The effect of sodium chloride and temperature on the rate and extent of growth of Clostridium botulinum type A in pasteurized pork slurry

A selective medium was used to enumerate Clostridium botulinum growing in the presence of natural spoilage organisms in a model cured pork slurry. The growth responses of a mixed spore inoculum of six strains of Cl. botulinum type A were studied at 15°, 20° and 27°C with 1˙5, 2˙5, 3˙5 or 4˙5% (w/v) salt added (a_w range 0961–0990). Gompertz and logistic curves, which have a sigmoid shape, were fitted to the data and lag times, growth rates, generation times and time to maximum growth rates were derived. Variation in germination rates of the spores occasionally gave a falsely extended lag time resulting in an exceptionally high estimate for growth rate. Products containing 4˙5% (w/v) NaCl would be capable of supporting growth of proteolytic strains of Cl. botulinum , even at 15°C, although the lag period would be extended. In products where absence of Cl. botulinum cannot be assured additional preservative measures are essential. The information obtained provides a framework to investigate the effects of a wider range of additives or variables on the growth responses of Cl. botulinum .     

The effect of temperature on the growth of non-proteolytic type B Clostridium botulinum

The effect of temperature between 4 and 35°C on the growth rate of a non-proteolytic type B strain of Clostridium botulinum was examined. Growth was in culture media at pH 6.7 and was measured by viable counts using the Most Probable Number (MPN) method. Doubling times were derived from the curve fitting model of Baranyi et al. (1992) and ranged from 42.3 h at 3.9°C to 22 min at 35°C.     

Inhibitory effect of combinations of heat treatment, pH, and sodium chloride on a growth from spores of nonproteolytic Clostridium botulinum at refrigeration temperature.

Nonproteolytic strains of Clostridium botulinum will grow at refrigeration temperatures and thus pose a potential hazard in minimally processed foods. Spores of types B, E, and F strains were used to inoculate an anaerobic meat medium. The effects of various combinations of pH, NaCl concentration, addition of lysozyme, heat treatment (85 to 95 degrees C), and incubation temperature (5 to 16 degrees C) on time until growth were determined. No growth occurred after spores were heated at 95 degrees C, but lysozyme improved recovery from spores heated at 85 and 90 degrees C.     

The effect of temperature on the germination of single spores of Clostridium botulinum 62A

Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability). The experiment was repeated at five different temperatures between 20°C and 37°C to look at the effect of temperature on the biovariability of the spore germination. Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature. The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g. permeabilities) or molecules (e.g. lytic enzymes) in the spore population. The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influenced by different environmental factors such as temperature and other factors that influence germination.     

The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum

It has been reported that non-proteolytic strains of Clostridium botulinum will grow at 3.3 degrees C, and they are therefore of concern in relation to certain chilled foods. The effects of combinations of inhibitory factors may be used to reduce the risk of growth of these bacteria in foods. The combined effect of pH values between 4.8 and 7.0, temperatures between 6 degrees and 30 degrees C, and sorbic acid concentrations up to 2270 mg/l on the probability of growth from a single spore of non-proteolytic, type B strains in a culture medium has been determined. A mathematical model has been developed that enables the effect of varying combinations of these factors on the probability of growth of non-proteolytic, type B Cl. botulinum to be predicted.     

The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum

L und , B.M., G raham , A.F., G eorge , S.M. & B rown , D. 1990. The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum. Journal of Applied Bacteriology 69 , 481–492.
It has been reported that non-proteolytic strains of Clostridium botulinum will grow at 3.3°C, and they are therefore of concern in relation to certain chilled foods. The effects of combinations of inhibitory factors may be used to reduce the risk of growth of these bacteria in foods. The combined effect of pH values between 4.8 and 7.0, temperatures between 6° and 30°C, and sorbic acid concentrations up to 2270 mg/1 on the probability of growth from a single spore of non-proteolytic, type B strains in a culture medium has been determined. A mathematical model has been developed that enables the effect of varying combinations of these factors on the probability of growth of non-proteolytic, type B Cl. botulinum to be predicted.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH ≥ 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12° and 16°C and pH values between 5·2 and 5·5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5·5 and incubated at 16°C, corresponding to a probability of growth from a single spore within 14 d of 1·6 × 10^-5. The probability of growth in 28 d in the remaining conditions was <9 × 10^-6. After transfer of inoculated media from 12° to 30°C growth occurred at pH 5·2–5·5 within 19 d. After transfer of inoculated media from 12° to 20°C growth occurred at pH 5·5 and 5·4 but not at pH 5·3 or 5·2 in 40 d. Growth at pH 5·2–5·5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH greater than or equal to 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12 degrees and 16 degrees C and pH values between 5.2 and 5.5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5.5 and incubated at 16 degrees C, corresponding to a probability of growth from a single spore within 14 d of 1.6 x 10(-5). The probability of growth in 28 d in the remaining conditions was less than 9 x 10(-6). After transfer of inoculated media from 12 degrees to 30 degrees C growth occurred at pH 5.2-5.5 within 19 d. After transfer of inoculated media from 12 degrees to 20 degrees C growth occurred at pH 5.5 and 5.4 but not at pH 5.3 or 5.2 in 40 d. Growth at pH 5.2-5.5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

Clostridium botulinum type A growth and toxin production in media and process cheese spread

We found that Clostridium botulinum type A grew well and produced toxin in media with a water activity (a(w)) of 0.972 or 0.965 and a pH of 5.7, but no growth or toxin production was observed at or below an a(w) of 0.949 during incubation at 30 degrees C for 52 to 59 days. a(w) and pH values of media were adjusted to those of cheese spreads commercially produced. Solutes used to adjust a(w) included combinations of NaCl, cheese whey powder, emulsifying salt, sodium tripolyphosphate, and glycerol. In agreement with results obtained for media, toxin was produced in samples of cheese spread (a(w), 0.970; pH, 5.7) at 30 to 70 days of incubation at 30 degrees C.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5·2 with HCl and incubated at 30°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10–20 weeks. Citric acid concentrations of 10–50 mmol/l at pH 5·2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10⁶. The citric acid also reduced the concentration of free Ca²⁺ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5·2 could be prevented by the addition of Ca²⁺ or Mg²⁺ and greatly reduced by Fe²⁺ and Mn²⁺. The addition of Ca²⁺, but not of the remaining divalent metal ions, restored the concentration of free Ca²⁺ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca²⁺. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5·2. The effect of citric acid and Ca²⁺ at pH 5·2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

The effect of incubation time and temperature on growth of Escherichia coli on gradient plates containing sodium chloride and sodium nitrite

Two-dimensional gradient plates are a convenient way of screening antimicrobial effects of preservative factors acting in combination across a broad range of physical and chemical conditions. We report the effects of sodium chloride, sodium nitrite and incubation temperature on the growth of Escherichia coli by staining, laser densitometry and computer graphics. Staining not only more easily distinguished the growth area but also gave an indication of the viability of cells present. 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride was the more useful of the two stains used. Inhibitory concentrations of sodium chloride decreased with reduced incubation temperature. The response of E. coli to combinations of salt and nitrite on gradient plates was very similar to its response in liquid medium.     

The effect of incubation temperature, sodium chloride and ascorbic acid on the growth kinetics of Aeromonas hydrophila

The effects of temperature, sodium chloride and ascorbic acid on the aerobic growth kinetics of a clinical strain of Aeromonas hydrophila were evaluated. At 5°C, ascorbic acid (1 mmol l^-1) and sodium chloride (3% w/v) inhibited the growth of the organism. At 10°C, ascorbic acid depressed only the maximum population densities (A) by approximately 2 log cycles, but not maximum specific growth rate (μ_m) or the lag time (Λ). On the contrary, NaCl caused A to increase, with the effect being greatest when the NaCl content was 1.5%. Temperature increase from 10 to 15°C resulted in an approximate doubling of μ_m and unexpectedly an apparent increase in Λ However, this apparent increase resulted from the particular manner in which the lag phase was mathematically calculated.     

Behaviour of proteolytic Clostridium botulinum types A and B near the lower temperature limits of growth

Summary The behaviour of spores of Clostridium botulinum type A and proteolytic C. botulinum type B has been studied in cooked meat medium at 10°C, 12°C, 15°C, and 20°C, using mixed cultures (9 groups of in total 41 strains) and pure cultures (41 strains).At 10°C a decrease of 1–1.5 log cycles for type B and of 2–4 log cycles for type A Clostridia was observed. Neither growth nor toxin formation could be demonstrated.At 12°C spores of some strains developed and formed toxin with 3–4 weeks, whereas other strains did not develop within 7 weeks.At 15°C growth and toxin formation could be observed within 1 week, whereas at 20°C toxin was formed mostly within 2 or 3 days. Incubation at 10°C prior to incubation at 20°C seemed to have some effect on the lag time.