Freeze-thaw effect on selected fecal indicator bacteria : Escherichia coli and Enterococcus faecalis / by Nicole A. Hawdon.

Abstract

The survival of two selected fecal indicator organisms, two strains of Escherichia coli, a Gram-negative bacterium, and two strains of Enterococcus faecalis, a Gram-positive bacterium, after freezing and thawing successively for five cycles was determined using a drop plating method. It was found that all bacterial strains, when an initial concentration of 1.0 x E +08 was used, showed significant decreases in their ability to be cultured (p < 0.05) when frozen and thawed at -15C, compared to three other freezing temperatures, -7, -30 and -80C, when all four were frozen for 24 hours. In addition, the number of culturable cells at -7, -30, or -80C were not significantly different from each other (p > 0.05). The differences in cell inactivation between the two strains of each species of bacteria tested at all temperatures was not significantly different after five freeze-thaw cycles; while the difference between species was shown to be significant, depending on the temperature and condition tested (p < 0.05). When comparing small sample volume sizes (100l) to larger sample volume sizes (100ml) the observed differences were that Escherichia coli strains showed a decrease in cell culturability at both -7C and -15C when cycled in the larger volume; whereas, Enterococcus faecalis strains showed a decrease in cell culturability at -7C and an increase in cell culturability at -15C when cycled at the larger volume. Additional studies investigating culturability, cell wall integrity, and membrane damage of the bacterial strains were conducted using 100ml samples, cycled at -7, -15, and -30°C, and evaluated by three microbiological methods: drop plating, epi-fluorescent microscopy, and flow cytometry, respectively. In all instances, the plate counting method indicated that there was a decrease in cells that were culturable. Results from flow cytometry indicated a smaller decrease in cell culturability, followed, lastly, by results using the epi-fluorescent microscope. Thus, these studies would suggest that the most damage that occurs to frozen and thawed cells, when cycled five times at -7,-15 or -30°C, would be due to damages occurring at the cellular level, rather than damages occurring on the cell envelope, since less cells were able to uptake nutrients from the culture plates.