Germination of Single Bacterial Spores

Changes in refractility and optical density occurring in individual spores of Bacillus cereus T and B. megaterium QM B1551 during germination were investigated by use of a Zeiss microscope photometer. The curves revealed that the germination process in single spores had two distinct phases; an initial rapid phase was followed by a second slower phase. Under the experimental condition employed, the first phase of germination of B. cereus spores lasted for approximately 75 +/- 15 sec, whereas the second phase lasted for 3 to 4.5 min. In B. megaterium spores, the first phase was observed to last for approximately 2 min and the second phase for more than 7 min. The duration of the second phase was dependent on conditions employed for germination. The kinetics of the first phase were strikingly similar under all conditions of physiological germination. Time-lapse phase-contrast microscopy of germinating spores also revealed the biphasic nature of germination. It was postulated that the first phase represents changes induced by an initial partial hydration of the spore and release into the medium of dipicolinic acid, whereas the second phase reflects degradation of the cortex and hydration of the core.     

Emerging Applications of Bacterial Spores in Nanobiotechnology

Bacterial spores are robust and dormant life forms with formidable resistance properties, in part, attributable to the multiple layers of protein that encase the spore in a protective and flexible shield. The coat has a number of features pertinent to the emerging field of nanobiotechnology including self-assembling protomers and the capacity for engineering and delivery of foreign molecules. This review gives an account of recent progress describing the use of the spore, and specifically, the spore coat as a vehicle for heterologous antigen presentation and protective immunization (vaccination). As interest in the spore coat increases it seems likely that they will be exploited further for drug and enzyme delivery as well as a source of novel self-assembling proteins.     

Biochemical Studies on Germination of Bacterial Spores

The initiating mechanism in the germination of Bacillus thiaminolyticus spores was studied with ¹⁴C-L -alanine. A characteristic pattern of incorporation of L -alanine into the spores was observed during the early stages of germination with two incorporation peaks, one occurred just after contact with L -alanine (first incorporation) and the other 5 min later (second incorporation). L -Glutamine, L -valine, or L -serine substituted for the incorporation of L -alanine during the first stage of germination. Although, L -alanine taken up during the first incorporation phase was extractable with trichloroacetic acid (TCA), that taken up during the second incorporation phase was not extractable. The distribution of radioactivity showed that incorporated L -alanine was located in the spore coat, mainly in the paracrystal fraction. The radioactive material which remained in the germination medium or was extractable from the spore coat fraction with TCA treatment or pronase digestion was identified as alanine. Significance of incorporation of L -alanine and its location in the spore in reference to the initiation of germination is discussed.     

Scanning Electron and Phase-Contrast Microscopy of Bacterial Spores

The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.     

Chemical States of Bacterial Spores: Dry-Heat Resistance

Mature bacterial spores can be manipulated by chemical pretreatments between states sensitive and resistant to dry heat. The two chemical forms of the spore differ in dry-heat resistance by about an order of magnitude. Log survivor curves for each chemical state were approximately straight lines. The temperature dependence of dry-heat resistance for each chemical state was similar to that usually found for dry-heat resistance. A method of testing spore resistance to dry heat has been designed to minimize artifacts resulting from (i) change of chemical state during the test, (ii) effects of water vapor activity, (iii) incomplete recovery of spores from the test container and clumping of spores. Implications of the existence of different chemical resistance states for experimental strategy and testing of dry-heat resistance are discussed.     

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.     

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.     

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.     

Liquid Chromatographic Determination of Dipicolinic Acid from Bacterial Spores

Dipicolinic acid was determined by reverse-phase liquid chromatography. Elution was with 0.2 M potassium phosphate, pH 1.8, containing 1.5% tert-amyl alcohol or higher concentrations of lower alcohols or acetonitrile. The normal analytical range was 50 to 1,000 μM, which is equivalent to 0.1 to 1 mg of spores per ml with a relative standard error of 2 to 4% and a detection limit of <100 pmol. Dipicolinic acid was fully extracted from spores by heating at pH 1.8 for 10 min at 100°C. Sporulating cultures may be analyzed in less than 20 min without separation of cells from media. Liquid chromatography was also used to detect dipicolinic acid in more complex substrates, e.g., guinea pig feces containing Metabacterium polyspora spores and canned food. Dipicolinic acid could be detected in unspoiled canned salmon containing <10⁶ added Bacillus cereus spores per g.     

Bacterial Spores Survive Treatment with Commercial Sterilants and Disinfectants

This study compared the activity of commercial liquid sterilants and disinfectants on Bacillus subtilis spores deposited on three types of devices made of noncorrodible, corrodible, or polymeric material. Products like Renalin, Exspor, Wavicide-01, Cidexplus, and cupric ascorbate were tested under conditions specified for liquid sterilization. These products, at the shorter times indicated for disinfection, and popular disinfectants, like Clorox, Cavicide, and Lysol were also studied. Data obtained with a sensitive and quantitative test suggest that commercial liquid sterilants and disinfectants are less effective on contaminated surfaces than generally acknowledged.