Thermal Inactivation Characteristics of Bacterial Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus stearothermophilus (1518) spores and putrefactive anaerobe (PA) 3679 (NCA) spores suspended in skim milk were determined after treatment in pilot-plant ultrahigh-temperature (UHT) processing equipment. Temperature-survivor curves were constructed from survival data to emphasize the critical nature of temperature control in process evaluation. Time-survivor curves for PA 3679 spores were concave upward, and decimal reduction time (DRT) curves for these spores supported the observation of a protective response occurring at the longest exposure times. However, exposure time did not markedly affect the extremely high z(D) value obtained for PA 3679 spores. The substitution of Gelysate for Trypticase and Thiotone as the peptone in the sporulation medium increased the relative heat resistance of B. stearothermophilus spores, but lowered the z(D) value from 16 F to 12 F. The DRT curves in all cases were linear, but the z(D) values observed in this study differed considerably from those reported by other workers.     

Efficacy of the Inactivation of Bacterial Spores in White Petrolatum and a Hydrophilic Ointment by Gamma Irradiation

To evaluate the possibilities of using gamma irradiation for the sterilization of ointments, the effect of irradiation on spores of Bacillus pumilus and Bacillus sphaericus in dry material and in two different kinds of ointments was studied. The results indicate that for sterilization purposes irradiation was less effective in white petrolatum as compared to irradiation in the dry state. No such protective effect was found in a hydrophilic ointment. Accordingly, the sterilization dose needed for the sterilization of an ointment can be decided upon only after inactivation experiments with suitable test organisms in the actual preparation.     

Thermal Inactivation Characteristics of Bacillus subtilis Spores at Ultrahigh Temperatures

The thermal inactivation characteristics of Bacillus subtilis A spores suspended in skim milk with the use of large-scale ultrahigh temperature (UHT) processing equipment were investigated in terms of survival as measured with two plating media. Data on survival immediately after UHT treatments were recorded in temperature-survivor curves, time-survivor curves, and decimal reduction time (DRT) curves. The temperature-survivor curves emphasized that inactivation is accelerated more by increases in the treatment temperature than by increases in the exposure time. Time-survivor curves and DRT curves were not linear. Generally, exceedingly concave time-survivor curves were observed with the standard plating medium; however, only slightly concave curves were observed when CaCl(2) and sodium dipicolinate were added to the medium. For a given UHT sample, larger D values were obtained by use of the medium with the added CaCl(2) and sodium dipicolinate. The DRT curves of all data were concave and appeared to have two discrete slopes (z(D) values). The z(D) values observed in the upper UHT range (above 260 F; 127 C) were twice those observed at lower test temperatures.     

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.     

Kinetics of Death of Bacterial Spores at Elevated Temperatures

The kinetics of death of Bacillus stearothermophilus spores (FS 7954) suspended in phosphate buffer (pH 7) were studied over a temperature range of 127.2 to 143.8 C and exposure times of 0.203 to 4.150 sec. These short exposure were achieved by use of a tubular flow reactor in which a suspension of spores was injected into a hot flowing stream at the entrance of the reactor. Thermal equilibria of the suspension with the hot stream was achieved within 0.0006 sec. After flow through a fixed length of reactor, the stream containing the spores was cooled by flash vaporization and then assayed for viable count. The death rate data were fitted by a logarithmic expression. However, logarithmic death rate was only approximated in the tail or high-kill regions of exposure. Death rate constants obtained from this portion of the data were found to correlate by Arrhenius as well as Absolute Reaction Rate Theory relationships. Thermal-death time curves were found to correlate the data rather poorly. The activation energy and frequency constant for an Arrhenius relationship fit of the data were found to be 83.6 kcal/gmole and 10^47.2 min^-1, respectively. The standard enthalpy and entropy changes for an Absolute Reaction Rate Theory relationship fit of the data were found to be 84.4 kcal/gmole and 157 cal/gmole-K, respectively.     

Inactivation of Strains of Salmonella senftenberg by Gamma Irradiation

S ummary : Strains of Salmonella senftenberg isolated from Norwegian herring meal and strain 775W were exposed to gamma radiation from a ⁶⁰Co source. When they were irradiated in phosphate buffered saline solution, the average D₁₀ values for all the strains was 19·3 krad. On irradiating strains 56 and 775W in herring meal the D₁₀ values were 192·1 and 188·5 krad, respectively, thus indicating that the suspending medium had a great effect on the radiation resistance of the organisms. Radiation doses of the order of 0·8–1·3 Mrad are recommended for the decontamination of herring meal.     

Inactivation of Mycobacterium avium subsp. paratuberculosis in Cow's Milk by Means of High Hydrostatic Pressure at Mild Temperatures

Two strains of Mycobacterium avium subsp. paratuberculosis (3644/02 and ATCC 19698) were inoculated (approximately 6 log CFU/ml) into sterilized milk to evaluate inactivation by high hydrostatic pressure. Reductions of M. avium subsp. paratuberculosis increased with pressure level. Significant differences were also found between M. avium subsp. paratuberculosis strains and between the media used. Average reductions of 4 log CFU/ml after treatment with 500 MPa are comparable to those caused by thermal treatments.     

Photoprotection by Dipicolinate Against Inactivation of Bacterial Spores with Ultraviolet Light

The resistance of three types of Bacillus cereus T spores to ultraviolet radiation corresponded to their dipicolinic acid (DPA) content. Photoprotection against ultraviolet light was observed in DPA-containing spores and in DPA-less spores irradiated through calcium dipicolinate.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: II. Factors Affecting Rates of Inactivation by Phenolic Bactericides

Aqueous suspensions of Bacillus subtilis NCTC 8236 spores, surviving 150,000 or 300,000 rad of gamma irradiation under air from a cesium-137 source, exhibited an enhanced rate of inactivation, compared to nonirradiated spores, when heated with different phenolic bactericides. The apparent magnitude of the enhanced inactivation rate, observed from survival curves, increased progressively with the irradiation dose applied and diminished progressively as the severity of heat treatment with 0.2% chlorocresol was increased either by raising the temperature from 70 to 90 C or reducing the pH from 8 to 6. The enhanced inactivation rate was unaffected when the concentration of sodium chloride added to 0.2% chlorocresol was altered from 0.4 to 0.8%. The enhancement effect was also observed when the heat treatment was carried out with 0.5% phenol and 0.3% m-cresol.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: III. Factors Affecting Rates of Inactivation by Phenylmercuric Nitrate

Aqueous suspensions of Bacillus subtilis NCTC 8236 spores, surviving gamma irradiation from a cesium-137 source, exhibited an enhanced rate of inactivation compared to nonirradiated spores when heated with 0.04% phenylmercuric nitrate. The enhanced rate of inactivation, observable from survival curves, was noted when spores were irradiated with 150,000 rad under air in either the presence or absence of the bactericide. The magnitude of the enhanced inactivation rate increased as the irradiation dose under air increased from 150,000 to 300,000 rad. The inactivation rates of spores surviving irradiation with 150,000 rad under either oxic or anoxic conditions did not exhibit a simple quantitative relationship. The enhancement effect was observed when the severity of the heat treatment was increased by either reducing the pH from 8 to 6 or raising the temperature from 70 to 90 C.     

Thermoradiation Inactivation of Naturally Occurring Bacterial Spores in Soil

Samples of soil collected from the Kennedy Space Center near the spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilzation techniques. The inactivation kinetics of the naturally occurring spores in soil were investigated by using dry heat and ionizing radiation, first separately and then simultaneously. Dry-heat inactivation kinetics of spores was determined at 105 and 125 C; radiation inactivation kinetics was determined for dose rates of 660 and 76 krads/h at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C, with a dose rate of 76 krads/h. Combined treatment was found to be highly synergistic, requiring greatly reduced radiation doses to accomplish sterilization of the population.     

Bacterial Cell Production from Hexadecane at High Temperatures

On mineral medium with hexadecane as the sole carbon source, stable mixed bacterial enrichment cultures were obtained from soil inoculum at 25, 35, 45, 55, and 65 C. Cell yields (grams of dry cells per gram of hexadecane) were determined for each of the enrichment cultures grown at the temperature at which they were enriched, and also for the 55 and 65 C cultures grown at various temperatures. In all cases, cell yields decreased with increasing growth temperature. The highest yield obtained at 65 C was 0.26, and the lowest yield obtained at 25 or 35 C was 1.02. Slower growth was observed at higher temperatures.     

Comparative Resistance of Nonsporogenic Bacteria to Low-Temperature Gamma Irradiation

A total of 36 microorganisms, comprising 19 species of 11 genera, were screened for radiation resistance with (60)Co gamma rays at a radiation temperatore of -80 +/- 2 C in phosphate buffer (pH 7.0) under vacuum. Micrococcus radiodurans was the most resistant organism. An initial population of 2.8 x 10(5) cells per dose of this species survived 2.4 but not 2.7 Mrad. Of the remaining 18 species with initial populations of about 10(6) cells per dose, Streptococcus faecium survived 0.9 to 1.5 Mrad, depending on the strain tested. S. faecalis QM survived 0.9 but not 1.2 Mrad. S. faecalis 1539 and Alcaligenes faecallis survived 0.6 but not 0.9 Mrad. Three species of Salmonella, one strain each of Escherichia coli, Streptococcus lactis, and Aerobacter aerogenes survived 0.3 but not 0.6 Mrad. The remaining 22 bacteria did not survive 0.3 Mrad, the lowest dose tested. Detailed survival curve determinations for four strains of S. faecium, the most resistant of the test bacteria of public health significance, indicated the following order of resistance at -80 C: alpha21 > theta12 = F(6) > FEC. Each strain produced two exponential survival curves with different slopes, the breaks occurring at 0.3 to 0.5 Mrad. The D values (doses which reduce the microbial population by 90%) of the more resistant cell fractions were two- to three-fold higher than the more sensitive cell fraction. The resistance of strain alpha21 was determined at different radiation temperature (+5, -30, -80, -140, -196 C). The D value-radiation temperature relationship followed a quadratic equation. Computations of E(a) and Q(10) values (activation energy and temperature coefficient, respectively) showed a very small thermodynamic effect on radiation death. An Arrhenius evaluation of the temperature effect on cell kill indicated that there was no simple physicochemical mechanism which might explain the change in D value as a function of temperature.     

Resistance of Bacillus subtilis Spores to Inactivation by Gamma Irradiation and Heating in the Presence of a Bactericide: I. Suitability of Viable Count Procedures

A statistical evaluation of viable count procedures utilized for obtaining treatment survival curve data for Bacillus subtilis NCTC 8236 spores is described. Within the various recovery conditions tested, incubation on nutrient agar containing 1% dextrose for 48 hr at 37 C was found to promote the highest count of viable spores surviving a variety of bactericidal treatments involving gamma irradiation, heat, and chlorocresol. The count of viable spores on the medium was not significantly altered when the dextrose was added to the nutrient agar either before autoclaving or aseptically at 50 to 55 C from a solution sterilized by filtration. The volume of medium which promoted the highest count of viable spores was 20 ml per 85 mm of diameter in disposable plastic plates. Counts of viable spores were reproducible on successive batches of media. The carry-over of variable concentrations of chlorocresol into the medium from serial dilutions affected the count of viable spores. Spores in the aqueous stock suspension used for all experiments were uniformly distributed after shaking and did not diminish significantly in viability after 16 months of storage at 5 C. Grouping of indexes of dispersion, calculated from quintuplicate plate colony counts, indicated that the suitability of the viable count procedures, employed for the enumeration of spores surviving the various bactericidal treatments, tended to diminish as the level of spore inactivation exceeded 95%.     

The Combined Effect of Hydrogen Peroxide and Ultraviolet Irradiation on Bacterial Spores

Ultra-violet (u.v.) light irradiation of spores of Bacillus subtilis in the presence of hydrogen peroxide produced a rapid kill which was up to 2000-fold greater than that produced by irradiation alone. A kill of 99–99% was produced by 30s u.v. irradiation of spores of 6 strains of Bacillus and Clostridium in the presence of hydrogen peroxide 1.0 g/100 ml but with the more resistant spores of 9 further strains, irradiation in the presence of hydrogen peroxide 2–5 g/100 ml followed by mild heating was required.     

Effect of Cell Moisture on the Thermal Inactivation Rate of Bacterial Spores

Thermal inactivation rates were determined for two strains of Bacillus subtilis var. niger spores after equilibration to various relative humidity (RH) levels. In these tests, small thin stainless-steel squares were each inoculated with a drop of spore suspension and equilibrated to 11, 33, or 85% RH. Following equilibration, the squares were placed on a hot plate preheated to 108, 125, 136, 164, or 192 C for various exposure times and then assayed for surviving organisms. The results revealed that spores of the A strain of B. subtilis were least resistant if preequilibrated to 11% RH and most resistant if preequilibrated to 85% RH. The same trend was obtained at all temperatures except 192 C, at which, no difference was noted, probably because the rapid kill time approaches the heat-up time of the stainless-steel square. The B strain of B. subtilis spores showed an opposite RH effect; that is, the cells preequilibrated to 11% RH were the most resistant. Because the two strains of spores were grown on different media, further studies were conducted at 136 C after subculturing the cells on different media. When the B strain was subcultured on the A strain medium, the pattern was reversed; the cells preequilibrated to low RH were then least resistant. Although it was not possible to reverse these cells to the original pattern by subculturing on the original B strain medium again, the pattern was altered to the point that there was no significant difference in heat resistance of these cells regardless of the preequilibration RH. The same result was obtained when the A strain was grown on the B strain medium; that is, the thermal resistance could not be reversed, but it was altered from the point where the low RH equilibrated cells were least resistant initially to the point where there was no significant difference in any of the cells regardless of what RH was used for preequilibration. The thermal resistance of spores seemed to be dependent on (i) the medium on which the spores are grown, (ii) the RH on which they are exposed before heating, and (iii) some genetic characteristic of the cell.     

Characterization of Bacillus pumilus E601 Spores After Single Sublethal Gamma Irradiation Treatments

Eighteen survivor strains of Bacillus pumilus E601 have been isolated after single sublethal irradiation treatments with ⁶⁰Co. Primary isolation was based on the loss of motility and pellicle formation. However, with subsequent subcultivation, eight isolates reverted back to the standard of exhibiting motility and pellicle formation. Characteristics of the isolates include alterations in spore radiation resistance and in the amino acid requirements for spore germination and outgrowth. Other alterations in cultural and physiological characteristics were found. Three of the isolates were asporogenous.     

Examination of Peak Power Dependence in the UV Inactivation of Bacterial Spores

We examine whether the rate of delivery of photons from a UV radiation source has an effect on the inactivation of spores. We directly compare the output of a high-peak-power UV laser source at 248 nm to a low-power continuous lamp source (254 nm) in the inactivation of Bacillus subtilis spores. The two UV sources differ by a factor of 10⁸ in peak power. Contrary to previous reports, no clear differences in spore survival were observed.