Total Coliform: Multiple Tube Fermentation Technique
This method is for the determination of the presence of a member of the coliform group in ground water and surface water. The coliform group, as analyzed for in this procedure, is defined as all aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hr at 35°C.
The method is a multiple-tube fermentation technique which is a three-stage procedure in which the results are statistically expressed in terms of the Most Probable Number (MPN). The three stages are -- the presumptive stage, confirmed stage, and completed test. For the analysis to be accurate, a five-tube test is required.
In the presumptive stage a series of lauryl tryptose broth primary fermentation tubes are inoculated with graduated quantities of the sample. The inoculated tubes are incubated for 24 hours and examined for gas formation. Incubation is continued for the tubes that have no gas formation and examined again at the end of 48 hours. Formation of gas in any amount within the 48 hour (+/- 3 hrs) is a positive presumptive test.
In the confirmed stage any primary fermentation tubes that showed gas formation during the 24 and 48 hour periods. Fermentation tubes containing brilliant green lactose bile broth are inoculated with medium from the tubes showing a positive result in the presumptive test. Inoculation should be performed as soon as possible after gas formation occurs. The inoculated tubes are incubated for 48 hours. and any gas formation at any time in the tube indicates a positive confirmed test.
The completed test is performed on all samples showing a positive result in the confirmed test and can also be used as a quality control measure on 20% of all samples analyzed. One or more plates of eosin methylene blue are streaked with sample and then incubated for 24 hours. After incubation, one or more typical colonies are transferred to a lauryl tryptose broth fermentation tube and a nutrient agar slant. The fermentation tubes and agar slants are incubated for 24 hours or for 48 hours if gas is not produced. From the agar slants corresponding to the fermentation tubes in which gas formation occurs, gram-stained samples are examined microscopically. The formation of gas in the fermentation tube and the presence of gram-negative, non-spore-forming, rod-shaped bacteria in the agar culture may be considered a satisfactorily completed test, demonstrating the positive presence of coliform bacteria in the sample.