CHAPTER 4: AIRBORNE ENDOSPORE BIOBURDEN AS AN INDICATOR OF SPACECRAFT CLEANLINESS SPACECRAFT CLEANLINESS
4.4 Results
holes have each received 1 or more particles, the next particle has but 1 chance in 10 of going into an unpenetrated hole. Therefore, on average, 10 additional particles would be required to increase the number of positive holes by 1.
easily be transported by moderate air flow over the surface of the biofilm. It was determined that greater that 200 bacterial endospores could be recovered from 1000L of air passed over the biofilm. This finding demonstrated that under laboratory conditions, surface microbial biofilm can release bacterial endospores that can be transported through the air, resulting in the airborne spread of potentially dangerous microorganisms from surfaces.
4.4.3 Correlation of airborne and surface endospores in a closed laboratory environment A total of 105 cfu of B. subtilis endospores were dispersed and aerosolized using a nebulizer inside a 12 L AtmosBag with a surface area of 68.6 cm × 76.2 cm. A computer exhaust fan was installed the AtmosBag to emulate natural air turbulence. Air and surface sampling were carried out to assess the airborne and total surface biomass after 30, 120, 240 min and then on a daily basis after 4 days (Figure 4.3). Immediately after aerosolization, ~30% of the initial bacterial endospore inoculum were suspended in air and only 30% were deposited on surfaces. After 1 day of settlement, we have observed a ratio of airborne to surface endospores to be ~1:4, with 8% airborne and 33% on the surface. Passive air sampling was evaluated by placing fallout plates inside the AtmosBag in triplicates at various point of time for an hour, 3 facing upward and 3 mounted 10 cm upside down. The rate of passive endospore infall was measured to be 0.632 cfu/cm2hr and up-rise rate was measured to be 0.106 cfu/cm2hr.
4.4.4 Correlation of AEB and total biomass in indoor environments
In Figure 4.4, the bacterial counts represent the heterotrophic vegetative portion because the endospore population in most of the cases was extremely low. The data show a weak positive correlation between airborne and surface bacteria. We can see that most of the data points align diagonally, showing an airborne to surface bacteria ratio of around 10 to 100. Heavily used and lightly used laboratories demonstrated a significant difference in terms of airborne and surface bacteria (p < 0.01). Laboratories also exhibit a higher bacterial counts then offices (p < 0.0001). Inhabited and uninhabited offices do not show a statistically significant difference (p = 0.1239). The level I biohazard cabinet shows extremely low concentration as expected, because the purpose of a bio-hood is to keep the air inside free from contamination. The ductless fume hood does not have low concentrations because it is designed to keep harmful chemical vapor inside the hood filter and is not a sterile environment. The data from different locations form distinct clusters, especially the distinction between labs, offices and the biohazard cabinet. The ductless fume hood showed a greater variation because its function is to protect the operator by drawing ambient air inside the hood. In the lab where anaerobic endospores research was executed, there is corresponding increase in airborne and surface proportion of anaerobic bacteria from the fluid thioglycollate cultures.
Preliminary indoor sampling showed a week correlation between human activity and airborne/surface bacterial concentration. To further testify this hypothesis, we compared these with the results of sampling in the Environmental Control and Life Support System Module at Marshall Space Flight Center support the hypothesis that bacterial spore bioburden can represent the total microbial content of an environmental
system. Time-course data indicated an increase in total microbial counts during the time of occupation and a decrease after the cabin was not in use. Data obtained from three different types of bacterial growth media (R2A, RBA and TSA) did not show any significant trends related to the type of media used for either the one day time course (day 6) or the 15 day time course of sampling. RBA and R2A counts showed slight increases with a single peak in each at 14:00 on day 6. Sampling data demonstrated a significant difference in the total CFUs obtained from each of the methods used. Fallout plate data shows no immediate trend across the plate types but with a significant increase over time.
Surface sampling done by RODAC plating collected data on the total CFU on a surface the approximate area of a 100mm diameter circle. Surface testing indicates a general increase in surface microbial content from an average of 3 cfu plate-1 to 19 cfu plate-1. Spore content of the air samples was indicative of the total microbial bioburden (Figure 4.5).
4.4.5 Correlation of AEB and total biomass in outdoor environments
Figure 4.6 shows the microbiological sampling results with SAS in the Atacama Desert and corresponding weather conditions measured by a portable weather station. Diurnal variation in the number of culturable bacteria was observed, positively correlated with relative humidity and wind speed, and inversely correlated with the air temperature and UV irradiation. Fallout plates made of PDMS were used. No CFU was recovered during the 12 hours of daytime. 4 ± 2 CFU plate-1 were obtained during the 12 hours at night.
The intense UV irradiation can be the cause for the inactivation of airborne bacteria during the day.
4.4.6 Comparison of biofilm-forming environmental-strain B. subtilis and lab-strain B.
subtilis endospores
The ability to form fruiting bodies could be passed on over 3 generations with intermittent sporulation, germination and outgrowth. Cultures of bacterial endospores (B.
subtilis 3160) were grown on TSA bacterial growth medium. Two weeks after the culture was initiated, to allow time for the formation of bacterial endospores on the surface of the biofilm, an SAS air sampler was used to measure the concentration of spores which could easily be transported by moderate air flow over the surface of the biofilm. It was determined that approximately 100 endospores could be recovered from 1000 L of air passed over the biofilm. A strain of B. subtilis that form biofilm and delicate aerial structure was isolated from Mojave Desert (Figure 4.7). Two weeks after colony formation on TSA, approximately 1000 endospores were recovered when 1000 L of air passed over the culture. This finding demonstrated that under laboratory conditions, surface microbial biofilm can release bacterial endospores that can be transported through the air, resulting in the airborne spread of potentially dangerous microorganisms from surfaces. No significant differences were observed in the unit DPA content and germination properties between these endospores.