CHAPTER 6: TIME-GATED LUMINESCENCE MICROSCOPY OF SURFACE BACTERIAL SPORES AS A RAPID BIOBURDEN ASSESSMENT OF
6.5 Discussion and Conclusion
(r = 0.9998). D-values were calculated as 12.68 ± 0.67 min and 13.39 ± 0.77 min using µEVA and NASA standard assay, respectively.
6.4.4 Monitoring of endospore inactivation to oxygen plasma
Figure 6.5b shows the microbial survivor curve followed first-order exponential inactivation kinetics from 0 to 30 min with good fitting to linear plots (R2 = 0.9852 for µEVA and R2 = 0.9884 for NASA standard assay). A sudden drop in germinability and culturability took place after 30 min. The two methods were highly correlated (r = 0.9997). The log-linear regime yielded D-values of 14.03 ± 1.56 min and 16.86 ± 3.96 min using µEVA and NASA standard assay, respectively.
spacecraft contamination is known to come from soil. Bypassing the lengthy outgrowth and colony-forming phases, µEVA proves to be a rapid and efficient assay combines time-gating and lanthanide luminescence photochemistry to detect germinable Bacillus endospores on surfaces. The unique photophysical and chemical characteristics of Tb- DPA make µEVA a powerful analytical tool for bioburden assessment in exobiological explorations.
Dry heat is currently used for the terminal decontamination process for spacecrafts. We have compared the inactivation kinetics of Bacillus endospores using µEVA and heterotrophic plate counts. In view of the increasing number of heat-liable electronics, there is an urging need to develop low-temperature sterilization regimen for spacecraft decontamination. Vaporized hydrogen peroxide and oxygen plasma are two potential candidates as both methods operate at relatively low temperature, around 45°C, and produce non toxic by-products towards the end of the sterilization process. We have compared the inactivation profile using µEVA and NASA standard assay on Bacillus endospores impregnated on metal strips. A high degree of correlation exists between the loss of germinability and culturability with respect to these two inactivation regimens.
The probability of obtaining a sterile spacecraft is enhanced significantly if the level of microbial contamination is relatively low prior to the terminal heat treatment.
Therefore, spacecraft hardware is assembled inside class 100 clean rooms where microbial contamination can be minimized. Poisson and Gaussian distributions are observed in the pure endospore suspension recovery from coupon experiment, but not from the class 100 clean room sampling. A much greater degree of heterogeneity has been observed in the latter case, characterized by aggregation and patchiness. Some kind
of territorialism may favor the presence of microbial hot spots in the clean room.
Variation may also be attributed by the larger variety of heat-shock survivors as revealed by the plate count results. At least 3 distinct colony morphologies and different germination kinetics have been observed in the clean room samples.
A quantitative relationship has been established between the two assays. µEVA and NASA standard assay are found to correlate in a 2:1 ratio at low inoculum regime and 1:1 at high inoculum regime. One plausible reason is that one of the intermediate extraction processes selects preferentially for germinable-but-not culturable endospore. It may give rise to the observed discrepancy, which becomes proportionately apparent at low concentrations. In general, more germinable endospores are recovered than culturable ones which may suggest that (1) germinable endospores endure heat shock, swabbing, sonication and vortexing processes better; (2) endospores lose their ability to form colonies during the sampling and extraction procedures. Endospores residing in a clean room environment are reported to be more resistant22,23. The first point is further substantiated by our clean room sampling results in which most of the germinable heat- shock survivors can form colonies.
The culturable endospore recovery rate, albeit low, is consistent with similar studies reported in the literature24-26. While the swab-rinse technique is not the most effective way to collect and extract microbes in suspension, it is adopted by the NASA standard assay and is thus used throughout this study. Both methods report a relatively high standard deviation. Angelotti et al. proposed that high variance in recovery was not inherent in the sampling, but was attributed by the intermediate extraction process27. So, we have proposed the use of PDMS roller in the collection and analysis of surface
endospores in future studies. The adhesive PDMS can readily collect endospores on a surface and can subsequently be pressed against a germination substrate for direct time- gated imaging, obviating steps such as resuspension, vortexing and sonication. Since µEVA is an endospore-specific method, there is no need to carry out an additional heat shock process to remove vegetative bacteria. As indicated in our data, the difference between heat shock and non heat shock µEVA data is statistically insignificant (p = 0.8064). This newly proposed PDMS sampling methodology is expected to facilitate the efficiency, precision and speed in the enumeration of surface endospores.
For a given population of endospores, a subset will germinate, and a subset of that population capable of germination will form colonies. The actual viable endospore population will fall between the populations capable of outgrowth and germination. The culture-based NASA standard assay will underestimate the viable endospore bioburden, due to viable-but-non-culturable (VBNC) populations; on the other hand, µEVA will overestimate the viable endospore bioburden since not every germinable endospore eventually develops into a visible colony. Therefore, µEVA and NASA standard assay are complementary methods in evaluating the upper and lower limits of bioburden level in planetary protection endeavors. In conclusion, we demonstrated that a basic microscope setup with time-gating image system is an effective tool for endospore detection, and the use of a solid substrate to induce Bacillus endospore germination and detect subsequent release of DPA, thus opening up new avenues for exploring the rich biology of endospores. We envision the µEVA as a general fluorescence lifetime imaging technique will contribute to the construction of a complete and high-throughput detection
system for endospores, providing insights into sterilization validation, astrobiology and planetary protection.