Thus far, spore transfer had been successful from the polycarbonate membrane onto stainless steel, aluminum, and to some extent, glass. In order to image the endospores under an ESEM (environmental scanning electron microscope), the spores were transferred onto a 4-mmdiameter, mirror-polished, stainless steel ESEM tab. For the spectroscopic and irradiation procedures in the Planetary Ice Group, it has also been necessary to transfer a highly concentrated, homogenous layer of spores onto a 1/2- or 1-in. (≈1.3- or 2.5-cm) aluminum mirror. Various other methods have been developed and tested for statistical spore deposition and transfer, but transfer was previously prone to uneven coverage due to poor contact, as well as visible microdroplets from oversaturation of the backing filter contact or non-homogeneity on a larger scale. A complete, reproducible method follows to avoid these issues and ensure quantitative predictions and uniformity.

Using a standard vacuum filtration setup with a 47-mm-diameter funnel and sterile materials, the desired number of spores is deposited onto the polycarbonate membrane with approximately 150 mL of sterile water and additional water to remove any remaining spores from the inside of the solution tube. It is easier to work with total spore counts in this case, rather than concentrations. The solution is mixed thoroughly with vortex and/or sonication before pouring into the filtration device. A 47-mm-diameter backing filter is wetted with deionized water to a point of saturation such that the filter is uniformly saturated, yet no additional water remains on the surface or beneath the filter. The spore-covered polycarbonate membrane is saturated directly by placing it onto the backing filter. It should adhere to the backing filter smoothly with no air bubbles, but not so that it is enveloped in excess water. Using two pairs of tweezers to ensure a firm grasp on the membrane, the saturated membrane is placed spore-side down onto the desired substrate. It should be allowed to stick evenly to the surface. For the 4-mmdiameter ESEM tabs, it is easiest if these have been previously attached with carbon tape to a larger ESEM stub. The membrane and substrate are blown with N2 gas so that it dries quickly and evenly. The membrane is removed, and the spores should remain on the mirror.

It has been determined in this method that the concentration required to form a 0.9 monolayer coverage is 1.17 × 108 spores/cm2. In addition, glass (microscope slides) is currently being investigated as a substrate that would be particularly useful for biological applications. A quantitative analysis of the spore transferring and recovering methods using culture counts is in progress to show that the density prediction based on the initial concentration of spore solution in the filtration process is accurate.

This work was done by Adrian Ponce, Arin R. Greenwood, and Aaron C. Noell of Caltech for NASA’s Jet Propulsion Laboratory. For more information, contact This email address is being protected from spambots. You need JavaScript enabled to view it.. NPO-48810