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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.
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.
A method for accurately determining urinary tract infections in man is introduced. The method is based on adenosine triphosphate (ATP) concentration in urine samples after removing nonbacterial ATP. Adenosine triphosphate concentration is measured from the bioluminescent reaction of luciferase when mixed with ATP. An examination was also made of the effectiveness of rupturing agents on monkey kidney cells Candia albicans, a Rhodotorula species, and a Streptomyces species in determining whether these cells could contribute ATP to the bacterial ATP value of a urine sample.
An experimental sterilization facility was developed to simulate conditions that will be encountered during terminal sterilization of space vehicles. The system consists of a temperature controlled oven with a nitrogen gas stream containing a known concentration of water. Moisture analyzers are utilized to monitor the gas flowing over spore samples contained in the oven. In its original configuration, no provision was made for the control of water vapor during the sterilization cycle. Because moisture profoundly influences the thermal inactivation of bacterial spores, an upper limit for the moisture content in the gas used to sterilize the space vehicle was established (25% RH at 0 C STP). Accordingly, a controller was developed and installed to provide these conditions in the experimental sterilization facility.
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 mechanism for thermal inactivation of bacterial spores under moist or dry heat was studied. Experimental conditions were established relating to spore loss of heat resistance and loss of optical density as a measure of the rate and extent of germination in spore suspensions. Events occurring during germination were correlated with phase darkening (refractility and non-refractility of spores), stainability characteristics of heat and non-heat treated spores, morphological characteristics, and studies on swelling of spores by an increase in packed cell volume.
Statistical considerations in designing sterilization experiments are discussed in terms of the basic design, pooling of runs, and process assessment.
The uses of scanning electron microscopy in assessing changes that occur in spores exposed to wet and dry heat cycles at elevated temperatures were examined. Several species of Bacillus and other nonspore-forming species of organisms were used for the experiment. Surface morphology of viable and nonviable organisms was clearly detectable by this method, making it a potentially useful technique for investigating microbial inactivation on space vehicle surfaces and components. Micrographs of the spores and bacterial cells are provided.
Dry heat sterilization of spacecraft was investigated by studying the production of spore crops, and thermal inactivation of the spores, and bacillus subtillus. Spore assays were made by conventional plate count methods, and survival curves for the spores are presented. The results indicate that the inherent resistance of spores from a parent cell can be maintained.
It is shown that airborne bacteria can maintain metabolic functions in a suitable atmosphere. It is theorized that particles in the Jovian atmosphere would have physical half-lives of 10 to 1500 years, depending upon which of two turbulent models is chosen.
C-14 metabolic studies on an aerosol mixture of Escherichia coli and a coliphage are indicative of phage production.
A series of experiments was conducted to determine the dry heat resistance of microorganisms in soil obtained from Denver Colorado, Pasadena California, Kennedy Space Center Florida, and Cincinnati Ohio. The results of the KSC terminal sterilization cycle experiment are given in graphs. The average number of viable organisms per m1 was calculated for 18 replicate soil samples for each sample area and points plotted equivalent to 30 hr exposure at 112 C. The result showed a reduction of 3 logs from the initial population for both KSC and Cincinnati soil samples. Results from other areas are given in graphs.
Efforts to show growth activity of anaerobic bacteria in aerosolized particles suspended in anaerobic gas (N2) are described. Evidence of spore generation in nitrogen was obtained. Results are discussed.
Dry heat treatment is specified as the preferred means for the terminal sterilization of spacecraft and for decontamination of spacecraft components. The presence of organisms highly resistant to dry heat in soil and fallout around assembly and industrial manufacturing areas is shown. The dry heat survival characteristics of the Cape Kennedy isolate 4-6 B. brevis spores is demonstrated. The presence of hardy organisms from soil samples obtained from geographical areas of the United States is shown. A resistant fraction appears to occur in low numbers in a soil sample. The heat resistance characteristics of 4-6 B. brevis and B. subtilis var. niger spores are compared. Their morphological characteristics are compared by scanning electron microscopy.
Spores of Bacillus subtilis var. niger were heat treated in aqueous suspension at 90 C, and observed for morphological changes and loss of viability. The 5 logs reduction that occurred in broth at 90 min required 210 min in buffered water. Five characteristic changes observed after spores were exposed 120 min at 90 C in buffered water were: (1) 90% loss of spore viability, (2) 5% stainability, (3) 76% increase in spore size (as observed by scanning electron microscopy), (4) 21% of spore areas remaining refractile, and (5) an increase of 77% in packed cell volume (PCV). Stainability and PCV changes were recognized only after secondary exposure in broth. Extended heat exposure (3 h at 90 C) resulted in 99% loss of spore viability and 99% loss of stainability. After 4 hours of heat exposure, 90% of the cells disintegrated. These results suggest that early germinal changes occurr concurrently with the early changes in the heat susceptibility of dormant spores.
Statistical techniques which have relevance to studies on the thermal inactivation on bacterial spores are discussed.