Performance of media for recovering stressed cells of Enterobacter sakazakii as determined using spiral plating and ecometric techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55 degrees C for 5 min), freezing (-20 degrees C for 24 h, thawed, frozen again at -20 degrees C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21 degrees C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Performance of Media for Recovering Stressed Cells of Enterobacter sakazakii as Determined Using Spiral Plating and Ecometric Techniques

A study was done to determine the performance of differential, selective media for supporting resuscitation and colony development by stressed cells of Enterobacter sakazakii. Cells of four strains of E. sakazakii isolated from powdered infant formula were exposed to five stress conditions: heat (55°C for 5 min), freezing (−20°C for 24 h, thawed, frozen again at −20°C for 2 h, thawed), acidic pH (3.54), alkaline pH (11.25), and desiccation in powdered infant formula (water activity, 0.25; 21°C for 31 days). Control and stressed cells were spiral plated on tryptic soy agar supplemented with 0.1% pyruvate (TSAP, a nonselective control medium); Leuschner, Baird, Donald, and Cox (LBDC) agar (a differential, nonselective medium); Oh and Kang agar (OK); fecal coliform agar (FCA); Druggan-Forsythe-Iversen (DFI) medium; violet red bile glucose (VRBG) agar; and Enterobacteriaceae enrichment (EE) agar. With the exception of desiccation-stressed cells, suspensions of stressed cells were also plated on these media and on R&F Enterobacter sakazakii chromogenic plating (RF) medium using the ecometric technique. The order of performance of media for recovering control and heat-, freeze-, acid-, and alkaline-stressed cells by spiral plating was TSAP > LBDC > FCA > OK, VRBG > DFI > EE; the general order for recovering desiccated cells was TSAP, LBDC, FCA, OK > DFI, VRBG, EE. Using the ecometric technique, the general order of growth indices of stressed cells was TSAP, LBDC > FCA > RF, VRBG, OK > DFI, EE. The results indicate that differential, selective media vary greatly in their abilities to support resuscitation and colony formation by stressed cells of E. sakazakii. The orders of performance of media for recovering stressed cells were similar using spiral plating and ecometric techniques, but results from spiral plating should be considered more conclusive.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

Repair and Enumeration of Injured Coliforms in Frozen Foods

Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA.     

COMPARISONS OF DIFFERENT MEDIA FOR COUNTING SUGAR TOLERANT YEASTS IN CONCENTRATED ORANGE JUICE

SUMMARY: To select and count the sugar tolerant yeasts which ferment sixfold concentrated orange juice, a high sugar agar medium was developed which contains 50% of glucose, 1% of citric acid and 1% of Tryptone; it is incubated for 4–5 days at 25°.
The medium has disadvantages: it is troublesome to prepare, and colonies grow slowly and are translucent. These properties result directly' from the high sugar concentration, on which the selective action of the medium depends.
Counts on this medium have been compared with those on potato dextrose or nutrient dextrose agars (with 2% and 1% of glucose respectively), with yeasts isolated from fermenting concentrate, in pure culture, and under various practical conditions. As a rule, the counts were virtually the same on the different media; nutrient dextrose agar occasionally failed to record small numbers of these yeasts. If the two low sugar media were acidified to pH 3·5 the counts were reduced.
Potato dextrose agar recorded, besides the above yeasts, sugar intolerant yeasts entering from dirty machines or through bad canning practice: nutrient dextrose agar recorded bacteria in addition. The difference between parallel plating on these media and on the high sugar medium thus yielded useful information about sources of casual contamination.
It is suggested that the above would also be largely applicable to other sugar-rich concentrates of not less than 50° Brix.     

Ampicillin-dextrin agar medium for the enumeration of Aeromonas species in water by membrane filtration

Published selective media were evaluated for the isolation of Aeromonas spp. from environmental samples by membrane filtration. Satisfactory recoveries were obtained only with mA agar (Rippey & Cabelli) and dextrin-fuchsin-sulphite agar (Schubert), but neither was sufficiently selective. The positive aspects of these two media were combined in a new medium, ampicillin-dextrin agar. Recovery from pure cultures and environmental samples was optimal at an ampicillin concentration of 10 mg/l and incubation for 24 h at 30°C under aerobic conditions, and specificity was high (i.e. confirmation rate usually <90%, no false negative colonies encountered). The medium can also be used for isolation of Aeromonas spp. from sea water provided that the vibriostatic agent 0/129 is added at 50 mg/1.     

Comparison of selective agars for the isolation and identification of Klebsiella oxytoca and Escherichia coli from environmental drinking water samples

Various selective media were assessed for their ability to detect and differentiate Klebsiella oxytoca and Escherichia coli in environmental water samples. Only two, Membrane Lauryl Sulphate agar and Deoxycholate Agar, could differentiate the two coliforms from each other and from the 'background' heterotrophs in water and this was a consequence of E. coli's ability to grow at 44°C and 37°C whereas Kl. oxytoca could only grow at 37°C. Modified M-FC medium effectively differentiated Kl. oxytoca but not E. coli in environmental samples. Other media characterized the different coliforms in pure culture but failed to do likewise in environmental samples. For example, pure cultures of E. coli fluoresced when MUG was added to the medium but single colonies on a mixed species plate failed to do so. MT7 agar distinguished the two coliforms from water heterotrophs but not from each other.     

A comparison of media and methods for the recovery of Yersinia enterocolitica and Yersinia enterocolitica-like bacteria from milk containing simulated raw milk microfloras

A number of plating and enrichment media proposed for the isolation of Yersinia enterocolitica from foodstuffs were examined for their ability to recover the type strains of Y. enterocolitica sensu stricto, Y. intermedia, Y. frederiksenii and Y. kristensenii. Nine selective plating media were evaluated for the quantitative recovery of the type strains in pure culture, and their inhibition of other organisms typical of both milk and enteric microfloras. Cefsulodin-irgasan-novobiocin (CIN) agar, incubated for 48 h at 25°C, allowed a high recovery of all the Yersinia spp. and was the most selective medium. The same four type strains were added to UHT milk that had been previously inoculated with bacteria to simulate either freshly drawn or cold stored milk microfloras. Twenty-six enrichment procedures (including cold enrichment, selective enrichment at higher temperatures, two-step procedures and a post-enrichment alkali treatment) were assessed for the efficiency of recovery of the Yersinia spp. Pre-enrichment in trypticase-soy broth (TSB) for 24 h at 22°C followed by selective enrichment in bile-oxalate-sorbose (BOS) medium for 5 d at 22°C and plating on CIN agar (48 h at 25°C) allowed the greatest increase in the numbers of Yersinia spp. and maximum inhibition of the competing microflora.     

Stability of plasmids in five strains of Salmonella maintained in stab culture at different temperatures

Four strains of Salmonella berta and one of Salm. enteritidis were stored as stab cultures in sugar-free agar at 5°, 22° and 30°C and in 15% glycerol at—80°C. The stability of the plasmid profiles in each of the strains was monitored over a period of 2·5 years.
Plasmid profiles were stable in all strains stored at—80°C, and only six of 450 colonies examined from strains kept in sugar-free agar at 5°C had lost plasmid molecules. Seventy of 440 colonies from stab cultures that were kept at 22°C, and 71 of 440 colonies at 30°C showed changed plasmid profiles. The total number of plasmids lost increased with time, and occasionally, more than one plasmid molecule was lost in the same strain.
The virulence associated plasmid of Salm. enteritidis was remarkably stable as it was maintained in all colonies examined at all temperatures investigated. Likewise, no change in Sma I restriction profile was observed in this plasmid molecule at any temperature.     

SGAP-10C agar for the isolation and quantification of Aeromonas from water

Glutamate starch penicillin (GSP) medium was used for the simultaneous isolation of Pseudomonas and Aeromonas . Modifications to reduce the number of Pseudomonas and background flora and to improve the recovery of Aeromonas from water samples are described. The original medium was modified by adding glucose and ampicillin. The addition of 10 μg/l of C-glucose to the medium (SGAP-10C) permitted better recuperation of stressed cells of aeromonads and the ampicillin reduced the numbers of Pseudomonas . The best temperature for the recovery of aquatic aeromonads was 28°C. The recovery of different species of Aeromonas on SGAP-10C was 93%. The selectivity of the medium was validated because 95·5% of 28 colonies tested with an Aeromonas -like morphology belonged to the genus Aeromonas . Moreover, when 45 strains of different genera were cultured on the medium, only Vibrio alginolyticus presented a confusing morphology. When the SGAP-10C was compared with GSP with 45 river samples, the new medium gave a significantly better recovery of Aeromonas spp., especially when large numbers of Pseudomonas spp. were present. SGAP-10C used at 28°C and 48 h was an efficient selective medium for the isolation of Aeromonas from fresh waters.     

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-β- d -glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44°C but recoveries were better at 41°C.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37 degrees and 42.5 degrees C (thermotrophs) and 30 degrees C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37 degrees and 42.5 degrees C. At 42.5 degrees C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37 degrees C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42.5 degrees C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

The Recovery of Sublethally Injured Escherichia coli from Frozen Meat

Sublethal injury to Escherichia coli , measured as the inability of surviving cells to grow on media containing bile salts, was monitored during frozen storage on meat at —5, —10 and —20° C. More rapid increases in injury occurred at the higher subzero temperatures and log phase cells were more susceptible than those in the stationary phase of growth. Repair of injury in non-selective liquid media took between 2 and 6 h at 25° and was often accompanied by an increase in total viable count. Incubation for a fixed period in broth was, therefore, unsuitable for the quantitative recovery of freeze-injured Esch. coli. Resuscitation on membrane filters avoided confusing repair of injury with multiplication of uninjured or repaired cells. The mean recovery of injured cells following incubation on membranes for 4 h at 35°C on tryptone soya agar, was 94%.     

The effect of freezing and the influence of isolation medium on the recovery of pathogenic fungi from sputum

The primary objective of this study was to determine whether freezing sputa in dry ice had any effect on the recovery of pathogenic fungi. Sputa seeded with each of five fungi (Histoplasma capsulatum, Blastomyces dermatitidis, Cryptococcus neoformans, Coccidioides immitis, and Aspergillus fumigatus) were frozen and stored for 24, 48, and 72 hours on dry ice. H. capsulatum was killed, and only a few colonies of B. dermatitidis and C. neoformans were isolated from these sputa. However, A. fumigatus and C. immitis withstood the effects of freezing. A second objective was to compare the recovery of all five fungi from seeded sputa stored at room temperature for 24, 48, and 72 hours, on yeast extract-phosphate agar with NH₄OH and on Sabhi agar. The yeast extract-phosphate agar with NH₄OH was superior to Sabhi agar, for the isolation of all fungi studied, except A. fumigatus.     

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4 degrees C, freezing at -20 degrees C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheep's blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and -20 degrees C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.     

Growth Characteristics, Bile Sensitivity, and Freeze Damage in Colonial Variants of Lactobacillus acidophilus

Rough (R) and smooth (S) colonial variants were isolated from a heterogeneous culture of Lactobacillus acidophilus RL8K. R and S types were stable upon repeated transfer on agar, but revertant colonies did appear after broth transfers. When propagated in commercial MRS broth, R and S cultures showed similar growth characteristics, and both cell types were insensitive to freezing and frozen storage at −20°C. Alternatively, during growth in scratch MRS broth, R cultures shifted to a reduced rate of growth during the late logarithmic phase. R cells grown under these conditions were susceptible to death by freezing and injury at −20°C. Microscopically, R cells were observed as long gram-positive rods with small nonstainable blebs protruding from the cell wall. In bile sensitivity studies of R and S cells plated on MRS agar plus oxgall, the S culture was resistant to 1% bile, whereas the R culture was sensitive to 0.6% bile. Differences in the bile resistance and freeze damage of R and S cells suggest that colonial and cellular morphologies are important considerations for the selection of Lactobacillus strains as dietary adjuncts and for the development of growth conditions for preparing frozen concentrated cultures from either cell type.     

Role of Suspending and Recovery Media in the Survival of Frozen Shigella sonnei

Shigella sonnei was frozen at -20 C in saline, nutrient broth, and milk, and plated, after thawing, upon synthetic medium, nutrient agar, and blood heart infusion agar. There was a difference in the numbers of cells recovered when the frozen and thawed cells were grown on different media. The synthetic medium was unable to recover cells injured by freezing or did so only poorly compared to the complex media. The addition of meat extract, peptone, or Casamino acids to the synthetic medium improved its ability to recover injured cells as measured by bacterial colony counts. This is suggestive of metabolic injury caused by the freezing processes since the cells which survived freezing required an enriched medium for growth. In this paper the term metabolic injury is used to express a change in the nutritional requirements of the organisms which resulted in an increase in growth factor requirements. Freezing the cells in saline resulted in greater injury compared to cells frozen in nutrient broth or milk; this suggested that these suspending agents possessed some protective quality. The metabolic injury increased with an increase in the length of time the cells were held in the frozen state.     

The enumeration of sublethally injured populations of Staphylococcus aureus in foods

Substantiating earlier investigations, pure cultures of Staphylococcus aureus were found to be equally well recovered on Baird-Parker agar at 37°C as at 42°C, whereas Micrococcus spp. are suppressed at the latter temperature to an extent exceeding 5 log₁₀ cycles. It was also established that egg yolk dissimilation by Staph. aureus is intensified at 42°C. Heat treated (60°C) populations of Staph aureus were quantitatively recovered on Baird-Parker agar at 42°C, though acid-injured populations were not. Acid-injury (2% lactic acid at 37°C) could be completely restored by solid medium repaiar during at least 6 h at 23°C on tryptone soya peptone yeast extract egg yolk pyruvate agar. Pure culture studies were confirmed in surveys on trade samples of foods.