A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

The effect of temperature on the growth of strains of Kloeckera apiculata and Saccharomyces cerevisiae in apple juice fermentation

The influence of temperature (10°C and 25°C) on the survival and growth of Saccharomyces cerevisiae and Kloeckera apiculata was examined in mixed and pure cultures during fermentation in apple juice. The growth reached by S. cerevisiae did not seem to be affected by temperature and the presence of K. apiculata . However, the growth and survival of K. apiculata , both in single and mixed cultures, were substantially enhanced at 10°C. The highest amount of ethyl acetate was produced by K. apiculata in pure culture at 10°C. Nevertheless, this concentration was lowest when both yeasts were fermented together at 10°C and 25°C.     

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.     

Nitrogenase activity (acetylene reduction) in root-associated, cold-climate species of Azospirillum, Enterobacter, Klebsiella and Pseudomonas growing at various temperatures

Abstract 23 Strains of diazotrophic root-associated bacteria isolated from various parts of Finland were tested for nitrogenase activity during growth at various temperatures. Nitrogenase activity was optimal at 20–37°C in cultures of Klebsiella pneumoniae , and at 14–20°C in cultures of Klebsiella terrigena and Enterobacter agglomerans . Strains of K. terrigena and E. agglomerans showed no activity at 37°C, and K. pneumoniae only minimal or no activity at 14°C. Azospirillum lipoferum exhibited high nitrogenase activity at both 28–37°C, but less than 25% of optimal activity at 20°C and no activity at 14°C. Pseudomonas sp. expressed nitrogenase activity at 14–28°C. None of the strains manifested nitrogenase activity at 4 or 42°C. There were only small local variations within a species between strains isolated at different locations.     

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.     

Methanol metabolism in thermotolerant methylotrophic Bacillus species

Abstract For a number of years we have tried to isolate versatile methylotrophic bacteria employing the ribulose monophosphate (RuMP) cycle of formaldehyde fixation. Recently this has resulted in the development of techniques for the selective enrichment and isolation in pure culture of Bacillus strains able to grow in methanol mineral medium over a temperature range between 35 and 60°C. At the optimum growth temperatures (50–55°C), these isolates display doubling times between 40 and 80 min. The metabolism of the strains studied is strictly respiratory. Methanol assimilation is exclusively via the RuMP cycle variants with the fructose bisphosphate (FBP) aldolase cleavage and transketolase (TK)/transaldolase (TA) rearrangement. Whole cells were unable to oxidize formate, and no activities of NAD-(in)dependent formaldehyde and formate dehydrogenases were detected. Formaldehyde oxidation most likely proceeds via the so-called dissimilatory RuMP cycle. The initial oxidation of methanol is catalyzed by an NAD-dependent methanol dehydrogenase present as an abundant protein in all strains. The enzyme from Bacillus sp. C1 has been purified and characterized.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

Incidence of Enterotoxigenic Clostridium perfringens in Healthy Humans in relation to the Enhancement of Enterotoxin Production by Heat Treatment

Enterotoxin production was greatly enhanced in two of five food poisoning strains of Clostridium perfringens subjected to heat treatment prior to incubation in Duncan and Strong sporulation medium. Heating was carried out on three successive cultures of each strain, the optimum temperature for treatment being 85 °C for one strain and 95 °C for another: on each occasion cultures were heated for 20 min. The triple heat treatment procedure was used in testing strains of Cl. perfringens isolated from faeces of healthy human subjects for production of enterotoxin. Eleven of 35 (31%) individuals were found to be carriers of enterotoxigenic strains, the isolates producing more than 0·1 μ/ml of enterotoxin. Six of the 11 enterotoxigenic strains were killed by heating at 95 °C but one isolate produced more enterotoxin following treatment at this temperature than after heating at 75 °C.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Comparison of the Effects of Liquid Medium Repair and the Incorporation of Catalase in MacConkey Type Media on the Recovery of Enterobacteriaceae Sublethally Stressed by Freezing

Nine pure cultures of species of Enterobacteriaceae were stressed by rapid freezing in tryptone soya broth (TSB) to — 22°C and subsequent storage at that temperature for 7 d. About one to two log cycles kill and at least one additional log cycle sublethal impairment was achieved. Numbers of colonies of these cultures in poured plates of violet red bile glucose (VRBG) agar, with 67 u/ml of catalase added at 47°C, were only slightly higher than those in plain VRBG, both incubated overnight at 30°C. Two hours incubation of TSB suspensions at 17–25° C resulted in almost complete restoration of the ability of cells to develop colonies in VRBG, without, however, leading to any significant multiplication.
Similar experiments with 32 samples of frozen minced meat, 27 samples of frozen surface water, 18 of frozen chicken liver and 14 of fresh sausage substantiated the results obtained in the studies on pure cultures.
In the experiments with the nine pure cultures the influence of the nutrient composition of the solid enumeration media: 'minimal' agar, TSB agar (TSBA) and Mueller-Hinton agar with Polyvitex nutrient supplement (MHA), on the recovery of Enterobacteriaceae stressed by freezing was also studied. Colony numbers in TSBA and MHA were virtually identical. The glucose mineral salts medium led to lower recovery, indicating that so-called 'minimal medium recovery' of stressed bacterial populations is not a common phenomenon.     

Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

Five psychrophilic sulphate-reducing bacteria (strains ASv26, LSv21, PSv29, LSv54 and LSv514) isolated from Arctic sediments were examined for their adaptation to permanently low temperatures. All strains grew at −1.8°C, the freezing point of sea water, but their optimum temperature for growth ( T _opt) were 7°C (PSv29), 10°C (ASv26, LSv54) and 18°C (LSv21, LSv514). Although T _opt was considerably above the in situ temperatures of their habitats (−1.7°C and 2.6°C), relative growth rates were still high at 0°C, accounting for 25–41% of those at T _opt. Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2–9°C above T _opt. In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between −1.8°C and T _opt. For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0°C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields at in situ conditions.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

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.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Development and evaluation of a starter culture for the industrial production of gari

Gari starter cultures (Gastat) were developed by mixing pure single strains of the organisms that ferment cassava. They were propagated and maintained as granules on dried cocoyam slurry. The cultures were tested for fermentative and acid-producing activity. The acidity produced at 30°C varied from 0.07% to 0.85% lactic acid with maximum levels occurring after 48 h. High levels of reducing sugar were produced during the first 24 h. The amounts produced were about 50% more than those from the self-inoculated cassava. The quality of the gari produced by the starter cultures was good and well accepted. The texture was similar to that produced by natural fermentation. These results highlight the possibility of using starter cultures in the large-scale production of gari.     

Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods

A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sterigmatomyces and Trichosporon , and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic activities of 50 psychrotrophic strains were studied by use of skim milk within the temperature range of 0–42°C. All the strains showed various degrees of proteolysis. In particular, Candida lipolytica. Trichosporon pullulans and Candida scottii were active species at low temperatures. Sensory spoilage due to the proteolytic yeasts were observed in mackerel homogenates stored at 10°C. C. lipolytica -inoculated homogenates caused spoilage with ammoniacal odours after 1 week of storage. Values of total volatile basic nitrogen at 10°C were highest with C. lipolytica among 35 strains tested, followed by Tr. pullans. Trichosporon cutaneum, C. scottii, Rhodotorula glutinis and Cryptococcus luteolus. Proteolytic psychrotrophic yeasts were widely distributed in raw seafoods.     

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.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

THE INFLUENCE OF CERTAIN MANUFACTURING PROCESSES ON THE STAPHYLOCOCCUS AUREUS CONTENT OF SPRAY-DRIED MILK

SUMMARY: Dried milk was prepared from milks inoculated with pure cultures of Staph. aureus , using a small pilot-type batch vacuum evaporator and spray drier. Vacuum evaporation reduced the numbers of Staph. aureus in most experiments but one strain was capable of multiplication at evaporating temperatures up to about 43°. Above 49° the organisms declined rapidly and at 56° they were eliminated. Residual staphylococci present in the warm concentrated milk feeding the drier increased in some experiments but declined markedly in others. Staphylococci added directly to warm concentrated milk always proliferated. A small proportion of staphylococci survived spray drying, even with high hot air temperatures. Different strains varied in their susceptibility to different stages of the whole process.