Coliforms, Escherichia coli, and E. coli 0157:H7

The first HGMF method for total and fecal coliforms was an adaptation of the water microbiology fecal coliform test (27). Both counts were carried out using mFC Agar without rosolic acid. The total coliform test was incubated at 35°C; the fecal coliform test consisted of a preliminary resuscitation step followed by incubation of mFC Agar at 44.5°C. The companion test for E. coli was a direct adaptation of the Anderson and Baird-Parker membrane-spread method previously mentioned. These methods were subjected to validation studies in 1983 and were recognized as Official Methods by the Association of Official Analytical Chemists (now AOAC International) (28).

Developments in E. coli differential test procedures, notably the introduction of 4-methylumbelliferyl-/?-d-glucuronide (MUG) as a differential reagent, prompted a redesign of the coliform and E. coli methods. In the newer method, a single HGMF was used for both the coliform and E. coli enumerations, eliminating the need for a 4-h resuscitation step. The entire analysis is complete in 24 h. This improved coliform/E. coli procedure was subjected to extensive in-house validation, followed by an AOAC-sponsored collaborative study (29,30). These studies demonstrated that the redesigned HGMF method produced quantitative total coliform and E. coli results that were not significantly different from the three-tube MPN method results over a wide range of food products. Furthermore, the confirmation rate of E. coli colonies on the HGMF method was in excess of 98% in the precollaborative study and more than 99% in the collaborative study. Based on combined results of both studies, the AOAC accorded "Official Action" status to the new procedure in 1990 (31).

The presence of E. coli 0157:H7 in the food supply has become increasingly of concern. This E. coli serotype produces a negative MUG reaction and, therefore, is not detected by any E. coli enumeration procedures based on ¡3-glucuronidase activity. It will also not grow in some culture media at elevated temperatures, making it more difficult to detect using the conventional three-tube MPN E. coli method. Several researchers have developed HGMF methods for E. coli 0157:H7. The earliest of these described the development of HC Agar, a selective and differential culture medium for presumptive enumeration of E. coli 0157:H7 (32). HC Agar relied on three differential reactions—MUG, indole, and sorbitol—to differentiate E. coli 0157:H7 from other Enterobacteriaceae, including other J?. coli. Two other methods employed enzyme-labeled antibody procedures to detect or enumerate E. coli 0157:H7 (33,34).

Recently, SD-39 Agar was developed for use in conjunction with the HGMF to detect and enumerate presumptive E. coli 0157:H7. The medium is based on three biochemical reactions that are read simultaneously: lysine decarboxylase, sorbitol fermentation, and ^-glucuronidase production. Selectivity is provided by the presence of mo-nensin and novobiocin and by incubation at 44 to 44.5°C. E. coli 0157:H7 tolerates the elevated temperature because of the presence of NaCl in the culture medium. Confirmation is accomplished by subculturing presumptive positive colonies and carrying out 0157 and H7 serological tests. This method was validated in a comprehensive pre-collaborative study and then in an AOAC-sponsored collaborative study (35,36). Results obtained by the HGMF method were either significantly higher than the reference method or not significantly different from the reference method, depending on the food product. The serological confirmation rates of presumptive positive E. coli 0157:H7 were 99% in the precollaborative study and 94% in the collaborative study. Based on the combined results of both studies, the method was accorded First Action in 1997 (36).

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Sleeping Sanctuary

Salvation For The Sleep Deprived The Ultimate Guide To Sleeping, Napping, Resting And  Restoring Your Energy. Of the many things that we do just instinctively and do not give much  of a thought to, sleep is probably the most prominent one. Most of us sleep only because we have to. We sleep because we cannot stay awake all 24 hours in the day.

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