Factors Affecting Resistance to Heat and Recovery of
Heat-Injured Bacteria 1
Market Quality Research Division, USDA, Beltsville, Maryland 20705
University of Maryland, Department of Dairy Science, College Park 20740
Abstract
An unidentified Pseudomonas sp. isolated
from pasteurized milk stored at 4 C ap-
peared killed by a heat treatment of 55 C
for 30 rain, since no colonies formed on
trypticase soy agar plates during incuba-
tion for 48 to 72 hours. However, when
the heated bacteria were held in trypticase
soy broth for 48 to 72 hours at 20 C or
two to three weeks at 4 C, some cells re-
covered ability to grow normally, t t e a t
resistance and recovery were affected by
the physiological state of the bacteria and
the nature of the heating and the recovery
medium. More complex heating media,
including milk whey, stabilized the bac-
teria to heat, and favored recovery. Re-
covery was also found with five species of
Salmonella and is probably a general
phenomenon after heating at time and
temperature combinations just above those
which are apparently lethal.
The limitation of shelf life of pasteurized
milk by the growth of so-called psychrophilic
bacteria during refrigeration is one of the most
serious problems facing the dairy industry. The
presence of psychrophilic bacteria in pasteur-
ized milk is usually attributed to post-pasteuri-
zation contamination; yet, it is frequently diffi-
cult to demonstrate psychrophilic bacteria in
freshly pasteurized milk. However, they com-
nmnly a p p e a r in substantial numbers in the
same s~mples after storage for a few days.
W i t t e r (31), after an extensive review of the
literature on psychrophilic bacteria, concluded
that psychrophiles do not survive pasteuriza-
tion. However, large numbers of psychrophiles
are found after 10 to 16 days of storage at
refrigeration temperatures (3, ]6, 17, 25, 29,
30). This suggests the possibility that bacteria
apparently killed by heating may recover their
ability to grow after extended storage.
Received for publication October 1, 1968.
1 Mention of specific trade names is made f o r
identifieatio~ purposes only and does not imply any
endorsement by the U.S. Government.
608
ROGER DABBAH and W. A. MOATS
and
J. F. MATTICK
The usual criterion of death of microorga-
nisms is failure to reproduce on plate count
agar, although as early as 1933 Rahn and Barnes
(24) believed that this was insufficient, and
Chambers et al. (4) showed that incubation of
heat-treated bacteria in lactose broth for 48-96
hr before inoculation on plate count agar was
necessary for their detection.
Detection of bacteria following stress has
been studied extensively. I n general, heating
(9, 20, 21) or freezing (2, 22, 26) of bacteria
results in supplementary nutritional require-
ments for recovery. F o r example, recovery of
bacteria injured by freezing is brought about
by the activity of specific peptides in trypti-
case (19, 28), and a factor present in yeast
extract promoted recovery of heat-treated psy-
chrophilie bacteria (11).
Variations in heat resistance of a given or-
ganism can result from differences in heat
treatment and recovery conditions (10) and
differences in temperature before heat treat-
ment (8).
Delayed growth of bacteria following heat
treatment has been explained on the basis of
attenuation (1) or dormancy (6). Other
workers have concluded that the bacterial cells
are injured by heat and suggested that the
nature of the damage is enzyme inactivation
(5), damage to the osmotic barrier and dis-
ruption of the balance of reaction rates (]8,
23), or structural changes in the protoplasm
(12). Jackson and Woodbine (15) are of the
opinion that the only effect of sublethal heat
treatment is to delay growth.
Chambers and co-workers (4) and Oarvie
(7) believe that recovery of bacteria following
stress does not occur and that subsequent growth
results from multiplication of a very small num-
ber of survivors. On the other hand, I-Ieinmets
and co-workers (13) and Iandolo and Ordal
(14) believe that recovery occurs in the absence
of growth and that it involves enzymatic ac-
tivity directed toward repair of injury.
This p a p e r shows that some cells of a psychro-
philic species of bacteria recovered and grew
normally after incubation under favorable c o n -
 HEAT RESISTANCE
ditions following an apparently lethal heat
treatment. Factors affecting heat resistance and
recovery are discussed.
Materials and Methods
Bacterial c~lt~re. The test organism was a
psychrophilic culture isolated from commer-
cially pasteurized milk held at refrigeration
temperature for 16 days. I t was a gram-nega-
tive rod, polar flagellated, and motile; it pro-
duced acid from glucose and slightly from
arabinose, with no gas production. No acid
or gas was produced from sorbitol, xylose, mal-
tose, trehalose, inositol, duleitol, mannitol, sali-
cin, rhamnose, raffinose, lactose, and sucrose;
indole production was negative; there was no
formation of nitrites from nitrates or produc-
tion of hydrogen sulfide. The isolate has an
optimum growth temperature of" 20 C, good
growth at 0 and 5 C, but will not grow at 37 C.
It has been identified only as a species of"
Pseudomonas.
Heating treatment. Fifteen milliliters of' the
bacterial suspension were introduced aseptically
into a 20-ml sterile glass ampule, sealed and
completely immersed in a thermostatically con-
trolled water bath. A control ampule contain-
ing a thermocouple linked to a Thermistemp
Tele-Thermometer (Yellow Springs Instrument
Co., Inc.) was used, and time of heating was
measured when the temperature of the sus-
pension in the control ampule reached 55 C.
After 30 rain at 55 C, the ampules were im-
mez~ed in ice-water for 3 rain. Ampules were
then opened with a hot wire glass cutter and
5 ml of the heated suspension transferred to
95 ml of unheated trypticase soy broth unless
otherwise indicated. Heated bacteria were
plated on tryptiease soy agar immediately after
heat treatment and every 24 up to 240 hr of
incubation at 20 C. The plates were incubated
at 20 C for 48 ± 2 hours.
Preparation of sterile whey. Raw whole milk
was warmed to 40 C and 10 ml of 5% acetic
acid added to each 100 ml. The whey was fil-
tered with suction through a layer of Cetite 545
and filter paper in a Biichner funneh The fil-
trate was neutralized to p H 6.8, Celite 545
added, and the mixture refiltered; the whey
was then sterilized by passing through a Seitz
filter.
Media. Tryptiease soy broth, tryptiease, and
phytone were obtained from the Baltimore Bi-
ological Laboratory, Baltimore, iVfaryland. Ba-
sal medium is the formula of Heather and
Vandcrzant (11).
OF BACTERIA 609
Experimental Results
The initial experiments were conducted to
determine if bacteria apparently killed by heat-
ing could, in fact, recover and grow in a fa-
vorable medium. The bacteria were heated for
30 rain at 55 C in tryptiease soy broth and
survivors enumerated on trypticase soy agar.
The heated suspension was transferred to fresh
broth, incubated at 20 C, and examined daily
for evidence of growth. The bacteria tested
included Pseudomonas fl~wrescens from our
laboratory collection, Pseudomonas fragi, an
Achromobacter species supplied by Dr. J. A.
Alford, USDA, Meat Laboratory, Beltsville,
Maryland, our Culture 18 isolated from pas-
teurized milk, and a subculture of Culture 18
which had recovered from an earlier heating
experiment. Under the particular conditions
studied~ only Culture ]8 showed the recovery
phenomenon as shown in Figure I ( D ) . For
convenience in illustration, the log of no de-
tectable survivors is plotted as 0. No survivors
were noted for 48 hr, but after 48 hr of incuba-
tion in tryptiease soy broth, survivors appeared
and grew normally. Figure I ( E ) shows the
same experiment conducted with bacteria which
recovered from the first heat treatment, show-
ing identical results, indicating that the bacteria
which recovered are the same as the originals
and that no selection for heat resistance oc-
curred. P. fluorescens (Fig. l-A) did not sur-
vive the heat treatment and no recovery
occurred. Some cells of the P. fragi and Achro-
mobacter species survived heat treatment and
grew normally with no lag after heating (Fig.
1-B and C). Culture 18 was, therefore, selected
as the test organism for further studies.
Criticisms have been made of this type of
experimenL on the grounds that the numbers
of survivors were too small to detect during the
recovery period or that they were contaminants
introduced during sampling. To test these criti-
cisms, loopfuls of 24-hr Culture 18 were inocu-
lated into ampules containing" trypticase soy
broth and the ampules sealed and incubated for
24 hr. 5lost of the ampules were then heated
at 55 C for 30 rain. Immediately after heat
treatment and every 24 hr up to 240 hr of incu-
bation at 20 C, duplicate ampules were opened
and all the contents plated on trypticase soy
agar in 1-ml portions. This technique elimi-
nated any possibility that uninjured survivors
would be overlooked and also eliminated any
possibility of contamination. Some bacteria
were observed in the heat-treated ampules after
96 hr (Fig. 2-]3), but neither the heat-treated
nor unheated cultures showed any multipliea-
J. DAII~Y SCIENCE VOL. 52, NO. 5
610 D A B B A H E T AL.

A D

8m

6.
4-

0 | ,f. .~ ; '-'--,d, ,L A----k--A---~

MI

<
_J
Q. 8. f
< 6,
qJ) f A'~A~A"~'4"A'~
I--

Z
41
0
I,,-

Z
0v
____/
u_

0 I0.,~
S. IA"
I,,,- -----o---- NOT HEATED
6-
< A----" HEATED
o 4.
0
.J

2.

°i I I l I I I I I '1 "l' " i

48 96 144 192 240 4~8 ' 96 ' 1~4 ' 142 '2~0
HOURSOF INCUBATION
FIG. 1. Growth curve of various psychrophilie bacteria before and a f t e r heat treatment at 55 C for
30 min.
A. P. fluorescens; B. P. frag~; C. Aehramobacter sp.; D. Pseudomonav sp. (Culture 18) ; E. subcul-
ture of heat-trea~ed and recovered Culture 18.
For convenience in illustrating, the logarithm of no detectable survivors is plotted as 0.

J. DAIRY SCIENCE VOL. 52, No. 5

 HEAT RESISTANCE OF BACTERIA 611

10.
/ - - o.
A
8.

6- /../ ,/
Md
I-- m 1
/ ..... heated
a. ! not heated
2. /
1
1
I-- 0- ~o --o
Z
O
I--
Z 10.

O
O B.
M.
O 6.
o tsb
• basal
~"
I--
4
/ "~ 0--- - - 0 - -
/
'~ 2, /
O
I
O /
0- - - ~ _-_-_p_: -o

i i
I I I I l I i I I
48 96 144 192 2 0

HOURS OF INCUBATION
t~im 2. Growth curve of heated and unheated Pseudo~onas sp. (Culture :18) when incubaLed at 20 O
in the heating medium (tsb: trypticase soy broth, or basal: basal minimal medium). Each count of the
heated bacteria represents the average count per millillter of the whole content o2 duplicate ampules
(30 n~).
A. Initial inoculum of 105/m].
B. Initial inoculum of 101°/rid.
l~or convenience in illustrating, the ]ogaxithm of no detectable survivors is plotted as 0.

tion. This is proof that bacteria can recover Factors involved in heat resistance. The
under conditions unsuitable for multiplication. physiological state of the bacteria, the type o2
I n a similar experiment (Fig. 2-A), where heating medium, and type of recovery medium
the initial inoculum was 105 to 106 rather than used all influenced recovery.
109 to 101°, recovery was faster (48 to 72 hr) Bacteria (Culture 18) from 14- to 16-hr
and multiplication did take place after recovery. cultures did not recover, whereas those from
No recovery was observed at either level of 24-hr cultures did, in line with the generally
inoculum when basal medium was used instead accepted fact that actively growing bacteria
of trypticase soy broth in these experiments are less heat resistant than resting bacteria.
(Fig. 2-A and B). When bacteria from Culture 18 were grown in
J. DAI~Y SCIENCE VOL. 52, NO. 5
612 D A B B A H ET AL.
milk whey and heated in it, some survivors
were found immediately after heating; none
was found immediately after heating when the
bacteria were grown in trypticase soy broth
and heated in the whey. This shows that the
growth medium can affect heat resistance. Sur-
vivors also were found in shaken trypticase soy
broth cultures immediately after heating,
whereas none was found in heated stationary
cultures.
Effect of heati~g medium. I t was observed
that more complex heating media resulted in
shorter recovery times after incubation in tryp-
ticase soy broth, indicating that the complex
media protected the bacteria from heat injury.
In the complete trypticase soy broth medium
containing trypticase, phytone, glucose, sodium
chloride, dipotassium phosphate, and glucose,
recovery occurred after 48 hr of incubation at
20 C. Omission of sodium chloride, or glucose,
or both, resulted in recovery after 96 hr. Ab-
sence of trypticase from the heating medium
resulted in no recovery up to 240 hr of incu-
bation at 20 C in fresh trypticase soy broth,
indicating the protective effect of trypticase
during heat treatment. I n the absence of phy-
tone, recovery occurred after 216 hr of incuba-
tion and no recovery was observed after heating
in the basal medium. Milk whey was slightly
less effective than trypticase soy broth in sta-
bilizing the bacteria to heat, as recovery oc-
curred after 72 hr of incubation. I t was also
observed that if bacteria were spun down and
resuspended in basal medium without washing,
the small amount of trypticase soy broth re-
maining with the bacteria gave significant pro-
tection from heat.
Effect of recovery n~edium. I n trypticase soy
broth, the most favorable medium for recovery,
bacteria were detected after 48 hr, whereas no
recovery occurred when they were incubated in
basal medium after heating in broth. When
bacteria were washed after heating and resus-
pended in the supernatant in which they were
heated, recovery was no faster than when they
were suspended in the fresh broth, indicating
that no recovery-promoting agents were leached
out of the cells during heating. Recovery was
much slower in milk whey, requiring more than
144 to 168 hr, as compared to more than 48
to 72 hr in trypticase soy broth after heating
in this broth. No recovery was observed after
heating in a basal broth medium supplemented
by trypticase and glucose and incubating in the
same fresh medium, or after heating in trypti-
case soy broth and incubating in the basal sup-
plemented medium. This indicates that trypti-
case alone in the liquid recovery broth was in-
J. DAIRY SCIEI~CE VOL. 52, NO. 5
effective in promoting recovery. These recovery
experiments were all carried out by incubation
at 20 C; however, it was determined that re-
covery could also take place at 4 C in trypti-
case soy broth, although holding for two to
three weeks was required.
Discussion
The results obtained show unequivocally that
some bacterial ceils that appear killed by heat
may recover and grow normally after incuba-
tion for long periods in a favorable medium.
Plate counts on trypticase soy agar incubated
for 48 to 72 hr showed no survivors in the broth
cultures for periods of up to nine days, after
which survivors appeared and grew normally.
I t is possible that colonies might have formed
on trypticase soy agar plates if they had been
incubated longer. I t was proven that no viable
survivors were present in the ampules after
heating by plating the entire contents of the
ampule. This eliminated any possibility of
overlooking a small number of survivors. While
it is possible that a small number of uninjured
survivors might be missed immediately after
heating, they would certainly multiply suffi-
ciently in 24 hr to be readily detectable. How-
ever, no survivors were noted for as long as
nine days, so that possibility of uninjured sur-
vivors clearly can be ruled out. Accidental re-
contamination is another possibility. This was
ruled out by carrying out the experiment in
sealed ampules so that recontamination was
unlikely.
The recovery period can be considered to be
an extended lag period resulting from severe
injury to the bacterial cells. I t differs from the
usual concept of a lag period in being longer
and in that no viable cells can be detected dur-
ing the recovery period, whereas viable cells
can be demonstrated in a lag period but they
fail to multiply. The recovery phenomenon
was also found with five species of Salmonella,
as well as with the test culture under the par-
ticular experimental conditions used. I t is
probable that this phenomenon is a general
one and will occur with all types of bacteria,
following heating under certain combinations
of time and temperature just below the point
at which there are no surviving cells. The tem-
perature used with the test culture was 55 C,
considerably below the normal pasteurization
temperature of 62 C, and recovery could not be
demonstrated after heating to 60 C for 30 rain.
However, it is conceivable that recovery could
occur after heating at pasteurization tempera-
ture, if the strain of bacteria used were slightly
more heat-resis.tant. I t should further be em-
 HEAT RESISTANCE OF BACTERIA 613

p h a s i z e d t h a t these e x p e r i m e n t s were conducted (4) Chambers, C. W., H. H. Tabak, and P. W.

in samples of only 15 ml. I n commercial pro- Kabler. 1967. Effect of Krebs cycle metabo-
cessing i n v o l v i n g large volumes, the p r o b a b i l i - lites on the viability of Escheriehia colt
ties of s u r v i v a l a n d reeove13r are increased in treated with heat and chlorine. J. Bscteriol.,
p r o p o r t i o n to the l a r g e r total n u m b e r of bac- 73 : 77.
(5) Curran, H. R., and P. R. Evans. 1937. The
t e r i a present. Most of the samples were incu-
importance of enrichments in the cultivation
b a t e d at 20 C; however, it also was shown t h a t of bacterial spores previously exposed to
recovery could occur a t 4 C, a l t h o u g h the hold- lethal agencies. J. Baeterlol., 34: 179.
ing time r e q u i r e d was two to t h r e e weeks, (6) Dahlberg, A. C. 1946. The rela.tionship of
r a t h e r t h a n the 48 to 72 h r r e q u i r e d at 20 C. the growth of all bacteria and coliform
The m a x i m u m h o l d i n g t i m e used in these bacteria in pasteurized milk held at refrig-
e x p e r i m e n t s was ten days. R e c o v e r y in some eration temperature. J. Dairy Sci., 29: 651.
eases r e q u i r e d the full ten days, a n d it seems (7) Gnrv~e, E. I. 1955. The growth of Escheri-
chia colt in buffer substrate and distilled
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water. J. Bacterio]., 69: 393.
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p a s t e u r i z e d milk a f t e r 20 days, m i g h t be ex- philes to different incubation temperature.
p l a i n e d on the basis o f recovery. J. Dairy Sol., 42: 1097.
The t y p e of m e d i m n in which the b a c t e r i a (9) Hansen, 1'C I-I., and H. Riemann. 1963. Fac-
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used, all were f o u n d to affect h e a t resistance sDoring organisms. J. Appl. Bacteriol.,
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