Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Environmental Stresses on Spore Populations of Bacillus stearothermophilus

Heat-shocking spores at 110 C in 20% sucrose solutions decreased the percentage of the rough variant in a mixed population (rough and smooth variants) of strain M. Heat-shocking spores of the rough variant of strain NCA 1518 in 20% sucrose produced a decline in the number which germinated, whereas the smooth variant of strain NCA 1518 increased in the number which germinated. By the use of phase microscopy and plate counts, from the same incubated spore suspension in distilled water, heat-induced dormancy was demonstrated at 52 C. Dormancy also occurred in 20% sucrose solutions when held at room temperatures. Heat-shocking spores of strain M in 20% sucrose solutions and plating immediately after 24- and 48-hr holding periods at 25 C produced a decline in the total population with the percentage of rough variant increasing with time. A second heat shock produced only an increase in the rough variant.     

Effect of Heat on Spores of Rough and Smooth Variants of Bacillus stearothermophilus

Spores of variants of Bacillus stearothermophilus were subjected to activating and lethal temperatures. Spore suspensions which were incubated longer contained a higher percentage of spores of the rough variant. The effect of sublethal heat on spore suspensions containing mixed variants (rough and smooth) was difficult to measure at sublethal temperatures (110 C), since the rough variant was not as heat-resistant. While the rough variant was activated in a shorter time, the smooth variant was not activated; when the smooth variant was activated, the rough was killed. A higher percentage of the smooth variant was forced into dormancy after being held at 50 C for 30 hr than the rough variant. When mixed populations were subjected to a lethal temperature (120 C), the curves only reflected the smooth variant. Since the curves which represented the smooth variant or mixtures containing the smooth variant were not linear, this was thought to be due to activation overbalancing the lethal effect. This research emphasized the importance of variants in explaining differences in spore resistance among spore suspensions of the same strain.     

Response of Micromonospora echinospora (NCIMB 12744) spores to heat treatment with evidence of a heat activation phenomenon

The effects of heat treatment on spores of the actinomycete Micromonospora echinospora were investigated. The percentage of culturable spores in untreated spore stocks was found to be approximately 20%. A 60 degrees C treatment of spores in phosphate buffer for 10 min led to an approximately five-fold increase in the number of culturable units. This indicated that a large proportion of the spores were constitutively dormant. Within 10 min and in the absence of an external energy-yielding substrate, the heat treatment was found to stimulate spore respiration suggesting that endogenous storage compounds were being utilized. Heating spores at 70 degrees C shortened the time period required for activation; holding times greater than 10 min, however, resulted in a reduction of culturable cells. Classic thermal death characteristics were seen at temperatures of 80 degrees C and above with D-values of 21.43, 2.67, 0.45 and 0.09 min being recorded at 70, 80, 90 and 100 degrees C, respectively. Spores of this organism, while being weakly heat resistant in comparison with bacterial endospores, are significantly more resistant than vegetative cells.     

Use of Calcium Dipicolinate for Enumeration of Total Viable Endospore Populations Without Heat Activation

A method, based on germination with 50 mm CaCl₂ and 40 mm sodium dipicolinate (Na₂DPA), was developed for the determination of total viable counts of bacterial spores requiring heat activation. Incorporation of these germinating agents into Tryptone Glucose Extract Agar permitted plate-count enumeration of essentially 100% of Bacillus subtilis strain 5230 spores without a heat treatment. Other spore suspensions were surveyed for their response to CaCl₂ and Na₂DPA, and for the subsequent removal of the heat-activation requirement for enumeration of maximal populations.     

Effects of Temperature on Activation, Germination, and Outgrowth of Bacillus megaterium Spores

The effects of temperature on the activation, glucose-induced germination, and outgrowth of Bacillus megaterium QM B1551 spores were investigated. There was no evidence for discontinuities in the response of spores to temperature in these processes reflecting reported thermal anomalies in the physical structure of water. Increasing the temperature of heat activation (aqueous suspensions, 5 min) increased the germinability of spores. Activation, as measured by extent of germination, was optimal after heating at 62 to 78 C, and the rate of spore germination was maximal after heat activation at 64 to 68 C. Increasing the temperature of activation above 68 C depressed the germination rate and increased the time lag before this rate was reached. Germination occurred over a wide range of temperatures, but was optimal between 28 and 38 C. The highest rate of germination was at 38 C; at lower incubation temperatures, the maximum attained rate was lower and the lag in attaining this rate was extended. Outgrowth (postgerminative development through the first cell division) of the germinated spores in Brain Heart Infusion (BHI) occurred in at least two phases-a temperature-dependent lag phase followed by a relatively temperature-independent phase of maximum outgrowth rate, during which increase in optical density was a linear function of time. Outgrowth time (time required for doubling of the initial optical density), essentially dependent on the time for completion of the lag phase, was shortest at temperatures between 34 and 40 C. The temperature-dependent lag phase was completed in a rich medium (e.g., BHI) but not in the glucose germination medium, suggesting that the endogenous reserves of the germinated spore were inadequate to support the metabolic synthetic events occurring during this period.     

The Effects of Heat Activation on Bacillus Spore Germination,with Nutrients or under High Pressure,with or without Various Germination Proteins

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores'' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75°C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the l-asparagine–glucose–fructose–K⁺ mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores'' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores'' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.     

Heat activation of Bacillus spores by the use of microwave irradiation

A study was conducted in which microwave irradiation and conventional waterbath treatment were compared as to their efficiency for heat-activating Bacillus spores. Spore suspensions were prepared from B. brevis, B. cereus, B. licheniformis, a lysogenic strain of B. megaterium (NRRL-B-3695), two strains of B. stearothermophilus, and B.Subtilis. Suspensions were either irradiated for 30 sec in a microwave oven, or conventionally heat-treated in the waterbath for 60 min at 60 degrees C, the serially diluted and plated onto nutrient agar. Colonies of each species from each treatment were isolated, and cultures were inoculated into several biochemical media. Spore suspensions heat-activated by microwave irradiation resulted in plate counts that were from 3% to 24% greater than from suspension heat-activated by conventional mean (60 degrees C for 60 min). There were no observed alterations in biochemical activities in any of the representative colonies from either of the two treatments. No induction of bacteriophage from lysogenic B. megaterium NRRL-B-3695 was observed in colonies from either of the two treatments. Microwave irradiation appears to be more efficient, less time-consuming, and at least as effective as heat activation by conventional waterbath treatment for Bacillus spores.     

Novel strains of Moorella thermoacetica form unusually heat-resistant spores

Two strains of Moorella thermoacetica, JW/B-2 and JW/DB-4, isolated as contaminants from autoclaved media for chemolithoautotrophic growth containing 0.1% (wt/vol) yeast extract, formed unusually heat-resistant spores. Spores of the two strains required heat activation at 100 degrees C of more than 2 min and up to 90 min for maximal percentage of germination. Kinetic analysis indicated the presence of two distinct subpopulations of heat-resistant spores. The decimal reduction time (D10-time=time of exposure to reduce viable spore counts by 90%) at 121 degrees C was determined for each strain using spores obtained under different conditions. For strains JW/DB-2 and JW/ DB-4, respectively, spores obtained at approximately 25 degrees C from cells grown chemolithoautotrophically had D10-times of 43 min and 23 min; spores obtained at 60 degrees C from cells grown chemoorganoheterotrophically had D10-times of 44 min and 38 min; spores obtained at 60 degrees C from cells grown chemolithoautotrophically had D10-times of 83 min and 111 min. The thickness of the cortex varied between 0.10 and 0.29 microm and the radius of the cytoplasm from 0.14 to 0.46 microm. These spores are amongst the most heat-resistant noted to date. Electron microscopy revealed structures within the exosporia of spores prior to full maturity that were assumed to be layers of the outer spore coat.     

Heat resistance of Paecilomyces variotii in sauce and juice

It has been demonstrated that some anamorphic fungi ( Paecilomyces variotii, Fusarium sp) could cause spoilage of food products after pasteurisation. Four food-borne and one clinical isolate of P. variotii were cultivated on one solid medium and three liquid media. Their survival after heating at 80–100˚C for 0.25–15 min in sterile distilled water and curry sauce or fruit juice was investigated. Heat resistance was determined by the thermal death method in a thermostatically-controlled oil bath. The most resistant spores of P. variotii from curry sauce cultivated on malt extract agar survived 100˚C for 0.5 min in sauce; cultivated in curry sauce survived 100˚C for 15 min in water and cultivated in malt broth survived 100˚C for 5 min in water and sauce. The most resistant spores of P. variotii from juice cultivated on malt extract agar were able to survive 100˚C for 15 min in water; cultivated in juice survived 100˚C for 0.5 min in juice and suspensions from cultivation in malt broth survived 100˚C for 1.5 min in juice. Spores of the clinical strain of P. variotiifrom malt extract agar survived 95˚C for 0.33 min in water, and orange juice cultures survived 96˚C for 10 min in orange juice. It was thus found that P. variotii strains cultivated in food were better adapted to heat stress, suggesting that fungal biomass suspensions were able to survive the higher temperatures for longer time intervals than spore suspensions. Journal of Industrial Microbiology & Biotechnology (2000) 24, 227–230. Received 02 June 1999/ Accepted in revised form 05 December 1999     

Inactivation of Geobacillus stearothermophilus Spores by High-Pressure Carbon Dioxide Treatment

High-pressure CO₂ treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO₂ treatment at 30 MPa and 35°C, to high-hydrostatic-pressure treatment at 200 MPa and 65°C, or to heat treatment at 0.1 MPa and 85°C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO₂ treatment. We also investigated the influence of temperature on CO₂ inactivation of G. stearothermophilus. Treatment with CO₂ and 30 MPa of pressure at 95°C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95°C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95°C for 120 min had little effect. The activation energy required for CO₂ treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO₂ treatment of G. stearothermophilus spores, CO₂ treatment at 95°C was more effective than treatment at 95°C alone.     

Activation ofDictyostelium discoideum spores with guanidine and methyl derivatives of urea

A majority ofDictyostelium discoideum spores were activated with guanidine hydrochloride and tetramethylurea treatments. Dimethylurea could be utilized over a wide range of concentrations to activate spores. The minimal concentration was 2 M dimethylurea employed for 45–60 min, and the maximal concentration was 5 M dimethylurea employed for 20–30 min. Moderate overstimulation with dimethylurea resulted in an increase in the postactivation lag time, while severe overstimulation caused lysis and death of the spores. Partial spore deplasmolysis was a requirement for activation with dimethylurea at 23,5°C; deplasmolysis and activation did not occur at 0°C. The time required to produce an LD₅₀ was twice the time required for optimal activation when spores were treated with high concentrations of urea derivatives. A correlation was found for the hydrophobicity of the urea family of compounds and the molar concentration required for maximal activation with a 30-min treatment (2 M tetramethylurea, 5 M dimethylurea, and 8 M urea).     

Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment

High-pressure CO2 treatment has been studied as a promising method for inactivating bacterial spores. In the present study, we compared this method with other sterilization techniques, including heat and pressure treatment. Spores of Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, and Geobacillus stearothermophilus were subjected to CO2 treatment at 30 MPa and 35 degrees C, to high-hydrostatic-pressure treatment at 200 MPa and 65 degrees C, or to heat treatment at 0.1 MPa and 85 degrees C. All of the bacterial spores except the G. stearothermophilus spores were easily inactivated by the heat treatment. The highly heat- and pressure-resistant spores of G. stearothermophilus were not the most resistant to CO2 treatment. We also investigated the influence of temperature on CO2 inactivation of G. stearothermophilus. Treatment with CO2 and 30 MPa of pressure at 95 degrees C for 120 min resulted in 5-log-order spore inactivation, whereas heat treatment at 95 degrees C for 120 min and high-hydrostatic-pressure treatment at 30 MPa and 95 degrees C for 120 min had little effect. The activation energy required for CO2 treatment of G. stearothermophilus spores was lower than the activation energy for heat or pressure treatment. Although heat was not necessary for inactivationby CO2 treatment of G. stearothermophilus spores, CO2 treatment at 95 degrees C was more effective than treatment at 95 degrees C alone.     

Combined Effects of High Hydrostatic Pressure and Temperature for Inactivation of Bacillus anthracis Spores

Spores of Bacillus anthracis are known to be extremely resistant to heat treatment, irradiation, desiccation, and disinfectants. To determine inactivation kinetics of spores by high pressure, B. anthracis spores of a Sterne strain-derived mutant deficient in the production of the toxin components (strain RP42) were exposed to pressures ranging from 280 to 500 MPa for 10 min to 6 h, combined with temperatures ranging from 20 to 75°C. The combination of heat and pressure resulted in complete destruction of B. anthracis spores, with a D value (exposure time for 90% inactivation of the spore population) of approximately 4 min after pressurization at 500 MPa and 75°C, compared to 160 min at 500 MPa and 20°C and 348 min at atmospheric pressure (0.1 MPa) and 75°C. The use of high pressure for spore inactivation represents a considerable improvement over other available methods of spore inactivation and could be of interest for antigenic spore preparation.     

Effects of temperature on the spores of thermophilic actinomycetes

The effects of temperature on the germination properties of spores of thermophilic actinomycetes were examined. Temperatures above and below the growth temperature of 55° C were found to produce marked changes in the germination properties of spores. High temperatures caused reductions in the germinative activities of spores. However, heated spore populations regained original germinative activities after maintaining them for suitable periods of time at 25°C. Recovery from the effects of heat on spore germination was also observed at 4°C, but at a much slower rate compared with 25°C. Spores of two strains of thermophilic actinomycetes, grown and prepared at 55°C, failed to germinate. Storage of dormant (nonactivated) spore populations at different temperatures demonstrated a low temperature requirement for the activation of these spores; while little or no activation occurred at 55°C, rapid activation took place at 25°C. Heating the spores at 80°C for 30 min slightly delayed the activation (rates) of spores at 25°C. The requirement of low temperature for spore activation was strain dependent and was influenced by the composition of the germination medium.     

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).     

Production and characterization of pure Clostridium spore suspensions

Aims:  A general protocol was derived for optimizing the production of pure, high concentration Clostridium endospore suspensions.
Methods and Results:  Two sporulation methods were developed that yielded high concentrations of notably pure Clostridium sporogenes , C. hungatei and C. GSA-1 (Greenland ice core isolate) spore suspensions (10 ml of 10⁹ spores ml⁻¹ with >99% purity each). Each method was derived by evaluating combinations of three sporulation conditions, including freeze drying of inocula, heat shock treatment of cultures, and subsequent incubation at suboptimal temperatures that yielded the highest percentage of sporulation. Pure spore suspensions were characterized in terms of dipicolinic acid content, culturability, decimal reduction time ( D ) value for heat inactivation (100°C) and hydrophobicity.
Conclusions:  While some Clostridium species produce a high percentage of spores with heat shock treatment and suboptimal temperature incubation, other species require the additional step of freeze drying the inocula to achieve a high percentage of sporulation.
Significance and Impact of the Study:  Pure Clostridium spore suspensions are required for investigating species of medical and environmental importance. Defining the conditions for optimal spore production also provides insight into the underlying mechanisms of Clostridium sporulation.     

Mathematical Model of Thermal Destruction of Bacillus stearothermophilus Spores

The experimental survival curves of Bacillus stearothermophilus spores in aqueous suspension, for six constant temperatures ranging from 105 to 130°C, displayed an initial shoulder before a linear decline. To interpret these observations, we supposed that, before the heat treatment, the designated spore suspension contained a countable and mortal N₀ population of activated spores and an M₀ population of dormant spores which remained masked during spore counting and had to be activated before being destroyed by heat. We also hypothesized that the mechanisms of both activation and destruction are, at constant temperature, ruled by first-order kinetics, with velocity constants k_A and k_D, respectively. Mathematical analysis showed that this model could represent not only our experimental survival curves, but also all other shapes (linear and biphasic) of survival curves found in the literature; also, there is an inherent symmetry in the model formulation between the activation and destruction reactions, and we showed that the dormancy rate (τ = M₀/N₀) is the only parameter which permits a distinction between the two reactions. By applying the model to our experimental data and considering that the dormancy rate is not dependent on the treatment temperature, we showed that, for the studied suspension, the limiting reaction was the activation reaction.     

Effect of Carbohydrates in Phosphate Buffer on Germination of Bacillus stearothermophilus Spores

Among strains, and among spore suspensions of the same strain, different spore-germination responses were observed when spores were heated in monosaccharides, disaccharides, and polysaccharides in 0.0083 m phosphate buffer (pH 7.1). It was hypothesized that these differences were due to rough and smooth variants in the spore population and to variation in the osmosensitivity of spores of variants within the population when subjected to a heat shock of 110 C.     

Growth from Spores of Nonproteolytic Clostridium botulinum in Heat-Treated Vegetable Juice

Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation.