Occurrence and Thermoresistance of Spores of Psychrophilic and Psychrotrophic Aerobic Sporeformers in Soil and Foods

Spores of psychrotrophic (able to grow at 5°C) aerobic sporeformers occurred in soil in high numbers (2 × 10³-5 × 10⁶/g), whereas psychrophilic (able to grow at 0°C) spores were present at significantly lower levels (500–10⁵/g). Psychrotrophic spores were absent in herbs and spices: in pasteurized meals prepared industrially their numbers varied from <10 to 1000/g. For spores harvested from Trypticase Soy Agar (TSA), the heat resistance of the cold-tolerant sporeformers was low with D _90°C-values from 1–11 min. The recovery of heated psychrophilic spores on this medium at 5°C was equal to their recovery at 20°C. However, the recovery of heated psychrotrophic spores was lower at 5°C than at 20°C, whereas unheated spores gave the same counts at both temperatures. The heat resistance of naturally occurring spores of cold-tolerant sporeformers washed from soil was comparable with the resistance of spores formed on TSA.     

The Effect of Incubation Temperature on the Recovery of Spores of Bacillus subtilis 8057

S ummary . The recovery of Bacillus subtilis spores was studied after different heat treatments at 95° and incubation at different temperatures in roll tubes in a gradient temperature incubator. Plate count agar and brain–heart infusion agar were used in the roll tubes. Unheated spores showed similar recoveries at 16–48° whereas heated spores had an optimum recovery temperature of c. 30.9. The rate of germination of untreated spores was greatest at c. 41° and ceased at 50°. Heated spores germinated at 52°5°, suggesting that recovery of heat-treated spores is not limited by their ability to germinate. Outgrowth of spores at different incubation temperatures was similar for germinated and ungerminated spores. Accordingly it is outgrowth rather than germination which is sensitive to temperature.     

Amino Acid Enhancement of Recovery in Dry-heat Damaged Spores of Bacillus subtilis

Spores of Bacillus subtilis MD2 and var. niger were dry-heat damaged at 150°, 160° and 170°C and recovered on media of increasing complexity. The greater the heat dose the more marked was the effect of amino acid supplements on recovery. For strain MD2 maximum germination and outgrowth of unheated spores could be obtained on a minimal salts + glucose medium with alanine, aspartic acid, glycine and methionine; the latter three amino acids served to enhance growth, not germination. The recovery of heat-damaged spores was significantly increased by adding valine plus isoleucine or arginine or glutamine. The increase was probably due to the use of valine and isoleucine as substrates of NAD-linked dehydrogenases to generate reducing power and serve as NH₃-donor, initiating germination in spores which were unable to germinate as a result of inactivation of alanine dehydrogenase. Valine or isoleucine added singly suppressed recovery by feedback inhibition of the pathways to both these amino acids during outgrowth.     

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).     

Repair and calcium dipicolinate release of bioindicators in propylene glycol and propylene glycol—water mixtures

B. PHILIPP AND H. SUCKER. 1992. The heat sterilization of spores of Bacillus stearothermophilus ATCC 7953 in propylene glycol (PG) and PG—water mixtures was investigated. Unusual non-logarithmic survival curves with a marked long shoulder, designated as the lag-time, were observed at the maximum of resistance. When the number of colony-forming units were determined after intervals of incubation, the growth curves of spores previously treated at 121°C in PG or PG-4% water varied considerably. Spores receiving a heat treatment that was longer than the lag-time showed no growth. When the heat treatment was shorter than the lag-time, spores showed a delayed growth phase. When spores received heat treatment that was long but which fell within the lag-time, there was a greater decrease in spore counts before the exponential growth phase began. In PG and PG with low water concentrations, a suppressed release of the specific spore substance calcium dipicolinate was observed during sterilization at 121°C of B. stearothermophilus ATCC 7953 and B. subtilis var. niger ATCC 9372. Calcium dipicolinate release, however, was observed in water.     

The effect of recovery medium on the estimated heat-inactivation of spores of non-proteolytic Clostridium botulinum

Heating spores of non-proteolytic strains of Clostridium botulinum at 85°C, followed by enumeration of survivors on a highly nutrient medium indicated a 5 decimal kill in less than 2 min. The inclusion of lysozyme or egg yolk emulsion in the recovery medium substantially increased apparent spore heat-resistance, with as little as 0.1 μg lysozyme/ml sufficient to give an increase in the number of survivors. After heating at 85°C for 2 min between 0.1% and 1% of the spores of 11 strains (5 type B, 4 type E, 2 type F) formed colonies on medium containing 10 μg lysozyme/ml. Enumeration of survivors on a medium containing lysozyme showed that heating at 85°C for 5 min resulted in an estimated 2.6 decimal kill of spores of strain 17B (type B). These findings are important in the assessment of heat-treatments required to ensure the safety with respect to non-proteolytic Clostridium botulinum of processed (pasteurized) refrigerated foods for extended storage such as sous-vide foods.     

Survival and growth of Bacillus cereus in bread

Bread doughs were artificially inoculated with spores of six Bacillus cereus strains at different inoculum levels and counts of survivors in bread determined during storage at 27·5°C. No B. cereus were isolated from the centre crumb of 400 g loaves when the dough contained less than 10⁴ spores/g whereas with 800 g loaves survival occurred with doughs containing 5·0 times 10³ spores/g. With all strains there was a period of at least 24 h before multiplication took place in the bread. The inclusion in dough of 0·2% of calcium propionate, based on flour, effectively delayed germination and subsequent multiplication of B. cereus spores. It is concluded that the risk of food poisoning due to the presence of B. cereus in bread is minimal.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Biological indicators for low temperature steam and formaldehyde sterilization: effect of variations in recovery conditions on the response of spores of Bacillus stearothermophilus NCIMB 8224 to low temperature steam and formaldehyde

Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml^-1 formaldehyde at temperatures between 63° and 83°C The data indicate that LTSF-exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.     

The vitamin requirements of Staphylococcus cohnii

In heat-resistance studies with spores of Clostridium sporogenes BC-2, an improved recovery medium was needed for severely heat-damaged spores as the used previously--Wynne medium in Miller-Prickett tubes--did not allow accurate counts of spores because of gas formation and disruption of agar. Initial test with pour plates of Viande-Leyure medium containing egg-yolk gave much increased counts for spores previously heated for 50 min at 112 degrees C; this increase was attributed to the presence of egg-yolk. Addition of egg-yolk to Reinforced Clostridial Agar, All-Culture Medium and Trypticase Soy Agar showed that Trypticase Soy Agar with egg-yolk was the best recovery medium. For the final formulation, the value of supplementation with cysteine-HCl and methylene blue was also shown. The resultant Egg-yolk Trypticase Soy Agar is conveniently prepared from BBL Trypticase Soy Agar (40 g) with the addition of 0.4 g/l cysteine-HCl, 4 mg/l of methylene blue and 2% Oxoid egg-yolk emulsion aseptically to the melted basal medium. For optimal spore counts, pour plates are incubated anaerobically for 5--7 d at 30 degrees C.     

Growth of moulds inoculated into commercial mineral water

The growth of mould spores of Penicillium sp. and Cladosporium sp. inoculated in a commercial mineral water product was studied. The strains had been isolated as fungal foreign bodies in commercial mineral waters. In product A, which was not originally sterilized and was contaminated with psychrophilic bacteria, the inoculated mould spores of the strains did not grow; no increases in viable colony counts or beta-glucans concentration in the samples were observed during storage. In a sterilized product A, inoculated spores of the strains grew into visible foreign bodies. The viable colony counts and the beta-glucans concentration in the samples increased during storage. These results showed that in a sterilized mineral water product, mould spores could grow into visible foreign bodies.     

Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef

Pieces of fresh beef were inoculated with three strains of Campylobacter jejuni . The meat was then allocated to three treatments: (a) vacuum packaged, (b) packaged in an atmosphere of 20% CO₂+ 80% N₂, and (c) packaged into sterile Petri dishes in anaerobic cultivation boxes, which were filled with a gas mixture of 5% O₂+ 10% CO₂+ 85% N₂. The packaging material in the first two treatments was PA 80/PE 100–PE 100/PA 80/PE 100. The survival of Campylobacter cells was followed at 37°C, 20°C and 4°C for 48 h, 4 days and 25 days, respectively. At 37°C the counts of two Campylobacter strains increased in each package treatment for 48 h. At 20°C and at 4°C the counts of the same two strains decreased by 1 to 2 log units and 0.5 to 1 log unit, respectively, during storage. The survival of the two strains was about the same in all package treatments. The third strain was the most sensitive of the strains studied. At 37°C its numbers increased only in the optimal gas atmosphere; at 20°C the strain was not detectable after 24 to 48 h storage and at 4°C after 4 days storage. The aerobic plate counts were determined for all samples at the same time as Campylobacter counts. The high indigenous bacterial numbers of the meat samples did not appear to have a great effect on the survival or growth of campylobacters.     

Identification of a Pea Component Stimulatory for Heat-Stressed Putrefactive Anaerobe 59-123 Spores

Pea extract contains a factor which improves recovery counts of heat-stressed putrefactive anaerobe spores in a complex medium up to threefold. The factor is heat-stable and nondialyzable. Most of the active principle is found in the precipitate which forms during storage of pea extract at 4 C. The precipitate disperses upon heating, is high in starch content, and retains activity after extraction with organic solvents and water. Treatment of pea extract with alpha-amylase results in complete destruction of the active principle. These observations indicate that starch is the factor in pea extract responsible for increased recovery counts of heat-stressed putrefactive anaerobe spores.     

Kinetic resolution of different recovery phases of photoinhibited photosystem II in cold-acclimated and non-acclimated spinach leaves

Leaf discs from spinach were exposed to a photon flux density of 1250 μmol m⁻²s⁻¹ at 5°C for 2 or 3 h in ambient air. Photoinhibition of photosystem II (PS II) was measured by means of chlorophyll fluorescence. Recovery of photosystem II was followed at 6°C and 20°C in low light or darkness for periods up to 12 h.
The experimental setup allowed kinetic resolution of different phases of recovery. The experiments revealed a temperature dependent dark recovery phase and two distinct light- and temperature dependent phases: (1) A relatively fast, light dependent recovery phase occurred in parallel with partial recovery of basic fluorescence at 6°C and 20°C. A population of PS II centers with very slow fluorescence induction kinetics, which had accumulated during photoinhibition treatment, disappeared during this phase. This fast recovery phase is proposed to represent reactivation of photoinhibited PS II, without dissassembly or incorporation of new D₁-protein. (2) A relatively slow light-dependent recovery phase took place at 20°C, but not at 6°C. In the presence of the chloroplast translation inhibitor streptomycin, part of the 2nd phase was inhibited. This phase is proposed to involve assembly of new Photosystem II centers, which is partly dependent on de novo synthesis of D₁-reaction center protein, but presumably is also using a preexisting pool of D₁-protein. Cold acclimation of the leaves resulted in a decreased sensitivity for photoinhibition of photosystem II. Recovery of photoinhibited photosystem II at 6°C of the cold-acclimated leaves was faster than in non-acclimated leaves, but this effect can be ascribed to diminished photoinhibitory damage.     

Relationship between Heat Activation and Percentage Colony Formation for Bacillus stearothermophilus Spores: Effects of Storage and pH of the Recovery Medium

Colony counts of unheated spores were higher in a medium at pH 5·9 than in media of higher pH. The pattern was reversed as the spores were heated. Slide germination studies showed that about 8% or less of unheated spores germinated in slide culture. Optimal heat activation resulted in about 50% germination. The colony counts of heat activated spores dropped significantly upon storage for 9 months, but those of unheated spores did not.     

Effects of Long‐Term Storage Methods on the Infectivity of Urediospores of Phakopsora pachyrhizi

A method for long‐term storage of spores of Phakopsora pachyrhizi was optimized. Three methods with different procedures for spore harvest and four different reactivation methods (varying in hydration or using heat shock) were analysed for the suitability for long‐term storage at ?80°C. All conservation methods as well as all reactivation methods lead to the infection of soybean leaves after 1 year of storage. Regarding efficiency and labour input, the most recommended method is to tap off spores from infected and sporulating leaves with subsequent dehydration before storage at ?80°C. Because hydration or heat shock steps did not provide any advantages, spores can be suspended in Tween water directly after storage and used as inoculum.     

Prediction of the shelf-life of pasteurized milk at different storage temperatures

Initial psychrotroph counts determined by a Most Probable Number technique were correlated with shelf-lives of pasteurized milk determined at a number of storage temperatures. The initial psychrotroph count was also correlated with a bacterial count carried out on milk agar containing crystal violet penicillin and nisin after previous incubation of the milk at 15°C for 25 h.
Pre-incubation counts carried out at a variety of temperatures and on a variety of media were examined for their relation to shelf-life. Shelf-lives at four pre-set temperatures (2, 6, 10 and 14°C) could best be predicted by pre-incubation of pasteurized milk at 15°C before inoculation on milk agar.
An equation which allows prediction of shelf-life of pasteurized milk at any storage temperature is described.     

Influence of acidification and type of acidulant of the recovery medium on Bacillus stearothermophilus spore counts

Unheated and heated (1 min at 121°C) Bacillus stearothermophilus spores were grown in non-acidified (pH 7) and acidified recovery medium at different pH levels (6.7, 6.5, 6.2 and 5.9) using citric acid or glucono-δ-lactone (GDL) as acidulants. It was observed that the lower the pH, the lower the proportion of micro-organisms which recovered. The level of recovery was not the same for both acidulants, meaning that at the same pH level citric acid showed a greater inhibitory effect that glucono-δ-lactone. Heat treatment sensitized spores to pH effect. A linear relationship of the behaviour of spores with each acidulant is proposed.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.