a fairly good, but incomplete

agreement was obtained. For example, Lewis gt gt. (1965)

 

found that the treatment of B. gtearothermophilus spores at
pH 1.5 and 25 C for 80 minutes increased the colony count
from 18% of total count of spores to 80%, and the heat
resistance of spores was greatly reduced. When these acti-

vated spores were exposed to pH 9.7 solution, 0.02 M Ca++,

33

dormancy and heat resistance was restored. Results obtained

with §t_stearothermophi1gg_E-2 revealed over 99.9% activa-

 

tion (of the total count) when the spores were treated at
pH 1.1 and 60’C for 60 minutes. This higher activation
effect is probably due to the combined effect of higher
temperature (60 C) and lower pH (1.1) used. However, even
at pH higher than 1.5 (pH 2.0), more activation of gt
stearothermophilus E-2 spores was obtained (99.9%).“ These
findings indicate that effect of temperature is important
in activating spores at low pH.

In another investigation, Brown gt _t. (1968) reported

that the presence of 0.5 N hydrochloric acid at 25 C in-

creased the colony count of Bt stearothermophilus (NCIB

 

8919) spores to the total microscopic count. Although the
time of exposure was not stated in their work, their data
show that maximum activation occurred at 28-30 minutes, and
that longer exposure to the acid reduced the colony count.
However, 60 minutes were required to obtain maximum activa-
tion at pH 1.1-2.0 in this study. This difference is
probably due to the higher acid concentration, resulting in
a lower pH, used by Brown gt gt. (1968). The reduced colony
counts which were observed when the spores of gt stearo-
thermophilus E-2 spores were treated over 60 minutes may

be due to inhibition or loss of certain factor(s) which
trigger germination or outgrowth. Destruction of the spores
is also another possibility. However, microscopic exami-

nation of activated spores showed that <l.0% of the total

34
count of spores were converted to phase dark spores even
after 120 minutes at pH 2.0 and 60 C. .

These results also indicate that at this specific
temperature and pH a critical time is involved, after which
the amount of activation as measured by the plate count
method was reduced. When the amount of activation was
determined by measuring the heat sensitive spores no reduc-
tion of activation over time was observed, which suggests
that activation has increased but the ability of spores to
form colonies on NAM was impaired.

Results on enumeration of thermophilic flat sour
spores in flour samples indicated that partial activation
occurred when the spores were incubated for 60 minutes at
pH 1.1 and 60 C. These results also suggested that some
of the spores failed to germinate due to interference of
some of the components of flour, such as minerals and salts,
with germination (Halmann, 1962 and Keynan gt gt., 1965) or
interference of carbohydrates with activation (Fields and
Finely, 1964).

Bacterial spores contain relatively high concentra-
tions (4-15% dry weight) of DPA (Halvorson and Howitt,
1961). Also maturation and development of heat resistance
in the bacterial spore parallels the accumulation of calcium
in the spore (Vinter, 1956). During germination: both Ca++
and DPA were released along with the spore peptides (Powell

and Strange, 1953).

35

Alderton and Snell (1963) reported that divalent
cations such as Ca++ and Mg++ can be replaced by hydrogen
ions, in a spore cation exchange system, resulting in a
lowered heat resistance for H-form spores. The sites of
exchange of these cations, which affect heat activation and
heat resistance of mature spores, have not been identified

(Alderton and Snell, 1963; Lewis gt 1., 1965). As stated

by Gould and Dring (1975), much circumstantial evidence
supports the hypothesis that heat resistance of bacterial
spores results partly from the relative dehydration of the
central protoplast or core. The relatively "dry" core might
be achieved by compressive contraction of the cortex caused
by the cation load on the cation exchange system of the
spore (Alderton and Snell, 1963; Lewis et al., 1965).
However, a more recent hypothesis suggested that heat resis-
tance of bacterial spores results from expansion of the
cortex caused by the electrostatic repulsion of negatively
charged groups on the cortex peptidoglycan. The results of
this study indicate that Ca++ plays an important role in
determining the heat resistance of the spores and, the 1
treatment at low pH and mild temperature conditions reduces
the heat resistance and activates the spores. This might
lead to the conclusion that peptidoglycan of the cortex and
DPA present in the spore system are acting like an ionic
buffer system which maintains a low pool concentration of

Ca++ within the spore protoplast, thus maintaining dormancy

and heat resistance of the spore (Gould and Dring, T974).

36

This hypothesis is supported by the fact that when the
spores were treated at low pH in this study, dormancy and
heat resistance of the spores were lost, and the observation
made by Hanson _t gt. (1972) that DPA-negative mutant spores
lost their heat resistance and dormancy rapidly on storage.
Further evidence for the role of DPA in maintaining dormancy
was reported by Zytkovicz and Halvorson (1972) who showed
that DPA-less mutant spores of B. cereus, B. megaterium and
B. subtilis were unable to germinate with customary germi-
nants. These results suggest that Ca++ and DPA were needed

for maintaining the heat resistance and the dormant state

of bacterial spores.

CONCLUSIONS

Some of the conditions for activation of gt stearo-
thermoghilus E-2 were studied. A pH of 1.1-2.0 and a tem-
perature of 60 C for 60 minutes were the best conditions for
activation, while treatment at 70 C resulted in faster
activation.

Activation of spores under these conditions was
paralleled by transformation of spores to a more heat sen-
sitive form. Thus, the degree of activation could be
measured by determining the amount of heat sensitive spores
provided that activation was 390%. Heating at 86 C was
used in this investigation to determine the proportion
of heat sensitive spores. This provided a more accurate
method for determining the degree of activation than the
direct plate counting method. Activation of gt stearo-
thermophilus E-2 spores at low pH may be used under certain
conditions as a substitute for a severe heat shock.

More research is needed to determine the exact time-
temperature-pH combination(s) which should be used to obtain

optimal activation.

37

BIBLIOGRAPHY

BIBLIOGRAPHY

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