Pathways of Glucose Metabolism by Rough and Smooth Variants of Bacillus stearothermophilus

The radiorespirometric method was used to study the pathways of glucose metabolism in the rough and smooth variants of Bacillus stearothermophilus NCA 1518. The Embden-Meyerhof (EM) pathway was more active in the smooth variant than in the rough variant. The participation of the hexose monophosphate shunt (HMP) and EM pathways in the smooth variant was calculated as 4.2 and 95.8%, respectively. The rough variant utilized glucose via the EM pathway exclusively or in combination with a pathway other than the HMP pathway. The estimated efficiency of the tricarboxylic acid system in the rough and smooth variants was 81.3 and 4.9%, respectively.     

Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores

Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.     

Heat Shock Affects Permeability and Resistance of Bacillus stearothermophilus Spores

[This corrects the article on p. 2517 in vol. 54.].     

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.     

Sporulation and Heat Resistance of Bacillus stearothermophilus Spores Produced in Chemically Defined Media

The effect of amino acids on sporulation is discussed. Heat-resistant spores were produced in a chemically defined medium.     

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.     

Effect of Soybean Casein Digest Agar Lot on Number of Bacillus stearothermophilus Spores Recovered

In recent years it has become increasingly apparent that Bacillus stearothermophilus spores are affected by various environmental factors that influence the performance of the spores as biological indicators. One environmental factor is the recovery medium. The effect of different lots of commercial soybean casein digest agar on the number of colony-forming units per plate was examined in two series of experiments: (i) several lots of medium from two manufacturers were compared in single experiments, and (ii) paired media experiments with four lots of medium were carried out and yielded three-point survivor curves. The results demonstrate that commercial soybean casein digest agar is variable on a lot-to-lot basis. The variation was lowest when recovering unheated or minimally heated spores and increased greatly with the severity of heating.     

Studies on Variants of Bacillus stearothermophilus Strain NCA 1518

The heat resistance, fermentation reactions, nutritional requirements, and phage sensitivity of 18 selected morphological variants of Bacillus stearothermophilus NCA 1518 were studied. It was found that when smooth variants mutated to rough colonial morphology, there was no concurrent change in fermentation reactions, nutritional requirements, or heat resistance. The smooth variant, and the rough mutants derived directly from it, presented a uniform pattern of biochemical capabilities which differed from the pattern presented by the rough variants isolated from the same stock culture. This led to the conclusion that the smooth and rough types previously observed in stocks of B. stearothermophilus NCA 1518 either were carried in the stock since the original isolation or represent a very profound and uncommon mutation, or that one of the variants has been introduced into the stock culture from an extraneous source sometime in the past.     

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.     

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.     

Fatty Acids from Vegetative Cells and Spores of Bacillus stearothermophilus

In spores of Bacillus stearothermophilus produced at 45 and 55 C, branched-chain fatty acids predominated in the former and straight-chain acids in the latter.     

Effect of Calcium in Assay Medium on D Value of Bacillus stearothermophilus ATCC 7953 Spores

The D value of commercial biological indicator spore strips using Bacillus stearothermophilus ATCC 7953 was increased by higher calcium concentrations in assay media. The calcium concentration in assay media varied among the manufacturers. The calcium concentration in assay media is an important factor to consider to minimize the variation of D value.     

Effect of Ultrasonic Waves on the Heat Resistance of Bacillus cereus and Bacillus licheniformis Spores

Heat resistance of Bacillus cereus and Bacillus licheniformis spores in quarter-strength Ringer solution decreases markedly after ultrasonic treatments which are unable to kill a significant proportion of the spore population. This effect does not seem to be caused by a loss of Ca(2+) or dipicolinic acid. The use of ultrasonics to eliminate vegetative cells or to break aggregates in Bacillus spore suspensions to be used subsequently in heat resistance experiments appears to be unadvisable.     

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.     

Relationship between Heat Activation and Percentage Colony Formation for Bacillus stearothermophilus Spores: Effects of Storage and pH of the Recovery Medium

Colony counts of unheated spores were higher in a medium at pH 5·9 than in media of higher pH. The pattern was reversed as the spores were heated. Slide germination studies showed that about 8% or less of unheated spores germinated in slide culture. Optimal heat activation resulted in about 50% germination. The colony counts of heat activated spores dropped significantly upon storage for 9 months, but those of unheated spores did not.     

Heat Resistance and Population Stability of Lyophilized Bacillus subtilis Spores

Bacillus subtilis 5230 spores were lyophilized in 0.067 M phosphate buffer and stored at 2 to 8°C for 9 to 27 months. The lyophilized spores were reconstituted with buffer or 0.9% saline, and the heat resistance was determined in a thermoresistometer. Lyophilization had no effect on the heat resistance of the spores but did result in a slight decrease in population (≤0.3-logarithm reduction). The lyophilized spores maintained heat resistance and population levels over the test periods. The D-values ranged from 0.44 to 0.54 min at 121.1°C, and the z-values ranged from 6.1 to 6.6°C. Lyophilization was concluded to be an acceptable alternative for storage of bacterial spores that are to be used as biological indicators in sterilization processes.     

Heat shock affects permeability and resistance of Bacillus stearothermophilus spores

Heat shock of dormant spores of Bacillus stearothermophilus ATCC 7953 at 100 or 80 degrees C for short times, the so-called activation or breaking of dormancy, was investigated by separating the resulting spores by buoyant density centrifugation into a band at 1.240 g/ml that was distinct from another band at 1.340 g/ml, the same density as the original spores. The proportion of spores at 1.240 g/ml became larger when the original dormant spores were heated for a longer period of time, but integument-stripped dormant spores were quickly and completely converted to spores with a band at 1.240 g/ml. The spores with bands at both 1.240 and 1.340 g/ml were germinable faster than the original dormant spores and thus were considered to be activated. The spores with a band at 1.240 g/ml, which were considered to be fully activated, were apparently permeabilized, with a resulting complete depletion of dipicolinic acid, partial depletion of minerals, susceptibility to lysozyme action, permeation of the gradient medium, changed structural appearance in electron micrographs of thin-sectioned spores, and partly decreased heat resistance (D100 = 453 min) compared with the original dormant spores (D100 = 760 min). However, the fully activated spores with a band at 1.240 g/ml, although devoid of dipicolinic acid, still were much more resistant than germinated spores or vegetative cells (D100 = 0.1 min). The spores with a band at 1.340 g/ml, which were considered to be partly activated, showed no evidence of permeabilization and were much more heat resistant (D100 = 1,960 min) than the original dormant spores.(ABSTRACT TRUNCATED AT 250 WORDS)