Development of a thin tube cultivation method for determining coliform counts in milk

A new rapid coliform-monitoring method using thin tube cultivation (TTC) was developed. One hundred microliters of modified selective medium (1.5% agar) and 100 microl of sample were mixed in thin cultivation tubes, yielding a semisolid mixture of 0.75% agar. The tubes were incubated at 37 degrees C and checked for a positive reaction (gas production from lactose fermentation and color changes from acid production) at 30-min intervals. The positive reaction times for TTC were compared with actual coliform numbers. The first experiment was performed to validate the TTC method using pure coliform cultures (the Escherichia coli group, the Enterobacter cloacae group. and the Klebsiella pneumoniae group). The results indicated that the TTC method saved about 10 to 14 h of incubation time compared with the conventional plating method. When coliforms were present in small numbers, such as I to 1.5 log10 CFU/ml, the TTC method detected their presence within 10 h. When highly contaminated samples were assayed (6 log10 CFU/ml), the TTC method detected the presence of coliforms within 4 to 5 h. In a food system experiment, pasteurized skim milk inoculated with a coliform mixture was assayed by the TTC method. The coliforms in the milk were quickly detected, resulting in a savings of 11 to 14 h compared with the conventional plating method. Cold-injured coliforms were studied to evaluate the TTC system. The recovery and estimation of injured coliforms by the TTC method strongly agree with those of the conventional plating method. The TTC system was also applied to ascertain the levels of natural coliforms in noninoculated milk. The TTC system reduced incubation times by about 11 to 14 h compared with the conventional plating method. According to these data, this method is a rapid, simple, accurate, and economical way to monitor and estimate total coliforms in milk.