APPLIED MICROBIOLOGY, Jan. 1969, p. 78-82 Vol. 17, No.

1
Copyright ( 1969 American Society for Microbiology Printed in U.S. A

Heat Resistance of Salmonella: the Uniqueness of

Salmonella senftenberg 775W
HENRY NG, HENRY G. BAYNE, AND JOHN A. GARIBALDI
Western Regional Research Laboratory, Agricultural Research Service,
U.S. Department ofAgriculture, Albany, California 94710
Received for publication 17 October 1968

Of approximately 300 cultures of Salmonella, representing 75 different serotypes,

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none was found to be as heat-resistant as S. senftenberg 775W. However, S. blockley
2004 was 5 times more heat-resistant and S. senftenberg 775W was 30 times more
heat-resistant than S. typhimurium Tm-i, the reference strain in this study. All other
strains of Salmonella tested, including 19 strains of S. senftenberg and 7 strains of
S. blockley, had decimal reduction times at 57 C of about 1 min, equivalent to that
of the reference organism, Tm-1. As observed in other bacterial species, strain 775W
is more heat-sensitive in the log phase than in the stationary phase of growth. Cells
from cultures grown at 44 C were more heat-resistant than those grown at either 35
or 15 C; the medium of growth, whether minimal or complex, made no appreciable
difference in heat resistance. Cells from cultures limited by a carbon source were
killed at a much slower rate than those limited by a nitrogen source and exhibited a
1-hr lag at 55 C before a significant rate of kill was attained. For any given set of
growth conditions, strain 775W was always more heat-resistant than another strain
of S. senftenberg, 197B, which has normal heat resistance.

The thermal resistance of the genus Salmonella
is a matter of great concern to all persons con-
cerned with public health and to food processors
and handlers. Although numerous reports, as
cited by Bayne et al. (4), indicate that salmonellae
are relatively sensitive to heat, one strain of S.
senftenberg, 775W, represents an exception.
This organism was first mentioned by Winter
et al. (19) with three other H2S-negative strains
of S. senftenberg in heat resistance studies. It
survived almost 5 min of heating at 60 C in liquid
egg. Later Solowey, Sutton, and Calesnick (15),
designating the organism for the first time as
775W, showed it to have a decimal reduction
time (DRT) at 61 C of 1.1 min in liquid whole
egg and 1.19 min in a tryptose broth as compared
to DRT values of less than 1 min at 58 C for other
Salmonella cultures. Despite its unusual heat
resistance, this organism, isolated from egg
powder along with 149 other Salmonella, has
not been adequately described. The only mention
of its taxonomic position was the serological
typing by E. H. Spaulding of the School of Medi-
cine, Temple University, Philadelphia, Pa. (19).
Although extensively used in several heat resis-
tance studies (1-3, 10, 12, 16), its identity was
neither questioned nor confirmed.
In view of the uniqueness and potential public
health significance of S. senftenberg 775W, we will
78
present a more thorough description of its bio-
chemical properties, ascertain the frequency of
occurrence of other Salmonella equally heat-
resistant, and determine how variations in
growth conditions, such as age of culture, growth
medium, and growth temperature, affect its heat
resistance and that of a strain of the same serotype
having normal heat resistance.
MATERIALS AND METHODS
Media. Generally, the medium was Trypticase Soy
Broth (BBL) supplemented with 2% (w/v) yeast
extract (Difco; TSB-YE) or Trypticase Soy Agar
similarly supplemented (TSA-YE). It should be noted
that commercial TSA differs from TSB not only in
the presence of agar but in the absence of 0.25% (w/v)
dextrose and 0.25% (w/v) dipotassium phosphate.
The minimal medium used was 56 (11), having the
following composition (per liter): 13.6 g of KH2PO4,
2.0 g of (NH4)2SO4, 0.01 g of CaCI2, 0.2 g of MgSO4i
7H20, and 0.0005 FeSO4. 7H20. The pH was adjusted
to 7.4 with NaOH. Glucose was autoclaved separately
and added aseptically to the sterile medium to give a
final concentration of 0.2% (w/v). In the nitrogen- and
and carbon-limited studies with S. senftenberg 775W,
the concentration of (NH4)SO4 and of glucose was
reduced to limiting levels 0.016 and 0.05%. respec-
tively, in the same minimal medium.
Bacterial strains. The Salmonella cultures were
obtained from (i) the National Communicable Disease
Center, Atlanta, Ga., (ii) The Microbiology Labora-
VOL. 17, 1969 HEAT RESISTANCE OF SALMONELLA 79
tory, California Department of Public Health, Berke- culture as S. senftenberg according to the Kauf-
ley, and (iii) our own collection. These were main- mann-White Schema (9).
tained as stab cultures in Cystine Trypticase Agar Thermal resistance of some Salmonella sero-
(BBL) and were carried on TSA-YE slants. types. Two hundred and ninety-six salmonellae
Growth of cultures. Cultures were generally grown of approximately 75 different serotypes were
in 250-ml Erlenmeyer flasks which had been modified screened for heat resistance and were compared to
by the addition, as sidearms, of matched 16-by 125-
mm test tubes. The flasks containing 25 ml of medium S. typhimurium Tm-1. Only results obtained on
were incubated at 15, 35, or 44 0.5 C on a water those serotypes of which 10 or more strains were
bath shaker (Research Specialties, Richmond, Calif.). tested are presented in Table 1. A minimum, a
Growth was measured by insertion of the sidearms maximum, and an average ratio among strains are
on the flask into a Klett colorimeter equipped with a given. No average ratio exceeded a value of two;
red filter (no. 66). The turbidity readings were con- i.e., no serotype on the average was more than
verted to dry weight by reference to a graph relating twice as resistant as S. typhimurium Tm-1. Even

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dry weight to Klett readings. the most heat-resistant strain of any one sero-
Viable counts. The number of viable cells was deter-type, e.g., one strain each of S. heidelberg, S.
mined by appropriately diluting samples in TSB-YE
and spread-plating a sample onto TSA-YE plates pullorum, S. manhattan, and S. tel-viv (the latter
which had been allowed to dry at room temperature two not shown on the table), was only slightly
for about 48 hr. The plates were incubated at 37 C more than twice as resistant as S. typhimurium
overnight. (Longer incubation periods did not signi- Tm-1. An exception was a strain of S. blockley
ficantly increase the counts.) 2004, which was consistently about five times as
Heating of cells. A 1-ml amount of cultures was resistant. Although not indicated in Table 1, six
introduced into milk dilution bottles containing 99 ml other strains of S. blockley had only an average
of TSB-YE which had been equilibrated at 55 -4 0.05 ratio of about 1. The survey included 19 strains of
C for about 0.5 hr. Samples were then removed at S. senftenberg other than 775W, among which
various intervals to determine survivors by viable
counts. Plotting the log of survivors against time were both H2S-positive and -negative strains;
allows the estimation of the DRT (DRT and D value none was significantly more heat-resistant than
are used interchangeably), which is the time, in S. typhimurium Tm-1. The decimal reduction
minutes, required to reduce the population by 90%. times for the chief strains at 57 C and pH 6.8 were:
Screening for thermal resistance. A relatively rapidS. typhimurium Tm-i, 1.2 min; S. blockley 2004,
procedure was devised to screen a large number of 5.8 min; and S. senftenberg 775W, 31 min.
cultures for heat resistance. It consisted of diluting 1 Effect of age of culture and temperature of
ml of each culture grown for 2 days at 35 C in TSB- growth on heat resistance. All prior studies on
YE into 99 ml of ice-cold medium and dispensing 2 ml thermal resistance of S. senftenberg 775W were
of the diluted cells into each of two 13- by 100-mm carried out on relatively old cells. It is well known
screw-cap test tubes. One tube of each culture was
kept on ice, whereas the other was immersed up to the that with other bacteria, old cells are more heat-
cap in a bath at 57 0.05 C for 10 min. After cooling resistant than young cells (13, 17). Therefore, it is
the heated tubes in an ice bath, the viable cell count conceivable that the extreme heat tolerance here-
was determined on the contents of both the heated
and the unheated tubes. DRT values were calculated
from this single time period and were compared with TABLE 1. Heat resistance of several Salmonella
the value obtained by similarly treating S. typhimurium serotypes as compared with S. typhimurium
Tm-1, a strain of normal heat resistance. Tm-i
RESULTS Ratio of heat resistance to
heat resistance of
No. of S. typhimurium Tm-i
Characteristics of S. senftenberg 775W. S. Serotype strains
tested -____

senftenberg 775W is a gram-negative, nonspore- Mifli- Mean Maxi-

forming, motile rod. It did not produce indole, mum mum
H2S, or urease. When grown on Triple Sugar Iron
Agar, it produced an alklaine slant with acid and S. dublin .......... 10 O.6 0.9 1.3
gas in the butt. It did not ferment lactose, sucrose, S. gallinarum. 11 0.7 1.0 1.7
salicin, inositol, adonitol, glycerol, or raffinose, S. heidelberg. 15 0.9 1.3 2.2
S. muenchen.
but did ferment sorbitol and glucose. It grew in S. newport ....... 10 1.0 1.2 1.6
11 0.9 1.1 1.6
Simmon's Citrate medium but not in malonate S. oranienburg . .. 14 0.7 1.1 1.8
broth. It produced a lysine decarboxylase. The S. pullorum ....... 14 0.7 1.2 2.2
cells agglutinated the group E polyvalent anti- S. senftenberg4 ... 19 0.6 1.2 1.8
serum and the single 0 (somatic) factor 19. The S. typhimurium.... 17 0.8 1.2 1.7
flagellar antisera g, s, and t were agglutinated.
These results clearly confirm the identity of the a Does not include S. senftenberg 775W.
80 NG, BAYNE, AND GARIBALDI APPL. MICROBIOL.

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10
0 20 10 20 20 40 60 80
Minutes Minutes Minutes
FIG. 1. Survival curves for S. senftenberg at 55 C. Strain 775W grown at 15 C (a), at 35 C (b), and at 44 C
(c). Strain 197B grown at 15 C (d), at 35 C (e), and at 44 C (f). Symbols: 0, log-phase cells; *, stationary cells.
tofore reported for S. senftenberg 775W may be difference not demonstrable in cells of 197B,
mainfested only when the culture is in the sta- which were equally resistant in both growth
tionary phase, and normal heat resistance may be media.
exhibited in the log phase of growth. Figures la, b, Effect of carbon and nitrogen limitation on heat
and c show that, at all growth temperatures tested, resistance. Since the foregoing experiments indi-
S. senftenberg 775W was indeed more heat-sensi- cated that cells in the stationary phase of growth
tive in the log phase than in the stationary phase; are more heat-resistant than those in the expo-
however, compared to another strain, 197B (Fig.
id, e, f), which has normal heat resistance, it was
still more heat-resistant in both log and stationary
phases. Furthermore, strain 775W was more heat-
resistant than 197B at all growth temperatures. In
addition, the heat resistance of both strains was a
function of growth temperature; the higher the
temperature, the more resistant were the cells. The
cells which had been grown at 44 C and had sur-
vived the heating (Fig. ic) were reisolated and
0~~~~~~
allowed to grow at 35 C and again were tested
for heat resistance. They were found to have a 24
DRT characteristic of cells grown at 35 C. Thus, vi
neither did growth of the culture at 44 C bring
about a heat-resistant mutant nor were the sur-
vivors of a heat treatment any more resistant than
the original cells.
Effect of growth medium on heat resistance. The
heat resistance of S. senftenberg strains 197B and
775W, grown for 48 hr in either a minimal me- *0 20 40 60 80 100 120
dium or in the TSB-YE, is shown in Fig. 2. Strain Minutes
775W was more resistant than 197B regardless of FIG. 2. Efiect of growth medium on heat resistance
growth medium. Cells of 775WY however, ap- ofS. senftenberg. Strain 775W grown in TSB-YE (a)
peared to be more resistant when grown in a and minimal medium (0). Strain 197B grown in TSB-
complex medium than in the minimal medium, a YE (0) and in minimal medium (0).
VOL. 17, 1969 HEAT RESISTANCE OF SALMONELLA
5
0 N-limited
>4 N-limited (S+7kr.)
(S+2hr)
o
3
i to, within experimental error, that of 775W. It
2
0 20 40 60 80 100 120 40
Minutes
FIG. 3. Eject of substrate limitation on heat resist-
ance of S. senftenberg 775W. Cells starved of carbon
source for 2 hr (0), and cells starved of nitrogen for 2
hr (E) and for 7 hr (U).
nential phase, it is important to ascertain whether
cells from cultures which go into the stationary
phase as a result of carbon limitation are different
in heat resistance from those limited by nitrogen.
Figure 3 shows that cells of 775W grown at 35 C
in a medium limiting in ammonium sulfate as the
sole nitrogen source and those grown in a medium
limiting in glucose as the sole carbon and energy
source exhibited survivor curves of different
shapes. The cells subjected to heating at 55 C,
after having entered the stationary phase for
approximately 2 hr as a result of nitrogen limita-
tion, were killed immediately at a constant ex-
ponential rate, whereas cells at the same stage of
growth limited by carbon were killed at a much
slower initial rate prior to attaining the same rate
as the nitrogen-limited cells. Furthermore, ex-
tending the period of nitrogen limitation to 7 hr
did not alter the heat resistance characteristics of
the cells.
DISCUSSION
The results of the present study indicate that S.
senftenberg 775W is indeed an unusual and rare
organism. Although the method used for screen-
ing the several hundred cultures is approximate, it
appears to be reliable for the purpose of compar-
ing heat resistance among organisms and it is
simple and rapid. Several of the cultures tested
in this survey had been isolated from pasteurized
egg products, but they proved to be no more heat-
resistant than the average. It appears, therefore,
that Salmonella recovered from these pasteurized
products resulted from recontamination or
improperly operating equipment. On the other
81
hand, the somewhat heat-resistant culture of S.
blockley 2004 was isolated from a human sal-
monellosis case by the California Department of
Public Health.
A strain of Salmonella as heat-resistant as S.
senftenberg 775W was reported (6) after com-
pletion of the present work. This strain of S.
senftenberg (designated strain S8 in the authors'
culture collection) had heat resistance identical
is a coincidence that the two strains are of the
same serotype. Limited tests performed in this
laboratory have failed to distinguish between the
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two strains. The possibility exists, therefore, that
they may be the same organism even though 775W
60 was isolated from dried eggs in the United States,
whereas S8 was isolated from home-killed meat
in England. Although both strains of heat-re-
sistant Salmonella are of the same serotype, our
work and that of Solowey et al. (15) and David-
son et al. (6) showed that not all S. senftenberg
strains are heat-resistant nor are all heat-resistant
salmonellae S. senftenberg. Furthermore, the abil-
ity to produce H2S is not correlated with heat
resistance.
Factors which affect heat resistance of bacteria
during heating should be distinguished from those
effective before heating, such as growth conditions
of the cells. Hansen and Riemann (8) have amply
studied and reviewed the former, whereas the
latter have not been studied as thoroughly.
The effect of age of culture on heat resistance
was first reported by Sherman and Albus (13)
and has since been confirmed (17). The results of
the present study unequivocally demonstrate that
S. senftenberg 775W is more resistant than other
strains of Salmonella, not only in the stationary
growth phase, as demonstrated so often, but also
in the exponential growth phase. However, as was
expected, the stationary-phase cells are many
times more resistant than exponential-phase cells.
The medium in which the cells are grown seems
to have little or no influence on their heat resis-
tance; those grown in a complex medium may be
slightly more resistant than those grown in a
glucose minimal medium.
The temperature at which cells are grown ap-
pears to have a larger influence on their thermal
resistance. Reports concerning this variable have
been conflicting. Sherman and Cameron (14)
found that cells grown at lower temperatures
were more heat-resistant, and Claydon, cited by
Hansen and Riemann (8), observed that Strepto-
coccus lactis exhibited greater heat resistance
when grown at 10 C than when grown at higher
temperatures. However, Elliker and Frazier (7)
found that E. coli survived heat treatment better
when grown at 38.5 or 40 C than when grown at
82 NG, BAYNE, AND GARIBALDI
28, 30, or 30.5 C. White (18) also found higher
heat resistance in cells of Streptococcus faecalis
grown at 45 C than at 27 C. Our results agree with
those of Elliker and Frazier and White.
A most significant observation is that heat
resistance of Salmonella can vary widely, depend-
ing on strain differences, age of cell, and tempera-
ture of growth, from a low DRT of 0.55 min for
197B grown in the log phase at 15 C to a high of
42 min for 775W grown to the stationary phase at
44C.
The kinetics of killing observed in our experi-
ments were complex, and interpretation of the
data is difficult. The survivor curves took on a
variety of shapes. There has been much discussion
and speculation on this subject, e.g., reference 5,
but its causes are unknown. In our experiments,
the curve representing an initial slow rate followed
by a more rapid killing rate was most frequently
obtained when S. senftenberg 775W was grown to
a stationary phase either in a complex medium or
in a minimal medium where glucose is limiting.
However, when cells were grown in a mini-
mal medium limited by nitrogen source, an
immediate exponential killing rate was obtained.
The lag in kill can be 1 hr or more at 55 C so
that the usual explanation of come-up time for
this type of multihit kinetics is inadequate since,
experimentally, the heating medium was allowed
to reach temperatures before cells were added.
Another explanation often advanced is that cells
are clumped so that many units must be inacti-
vated before a kill is detected. The magnitude of
the lag, however, would require clumps of the
order of hundreds of cells, an unlikely event. At
present, no explanation can be offered for the lag
in killing.
The other kind of kinetics usually observed
with log-phase cells is one in which an initial
rapid kill is followed by a slower kill. This can be
explained by the presence of a heterogeneous
population in which a small percentage of
cells is more heat-resistant, for example, those
which failed to enter the exponential phase but
remained in the more resistant stationary or lag
phase.
Work now in progress to determine why S.
senftenberg is so much more heat-resistant than
the other salmonellae may reveal what factors
contribute to the heat resistance of bacteria and
may reveal the mechanism by which heat kills
bacteria.
APPL. MICROBIOL.
ACKNOWLEDGMENT
We gratefully acknowledge the technical assistance of Catherine
Powers, Califoria State Department of Public Health, Berkeley,
who performed the biochemical and serological tests on S. senften-
berg 775W.
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