Title: Testing Aspects of Freezing Preservation on Oral Bacterial Viability
Grant McCaulley (Presenter)
University of Iowa
Jeffrey Banas, University of Iowa College of Dentistry
Anne Welhaven, University of Iowa
Fang Qian, University of Iowa College of Dentistry
Objectives: A cryoprotectant (storage media) is used to improve retention of viability in frozen bacteria. The purpose of this study was to determine if storage media type affects bacterial viability. In addition, the effects of repeated freezing/thawing on bacterial viability were examined.
Methods: The bacteria used were a pure culture of Streptococcus mutans (SM) and a dental plaque (DP) sample. Six media were used; trypticase-soy broth with yeast extract (TSB-YE) and 0.1% L-cysteine plus the following: glycerol at concentrations of 10%, 15%, 20%, or 25%, skim milk at 10%, or combined 10% glycerol and 10% skim milk. SM and DP samples were quantified initially and after each freeze/thaw cycle. Additional SM samples preserved in media with 10% glycerol were quantified at 5 freeze/thaw time points.
Results: Fluctuations in the viable counts were seen from time point to time point for each media used. Overall, retention of viability appeared to be optimal in 10% glycerol and averaged 102% for SM and 87% for DP after 5 freeze/thaw cycles each. For SM stored in 10% glycerol there was a significant decrease in viability from times 0 to 1 (p=0.0108), followed by a significant increase from 1 to 5 (p=0.0051), showing an overall increase in viability from times 0 to 5. Similar initial decreases and subsequent increases in viability were seen with repeated freezing/thawing of DP in all media used.
Conclusions: The lack of a major difference in viability using different media suggest that other factors, such as time and freezing/thawing protocols, may have a larger impact on retention of viability than the media used. The fluctuations in viability measurements from time point to time point, especially apparent increases in viability, may reflect increased fragility of bacterial chains or clusters upon freezing/thawing.