Thermal resistance of Bacillus subtilis var. niger in a closed system.

The heat resistance of Bacillus subtilis var. niger has been measured from 85 to 125 degrees C using moisture levels of percent relative humidity (%RH) less than or equal to 0.001 to 100 in a closed system. Five curves have been presented to characterize the thermal destruction, using thermal death times defined as F values at a given combination of three moisture and temperature conditions. Reductions of 99.99% (4-log10 cycles) of the initial population were estimated for the three moisture conditions. At 110 degrees C, the expected time for a 4-log10 reduction was 1.1 h at %RH = 100, 3.1 h at %RH less than or equal to 0.1 and 54 h at %RH = 10.7. Goodness-of-fit tests to examine the adequacy of three polynomial models failed to indicate a trend. The linear model (from which estimates of D are obtained) was satisfactory for estimating the thermal death times (%RH less than or equal to 0.1) in the plate count range. The estimates based on observed thermal death times and D values for the %RH = 100 diverged so that D values generally gave a more conservative estimate over the temperature range 90 to 125 degrees C. Estimates of ZF and ZL ranged from 32.1 to 58.3 degrees C for the %RH less than or equal to 0.1 and 100. A ZD value of 30.0 was obtained for data observed at %RH less than or equal to 0.1. The ZF results were obtained from plotting observed log times to achieve a 99.99% reduction in the initial population versus temperature. Estimates of ZL and ZD were obtained by using linear estimates of L100 approximately equal to 4D and D values in a similar plot.     

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.     

Thermal Activation and Dry-heat Inactivation of Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger

Spores of Bacillus subtilis MD2 and Bacillus subtilis var. niger were heat activated for different times at 60° and 80°C. Strain MD2 required considerable heat activation while B. subtilis var. niger did not. Maximum germination rates increased with heat activation dose and declined subsequently without loss of germinability. Germination rates and percentages were considerably greater in tryptone glucose extract (TGE) than in nutrient broth. The addition of 2°° dimethyl sulphoxide did not increase germination in nutrient broth. The spores of var. niger are more resistant to dry-heat than MD2 although they are less resistant to moist heat. Survivor curves in the dry-heat range 140°-170°C gave D-values from 4–123 to 0.106 min for MD2 and 5.679 to 0.233 min for var. niger recovered on TGE agar. D-values were lower on poorer media. The z-values for MD2 and var. niger on TGE were 18.7°C and 21.25C respectively.     

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.     

Foam Separation of Pseudomonas fluorescens and Bacillus subtilis var. niger

An experimental investigation established the effect of the presence of inorganic salts on the foam separation of Pseudomonas fluorescens and of Bacillus subtilis var. niger (B. globigii) from aqueous suspension by use of a cationic surfactant. For P. fluorescens, 5.0 mueq/ml of NaCl, KCl, Na(2)SO(4), K(2)SO(4), CaCl(2), CaSO(4), MgCl(2), or MgSO(4) produced increases in the cell concentration in the residual suspension (not carried into the foam) from 2.9 x 10(5) up to 1.6 x 10(6) to 2.8 x 10(7) cells per milliliter (initial suspensions contain from 3.3 x 10(7) to 4.8 x 10(7) cells per milliliter). The exceptional influence of magnesium was overcome by bringing the cells into contact first with the surfactant and then the salt. For B. subtilis, the presence of 5.0 mueq/ml of any of the eight salts increased the residual cell concentration by one order of magnitude from 1.2 x 10(4) to about 4.0 x 10(5) cells per milliliter. This occurred regardless of the sequence of contact as long as the surfactant contact period was sufficient. The presence of salts increased collapsed foam volumes with P. fluorescens and decreased collapsed foam volumes with B. subtilis.     

Electron microscopy of isolated cell walls of Bacillus subtilis var. niger

Isolated cell walls of Bacillus subtilis have a striated appearance in the electron microscope. The structure persists when teichoic acids are removed. It is inferred that the structure bears on the arrangement of the peptidoglycan chains.     

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.     

The wall content and composition of Bacillus subtilis var. niger grown in a chemostat

Bacillus subtilis var. niger was grown in a chemostat with various growth limitations and at various growth rates. The wall content and composition of the organism grown under these conditions were determined. The wall content, expressed as a percentage of the dry weight of organisms, varied with the growth rate. Analysis of wall samples showed that their composition also varied, particularly with respect to the phosphorus content. Wall samples extracted with trichloroacetic acid under carefully controlled conditions were found to contain various amounts of phosphorus, this being present as a glycerol phosphate polymer containing hexose (glucose and in some cases galactose), i.e. a teichoic aid. Teichoic acids were present in the walls of organisms grown under all conditions except when phosphorus limited growth. Then a different anionic polymer, composed of glucuronic acid and N-acetylgalactosamine (a teichuronic acid), was present. Under the specific growth conditions at pH7.0 and 35 degrees C in a chemostat, teichoic acid and teichuronic acid appeared to be mutually exclusive.     

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.     

四溴甘脲对枯草杆菌黑色变种芽胞损伤作用的电镜观察

为探索四溴甘脲消毒剂杀灭细菌的机理,采用透射电镜技术对四溴甘脲消毒剂处理过的枯草杆菌黑色变种芽胞的超微结构进行了分析和比较.结果显示,以含有效溴274mg/L的四溴甘脲消毒剂作用30min,可使枯草杆菌黑色变种芽胞杀灭率达到100%.在透射电镜下观察到,经该消毒剂作用的枯草杆菌黑色变种芽胞壳质破损断裂明显,壳内结构模糊,核心溶解,有的芽胞近似空壳.结果显示,四溴甘脲消毒剂杀灭芽胞效果优于普通含氯消毒剂,对细菌芽胞超微结构破坏明显.     

Influence of Spore Moisture Content on the Dry-Heat Resistance of Bacillus subtilis var. niger

The dry-heat resistance of Bacillus subtilis var. niger spores located in or on various materials was determined as D and z values in the range of 105 through 160 C. The systems tested included spores located on steel and paper strips, spores located between stainless-steel washers mated together under 150 inch-lb and 12 inch-lb of torque, and spores encapsulated in methylmethacrylate and epoxy plastics. D values for a given temperature varied with the test system. High D values were observed for the systems in which spores were encapsulated or under heavy torque, whereas lower D values were observed for the steel and paper strip systems and the lightly torqued system. Similar z values were obtained for the plastic and steel strip systems (z(D) = 21 C), but an unusually low z for spores on paper (z(D) = 12.9 C) and an unusually high z for spores on steel washers mated at 150 inch-lb of torque (z(D) = 32 C) were observed. The effect of spore moisture content on the D value of spores encapsulated in water-impermeable plastic was determined, and maximal resistance was observed for spores with a water activity (a(w)) of 0.2 to 0.4. Significantly decreased D values were observed for spores with moisture contents below a(w) 0.2 or above a(w) 0.4. The data indicate that the important factors to be considered when measuring the dry heat resistance of spores are (i) the initial moisture content of the spore, (ii) the rate of spore desiccation during heating, (iii) the water retention capacity of the material in or on which spores are located, and (iv) the relative humidity of the system at the test temperature.     

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.     

Resistance of Bacillus subtilis var. niger Spores Occluded in Water-insoluble Crystals to Three Sterilization Agents

The resistance to destruction of spores of Bacillus subtilis var. niger occluded in crystals of calcium carbonate and exposed to ethylene oxide and moist and dry heat was determined and compared with the destruction of unoccluded spores. Occluded spores could not be inactivated with ethylene oxide. Resistance to inactivation was approximately 900 and 9 times higher for occluded than for unoccluded spores subjected to moist and dry heat, respectively, at 121 C. The protective effect may be due either to the unavailability of oxygen for destruction by oxidation or to inhibition of the loss of essential cell constituents by vaporization. Evidence also implicates the crystal structure as a thermal conductivity barrier. Occluded spores retained viability over a 3-year period compared with unoccluded spores which decreased over 90% during this period. Occluded spores in insoluble materials are seldom encountered in the technology of sterilization, but could be the most critical factor in the sterilization of interplanetary vehicles. Entrapped spores in insoluble materials are usually difficult to detect, and are very stable as well as extremely resistant to destruction by heat and ethylene oxide.     

Control of teichoic acid and teichuronic acid biosynthesis in chemostat cultures of Bacillus subtilis var. niger

1. Quantitative determination of the anionic polymers present in the walls of Bacillus subtilis var. niger organisms undergoing transition, in a chemostat culture, from either Mg(2+)-limitation to PO(4) (3-)-limitation or K(+)-limitation to PO(4) (3-)-limitation showed that teichuronic acid synthesis started immediately the culture became PO(4) (3-)-limited and proceeded at a rate substantially faster than the rate of biomass synthesis. 2. Simultaneously, the cell-wall teichoic acid content diminished at a rate greater than that due to dilution by newly synthesized wall material, and fragments of teichoic acid and mucopeptide accumulated in the culture extracellular fluid. 3. Equally rapid reverse changes occurred when a PO(4) (3-)-limited B. subtilis var. niger culture was returned to being Mg(2+)-limited. 4. It is concluded that in this organism both teichoic acid and teichuronic acid syntheses are expressions of a single genotype, and a mechanism for the control of synthesis of both polymers is suggested. 5. These results are discussed with reference to the constantly changing environmental conditions that obtain in a batch culture and the variation in bacterial cell-wall composition that is reported to occur throughout the growth cycle.