‘—
——v—-—__ w—v vvvw

 

 

THE EFFECT OF POPULATION LEVELS AND
EXPOSURE TIMES AND TEMPERATURES ON

GROWTH CLMEVEg Q? MS 102

Thesis {or ”19 Degree of M. S.
MICHIGAN STATE UNIVERSITY

Ormond Charles Pailthorp
1958

THESIS

 

THE EFFECT OF POPULATION LEVELS AND EXPOSURE TIMES
AND TEMPERATURES ON GROWTH CUEVES 0F MS 102

I. The lag phase of MS 102 with constant papulation and
variable heat.

II. The lag phase of MS 102 with constant heat and variable
population.

By
03mm: CHARLES PA ILTHORP

AN ABSTRACT

Submitted to the College of Agriculture
Michigan State University of Agriculture and '
Applied Science in the partial fulfilhment
of the requirements for the degree of

MASTER OF SCIENCE
Department of Dairy

1958
/

Approved a! s. , . were.“ .L

/ ‘ 4 / fi
a. . 1‘51. ,u/4__/./A 1“ 7

ABSTRACT

Selected population levels of Micrococcus varians sp.

 

MS 102 were heated at 61°C. for 30 minutes, 69°C. for 17
seconds, 76°C. for 17 seconds, 81°C. for 5 seconds and 82°C.
for 5 seconds. Growth curves were constructed for survivor
papulations in milk; a geometrical method of lag measure-
ment was used to estimate the lag times for all growth
curves. Control lag times for the various cell levels were
established simultaneously for use as norms for MS 102 in
milk.

At high initial inoculum levels (100,000 to 200,000
cells per ml.) the lag times with unheated cells were
comparable to those obtained after heat treatment. At the
temperatures cited above, the lag phases were progressively
' extended beyond control lag phases by elevating the pro-
cessing temperature. This temperature - lag time relation-
ship prevailed at medium and low survival levels (10,000 to
20,000 and 1,000 to 9,000 cells per ml. respectively), but
not at high populations of survivors after heating. A

notable exception occurred at low initial cell papulations

ORMDND CHARLES PAILTHORP

after Low-temperature Long-time treatment when the lag
time (42 hours) equalled that obtained after 82°C. for 5
seconds.

The maximum lag times (1.5 to 2.0 days) were
obtained with medium, low and very low survivor populations
after heat treatment at 82°C. for 5 seconds (the highest
temperature studied).

Control growth curves of MS 102 in milk revealed
progressively shorter lag phases for initial populations
above 1,000 cells per ml.; below this papulation the lag
phase increased gradually as the initial cell concentration
was reduced.

Lag times for high survivor levels were not sig-
nificantly altered by the heat treatments explored. At
high concentrations of survival the size of the initial
papulation appeared to affect the lag times in a manner
similar to that observed for unheated controls. The
combined lag extending effects of lower papulations and
heat treatment seemed to account for the appreciable lag
time extensions obtained for medium and low survivor papu-

lations.

THE EFFECT OF POPULATION LEVELS AND EXPOSURE TIMES

AND TEMPERATURES ON GROWTH CURVES OF MS 102

I. The lag phase of MS 102 with constant population and
variable heat.

11. The lag phase of'MS 102 with constant heat and variable
population.

BY
ORMOND CHARLES PAIETHORP

A.THESIS
Submitted to the College of Agriculture
Michigan State university of Agriculture and
Applied Science in the partial fulfillment
of the requirements for the degree of
‘MASTER OF SCIENCE
Department of Dairy

1958

- :67”

3/; , 9" J ..
13,. 7543

'To Jacqueline, Nanette
and

my Parents

ACKNOWLEDGMENTS

The writer wishes to make known those who provided
timely assistance, and particularly those professors who
offered sincere words of direction and kindness in addition
to valuable suggestions pertaining to the subject matter.

My grandparents,er. and Mrs. R. J. Burch, through
their successful endeavors in the Michigan lumber industry
and their desire to share with their grandson, have pro-
vided additional incentive for him to attain the goals
ahead.

Dr. L. G. Harmon constantly provided personal and
professional advice, but in such a manner as to allow the
writer freedom in choosing his way.

Dr. E. W. Cook, and others at Centre College, opened
the world of biology to the writer and in so doing provided
the initial but lasting incentive to attain scientific
goals without losing zest for human relations.

Without the direction of Dr. Ou'w. Kaufmenn, the
technical effectiveness of this effort might have been lost.
Dr. Kaufmann's constant quest for basic knowledge of the
subject was contagious, thus strength was imparted to this

worker.

TABLE OF CONTENTS

Page

Introduction . . . . . . . . . . . . . . . . . . . . 1

Literature Review . . . . . . . . . . . . . . . . . 3
The Lag Phase of Bacterial Growth . . . . . . . 3
The Heat Tolerant Micrococcus varians sp.

MS 102 O O O O O O O O O O O O O ..... O O 9

Experimental Procedures . . . . . . . . . . . . . . 11

Normal Growth Curves at Adjusted Population
Levels . . . . . . . ..... . . . . . . . 11

Post Heat Shock Growth Curves . . . . . . . . . 16

Results and Discussion . . . . . . . . . . . . . . . 41

Effect of Heat Treatment on the Lag Phase at
Selected Cell Concentrations . . . . . . . . . 41

‘Bffect of Survivor Cell Concentrations on the
Lag Phase . . . . . . . . . . . . . . . . . . . 45

General Considerations Evolving from the Lag
Extension Studies . . . . . . . ...... . . 47
Summary . . ....... . . . . ........ . 49

Literature Cited . . . . . . . . . . . . . . . . . . 50

ii

TABLES

Page

I. Plate counts of milk samples containing
a high initial cell concentration of MS 102
after heat treatment at 61° C. ,69° C. or .
76°C. . . . . . . . . . . . . . . . . . . . 20

II. Plate counts of milk samples containing a
medium initial cell concentration of MS 102
after heat treatment at 61°C., 69°C. or
76°C. . . . . . . . . . . . . . ..... . 22

III. Plate counts of milk samples containing a
low initial cell concentration of MS 102
after heat treatment at 61°C., 69°C. or
76°C. . . . . . . . . . . . . . . . . . . . 24

IV. Plate counts of milk samples containing a
low or medium initial cell concentration
of MS 102 after heat treatment at 81°C. or
82°C. . . . . . . . . . . . . . . . . . . . 26

V. Plate counts of milk samples containing a
low initial cell concentration of MS 102
after heat treatment at 81°C. or 82°C. . . . 27

VI. Plate counts of milk samples containing a
very low initial bacterial pogulation after
heat treatment at 81°C. or 82. . . . . 28

VII.‘ Plate counts of milk samples containing
initial cell concentrations selected for
the construction of control growth curves
showing rate of growth in milk . . . . . . 35

iii

TABLES continued

Page
VIIb. Plate counts of milk samples containing
extremely low initial cell concentrations
selected for the construction of control
growth curves in milk . ..... . . . . . 36

VIII. Plate counts of milk samples containing
extremely low initial cell papulations . . 38

IX. The effect of selected heat treatments on
the total lag phase of MS 102 . . ..... 39

X. The effect of selected heat treatments on
the extension of the lag phase of MS 102 . 40

iv

FIGURES

The procedure used to secure the desired cell
concentrations before heat treatment . . . . .

A diagram of the dilution procedure utilized to
obtain extremely low cell concentrations prior
to establishing unheated control growth

StUdies O O O O O O O O O O O O O O O .....

Effect of heat shock on the growth curve of

survivors. (High initial cell concentration) . .

Effect of heat shock on the growth curve of

survivors. (Medium initial cell concentration) .

Effect of heat shock on the growth curve of*
survivors. (Low initial cell concentration)

Growth curves of MS 102 cells which survived

81°C. or 82°C. for 5 seconds . . . . . . . . . .

Effect of heat on the lag phase of MS 102.

(Average initial population - 19,000/ml.) . . .

Effect of heat on the lag phase of MS 102.
(Average initial population : 2000Am1.)

Page

13

21

23

25

29

30

31

FIGURES continued

Page
9. Effect of the survivor cell concentration on
the growth curve after 61°C. for 30 minutes . . 32

10. Effect of the survivor cell concentration on
the growth curve after 76°C. for 17 seconds . . 33

11. Effect of the survivor cell concentration on
the growth curve after 69°C. for 17 seconds . . 34

12. Effect of initial cell concentration on the
lag phase of control growth curves of
unheated cells . . . . . . . . . . . ..... 37

vi

INTRODUCTION

In the interest of public health, several pasteuri-
zation standards foerilk have been developed and.approved
by the various regulatory agencies. Initially, Low-temper-
ature Long-time (LTLT) (143°F. for 30 minutes) milk pasteur-
ization prevailed; eventually, a High-temperature Short-
time (HTST) (161°F. for 16 seconds) process was accepted.
The consumer's protection from.pathogens was paramount
while the keeping quality or shelf life was less significant.

Continued research revealed the presence, in milk and
other foods, of heat tolerant bacteria capable of propa-
gating at post-heat storage temperatures used for these
foods. The shelf life of milk and its products was shown
to be partially dependent on the type and numbers of thermo-
duric bacteria surviving pasteurization. Instances of de-
layed bacterial spoilage were reported. One plausible ex-
planation was that injury to the cell during heating was
followed by a recovery or lag period and physiological
adjustment-which preceded logarithmic growth. Research has
demonstrated the extension of the cellular recovery period
by controlled exposure of the cells to physical and chemical

agents.

The study reported herein was designed to supplement
the information concerning the reaction of bacteria to
various levels of "heat shock." The roles of population
size, and time and temperature as precursors of extended

bacterial latency were examined.

LITERATURE REVIEW

The Lag Phase of Bacterial Growth

Porter (18) acknowledged that Muller probably first
noted the lag phase of bacterial growth in 1895. The lag
phase is described by Thomas and Grainger (24) as a period
during which the population level is nearly constant.
Hinshelwood (12) described lag more specifically as the
elapsed time between inoculation and the stage of maximun
cell division. True lag was differentiated as the elapsed
time between inoculation and initial cell growth; apparent
lag was described as the entire period from the initial
inoculation until the onset of logarithmic growth. Both
of these lag phases appear to be similar. Smith and Martin
(23), as well as Sarles gt §_]._. (21), also defined the bac-
terial lag phase as a time lapse between initial inocula-
tion and the onset of the logarithmic growth rate. Another
description of lag was presented by Dubos (5) who referred
to the lag period of a culture as the time during which
there was an increase in, cell volune in the absence of cell

division .

Several explanations for bacterial latency have been
presented. Winslow (28) employed cell counts at selected
time intervals to demonstrate two distinctly different
phenomena of lag. He reported an adjustment period in-
volving bactericidal or bacteriostatic action and a period
during which the cell mass increased rapidly while propa-
gation was retarded. Hershey (ll) determined that
bacterial latency was created by a pause in the fission
of smaller cells which later would undergo division when
they had attained adult proportions on the new substrate.

Experimental studies of Chesney (3) were designed
to secure information on the metabolic activities of se-
lected pneumococci. He concluded that cell alteration was
reflected as a deviation in lag from the normal for a
constant set of conditions. Such alterations were believed
to have resulted from the lack or absence of a metabolic
factor essential to normal physiological activity. Cell
injury merely resulted from the direct or indirect expo-
sure of the cells to their metabolic wastes. The extent
of this injury would be measured by the lag extension.
Where cell damage was great, death would probably follow.
Chesney concluded that lag evidenced current or previous
exposure to unsuitable nutritional sources.

-4-

Many factors have been described, any one or combi-
nation of which.wou1d conceivably alter the period of
latency. Substrate constituents may be inadequate to
nourish normal growth (3, 8, 12). Hydrogen ion concen-
tration affects the growth curve (12). The cautious
control of pH is advisable during any studies of growth
characteristics. Topley and Wilson (26) include the size
of inoculum.among factors considered essential to normal
lag and growth. Within certain limits, the inoculum level
may show effect upon the growth characteristics. Hershey
(11) observed the effect of papulation levels on the latent
period of Escherichia £213. The lag was not affected by
the size of seeding when the inoculum was small. Pure
cultures involving concentrations above 107 cells per ml.
in fresh broth resulted in an unfavorable environment which
caused fission of smaller cells, thus shortening the re-
sulting latent period. At the same time, the rate of
increase in cell size was retarded. .

The growth stage of parent cells, freshly trans- '
ferred to a suitable medium, affects the lag phase of their
progeny (26); in addition, the duration of the latent phase

reportedly decreases as the frequency of transfer increases.

-5-

As the Optimum growth temperature for an organism
is approached, the lag portion of the growth curve more
closely approximates the normnwhen all other controlling
factors are maintained at Optimum (12, 26). Appreciable
variations in incubation could thus alter the results of
controlled studies on bacterial growth.

Specific genera of organisms may possess a typical
lag phase; that is, a time lag which is particularly long
or short relative to latent phases of other bacterial genera.
The coli-typhoid group has been observed to reach the loga-
rithmic phase of growth very rapidly (26).

At least two methods of lag measurement have been
proposed. Hershey (9) calculated lag as an increase in
bacterial size or cell volume. He assumed an average
maximum size for the adult cell of each species and derived

the formula below:

L - l . log 'égg
M log 2 380

where L - time for 501 population increase
Mi- generations per hour assuming binary fission
Sa - average size of adult cells

So - average cell size initially

-6-

Aerated cultures provided stable cells when all oxidizable
matter had been utilized. These stabilized cells were
used to construct growth curves from which the required
values were obtained.

Hinshelwood (12) proposed a method for lag measure-
ment. This was recently evaluated by Finn (7) who con-
sidered the method comparable to that for measuring rela-
tive lag.

Changes in the lag phase of bacterial growth have
been observed under various experimental conditions.
Eijkman (6) heated g. gg1_i_ at 125.6°F. for o to 35 minutes.
Suspension of cells heated for 6 to 35 minutes showed no
growth at 3 days; all but the 35 minute sample showed bac-
terial growth at 13 days. Lawton and Nelson (16) explored
the influence of heat and chlorine on the growth of se-
lected psychrOphiles. Survivors of a culture partly
destroyed by heat displayed increased lag when.incubated
at 5 or 10°C. Incubation at 25°C. or less resulted in
less extension of lag. Extension of the latent period was

demonstrated using Pseudomonas fluorescens and Pseudomonas

 

geniculata isolated from milk.
The effect of plating media on the recovery of heat
treated Pseudomonas fluorescens was observed by Heather

-7-

and van der Zant (8). Plate counts on more complex media
yielded more survivors. The addition of glutamic acid,
proline, histidine, glycine and.methionine, normal chemical
components of milk, to plating media enabled heat shocked
cells to propagate id greater numbers on synthetic sub-
strates. Studies of factors limiting bacterial growth led
Hershey (9) to report that bacteria surviving heat showed
a progressively lengthened lag phase. unexposed cells and
cells exposed to 56°C. were compared via growth character-
istics. This worker concluded that extended latency
resulted from injury, rather than from the survival of
specially adapted cells occurring in small numbers. Ex-
tension of the lag phase of Escherichia ggligwas demon-
strated.

Kaufmann and Andrews (14) established the thermal
destruction rates of selected Pseudomonads to determine
their heat resistance. Experimental results indicated that
both growth rate and contamination level are important in
psychrophilic spoilage of milk. It was Concluded that the
initial population level at contamination may be less sig-
nificant than the growth rate of the organimm in so far as

shelf life is concerned.

Mossel and M01 (17) incubated coffee milk at 32°C.
and noted that slight curdling occurred after 11 days
(initial controls were negative). Obvious spoilage
resulted after 14 days, at which time Bacillus coagulans

was isolated.
The Heat Tolerant Micrococcus varians sp. MS 102

Barber (1, 2) reviewed the history and character-
istics of Micrococcugvarians sp. MS 102.. The organism was
isolated from pasteurized milk at the National Dairy
Research Laboratories. Studies revealed the ability of MS
102 to resist 180°F. for 15 seconds. Growth on N-Z-Amine
Agar produced easily observed golden-yellow colonies,
possessing a thermal death time curve slape (in ice cream
mix), of ll.4°F. compared to 12.6°F. for Mycobacterium
tuberculosis. Barber also discussed the stability of MS
102, its uniform resistance to heat, and ease of enumera-
tion as the reasons for its use in the experimental heat
treatment of bacteria.

Speck ggugl. (22) used MS 102 to test the efficiency
of High-temperature Short-time pasteurization of ice cream
mix. The results of these heat treatment evaluations
follow: l80°P.fiL.seconds... 82.9 per cent survival,

-9-

185°F./l second ... 94.1 per cent survival, l90°F./l second

. 20.9 per cent survival. Tryptone Glucose Meat Extract
Agar was employed as the recovery medium, and incubation
was at 35°C. for 48 hours. The work of Read £5.21, (19)
showed clearly the effect of "come-up time heat" on MS 102.
At 88.8°C. for 0.25 second 99.9 per cent destruction was
obtained. An equivalent kill occurred at 88.1°C. for 0.5
second. Mere recent studies by Read 35 21° (20) indicated
99.9 per cent destruction of MS 102 at l9l.9°F. and l90.6°F.
for 0.25 and 0.50 second, respectively, using milk as the
suspensory medium. Within the l90.6° to l9l.9° F. range,
the pH of the milk was not significantly altered, and

little protein denaturation was noted.

-10-

EXPERIMENTAL PROCEDURES
Normal Growth Curves at Adjusted Population Levels

Preparation _o_f_ TE 1.92 _(_:_e_l_l_.g. Actively propagating cells of
Micrococcus varians species MS 102 (1) from a 24-hour cul-
ture were transferred to N-Z-Case agar slants prepared
according to Tobias £5 21, (25). The composition of the
medium was as follows:

Yeast Extract ..........

N-Z-Case* ..............
Glucose ................

ouwkuuo
BINOQODGDmH?

Ho

Slants were incubated 24 hours at 32°C. 3 1°C. and then re-
frigerated. After 48 hours at 6°C. 1 1°C. the culture was
transferred to 150 ml. prescription bottles and incubated
for 24 to 26 hours at 32°C. prior to harvesting cells for
growth studies in milk. Thirty ml. of agar were dispensed
into each bottle to provide a reasonably constant surface

area.

 

*This preparation was obtained from Sheffield
Chemical, Nerwich, New York.

The 24 to 26 hour cultures of MS 102 were harvested
using two 20 m1. portions of sterile, buffered distilled
water. An inoculating needle was employed to tease cells
away from the slant surface. The cell suspension was
filtered through sterile cotton to remove clumps of agar
and transferred to a chilled Waring blendor jar. Following
30 seconds of blending, the suspension was transferred to
a dilution bottle. Cell loss during decantation was mini-
‘mized by rinsing the blendor with sterile water. The final
volume of suspended cells was adjusted to 100 ml. and re-
frigerated at 6°C. t 1°C. until appropriate dilutions were

made for normal growth curve studies in milk.

Dilution Scheme, Cell dilution methods were devised to
yield desired populations per ml. Flow diagrams for all
dilution procedures are given in Figure 2. Control growth
studies were made in 150 m1. prescription bottles con-
taining sterile, homogenized milk (3.5 per cent butterfat).

Some difficulty was encountered when the milk tended
to display caramelization after autoclaving. This problem
'was avoided by autoclaving the milk at 121°C. for 10
'minutes under 15 pounds of pressure.

Water for dilution blanks and cell washing was

buffered to pH 7.0 and sterilized at 121°C. for 15 minutes
-12-

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-13-

(2,000,000,000) (200,000,000)

 

 

 

 

 

 

 

‘100 ml. HO?*

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 2. A diagram of the dilution procedure utilized to
obtain extremely low cell concentrations prior to establishing un-
heated control growth studies.

*Buffered water. Except where otherwise noted,
sterile milk was the diluent.

**Numbers in parentheses indicate the calculated
cells per ml. at the various levels of dilution.

-m-

 

under 15 pounds of pressure. Cell yields per slant approxi-
mated two billion cells per ml. after dilution to 100 ml.

To assure uniform, accurate counts, cell suspensions
were shaken 25 times in accordance with accepted methods

before appropriate dilutions were made.

8

Incubation Procedure £95 M 925293. MS 102 was grown
in milk which was tempered to 32°C. prior to inoculation.
Incubation was interrupted only to take samples for plating
at predetermined intervals. Since the lag was measured
according to the graphical method proposed by Hinshelwood
(12) and supported by Finn (7), it was necessary to obtain

well defined lag segments of all growth curves.

Plating Procedure. Plate count agar was selected for
plating. Samples containing less than one organism.per ml.
were plated in adequate volume to yield final counts of one
or more cells per 10 ml. whenever possible. If less than
one cell per ml. was encountered in low population studies,
the result was converted to a whole number. Plates were
poured in duplicate. When exceptionally low counts were
encountered, the milk was distributed in several plates.
The colonies counted per group of plates were divided by
the volume plated to determine the count per ml. When

-15-

necessary, as previously discussed, counts of less than one
cell per ml. were converted to whole numbers which repre-
sented larger aliquots. For example, 0.5 cell per ml.

would convert to 5 cells per 10 ml.
: Post Heat Shock Growth Curves

Procedures used for the preparation of cells, incu-
bation of heated cells, plating, incubating and counting
were the same as described under the previous section.
Uniform.procedures were selected so that growth curves of

heated and unheated MS 102 populations could be compared.

Dilution Scheme. A flow diagram outlining the procedures

 

employed to obtain cell dilutions of 1:10 and 1:100 is
presented in Figure l. The method diagrammed was also used
in obtaining dilutions for growth curves after heat treat-
ments at 81°C. and 82°C. Sterile, homogenized milk was
used in the dilution procedure to obtain desired initial
bacterial populations for all growth curves. ‘Whenever
survivor growth trials were made on heat treated cells,
corresponding control growth curves were established on

unheated cells.

-15-

W 9_f_ Inoculation £93; gga_t_ Treatment. Eighteen ml. of
sterile milk were added to sterile test tubes. The tubes
and contents were brought to the required temperature before
the organisms were injected. A thermometer well, contain-
ing 18 m1. of milk, permitted evaluation of the product
temperature prior to inoculation and timing. Come-up time
was further minimized by tempering the inoculum to 43°C.
before injection into the heating medium.

Two m1. of inoculun were injected into the heated
milk using a 5 m1. syringe with a six inch, 18 gauge needle.
By ejecting the bacterial suspension while moving the
needle toward the bottom of the tube, efficient cell dis-
persion was possible. Sterile gauze pads were utilized to
wipe inoculun from the outside of the needle as bubbles
were expelled from thesyringe barrel. Timing comenced at
the instant the inoculum was added. At the conclusion of
the desired holding time, each tube was promptly plunged
into a solution of 29 per cent calcium chloride which had
been stored at -23°C. for 24 hours prior to use, as recom-
mended by Kaufmann 9.9 51. (15) . Hand agitation for 15
seconds hastened cooling of the heated contents of the
tubes. The smnples were held at -12°C. for 5 minutes
before plating. All specimens (Figure l) were brought to

-17-

room temperature prior to sanlpling to assure accurate

measurement.

Heating Equipment. A Magni Whirl Full Visibility Jar Bath*

was used to maintain the required temperatures.

Methods g§_Measurement. Holding times are estimated to be
accurate to within 3 1 second. Menual operations were
standardized to yield uniform tests. Temperatures were
‘measured with two factory calibrated Centigrade thermo-
‘meters. The relatively large volume of inoculum at 43°C.
caused a demonstrable reduction in holding temperatures.
A comparison of minimum temperatures calculated calori-
metrically, and experimental values obtained each 5 seconds
with a recording potentiometer, showed close agreement.
Since the minimum.temperature for all treatments was evi-
denced at 5 seconds, the minimum.temperatures were selected
for reporting and discussion.

The minimum and maximum temperatures for each treat-

ment follow:

 

*Manufactured by the Blue M Electric Company, Blue
Island, Illinois.

-18-

Temperature of Milk Minimun Calculated Temperature

before Inoculation ‘ after adding 2 ml. of Inoculum
1' 1°C.
87°C . ...... . ................ 82°C
86°C. ............. ......... ... 81°C.
80°C .................. .. . 76°C
72°C .................. .. ..... 69°C
61°C. ....... ......... ... ...... 60°C

The recording potentiometer employed in this work first
registered the temperature 5 seconds after the addition of
the inoculum. That part of the heating curve which oc-
curred between injection of the inoculum and 5 seconds of
holding may be highly significant at the higher tempera-
tures of treatment. Instruments were not available which
would give a temperature response in less than 5 seconds,
therefore it was impossible to evaluate this segment of

the heating curve.

-19-

TABLE I

Plate counts of milk samples containing a high initial

cell concentration of MS 102 after heat treatment

 

 

 

 

 

 

eat 61°C., 69°C. or 76°C.
TRIALI*
unheated 69°C. 76°C. 61°C.
(control) 17 sec. 17 sec. 30 min.
Time Time
(hrs.) (hrs.)

.Av. count Av. count Av. count Av. count

per ml. per ml. per ml. per ml.

(x1000) (x1000) (x1000) (x1000)

0 180 230 220 0 180

9 170 210 180 13 ~ 180

18 290 210 220 21 200

24 680 300 410 27 260

31 9200 700 6800 33 1300

36 17000 3900 8300 37 2100

46 19000 5300 3900 44 3400

- - - - 50 52000

TRIAL II

0 210 240 250 0 190

10 230 270 260 13 260

15 390 310 270 21 330

20 450 500 290 27 370

25 5500 1600 260 33 1100

32 9000 14000 380 37 2800

37 26000 15000 1700 44 8700

42 16000 19000 1800 50 35000

48 840 960 810 - -

 

 

 

 

 

different generations of MS 102.

-20-

*
Trials I and II represent separate tests run on

 

LOG NO. PER ML.

LOG N0. PER ML.

 

 

4 e
3 _ TreoImsnI - None
0 TRIAL I
2f - TRIAL 11
OT _1 J l l I
I0 20 30 4O 50
HRS.
8 _

 

 

4 I.
3 _ 69° C./I759c.
o TRIAL I
2 " o TRIAL II
T l l l
0 IO 20 30

HRS.

 

 

 

I- 6I o Cfl/3OITIII'I
0 TRIAL I
b O TRIAL U
i l L l L I
0 IO 20 30 4O 50
HRS.

 

 

 

I‘ 76° C./I7sec.
o TRIAL I
T o TRIAL II
i l l l l I
0 IO 20 30 40 50
HRS.

Figure 3. Effect of heat shock on the growth curve of
survivors. (High initial cell concentration).

TABLE 11

Plate counts of milk samples containing a medium initial
cell concentration of MS 102 after heat treatment

at 61°C., 69°C. or 76°C.

 

 

 

 

 

 

 

 

 

 

mm. 1*
unheated 69°C. 76°C. 61°C.
(control) 17 sec. 17 sec. 30 min.
Time Time
Khrs.) (hrs.)
Av. count Av. count Av. count Av. count
per ml. per ml. per ml. per ml.
‘(x1000) (x1000) (x1000) (x1000)
0 14 21 17 0 17
9 13 19 l4 13 37
18 30 16 9 21 18
24 80 49 200 27 720
31 5600 274 1500 33 2600
36 8400 1600 4300 37 8700
46 14000 3000 8300 44 11000
- - - - 50 9200
mm. TI
0 19 19 18 0 13
10 19 19 9 l3 17
15 29 22 10 21 15
20 140 250 12 27 600
25 460 410 206 33 2500
32 5600 5400 4800 37 6200
37 7200 5400 19000 44 7600
42 14000 6500 9700 50 8100
48 3400 6900 11000 - -

 

*Trials I and II represent separate tests run on
different generations of MS 102.

-22-

 

LOG N0. PER ML.

LOG N0. PER ML.

 

 

 

Bf- I
I o
O
k c
3 _ Treatment -None _ 6| ° C./ 30min.
0 TRIAL I 0 TRIAL I
2* o TRIAL II o TRIAL 1:1

 

 

I-
oi I I I i J I I l I
O

I I
I0 20 30 4O 50 IO 20 30 4O 50
HRS. HRS.

 

3 _ 69° C./I7sec. _ 76° C./l7sec.
0 TRIAL I 0 TRIAL I
2*- . TRIAL n ‘ 0 TRIAL]!

 

 

I J l J i l l l l I

I0 20 30 4O 50 0 IO 20 30 4O 50
HRS. HRS.

Figure II. Effect of heat shock on the growth curve of
survivors. (Medium initial cell concentration).

-23-

TABLE III

Plate counts of milk samples containing a low initial
cell concentration of MS 102 after heat treatment

at 61°C., 69°C. or 76°C.

 

 

 

 

 

 

 

 

 

 

TRIAL 1*
unheated 69°C. 76°C. 61°C.
(control) 17 sec. 17 sec. 30 min.
Time Time
(hrs.) (hrs.)
Av. count Av. count Av. count Av. count
per ml. per ml. per ml. per ml.
(x1000) (x1000) (x1000) (x1000)
0 2.0 1.1 1.8 0 2.2
9 1.7 1.8 1.5 13 1.9
18 1.9 1.7 1.4 21 2.6
24 40.0 1.8 1.5 27 3.0
31 3200.0 19.0 7.0 33 3.1
36 3700.0 420.0 400.0 37 2.9
46 15000.0 1400.0 5500.0 44 4.5
- - - - 50 1400.0
TRIAL II
0 2.4 2.3 1.7 0 1.8
10 2.0 2.1 1.9 13 1.5
15 2.7 2.6 1.9 21 1.7
20 2.2 20.0 2.1 27 1.5
25 57.0 280.0 1.7 33 2.1
32 2400.0 1100.0 3.7 37 1.8
37 ‘ 5100.0 1100.0 360.0 44 2.5
42 3300.0 6500.0 2700.0 50 1700.0
48 41000.0 2900.0 2300.0 - -

 

*Trials I and 11 represent separate tests run on
different generations of MS 102.

-24-

 

 

 

LOG N0. PER ML.

LOG N0. PER ML.

 

O.

 

Treatment -None

 

2 ‘ o TRIAL I
i 0 TRIAL II
0 I I I I
IO 20 30 40 50
HRS.
8T

 

 

 

69° C./I7$ec.
2 o TRIAL I
o TRIAL II
0 I I 4 I I
I0 20 30 4O 50
HRS.

I—

i o TRIAL II
I I

O

 

6| ° C./30min.
o TRIAL I

 

I

IO 20 30
HRS.

I I
40 50

 

 

 

76 ° C./I7sec.
" o TRIAL I
i o TRIAL II
I I I I I
0 IO 20 30 40 50
HRS.

Figure 5. Effect of heat shock on the. growth curve of

survivors .

-25-

(Low initial cell concentration).

TABLE IV

Plate counts of milk samples containing a low or medium
initial cell concentration of MS 102 after
heat treatment at 81°C. or 82°C.

 

 

 

 

 

TRIAL 1*

I l Uhheated control 81°C.75 sec. EZEC.Z§ sec.|
Time Av. count Av. count. Av. count
(hrs.) per ml. per ml. per m1.

(x1000) (x1000) (x1000)
0 210 21.0 2.7
12 200 6.4 1.9
18 590 6.7 1.6
25 1400 7.4 2.0
30 1900 7.6 1.8
35 . 2300 960.0 22.0
'39 2800 3000.0 28.0
43 7400 14000.0 31.0
50 17000 12000.0 240.0
55 - 15000.0 1000.0
TRIAL II
0 (same as above) 140 130
12 250 38
18 180 88
25 1100 200
30 1900 1400
35 2300 1800
39 2600 3000
43 5000 11000
50 4000 11000
55 8500 13000

 

 

 

 

*Trials I and 11 represent separate tests run on
different generations of MS 102.

-26-

TABLE V

Plate counts of milk samples containing a low initial
cell concentration of MS 102 after heat treatment
at 81°C. or 82°C.

 

 

 

 

 

TRIAL 1*

I I unheated control 1 C. 5 sec. §23C.ZS sec.I
Time Av. count Av. count . Av. count
(hrs.) per ml. per ml. per ml.

(x1000) (x1000) (x1000)
0 16 3.7 1.4
12 20 1.3 .63
18 870 1.2 .82
25 3500 1.1 .81
30 . L.A.** 1.3 .70
35 8100 1.5 .72
39 40000 3.2 1.0

. 43 - 9.6 3.3
50 - 370.0 16.0
55 - 1700.0 890.0
TRIAL II

0 (same as above) 5.9 .59
12 1.7 .38
18 1.4 .22
25 1.4 .31
30 1.4 .25
35 4.4 .64
39 42.0 .58
43 540.0 .60
50 sample sample
55 depleted depleted

 

 

 

 

tTrials I and II represent separate tests run on
different generations of MS 102.

**Laboratory Accident.
-27-

TABLE VI

Plate counts of milk samples containing a very low
initial bacterial papulation after
heat treatment at 81°C. or 82°C.

 

 

 

 

 

 

TRIAL 1*
unheated control 81 C. 5 sec. 8 C. 5 see.
Time Av. count Av. count Av. count
(hrs.) per ml. per ml. per ml.
(x1000) (x1000) (x1000)
0 ' 1.9 .19 .35
12 . 1.9 .04 .012
18 1.5 .13 .019
25 32.0 .07 .015
30 720.0 .06 .012
35 2900.0 .22 .020
39 3600.0 .30 .016
43 5800.0 .50 .030
50 - .46 -
55 - - -
TRIAL II
0 (same as above) .055 L.A.**
12 .038
18 ' .043
.25 . .032
30 .031
35 .039
39 .050
43 .052
50 .020
55 .010

 

 

 

*Trials 1 and 11 represent separate tests run on
different generations of MS 102.

**Laboratory Accident.

-23-

LOG N0. PER ML.

LOG N0. PER ML.

  
 
  
 

Initial cell cane/ml.

 

 

1 <>-I30,000
IIIA" 2,700
lilo-"L400
11 c ----- 590
1:10 ------ 35

Heat treatment - 82°C. / 5 sec.

 

I I l' I J J
0 IO 20 30 4O 50 60

 

HRS.
. II
7’ I
6' III
5<

Initial cell conc./ml.

 

qb‘ 1 <>-I40,000
na--2I,000
man-3, 700
I. ----- ISO
2110 ------ 55

 

 

Heat treatment - 8|°C./5 sec.

 

l I I I I I
0 IO 20 30 4O 50 60
H RS.

Figure 6. Growth curves of MS 102 cells which survived
81°C. or 82°C. for 5 seconds.

-29-

 

 

 

 

 

 

F TREATMENT ‘
O No heat ¢
0 6|°C./3O min.
7- O 69°C./I7sec.
A 76°C./I7sec. /
¢ 8|°C./5 sec. '
.5 6" ¢
2
o:
'ci‘
0. L.
2
L9
0
.4
5b—
' /
/
‘Oo¢r -3 -0’ o
\\\
4r\ ‘
\
\\\ m ¢ ¢ NOTE: DATA ARE
‘4? Y FROM TABLES
11 AND m-
f 1 1 1 J 1
0 IO 20 3O 40 50

HRS.

Figure 7. Effect of heat on the lag phase of MS 102.
(Average initial population - 19,000/n1.)

-30..

TREATMENT ‘

 

 

 

 

 

 

 

 

 

 

- O No heat 4.
O 6|°C./30 min.
0 69°C./l7sec. .
A 76°C./l7 sec. 0 1
6r- ¢ 8|°C./5 sec. /
O 82°C./5 sec. ..
0 ¢
.i
2
0: !5__
‘i‘
0'
2:
m
C) _.
.1
0
4r— 1'
0
‘£\ . .
w \
A0. '3 3 Cb
5‘ ‘ ' " 3235...-
3 \\\ ‘9 FROM TABLES
‘~~_ _ m '3 mANDIIz
‘%i 1 C) .1 ‘? I 1
0 IO 20 30 4O 50
HRS.

Figure 8. Effect of heat on the lag phase of HS 102.
(Average initial population - 2000/1011.)

-31-

 

 

LOG N0. PER ML.

2 _ Av. initial no. per. ml.

 

 

o‘I-ISHDJDCKJ
|_ on-15,000
AIR-L500
0 IO 20 30 40 50
HRS.

Note: Data are from Tables I, II
and III

Figure 9. Effect of the survivor cell concentration on
the growth curve after 610C. for 30 minutes.

-32-

 

 

 

 

 

 

 

 

8"
7' o ' ..
t [H
O
6“
J ., . - ° '2 ‘
=5 55_
gt: .
C5 4' .
2 N A A A ‘
8 3 ‘ ‘
.1
2t- Av. initial n0. per. ml.
1-240900
'_ II-l8,000
IUI-l,8CHD
0 IO 20 30 40 50
tHQS.

Note: Data are from Tables I, II
and III

Figure 10. Effect of the survivor cell concentration on
the growth curve after 760C. for 17 seconds.

-33-

 

 

 

 

 

 

.J
EB
Q:
ht
Q.
‘2’
Q)
C)
a
2 _ Av. initial no. per. ml.
I-24OQOO
I l— H-Z0,000
IflI-4,7CK)
O I I l I J
IO 20 30 4O 50
FHRS.

Note: Data from Tables I, II, III

Figure ll. Effect of the survivor cell concentration on
the growth curve after 69°C. for 17 seconds.

-3h-

TABLE VII

Plate counts of milk samples containing initial
cell concentrations selected for the
construction of control growth
curves showing rate of
growth.in milk

 

 

 

 

Time Count Count Count Count
(hrs.) per ml. per ml. per ml. per ml.
(x1000) (x1000)
0* 0* 13* F‘"
0 12 1.1 150 18
4 14 1.0 150 18
9 16 1.2 170 14
12 19 1.2 200 16
18 41 1.1 200 14
22 630 2.4 200 10
26 5000 4.8 260 40
33 13000 700 1000 260
36 17000 3500 2900 720
42 - 8000‘ 18000 3100
3* ' A*
0 210 1800
10 230 1600
15 390 3600
20 450 3500
25 5500 3300
32 9000 4900
37 2600 6200
39 L. A.** 4800
42 16000 -
48 840 -

 

*Refers to the correspondingly designated growth
curve of Figure 12.

**Plates at 39 hours were contaminated.
-35-

TABLE VIIb

Plate counts of milk samples containing extremely
low initial cell concentrations selected for
the construction of control growth
curves in milk

 

 

Time Count Count Count Count

(hrs.) per :1. per ml. per 10 ml. per 10 ml.
0 4 4 4 4
10 4 3 8 no recovery
21 5 4 8 " "
25 5 6 4 " "
30 4 4 no recovery " "
38 3 3 n n n n
48 no recovery no recovery " " " H
53 H H N H H II H H
72 H H H H H H II n
78 H H H H H H H II
84 H H H H H H H . H
96 I! H H H H II I! I!
99 H H H H H H H H

104 H H H I! I! H H H

 

 

 

Note: The dilution scheme followed to obtain these
extremely low initial cell concentrations is
presented in Figure 2.

*Refers to the correspondingly designated growth curve
of Figure 12.

U);

it...

 

 

 

 

LOG N0. PER ML.

 

 

 

 

  

 

 

 

Initial cell cone/ml.
A O - l,800,000

 

Ii ‘1 I
2 | 80—- 2:0,000
l CO---IZ,OOO
I DO--- I, IOO
I EA ----- I50
Q Q ‘ 1 F6 —————— I8
I w I 0 5‘ ““““ ‘4
I
: 2.; N
\
I I I J l \(I l
0 IO 5 20 3O 4O 50
Log time HRS.

Figure 12. Effect of initial cell concentration on the
lag phase of control growth curves of unheated cells.

-37-

TABLE VIII

Plate counts of milk samples containing extremely
low initial cell papulations

 

 

 

 

 

Triplicate samples
Time ~
1,, E1 E2 153 I X1 i2 x3 I Y1 Y2 Y3
hours I
Colonies per 10 m1. plated
0 5 7 3 4 4 3 no recovery
12 0 1 1 0 1 1 " "
22 2 0 0 0 0 1 " "
33 6 2 0 no recovery " "
42 20 o 2 H H H H
60 10 o 0 H H H H
72 no recovery " " " "
96 H H H II N H
13 days n n n n n n

 

 

 

 

 

Note: Samples E, x and Y'were designed to contain 2, 1
and 0.1 cell per m1. as shown in Figure 2.

-33-

TABLE IX

The effect of selected heat treatments on the
total lag phase of MS 102

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Initial IBnheated 61°C. 69°C. 76°C. 81°C. 82°C.
population control 30 min. 17 sec. 17 sec. 5 sec. 5 sec.
range
per'ml.
Lag in Hours
210,000 17 25 23 25 19 21
18,000 16 21 20 21 31 42
1,800 20 42 22 30 35 42
190 28 - - - - 50*
100 25 - - - 52* 44*

 

*The lag phase was
Depletion of the sample

growth curve.

Note:

longer than the time indicated.
prevented continuation of the

An initial cell concentration of 2700 per ml.

was included in the medium range since, of the
data obtained, it most nearly approximated the
limits defined.

-39-

 

 

m‘a

_‘.r ...» I

TABLE X

The effect of selected heat treatments on the
extension of the lag phase of MS 102

 

 

 

 

 

 

 

 

 

 

 

Temperature Initial population per m1. after heating
and time .
-0f 210,000 I 18,0001 1, 0 190
treatment ‘
Extension of the lag in hours (beyond control)
69°C./17 sec. 6 4 2 * *
|61°C./30 min. 8 5 22 * *
76°C./l7 sec. 8 5 1o * *
I51°c. IS sec. 2 15 15 * 27
Isz°c. /5 sec. 4 26 22 22 19

 

 

 

 

 

 

 

*No determinations were made.

-40-

 

 

RESULTS AND DISCUSSION

Effect of Heat Treatment on the Lag Phase at

Selected Cell Concentrations

The initial papulations obtained after heat treat- I
ment are referred to as high in the range of 100,000 to I
200,000 cells per m1., medium in the range of 10,000 to .
20,000 per 1.1., low in the range of 1,000 to 9,000 per m1. 3
and very low in the range of 100 to 900 cells per ml.

Individual growth curves of survivor populations
after the various heat treatments are shown in Figures 3,
4, 5, 6, 7 and 8; the average curves are shown in Figures
9, 10 and 11. The total 'lag times calculated from the
growth curves of survivor and unheated control populations
are summarized in Table IX. The hours of lag extension,
beyond their respective controls, are presented in Table X.

‘With high initial populations, the total lag times
after heating at 61°C. for 30 minutes or 69°C. for 17
seconds were 25 and 23 hours respectively, as compared to
17 hours for the control. It is apparent that these two

treatments only extended the lag phase of MS 102 about 6

to 8 hours. It is interesting to note that both of these
procedures are approximately equivalent in their effect on
the lag time when high initial cell concentrations are used.
Little or no lag extension over the unheated control oc-
curred on heating to 81°C. or 82°C. for 5 seconds. The
effect of massive initial inoculation on the lag phase of

growth (26) may have limited the effects of "heat shock" |

at the temperatures used in this instance.

"t‘J . .'

The lag time for medium survivor papulations was

 

slightly greater than the control after all heat treatments
(Figures 4, 6 - curve 11). At 81°C. for 5 seconds and at
82°C. for 5 seconds the total lag periods were 31 and 42
hours respectively. This represents an extension of the
lag time by at least 26 hours at the higher temperature.

At this population level the total lag times of the samples
subjected to 61°C. for 30 minutes and 69°C. for 17 seconds
were 20 and 21 hours; this represents an insignificant
increase in lag of 4 and 5 hours, respectively. Since the
population was constant, the only variable factor which
might account for the obvious increase in lag time would be
the heat treatment; the increase in lag phase appears to be
a direct result of "heat shock." Conditions which approxi-
mated the standard pasteurization process for milk failed

-42-

to induce an appreciable extension of the lag phase; treat-
ments which tended slightly in the direction of ultra high
temperature processing yielded a lag phase of approximately
31 to 42 hours.

Very low post-heat populations (Figure 6 - curves V
and VI), showed lags of 44 to 52 hours after treatment at
82°C. and 81°C. respectively for 5 seconds. Undoubtedly
longer lag phases would have been obtained had it been

possible to continue the experiment.

 

At low papulation levels the total lag time was 42
hours after heat treatment at both 82°C. for 5 seconds and
61°C. for 30 minutes,.as compared to 20 hours with the un-
heated control; this is an increase of 22 hours. At 69°C.
for 17 seconds the increase was very slight. No definite
explanation can be given at this time for the very long
(22 hour) extension of the lag phase observed for low
concentrations of survivors after 61°C. for 30 minutes and
82°C. for 5 seconds, particularly in view of the relatively
low lethality of the LTLT treatment. A temperature of 69°C.,
which is higher than 61°C., did not induce an appreciably
extended lag phase. This would suggest a minimum tempera-
ture for the occurrence of the "heat shock" phenomenon under
certain conditions; the minimum temperature would probably

-43-

vary with different organisms. The lack of any appreciable
extension of the lag time at 69°C. for 17 seconds at low
population levels would tend to minimize the shelf life of
a product.

Following heat treatment at 81°C. and 82°C., a
gradual decrease in cell count was noted during the first
12 hours of the lag phase (Figures 6, 7 and 8). With the
former, the populations obtained by the standard plate
counts were reduced about 60 per cent between 0 time and
12 hours of incubation (Tables IV, V and VI); at the latter
temperature the average papulation decrease was 57 per cent
in a similar period of time. This post-heat-death may have
resulted from a nutritional difference between the standard
plating agar and the growth.medium.(milk). Post-heat cell
counts were made immediately after heat treatment using
Plate Count Agar (PCA). If the essential recovery factors
of a nutritional or physio-chemical nature were supplied by
PCA, but not by milk, then greater recovery of injured cells
would be expected on plates poured at zero time. During
the first 12 hours of incubation in milk, some of the
injured cells may have gradually lost their viability due
to the absence of essential recovery factors. The PCA may
have provided the readily available amino acids essential

-44-

to the recovery of the heat injured cells. Although milk
proteins contain the essential amino acids, the amino acids,
23; 22, may not be as readily available for assimilation.
Injury to the semipermeable membrane may also be involved
in the gradual death of cells due to abnormal osmotic

balance.
Effect of Survivor Cell Concentrations on the Lag Phase

Normal growth curves of MS 102 at selected cell
concentrations are presented in Figure 12. unheated control
growth curves with initial cell populations above 1,100 per
ml. showed progressively shortened lag phases; below this
level the lag phase appeared to be progressively extended.
Results of studies for extremely low initial levels of
inoculum are presented in Tables VIIb and VIII. Uniform
growth.was not obtained at initial papulations below 10
cells per ml.

The results pertaining to the effect of population
on the lag phase after heat treatment are given in Figures
9, 10, 11 and Table X. At high and medium levels of
survival the lag times of samples heated to 61°C. for 30
minutes, 69°C. for 17 seconds and 76°C. for 17 seconds were
similar. At all high levels of papulation the heat

-45-

treatment did not materially increase the duration of the
_lag phase; at medium cell concentrations the effect of heat
was slight with respect to lag extension. Exceptionally
short lag times occurred in samples heated to 81°C. and
82°C. for 5 seconds at high initial cell concentrations,

as compared to the lag obtained at medium and low papu-
lations. Since the lag times for high cell levels at these
temperatures (19 and 21 hours) are similar to the 17 hour
lag time of the unheated control, perhaps the lag reduction
effect of high populations overbalanced the lag extension
effect of the heat treatment employed.

Among samples heated to 61°C. for 30 minutes the
longest lag time occurred at the law initial cell concen-
tration. In all cases, the lag times for high and medium
cell levels after 61°C. for 30 minutes were similar. Lag
times for MS 102 organisms surviving 69°C. for 17 seconds
at high and medium population levels were also similar (23
and 20 hours respectively). A marked contrast occurred at
82°C. for 5 seconds at which the lag time was much shorter
for high concentrations of cells than for medium or low
surviving populations (21, 42 and 42 hours respectively).
At 81°C. and 82°C. for 5 seconds the effect of "heat shock"
on the lag phase with high populations seems small compared

-46-

.A'I' '

II {.III \II

to the effect on lower concentrations of cells.

General Considerations Evolving from

the Lag Extension Studies

The keeping quality of food products depends, to
some extent, on the duration of the lag phase of the
bacteria which survive the thermal process to which the
product was exposed. According to‘Williams (27) aerobic
spore formers, heat resistant micrococci, streptococci and
corynebacteria are the four genera most likely to cause
post-pasteurization spoilage in milk and related foods.

The results reported herein show that the lag phase
of growth of'MS 102, subjected to 82°C. for 5 seconds, is
considerably longer than that obtained in whole milk after
exposure at 69°C. for 17 seconds (conditions approximating
HTST). The limitations of equipment prevented a study of
the "heat shock" phenomenon at temperatures between 82°C.
and 143°C. If the lag phase increases considerably at
these higher temperatures, the storage-life of milk might
»be materially extended. Preliminary studies by Kaufmann
(13) on whole milk pasteurized at high temperatures (97°C.)
indicate that the lag period, pg; 32, may be as long as 2
or 3 weeks when milk is stored at 4 to 7°C. On the basis

-47-

of the studies reported here, and those of Kaufmann (13),

it is conceivable that processing at ultra high temperatures
might result in long lag periods for spoilage bacteria which
survive and increased storage life for thermally processed
foods.

The literature contains several reports of un-
explained, delayed bacterial growth after heat treatment.
Day (4) reported false negative tests for psychrophiles
unless the milk was held at least 3 days. Such delays
after HIST processing may represent the normal growth
process for certain psychroPhiles from post-heat contami-
nation, or may indicate an extended lag time after heat
treatment. Eijkman (6) reported that a suspension of g.
‘ggli cells, heated for 6 to 35 minutes at 125.6°F. showed
no growth after 3 days, but all except the sample heated
for 35 minutes contained viable cells after incubation for
15 days. Heather and van der Zant (8) demonstrated iné
creased lag for some pseudomonads after heat treatment at
135°F. for l to 8 minutes. Mbssel (l7 ) observed the
curdling of coffee milk (processed at 110°C. for 30 minutes)
after 11 days at 32°C. The delays observed in these in-
stances may be associated in part with an extension of the

lag phase which exists after exposure to heat.

-43-

SUMMARY
The Effect of Heat Treatment on the Lag Phase of MS 102

Selected cell concentrations of MB 102 were heated
in milk at 61°C. for 30 minutes, 69°C. for 17 seconds,
76°C. for 17 seconds, 81°C. for 5 seconds and 82°C. for 5
seconds. The lag phases of growth curves of surviving
bacteria, propagated in whole milk at 32°C., were extended
beyond the lag times observed in unheated controls. The
lag extension effect was observed at medium and low
survivor papulations (10,000 to 20,000 and 1,000 to 9,000
.cells per ml. respectively), but not at high survivor cell
concentrations (100,000 to 200,000 cells per ml.).

medium population levels subjected to 61°C. for 30
minutes or 69°C. for 17 seconds showed lag times of 21 and
20 hours respectively. These periods of lag were approxi-
mately one half as long as the lag time (42 hours) observed
after heating cells at 82°C. for 5 seconds.

At low population levels subjected to 82°C. for 5
seconds or 61°C. for 30 minutes the lag periods (42 hours)
were much greater than the lag time (22 hours) after 69°C.

for 17 seconds.

 

LITERATURE CITED

(1) Barber, F. W. No Hold Pasteurization Makes Progress.
Food Eng., .2_5_: 62. 1953.

(2) Barber, F. W. and Hodes, H. P. A Culture Suitable for
Use in Evaluating Methods of Pasteurizing Milk.
Bact. Proc., p. 25. 1950. (Abstract).

(3) Chesney, A. M. Latent Period in the Growth of Bacteria.
J. Expt. Med., _2_4_: 387. 1916.

(4) Day, E. A. and Doan, F. J. A.Test for the Keeping
Quality of Pasteurized‘Milk. J. Milk and Food
Tech., 12: 63. 1956.

(5) Dubos, Rene, J. The Bacterial Cell. Harvard Univer-

sity Press, Caisridge, Mass. 1945.

(6) Eijkman, C. Die Uberlebungskurve bei Abtotung von
Bacterien durch Hitze. Biochem. Z.,'ll: 12. 1908.

(7) Finn, R. K. 'Measurements of Lag. J. Bact., 29; 352.
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-51-

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