A comparative analysis was carried out between an emerging cryogenic grinding method and a conventional wetchemistry/ bead-beating endospore disruption approach. After extensive trial and error, it was determined that a regimen of three cryogenic grinding cycles of 2 minutes each was optimum for downstream DNA recovery. Spores embedded in ice exhibited a mere 1-log reduction in recovery following cryomilling for up to 30 minutes. The observed total spore-borne DNA recovery was quite impressive, as well established, streamlined techniques for extracting DNA from endospores typically recover, at best, ≈10% of the molecules present. To facilitate the nucleic-acid-based testing required to detect and quantify DNA and endospores recovered, this innovation implements cryogenic grinding procedures followed by qPCR (quantitative polymerase chain reaction) methods to verify this novel capture technique.

The extraction of DNA from hardy microorganisms, bacterial endospores in particular, usually requires some form of pretreatment, such as mechanical agitation, freeze/thaw cycling, enzymatic lysis cocktails, and/or chemical species to promote the degradation and breaking open of the protective exosporia and hardy spore coats. In this innovation, a heuristic means of cryogenic milling with a SamplePrep 6870 cryogenic grinder is employed to purified endospore stock suspensions.

Upon achieving optimum grinding conditions (e.g. cycle time, cycle periodicity), this cryogenic means of endospore disruption is far superior to conventional mechanical abrasion strategies (such as bead-beating). This is likely a consequence of the absence of accrued heat and noxious chemicals, concomitant with standard pretreatment regimens, which degrade naked DNA molecules upon spore rupture. Cryogenic grinding proved to be an effective and efficient means of assaying intact bacterial endospores. This approach facilitates more controlled parameters for biomaterial extraction, particularly from low-density matrices (e.g. regolith, cleanroom surfaces), and thereby potentially increases DNA yield from the desired target biological constituent. This superior means of mechanical abrasion has widespread microbiological applications, ranging from environmental diversity assessment to single cell gene expression studies.

This work was done by Myron T. La Duc, James N. Benardini, Kasthuri J. Venkateswaran, and Wayne N. Schubert of Caltech for NASA’s Jet Propulsion Laboratory. NPO-48882