Assessing freeze-thaw and high pressure low temperature induced damage to Bacillus subtilis cells with flow cytometry

The effect of high pressure low temperature (HPLT) treatment of Bacillus subtilis PS832 in the domain of Ice I-III was studied. Flow cytometry (FCM) forward and sideward scatter measurements gave no indication for HPLT induced complete rupture of cells. The single cell population was gated using FCM in combination with propidium iodide (PI) and carboxyfluorescein diacetate. Results show that membrane damage was inflicted upon a significant subpopulation of cells. Of the remaining cells an increase in severely stressed and ghost cells was found. Positive PI staining was indicative for nearly complete loss of viability, while impaired esterase activity was not indicative for absence of viability. We confirmed that Ice I-Ice III phase transition, characteristic for a − 25 °C/250 MPa treatment led to a > 4 log inactivation of vegetative B. subtilis. However, such a treatment did not influence the robustness of survivors. Finally, cells that survived either freeze-thaw or HPLT did not sporulate upon entry into stationary phase.
Industrial relevance: The single cell physiology data represented here, underpin that industrial application of this preservation technology is best done at conditions where Ice I-Ice III phase transitions occur. Importantly, the data also shows that compared to (just) a freeze-thaw cycle at − 25 °C, such treatment does not lead to less robustness (outgrowth capability) of survivors.