Environmental Engineering Reference
In-Depth Information
in the process. In waste testing, the procedure normally consists of presump-
tive, confirming, and completed phases. It is recognized as the method of
choice for any samples that may be controversial (i.e., enforcement related).
When multiple tubes are used in the fermentation technique, results of
the examination of replicate tubes and dilutions are reported in terms of the
most probable number (MPN) of organisms present. This number, based on
certain probability formulas, is an estimate of the mean density of coliforms
in the sample. Coliform density provides the best assessment of water treat-
ment effectiveness and the sanitary quality of untreated water.
The production of gas in a single fermentation tube may indicate the pres-
ence of coliforms, but it gives no indication as to the concentration of bacteria
in the sample; that is, a coliform count cannot be obtained directly. The gas
bubble created could be the result of one bacterium or millions. The preci-
sion of each test depends on the number of tubes used. The most satisfac-
tory information will be obtained when the largest sample inoculum shows
no gas at all in a majority of the tubes. Bacterial density can be estimated
mathematically or derived from a table using the number of positive tubes
in multiple dilutions. The number of sample portions selected is governed by
the desired precision of the result. MPN tables are based on the assumption
of a Poisson (random) distribution; however, if the sample is not adequately
shaken before the portions are removed or if clumping of bacterial cells
occurs, the MPN value will underestimate the actual bacterial density.
The technique utilizes a two-step incubation procedure. The sample dilu-
tions are first incubated in lauryl (sulfonate) tryptose broth for 24 to 48 hours
(presumptive phase). Positive samples are then transferred to EC broth and
incubated for an additional 24 hours (confirming phase). Positive samples
from this second incubation are used to statistically determine the MPN
from the appropriate reference chart.
A single-media, 24-hour procedure is also acceptable. In this procedure,
sample dilutions are inoculated in A-1 media and incubated for 3 hours at
35°C, then incubated for 21 hours at 44.5°C. Positive samples from these inoc-
ulations are then used to statistically determine the MPN value from the
appropriate chart.
Fecal Coliform MPN Presumptive Test Procedure
1. Prepare dilutions and inoculate five fermentation tubes for each
dilution.
2. Cap all tubes and transfer to incubator.
3. Incubate 24 ± 2 hr at 35 ± 0.5°C.
4. Examine tubes for gas.
Gas present (positive test)—transfer.
No gas—continue incubation.
5. Incubate for a total time of 48 ± 3 hr at 35 ± 0.5°C.
