Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

J. appl. Bact. 1976, 40, 365-374

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

JENNIFER M.WILSON* AND R. DAVIES

National College of Food Technology, St George's Avenue, Weybridge KT13 ODE, England

Received 10 December 1975 and accepted 9 january 1976

Exposure of SalmonelIa sertjiienberg 4969 to sublethal heating in phosphate buffer, pH 7 '0, at 52" produced thermally injured cells characterized by their relative inability to form colonies on trypticase soy yeast extract agar compared to minimal medium (M9) agar. During subsequent incubation at 37" in liquid media, more injured cells were capable of repair in M9 than in nutrient media used for pre-enrichment purposes. M9 was superior to lactose broth as a liquid holding medium to restore the ability of injured cells to grow on both rich and selective agar media. The addition of food products produced a more favour- able environment for the repair of thermally injured cells in M9 rather than lactose broth. Pre-enrichment in M9 was 100 times more effective than using lactose broth as the preliminary step in the detection of S. senftenberg in laboratory pasteurized liquid egg albumen.

IT IS WIDELY recognized that micro-organisms which have been exposed to sub-lethal physical stress may sustain cellular damage of a type which can subsequently be repaired if suitable environmental conditions are provided (Rosenthal & Iandolo, 1970; Ordal, 1971 ; Tomlins &Ordal, 1971 ; Ray &Speck, 1972; Warseck, Ray & Speck, 1973). Injury, for example, may be manifest as additional nutritional requirements (Heater & van der Zant, 1957; Straka & Stokes, 1959) or as increased sensitivity to selective agents (Clark & Ordal, 1969; Tomlins & Ordal, 1971; Maxcy, 1973). Current isolation procedures, therefore, used to detect salmonella in processed foods recognize that injured cells may be present, and generally include a preliminary resuscitation in non-selective, nutrient medium (Thatcher & Clark, 1968 ; Mossel & Ratto, 1970 ; Ray, Jezeski & Busta, 1972). Media used for this purpose include: lactose broth (North, 1961; Hall, Brown & Angelotti, 1964; Ray et al., 1972); nutrient broth (Hobbs, 1963; Corry, Kitchell & Roberts, 1969) and tryptic soya broth (Clark & Ordal, 1969; Mossel & Ratto, 1970; Van Schothorst & Van Leusden, 1972; Wilson, Andrews & Poelma, 1975).

In the case of sub-lethal heat stress, however, Gomez, Sinskey, Davies & Labuza (1973) obtained a higher recovery of thermally injured S. typhimurium LT2 on a minimal medium agar than on trypticase soy yeast extract agar. The phenomenon was designated as 'minimal medium recovery (MMR)', in accord with radiobiological practice, and the apparent toxicity of a rich nutritional environment to heated cells has since been attributed to the development of DNA-lesions (Gomez & Sinskey, 1973).

* Present address: BFMIRA, Randalls Road, Leatherhead, Surrey.

P651

366 J . M. WILSON AND R. DAVIES

I i l this investigation we have compared the recovery of thermally injured salmonellae held in minimal medium and in nutrient media recommended for pre-enrichment purposes. Our definition of MMR is, therefore, extended to include the restoration of viability to cells held in liquid minimal medium in addition to the more conventional interpretation of enhanced cell recovery on solid media.

We have examined the effect of the presence of food materials on MMR and have considered its potential application within procedures of detecting thermally stressed salmonellae which have been grown, heated and recovered in the presence of a food product.

Materials and Methods

Organism

Salmonella senftenberg (NCTC 4969) was obtained from the National Collection of Type Cultures, London.

Media

Cultures were grown in Oxoid Tryptone Soya Broth supplemented with 0.5 % Oxoid Yeast Extract (TSY). Pre-enrichment media used were: Oxoid Lactose Broth (LB); Oxoid Nutrient Broth (NB); TSY and minimal medium (M9). [The latter, being a modification of the chemically defined medium of Adams (1 959), contained (g/l) : NazHP04, 7.0; KHeP04, 3.0; NaCI, 0.5; NH4CI, 1.0; MgS047H20, 0.25; glucose 2.0; pH 7.0.1 Standard Oxoid enrichment media used were Selenite Broth (CM395) and Tetrathionate Broth (CM29). Media used for viable cell counts were: M9 or TSY with the addition of 1.5 % agar; Difco Bismuth Sulphite Agar (BSA); Oxoid (CM329) modified Brilliant Green Agar (BGA) and Oxoid (CM227) Hynes modified Desoxycho- late Citrate Agar (DCA).

Preparation of cell suspensions

TSY broth cultures were incubated at 37" in a water bath with rotary shaking at 80 rev/min. Cells were harvested by centrifuging during mid-exponential phase (4 h) or stationary phase ( I 6 h), washed twice and resuspended in 0.067 mol/l phosphate buffer (pH 7.0) at a concentration of 1-0-5.0 x lo9 cells/ml. All manipulations were carried out at 37" to avoid cold-shock.

Heat treatmePtt

The heating vessel was a 250 ml three-necked distillation flask immersed to the neck in a water bath and stirred mechanically. Cells were heated by adding 0.5 ml of cell suspension to 49.5 ml of heating menstruum pre-equilibrated and held at the appropriate temperature (or at 37 * 0.05 to provide an uninjured control population). Samples of I ml were removed at intervals for serial dilution in phosphate buffer maintained at 37". Duplicate 0.1 ml samples from appropriate dilutions were spread on the surface of pre-dried plates of M9, TSY or differential agar media. The plates were incubated at 37" and counted after 48 h.

Where a standard heat treatment was required, exponential and stationary phase

MINIMAL MEDIUM RECOVERY OF HEATED SALMONELLA 367

cells were heated in phosphate buffer (pH 7) at 52 k 0.05" for 15 and 35 min, res- pectively. These treatments produced 2 to 3 decimal reductions in cell survival on TSY agar.

Liquid holding conditions

After the standard heat treatment, 1 ml samples from the heating flask were diluted 1 : 20 into the chosen liquid holding medium, held a t 37". Repair of injured cells, assessed as increased recovery on TSY agar and designated as liquid holding recovery (LHR), was followed by removal of 1 ml samples at intervals for serial dilution and surface plating.

Food products were added to liquid holding media before the addition of inoculum. Fresh egg albumen and whole liquid egg were prepared aseptically (Anellis et al., 1954) and each sample was blended for 30s in a Stomacher 400 (A. J. Seward, Suffolk, England) before addition to the liquid holding medium at a concentration (v/v) of 10% or 25 %. Dry food products, obtained commercially, were added in similar concentrations (w/v) before thorough mixing on a Vortex mixer. The final pH was adjusted to 7.0 using sterile solutions of hydrochloric acid or sodium hydroxide.

Bone meal samples were autoclaved at 121" for 15 min before use.

Laboratory pasteurization of egg albumen

Fresh egg albumen was prepared as described previously and inoculated with S. senftenberg. Cultures were grown in mid-exponential phase (6 h) at 37", harvested by centrifuging, resuspended in 0.5 ml egg albumen and pasteurized for 4 min by addition to 19.5 ml albumen pre-heated and held at 57.4" (Hobbs, 1974). The pH was maintained at 7-0 during all stages to facilitate growth and prevent additional injury due to pH changes.

Results

Except where otherwise indicated all data relate to mid-exponential phase cells.

Survival at 52"

The colony forming ability of S . senftenberg on rich, minimal or differential media after heat treatment at 52", is shown in Fig. 1. BGA, BSA and DCA were found to be no more inhibitory to injured cells than was the rich medium, TSY. This observation differs from that of Clark & Ordal (1969). However, during the first 30 min of heat treatment, higher recovery of thermally injured cells was obtained by plating on M9 agar. This result, for TSY grown cells, is similar to that observed for minimal medium grown cells of S. typhimurizrm LT2 (Gomez et al., 1973).

Liquid holding recovery

It has been observed frequently that thermally injured cells exhibit an extended lag period before resuming growth in a nutrient liquid medium (Kaufman, Harman, Pailthorp & Pflug, 1959; Jackson & Woodbine, 1963). I t was, therefore, necessary to determine the period of liquid holding during which increased recovery could be

368 J. M. WILSON AND R. DAVIES

0 15 30 45 i

Time IP phosphate buffer i p H 7 0 ) at 5 2 O C (min)

Fig. 1 . Survival of S. senjienberg 4969 heated in phosphate buffer (pH 7) and plated on various recovery media. Surviving fraction when plated on: 0, TSY agar; 0 , M9 agar; A, DCA; 0, BSA; V, BGA.

attributed to repair before resumption of cell division. Fig. 2 shows the changes in recovery of standard heat-injured cells, held in M9 and lactose broth, as seen by plating on TSY agar. A rapid increase in cell recovery from both media occurred during the first 2 h of liquid holding. In the case of M9, this increase was much greater than could be expected from the rate of cell division (mean generation time:

I 0 3

0'

0 2 4 6 8 10 I I I I I

Holding time after heat treatment (h )

Fig. 2. Effect of liquid holding on the viability of heated or unheated S. senjienberg 4969 plated on TSY agar. Cells were heated in phosphate buffer (pH 7) at 52" for 15 min (heated cells) or at 37" for 15 min (unheated cells). Heat treated cells were then diluted (I : 20) into: 0, M9 medium; A, lactose broth. Unheated cells were also diluted (1 : 20) into; 0, M9 medium; A, lactose broth. The liquid holding cultures were incubated at 37" and plated periodically on TSY agar.


52 min) exhibited by the unheated control population. Following a period during which recovery remained relatively constant, further increases occurred consistent with normal outgrowth of the surviving cell populations. It would, therefore, appear that the increases in recovery observed during the first 3 h of holding in lactose broth or in M9 reflect repair of injured cells.

As shown in Fig. 3, minimal medium clearly provided more beneficial conditions

1

I I 60 120 180

Holding time after heat treatment ( m i d

Fig. 3. Effect of liquid holding recovery media on the viability of heat injured S. senftenberg 4969. Cells were heated in phosphate buffer (pH 7) at 52" for 15 min, diluted (1 : 20) into various holding media, incubated at 37" and periodically plated on TSY agar. Surviving fractions after holding in: 0, TSY; 0 , M9 medium; 0, nutrient broth; V, lactose broth; A, selenite broth.

for the repair of injured cells than did various nutrient pre-enrichment media and more than half the initial population could be recovered after 2 h holding in M9. Of the standard pre-enrichment media used, only lactose broth provided suitable conditions for repair. The initial decline in survivors from nutrient broth or TSY indicates that both these media were toxic to a proportion of the injured population.

Selenite broth, however, was extremely toxic to injured cells as shown by the 100-fold decrease in survivors during the first 15 min of liquid holding. Recovery from tetrathionate broth was similar to that from TSY.

EfSect of LHR on ability to grow on selective agar media

Repair in liquid minimal medium also restored the ability of injured cells to grow on BGA, BSA and DCA selective media as shown in Table 1. Incubation in lactose broth likewise facilitated some increased recovery on these media, though to a considerably lesser extent, and holding in TSY broth resulted in loss of viability rather than increased recovery.

Infruence of ,food products on LHR

Fig. 4 shows typical results for the repair of standard heat-injured cells in M9 or


TABLE 1 EfSect of plating medium and liquid holding medium on viable count of

thermally injured" S. senftenberg 4969

Viable countlml ( x lo4) ,

Holding medium/holding time (h)

M9 LB TSY A

7 \

Plating No ------- r------ medium holding 1 3 1 3 1 3

TSYA 8.1 1400 2400 120 380 2.6 13.0 BGA 7.5 1500 2500 190 380 3.2 10.0 BSA 18.0 960 980 640 660 7.2 6.0 DCA 9.0 290 390 12 27 0.7 1 . 1

~ _ _ ~ ~ - _ _ _ ~

* Cells were heated in phosphate buffer, pH 7.0, at 52" for 15 min.

I I00

t I

A i

-0-0 I I " I I I I I

60 120 180 Holding time after heat treatment (rnin)

Fig. 4. Effect of food materials on the liquid holding recovery of heat injured Xsenftenberg 4969. Cells were heated in phosphate buffer (pH 7) at 52" for 15 min, diluted (1 : 20) into lactose broth or M9 liquid holding media containing added food materials, incubated at 37" and periodically plated on TSY agar. Surviving fractions after holding in: 0, M9 medium+ 10% (w/v) bone meal; A, M9 medium+ 10% (w/v) dried skim milk; ., M9 medium+ 10% (v/v) fresh egg albumen; 0, lactose broth+10% (w/v) bone meal; A, lactose broth+ 10% (w/v) dried skim milk; 0, lactose broth+ 10% (v/v) fresh egg albumen.

lactose broth to which 10% of various food products had been added. Further examples are given in Table 2. Bone meal or skim milk powder had little effect on repair in minimal medium (MMR) but the environment produced by combination with lactose broth was toxic to injured cells. The addition of fresh whole liquid egg did not affect LHR repair in either medium which remained similar to that shown in the absence of food (Fig. 3). Fresh egg albumen, however, increased the number of survivors recovered from both media with more than 80% of the initial population being recovered from the M9 egg albumen mixture after 3 h incubation.

Table 2 also shows that complete recovery was obtained where a higher concentration (25%v/v) of fresh egg albumen was added to M9 whereas increasing the

MINIMAL MEDIUM RECOVERY OF HEATED SALMONELLA 37 1

TABLE 2 Effect of food materials on the liquid holding recovery of thermally

injured" exponential phase cells of S . senftenberg 4949 ~ ~ ~ -~

Percentage recoveryt after 3 h LHR in A r

Food material M9 plus LB plus M9 plus LB plus addedf 10% food 10% food 25 % food 25% food

Bone meal 20.8 0 .2 1 0 . 1 < 0 * 1 Skim milk powder 27.0 1 . 6 13.0 3.5 Dried egg albumen 20.0 - 24.0 7.2 Dried whole egg 27.0 3.7 51 $0 1.7 Fresh egg albumen 83.0 6.0 107.0 38.0 Fresh whole egg 6 6 . 0 19.0 109.0 50.0

~ ~ ~~~

* Cells were heated in phosphate buffer, pH 7.0, at 52" for 15 min. 7 Viable count on TSY agar expressed as percentage of unheated control viable count. f Dry materials added as % (w/v); liquid materials as % (v/v).

concentration of other food products decreased MMR. This latter effect was most evident with bone meal where MMR was markedly inhibited.

Similar experiments were carried out using cultures grown to stationary phase (16 h) in TSY broth and heated at 52" for 35 min. Repair in minimal or nutrient media, with or without addition of food products is summarized in Table 3.

TABLE 3 Effect of food materials on the liquid holding recovery of thermally injured" stationary

phase cells of S. senftenberg 4949 ~ ~~~~

Percentage recovery? after 10% 3 h LHR in

addedf M9 LB Food material

Bone meal 4.8 <0.1 Skim milk powder 2.2 0.4 Fresh egg albumen 4.0 1 . 1 Fresh whole egg 4.4 0.4 No additions 1.8 0.8

* Cells were heated in phosphate buffer, pH 7.0, at 52" for 35 min. t Viable count on TSY agar expressed as percentage of unheated control viable count. f Dry materials added as % (w/v); liquid materials as % W ) .

In comparison with exponential phase cultures, less repair was observed in both holding media. In the presence of 10% food products, however, M9 generally gave enhanced LHR compared to holding in lactose broth. The combination of lactose broth and bone meal was again found to be relatively toxic to thermally injured cells.

28


Application of MMR to pasteurized liquid egg albumen

The simulation of practical conditions, where S. senftenberg 4969 was allowed to grow at 37" to mid-exponential phase, pasteurized at 57" for 4 min and recovered in either M9 or lactose broth, all in the presence of fresh liquid egg albumen, gave the liquid holding recovery data shown in Fig. 5. Addition of pasteurized fresh egg albumen at

'00, 1

60 120 180 Holding time after heof treatment (min)

Fig. 5. Liquid holding recovery of heat injured S. senftenberg4969 from laboratory pasteurized egg albumen. Cells were grown to exponential phase in fresh egg albumen (pH 7) which was then heated at 57.4" for 4 min. The heated albumen was then added at 10 % (v/v) to lactose broth or M9 liquid holding media, incubated at 37" and periodically plated on TSY agar. Surviving fractions after holding in: 0, M9 medium; 0, lactose broth.

10% (v/v) to M9 followed by incubation at 37" for 3 h led to the recovery of x lo5 more survivors than could be detected immediately after heat treatment. In comparison, similar incubation in lactose broth increased recovery by a lesser factor of x 103.

Discussion The results obtained in this investigation, for S. senftenberg 4969, essentially substantiate those of Gomez et al. (1973) who introduced the concept of 'minimal- medium-recovery' for thermally injured salmonellae. A significant difference, however, is that we find that the phenomenon is not limited to minimal medium grown cells but can also be demonstrated for cells grown in complex medium or in liquid egg. Thus we question the hypothesis that MMR may be simply interpreted as a heat-induced exacerbation of a cell's normal sensitivity to sudden changes to a richer nutritional environment.

The study provides further evidence, however, that potentially expands the practical significance of MMR beyond the confines of the particular conditions of its original model system. The introduction of food ingredients into the liquid holding systems, containingstandard thermally injuredcells revealed that repair during holding in minimal medium can be enhanced, particularly by fresh egg albumen, to a point where 100 %


of the heated population can be recovered (Table 2). Holding in lactose broth, on the other hand, contrary to the findings of Hall et al. (1964), is not only less effective but in the case of admixture with bone meal samples, as shown in Fig. 4, can be initially toxic to injured cells. The further application of MMR to the detection of S. senftenberg in pasteurized fresh egg albumen, as indicated in Fig. 5, has even greater significance. The cells were allowed to grow in the egg albumen before exposure to a commercially recommended heat treatment (Hobbs, 1974) more severe than that used in the model system, yet which achieved only a disconcertingly small reduction in viability (slightly more than one decimal reduction factor) when evaluated by minimal medium pre-enrichment.

It must be cautioned, of course, that the MMR phenomenon demonstrated in this study relates primarily to the detection of salmonellae which were allowed to grow to early or mid-exponential phase before heat treatment. This may be relevant to a test evaluation of a thermal process using artificially inoculated raw material (Garibaldi, Ljichi & Bayne, 1969) but it could be argued that it would imply an unlikely degree of gross malpractice in a food-processing situation. The limited data for stationary phase cells, however, as shown in Table 3, provide no evidence of a reduced recovery of injured cells by pre-enrichment in minimal medium and, indeed, M9 is shown to be superior to lactose broth when used for this purpose in the presence of food materials. The results shown in Table 1, furthermore, confirm that holding injured cells in minimal medium facilitates a returned tolerance to selective media as reported for stationary phase cells of S. typhimurium held in various pre-enrichment media (Clark & Ordal, 1969) or in citrate minimal medium (Tomlins & Ordal, 1971). Thus it seems reasonable to speculate that minimal medium pre-enrichment should consistently and significantly improve the detection of naturally occurring contaminant salmonellae in sub-lethally heated foods. The degree of advantage over media currently employed for this purpose, however, will depend on the previous growth history of the organisms present, the type of food involved and the amount of food added to the liquid pre- enrichment medium.

References ADAMS, M. H. (1959). Bacteriophages. New York : Interscience Publishers. ANELLIS, A., LUBAS, J. & RAYMAN, M. M. (1954). Heat resistance in liquid eggs of some

strains of the genus Salmonella. Fd Res. 19, 377. CLARK, C. W. & ORDAL, Z. J. (1969). Thermal injury and recovery of Salmonella typhimurium

and its effect on enumeration procedures. Appl. Microbiol. 18, 332. CORRY, J. E. L., KITCHELL, A. G. & ROBERTS, T. A. (1969). Interactions in the recovery of

Salmonella typhimurium damaged by heat or gamma radiation. J. appl. Bact. 32, 415. GARIBALDI, J. A., IJICHI, K. & BAYNE, H. G. (1969). Effect of pH and chelating agents on

the heat resistance and viability of Salmonella typhimurium Tm-1 and Salmonella senftenberg 775W in egg white. Appl. Microbiol. 18, 318.

GOMEZ, R. F. & SINSKEY, A. J. (1973). Deoxyribonucleic acid breaks in heated Salmonella typhimurium LT2 after exposure to nutritionally complex media. J. Bact. 115, 522.

GOMEZ, R. F., SINSKEY, A. J., DAVIES, R. & LABUZA, T. P. (1973). Minimal medium recovery of heated Salmonella typhimurium LT2. J. gen. Microbiol. 74, 267.

HALL, H. E., BROWN, D. F. & ANGELOTTI, R. (1964). The quantification of salmonellae in foods by using the lactose preenrichment method of North. J. Milk Fd Technol. 27,235.

HEATER, C. D. & VAN DER ZANT, W. C. (1957). Effect of the plating medium on the survival of heat-treated cells of Pseudomonas Jluorescens. Fd Res. 22, 164.


HOBBS, B. C. (1963). Techniques for the isolation of salmonellae from eggs and egg products. Annls. Znst. Pasteur 104, 621.

HOBBS, B. C. (1974). Food Poisoning and Food Hygiene. London: Edward Arnold (Publishers) Ltd.

JACKSON, H. & WOODBINE, M. (1963). The effect of sub-lethal heat treatment on the growth of Staphylococcus aureus. J. appl. Bact. 26, 152.

KAUFMANN, 0. W., HARMAN, L. G., PAILTHORP, 0. C. & PFLUG, I. J. (1959). Effect of heat treatment on the growth of surviving cells. J. Bact. 78, 834.

MAXCY, R. B. (1973). Condition of coliform organisms influencing recovery of subcultures on selective media. J. Milk Fd Technol. 36, 414.

MOSSEL, D. A. A. & RATTO, M. A. (1970). Rapid detection of sublethally impaired cells of Enterobacteriaceae in dried foods. Appl. Microbiol. 20, 273.

NORTH, W. R. (1961). Lactose pre-enrichment method for isolation of salmonella from dried egg albumen. Appl. hficrobiol. 9, 188.

ORDAL, Z. J. (1971). Symposium on the restoration of sublethally impaired bacterial cells in foods. I11 Influence of thermal stress. J. Milk Fd Technol. 34, 549.

RAY, B., JEZESKI, J. J. & BUSTA, F. F. (1972). Isolation of salmonellae from naturally con- taminated dried milk products. 111 Influence of pre-enrichment conditions. J. Milk Fd Technol. 35, 607.

RAY, B. & SPECK, M. L. (1972). Repair of injury induced by freezing Escherichia coli as influenced by recovery medium. Appl. Microbiol. 24, 258.

ROSENTHAL, L. J. & IANDOLO, J. J. (1970). Thermally induced intracellular alteration of ribosomal ribonucleic acid. J. Bact. 103, 833.

STRAKA, R. P. & STOKES, J. L. (1959). Metabolic injury to bacteria at low temperatures. J. Bact. 78, 181.

THATCHER, F. S. & CLARK, D. S. (1968). Micro-organisms in Foods-Their Significance and Methods of Enumeration. University of Toronto Press.

TOMLINS, R. I. & ORDAL, Z. J. (1971). Requirements of Salmonella typhimurium for recovery from thermal injury. J. Bact. 105, 512.

VAN SCHOTHORST, M. & VAN LEUSDEN, F. M. (1972). Studies on the isolation of injured salmonellae from foods. Zentbl. Bakt. Parasitkde. Abt. Z 221, 19.

WARSECK, M., RAY, B. & SPECK, M. L. (1973). Repair and enumeration of injured colifornis in frozen foods. Appl. Microbial. 26, 919.

WILSON, C. R., ANDREWS, W. A. & POELMA, P. L. (1975). Evaluation of cultural methods to isolate Salmonella from pressed yeast and dried inactive yeast. J. Milk Fd Technol.38,353.

Minimal Medium Recovery of Thermally Injured Salmonella senftenberg 4969

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