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.     

Injury and repair in biocide-treated spores of Bacillus subtilis

Abstract Bacillus subtilis NCTC 8236 spores exposed to appropriate concentrations of test biocides (glutaraldehyde, two iodine and two chlorine preparations) were able to repair injury if subsequently held in nutrient broth at 37°C but not in broth at 22°C, sterile filtered water at 4, 22 or 37°C or germination medium at 37°C. Repair appeared to occur primarily during outgrowth and was initiated soonest for iodine-treated spores and latest for glutaraldehyde-treated ones.     

Dry Heat Inactivation of Bacillus subtilis var. niger Spores as a Function of Relative Humidity

Dry heat sterilization of Bacillus subtilis var. niger spores at 105 C is enhanced in the relative humidity range 0.03 to 0.2%. D-values of 115 and 125 C are predicted by a kinetic model with parameters set from 105 C data. These predictions are compared to observations.     

Dry-Heat Destruction of Bacillus subtilis Spores on Surfaces: Effect of Humidity in an Open System

Bacillus subtilis var. niger spores were tested for dry-heat resistance on stainless-steel strips hung in an oven. Heat resistance was dependent on the relative humidity before and during treatment, which in turn affected the water content of the spores. Higher humidities increased the heat resistance of the spores. D-values ranged from 16.1 min for spores conditioned at <2% relative humidity (RH) and treated at 0.34% RH to 37.6 min for spores conditioned at 89% RH and treated at 1.1% RH. The y-intercept of the regression line ranged from 6.94 x 10(4) for spores conditioned and treated at the low humidities to 2.00 x 10(5) for spores conditioned at 89% RH and treated at 0.34% RH. For a constant value of N(0), the y-intercept appears to be lowered by low-humidity conditions. The statistic log y(0)/log N(0) is used to measure the downward displacement of the regression line. Values obtained in this experiment range from 0.90 for spores conditioned at <2% RH and treated at 0.34% RH to 1.04 for spores conditioned at <2% RH and treated at 1.1% RH. A combination of linear regression and analysis of variance methods was used for data analysis. The former estimates D-values and y-intercepts, whereas the latter is sensitive to differences between treatments.     

Thermal Death of Bacillus subtilis var. niger Spores on Selected Lander Capsule Surfaces

Dry-heat sterilization of planetary lander capsules requires a knowledge of the thermal resistivity of microorganisms in the environment to which they will be subjected during sterilization of the space hardware. The dry-heat resistance of Bacillus subtilis var. niger spores on various lander capsule materials was determined at 125 C. Eight surface materials were evaluated, including a reference material, stainless steel. Survivor curves were computed, and decimal reduction times (D values) were obtained by a linear regression analysis. In four tests on stainless steel, the average value of D at 125 C was 17.07 min. The D values for the other seven materials tested ranged from 18.64 min on magnesium surfaces to 20.83 min on conversion-coated magnesium. Of the materials evaluated, the results indicate that there is only a significant difference in the thermal resistance of B. subtilis var. niger spores on conversion-coated magnesium and conversion-coated aluminum from that on the reference material, stainless steel. The differences in D values for all the test surfaces may be the result of variations in test procedures rather than the effect of the surfaces on the thermal resistivity of the spores.     

The effect of visible radiations on the germination and outgrowth of Bacillus spores

The effect of visible radiations on the germination and outgrowth of spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger was determined by direct observation of populations irradiated on the surface of nutrient agar. Little effect on germination (phase darkening) was found but white light prevented outgrowth of some and retarded it for all spores. Different wavebands in the visible spectrum differed in their effect on outgrowth, the greatest retardation being found for the shorter wavelengths, 410–570 nm. Outgrowth in dark controls was always greater both in number of spores outgrown and rate of outgrowth. The results are consistent with others, suggesting an effect of singlet oxygen generated from endogenous photosensitizers by visible radiation.     

Effects of temperature and heat activation on germination of individual spores of Bacillus subtilis

Phase intensity changes of individual germinating spores of Bacillus subtilis were determined by phase-contrast light microscopy and image analysis. Two germination phases were investigated. The length of the time period before a change in phase brightness was evident and the duration of the phase intensity change until a constant greylevel was maintained. The incubation temperature (37 and 20 °C) and heat activation (10 min at 65 °C) had a distinct effect on both phases. At 37 °C, spores of B. subtilis 604 started to show a decrease in brightness in l -alanine buffer after 3–39 min and needed 10–39 min to complete the phase change. At 20 °C, lag times of 10–100 min were observed and the spores needed 30–100 min to reach a constant greylevel. Heat activation and subsequently exposure to l -alanine buffer at 20 °C reduced the lag phase to 6–90 min and the phase change was finished after 30–60 min. Our results indicate enzymatic involvement before and during the phase intensity change of germinating spores.     

Dry-Heat Resistance of Bacillus subtilis var. niger Spores on Mated Surfaces

Bacillus subtilis var. niger spores were placed on the surfaces of test coupons manufactured from typical spacecraft materials (stainless steel, magnesium, titanium, and aluminum). These coupons were then juxtaposed at the inoculated surfaces and subjected to test pressures of 0, 1,000, 5,000, and 10,000 psi. Tests were conducted in ambient, nitrogen, and helium atmospheres. While under the test pressure condition, the spores were exposed to 125 C for intervals of 5, 10, 20, 50, or 80 min, with survivor data being subjected to a linear regression analysis that calculated decimal reduction times. Differences in the dry-heat resistance of the test organism resulting from pressure, atmosphere, and material were observed.     

Ultrahigh-Temperature Activation of a Low-Temperature Bacillus subtilis Spore Germination System

Exposure of Bacillus subtilis A spores to 115 to 125 C for several seconds activated a low-temperature germination system that remained dormant after a heat treatment that activated the normal l-alanine- and glucose-stimulated germination systems. The low-temperature germination system was characterized by an optimum temperature lower than that of the l-alanine or glucose germination systems (30 C versus 45 C), germination in the absence of exogenous germination stimulants, and the capacity for heat-induced deactivation and subsequent reactivation. The rates of activation at 115 to 125 C were exponential and were not influenced by a previous heat treatment that activated the l-alanine- or glucose-stimulated germination systems. Although activation of the low-temperature germination system was accompanied by suppression of l-alanine-stimulated germination, it did not appear to be a modification of the l-alanine germination system.     

Thermal inactivation kinetics of Bacillus subtilis spores suspended in buffer and in oils

The heat resistance of Bacillus subtilis 5230 and A spores freeze dried and suspended in buffer or oils was investigated. As expected, spores were more resistant to heat when suspended in oils than in buffer. This was ascribed to the low a _w of oils and to their content of free fatty acids. Linear survivor curves were obtained for spores suspended in buffer at 105°C or above and for B. subtilis A spores suspended in a vegetable oil. However, the survivor curves of the spores suspended in mineral oil (strain 5230) or olive oil (both strains) were concave upward with a characteristic tailing. The tailing could not be ascribed to spore clumping or to a specific heat injury that can be circumvented by Ca-dipicolinate. It is possibly due to another mechanism of injury or to the activation at high temperature of a normally dormant germination system.     

Resistance of Spores of Bacillus subtilis var. niger on Kapton and Teflon Film to High Temperature and Dry Heat

To determine parameters that would assure sterility of a sealed seam of film for application in "split-seam entry," spores of Bacillus subtilis var. niger were sprayed onto pieces of Kapton and Teflon film. Short-time, high-temperature (200 to 270 C) exposures were made with film pieces between aluminum blocks in a hot-air oven, and the D and z values were determined after subculture of surviving spores. The use of Kapton film allowed the study of high temperatures, since it is not heat sealable and could be used to make thin packages for heat treatment. Spores on Teflon were dry-heat treated in a package designed to simulate an actual seam to be sealed. The z values of 29.1 C (52.4 F) for spores on Kapton and 139 C (250.4 F) for spores on Teflon were calculated.     

A note on the effect of storage on the chemical resistance of spores of Bacillus subtilis SA22 and Bacillus subtilis globigii B17

Spores of Bacillus subtilis SA22, harvested from nutrient agar after 9 d at 30°C and stored in distilled water at 4°C, were unaltered in their resistance to 17.7% hydrogen peroxide or 0.04% peracetic acid after storage for up to 134 weeks. Three spore crops of B. subtilis globigii were unaffected by storage for up to 134 weeks with respect to 17.7% hydrogen peroxide resistance but were significantly more resistant to 0.04% peracetic acid following storage.     

Survival of Microorganisms in a Simulated Martian Environment: II. Moisture and Oxygen Requirements for Germination of Bacillus cereus and Bacillus subtilis var. niger Spores1

The effects of moisture and oxygen concentration on germination of Bacillus cereus and B. subtilis var. niger spores were investigated in a simulated Martian environment. Less moisture was required for germination than for vegetative growth of both organisms. A daily freeze-thaw cycle lowered moisture requirements for spore germination and vegetative growth of both organisms, as compared with a constant 35 C environment. Oxygen had a synergistic effect by lowing the moisture requirements for vegetative growth, and possibly germination, of both organisms. Oxygen was not required for spore germination of either organism, but was required for vegetative growth of B. subtilis and for sporulation of both organisms.     

Resistance of Bacillus Spores to Combined Sporicidal Treatments

S ummary . Moist heat at 82° (100° for Bacillus stearothermophilus ) and solutions of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride at 24° separately showed no sporicidal activity against B. pumilis, B. stearothermophilus, B. subtilis and B. subtilis var. niger . Spores of the last organism were the most sensitive to γ radiation, the D value being 0.16 Mrad. Prior irradiation with a dose of 0.16 Mrad brought about only a slight increase in the sensitivity of the spores to moist heat. The presence of bactericide during irradiation did not affect radiation resistance. Inactivation rates were greater when the spores were heated in the presence of a bactericide than in aqueous suspension and benzalkonium chloride was more active than chlorocresol. Chlorocresol enhanced the heat activation of B. stearothermophilus at 100°. Irradiation in the presence of 0.2% w/v chlorocresol or 0.01% w/v benzalkonium chloride had no effect on the subsequent resistance of the spores when heated in the presence of these bactericides. It is concluded that it is unlikely that combinations of moist heat, radiation and bactericides, each less severe than when used in an accepted sterilization process, will lead to an alternative process which, while less damaging to the materials being sterilized, would still maintain the accepted standards of freedom from contamination.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis

The influence of different sporulation temperatures (30, 37, 44 and 52°C) upon heat resistance of Bacillus subtilis was investigated.
Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30–44°C. Sporulation at 52°C did not show any further increase in heat resistance.
This effect was constant over all the range of heating temperatures tested (100–120°C). z value remained constant ( z = 9°C).
Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52°C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Heat-induced resistance of Bacillus stearothermophilus spores

Spores of Bacillus stearothermophilus CNCH 5781 were suspended in distilled water or nutrient medium. A 28 μl aliquot of each was inoculated into haematocrit capillaries and subjected at different time intervals to sublethal temperatures of 63° or 100°C as heat activation for germination. This was followed by heat treatment at 121·1°C and the heat parameter D _121·1 was measured. Contrary to standard observations, heat resistance was observed to increase following activation, a phenomenon which we named 'heat-induced resistance'.     

Effect of pH on Sporulation of Bacillus Stearothermophilus

An improved broth medium was developed for high growth yields of Bacillus subtilis var. niger NCIB 8649, Bacillus cereus NCIB 9373, and Bacillus stearothermophilus NCIB 8919 and ATCC 7953. Sporulation was abundant (1.1 times 10-8 B. subtilis var. niger and 9.2 times 10-7 B. cereus per ml) at an initial pH of 7.0. Sporulation of both strains of B. stearothermophilus took place (1.9 times 10-7 and 2.4 times 10-7/ml, respectively) in this medium when initial pH values of 7.7 to 8.7 were used.     

Germination of spores of Bacillus subtilis var. niger following exposure to gaseous ethylene oxide

Spores of Bacillus subtilis var. niger exposed to a lethal dose of ethylene oxide (ETO) germinated freely under a variety of nutritional conditions. Outgrowth, however, did not occur. Good germinants for ETO-exposed spores were (in order of decreasing effectiveness) alanine, valine, cysteine, isoleucine and histidine. Asparagine, while a germinant for unexposed spores, did not allow the germination of ETO-exposed spores. Mixtures of amino acids were no more effective than alanine alone. Exposure to ETO lowered both the rate and amount of germination but the effect was much less than that on viability. A linear relationship was obtained by plotting germination in glucose alanine against survivors on a logarithmic scale. This relationship did not occur when valine was the germinant.     

Thermal Inactivation of Aerosolized Bacillus subtilis var. niger Spores

A hot-air sterilizer capable of exposing airborne microorganisms to elevated temperatures with an almost instantaneous heating time was developed and evaluated. With this apparatus, aerosolized Bacillus subtilis var. niger spores were killed in about 0.02 sec when exposed to temperatures above 260 C. This is about 500 times faster than killing times reported by others. Extrapolation and comparison of data on the time and temperature required to klll B. subtilis var. niger spores on surfaces show that approximately the same killing time is required as is necessary for spores in air, if corrections are made for the heating time of the surface.     

A note on the effect of sporulation conditions on the resistance of Bacillus spores to heat and chemicals

Spores of Bacillus subtilis SA22 harvested after 22 d incubation on nutrient agar at 30°C were more resistant to 0–04% peracetic acid at 20°C than spores harvested following 2 d incubation. Similarly, spores of B. subtilis globigii B17, harvested after 7 d incubation on a sporulation agar were up to 10 times less resistant to 0.04% peracetic acid at 20°C than spores harvested after 35 d incubation. An increase in resistance to heating at 100°C and to exposure to 17.7% hydrogen peroxide at 20°C occurred as the age of B. subtilis SA22 spores prior to harvesting increased, whereas differences in resistance were not observed with spores of B. subtilis globigii B17.