THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water

Introduction of ampicillin dextrin agar (ADA) has revealed problems in details of the preparation. The final pH of the medium varied substantially between different laboratories. Measuring temperature has a pronounced effect on the pH (0·7 units lower at 50°C than at 6°C). Addition of agar during medium preparation resulted in a fall in pH of 0·5 units. If poured plates were stored in the refrigerator, the pH was reduced by 0·1–0·4 units, in particular during the first day. Recovery of Aeromonas from pure cultures and naturally polluted samples was unaffected by variation in pH between 7·1 and 8·3 but colony differentiation was optimal at a higher pH. The use of ADA at a final pH of 7·8 ± 0·2 (at 25°C) is recommended. Different types of dextrin differed in respect of solubility, fermentability and colony differentiation. Optimal results were obtained with Difco 161 and Merck 3006.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

METHODS FOR THE ISOLATION OF FAECAL STREPTOCOCCI (LANCEFIELD GROUP D) FROM BACON FACTORIES

SUMMARY: Two methods have been developed for isolating and enumerating group D faecal streptococci from localities such as bacon factories where they are heavily outnumbered by other organisms. The first depends on presumptive counts in a Lab-Lemco-peptone-glucose broth (pH 6·0) containing 0·1% of thallous acetate with confirmation by streaking on tetrazolium agar. The other method involves direct plating on tetrazolium-glucose agar (pH 6·0) containing 0·1% of thallous acetate. On the tetrazolium medium differentiation can be made between Streptococcus faecalis and its variants zymogenes and liquefaciens and the other group D organisms, Strep. faecium, Strep. durans and Strep. bovis .     

The effect of recovery conditions on the apparent heat resistance of Bacillus cereus spores

The effect of recovery media and incubation temperature on the apparent heat resistance of three ATCC strains (4342, 7004 and 9818) of Bacillus cereus spores were studied. Nutrient Agar (NA), Tryptic Soy Agar (TSA), Plate Count Agar (PCA) and Milk Agar (MA) as the media and temperatures in the range of 15–40°C were used to recover heated spores. Higher counts of heat injured spores were obtained on PCA and NA. The optimum subculture temperature was about 5°C below the optimum temperature for unheated spores. No significant differences in heat resistance were observed with the different recovery conditions except for strains 4342 and 9818 when MA was used as plating medium.
Large differences in D -values were found among the strains ( D ₁₀₀=0·28 min for 7004; D ₁₀₀=0·99 min for 4342; D ₁₀₀= 4·57 min for 9818). The 7004 strain showed a sub-population with a greater heat resistance. The z values obtained for the three strains studied under the different recovery conditions were similar (7·64°C 0·25).     

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.     

Effects of sporulation pH on the heat resistance and the sporulation of Bacillus cereus

Spores of Bacillus cereus ATCC 7004, 4342 and 9818 were obtained in nutrient agar at several pH from 5·9 to 8·3. The optimum pH for sporulation was around 7, but good production of spores was obtained in the range 6·5–8·3. With all three strains, D -values clearly dropped with sporulation pH, decreasing by about 65% per pH unit. z -Values were not significantly modified ( P > 0·05) by this factor. Mean z -values of 7·13 °C ± 0·16 for strain 7004, 7·67 °C ± 0·04 for 4342 and 8·80 °C ± 0·64 for 9818 were obtained.     

Size at hatching and incubation period of Sympterygia bonapartii (Müller & Henle, 1841) (Chondrichthyes, Rajidae) bred in captivity at the Temaiken Aquarium

Two mature Sympterygia bonapartii held in captivity laid a total of 136 egg cases, 33 of them were incubated at constant temperature (16·5° C), salinity (36) and photoperiod (12L:12D). The hatching rate was 100%. Juvenile fish hatched after 135 ± 10 days (mean ± s . d .) and the mean total length, disc width and mass at hatching were 140 mm, 86 mm and 15·1 g respectively.     

Note: Influence of different heating media on thermal resistance of Neosartorya fischeri isolated from papaya fruit

E. RAJASHEKHARA, E.R. SURESH AND S. ETHIRAJ. 1996. A heat-resistant mold identified as a strain of Neosartorya fischeri was isolated from microbiologically spoiled papaya fruits. The optimum heat activation temperature and time for the ascospores of the test mold was found to be 80°C for 15–30 min. The decimal reduction times ( D -values) at 85°, 87° and 89°C in phosphate buffer (pH 7·0) as heating medium were 35·25, 11·1 and 3·90 min respectively and hence the calculated z -value was 4·0°C. In grape and mango juices as heating media, the D _80°C and the D _85°C values were increased as the °Brix level raised from 10 to 45. In commercial fruit juices of mango, orange, pineapple and mango-pineapple blend as heating media D _85°C values were greater than those observed for phosphate buffer.     

Thermally induced phenotypic plasticity of swimming performance in European sea bass Dicentrarchus labrax juveniles

The vulnerability of embryonic and larval stages of European sea bass Dicentrarchus labrax to environmental temperature and the longer-term consequences for the early juveniles was demonstrated. This phenotypic plasticity was highlighted by subjecting D. labrax at 15·2 ± 0·3 or 20·0 ± 0·4° C (mean ± s . d .) up to metamorphosis and then at the same temperature (18·5 ± 0·7° C). After 4–6 weeks at the same temperature, the measurement of critical swimming speed at four exercise temperatures (15, 20, 25 and 28° C) showed a significantly higher swimming capacity in the fish initially reared at 15° C than for fish initially reared at 20° C. This performance was correlated with significant differences in the phenotype of red muscle. Thermally induced phenotypic plasticity was clearly demonstrated as an important mechanism controlling swimming performance in early juveniles of D. labrax .     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.     

Effect of the ethanol content of beer on the heat resistance of a spoilage Lactobacillus

D -values of a heterofermentative beer spoilage lactobacillus were measured at 55°C, 60°C and 65°C in beers containing <0·05% to 4·4% v/v ethanol. Z -values for the different beers varied between 9·17 and 12·13°C. At each temperature an increase in ethanol reduced the measured D -value. The maximum, 5·01 min was observed in alcohol-free beer (<0·05%) at 55°C and the minimum, 0·31 min, at 60°C and 65°C in beer containing 4·4% ethanol. D -values could be increased by prior growth in the presence of ethanol. They could be reduced by adding ethanol to alcohol-free beer or by increasing its hop extract content. The implications for the pasteurization of low-alcohol beers are discussed.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Heat resistance of different serovars of Listeria monocytogenes

Twelve Listeria monocytogenes strains representing seven serovars were heat-treated in physiological saline by a glass capillary tube method. Five strains were treated at 58°, 60° and 62°C, three at 60°, 62° and 64°C and four at 60°C. Heat-treated bacteria were recovered on blood agar in two ways: (1) incubation at 37°C for 7 d; and (2) preincubation at 4°C for 5 d, followed by incubation at 37°C for 7 d. D and z values were determined. Better average recovery and higher D values were obtained when the preincubation procedure was used. The final evaluations of the heat resistance properties of the strains were therefore based on values for preincubated samples. D values recorded at 58°, 60°, 62° and 64°C for preincubated samples were 1.7–3.4, 0.72–3.1, 0.30–1.3 and 0.33–0.68 min, respectively. z values determined were 5.2–6.9°C. D values were compared statistically. Significant differences in heat resistance were noted both between serovars and between strains belonging to the same serovar.     

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.     

The chilling sensitivity of root ammonium influx in a cultivated and wild tomato

A chilling episode of a few hours damaged root ammonium absorption in a cultivated tomato ( Lycopersicon esculentum cv. T-5), but not in a wild congener from high altitudes ( Lycopersicon hirsutum LA1778). In the cultivar, ammonium influx was strongly temperature dependent and showed the residual effects of chilling, whereas ammonium efflux was nearly temperature invariant and showed no persistent effects. A 2 h exposure to 5 °C significantly depressed subsequent ammonium absorption at 20 °C, and about 12 h at 20 °C was required for recovery. For both the cultivated and wild species, rerooted cuttings were slightly less sensitive to chilling than seedlings. The relative inhibition (mean ± SE) of ammonium absorption before and after chilling was 58·4 ± 2·5% for the cultivated species and 29·0 ± 9·1% for the wild species. The F₁ hybrid between the species showed a relative inhibition of 52·4 ± 3·6%, suggesting that chilling sensitivity may be dominant. In a backcross of the hybrid to L. esculentum , the phenotypic distribution of the relative inhibition of ammonium absorption indicated that this trait is segregating.     

The critical thermal limits for juvenile Arctic charr Salvelinus alpinus

The chief objective was to determine the critical thermal limits for alevins, fry and parr of Arctic charr, Salvelinus alpinus , (L.) from four races living in Windermere (northwest England). The experimental fish were reared in a hatchery but were the progeny of wild parents. As comparisons between tethal temperatures at four acclimation temperatures (5, 10, 15, 20° C) revealed few significant racial differences, the data were pooled to estimate the lethal values for survival over 7 days (incipient lethal temperature) and over only 10 min (ultimate lethal temperature) for each life stage. Upper lethal values increased with acclimation temperatures for alevins but this effect was negligible for fry and parr, Alevins were generally less tolerant than fry and parr at lower, but not higher, acclimation temperatures; e.g. after acclimation at 5° C, mean upper ultimate values were 23·3, 25·1 and 25·7° C and mean upper incipient values were 18·7, 21·5 and 21·5° C for alevins, fry and parr respectively; after acclimation at 20° C, mean upper ultimate and incipient values were 26·2, 26·1 and 26·6° C and 20·8, 20·8 and 21·6° C for alevins, fry and parr respectively. The area of the temperature tolerance polygon (expressed as ° C²) for juvenile Arctic charr is amongst the lowest recorded for salmonids; being 409, 439 and 461° C² for alevins, fry and parr respectively. These low values are due to lower upper tolerance limits, not high lower tolerance limits; the latter being close to 0° C (<1°C for parr and fry, <0·3° C for alevins) at all acclimation temperatures. Arctic charr are therefore amongst the least resistant of salmonids to high temperatures but probably the most resistant to low temperatures.     

The influence of thermoregulation on behavioural recovery from exercise in a lizard

1. Past work on the thermal preferences of Dipsosaurus dorsalis (Biard & Giard) has indicated that intense, exhaustive exercise causes these lizards to select a body temperature (33·5 °C) which is cooler than their preferred activity temperature of 40°C during the first 1–2 h of exercise recovery.
2. In order to test the hypothesis that the thermal regime selected by exhausted D. dorsalis is beneficial to the process of exercise recovery, lizards were forced to undergo both exhaustive and sprinting exercise at their preferred body temperature of 40°C. The peak speeds attained and the total distances travelled by these animals during these two different exercise protocols were measured and the animals were then forced to undergo a second bout of either sprinting or exhaustive exercise, following a 30–330 min recovery at either 20°C, 40°C or under a variable thermal regime which duplicated that selected by animals following exercise.
3. Animals recovering at a constant 40°C regained their ability to repeat exhaustive activity in less than 85 min, while animals recovering under the other two thermal regimes required between 85 and 100 min of recovery to be able to repeat this activity. Animals recovering at both 40°C and under the variable thermal regime regained their ability to repeat sprint behaviour within 60 min of recovery, while animals recovering at 20°C required more than 100 min of recovery to be able to repeat sprint behaviour.
4. These results formed the basis of the conclusion that the post-exercise behaviour selected by D. dorsalis retards the rate at which the animals recover their ability to repeat exhaustive exercise when compared with recovery at a constant 40°C but does not retard their ability to repeat sprint exercise.