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Thermal inactivation curves of Bacillus subtilis spores for decontamination of interplanetary space vehicle components
Effects of thermoradiation inactivation on Bacillus subtilis var. niger at various temperature, and natural spores in soil
A quantitative means was developed to investigate the sensitivity of current spacecraft sterilization plans to variations in D-values. A quantitative expression was derived to represent the distribution of D-values among a population of naturally occurring organisms. An investigation was made of (1) the inactivation of both Bacillus subtilis var. niger spores and Cape Kennedy soil spores by gamma-radiation at room temperature in a nitrogen environment, and (2) the thermoradiation resistance of Cape Kennedy soil spores at elevated temperatures below 125 C. The relation between standard survival experiments with bacterial spores in soils and results obtained on spacecraft surfaces is discussed. Sporocidal properties of aqueous formaldehyde can be increased by elevating the temperature.
Although Bacillus subtilis var. niger is the standard test organism for NASA planetary quarantine sterilization studies, it was found that some naturally occurring soil organisms are more heat resistant. The separation of these organisms from soil particles is described. Experiments are discussed which were designed to show that the heat resistance is a natural characteristic of the organisms, rather than a condition induced by the clumping effect of agglomerated particles and organisms.
The efficiency of a biodetection grinder, used to recover buried contamination, was tested using spacecraft components and laminated polystyrene strips containing Bacillus subtilis var. niger spores. The surfaces were decontaminated before tests. Results are given in tabular form. Tables are also given for heat resistance of bacteria spores, prevalence of bacteria in spacecraft before launch, and the types of bacteria found in Apollo 15 spacecraft components and command modules.
A set of conditions in which 90 C was a more lethal temperature than 125 C for the destruction of Bacillus subtilis var. niger was identified as a function of relative humidity, with maximum effectiveness at 100% R.H. A systematic study of the influence of head-space moisture and temperature on the destruction of B. subtilis var. niger is reported.
Data showing that 90 C was a more effective temperature than 125 C for the destruction of Bacillus subtilis var. niger when the head-space moisture was fixed at 474 micrograms H2O/ml have been confirmed and are summarized. The influence of head-space moisture at 90 C was investigated and it was observed that as the head-space moisture was increased 474 micrograms H2O/ml, there was a corresponding increase in lethality of the system. This moisture level corresponded to 100% relative humidity at 90 C, and additional water in the cans should have had no effect. A profound descrease was observed in the destruction of the organisms when additional water was added. The discontinuity observed in these data is troublesome and must be resolved as the first step in an orderly exploration of low temperature inactivation of B. subtilis var. niger.
With missions to Jupiter, the spacecraft will be exposed for extended duration to solar wind radiation and the Jovian trapped radiation belt. This study is designed to determine the effect of these radiation environments on spacecraft bacterial isolates. The information can be used in the probability of contamination analysis for these missions. A bacterial subpopulation from Mariner Mars 1971 spacecraft (nine sporeforming and three nonsporeforming isolates) plus two comparative organisms, Staphylococcus epidermidis ATCC 17917 and a strain of Bacillus subtilis var. niger, were exposed to 2-, 12-, and 25-MeV electrons at different doses with simultaneous exposure to a vacuum of 0.0013 N/sqm at 20 and -20 C. The radioresistance of the subpopulation was dependent on the isolate, dose, and energy of electrons. Temperature affected the radioresistance of only the sporeforming isolates. Survival data indicated that spores were reduced approximately 1 log/1500 J/kg, while nonsporeforming isolates (micrococci) were reduced 1.5 to 2 logs/1500 J/kg with the exception of an apparent radioresistant isolate whose resistance approached that of the spores. The subpopulation was found to be less resistant to lower energy than to higher energy electrons.
A space molecular sink research facility (Molsink) was used to evaluate the ability of microorganisms to survive the vacuum of outer space. This facility could be programmed to simulate flight spacecraft vacuum environments at pressures in the .1 nanotorr range and thermal gradients (30 to 60 C) closely associated to surface temperatures of inflight spacecraft. Initial populations of Staphylococcus epidermidis and a Micrococcus sp. were reduced approximately 1 log while exposed to -105 and 34 C, and approximately 2 logs while exposed to 59 C for 14 days in the vacuum environment. Spores of Bacillus subtilis var. niger were less affected by the environment. Initial spore populations were reduced 0.2, 0.3, and 0.8 log during the 14-day vacuum exposure at -124, 34, and 59 C, respectively.
Aqueous formaldehyde is shown to exert both sporostatic and sporocidal effects on Bacillus subtilis spores. The sporostatic effect is a result of the reversible inhibition of spore germination occasioned by aqueous formaldehyde; the sporocidal effect is due to the temperature-dependent inactivation of these spores in aqueous formaldehyde. The physicochemical state of formaldehyde in solution provides a framework with which to interpret both the sporostatic and sporocidal properties of aqueous formaldehyde.
This study was initiated to determine the percentage of spores that would be expected to be released from the interior of solid materials by aeolian erosion on a planetary surface. Methyl methacrylate and Eccobond disks were fabricated so that each disk contained approximately 40,000 Bacillus subtilis var. niger spores. The disks were placed in a specially designed sandblasting device and eroded. Exposure periods of 0.5, 2 and 24 hours were investigated using filtered air to accelerate the sand. A series of tests was also conducted for a 0.5 hour period using carbon dioxide. Examination of the erosion products showed that less than 1% of the spores originally contained in the solids was released by aeolian erosion.
The sterilization parameters for the Viking lander of D sub 125 C = 30 minutes and z = 21 C for the exposed bioburden were derived from the experimental findings of several laboratories conducting thermal inactivation studies on Bacillus subtilis var. niger by dry heat. The moisture constraint, that the sterilizing gas shall be less than 25 percent relative humidity at standard conditions of 0 C and 760 mm Hg pressure, was added in recognition of the profound influence of water vapor on the time and temperature required for thermal inactivation of these spores. Data is presented demonstrating that the application of the moisture parameter does not significantly change the D sub 125 C and z values of 30 minutes and 21 C, respectively. Data are presented also to show the maximum influence that could be expected by decreasing the humidity to near zero percent relative humidity at 105, 113, and 125 C.
The thermal inactivation curve for Bacillus subtilis var. niger spores on the Viking lander is examined. Tests were conducted at 113 C and 25% RH, and over a wide range of temperatures using .001% RH and additions of P2O5 to dry the environment. Results show the 25% RH environment did not significantly reduce the survival curve, while the survival curves for spores treated under the drier .001% RH environment was reduced by a factor of 3.
Studies were made of atypical organisms found in Bacillus subtilis var. niger spore colonies. Efforts were aimed at: (1) determining the heat sensitivity of these atypical white spores treated under dry heat conditions and their influence on the nature of the survival curve, (2) preparing a new spore crop obtained from spore isolates by purification procedures, and (3) comparing spore crops obtained from Cape Kennedy (SSM-10) and Minnesota (Minn. sp. AAEF) with the old Cincinnati and new purified Cincinnati spore crop under dry heat conditions.
The survival of Bacillus subtilis var. niger spores suspended in solutions of sucrose and glycerol at calculated water activities and varying temperatures was studied. The overall results indicated that as the water activity of the liquid decreased from .99 to .85, the heat resistance of the spores increased. The nature of the substance controlling the water activity, and the history of the spores prior to treatment also had an affect on their heat resistance.
The development, design, and testing of a preliminary flight prototype potable water bactericide system are described. The system is an assembly of upgraded canisters composed of: (1) A biological filter; (2) an activated charcoal and ion exchange resin canister; (3) a silver chloride canister, (4) a deionizer, (5) a silver bromide canister with a partial bypass, and (6) mock-up instrumentation and circuitry. The system exhibited bactericidal activity against 10 to the 9th power Pseudomonas aeruginosa and/or Type IIIa, and reduced Bacillus subtilis by up to 5 orders of magnitude in 24 hours at ambient temperatures with a 1 ppm silver ion dose. Four efficacy tests were performed with a AgBr canister dosing anticipated fuel cell water. Tests show that a 0.05 ppm silver ion dose was bactericidal against 3 plus or minus 1 x 10 to the 9th power (5 plus or minus 1 x 10,000/ml Pseudomonas aeruginosa and/or Type IIIa in 15 minutes or less.
The Apollo 17 biostack experiment to establish the biological efficiency of individual heavy nuclei particles of galactic cosmic radiation are reported. The experiment theory, interaction of heavy nuclei particles with biologic matter, and the total dose of cosmic ionizing radiation are discussed along with the radiation effects of heavy nuclei on Artemia salina eggs, and Bacillus subtilis.
The experimental design of a study to evaluate the effect of different cleaning methods and storage conditions on the dry heat resistance of Bacillus subtilis var. niger spores is described and the results for the first evaluation are reported. Specifically, the synergistic effect which occurs when spores are subjected simultaneously to dry heat and gamma radiation so as to be able to specify thermoradiation sterilization cycles was investigated. Attempts were made to understand the underlying mechanism(s) that lead to spore death from this combination of stresses. Data cover: (1) the survival of spores on surfaces at various temperatures in a precisely controlled environmental system, (2) the rate of destruction of these spores at ambient temperature when subjected to gamma radiation, and (3) the rate of destruction of spores when they are subjected to combined gamma radiation and thermal stresses.