Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

Gas plasma, produced by a short high­voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme­linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2­4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75­142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO­), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

Kinetics of germination of individual spores of Geobacillus stearothermophilus as measured by raman spectroscopy and differential interference contrast microscopy.

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T(lag)) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T(release), and DT(release) (T(release) - T(lag)) was 1-2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T(lag) values. 4) Values of T(lag) and T(release) were heterogeneous among individual spores, but DT(release) values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T(lag) values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer T(lag) values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species.

Amyloid histology stain for rapid bacterial endospore imaging.

Bacterial endospores are some of the most resilient forms of life known to us, with their persistent survival capability resulting from a complex and effective structural organization. The outer membrane of endospores is surrounded by the densely packed endospore coat and exosporium, containing amyloid or amyloid-like proteins. In fact, it is the impenetrable composition of the endospore coat and the exosporium that makes staining methodologies for endospore detection complex and challenging. Therefore, a plausible strategy for facile and expedient staining would be to target components of the protective surface layers of the endospores. Instead of targeting endogenous markers encapsulated in the spores, here we demonstrated staining of these dormant life entities that targets the amyloid domains, i.e., the very surface components that make the coats of these species impenetrable. Using an amyloid staining dye, thioflavin T (ThT), we examined this strategy. A short incubation of bacillus endospore suspensions with ThT, under ambient conditions, resulted in (i) an enhancement of the fluorescence of ThT and (ii) the accumulation of ThT in the endospores, affording fluorescence images with excellent contrast ratios. Fluorescence images revealed that ThT tends to accumulate in the surface regions of the endospores. The observed fluorescence enhancement and dye accumulation, coupled with the sensitivity of emission techniques, provide an effective and rapid means of staining endospores without the inconvenience of pre- or posttreatment of samples.

Morpho-structural variations of bacterial spores after treatment in steam vacuum assisted autoclave.

This study intended to verify, through microbiological techniques and TEM investigations, the killing of bacterial spores after treatment in steam autoclave, and to propose strictly morphological considerations about the target of this sterilisation process. Autoclave is the most common device for sterilising instruments in order to prevent cross infections in dental offices. The autoclave efficiency has been improved in the last years and part of this improvement is related to both a better and more correct use of the autoclave system and to the technological innovations introduced in the last generation of devices. However, associations as ADA or CDC suggest to regularly verify the process of 'autoclaving' through biological indicators (BI). The most commonly used BI are made of spores strips or suspensions of Bacillus Subtilis (pb 168) and Bacillus Stearothermophilus (ATCC 10149). They visually prove, changing colours on enzymatic base, the death of micro-organism and if the physical parameters, necessary for sterilisation, have been achieved. These two strains of endospore-forming bacteria were processed and prepared following two different techniques: Karnovsky fixed and epon embedded--phosphotungstic acid fixed for direct observation. The kind and the extent of analysed modifications are extremely various: from deep lacerations, which changed the spore structure, to little clefts which let the cytoplasm go out.

Localized insertion of new S-layer during growth of Bacillus stearothermophilus strains.

Bacillus stearothermophilus strains PV 72 and ATCC 12980 carry a crystalline surface layer (S-layer) with hexagonal (p6) and oblique (p2) symmetry, respectively. Sites of insertions of new subunits into the regular lattice during cell growth have been determined by the indirect fluorescent antibody technique and the protein A/colloidal gold technique. During S-layer growth on both bacillus strains the following common features were noted: 1. shedding of intact S-layer or turnover of individual subunits was not seen; 2. new S-layer was deposited in helically-arranged bands over the cylindrical surface of the cell at a pitch angle related to the orientation of the lattice vectors of the crystalline array; 3. little or no S-layer was inserted into pre-existing S-layer at the poles, and 4. septal regions and, subsequently, newly formed cell poles were covered with new S-layer protein.

Steam sterilization of air turbine dental handpieces.

The efficacy of autoclaving high-speed dental handpieces was tested in two types of downward displacement steam sterilizers (instrument autoclaves), commonly used in the dentist's office. Eight series of experiments were performed with various sterilization schedules. Lubrication oils with or without antimicrobial agent were used in four of the series. Each handpiece was inoculated with 1 ml of a suspension containing equal amounts of saliva and spores of Bacillus stearothermophilus (approx. 10(6) spores/ml). Neither sterilization at 120-124 degrees C for 20 min nor at 134-136 degrees C for 10 min resulted in complete inactivation of the spores in series in which the instruments were tested without oil or with oil containing no antimicrobial agent. However, when the handpieces were lubricated with oil containing isopropanol and formaldehyde and the schedule 134-136 degrees C/10 min was used, no growth was observed. The results indicate that instrument autoclaves with built-in programs of 120-124 degrees C/20 min and 134-136 degrees C/10 min could have insufficient capacity to sterilize lubricated or unlubricated dental handpieces. The use of oils containing an antimicrobial agent can overcome this problem.

[Growth of obligate-thermophilic bacteria on a medium with paraffin]. / Razvitie obligatno-termofil'nykh bakterii na srede s parafinom.

The obligate thermophilic bacteria Bacillus stearothermophilus (the optimal growth temperature 55-73 degrees C) and Thermus ruber (the optimal growth temperature 60 degrees C) were isolated from hot water springs of the Kunashir Island for the first time in a liquid mineral medium with paraffin. Some of the bacteria belonging to Bac. stearothermophilus grew at a high rate in a liquid medium with paraffin (strain 16); other strains (12a, 12b) were capable of growing only together with Thermus ruber; some strains could grow only on a solid medium with paraffin (strains 14a, 14b).

Criteria for determining steam sterilization cycles for lubricated and nonlubricated mechanical equipment.

Requirements and procedures for determining steam sterilization cycles for lubricated and nonlubricated, complex, mechanical equipment are established. Factors affecting exposure times are discussed, as are procedures and data needed to determine the penetrability of steam into mechanical equipment, and the effect of lubricating oil in impeding steam penetration to the spore. The total effects of these factors in sterilizing times are presented. Sterilizers with a prevacuum cycle, which can remove air from the equipment efficiently and replace it with saturated steam, are recommended for complex mechanical equipment.