Download Minimal Medium Recovery of Heated Salmonella

Journal of General Microbiology (I973), 74, 267-274
Printed in Great Britain
267
Minimal Medium Recovery of Heated Salmonella typhimurium
LT2
By R. F. G O M E Z , A. J. S I N S K E Y , R. DAVIES" A N D T. P. L A B U Z A
Department of Nutrition and Food Science, Massachusetts Institute of Technology,
Cambridge, Massuchusetts 02139, U.S.A.
(Received 30 August 1972; revised 25 September 1972)
SUMMARY
Exponential phase Salmonella typhimurium LT 2, S. thompson and S. heidelberg
grown in a glucose-salts medium (M-9), harvested at 37 "C and heat-treated at
50 "C exhibit a higher recovery on M-9 agar than on Trypticase Soy agar with
yeast extract. This phenomenon is analogous to 'minimal medium recovery' of
irradiated bacteria. Supplementation of M-9 agar with yeast extract or Casamino
acids resulted in lower viable counts of heat-treated bacteria than on M-9 agar
alone. Heat-treated bacteria incubated at 37 "C in M-9 broth or distilled water
recovered their ability to grow on TSY agar. This recovery is inhibited by
hydroxyurea.
INTRODUCTION
Recovery of heat-treated micro-organisms depends upon the isolation or enumeration
media employed. For example, heated Staphylococcus aureus are less tolerant to salt
(Iandolo & Ordal, I 966). The practice of preliminary resuscitation in non-selective enrichment media has been proposed (Mossell & Ratto, 1970). This procedure should also allow
for the recovery of 'metabolically injured' cells (Straka & Stokes, 1959) which, after a variety
of physical stresses, are unable to multiply on simple defined media and require nutrient
supplementation. We show here, for heated Salmonella typhimurium LT 2, that the phenomenon of 'metabolic injury' depends on pretreatment growth conditions and very often the
opposite result can be obtained.
In accord with recent reports of thermally induced DNA breakage in Escherichia coli
(Bridges, Ashwood-Smith & Munson, 1969; Sedgwick & Bridges, 1972) and its restitution
(Woodcock & Grigg, I972), we present evidence of the repair of thermal damage and its
possible dependence on DNA synthesis.
METHODS
Organisms. Salmonella typhimurium strain D B 2 I , an isolate of LT 2, was obtained from
D. Botstein, Department of Biology, Massachusetts Institute of Technology. Salmonella
heidelberg and S. thompson were obtained from the collection of the Department of Nutrition and Food Science, Massachusetts Institute of Technology.
Growth media. TSY, BBL Trypticase Soy broth supplemented with 037L Difco Yeast
Extract. M-9, a modification of the chemically defined medium of Adams (1959) containing
(g/rooo ml distilled water): Na,HPO,, 7; KH,PO,, 3 ; NaCl, 0 . 5 ; NH,Cl, I ; MgSO,. 7H,O,
0.25; dextrose, 2.
* Present address: National College of Food Technology, University of Reading, St George's Avenue,
Weybridge, Surrey, U.K.
18
M I
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c 74
268
R. F. G O M E Z , A. J. S I N S K E Y , R. D A V I E S A N D T. P. L A B U Z A
At times bacteria were also plated on M-9 supplemented with nucleosides, vitamins or
amino acids. Nucleosides : adenosine, guanosine, thymidine, cytidine, and uridine (10 pg/ml
of each) ; vitamins : thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine, folic
acid, inositol, choline, biotin, p-aminobenzoic acid, vitamin B,, (10pgfml of each) ; amino
acids : glutamine, alanine, arginine, aspartic acid, cystine, glutamic acid, glycine, histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan,
tyrosine, valine, asparagine (10pg/ml of each).
Heat treatment. Bacteria from exponential phase cultures of Salmonella typhimurium
LT2, grown at 37 "C in TSY or M-9 broth, were harvested by centrifugation, washed and
resuspended in distilled water at 37 "C at density of 3-0to 6.0 x IO*bacteria/ml. The suspensions were incubated at 50 "C for various times, serial dilutions were made in phosphate
buffer (0.067 M, pH 7) at room temperature and 0.1 ml samples were spread on the surface
of TSY and M-9 agar plates at room temperature. The plates were incubated at 37 "C.
TSY plates were counted after 24 h and M-9 plates after 48 h.
Post-heat treatment incubation. An exponentially growing culture of Salmonella typhi~ M-9 was harvested, washed and resuspended in distilled water. All of these
murium L T in
operations were conducted at 37 "C to avoid cold shock. The suspension was divided into
two portions, one being held for 18 min at 37 "C (control culture), the other for 18 min at
50 "C. After 18 min both suspensions were diluted (I :20) into M-9, TSY or distilled water,
and incubated at 37 "C.Enumeration was performed on M-9 and TSY agar plates immediately after heat treatment and during the incubation period. When the post-heat treatment
incubation was done in water, the bacteria were filtered on to Millipore membranes (HA
0.45 pm; 25 mm), washed twice with distilled water and resuspended immediately after
heating to free them of leakage materials. Control experiments showed 100% recovery of
bacteria from the membranes.
Eflect of hydroxyurea after heat treatment. Hydroxyurea (Sigma Chemical Co., St Louis,
Missouri, U.S.A.) was recrystallized from absolute alcohol and introduced at a concentration of 0.1 M in the post-heat treatment M-9 incubation broth. To avoid any carry-over of
hydroxyurea on to the plating media, 3 ml samples of the broth were filtered through
Millipore membranes (HA 0.45 pm; 25 mm), washed twice with 5 ml of 0.067 M-phosphate
buffer (pH 7.0), resuspended in 3 ml of phosphate buffer and plated on M-9 and TSY agar
plates.
RESULTS
Fig. I shows that, when the culture was grown in TSY broth, the classical 'metabolic
iniury ' was observed after heat treatment, TSY agar yielding higher recovery of thermally
stressed bacteria than M-9 agar during the early stages of heat treatment. On the other hand,
when the culture was grown in M-9 before exposure to heat, reduction of viable counts on
TSY agar relative to M-9 was observed, particularly during the first 60 min of heating.
This phenomenon is similar to the 'minimal medium recovery' effect observed in radiation
sensitive organisms (Ganesan & Smith, 1968; Ganesan & Smith 1970). The effect was
independent of the composition of the suspending and heating menstrua (Table I) and
brain heart infusion and other chemically defined and undefined enriched media
behaved similarly to TSY (Table 2), with the exception of M-9 supplemented with
nucleosides or vitamins. 'Minimal medium recovery ' after heat treatment has been
demonstrated for several serotypes of salmonellas (Table 3). Neither Salmonella
heidelberg nor S. thompson showed any significant 'metabolic injury' when grown in TSY
broth. In general, 'minimal medium recovery' was the phenomenon most often observed.
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Recovery of heated salmonellas
1oo
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269
TSY grown culture
g
.-
Y
&g,
,=&.
10-3
>
.4
M-9 plates
-=-
*E
$ 10-4
1
TSY plates
10-5
0
.,
30
60
90
120
150
180
Minutes in water at 50 ' C
Fig. I . Survival curve of Salmonella typhimuvium L T ~in water at 50 "C. Dotted lines, culture
grown in TSY broth prior to heat treatment; solid lines, culture grown in M-g broth prior to heat
plated on TSY agar; A and
plated on M-g agar.
treatment; 0 and
+,
Table I . Efect of heating inen~truumon the plating eficiencies of M-9 and TSY agar
,
grown in M-9 after 25 min in water at 50 "C
for Salmonella typhimurium L T ~ previously
Surviving fraction
Heating liquid
Distilled water
0.067 M-PO,~-buffer
Skimmed milk
7-A-
M-9
2'0 x 10-1
6.2 x 1o-I
7.2 x 10-1
TSY
35x
2-3 x
3-7 x
IO-~
IO-~
IO-~
When the cultures were grown in M-9 medium, counts on M-9 agar were always significantly higher than on TSY agar even before harvesting and heating. This suggests that the
lower recovery of heated organisms on TSY compared with M-9 agar was not totally due to
thermal treatment but may reflect an inability of the cultures to cope with a shift up from
M-9 broth to TSY agar and that this situation is aggravated by heat treatments.
When the organisms were suspended in M-9 after heat treatment (Fig. 2) the control
culture showed an exponential increase in both TSY and M-g plate counts after 60 min,
the M-9 plate counts being slightly higher than the TSY plate counts. The M-9 plate counts
of the heat-treated culture remained constant for 4 h and then increased at the same rate
and in the same fashion as the control. The TSY plate counts of the heat-treated culture,
which initially showed the reduced viability effect, started increasing immediately and
reached the same level as the M-9 plate counts after 4 h of incubation. We interpret this as
resulting from the repair of heat-induced damage, as it is too rapid to be due to division in
M-9 medium.
The behaviour of heated and control organisms in TSY is shown in Fig. 3. The control
culture responded to the shift up from M-9 to TSY, after holding for 18 min at 37 "C in
distilled water, by a slight decrease in viability on both plating media. Exponential growth
18-2
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270
R. F. G O M E Z , A. J. S I N S K E Y , R. D A V I E S A N D T. P. L A B U Z A
Table 2. Effect of plating media on the recovery of Salmonella typhimurium LT2, previously
grown in M-9, after 25 min in water at 50 "C
Plating medium
Surviving fraction
M-9
M-g nucleosides ( I o pglml)
M-9 vitamins (10 ,ug/ml)
+
+
M-9+ Casamino acids ( 0 . 2 5 %)
M-9 + 20 amino acids (10,ug/ml)
M-9 + 0.5 % yeast extract
Brain heart infusion
TSY
M-9 2 % trypticase soy
+
I ' O X 10-1
3.0 x 10-1
9'4 x 1 0 - 2
2-4x I O - ~
I ' O X 10-2
5.0 x I O - ~
3.2 x I O - ~
1.5 x 10-*
1 - 2x I O - ~
Table 3. E f e c t of growth medium prior to heat treatment OYEthe plating eflciency on M-9
and T S Y agar for Salmonella heidelberg and S. thompson after heat treatment in water
at 50 "C
Organism
Growth
medium
Minutes
at 50 "C
M-9 plate counts
TSY plate counts
Salmonella heidelberg
M-9
0
15
10'0
0
1'0
25
1.5
I '4
45
TSY
90
Salmonella thompson
M-9
TSY
2.7
17.0
0
1.6
I5
45
4'2
10'0
0
0.74
25
0.70
I00
4'90
was resumed after I h. In contrast, holding in TSY after heat treatment resulted in a marked
decrease in the counts on M-9 agar and a small increase in the counts on TSY agar. In other
words, as far as it can be detected, holding in TSY broth resulted in the death of the vast
majority of heat-treated bacteria.
The results of incubation in distilled water after heating are shown in Fig. 4. Viable
counts of the control cultures remained constant for the duration of the experiment as did
M-9 plate counts of the heated culture, but TSY plate counts increased to within 70 yoof the
M-9 counts, indicating that the majority of bacteria were able to repair the heat-induced
damage without the aid of any exogenous materials.
Incubation in M-9 broth containing 0.1 M-hydroxyurea (Fig. 5) caused a decrease in the
recovery of heated bacteria from thermal injury; only 1 % of the 'heat-injured' cells
regained their ability to grow on TSY agar. In addition, 95% of the cells lost their ability
to grow on M-9 agar plates. Hydroxyurea at 0.1M was strictly bacteriostatic to control
cultures within the periods used in this experiment.
DISCUSSION
Although reports dealing with 'metabolic injury' are abundant (Nelson, I 943 ; Allwood
& Russell, 1966; Harries & Russell, 1966; Russell & Harries, 1968), our investigations have
shown that the opposite phenomenon, ' minimal medium recovery ', is observed when
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Reco very of heated salmonellas
27 1
M-9 plates
A
0
-
M-9 nlateq
-.-
-
~
~~
0
Heat-treated culture
~
60
120
180
240
300
Minutes in M-9 broth after heat treatment
360
420
Fig. 2 . Post-heat treatment incubation in M-9 medium. Bacteria were held in water at 37 "C(open
symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then diluted ( I :20) into M-9
broth at 37 "C and plated at intervals on TSY agar (0and 0 ) and M-9 agar (A and A).
1O ' O
10'
1 O8
s 10'
*
0
2
aJ
5
I106
5
10'
1 O4
0
I
40
I
I
I
I
I
80
120
180
240
300
Minutes in TSY broth after heat treatment
+
/
490
Fig. 3. Post-heat treatment incubation in TSY broth. Bacteria were either held in water at 37 ' C
(open symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then diluted (I : 2 0 ) into
TSY broth at 37 "C and plated at intervals on TSY agar (0and 0) and M-g agar (A and A).
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R. F. GOMEZ, A. J. S I N S K E Y , R. D A V I E S A N D T. P. L A B U Z A
I
Control culture
lo'
cr
El
3
lo6
-
TSY plates
eA
M-9 plates
-
0)
3
105
104
I
I
I
I
1
Fig. 4. Post-heat treatment incubation in distilled water. Bacteria were either held in water at
37 "C (open symbols) or at 50 "C (closed symbols) for 18 min. Both cultures were then filtered
through Millipore membranes (HA 0.45pm; 25 mm), resuspended in distilled water and plated at
intervals during incubation at 37 "C on TSY agar (0 and 0 ) and M-g agar (A and A).
1
1 OR
107
:
Y
3
c-
lo6
%
s
I
\
I
1 0 5 I-
104
With hydroxyurea
/-/
-
TSYplates
I
I
i
I
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I
Recovery of heated salmonellas
273
bacteria are grown in minimal media prior to heat treatment. Salmonella typhimurium L T ~ ,
grown in M-9 medium, is extremely sensitive to a shift up to either TSY broth or TSY agar
after sublethal heat treatment (Fig. 3). Sensitivity to a shift-up from M-9 to TSY broth
without heat treatment, as in Fig. 3, was not always observed.
These findings substantiate those of Labots (1959), who has reported that the addition
of yeast extract to milk containing sublethally heated coliform bacteria, previously grown
in milk, causes a decrease in viable numbers, and that dilution with water retarded the death
rate. Mukherjee & Bhattacharjee (1970) have observed an increase in counts on nutrient
agar during incubation in phosphate buffer of heat-treated Escherichia coli. However, they
did not report the high recovery on minimal media agar seen in our investigations (Fig. 2).
In radiation-sensitive strains of Escherichia coli ‘minimal medium recovery ’ has been
extensively studied by Ganesan & Smith (1968, 1971). They hypothesize that the phenomenon is due to a recovery process after ultraviolet irradiation, that is inhibited by complex
media and appears to require conditions which permit the synthesis of DNA. It should be
noted that ‘minimal medium recovery’ of irradiated bacteria is only observed in radiationsensitive strains.
Woodcock & Grigg (1972) and Sedgwick & Bridges (1972) demonstrated the presence of
single- and double-stranded breaks in the DNA molecule of Escherichia coli after heat
treatment and the former group showed the restitution of these during incubation in phosphate buffer. Our experiments indicate that hydroxyurea not only blocks repair of ‘thermal
injury’ but in addition it is bactericidal to heat-injured cells (Fig. 5). Hydroxyurea inactivates
protein fraction B 2 of the ribonucleotide diphosphate reductase system without significantly
affecting protein or RNA synthesis, thus depleting the cell of DNA synthesis precursors
(Krakoff, Brown & Reichard, 1968). If the effect of hydroxyurea is only that reported by
Krakoff et al. (1968), then deoxyribonucleotides are required for the repair of ‘thermal
injury’ and their absence results in the inactivation of heat-treated organisms. If DNA
breaks are thermally induced in Salmonella typhimurium LT 2 and subsequently repaired,
when provided with suitable conditions, then such a repair requires de novo accumulation
of deoxyribonucleotide diphosphates and possibly reincorporation into the DNA molecule.
The extreme sensitivity of M-9-grown bacteria to rich media after thermal treatment
questions the validity of the practice of pre-enrichment procedures in rich media for the
isolation of food-borne pathogens. The preprocessing history of the micro-organisms is
likely to affect their recovery in rich media and, if their physiological state resembles that of
M-9-grown bacteria, then a priori, the pre-enrichment step should lower the sensitivity of
the isolation procedure.
This work is Contribution no. 2027 from the Department of Nutrition and Food Science,
Massachusetts Institute of Technology and was supported by Public Health Service Grant
FD-000174-02.We thank Professor Dr D. A. A. Mossel of the Central Institute of Nutrition
and Food Research, Zeist, The Netherlands, and Dr Ir. 5. Stadhouders of the Netherlands
Institute for Dairy Research for their interest in this work. We appreciate the helpful
comments of Dr B. A. Bridges of the University of Sussex.
REFERENCES
ADAMS,
M. H. (1959). Bacteriophages, 1st edn., p. 446. New York: Interscience Publishers.
ALLWOOD,
M. C . & RUSSELL,
A. D. (1966). Some factors influencing the revival of heat-damaged Staphylococcus aureus. Canadian Journal of Microbiology 12, I 295-1297.
Downloaded from www.microbiologyresearch.org by
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274
R. F. G O M E Z , A. J. S I N S K E Y , R. D A V I E S AND T. P. L A B U Z A
BRIDGES,
B. A., ASHWOOD-SMJTH,
M. J. & MUNSON,R. J. (1969). Correlation of bacterial sensitivities to
ionizing radiation and mild heating. Journal of General Microbiology 58, I 15-124.
GANESAN,
A. K. & SMITH,K. C. (1968). Recovery of recombination deficient mutants of Escherichia coli
K-I2 from ultraviolet irradiation. Cold Spring Harbor Symposia on Quantitative Biology 33, 235-242.
GANESAN,
A. K. & SMITH,K. C. (1970). Dark-recovery processes in Escherichia coli irradiated with ultraviolet light. 111. Effect of rec mutations on recovery of excision-deficient mutants of Escherichia coli
K-12. Journal of Bacteriology 102,404-410.
GANESAN,
A. K. & SMITH,K. C. (1971). The duration of recovery and DNA repair in excision deficient
derivatives of Escherichia coli K-I2 after ultraviolet irradiation. Molecular General Genetics I13,
285-296.
HARRIES,
D.& RUSSELL,
A. D. (1966). Revival of heat-damaged Escherichia coli. Experientia 22, 803-804.
IANDOLO,
J. J. & ORDAL,J. Z. (1966). Repair of thermal injury of Staphylococcus aureus. Journal of Bacteriology 91, 134-142.
KRAKOFF,
I. H., BROWN,
N. C. & REICHARD,
P. (1968). Inhibition of ribonucleoside diphosphate reductase
by hydroxyurea. Cancer Research 28, 1559-1565.
LABOTS,
H. (1959).The behaviour of sub-lethally heated Escherichia coli in milk and other media. Proceedings
of X V International Dairy Congress 3, 1355-1360.
MOSSEL,
D. A. A. & RATTO,A. (1970). Rapid detection of sublethally impaired cells of Enferobacteriacea in
dried foods. Applied Microbiology 20, 273-275.
MUKHERJEE,
P. & BHATTACHARJEE,
S.B. (1970). Recovery of bacteria from damages induced by heat.
Journal of General Microbiology 60,233-238.
NELSON,
F. E. (1943). Factors which influence the growth of heat-treated bacteria. I. A comparison of four
agar media. Journal of Bacteriology 45, 395-403.
RUSSELL,
A. D . & HARRIES,
D. (1968). Factors influencing the survival and revival of heat treated Escherichiu
coli. Applied Microbiology 16,335-339.
SEDGWICK,
S . G. & BRIDGES,
B. A. (1972). Evidence for indirect production of DNA strand scissions during
mild heating of Escherichia coli. Journal of General Microbiology 71,191-193.
STRAKA,
R. P. & STOKES,
J. L. (1959). Metabolic injury to bacteria at low temperatures. Journal of Bacteriology 78, 181-185.
WOODCOCK,
E. & GRIGG,G. W. (1972). Repair of thermally induced DNA breakage in Escherichia coli.
Nature New Biology 237,76-79.
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