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.     

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.     

Microbial validation of vent filters

The Upjohn Company uses filtration to remove microorganisms and particulates from air and other gases which may come in contact with sterile products. To validate the microbial retentivity of these filters, they were challenged with an aerosol of Bacillus subtilis var niger spores. An aerosol challenge was used because it more closely simulated the use for which these filters were designed. The test apparatus was constructed of autoclavable components using a jet-type nebulizer and heated air mixing tube. Characterization of the aerosol particle size distribution with a particle size analyzer demonstrated that 80% of the particles had a diameter of x 3.0 times;m and that the particles had a mean mass diameter of 1.9 times;m with a geometric standard deviation of 1.8 times;m. Studies conducted with aerosols of Bacillus subtilis var niger spores demonstrated that the test apparatus could recover ca. 50% of the spores that were aerosolized. Hydrophobic filters from various manufactures were challenged with an aerosol of at least 10(8) spores of Bacillus subtilis. All filters tested could retain at least 10(9) spores when physical integrity of the filter was verfield.     

Limitations of Thioglycolate Broth as a Sterility Test Medium for Materials Exposed to Gaseous Ethylene Oxide

Although ethylene oxide is a reliable sterilizer, the process may be limited by diffusion. Thus, situations may exist where microorganisms are protected from the sterilizing gas. It is possible that the exterior of a substance may be sterilized, whereas the interior is not. We investigated three general types of materials in which this limitation of diffusion could occur: the bore of glass and plastic tubing, the center of cotton balls, and plastic adhesive film/paper backing interface. These materials were contaminated as close to their geometric center as possible with Bacillus subtilis var. niger spores occluded in crystals of sodium chloride. After exposure of the contaminated materials (except aluminum foil) to ethylene oxide, thioglycolate broth (a standard sterility-test medium) indicated sterility, whereas Trypticase Soy Broth indicated nonsterility. It is likewise possible that aerobic microorganisms, surviving in or on material after exposure to dry heat or steam sterilization processes, would not be recovered by thioglycollate broth. Entrapped aerobic organisms will probably not grow out in the low oxygen tension zone of an anaerobic medium such as thioglycollate broth. It is recommended than an aerobic medium such as Trypticase Soy Broth be used concurrently with thioglycolate broth for sterility testing.     

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.     

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.     

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.     

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.     

Graphical Procedure for Comparing Thermal Death of Bacillus stearothermophilus Spores in Saturated and Superheated Steam

The thermal death curve of dried spores of Bacillus stearothermophilus in saturated steam was characterized by three phases: (i) a sharp initial rise in viable count; (ii) a low rate of death which gradually increased; and (iii) logarithmic death at maximal rate. The first phase was a reflection of inadequate heat activation of the spore population. The second and third phases represented the characteristic thermal death curve of the spores in saturated steam. A jacketed steam sterilizer, equipped with a system for initial evacuation of the chamber, was examined for superheat during normal operation. Measurements of spore inactivation and temperature revealed superheat in surface layers of fabrics being processed in steam at 121 C. The high temperature of the fabric surfaces was attributed to absorption of excess heat energy from superheated steam. The superheated steam was produced at the beginning of the normal sterilizing cycle by transfer of heat from the steam-heated jacket to saturated steam entering the vessel.     

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.     

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.     

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.     

Inactivation of bacillus spores in dry systems at low and high temperatures.

A plot of the thermal resistance of Bacillus subtilis var. niger spores (log D value) against temperature was linear between 37 and 190 degrees C (z = 23 degrees C), provided that the relative humidity of the spore environment was kept below a certain critical level. The corresponding plot for Bacillus stearothermophilus spores was linear in the range 150 to 180 degrees C (z = 29 degrees C) but departed from linearity at lower temperatures (decreasing z value). However, the z value of 29 degrees C was decreased to 23 degrees C if spores were dried before heat treatment. The straight line corresponding to this new z value was consistent with the inactivation rate at a lower temperature (60 degrees C). The data indicate that bacterial spores which are treated in dry heat at an environmental relative humidity near zero are inactivated mainly by a drying process. By extrapolation of the thermal resistance plot obtained under these conditions for B. subtilis var. niger spores, the D value at 0 degrees C would be about 4 years.     

Novel Multi-Slit Large-Volume Air Sampler

Scientific investigators who are interested in the various facets of airborne transmission of disease in research laboratories and hospitals need a simple, continuous, high-volume sampling device that will recover a high percentage of viable microorganisms from the atmosphere. Such a device must sample a large quantity of air. It should effect direct transfer of the air into an all-purpose liquid medium in order to collect bacteria, viruses, rickettsia, and fungi, and it should be easy to use. A simple multi-slit impinger sampler that fulfills these requirements has been developed. It operates at an air-sampling rate of 500 liters/min, has a high collection efficiency, functions at a low pressure drop, and, in contrast to some earlier instruments, does not depend upon electrostatic precipitation at high voltages. When compared to the all-glass impinger, the multi-slit impinger sampler collected microbial aerosols of Serratia marcescens at 82% efficiency, and aerosols of Bacillus subtilis var. niger at 78% efficiency.     

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 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.     

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.     

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.     

Simple Liquid Scrubber for Large-Volume Air Sampling

A new large-volume air sampler called the "simple liquid scrubber" is described. It can recover a high percentage of microorganisms from large volumes of air, up to 950 liters/min, and concentrate them into a small volume of liquid at a ratio of about 400,000 to 1. The principle of operation of the scrubber is based on the production of a fine mist in a rapidly moving airstream with ultimate collection of the airborne particles by impingement into the film of liquid formed upon impaction of the mist droplets on the scrubber walls. The scrubber compared favorably with the all-glass impinger (AGI-30) and the slit sampler in tests with the normal flora and with experimental aerosols of Bacillus subtilis var. niger spores.     

Effect of combined heat and radiation on microbial destruction.

A series of experiments at several levels of relative humidity and radiation dose rates was carried out using spores of Bacillus subtilis var. niger to evaluate the effect of heat alone, radiation alone, and a combination of heat and radiation. Combined heat and radiation treatment of microorganisms yields a destruction rate greater than the additive rates of the independence agents. The synergistic mechanism shows a proportional dependency on radiation dose rate an Arrhenius dependency on temperature, and a dependency on relative humidity. Maximum synergism occurs under conditions where heat and radiation individually destroy microorganisms at approximately equal rates. Larger synergistic advantage is possible at low relative humidities rather than at high relative humidities.