Evaluating survival of Escherichia coli O157:H7 in frozen and thawed apple cider: potential use of a hydrophobic grid membrane filter-SD-39 agar method.

To determine the susceptibility of Escherichia coli O157:H7 to freezing and thawing in apple cider, methods that recover injured cells are needed for accurate enumeration. This study compared the ISO-GRID hydrophobic grid membrane filter (HGMF) SD-39 agar method to two other methods: a reference most probable number (MPN) method, and plating on sorbitol MacConkey agar (SMA). To determine numbers of injured cells, SMA spread plating was also compared to Trypticase soy agar (TSA) spread plating. Two strains of E. coli O157:H7 QA 326 and ATCC 43895, were inoculated into presterilized apple cider (10 ml) which was then frozen (-20 degrees C for 24 h). Samples were thawed at 4 degrees C for 4 h, or at 23 degrees C for 1.5 h, or in a microwave oven (700 W for 10 s). Substantial cell death (0.69- to 6.33 log10 CFU/ml decreases) and injury (0.70- to 2.38-log10 CFU/ml decreases) occurred during freezing and thawing. The extent of death and injury varied with strain and thawing method. The TSA spread plating method recovered the most cells while the HGMF method always recovered more viable cells than the reference MPN method and also either recovered significantly more (P < 0.05) cells or a not significantly different number of cells than SMA spread plating. Some injured cells of both strains were not counted by the HGMF method. Significant numbers of cells injured by freezing and thawing at 4 degrees C in apple cider were enumerated in the cider was diluted 1:2 Trypticase soy broth immediately before plating. Two epifluorescent microscopic methods showed that injury was not associated with loss of cell membrane integrity.