Recovery of bacterial spores dried on aluminium strips

Bacterial spores dried on aluminium strips are used in microbiological validation of packaging and processing systems. Vortex agitation and sonication in Butterfield's buffer, 70% ethanol or 0·1% Tween 80 were evaluated for ease of recovery of bacillus spores dried on aluminium strips to compare the concentration of dried spores to dilutions used to inoculate such strips. The highest recovery for Bacillus subtilis var. globigii spores was observed with sonication in 70% ethanol with average recovery close to the initial inoculum. The highest recovery for B. stearothermophilus spores was with sonication in Butterfield's buffer, averaging 0·8 log less recovery than the initial inoculum. Bacillus subtilis var. globigii spores were recovered from strips in greater numbers than B. stearothermophilus spores for all treatment medium combinations. Scanning electron microscopy revealed unrecovered spores adhering to strips after treatment. Recovery of B. subtilis var. globigii spores decreased with time over the 4 week storage period.     

Sterilization of soybean cotyledons by boiling in lactic acid solution

F. RUÍZ-TERÁN AND J.D. OWENS. 1996. The effect of pH on the heat resistance of Bacillus stearothermophilus spores at 100°C in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 sodium phosphate buffer was examined. At pH values of 7.0 and 6.0 spores survived 60 min exposure unharmed but at pH 4.3 and 3.0 they died with decimal reduction times (DRTs) of 27 min and 2.8 min, respectively. Death rates were similar in the presence or absence of hydrated soybean cotyledons. In the presence of phosphate buffer and cotyledons at mean pH 3.6 the DRT was 118 min but in the presence, in addition, of lactic acid it was 11 min. It is suggested that the enhanced death rate was due to toxic effects of undissociated lactic acid. Rhizopus oligosporus NRRL 2710 grew well on cotyledons, having pH values from 7.0 to 3.7, prepared by boiling for 60 min in the presence of 0.11 mol 1^-1 lactic acid and 0.2 mol 1^-1 phosphate buffer.     

Optimized dipicolinate activation for viable counting of Bacillus stearothermophilus spores

Spores of Bacillus stearothermophilus were exposed to calcium and sodium salts of dipicolinic acid (DPA) in phosphate and Tris acid maleate buffers over the range pH 4.5–10.0. The exposed spores were enumerated using a standard plate counting technique from which the kinetics of colony formation were determined and maximum colony counts were obtained for each condition examined. Exposure of the spores to calcium-DPA (50-40 mmol/l) in Tris acid maleate buffer pH 9.0 maintained at 10°C was found to produce an optimal response. Following this method the total viable population of a spore suspension was enumerated. This was demonstrated statistically using the Wilcoxon rank-sum test for significance. Calcium-DPA was found to produce activation in spores but further germinants and nutrients were required for colony formation. The Ca-DPA treatment was found to be effective in enumerating both naturally dormant spores and heat injured spores.     

Effect of disruption by sonication under different conditions on the activity of glucose dehydrogenase from resting spores of Bacillus subtilis

The activity of glucose dehydrogenase present in resting spores of Bacillus subtilis varied strikingly with the conditions for disrupting the spores by sonic treatment, namely, the time and strength of sonication, and the type and pH of the solution used for suspending the spores. When the resting spores were sonicated for 30 min at a current of 1.45 A in 100 mM phosphate buffer in the range of pH 6.0 to 6.6 or in deionized water, the enzyme activity of the former suspension was approximately 10 times higher than that of the latter suspension. However, the enzyme activity of the latter was markedly stimulated in the presence of sodium chloride. The glucose dehydrogenase from resting spores disrupted in 100 mM phosphate buffer (pH 6.6) was a salt-independent, active enzyme with a molecular weight of about 120,000, whereas the enzyme from resting spores disrupted in deionized water was a salt-dependent, inactive one with a molecular weight of about 55,000. A high concentration of dipicolinic acid strongly inhibited activation by a salt of inactive glucose dehydrogenase from resting spores in deionized water, suggesting one of its several important roles in vivo.     

The Effect of Sodium Chloride on Heat Resistance and Recovery of Heated Spores of Bacillus stearothermophilus

Increasing concentrations (2, 4 and 8% w/v) of sodium chloride in the heating medium progressively reduced the heat resistance of spores of Bacillus stearothermophilus. Storage at 4° in water or in sodium chloride solutions had little effect on viable counts of unheated spores, but with the increase in sodium chloride concentration there was a reduction in the heat activation effect and a small decrease in heat resistance of the spores. Increasing the severity of heat treatment rendered spores increasingly sensitive to sodium chloride in the plating medium.     

Effect of simultaneous application of heat and pressure on the survival of bacterial spores

The effect of simultaneous application of moderate hydrostatic pressure (10-300 atm) and heat on the survival of the Bacillus stearothermophilus spores in a flow-through system was investigated. A high heterogeneity of the sensitization of spores to heat by pressure was found. A higher degree of reduction of heat resistance was observed at the low than at the high temperatures tested. The simultaneous application of moderate pressure and heat can not be applied for the preservation of liquid foods due to the extreme heterogeneity of spore sensitization to heat by pressure.     

Modelling the combined effects of pH,temperature and sodium chloride stresses on the thermal inactivation of Bacillus subtilis spores in a buffer system

AIMS: The inactivation of Bacillus subtilis 168 spores subjected to the combined stress of pH, temperature and sodium chloride in a buffer system was modelled. METHODS AND RESULTS: Bacillus subtilis 168 spore suspension in 50 mmol l-1 potassium phosphate buffer was heated in an open system using a block heater. A second order polynomial equation was used to describe the relationship between pH, temperature, sodium chloride concentration and the logarithm of the decimal reduction time (D-value) of the spores. Response surface graphs were constructed to predict the inactivation within the experimental domain. The data obtained were also compared with those reported for B. subtilis in different media and foods included in a large reference-based database of thermal inactivation (ThermoKill Database, TKDB R9100), which was constructed in the laboratory. CONCLUSIONS: All the variables studied seemed to have a significant effect on the inactivation of B. subtilis 168 spores in potassium phosphate buffer. The coefficient of determination, r2, and an analysis of the residuals from the model indicated the adequacy of the model to predict the inactivation of B. subtilis spores within the range of the experimental variables studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study will enable a better understanding of the inactivation of B. subtilis spores under the influence of the studied environmental variables. The model can be used by food industries to assess and monitor the shelf life of food products in the event of a chance contamination by B. subtilis spores.     

Germination of Spores of Bacillus stearothermophilus Induced by Analogues of Dipicolinate Di-Anion

The structural specificity required for induction of germination of spores of Bacillus stearothermophilus by analogues of dipicolinate in buffer at pH 5.5 is similar to that found previously with spores of Bacillus megaterium and calcium chelate salts at pH 8. 4H-pyran-2,6-dicarboxylate, but no other analogue tested, is as effective as dipicolinate di-anion.     

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.     

Effect of simultaneous application of heat and pressure on the survival of bacterial spores

C.G. MALLIDIS AND D. DRIZOU. 1991. The effect of simultaneous application of moderate hydrostatic pressure (10–300 atm) and heat on the survival of the Bacillus stearothermophilus spores in a flow-through system was investigated. A high heterogeneity of the sensitization of spores to heat by pressure was found. A higher degree of reduction of heat resistance was observed at the low than at the high temperatures tested. The simultaneous application of moderate pressure and heat can not be applied for the preservation of liquid foods due to the extreme heterogeneity of spore sensitization to heat by pressure.     

Inhibition by potassium ion of the pregerminative response to L-alanine of unactivated spores of Bacillus cereus T.

The effect of potassium ion on L-alanine-inosine-induced germination of unactivated spores of Bacillus cereus T was studied. Unactivated spores germinated in 0.1 M sodium phosphate buffer (NaPB), but not 0.1 M potassium phosphate buffer (KPB), at pH 8.0 and at 30 C. Inhibition of germination was also observed on incubation of unactivated spores in NaPB containing potassium chloride. Previously it was demonstrated that germination of unactivated spores involves at least two steps, one induced by L-alanine, and the other by inosine. Potassium ion seems to inhibit the response of the spores to inosine, because: (1) Spores that had been preincubated with L-alanine in NaPB or KPB, germinated in NaPB but not KPB in the presence of inosine. (2) During germination in NaPB, incorporation of L-[14C]alanine showed bimodal kinetics with a rapid first phase and a second continuous phase, but in KPB the second phase of incorporation did not occur. The events occurring before germination of unactivated spores are discussed with reference to the initiation of germination.     

Recovery of Spores of Bacillus stearothermophilus from Thermal Injury

Bacillus stearothermophilus grown in nutrient broth produced a product which promoted recovery from thermal injury of its spores. This phenomenon was observed with nutrient agar as the plating medium but not with a medium composed of Trypticase, Phytone, dextrose and phosphate (TPDP). Recovery of injured spores was greatest in such a medium if it contained starch or charcoal. Trypticase soy agar and dextrose tryptone agar were markedly inferior to TPDP medium.     

Biological indicators for low temperature steam and formaldehyde sterilization: the effect of defined media on sporulation, growth index and formaldehyde resistance of spores of Bacillus stearothermophilus strains

Preliminary screening was carried out on spores of 29 strains of Bacillus stearothermophilus to determine their potential as biological indicator organisms for low temperature steam and formaldehyde sterilization. Each strain was sporulated on four chemically defined media. Fourteen strains produced satisfactory sporulation on one or more of the media but there was considerable variation in the extent of sporulation. The growth index of the spores, which was dependent on both the strain of organism and the sporulation medium, ranged from 1% to 90%. The spores were appraised on the basis of their resistance to inactivation by 0.5% w/v formaldehyde in aqueous solution at 70°C. The survivor curves obtained could be characterized into five types on the basis of the shape of the curve. Only five strains of Bacillus stearothermophilus produced spores with the characteristics of high resistance, linear semi-logarithmic survivor curve and high growth index that would be required of a potential biological indicator organism.     

Chemical States of Bacterial Spores: Heat Resistance and Its Kinetics at Intermediate Water Activity

Bacterial spore heat resistance at intermediate water activity, like aqueous and strictly dry heat resistance, is a property manipulatable by chemical pretreatments of the dormant mature spore. Heat resistances differ widely, and survival is prominently nonlogarithmic for both chemical forms of the spore. Log survival varies approximately as the cube of time for the resistant state of Bacillus stearothermophilus spores and as the square of time for the sensitive state. A method for measuring heat resistance at intermediate humidity was designed to provide direct and unequivocal control of water vapor concentration with quick equilibration, maintenance of known spore state, and dispersion of spores singly for valid survivor counting. Temperature characteristics such as z, E(a), and Q(10) cannot be determined in the usual sense (as a spore property) for spores encapsulated with a constant weight of water. Effect on spore survival of temperature induced changes of water activity in such systems is discussed.     

The Effect of Freezing on the Radiation Sensitivity of Bacterial Spores

S ummary : Bacillus pumilus spores, irradiated under aerobic conditions, were inactivated exponentially at the same rate whether they were at room temperature (10–13°) in phosphate buffer or at -79° in phosphate buffer or in heart infusion broth.
Clostridium welchii spores were irradiated in Robertson's cooked meat medium under anaerobic conditions. With unheated spores, and those subjected to a heat shock before irradiation, the inactivation rate was the same at room temperature and -79°. The same applied to spores heat shocked after irradiation for doses up to 450 Krads, but above this dose level the spores irradiated frozen were more sensitive.
The effect of the heat shock, whether applied before or after irradiation, was to increase the number of survivors, and the proportionate increase appeared to vary with dose.     

The Effect of Halocarbon Aerosol Propellents on Bacteria

SUMMARY: The survival of Escherichia coli and spores of Bacillus subtilis var. niger and B. stearothermophilus suspended in halocarbon propellents for several weeks has been demonstrated. There was no significant loss of dipicolinic acid from the spores. Lethal action may be due to interaction of the propellents with cellular lipid.     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

Alkali cation effect on carbonyl-hemoglobin's and -myoglobin's conformer populations when exposed to freeze-concentration of their phosphate-buffered aqueous solutions

Freeze-concentrated aqueous phosphate-buffered (pH 6.8) solutions of carbonyl-hemoglobin (HbCO) and -myoglobin (MbCO) were investigated by Fourier-transform infrared spectroscopy for the effect of alkali cation on the population of conformers. When using sodium phosphates as buffer components, HbCO was transformed from conformer III (at approximately 1951 cm-1) which is the dominant form at ambient temperatures, into conformer IV (at buffer concentration at a given temperature. The conformational changes started slightly below the temperature where ice began to crystallize and the remaining solution became freeze-concentrated, and they were reversible for HbCO. For MbCO in 0.5 M sodium phosphate buffer solution, however, they were irreversible and MbCO denatured completely. When potassium phosphate salts were used for preparing the buffer at the same pH of 6.8, little or no transformation of conformer III into conformer IV was observed. The conformational changes induced by sodium salts are attributed to a decrease in pH and it is shown by infrared spectroscopy that during freeze concentration drastic changes in composition of the two buffer components H2PO4-/HPO(2)4- occur, the acid component increasing strongly relative to the base component. Supersaturation is also important because change from conformer III to IV requires a minimum concentration of sodium salts: whereas 0.1 M sodium phosphate buffer concentration shows a strong effect, 0.03 M concentration does not and therefore behaves like a potassium phosphate buffer.     

Effect of moderately acidic pH on heat resistance of Clostridium sporogenes spores in phosphate buffer and in buffered pea puree.

The effect of pH in the range 5.0 to 7.0 on the thermal destruction of spores of Clostridium sporogenes putrefactive anaerobe 3679 was examined by three methods: a capillary tube method in which spores were suspended in phosphate buffers, a thermoresistometer method in which spores were suspended in buffered pea puree adjusted to the same set of pH values, and a thermal death time can method in which spores were again suspended in buffered pea puree. The results indicated that increasing acidity is, in general, accompanied by decreasing heat resistance, although the pH effect was more pronounced at the higher than at the lower processing temperatures. Certain pH values appear to be critical, as they produced, in all three sets of experiments, effects which would not be predicted by the overall relationship between acidity and spore heat resistance. Differences between heat resistance in phosphate buffer as compared with that in pea puree adjusted to the same pH were also noted. D-values in buffer were found to be lower than those in pea puree, except at the highest temperatures coupled with the lowest pH values. The differences between buffer D-value and pea puree D-value were found to increase with increasing pH and with decreasing temperature. On the other hand, at all pH values examined, z-values determined in buffer were somewhat higher than those determined in pea puree adjusted to the same pH.     

The sporicidal activity and inactivation of chlorhexidine gluconate in aqueous and alcoholic solution

The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range. Activity was not observed at 20 degrees C even with concentrations as high as 10% chlorhexidine. Temperatures of 37 degrees-70 degrees C in combination with such high concentrations were required for reductions in spore viability. No viable spores were recoverable after 4 h contact at 55 degrees C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution. Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results. The spores of other Bacillus species examined (B. cereus, B. megaterium and B. stearothermophilus) proved to be considerably less resistant than those of B. subtilis. Presence of organic matter had little effect on the activity.