{ff-{ERMAL B‘ESTRUCTWN 'D-F ‘BACTERAL SPORES UNDER
GPEN SYSTEM DRYMEAT CONDITIONS

Thesis for the Stages a? M. 8.
3510mm SINK WWERSHY
KENNETH 3. mx

1%?

ABSTRACT

THERMAL DESTRUCTION OF BACTERIAL SPORES UNDER
OPEN SYSTEM DRY HEAT CONDITIONS

by Kenneth I. Fox

The sterilization of surfaces by dry heat presents
many unknown variables. The thermal resistance of the
most heat resistant organisms which could be potential
contaminants on various surfaces must be known. The ef-
fect of the test surface composition on the resistance of
the spores must be determined, and the effectiveness of
the heating procedure must be evaluated.

The purposes of this investigation were to study

the effect of Open system dry heating of Bacillus subtilis

 

Spores, and to determine the effects of gas flow rate on
the thermal resistance of the bacterial spores. The tests
were conducted under conditions of flowing air and nitrogen.
Survivor curve tests were run in a specifically de-
signed dry heat oven. This oven provided accurate temper—
ature control and also allowed air or nitrogen to pass over
the spores during the lethal treatment. Experiments were
carried out at various flow rates of the two gases (air
and nitrogen) and the data were expressed as survivor

curves from which the decimal reduction time (D value) was

Kenneth I. Fox

obtained. Linear regression analysis methods were used to
compute the slope of the survivor curves.

The results of these experiments indicated that at
temperatures below 283’F., increasing the gas flow rate
decreased the D value. However at 283’F., the effect of
increasing the gas flow rate was to increase the D value.

. The resistances observed in the nitrogen tests were about
the same as those obtained in the air studies. This seemed
to mitigate the possibility of oxidation by atmospheric
oxygen as being a major factor in dry heat thermal
destruction of bacterial spores.

These results seemed to indicate that as the gas flow
rate is increased the effect of temperature on the de—
struction rate of the spores is lessened, the 2 value
being very large. It is believed that the higher gas
flow rates cause a greater dehydration of the spores and
that spore moisture loss is the determining factor in dry

heat thermal resistance.

THERMAL DESTRUCTION OF BACTERIAL SPORES UNDER

OPEN SYSTEM DRY HEAT CONDITIONS

By

Kenneth I. Fox

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science

1967

ACKNOWLEDGMENTS

The author wishes to express his appreciation to
his major professor, Dr. I. J. Pflug, for his continued
interest and guidance throughout this study and to Dr.
M. C. Coleman and Dr. R. V. Lechowich who served on the
committee for their suggestions in editing this manu-
script.

Appreciation is extended to Dr. B. S. Schweigert,
Chairman, Food Science Department, for his interest and
support of this program; to Mrs. Helen Erlandson for her
assistance in the laboratory; and to Bruce D. Eder for

his help in editing the manuscript.

ii

to my parents

iii

ACKNOWLEDGMENTS
DEDICATION.
LIST OF TABLES

LIST OF FIGURES

Chapter
I.

II.

TABLE OF CONTENTS

INTRODUCTION. .
LITERATURE REVIEW . . . . . . . .
A. Characteristics of Spores . . . .
1. Composition Related to Heat
Resistance. . . .
2. Characteristics Under Wet Heat .
3. Characteristics of Dry Cells.
B. Kinetics of Bacterial Destruction
C. Destruction of Bacteria by Wet Heat.
1. General.
2. Factors Affecting Wet Heat
Resistance. . . .
a. Medium. . . . . . .
b pH . . . . . . . . .
c. Salts . . . . . . . .
d. Age of Cells. . . .
e. Growth and Sporulation Tempera-
ture O I O O I O O O O
D. Destruction of Bacteria by Dry Heat.

1.

General. . . . . . . .

iv

Page

ii
iii
vii

viii

LA)

C) \1 mzw

l3
13
1A
1A
1A
l2
15

15

Chapter

III.

IV.

Page
2. Factors Affecting Dry Heat
Resistance . . . . . . . . 17
a. Initial Number of Organisms . . 17
b. Water Activity . . . . . . 17
c. Menstruum . . . . . . . . 18
d. Support Medium . . . . . . 19
e. Effect of Test Atmosphere. . . 2l
3. Variability of Dry Heat Data . . . 22
EXPERIMENTAL PROCEDURE. . . . . . . . 2A
A. Design of Apparatus and Description of
Equipment. . . . . . . . . . . 2A
B. Calibration of Dry Heat Oven . . . . 32
1. Temperature. . . . . . . . . 32
2. Heating Time . . . . . . . . 32
3. Heating of Cups . . . . . . . 33
A. Cooling of Cups . 33
5. Temperature Variation of Cups With-
in the Same Bar . . . 36
6. Temperature Fluctuation Among Bars . 36
7. Operating Range of the Oven . 38
8. Calibration of Thermistor Amplifier. 38
C. Description of Operation of Oven . . . 39
D. Preparation of Spores. . . . . . . A0
1. Growth and Harvesting . . . . . A0
2. Initial Count . . . . Al
3. Preparation for Heating Tests . . A2~
E. Methods of Analysis . . . . . . . AA
F. Method of Linear Regression. . . . . A6
RESULTS AND DISCUSSION. . . . . . . . 50
A. Decimal Reduction Times . . . . . . 50
B. Thermal Resistance--z Values . . . . 6A

Chapter Page
C. Shape of Survivor Curves . . . . . 66

D. Possible Causes for Variability Among

Replicate Plate Counts. . . . . . 69

V. CONCLUSIONS. . . . . . . . . . . 72
APPENDIX . . . . . . . . . . . . . . 73
LITERATURE CITED . . . . . . . . . . . 85

vi

01

LIST OF TABLES

Lag Correction Factors, and f and 3 Values
for Cups Heated in Dry Heat Oven in Still
Air . . . . . . . . . . . .

Initial Spore Counts Throughout the Testing
Period . . . . . . . . .

D Values (min) for Individual Thermal
Destruction Tests Showing 80 Percent
Confidence Limits

D Values (min) for Individual Thermal
Destruction Tests Showing 95 Percent
Confidence Limits . . . .

Average Linear Regression D Values in
Minutes After Discarding Data Points Be—
lieved to be in Error . .

2 Values for Various Gas Flow Conditions.

vii

Page

3U

AA

52

53

SA
SA

Figure

|>-’
I
(3

\J'I

CD

10.

11.

LIST OF FIGURES

Page
Bottom Block of Dry Heat Oven . . . . . 27
Assembled Top and Bottom Blocks of Dry
Heat Oven. . . . . . . . . . . 27
Dry Heat Oven Showing Upper Position of
Sample Shelf. . . . . . . . . 28
ry Heat Oven Showing Petri Dish in
Position for Receiving Sample Cups . . 28
Diagramatic Pattern of Gas Flow Through
Dry Heat Oven . . . . . . . . . 29
chematic Block Diagram of Electrical
Circuit Controlling Dry Heat Oven. . . 31
Heating Curve for Sample Cup at 265° F.
in Still Air. . . . . . . . 35
Special Sample Bar Allowing Passage of
Six Thermocouple Wires . . . . . 37
Thermal Resistance Curve for Dry Heating
in Therva l Death Time Cans . . . . 56
Thermal Resistance Curve for Wet Heating
in Thermal Deat Time Cans . . . . . 57
Thermal Resistance Curve for Still Air in
Dry Heat Oven . . . . . . . . . 58
Thermal Resistance Curve for 1.4 cfm Air
Flow in Dry Heat Oven. . . . . . . 59
Thermal Resistance Curve for A.O cfm Air
Flow in Dry Heat Oven. . . . . . . 60
Thermal Resistance Curve for 1.4 cfm
Nitrogen Flow in Dry Heat Oven. . . . 6l

viii

Figure Page

12. Thermal Resistance Curve for 3.0 cfm
Nitrogen Flow in Dry Heat Oven . . . . 62

13. Sample Survivor Curve for Bacillus subtilis
in Dry Heat Oven (Still Air) at 2850 F. . 67

 

ix

I. INTRODUCTION

The wet heat destruction of bacteria has always been
important to the medical profession, the food industry and
the drug industry. Recently with the advent of the space
age, dry heat sterilization has become important for steri—
lizing interplanetary probes. The sterilization of space-
craft has provided a considerable impetus to dry heat
thermal resistance research.

Very little is known about factors affecting dry heat
ucticn of bacteria. The obvious parameters such as
time and temperature necessary for the destruction of

acterial spores are still not completely understood be-

(:7

cause another variable, moisture level, is implied. The<5
surfaces on which the bacterial spores are present have
considerable effects on their resistance to dry heat. The

atmosphere in which the spores are heated presents many

N

roblems to the scientist who tries to predict bacterial

r

resistance. The moisture content of the atmosphere, its
chemical composition and its pressure are all variables
which are still not completely understood in their relation—

ship to dry heat resistance of bacterial spores.

It is the purpose of this study to try to understand
and gain insight into two of these variables: (I) the

effect of nitrogen vs. air and (2) the gas flow rate.

II. LITERATURE REVIEW

A. Char cteristics of Spores

H)

 

;. Composition Related to Heat Resistance
The formation of spores is perhaps one of the most
outstanding characteristics of the bacterial genera

iacillus and Clostridium. The spores of these genera are

 

characterized as being highly refractile, and having high=v
density. These spores, by virtue of their dormant state
maintain greatly reduced metabolic activity. It has been
oostulated that in the genus Bacillus the spore consists
of a central cortex surrounded by a spore coat of protein,
iWarth, Ohye, and Murrell, 1962). The central cortex has
been shown by electron microscopy to be a layered matrix
'Mayall and Robincw, l957).

One compound which has been consistently found in
Lhe genus Bacillus is dipicolinic acid (DPA) (Church and

“Ivorson, 1939); the DPA content of Bacillus spores usually

ranges from 5 to lU percent dry weight (Murrell and Warth,
1965). This DPA is always bound by calcium ions so that
noInatter what the DPA content, the calcium-dipicolinic
acid ratio is approximately one (Murrell and Warth, 1965).

These authors (Murrell and Warth, 1965) correlated

*arious factors to heat resistance of the spores. They

developed multiple regression correlation equations for

seven variates, and co.related these variates to heat

resistance. These variates were: dipicolinic acid,
iiamincpimelic acid, hexosamine, spore weight, Mg-Ca, Ca,

Lg. Inese e/uations enable heat resistance to be pre—
iitted with a fair degree of precision.
The calcium content appears to have an effect on
.he heat resistance of bacterial spores; increasing calcium
cntent appears to increase the spore resistance to wet
heat (Curran, Brunstetter and Meyers, 1943). Sugiama
1951), Amaha and Ordal (1957) and others have observed
‘ne ia:t that calcium is needed in the development of heat
'esistant spores. Furthermore, d,e-diaminopimelic acid
:DAP) has also been shown to be a constituent of heat

resistant spores (Warth et al., 1962).

 

 

 

 

c-3r“:t_-ls‘l:s Inger et Feat
. fl - . A“ A. a} ,‘ 1" ’. ,I. a [I I.\ ‘, > ‘,_‘ ,
i 1 a a c aa' (ij/ DHVWCQ that the heat TESlStanctt

pres increases with increasing calcium ion

(i)

he effect of holding the spores

in calcium buffers demonstrated by Alderton, Thompson and
Snell (196A) may be to increase their calcium ion content
and subsequently their heat resistance. It is questionable
whether the resistance would increase when held in solutions
sther than calcium buffers. Alderton, Thompson and Snell

al96A) stated that holding of spores in sodium buffer at

pH 5.7 gave only minor enhancement of heat resistance,
but at higher pH the sodium ion was effective in conferring
heat resistance.

Because of the relationships between DPA content and
heat resistance, and observations that spore development
temperature affects heat resistance, the central cortex
appears to be functional in heat resistance. The moisture
content of the spores is believed to have an effect on
their heat resistance. Early investigators (Cohn, 1877;
Burke, 1923) thought that the spore contained a water im-
permeable membrane. This has been challenged by Murrell
and Scott (1958) and Black and Gerhardt (1962).

Henry and Friedman (1937) postulated a theory for the
heat resistance of bacterial spores, which claimed that
most of the water in the spore is bound water, and that
this bound water may be inactive in so far as its influence
on the coagulation of protein material by heat is concerned.
Friedman and Henry (1938) determined the bound water con—

w—

tent for spores and vegetative cells of Bacillus subtilis, 2.

 

§

negaterium, and B. mvcoides. They found that in all cases,

——-

 

the spores had a greater water binding capacity than did
the corresponding vegetative cells. Waldham and Halvorson
(195A) determined the relationship between equilibrium

vapor pressure and moisture content of bacterial spores.
/

They proposed that the spore is heat resistant because the *-
proteins in the spore are immobilized through linkages in—

volving the polar groups and some solid material.

Sadoff et al. (1965) investigated the effect of
various solvents on the properties of glucose dehydrogenase

from spores of Bacillus cereus in an effort to understand

 

the mechanism of its heat resistance in spores. These
authors found a million fold decrease in the heat re-
sistance of the glucose dehydrogenase in Yllg when spores
germinate. They found that this same range of heat re-
sistances can be produced with isolated enzymes when sub—
jected to various ionic environments. These experiments:
suggest that the heat resistance of bacterial endosporesi
may be a function of the heat resistance of the enzymes.“”
It is known that several enzymes which are present in
spores show greater heat resistance than the enzymes present
in the vegetative cells (Stewart and Halvorson, 195“).
There is also evidence that the more heat resistant bac-
terial spores are possibly more strongly disulfide bonded

in the protein layers of the cell (Murrell, 196A).

3. Characteristics of Dry Cells

 

Black and Gerhardt (1962) found evidence that the
spore consists of a central core which is kept relatively
low in moisture in the dormant spore. Since dry proteins
are more thermostable than moist proteins, this may have
some bearing on heat resistance. Black and Gerhardt (1962)
in their studies of the permeability of spores found that
the water is distributed unevenly, with the core having

little water and a high density. Finally these investigators

proposed that the core of the dormant spore exists as an
insoluble and thermostable gel, in which cross linkages
between macromolecules occur through stable but reversible
bonds so as to form a high polymer matrix with entrapped
free water.

The theory of a contractile cortex system in the
bacterial spore would provide a mechanism to dehydrate the
protoplast and a mechanism for maintaining this state.

The contractile cortex theory is compatible with the cur—
rent data available on water permeability of bacterial
spores (Lewis, Snell and Burr, 1960). Since it seems
probable that there is free passage of water into and out
of the bacterial spore (Black and Gerhardt, 1962), this
water need not be entirely excluded by mechanical pressure

from the cortex.

B. Kinetics of Bacterial Destruction

 

To discuss the death of bacteria, we must have some
suitable criterion of death.; Schmidt (195a) has stated
that the only single practical criterion of death of micro—
organisms is their failure to reproduce, when as far as is
known, suitable conditions for reproduction are provided.
If we assume that this definition of death is satisfactory
then we must inquire into what causes a cell to lose its
ability to reproduce.

Amaha and Sakaguchi (1957) have stated that the death

of bacterial spores by heat is a gradual chemical process

which proceeds step by step. Rahn (l9U5) proposed a
logarithmic order of death for bacteria. This theory
stated that the death of a cell is due to the inacti—
vation of a single critical molecule. He therefore
asserts that the death rate should follow the same
kinetics as does a monomolecular decay reaction.

The expression for this rate of death is then

 

Where,
N = number of organisms remaining alive
k = rate of destruction (constant)
t = time

By integrating this equation

k
IgfiL—kfdt

we obtain,

1n N = —kt + C

where C = constant of integration
using the initial conditions where t = O and N = N

0)

log N0 = C

log N - log N0 = —kt

Katzin, Sanholzer, and Strong (19A3) obtained the follow-
ing equation for a 90 percent reduction in the original

population:

k = x log NO/0.1NO

(‘le

Here they defined t as the decimal reduction time.
Schmidt (195A) changed the equation slightly by using D
for decimal reduction time and substituting it for 1/k

obtained

D = t/(log NO — log N)

This equation then suggests a straight line relation—
ship if we plot log N vs. t. This is the equation of the
Logarithmic order of death as it was originally proposed
by Rahn.

Much evidence appears to support the logarithmic
theory, (Madsen and Nyman, 1907; Watkins and Winslow, 1932;
Rahn, 19A5), however, many investigators have obtained
survivor curves which are not linear. A number of workers
have obtained wet heat survivor curves which were concave
downward (Amaha and Ordal, 1957; El-Bisi and Ordal, 1956;
Licciardello and Nickerson, 1963, Lechowich and Ordal,

1962 and Fox, Eder and Pflug, 1967). Other investigators
have found wet heat survivor curves which were concave up-

ward (Frank and Campbell, 1957; Walker, Matches and Ayres,

1961; Fox, Eder and Pflug, 1967).

10

Various explanations have been offered for these
nonlogarithmic curves. Rahn (19A5) suggests that curves
which are concave downward are caused by clumping of the
individual bacterial cells. Fox, Eder and Pflug (1967)
gave as a possible cause of their concave upward dry heat
survivor curves, the fact that a mixed culture might be
present. Rahn (19A5) suggests that curves which are con—
cave downward are caused by clumping of the individual
bacterial cells. El—Bisi and Ordal (1956) explained their
concave downward wet heat survivor curves as follows:
during relatively short heating times some of the viable
spores were not heat activated and as a result germination
did not occur when these samples were plated. At longer
times, the number not activated diminished. Because the
number of spores surviving at short times was underesti-
mated, the survivor curves did not drop logarithmically
until all viable spores were heat activated.

C. Destruction of Bacteria
by Wet Heat

 

 

1. General

 

Wet heat is defined as heating in an environment
of 100 percent relative humidity or an environment in
which the water activity is very close to 1.00. The water
activity is the moisture content of the spore which is in

equilibrium with the moisture in the surrounding atmosphere.

11

Wet heat refers to the heating medium being saturated
steam above 212° F. or water at temperatures below 212° F.

One of the first theories for the destruction of
bacteria was proposed by Lewith (1890). He proposed that
the coagulation of protein was the cause of death of bac-
terial spores for both wet and dry heat. His theory re—
garded the moisture content of the bacterial cell of extreme
importance in determining its heat resistance. He therefore
believed that bacteria were much more labile to wet heat
than to dry heat because of the greater thermolability of
moist proteins.

Rahn (1945) in explaining his logarithmic order theory ,
postulated that a single critical molecule was coagulated orI
denatured and this caused the cell to die. Ingraham (1962)
proposed that a hereditary factor in the cell was somehow
inactivated by the heat.

To understand the destruction of bacterial spores in
wet heat it is necessary to discuss some of the processes
involved in producing spores from vegetative cells. The
genus Bacillus forms spores under a variety of conditions.
These conditions of spore formation affect the heat resis-
tance of the final spores. The transformation of a vege-
tative cell into a spore is accompanied by a number of
changes in the physiology and biochemistry of the bacterial
cell. Although the complete mechanism of spore formation
has not been elaborated, a number of factors have been

shown to promote sporulation in the Bacillus species.

l2

Srinivasan (1965) found that extracts of spore formers
possess the ability to transform vegetative cells of B.
cereus to granulated cells having the property of sporu-
lating endotrophically i.e., sporulating in distilled water.
Amino acids appear to play an important role in the sporu—
lation process; Bernlohr (1965) found that during sporu-
lation there is an increase in the intracellular pool of
many amino acids. Isoleucine and valine are oxidized to
carbon dioxide during sporulation in B. licheniformis. Many
workers (Amaha and Ordal, 1957; Murrell and Warth, 1965)
have found that Mn++ ions will induce sporulation in the
genus Bacillus.

Before spores of bacteria will germinate, they must
be either heat activated or somehow chemically treated.
Rieman and Ordal (1961) showed that certain B. subtilis
spores can germinate without heat activation on alanine
initiation, merely by the addition of calcium and DPA.
Since the activity of alanine dehydrogenase has been found
to be very high in germinating spores, O'Connor and Halvorson
(1960) proposed that alanine and some of its analogues
initiate germination since they are deaminated by alanine
dehydrogenase. The method of heat shocking spores to
cwuise them to germinate was first used by Curran and Evans
(1945). Heat activation causes various changes in the
spcnres, stimulates germination, activates enzymes which
are <dormant in the resting spores, and changes some of the

requirements for germination. The exact temperatures and

13

times required for heat activation vary widely with the
particular type of bacterial spore (Busta and Ordal, 1964).
Keynan gB_a£. (1964) suggested that the activation changes
the tertiary structure of a protein responsible for the
maintenance of the dormant state of spores, and that this
is a type of reversible denaturation. However, if germi-
nation is not induced by placing the spores on a suitable
nutrient medium immediately after heat activation, the
spores will revert to the dormant state (Curran and Evans,
1945).

After activation has occurred, the spore loses its
refractility and assumes the characteristics of a vege-
tative cell. Levinson and Hyatt (1956) observed that
special nutritional requirements are necessary to convert
the spore into a vegetative cell and these requirements are
different than those for germination. At_the initiation of
outgrowth new proteins are formed which were not present in
the resting spore (Kobayashi gg_al., 1965). Finally, during
outgrowth, a new vegetative cell is produced with all the

characteristic properties.

1 "I Q‘\
\

\

2;“ Factors Affecting Wet Heat Resistance

 

a. Medium.--There are a number of factors which can
affect the observed wet heat resistance of bacteria. Frank
and Campbell (1955) have found that the survival time of
spores was longer using certain subculture media which

might indicate that a severe reaction is necessary to make

14

a spore incapable of reproduction in a nutritionally more
complete subculture medium.

b. pB.--The pH of the wet heat environment is very
important to the wet heat resistance. Most proteins and
other biological compounds have their maximum molecular
stability at or near pH 6.8. For this reason the observed
resistance of bacteria in an acid medium might be much
lower than at neutral pH.

c. Ba£3§.--Salts can play an important role in the
destruction of bacteria by wet heat. Since proteins, DNA,
and other macromolecules present in the bacterial cell are
largely stabilized by hydrogen bonds, any change in the
ionic strength of the environment may tend to rupture some
of these hydrogen bonds, and therefore lower the heat re-
sistance. Certain compounds such as quarternary ammonium
salts are believed to interfere with bacterial fermentation
or respiration by inhibiting the enzymes of carbohydrate
metabolism.

d. Age of Cells.—-The age of the cells may have some

 

effect on their exhibited heat resistance. Esty and Meyer
(1922) found that young moist spores are more resistant
than old spores under wet heat conditions, however, Williams
(1929) found no correlation between the age of the spores
and their resistance when several species of organisms were
examined.

e. Growth and Sporulation Temperature.--The temper-

amire at which the spores are grown appears to have some

15

effect on the resistance of the spores. Early studies

showed that the resistance of spores of Bacillus anthracis

 

increases with increasing growth temperature (Weil, 1899).
El-Bisi and Ordal (1956) found the resistance of Spores of

a strain of Bacillus coagulans to be greater with increased

 

sporulation temperature.

It can be safely said that many compounds will have
an effect on the heat resistance of bacterial spores. For
example, Sugiama (1951) found an increase in the heat
resistance of bacterial spores when heated in a menstruum
containing some soluble carbohydrate. Jensen (1945) found

an increase in the resistance of Streptocci sp. when heated

 

in a medium high in fat instead of a purely aqueous medium.

D. Destruction of Bacteria
by Dry Heat

 

 

1. General

 

The destruction of microorganisms by dry heat is cer-
tainly different in magnitude and may also be physiologically
different than destruction by wet heat. Unlike wet heat
which is precisely defined as heating in a medium consist-
ing of 100 percent relative humidity, dry heat is much less
readily defined and is usually taken to refer to the de-
struction of microorganisms in any environment where the
relative humidity is less than 100 percent or where the
water activity is less than 1. It is often assumed that

dry heat is an equally specific condition as is wet heat;

16

this is not true: dry heat includes an endless number of
combinations of very different conditions. Some of these
conditions are: (1) initial water content of organisms
before dry heating, (2) the nature of the liquid medium or
menstruum from which the organisms are dried, (3) the
relative composition of the gas atmOSphere in contact with
the organisms during dry heating, and (4) physical nature

of the material in contact or supporting the dry spores.

Va

Early studies on the dry heat resistance of bacterial

spores of Clostridium botulinum brought a new era into the

 

 

study of heat resistance of microorganisms (Tanner and Dack,
1922). The study of dry heat resistance was then relatively
slow due to the difficulty in defining dry heat and the
apparent greater variability of resistance to dry heat than
to wet heat. Later Collier and Townsend (1956) studied the

resistance of Bacillus stearothermophilus, B. polymyxa and

 

Putrefactive anaerobe 3679 under superheated steam. Pflug
(1960) performed dry heat resistance experiments on Bacillus
subtilis spores in superheated steam. It was found that
the resistance to superheated steam was much greater than
the resistance to saturated steam.

Pflug and Augustin (1961) found larger resistances
when spores of B. subtilis were exposed to dry hot air than
to steam. Jacobs, Nicholas and Pflug (1965) studied the

heat resistance of Bacillus subtilis spores in atmospheres

 

of different water content. They held the test spores in

17

thermal death time cans (Pflug and Augustin, 1961) main-
tained at water vapor contents ranging from 0 to 100 percent
controlled by various hydrated salts. Their results showed
that the resistance of the spores was very much dependent :
upon the moisture content of the atmosphere in which the
spores were heated.

.—""\
.' \_

52. ,Factors Affectinngry Heat Resistance

 

Murrell (1964) classified factors affecting dry heat
resistance as occurring before heating, during heating, and
after heating. The factors present during heating are
those which are pertinent to a study of dry heat resistance.

a. Initial Number of Organisms.--Sisler (1961) deter-

 

mined survivor curves for different initial numbers of spores

of Bacillus subtilis heated in wet steam at 250° F. and in

superheated steam at 320° F. The D value for the 106

7

 

Spores/m1 was greater than the D value for the 10 spores/ml
when tested in superheated steam. These differences were
not observed with wet steam.

b. Water Activity.--The effect of water activity has

 

been found in many cases to affect the exhibited resistance
of spores to dry heat (Murrell and Scott, 1957; Marshall,
Murrell and Scott, 1963). Recently Murrell and Scott (1966)
have reported a method in which they controlled the water
activity (aw) of the spores during the actual heat treat-
ment. These workers found a maximum resistance when the

controlling water activity was around 0.3. In all cases

18

they found a decrease in resistance as the water activity
approached either 0 or 1, the resistance being much lower
at water activity of 1.

c. Menstruum.--Fry and Greaves (1951) found that the

 

survival of spores during and after drying was very dependent
upon the liquid or menstruum from which they were dried.

They found that when bacterial cells were dried from plain
distilled water, their survival time in the dried state was
shorter than if the cells were dried out of some other sol-

utions. When they dried cells of Staphlococcus aureus from

 

glucose solutions, they found a much greater survival time.
These workers proposed that the glucose acts as some sort
of protective colloid. They claimed, however, that a much
more probable explanation of the sugar effect is that the
presence of glucose insures the retention of a certain amount
of moisture in the dry spores which is necessary for sur-
vival. Annear (1956) found that freeze dried bacteria could
be preserved for long periods of time by suspending them in
peptone solutions prior to freeze drying. He attributed
this increased survival to the fact that the peptone pro-
tects the cells from becoming too dry during the freeze dry-
ing process.

Heller (1941) found that significant decreases

occurred in the death rates of freeze dried Streptococcus

 

pyogenes and Escherichia coli when the organisms were dried

 

in a menstruum containing crystalline compounds of high

l9

solubility and which the organism was capable of utilizing

in normal fluid media. When Streptococci were dried in

 

the presence of colloidal substances, the death rates de-
creased with increases in the hydrophylic property and in
the protective colloid effect of these compounds.

d. Support Medium.——Another factor which appears to

 

influence the dry heat resistance of bacterial spores is
the support medium. Augustin (1964) found that the dry
heat resistance of Putrefactive anaerobe 3679 was higher on
tin than on aluminum. Pflug and Fox (1966) determined the

dry heat resistance of Bacillus subtilis spores on various

 

support media. They found that the highest resistance
occurred when the spores were heated on tin. Aluminum,
glass and filter paper showed respectively decreasing spore
resistances. The reason for the very low resistance of the
spores on the filter paper might be attributed to the fact
that on this medium the spore suspension diffused through
the paper and thus the spores were relatively scattered
apart as compared to clumping of the spores on the tin or
aluminum. It is possible that when the spores are very close
together as on the tin and glass, there might be some pro-
tective effect of the cells themselves. Another possibility
is that when the spores are clumped they might retain Just
enough water to permit them to survive longer (an effect

similar to that found by Fry and Greaves (1951)).

20

Many workers have experienced differences in resis—
tance of spores to dry heat when supported on different
surfaces (Angelotti, 1967; Fox and Pflug, 1967; Bruch,
1963). These workers in trying to determine sterilization
parameters for the National Aeronautics and Space Adminis-
tration (NASA) have found that one of the main problems in
this area is the recovery of the organisms from the surfaces.
At the present time there are many different methods avail—
able and in use for recovering the spores from the test
surfaces. Some of these include soaking the surface in
water solutions, shaking these solutions, scraping the
surface to loosen the spores, using ultrasonicators for re—
moving the spores, and breaking up the surface. Puleo 2243;:
(1966) have reported a system in which they used two methods
for removing spores from surfaces and within various solids.
In one method they pulverized the surface material (usually
a plaster—like material) in a Waring blender; in the other
:nethod they used a mortar and pestle. They found that the
best recovery was obtained using the mortar and pestle.
Other workers (Favero, 1966) have obtained satisfactory
.results using an ultrasonicator to remove the heated spores
from the test surface material.

The fact that the surface spores are very difficult
to remove may be the reason for some of the variation re-
ported in dry heat data. Bruch, Koesterer, and Bruch

(1963) determined the dry heat resistance of several

21

species of spores using various support media. These
investigators used paper, glass and sand for supporting

the dry spores for thermal destruction testing. They

found that in most cases the highest D value occurred when
the support medium was sand and the lowest D value occurred
using the paper strip. These investigators also determined
the z value for B. subtilis v. giggg in dry heat. They
found a z value of 49° F. for this organism. This is

quite high when compared with a z of 17° to 25° F. usually
obtained with this organism in wet heat.

e. Effect of Test Atmosphere.-—Pheil et a1. (1967)

 

studied the effect of various gas atmospheres on the dry

heat resistance of bacterial spores. They found that the
resistances of B. subtilis in air and carbon dioxide were
essentially the same, but higher in helium and nitrogen.
Oxygen gave values close to those obtained with air and
carbon dioxide. These results seem to lessen the possibility
of some type of oxidation mechanism being involved in dry
heat bacterial destruction.

Silverman (1966) found that the dry heat resistance
of various species of spores was greatly reduced when the
spores were heated in an atmosphere with dry air flowing
over the spores at the rate of 3 liters per minute. He
also found that the resistance increased as the moisture

Content of the air increased.

22

3. Variability of Dry Heat Data

 

At the present time the data resulting from dry heat
experiments are rather inconsistent and very difficult to
reproduce. Many workers have found difficulty in repli-
cating plate counts during various dry heat resistance
experiments (McDade, 1967; Angelotti, 1967; Fox, 1967).
These workers have found variability among replicate sam-
ples during dry heat testing to be as much as ten fold
and often greater. The causes of this variability have
not yet been explained. One of the most promising explan-
ations is the poor recovery of spores from surfaces exper-
ienced in most cases as previously discussed. Other factors
which might cause some variation have been listed by Bruch
(1963) as factors which appear to affect the dry heat resis—
tance of bacterial spores: (1) effect of spore carrier on
dry heat resistance of bacterial spores; (2) effect of
spore nutrition on dry heat resistance of bacterial spores;
(3) effect of sporulation environment on the dry heat re—
sistance of bacterial spores; (4) effect of recovery environ—
ment on the survival of spores exposed to dry heat; (5)
effect of exposure environment on microbial dry heat re-
sistance; (6) effect of combination of sterilizing agents
on the rate of microbial destruction.

The variability of dry heat resistance data is very
great as exemplified by the findings of the Spacecraft

Sterilization Advisory Committee. This committee found

23

decimal reduction times for B. subtilis v. 21522 heated

on stainless steel surfaces at 125° C. to vary from 8
minutes to 40 minutes when determined in various different
dry heat testing apparatus (Spacecraft Sterilization

Advisory Committee, 1967).

III. EXPERIMENTAL PROCEDURE

Design of Apparatus and Description
of Equipment

 

 

One of the primary objects of this research was to

provide an accurate method for heating the spores. Exist-

ing devices were not satisfactory, therefore a new heating

device was needed that would satisfy the following con—

ditions:

1.

The temperature of the spores should remain
constant throughout the test period. An
accuracy of plus or minus one degree F was
desired.

The device would hold six sets of samples, each
set consisting of six replicates, each set of
replicates removable from the oven without dis—
turbing the other samples.

Reproducible heating and cooling times are
needed; the come-up time of the samples should
be as short as practicable and the cool

down time of the samples after removal from the
oven as rapid as practicable.

The method of removing the samples from the oven

after heating must be aseptic.

24

25

5. Provision should be made for preheating air or

other gases used in gas flow tests.

A simple device was designed and constructed to meet
the above criteria; it was constructed and performed satis-
factorily. The apparatus or oven that was constructed can
be thought of as a large metal block 5 x 8.5 x 9 inches
with six slots for the six sets of samples. The large mass
of metal with its considerable thermal capacity simplifies
temperature control problems. Five sides of the block
were insulated leaving only the face of the block exposed.

The oven was constructed from two large aluminum
blocks each 2.5 x 8.5 x 9.0 inches. Figure l—a is a photo-
graph of the bottom block illustrating the heating chambers;
each chamber is fitted with a sample bar containing six
positions for samples. Each sample bar has a push rod made
of heat resistant material. The push rods are adhered to
the sample bars with epoxy resin.

Figure l-b shows the assembled top and bottom blocks;
each block contains two electrical cartridge heaters. A
Honeywell L7038 thermistor is located in the bottom block.
This thermistor, when used with a Honeywell R70810 temper—
ature controller (described later) gives : 0.3° F. tempera-
ture control over the range 220° F. to 320° F.

Slits l/l6-inch in width, 5—inches in length and
1-inch deep to heat the incoming gas are located in the
top block. The gas enters through an opening on the side

of the unit and goes into a manifold that feeds the six

26

sets of slits“ The gas flows to the rear of the oven
through the heating slits. Each set of slits has a hole

at the rear which carries the gas to the rear of the sample
heating chambers. The gas then flows over the spore sam—
ples and out the front of the oven. Figure 2 shows a
diagramatic view of the gas flow path through the oven.

An aluminum plate l/8-inch thick placed on top of the

upper block seals the gas heating area.

To minimize the heat loss from the unit the entire
device is located inside a 3/4—inch thick plywood box.
Spacers keep the plywood l/2-inch from the surface of the
aluminum.

Attached to the front of the oven is a movable shelf
that can be located in two positions. The first position
is directly under the sample bars as shown in Figure l-c.
With the shelf in this position, the sample cups are
placed in the bars. The shelf enables all 36 sample cups
to be loaded at the same time. The six bars are then
pulled into the oven simultaneously by means of the cross
bar located at the rear of the oven.

After loading, the shelf is moved to its second
position, about 1—inch below the previous level. In this
position the shelf can hold a sterile petri dish for re-
ceiving the sample cups. As shown in Figure l—d a bar
has been pushed out and the sample cups have fallen into
the sterile petri dish. The detailed operation of the

OVen will be described in a later section.

27

 

Figure 1-a (Top).--Bottom block of dry heat oven.
1-b (Bottom).--Assembled top and bottom
blocks of dry heat oven.

28

 

Figure 1-c (Top).—-Dry heat oven showing upper position

of sample shelf.
l-d (Bottom).—-Dry heat oven showing petri dish

in position for receiving sample cups.

29

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77

 

 

21:1?ij \

 

 

 

 

 

d!

I

 

 

whopcm mam

30

The temperature controller is a Honeywell Versa—
Tran Transistorized amplifier relay #R708lC, designed to
control a heating load. Using the L7038 thermistor sensor
it was possible to control the oven heaters so the oven
temperature variation was within a temperature differential
of approximately 0.3° F. This temperature controller
actuated a normally open 10 amp relay which in turn con-
troled the input current to the four heating elements;
the heating elements are 3/8—inch diameter x 6-inches long
and are rated at 250 watts at 120 VAC. Figure 3 shows a
schematic block diagram of the oven electrical control
circuit. Eighty minutes were required for the oven to heat
from room temperature to 260° F.; a timing device was in-
cluded in the electrical circuit so the oven could be auto—
matically turned on 2 hours before testing was to be started.

To study the effects of gas flow rates on the rate of
destruction of the bacterial spores a system was designed
to carry and meter the gas from the compressed gas cylinders
to the oven. The gas flow rate was controlled by a dia—
phragm pressure reducing valve attached to the gas cylinder.
The gas was metered using a previously calibrated rotameter.

It was important that the gas flowing over the spores
be at the same temperature as the spores. By inserting
thermocouples in the chambers within the oven Just above
the positions where the spores are held in the cups, it

was found that the gas temperature was always within 1° F.

31

thernfistor

 

 

 

 

heaters

 

 

 

 

 

 

 

 

 

 

 

#—

 

 

 

 

 

 

 

 

amplifier

thner

control

 

 

 

Figure 3.--Schematic block diagram of electrical
circuit controlling dry heat oven.

32

of the oven temperature. This was verified at both the

high flow rate (4 cfm) and the low flow rate (1.4 cfm).

B. Calibration of Dry Heat Oven

 

To calibrate the oven, it was necessary to determine
a number of parameters: temperature control cycle and
fluctuation, heating up time, heating and cooling times
of the sample cups, temperature variation among cups with—
in the same bar, temperature variation between bars, and.

operating range of the oven.

1. Temperature

 

The temperature of the oven was found to follow a
cyclical pattern; the magnitude of the fluctuation at
260° F. was about 0.5° F., and the cycle time was 5.5
minutes. Since the oven was provided with several thermo-
couple holes, it was possible to measure the temperature
at various points across the block. It was found that
the greatest temperature differential in the block was

about 1° F.

2. Heating Time

 

The oven required about 80 minutes to heat from an
initial temperature of 75° F. to an operating temperature
of 260° F. The heating curve (temperature vs. time) is

linear.

33

3. Heating of Cups

 

To determine the heating and cooling times of the
sample cups a small (30 gauge) thermocouple was soldered
in the bottom of a sample cup. This cup was then loaded
into the special bar and pulled into the oven. Using a
Honeywell Recording Potentiometer, the temperature was
recorded every 5 seconds during the heating of the cups.
These time-temperature data were then plotted by the
method described by Ball and Olson (1957). This method
involves plotting the temperature difference, heating
medium temperature-sample cup temperature, on inverted
semi-logarithmic coordinates versus heating time. The
temperature response parameter f is defined as the time
required for the resulting linear heating curve to traverse
one log cycle (Kopelman, 1966). The heating lag is ex-

pressed by

where Ta is the ordinate of the origin of the

asymptote
T1 is the temperature of the heating medium
T0 is the initial temperature of the sample

cups

4. Cooling of Cups

 

To determine how rapidly the cups cool after removal

from the oven into the sterile petri dish, cups with the

34

small thermocouples were pushed from the oven and allowed
to cool while the temperature was recorded on the potentio—
meter. These data were plotted as cooling curves (Ball and
Olson, 1957) and the fC (temperature response parameter for
cooling) and Jo (cooling lag) were calculated.

Figure 4 shows a sample heating curve for the cups.
Table 1 shows the f and 3 values of the sample cups for
various oven temperatures determined from the various heat—
ing curves and also the lag correction factors. The lag

TABLE 1.——Lag correction factors, and f and J values for
cups heated in dry heat oven in still air.

 

 

 

f f J J Lag Correction Factor (min)
Temp. h c h c
°F. (sec)(sec) z=10 20 30 40 60 80
255 117 80 0.71 1.0 1.8 1.3 0.9 0.67 0.28 0.02
265 120 91 0.75 1.0 2.5 1.9 1.5 1.4 0.87 0.61
275 110 78 0.70 1.1 1.9 1.4 1.0 0.80 0.45 0.20
285 135 84 0.33 1.0 1.8 1.1 0.67 0.40 —- ——

 

correction factor is the time which should be subtracted
from the actual heating time in a thermal resistance study
since it represents the initial heating time where no
significant lethality is accrued and the lethality of the
cool that occurs after the end of the heating time. The
lag correction factors were calculated by the method of

Pflug (1966). Since these values were relatively small,

35

 

264 3.

I

”ll!

 

255

f= 120 sec.
j = 0.75

 

165

 

TEMPERATURE,°F

1

III [I

 

 

 

I I I I I I I
25 50 75 100 125 150 I75

TIME-SEC.

Figure 4.--Heating curve for sample cup at 265° F. in
still air.

36

it was decided to discount them as being insignificant

in plotting the survivor curves.

5 Temperature Variation of Cups
Within the Sam Bar

 

 

To determine the temperature variation

of the six

cups within one bar a special bar was prepared with a

hole drilled as shown in Figure 5. A sample
30 gauge thermocouple soldered to the bottom
in each hole and the six pairs of lead wires
the hole in the bar through the push rod out

the oven and then to the potentiometer. The

cup with a

was placed
passed through
the rear of

bar was moved

into the oven, and after the cups had reached oven tempera-

ture the temperatures were recorded on the 12 point record—

ing potentiometer. The temperatures of the six cups within

the same bar agreed to within 0.5° F. It is
of this temperature difference may have been

thermocouples themselves.

6. Temperature Variation Among Bars

 

felt that part

due to the

To determine if there was any temperature variation

among the six bars, each bar was provided with a thermo—

couple inserted through the push rod into the thermocouple

hole at the rear of the bar. A set screw at

the rear of

the bar held the thermocouple in place. In this manner

each bar temperature could be measured each 5 sec. using

‘the recording potentiometer. It was found,after observing

tlie recorder output for this test for several hours, that

 

 

 

 

 

 

 

 

 

 

 

 

38

there were essentially no differences in bar temperatures.
Due to the method of printing of the temperatures and the
inherent error in the potentiometer only differences

greater than about 0.4° F. can be discerned.

7. OperatingfiRange of the Oven

 

As a final test it was necessary to determine the
limits of temperature within which the oven could be
operated. To do this the oven was set at different temper—
atures and the temperature fluctuation observed. It was
found that at all the temperatures tested from 230° F. to
315° F. the temperature control was within 0.4° to 1° F.
This fluctuation is due in part to the temperature profile

set up in the block. Temperatures above or below this

*3

J

a.ge were not tested since it was not desired to utilize

such temperatures.

8. Calibration of Thermistor Amplifier

 

t was necessary to calibrate the thermistor amplifier
so the temperature could be preset to within 3° F. The re—
maining fine control was accomplished using the potentio-
meter. A dial plate on the thermistor amplifier was simply
rotated and reset to assure calibration. In this manner it
was possible to set the temperature to within 3° F.

Since all the bar temperatures were the same the
monitoring of the system during dry heat testing was
accomplished by measuring the temperature of the third

bar.

39

C. Description of Operation of Oven

 

The following discussion will describe an actual de—

tailed operation of the oven under zero air flow conditions.

(For convenience this discussion will assume that the oven

is operated at 270° F. for a total time of 3 hours, and

that the samples will be withdrawn at 30—minute time inter-

vals.)

X“

The procedure is as follows:

Adjust temperature control (approximately : 3° F.).
Set preheating timer.

When oven stabilizes readjust control so that
oven cycles : 0.5° F. of the control temperature.
Load 36 samples.

At t = 0 all samples are drawn into oven and the
timer is started.

Shelf is lowered to unloading position.

Just before t = 30 a sterile petri dish is placed

At t = 30 the first push rod is operated to remove
samples which fall into petri dish.
Using sterile tongs the sample cups are trans-

ferred to sterile 10 ml water blanks.

Steps 8 and 9 are repeated for each time interval for

the duration of the test.

40

D. Preparation of Spores

 

1. Growth and Harvesting

 

The organism used in this study was Bacillus subtilis

 

5230 (15 p). This strain of B. subtilis will grow anaero-
bically in the presence of fermentable carbohydrate (Sisler,
1961). The original culture was provided by Dr. C. F.
Schmidt of Continental Can Company. He originally obtained
the culture from H. Curran as a 15 u strain.

A few m1 of the stock suspensionvmnmaheat shocked and
placed in five tubes of dextrose, tryptone, starch (DTS)
broth with bromcresol purple indicator. These tubes were
incubated for 24 hours at 98° F. after which a loop of
suspension was transferred to another 5 tubes of DTS broth.
After 24 hours incubation at 98° F., one loop of suspension
from each of these five tubes was inoculated onto five
plates of sporulation media which were then incubated for
108 hours at 98° F.

The sporulation media consisted of Difco nutrient
agar containing 0.5 percent glucose and 1 ppm Mn++ from
MnSOuaH2O. To prepare the media, 0.5 percent glucose was
added to the nutrient agar and then autoclaved for 15
minutes at 250° F. Then a solution of MnSOu-H2O (100 ppm)
was prepared, sterilized and added to the agar just prior

to pouring the plates. The plates were allowed to harden

before streaking.

41

After the plates had been incubated for the appro—
priate time, 50 m1 of sterile distilled water were poured
over each plate and an L-shaped glass stirring rod was
used to wipe the spores from the surface of the agar. The
water which now contained the spores was poured through a
sterile glass wool filter through a sterile funnel into
centrifuge bottles (250 ml). The surface of the agar was
washed once more with 20 ml of sterile water and this
solution was also poured into the centrifuge bottles. The
suspension was distributed throughout four centrifuge
bottles. These spores were then centrifuged for about 30
minutes at 2800 rpm, and the supernatant discarded. The
spores were then resuspended in sterile water, shaken to
break up any clumps of spores and centrifuged again. This
procedure was repeated four times. Finally the spores were
resuspended in M/l5 phosphate buffer, pH 7.0 and washed in
the buffer three more times. They were then stored in this

buffer at 358 F.

B. Initial Coung

 

 

The spore suspension was plated (pour plate) to deter-
mine the number of spores. A small sample was removed from
the stock bottle and heat shocked for 10 minutes at 212° F.
Appropriate dilutions were then made and the spores were
plated in duplicate. The media used for this determination
was dextrose, tryptone, starch agar (DTS). The plates

were incubated for 48 hours at 98° F. and the colonies were

42

counted. The counts showed that this stock solution con-
tained 8.5 x 108 spores/m1. Microscopic examination of
this culture showed that it consisted essentially of 100
percent spores as determined by Malachite Green-Safranin
spore stain.

A 1/100 dilution of this suspension was made using
the M/l5 phosphate buffer and poured into a small jar con-
taining glass beads and a magnetic stirrer. This jar was
used as the stock suspension for the entire set of experi—
ments. The magnetic stirrer and the glass beads were used
so that each time it was necessary to withdraw samples from
the jar, the contents could be mixed to break up any clumps
and distribute the spores evenly. This suspension was
plated to determine the number of spores and the count

showed that it contained 8.5 x 106 spores/ml.

3. Preparation for Heating Tests
Io prepare the spores for heat treatment, 0.01 ml of

an approXimately 107 spores/ml suspension was dispensed

L
(j)
H"
:5

a micrometer syringe (Roger Gilmont Instruments,
Inc.) into small 11 mm O.D. x 8 mm deep tin plate cups
(Pflug and Esselen, 1953). Prior to innoculation the cups
had been washed, placed in petri dishes, sterilized in the
autoclave at 250° F. for 30 minutes, and dried in an air
oven at 250° F. The inoculated cups were dried at 70° F.
for 24 hours at 29 in. mercury vacuum and stored in a

dessicator over CaCl2 until used.

43

To determine the initial number of organisms for
each test, five inoculated cups were dropped into each
of five screw cap dilution tubes containing 10 ml sterile
distilled water. These tubes were placed in a boiling
water bath for 10 minutes to heat shock the spores. After
the heat shock, the caps were tightened and the tubes were
shaken Vigorously for 10 minutes for removal of the spores
from the surface of the cups, and to distribute the spores
throughout the 10 ml suspension. A 1/100 dilution was
made of each tube and one m1 of the dilution was plated
(pour plate) with dextrose, tryptone, starch agar. When
the agar had solidified, a thin layer of DTS agar was
poured over the surface to prevent any spreading of surface
colonies. The plates were inverted and incubated for 48
hours at 98° F. The plates were counted, and the average
count of the five plates taken as the initial number of
organisms. The initial number varied slightly from test
to test over the period during which the experiments were
conducted. The initial number for various dates through—
out the testing program are given in Table 2.

For the heating studies, the spores were prepared as
outlined above, placed in the dry heat oven for the appropri—
ate heat treatment time, dropped into a sterile petri dish
and transferred into screw cap dilution tubes containing
10 ml of sterile distilled water. The tubes were shaken

vigorously for 10 minutes for removal of the spores from

44

TABLE 2.——Initial spore counts throughout testing period.

 

Initial Count

 

 

Date Spores/Cup
10—17 7.0 x 104

10—26 6.8

11-7 7.3

12-5 6.4

12—13 6.5

12—20 7.0

1-9 6.5

the surface of the cups. Appropriate dilutions were made

according to the particular heat treatment which the samples
had received, and one m1 of the dilution was plated (pour

plate) onto DTS agar as outlined above.

v—-\

E. Methods of Analysis

 

There are two general methods available for the
analysis of heat resistance data: Fraction Negative (FN)
and Survivor curve.

The Fraction Negative is a procedure in which the
bacterial suspensions to be tested are heated in replicate
tubes until the viable population in each tube has reached
the point where some of the tubes contain at least one
viable cell and some of the tubes are sterile. The method

of analysis consists of a most probable number technique

45

for determining the number remaining and from this value
the D value can be determined. This method has been ade-
quately described by Eder (1966).

The other common method and the one used in this
study consists of reporting the data as survivor curves.

In this method the number of viable organisms is counted
after various heating treatments and the number of viable
cells is plotted against the time. The plate count—
survivor curve method gives more information about the
order of death of the bacteria than does the FN analysis.
Furthermore the survivor curve does not depend upon any
assumptions as does the fraction negative method.

The principle advantage of the fraction negative
method of analysis is the fact that it allows one to calcu—
late the D value in the very low survival ranges. By using
survivor curve techinque we are limited to about 30 colonies
as a lower limit of survival.

In many sterilization experiments it is indeed the
low levels of survival that we are interested in. If this
is the case it would be a good idea to use the Fraction
Negative method in the low survival range and a standard
most probable number technique in the upper range. This
would generate a survivor curve which would range from the
initial number to a very low survival level.

The method used in this thesis involves plotting the
number of viable spores at various time intervals against

the time on semi-logarithmic coordinates.

46

The data were analyzed by means of linear regression
using the least square approach. The plate counts at the
various time intervals were typed onto punched computer
cards and the least square coefficients, i.e., the D value
(-1/slope) and the apparent initial number Na (ordinate
intercept) were calculated. Eighty percent and ninety-
five percent confidence limits were calculated on the D
value. To perform this analysis a program was written for
the Michigan State University CDC 3600 digital computer.
This program calculated for each experiment the D value,
the apparent initial number Na and the confidence limits on
the D value. It was found that the confidence limits of
some of the D values were large as shown by the analysis
which was a modification of the t test. These large confi-
dance limits were caused by the great variability in the

replicate plate counts at a given time.

F. Method of Linear Regression

 

The linear regression D value was calculated by the
least squares method of Dixon and Massey (1957). The method
will be described here in detail. Each survivor curve test
resulted in a series of six replicate plate counts for
each time interval. For the analysis, the plate counts
(corrected for dilution) represent the P values and the
times represent the x values. Each corrected plate count
was first converted to the corresponding logarithm so that

for this analysis:

47

Y = log P

The x values were left as natural numbers.

The following quantities are then calculated:

 

 

 

 

 

N
(1) Y = Z Xi/N N = total number
i=1 of data
points per
test
_ N
(2) Y = z Yi/N
i=1
N 2
z X 2 - 1‘1
i=1 i N
(3) 3x2 = N _ l variance of x
(4) SX = VSXZ standard devi-
ation of x
N 2
N E ii]
1—1
X Y - .
i=1 1 N
(5) 8y2 = N _ 1 variance of Y
(6) S = VS 2 standard devi-
y y ation of Y
N
N E XiZYi
1—1
2 XiYi — ————————
i=1 N
(7) b = N 2 slope of
Z X regression
N i=1 1 line
2 X 2 -
._ 1 N
i-l

(8) D =

since by definition

D =
- log Na/N
log N = —D—-

This is the

log

 

decimal
'3— reduction
time
t—
Na/N
= —t/D
+ log Na

equation of a straight line in the form of

Y = m x + b
where
log N = ordinate value
i-.-
- D — Slope
LOg Na = ordinate intercept
N
iilYi _ apparent
(9) log Na = N - bx initial
number
N _ 1 mean square
(10) Sy-x2 = fi—:—§ (Sy2 - b2 - sz) deviation of
sample points
from regres—
sion line
(11) Sy'x = VSy-x2 standard error

of estimate

(l2)

(l3)

|+

 

 

Sy-x

 

S
x

/N - l

where

confidence limits
of slope

t is the .975
percentile of the
t distribution
for N-l degrees
of freedom

confidence limits
of D value

IV. RESULTS AND DISCUSSION

A. Decimal Reduction Times

 

The data from all experiments were used to calculate
linear regression survivor curves, as previously described
under analysis. Table 3 summarizes the D values obtained
for the individual tests showing 80 percent confidence
limits for each D value. Table 4 shows the same D values
with their 95 percent confidence limits.

Table 5 shows the average linear regression D values
after discarding some tests which were felt to be in error.
In preparing Table 5, those D values which showed opposition
to the theory that as the temperature increases the D value

decreases, were discarded. The following D values were

 

 

discarded:
Temperature (°F.) Flow Condition D Value (min)
/”“\
{255; 0 flow air 110
275 1.4 cfm air 62
285 1.4 cfm air 109

 

In those cases where more than one D value was ob—
tained at the particular condition an average was taken

of the values.
50

51

On examining Table 5 we can see that the resistance
at 255° F. (D value) decreased as the flow rate was in-
creased. The D value obtained for the low flow rate (1.4
cfm) of nitrogen was the same as that of the high flow rate
of air. However, the high flow rate of nitrogen gave the
lowest D value of all.

We see a slight decrease in resistance between the
zero flow condition and the low air flow condition at 265° F.
However, there is no difference between the D value obtained
at the low air flow rate and the high air flow rate. The
D values for the nitrogen tests were lower than those for
air, but no difference was observed between the two flow
rates.

There appeared to be no difference in resistance be-
tween the still air condition and the flow conditions at
275° F. No difference was observed between the nitrogen
flow rates; however, the nitrogen D values were about 1/2
the value of the D values obtained in the air tests.

At 285° F. the effect of increasing the flow rate of
air has been to increase the D value slightly. For the
nitrogen tests there is a slight increase in D value with
higher nitrogen flow.

Table 6 summarizes the z values (temperature change
required to produce a ten fold change in D value) for the
various experimental conditions. The data for the closed
System dry heat and for the wet heat are from Fox, Eder

and Pflug (1967). These data are listed to show the

52

 

 

 

 

 

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50H no mma moa m:
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Aoa-mav Aos-oav Aomm-mac “Ha-sac Aaam-asv
mm m: flea mo moa
Aemmanaaav
mom

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ea ma SHH son one
Amflmuaav Ammmueoav
won and
Amomfiueomv
Ham

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FHA Hma mma mam OHH
Amemuaoav
mmm

.sco o.m .suo a.H .sc0 0.: .sco :.H .sc0 0 A.moV

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.mpfiEHH mocmpfimcoo ucmoemd

om wcfizonm mummp COHpQSmemU HMEemcu Hm3©H>HUcH pom AQHEV mosam> QI|.m mqmde

53

 

 

 

 

 

Ammfiuamv Aeaalamv Assansev Amsuomv
ss as as am mam
Ammmumev Aoaaummv Au- -mev Aamsmnomv Asm-amv
sea as mmH aoa m:
Aamauomv Amomumzv Aaaaumov
Hs mm em
msm
Aomuaav Aflsnmmv Asaaummv Ammanmev Aommnomv
sm we and mm sea
A.. uomv
mom
Amsaasmv Ammanaov Ammmumsv Amomumsv Au- nosv mom
sa ma SHH soa aea
Asmanoov Aammuomv
moa ::H
An. -emmv
Ham
Amaaummv Amamumoav Ammmumav Aasomumaav Aemmumsv mmm
sHH Hma aoa mam OHH
Amasausmav
mmm
.eho o.m .sco :.H .sc0 0.: .eho :.H .Eu0 0 A.sov
cowonpfiz cowonufiz LH< LH< AH< magpmnmqeoe

 

.mpHEHH mochHMQOO unmoema

mm meazoem momma concoswpmme flashers assessaeea sou Aeasv mmzam> a--.: mamas

54

TABLE 5.--Average linear regression D values in minutes
after discarding data points believed to be in error.

 

 

 

Temper— Air Nitrogen
ature

(°F.) Still 1.4 cfm 4.0 cfm 1.4 cfm 3.0 cfm
255 383 216 169 151 117
265 165 107 112 93 97
275 100 95 106 48 57
285 47 74 95 64 .77

 

 

TABLE 6.--z values for various gas flow conditions.

 

Flow Condition z value (°F.) D250 (min)

 

' dry heat closed system-

TDT can 31 600
1 wet heat closed system- ;
‘.TDT can 25 0.50 .
still air-dry heat oven 32 800
1.4 cfm air flow-dry heat oven 50 340
4.0 cfm air flow-dry heat oven 100 120

1.4 cfm nitrogen flow-
dry heat oven 42 270

3.0 cfm nitrogen flow-
dry heat oven 73 150

 

55

relative values of the open system resistances when com—
pared to the closed system and wet heat system.

Figures 6 through 12 show the thermal resistance
curves (plot of log D value versus temperature) for the
various heating conditions. Figures 6 and 7 are from
Fox, Eder and Pflug (1967).

It is important to realize that there is a major
difference between the open heating systems and the closed
systems. The open dry heat system provides a relatively
low humidity and it is probable that as the spores are
heated, they also become drier. For this reason the sur—
vivor curve has a slightly different meaning in the open
system than in the closed system. In the open system the
moisture content of the spores may not be constant through—
out the heating period; the spores may become drier as they
are heated due to the loss of moisture from the open system.
in the wet heat system, the spores are maintained at a
water activity near 1.0 throughout the heating time, and
in closed system dry heat methods there is an equilibrium
established between the water in the spores and the water
Japor in the atmosphere inside the container. If the
water activity of the spores were held constant during the
heating period we could be more certain that the D value
obtained was a true D value. However, it is not possible
at the present time to hold the water activity of the

spores constant and at the same time maintain an open

LOG D VALUE

56

 

 

 

5

4-

3_ Z : 31°F
D250 = 600 MIN

2..

1

 

 

200 250 330» 350
TEMPERATURE, °F

Figure 6. --Thermal resistance curve for dry heating in
thermal death time cans.

57

 

 

3

2..
m
:3
_I
E. ..

1_ Z=25°F
O 02”.:0'5 MIN
0
0
—l

o-

-1

 

 

 

150 150 280 210
TEMPERATURE,°F

Figure 7.--Thermal resistance curve for wet heating in
thermal death time cans.

58

 

 

 

 

 

UJ
3 2:32T'
§ 02502800 MIN
C3
0 1‘
o,
_J
I) I I I I
255 265 275 285

TEMPERATURE °F

Figure 8.--Thermal resistance curve for still air in
dry heat oven.

59

 

 

 

 

 

UJ
' :3
.J
< O
> 2:50 F
0 02502340 MIN
L9 1"
o
_J
0 I l u u
255 265 275 285

TEMPERATURE °F

Figure 9.--Thermal resistance curve for 1.4 cfm air
flow in dry heat oven.

6O

 

 

 

 

3
DJ
:>
_J
<
>
o z: 100 °F
0 1"“ 02502120 MIN
0
.J
0 I I l I
255 265 275 235

TEMPERATURE °F'

Figure lO.--Thermal resistance curve for “.0 Cfm air
flow in dry heat oven.

61

 

 
    

 

 

 

3
Z=42°F
m Dzso=270 MIN
2)
_l 2-
<[
:>
'0

L9
0
.J

1 I

I I I
255 265 275 235
TEMPERATURE °.F

Figure ll.--Thermal resistance curve for l.“ cfm nitrogen
flow in dry heat oven.

62

 

Z=73°F
Dzso=150 MIN

 
   

LOG D VALU E

 

 

 

I l I
255 265 275 285

TEMPERATURE ’F‘

Figure l2.--Therma1 resistance curve for 3.0 cfm
nitrogen flow in dry heat oven.

63

system. Murrell and Scott (1966) reported that they held
the water activity fairly constant throughout the heating
schedule, but to do this it was necessary to use sealed
test tubes containing certain salts to keep the relative
humidity constant.

If we assume (1) that as the flow rate of gas over
the spores is increased, the rate of dehydration of the
spores is accelerated and (2) inactivation of the spores
is increased by extreme dessication then it would seem that
spores are much more sensitive to thermal destruction in
the more dessicated state. Davis, Silverman and Keller
(1963), however, reported that up to 40 percent of the

initial number of organisms of g. subtilis var. niger could

 

survive for 5 days at 60° C. under ultra high vacuum
(approximately 10"10 Torr). At the same temperature these
workers found that under atmospheric pressure about 70 per—

cent of the initial number of spores remained viable for 5

days. It is apparent then from these studies that the ef-

('f

fee of extreme vacuum (dessication) does cause some death
of bacterial cells by itself. When these workers held the
same spores under the ultrahigh vacuum for 5 days at higher
temperatures, they found that at 880 C. less than 0.01 per—
;en; survived and at 100‘ C. and 107° C. there were no
survivors remaining.

The effect of nitrogen has been to give resistances

slightly lower than those obtained in air. It was

originally thought that if oxidation of dry bacterial

6M

spores was contributing some lethal effect, the nitrogen
should increase the resistance since there would be little

if any oxidation during the heating in the nitrogen. The
results show, however, that the resistance in nitrogen as
compared to air was higher only at 285° F. Since at this
temperature the D value of the spores heated in air generally
increased as the air flow rate increased we cannot infer
anything concerning oxidation as a cause of death. If
oxidation were occurring during dry heating in air we might
expect the D value to decrease as we increase the air flow

(as it did at temperatures below 285° F.), but we would

(I)
\
('f

xpec the D values under nitrogen to be very high. Pheil,

flu , Nicholas and Augustin (1967) suggested that if the

"U
0‘)

O

xggen content of the gas is responsible for the major
destructive effect in dry heat sterilization, then the dry
heat resistance should be less in oxygen. However, in
their studies these workers found that the resistance was

not measurably different between oxygen and air. These

aut ors also tested the resistance of Clostridium sporogenes

 

LU

and -a3illus subtilis under inert gas atmospheres. They

 

found that in general these inert gases (helium, nitrogen)

gave the largest D values.

B. Thermal Resistance--z Values

 

From Table 6 we can see that for both the nitrogen

and the air tests, the largest 2 value was obtained at the

65

highest flow rate which means that as the flow rate of
the gas increases, the thermal resistance becomes less
temperature dependent.

In the dry heat closed system using thermal death
time cans, the open system with zero air flow, and the
open system having low flow rate we have observed essen-
tially the same 2 values. The low flow rate of nitrogen
has given a slightly higher 2 value. Therefore in systems
where the rate of dehydration (rate of flow) of the spores
is relatively low (closed systems and low flow rate systems)
the main influencing factor of the thermal resistance is
the temperature.

Eder (1966) studied the resistance of Bacillus subtilis

 

spores in simulated flexible package seals. This study was
an effort to determine how the resistance of the spores is
affected when the spores are confined to a very small

closed system, i.e., the seal interface. He proposed that
if moisture loss from the spore during dry heating decreased
the resistance, then conditions which restrict the water
loss might increase dry heat resistance. He found that at
high temperatures (3050 F.) the resistance was higher in
simulated seals than on thermoresistometer cups, but at

lower temperatures the two resistances were about the same.

66

C. Shape of Survivor Curves

 

Figure 13 shows a sample survivor curve. This curve
is representative of those obtained for all the heating
tests. In all cases there is an apparent initial rapid
drop in number of survivors. This shape is generally
known as concave upward, or broken survivor curve. Alder-
ton, Thompson and Snell (196“) stated that their data sup-
ports the hypothesis that concave upward survivor curves
result from a gradual increase in heat resistance developed
during heating. In their experiments Bacillus megaterium
spores were first stripped of metallic ions by holding them
in nitric acid, then stored in calcium buffer solutions
and finally tested for heat resistance and Ca++ ion uptake
at preselected time intervals.

Another explanation of the characteristic shape of
these dry heat survivor curves may be explained as possibly
being due to the fact that the culture under observation
is non—homogeneous in resistance, and that there are mostly
very liable or low heat resistant organisms which are
killed very rapidly as indicated by the first portion of
the curve. The second portion of the curve could be due
to the higher heat resistance of the remaining organisms.
Stumbo (1965) gives two possible explanations for these
concave upward survivor curves. First he discusses the
mixed flora theory which has been mentioned. As a second

explanation he gives the flocculation theory. Flocculation

LOG VIABLE NUMBER

67

 

 

D=43 MIN.

 

 

 

 

2.-
1'5 3'0 4'5 5° 75
TIME. MIN.

Figure l3.-—Sample survivor curve for Bacillus subtilis
' in dry heat oven (still air)fat 285° F.

 

68

of the spores occurs early in the heating procedure and

. . . as clumping nears completion, the colony count

will tend to level off during the time the number of

surviving cells per clump is being reduced to one
cell per clump. Only when virtually all clumps in
which viable cells remain contain only one viable
cell per clump will the rate of reduction in colony
count assume that of a logarithmic order.

In testing the heat resistance of six species of
spores at various water activities (aw) ranging from O to
l, Murrell and Scott (1966) obtained survivor curves for
two species which were similar in shape to the dry heat
survivor curves found in this study. They attributed the
sudden initial decrease in viable number as a result of
damage during freeze drying of the spores.

It is possible that the culture used in the present
studies contained some spores which were very susceptible
to dehydration. Therefore these susceptible cells were
not destroyed during the initial drying procedure and

appeared in the initial count of the organisms. _However,

when the sample was placed in the dry heat oven, these

susceptible cells were dehydrated further and killed immedi-

ately, thus accounting for the sudden drop in the survivor
curve.

It is also possible that the spore suspension is
-essentially uniform but during the first few minutes of
dry heating, the heat energy induces changes in the spores

that cause the spores still viable to move into a more

resistant state. This explanation of developing resistance

69

seems to parallel Alderton, Thompson, and Snell's (l96U)
observations of an increase in resistance during heating
when stripped spores were heated in a system containing
cations.

The method of heat shocking the spores used in this

r search was that of Curran and Evans (19H5). The spores

(D

were suspended in distilled water and heated at 212° F.
for 10 minutes before being plated. It is possible that
since this heat shock was a wet heat treatment in reality
the count obtained by this method would not fall on the
straight line formed by the dry heated spores.

D. Possible Causes for Variability
Among Replicate Plate Counts

 

 

The large variability among replicate plate counts
has caused the confidence limits on the D values to be
very large in some instances. These large variations may
be caused by:

1. Variations in the original numbers of spores per
sample. This is a very improbable reason since
the spores were pipetted into each cup using an
accurate microsyringe and the initial counts
which were obtained before heating showed very
consistent results. Table 2 summarizes the
initial numbers obtained for the various heating

tests over a period of 3 months.

7O

2. Variations in flow rates over each sample cup
during actual heating treatments. Due to small
convection currents which are probably set up
in the sample chambers there may be some differ-
ences in the air flow rate within each cup.

3. Recovery of spores from cups. This is the most
promising explanation. The fact that the method
of plate counting depends upon the removal of
the spores from the test surface makes it neces—
sary that the same proportion of spores are re-
moved from all cups. It is quite possible that
during the heating process these spores become
"baked" onto the surface and that when the cups
are soaked for 10 minutes to remove the spores,
some spores are retained on the surface of the
cup. If the percentage retained on the surface
is not constant in each cup, this could explain
some of the variation.

An effort was made to correlate the variation in D
value with the date of the experiment. It was thought
possible that as the spores were stored in the stock sus—
pension throughout the testing period their resistance
might have changed. However, when the D values were
examined in relation to when they were obtained, no trend
could be seen. There were slightly more instances when

the D value dropped as storage time of the stock suspension

71

creased, however, there were not enough data available

reach any definite conclusions.

V. CONCLUSIONS

From the results of this study it is concluded that:
1. There is a difference in the response of bac-
terial spores when heated in dry heat open systems

and dry closed systems.

{\J

As the rate of gas flow is increased, the 2 value
of thermal destruction of the spores becomes
larger.

3. There appears to be no difference in the thermal
destruction in air versus nitrogen and therefore
oXidation of cellular components is probably not

a major factor in the dry heat thermal destruction

of bacterial spores.

J

The cecimal reduction tim (D value) obtained in

L

'3'

1o
-1

(D

y at system may have a slightly

LL

an open
different significance than the corresponding
D value obtained for wet heat and a closed dry
heat system. In the open system the composition
of the spores may not be constant throughout the

heating period.

72

APPENDIX

73

74

 

 

 

 

 

 

 

 

 

Run #2. Temperature = 285° F. Still Air. Date: 10-12
Time (min) Viable Counts
15 530 1670 1470 1560 190 780
30 220 340 330 320 300
45 260 330 no 90 7o
60 70 40 140 50 30 60
75 30 10 70 20 130
D value of regression line = 43.0 min.
Apparent initial number = 1576.3.
Run #3 Temperature = 275° F. Still Air. Date: 10—12
Time (min) Viable Counts
15 1800 1600 2000 800 1500 800
30 690 3600 4150 4520 2490 1120
45 680 340 620 230 870 3900
60 200 210 590 220 150 270
75 1010 900 3310
D value of regression line = 106.3 min.
Apparent initial number = 2211.0.
Fun #4 Temperature 2650 F. Still Air. Date: 10-17
Time (min) Viable Counts
15 5000 6000 5000 5000 3000 5000
30 4800 400 200 900 200 400
45 1330 2500 230 3800 1090 120
60 400 630 1100 3200 900 180
75 280 680 900 1790 1030 2210
90 390 1049 180 1740 5150 2450
D value of regression line = 203.9 min.
Apparent initial number = 1963.2.

75

 

 

 

 

 

 

 

 

 

Run #5. Temperature: 295° F. Still Air. Date: 10-17
Time (min) Viable Counts
10 3000 4000 3000
20 200 800 1700 300 500
30 470 310 20 930 320 320
40 20 140 120 250 70 400
50 160 O 10 110 10 290
60 140 240
fa
D value of regression line = 27.8 min.
Apparent initial number = 3445.8.
- 1
Run #6. Temperature: 275° F. Still Air. Date: 10-19
Time (min) Viable Counts
15 7900 4700 6600 4400 1500 2600
30 700 4000 700 8600 100 1900
45 1540 980 1790 200 2750
60 680 1010 2450 4700 2750
75 430 300 1250 1200 570
93 900 710 300 120 370
D value of regression line = 94.5 min.
Apparent initial number = 4189.4.
Run #7. Temperature: 265° F. Still Air. Date: 10-19
Time (min) Viable Counts
15 1000 2600 5800 13400 1700
30 340 6000 1470 4250 2560 1710
45 470 1020 4950 420 5200
60 120 60 410 100 2250
75 6950 6900 2700 1920 1400 920
D value of regression line = 149.1 min.

Apparent initial number = 3337.1.

76

 

 

 

 

 

 

 

 

 

Run #9. Temperature: 255° F. Still Air. Date: 10-24
Time (min) Viable Counts
30 3000 18000 5000 4000 5000 12000
60 3700 400 1300 800 1100 3300
90 8600 2000 2200 1600 100 1200
120 370 490 320 1900 8600 7650
150 4950 6150 130 1120 400 4900
180 1670 1370 3850 280 260 900
D value of regression line = 255.2 min.
Apparent initial number = 4295.0.
Run #10. Temperature: 255° F. Still Air. Date: 10-26
Time ( Viable Counts
20 5600 2500 7300 1900 4600 17500
40 210 1150 2010 1660 4100 1290
60 500 570 1720 6050 4000 5450
80 1060 450 620 750 640
100 720 340 410 1040 5850
12' 370 440
D value of regression line = 110.6 min.
Apparent initial number = 5243.7.
Bin #11 Temperature: 265° F. Still Air. Date: 11—1
Time (; Viable Counts
20 7500 14600 17000 21000 14500 2700
40 5400 810 8200 7200 10100 790
60 660 1720 600 5400 5600 1900
83 1340 810 560 1980 6750 7750
100 970 5100 1070 1680 1000 1540
120 960 390 750 6600 4600 5000
D value of regression line = 144.3 min.
Apparent initial number = 8316.3.

77

 

 

 

 

 

 

 

 

 

Run #12. Temperature: 285° F. Still Air. Date: 11-7
Time (min) Viable Counts
15 3100 710 2300 3300 2430
30 2450 1500 1490 510 1950 3750
45 1710 370 2750 1180 470 170
60 430 780 250 210 1120 1600
75 20 110 310 40 220 510
D value of regression line = 50.1 min.
Apparent initial number = 5626.2.
Fun # 15. Temperature: 275° F. 1.4 CFM Air Flow. Date: 11-30
Time (min) Viable Counts
15 490 420 1910 5850 3950 5250
30 1340 1540 3450 270 3050
45 1810 2420 140 2080 1020 640
60 370 1500 200 1670 2350 120
75 330 590 90 210 70 130
D value of regression line = 62.1 min.
Apparent initial number = 4129.7.
Fun #17. Temperature: 285° F. 1.4 CFM Air Flow Date: 12—5
Time (min) Viable Counts
15 3700 3500 700 2300 3100 500
30 1250 480 100 50 150 340
45 1230 190 190 40 340 1690
60 350 1020 630 130 80 90
75 740 260 770 380 180 200
D value of regression line = 108.8 min.
Apparent initial number = 1057.8.

78

 

 

 

 

 

 

 

 

 

Run #18 Temperature: 255° F. 1.4 CFM Air Flow Date: l2-6
Time (min) Viable Counts
20 12400 7000 12700 4300 12200 12100
40 1400 460 4000 2750 1030 1440
60 1000 990 960 2250 5150 6600
EC 1700 8000 6200 1260 7500 6950
100 7050 6700 6750 830 1900 6300
196 30 1280 160 600 6500 2950
D value of regression line = 216.2 min.
Apparent initial number = 5889.1.
bun #19 Temperature: 255° F. 4.0 CFM Air Flow Date: 12—6
Time (min) Viable Counts
15 9100 5300 13000 9100 7300 6400
30 2350 790 3750 6750 3800 2700
45 1600 7750 3500 3150 5550 7750
60 5150 930 6650 2050 7650 890
75 1000 490 5250 900 4900
90 1900 4400 1600 3150 5900 1900
D value of regression line = 169.2 min.
Apparent initial number = 6728.9.
Fun #20 Temperature: 265° F. 4.0 CFM Air Flow Date: 12—6
Time (min) Viable Counts
15 1300 5600 1400 12200 3600 1500
30 4400 1010 6650 850 3850 5500
45 2750 2000 2850 4000 650 4550
60 260 420 3400 500 1250 3300
75 3150 3200 1100 290 150 2300
D value of regresslin line = 107.1 min.
Apparent initial number = 4768.2.

79

 

 

 

 

 

 

 

 

 

Run #21 Temperature: 275° F. 4.0 CFM Air Flow Date: 12-7
Time (min) Viable Counts
15 3200 2900 7400 4400 5600 5500
30 4350 1900 760 830 1300 1120
45 4150 3900 700 4150 3750
60 710 1510 2200 3700 2350 790
75 1710 1550 930 1550 770 1390
D value of regression line = 140.9 min.
Apparent initial number = 4233.6.
Run #22 Temperature: 285° F. 4.0 CFM Air Flow Date: 12—8
Time (min) Viable Counts
15 1300 1000 1500 6900 1400 800
30 850 680 2400 150 480 2000
45 70 50 720 1140 2650 1080
60 270 1190 3910 1010 2290 150
75 1080 540 160 200 740 230
D value of regression line = 128.3 min.
Apparent initial number = 1591.6.
Hun #23 Temperature: 285° F. 4.0 CFM Air Flow Date: 12-8
Time (min) Viable Counts
15 1600 6000 900 1000 5000 2600
30 490 70 290 980 780 520
45 340 420 1430 2800 250 1600
60 690 120 190 80 220 900
75 660 630 50 60 10 1850
D value of regression line = 61.7 min.
Apparent initial number = 2636.1.

 

 

 

 

 

 

 

 

 

Run #24 Temperature: 275° F. 4.0 CFM Air Flow Date: 12-9
Time (min) Viable Counts
15 17500 11500 11000 3300 16000 13000
30 4250 8320 6500 780 1830 7600
45 7400 5250 5850 8300 5400 8850
60 2270 740 960 2200 1550
75 2000 1400 500 2600 2100
D value of regression line = 71.1 min.
Apparent initial number = 15368.6.
Fun #25 Temperature: 265° F. 4.0 CFM Air Flow Date: 12—12
Time (min) Viable Counts
15 8900 5100 2900 13800
30 1900 4100 5900 4200 4450 8350
45 450 3700 4900 5150 810 4300
60 4000 1520 5000 1700 3750 6700
75 1250 1600 380 4300 3650 4300
D value of regression line = 116.5 min.
Apparent initial number 7918.6.
Run #2 Temperature: 255° F. Still Air Date: 12-13
Time (min) Viable Counts
30 10700 6000 3500 17500 4100 6400
60 1500 15500 5100 2400 3200 4600
90 1240 490 5600 5250 4150 1550
120 6000 7050 1700 1500 2750 1900
150 3300 2150 3200 4200 5100 3500
180 2800 950 3600 5500 3400
D value of regression line = 510.6 min.
Apparent initial number = 5524.5.

81

 

 

 

 

 

 

 

 

 

Run #27 Temperature: 265° F. 1.4 CFM Air Flow Date: 12-14
Time (min) Viable Counts
15 3500 9800 12100 11800 6000 4100
30 900 4600 1250 5300 4600 540
45 3000 2200 4650 5700 1200 1600
60 1450 920 1060 1030 4400 490
75 1850 1150 3750 1100 1650
90 390 3900 520 900 3300 410
D value of regression line = 107.7 min.
Apparent initial number = 6277.4.
Run #28 Temperature: 285° F. 1.4 CFM Air Flow Date: 12—15
Time (min) Viable Counts
15 2200 6800 4800 5100 1100
30 1030 620 390 510 400 340
45 470 2200 1650 2450 270 2300
60 1400 1250 180 510 170 630
75 200 90 670 0 1850 100
90 60 310 320 500 1070 470
D value of regression line = 74.4 min.
Apparent initial number = 2902.2.
Run #29 Temperature: 285° F. 3.0 CFM N2 Flow Date: 12-19
Time (min) Viable Counts
15 300 4100 3000 1100 800 3700
30 1130 460 310 110 1450 680
46 220 1160 120 1600 160 470
60 1030 80 420 320 30 480
75 170 160 350 400 1200 420
90 70 330 300 230 130 610
D value of regression line = 107.6 min.

Apparent initial number

= 1298.8.

 

 

 

 

 

 

 

 

 

Run #32 Temperature: 275° F. 3.0 CFM N2 Flow Date: 12-20
Time (min) Viable Counts
15 500 1200 400 1400 9200 500
30 1440 420 720 190 470 560
45 860 200 80 130 320 220
60 230 240 60 730 2000 290
75 80 120 140 70 90 220
90 50 120 40 10 100 10
D value of regression line = 57.0 min.
Apparent initial number = 1963.0.
Run #33 Temperature: 265° F. 3.0 CFM N2 Flow Date: 12—21
Time (min) Viable Counts
20 3100 800 1000 10200 4500 10300
40 2200 250 860 470 450 1100
60 1050 140 480 70 250 4350
80 590 790 3350 190 130 3500
100 330 390 210 460 500 130
120 120 30 630 100 600 180
D value of regression line = 96.7 min.
Apparent initial number = 3025.5.
Run #3 Temperature: 255° F. 3.0 CFM N2 Flow Date: 12—21
Time (min) Viable Counts
20 2300 4900 2000 1900 6200 1400
40 500 790 2850 1700 700 360
60 4900 2150 710 150 260 890
80 190 2800 1010 620 150 640
100 340 440 950 470 280 470
120 630 310 290 300 400 60
D value of regression line = 116.9 min.

Apparent initial number

= 2833.3.

83

Run #35 Temperature: 275° F. 1.4 CFM N Flow Date: 12—22

 

 

2
Time (min) Viable Count
15 800 300 1100 700 1200 700
30 470 1040 40 460 300 1300
45 700 190 160 140 100 240
60 380 150 170 60 70 70
75 20 20 20 10 100 280

 

D value of regression line = 47.9 min.
Apparent initial number = 1653.6.

Run #36 Temperature: 265° F. 1.4 CFM N Flow Date: 12—27

 

 

2
Time (min) Viable Count
15 1600 2300 1700 3100 2600 1700
30 650 1110 840 420 520
45 650 450 1050 800 490 3350
60 500 80 80 610 190 740
75 230 240 1050 590 410 650
90 220 60 140 110 420 3550

 

D value of regression line = 92.8 min.
Apparent initial number = 2141.8.

Run #38 Temperature: 285° F. 1.4 CFM N Flow Date: 12—28

 

 

2
Time (min) Viable Count
15 700 1200 600 1600 400 700
30 50 90 420 330 390 510
45 640 1090 30 1400 310 100
60 70 30 150 240 1050 380
75 50 390 O 120 400 50

 

D value of regression line = 64.3 min.
Apparent initial number = 1124.3.

 

 

 

Run #40 Temperature: 255° F. 1.4 CFM N2 Flow Date: 1-2
Time (min) Viable Count
20 4300 5000 4400 4700 3900 2900
40 1010 820 2600 1000 430 1150
60 400 630 520 1250 530 730
80 470 260 250 690 550 570
100 230 720 750 850 190 1080
120 5400 460 790 320 710 510
D value of regression line = 151.1 min.

Apparent initial number

= 2562.3.

 

 

 

 

 

 

Run #42 Temperature: 275° F. 1.4 CFM Air Flow Date: 1—9
Time (min) Viable Count
15 3700 500 400 2500 4500 1400
30 320 180 530 510 1200 1090
45 280 1720 200 270 660 160
60 220 240 70 180 1420 210
75 150 500 20 290 350 120
90 110 2250 470 140 40 480
D value of regression line = 95.0 min.
Apparent initial number = 1355.2.
Run #43 Temperature: 285° F. 3.0 CFM N2 Flow Date: 1-9
Time (min) Viable Count
20 1400 2100 2500 1100 200 1200
40 120 300 190 220 1100 490
60 170 180 50 240 10
80 200 70 10 O 80 50
100 30 110 20 120 80 10
120 70 120 60 210 10 110
D value of regression line = 77.1 min.

Apparent initial number

= 953.0.

._ .___.... “V3

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