Homogeneous Bacterial Aerosols Produced with a Spinning-Disc Generator

Aerosols composed of viable particles of a uniform size were produced with a commercial spinning-disc generator from aqueous suspensions of Bacillus subtilis var. niger spores containing various amounts of an inert material, dextran, to regulate aerosol particle size. Aerosols composed of single naked spores having an equivalent spherical diameter of 0.87 mum were produced from spore suspensions without dextran, whereas aerosols produced from suspensions containing 0.001, 0.01, 0.1, and 1% dextran had median diameters of 0.90, 1.04, 1.80, and 3.62 mum, respectively. Such aerosols, both homogeneous and viable, would be useful for calibrating air sampling devices, evaluating air filter systems, or for employment wherever aerosol behavior may be size-dependent.     

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.     

Trajectory of aerosol droplets from a sprayed bacterial suspension

Simulated droplet trajectories of a polydispersed microbial aerosol in a laminar air flow regimen were compared with observed dispersal patterns of aerosolized Bacillus subtilis subsp. niger spores in quasilaminar airflow. Simulated dispersal patterns could be explained in terms of initial droplet sizes and whether the droplets evaporated to residual aeroplanktonic size before settling to the ground. For droplets that evaporated prior to settling out, a vertical downwind size fractionation is predicted in which the microbial residue of the smallest droplets settles the least, and is found in the airstream at about sprayer height, and progressively larger droplet residues settle to progressively lower heights. Observations of spore particle size distributions downwind from a spray source support the simulation. Droplet and particle size distributions near the source had three size fractions: one containing large, presumably nonevaporated droplets of greater than or equal to 7 microns in diameter, and two smaller fractions, with diameters of 2 to 3 microns (probably the residue of droplets containing more than one spore) and 1 to 2 microns (probably the residue from single-spore droplets). As predicted by the simulation, the aerosol settled and progressed downwind, with the number of small droplets and particles increasing in proportion to the height and distance downwind.     

Trajectory of aerosol droplets from a sprayed bacterial suspension.

Simulated droplet trajectories of a polydispersed microbial aerosol in a laminar air flow regimen were compared with observed dispersal patterns of aerosolized Bacillus subtilis subsp. niger spores in quasilaminar airflow. Simulated dispersal patterns could be explained in terms of initial droplet sizes and whether the droplets evaporated to residual aeroplanktonic size before settling to the ground. For droplets that evaporated prior to settling out, a vertical downwind size fractionation is predicted in which the microbial residue of the smallest droplets settles the least, and is found in the airstream at about sprayer height, and progressively larger droplet residues settle to progressively lower heights. Observations of spore particle size distributions downwind from a spray source support the simulation. Droplet and particle size distributions near the source had three size fractions: one containing large, presumably nonevaporated droplets of greater than or equal to 7 microns in diameter, and two smaller fractions, with diameters of 2 to 3 microns (probably the residue of droplets containing more than one spore) and 1 to 2 microns (probably the residue from single-spore droplets). As predicted by the simulation, the aerosol settled and progressed downwind, with the number of small droplets and particles increasing in proportion to the height and distance downwind.     

Effect of NO2 on Airborne Venezuelan Equine Encephalomyelitis Virus

Studies were conducted to determine the effect of nitrogen dioxide (NO(2)) on aerosol survival and biological decay rate of Venezulean equine encephalomyelitis (VEE) virus and spores of Bacillus subtilis var. niger. The NO(2) concentrations used in the experiments were 0.5, 5, and 10 ppm at 24 C and 85% RH. The survival of airborne VEE virus disseminated as particles 1 to 5 mum in diameter was significantly influenced by the presence of 5 ppm of NO(2). At this concentration, the biological decay rate increased threefold and the aerosol recovery and aerosol survival of the VEE virus were significantly lower than at 0.5 ppm or in the absence of NO(2). Airborne spores of B. subtilis were not significantly affected by as much as 10 ppm of NO(2).     

Aerosol stability of infectious and potentially infectious reovirus particles.

The aerosol stability of two particle forms, infectious and potentially infectious, of reovirus were examined under static conditions for a range of relative humidities at 21 and 24 degrees C. Virus aerosolization efficiency was determined for two methods of dissemination: Collison nebulizer and Chicago atomizer. Suspensions of Bacillus subtilis var. niger spores were added to reovirus preparations that included both particle forms and disseminated into a dynamic aerosol toroid to estimate the physical decay of the aerosols. At 90 to 100% relative humidity, both reovirus particle forms showed less than 10-fold loss of infectivity after 12 h of aging. At lower relative humidities the aerosol decay curve showed rapid initial decay followed by a markedly lower decay rate. Our findings reveal that reovirus particles are relatively stable in the airborne state.     

Microbiological Aspects of Ethylene Oxide Sterilization: IV. Influence of Thickness of Polyethylene Film on the Sporicidal Activity of Ethylene Oxide

Spores of Bacillus subtilis var. niger, dried on nonhygroscopic and hygroscopic surfaces, were enclosed in one of four thicknesses of low-density polyethylene film (2, 4, 6, and 20 mils). The surfaces were then placed in a specially designed thermochemical death rate apparatus and exposed to an ethylene oxide concentration of 600 mg/liter (at 54.4 C) and 50% relative humidity. Survival data, including both spore survivor curves and decimal reduction values (expressed as D values at 54.4 C-600 mg of ethylene oxide per liter), demonstrated similar survivor patterns when the B. subtilis var. niger spores were enclosed in low-density polyethylene films 2, 4, and 6 mils thick. A comparison of these patterns with those of spores enclosed in glassine and subjected to identical exposure conditions revealed only slight variations. The use of 20-mil polyethylene film greatly increased the time required to kill B. subtilis var. niger spores under the exposure conditions.     

Microbiological Safety Evaluation of an Industrial Refuse Incinerator

An industrial refuse incinerator was tested to determine minimal operating temperatures required to prevent release of viable microorganisms into the atmosphere. A liquid suspension of Bacillus subtilis var. niger spores was disseminated into the firebox as an aerosol, and dry spores mixed with animal bedding were dumped into the firebox. The minimal requirement for wet spores was 575 F (302 C) for the firebox air temperature and 385 F (196 C) for the firebrick refractory lining. When dry spores were used, these temperatures were 700 and 385 F (371 and 196 C), respectively.     

Comparison of aerosol and bioaerosol collection on air filters

Air filters efficiency is usually determined by non-biological test aerosols, such as potassium chloride particles, Arizona dust or di-ethyl-hexyl-sebacate (DEHS) oily liquid. This research was undertaken to asses, if application of non-biological aerosols reflects air filters capacity to collect particles of biological origin. The collection efficiency for non-biological aerosol was tested with the PALAS set and ISO Fine Test Dust. Flow rate during the filtration process was 720 l/h, and particles size ranged 0.246–17.165 μm. The upstream and downstream concentration of the aerosol was measured with a laser particle counter PCS-2010. Tested bioaerosol contained 4 bacterial strains of different shape and size: Micrococcus luteus, Micrococcus varians, Pseudomonas putida and Bacillus subtilis. Number of the biological particles was estimated with a culture-based method. Results obtained with bioaerosol did not confirmed 100% filters efficiency noted for the mineral test dust of the same aerodynamic diameter. Maximum efficiency tested with bacterial cells was 99.8%. Additionally, cells reemission from filters into air was also studied. Bioaerosol contained 3 bacterial strains: Micrococcus varians, Pseudomonas putida and Bacillus subtilis. It was proved that the highest intensity of the reemission process was during the first 5 min. and reached maximum 0.63% of total number of bacteria retained in filters. Spherical cells adhered stronger to the filter fibres than cylindrical ones. It was concluded that non-biological aerosol containing particles of the same shape and surface characteristics (like DEHS spherical particles) can not give representative results for all particles present in the filtered air.     

The effect of transition metal ions on the resistance of bacterial spores to hydrogen peroxide and to heat.

The presence of 10 microM-Cu2+ increased the lethal effect of hydrogen peroxide on spores of Clostridium bifermentans but not on those of Clostridium sporogenes PA 3679, Clostridium perfringens, Bacillus cereus or Bacillus subtilis var. niger. Cu2+ at 100 muM also increased the lethal effect of heat on spores of C. bifermentans but not on those of B. sutilis var. niger. The rate and extent of Cu2+ uptake by spores of C. bifermentans and B. subtilis var. niger were similar, but examination of unstained sections of spores by electron microscopy suggested that Cu2+ is bound by the protoplasts of spores of C. bifermentans but not of B. subtilis var. niger.     

Microbiological Aspects of Ethylene Oxide Sterilization: II. Microbial Resistance to Ethylene Oxide

The death rate kinetics of several sporeforming and nonsporeforming microorganisms, including radiation-resistant cocci, were determined by exposing them to a mixture of ethylene oxide and dichlorodifluoromethane (500 mg of ethylene oxide per liter, 30 to 50% relative humidity, and 54.4 C). Spore survivor curves obtained from tests of inoculated and exposed hygroscopic and nonhygroscopic carriers showed that the spores of Bacillus subtilis var. niger are more resistant to ethylene oxide than are spores of Clostridium sporogenes, B. stearothermophilus, and B. pumilus. The decimal reduction times (expressed as D values at 54.4 C-500 mg of ethylene oxide per liter) obtained under the test conditions for B. subtilis var. niger spores on hygroscopic and nonhygroscopic carriers exceeded the values obtained for the other organisms considered, both sporeformers and nonsporeformers. The decimal reduction times for the vegetative cells of the radiation-resistant organisms (Micrococcus radiodurans and two strains of Streptococcus faecalis) and the ATCC strain of S. faecalis demonstrated comparable resistance to ethylene oxide with the spores of C. sporogenes, B. stearothermophilus, and B. pumilus, but not those of B. subtilis var. niger.     

Resistance of Micro-organisms to Inactivation by Gaseous Ethylene Oxide

A simple method for the exposure of micro-organisms to ethylene oxide on membrane filters in a modified desiccator has been devised and used to study microbial resistance to the gaseous sterilant and the term ' R -value' is suggested to express this. The resistance of many known species and isolates has been assessed and compared. Several species of Bacillus were isolated from natural habitats and their spores were found to be more resistant than the strain of Bacillus subtilis var. niger (NCTC 10073) frequently used to monitor ethylene oxide sterilization. However, endospores of some bacterial species exhibited little resistance. Fungal spores and vegetative bacteria exhibited low resistance to the sterilant except after drying in organic material when they appeared more resistant than spores of B. subtilis var. niger. It was concluded that resistance to ethylene oxide did not correlate with resistance to heat, irradiation or other chemical disinfectants, or to the existence in the endospore form per se.     

Resistivity of Spores to Ultraviolet and γ Radiation while Exposed to Ultrahigh Vacuum or at Atmospheric Pressure

Viability studies were conducted on microbial spores subjected to ultrahigh vacuum (UHV) in the 10(-9) to 10(-10) torr range. After 5 to 7 days in vacuum, they were exposed to ultraviolet (UV) or to gamma radiation either while still under vacuum or in the presence of dried air. Among the four test organisms subjected to UHV and ultraviolet radiation, Aspergillus niger was the most resistant; Bacillus megaterium, B. subtilis var. niger, and B. stearothermophilus were about equally less resistant. All four spores were more sensitive to ultraviolet radiation when UHV-dried than when desiccant-dried. Of the four test organisms subjected to UHV and gamma radiation, B. megaterium proved to be the most resistant; A. niger was the least resistant; and the remaining two organisms were of intermediate resistivity. All four organisms were less radiation resistant when UHV-dried than when irradiated in their normally hydrated state, and all showed an increased radiosensitivity after vacuum drying when oxygen was present. In addition, spores of B. subtilis var. niger and A. niger were less radiosensitive when UHV-dried and irradiated in vacuum than when "wet" and irradiated in air, whereas the reverse relationship was observed for the remaining two organisms. Based on the fact that microbial contaminants can be readily shielded from UV light by soils, metal particles, etc., and considering that the levels of ionizing radiations reported to be present in interstellar space are generally lower than those used in these experiments, the decrease in radioresistivity imparted by UHV drying is not of a sufficient magnitude to sterilize dependably portions of a spacecraft while on a mission.     

An assessment of the Sartorius MD8 microbiological air sampler

Tests described in this paper show that gelatine membrane filters used in the MD8 microbiological air sampling system collected monodispersed aerosols between 0·7 and 1·0 μm containing viable Bacillus subtilis var. niger spores, with an efficiency of 99·9995%. Gelatine membrane filters linked to the MD8 control pump system were as effective as the well established Casella slit-to-agar device for collecting some viable bacteria, nebulized under controlled experimental conditions and naturally occurring airborne micro-organisms in a pharmaceutical plant. By using a long flexible hose connection to the control pump, the head could be positioned where sampling was required in locations remote from the pump exhaust, making it suitable for microbiological monitoring in critical locations such as laminar flow stations and isolators.     

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.     

Evaluation of laminar flow microbiological safety cabinets

The microbiological control efficiency of two class 100 laminar down-flow hoods was determined by using aerosols of Bacillus subtilis var. niger spores. The first unit challenged utilized a slanted eyelid to partially enclose the front work opening. This hood showed nearly perfect control of ambient organisms in the work area. It also gave a 10(6) or greater drop in the number of organisms passing out of the exhaust system. However, when the interior work area of the hood was challenged, significant numbers of spores penetrated the air barrier and escaped into the ambient air. A redesigned laminar flow hood was built incorporating a vertical eyelid and a reduced opening to the work area. This hood showed the same excellent characteristics for controlling ambient contamination. Exhaust system leakage was also extremely low. Air barrier efficiency for the newer hood was increased with lower amounts of spore penetration into the ambient air.     

Microbiological hazard from the exhaust of a high-vacuum sterilizer.

Data are presented which show the potential for release of viable microorganisms into the atmosphere from high-vacuum steam sterilizers during the evacuation cycle preceding application of steam under pressure. Bacillus subtilis var. niger spores, Serratia marcescens cells, and T1 coliphage disseminated into the sterilizer chamber as small particles from liquid suspensions, and dried spores of B. subtilis var. niger distributed on bulk discard materials were recovered from the atmosphere around pipes venting steam from the steam ejectors used to create chamber vacuum. Evaluation of the hazard involved is discussed, and the design, fabrication, and installation of a valved filter system for preventing release of viable microorganisms are presented. The filtration system utilized an F-700 water-resistant filter and was shown to eliminate the release of viable airborne microorganisms from a high-vacuum sterilizer. A method is presented for determining size requirements for an atmospheric vent filter in relation to the volume of a sterilizer.     

The Effect of Halocarbon Aerosol Propellents on Bacteria

SUMMARY: The survival of Escherichia coli and spores of Bacillus subtilis var. niger and B. stearothermophilus suspended in halocarbon propellents for several weeks has been demonstrated. There was no significant loss of dipicolinic acid from the spores. Lethal action may be due to interaction of the propellents with cellular lipid.     

Experimental Technique for Studying Aerosols of Lyophilized Bacteria

An experimental technique is presented for studying aerosols generated from lyophilized bacteria by using Escherichia coli B, Bacillus subtilis var. niger, Enterobacter aerogenes, and Pasteurella tularensis. An aerosol generator capable of creating fine particle aerosols of small quantities (10 mg) of lyophilized powder under controlled conditions of exposure to the atmosphere is described. The physical properties of the aerosols are investigated as to the distribution of number of aerosol particles with particle size as well as to the distribution of number of bacteria with particle size. Biologically unstable vegetative cells were quantitated physically by using ¹⁴C and Europium chelate stain as tracers, whereas the stable heat-shocked B. subtilis spores were assayed biologically. The physical persistence of the lyophilized B. subtilis aerosol is investigated as a function of size of spore-containing particles. The experimental result that physical persistence of the aerosol in a closed aerosol chamber increases as particle size is decreased is satisfactorily explained on the bases of electrostatic, gravitational, inertial, and diffusion forces operating to remove particles from the particular aerosol system. The net effect of these various forces is to provide, after a short time interval in the system (about 2 min), an aerosol of fine particles with enhanced physical stability. The dependence of physical stability of the aerosol on the species of organism and the nature of the suspending medium for lyophilization is indicated. Also, limitations and general applicability of both the technique and results are discussed.     

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.