I have seen several papers recently in which plate (viable) counts of bacteria were done on samples which had been frozen in liquid nitrogen and stored at -80 for weeks to months. Does someone know of a reference documenting this procedure?
This topic is often neglected. I just know that there are some papers comparing freezing vs. room-temperature storage for soil samples (PMID:20138194). It seems comprehensable that the optimal storage conditions depend somehow on the bacteria (of interest) itself and the soil type.
I just checked the bacterial load per qPCR after freezing at -80° and found no significant effect for sandy loam soil. However, I did not check the CFU.
Here are a couple of ‘older’ papers that touch on this topic:
Smittle, R. B., S. E. Gilliland, and M. L. Speck. 1972. Death of Lactobacillus bulgaricus Resulting from Liquid Nitrogen Freezing. Applied Microbiology 24:551-554.
Wellman, A. M. and G. G. Stewart. 1973. Storage of Brewing Yeasts by Liquid Nitrogen Refrigeration. Applied Microbiology 26:577-583.
Molecular quantification vs. CFU enumeration will (likely) yield very different outcomes...
I also used this method. You have to test whether it works in your system. I have heard rumors that bacteria from tropical soils where sensitive to freezing whereas soil bacteria from temperate regions are generally resistant. Rapid freezing of small portions minimizes the formation of disruptive ice crystals in the cells. Cell culture collections often use DMSO or glycerol to avoid the formation of ice crystals during freezing.
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