The number of Fecal Coliform in an effluent sample is reflected by how many colonies grew on the membrane during incubation and how much sample was filtered through the membrane. After incubation, remove the samples and count all of the blue colonies on each filter. Samples should ideally have at least 1 plate with 20 – 60 colonies; however, samples are not rejected if there is a countable number.
It is important to understand that the volume of sample filtered could be very small or up to 100 mL because the volume has to be adjusted for the expected Fecal Coliform concentration. For samples that are suspected to contain very high numbers of Fecal Coliform, a dilution is made that may contain only 0.1, 0.01, 0.001 mL/100mL of actual sample. Sometimes, it is necessary that dilutions are taken even further. For most wastewater treatment plant effluents, undiluted sample volumes of 10, 50 and 100 mL will commonly be filtered. If the sample is diluted, the actual volume of sample in the dilution (not the total volume, including dilution water) must be entered into the equation in order to obtain the correct CFU/100 mL density.
Fecal Coliform densities are reported using specific rules:
Filters having 20 – 60 colonies growing on them are preferred. (Report only the results from plates with 20 – 60 colonies when they occur).
If no plate has between 20 – 60 colonies, all the counts are added from the sample plates and divided by the total volume of sample filtered.
Samples with growth covering the entire plate with no distinct colonies are reported as confluent growth.
Samples with more than 200 colonies are reported as too numerous to count (TNTC).
Confluent growth and TNTC plates cannot be used for DMR reporting purposes (re-sampling is required).
If no sample plates have blue colonies and all Quality Control checks out, report result as < 1 CFU /100 mL.
For DMR purposes – < 1 CFU /100 mL = 1
Because the M-FC test is based on cultivating small numbers of microorganisms, many variables exist that can affect the accuracy of the test. The quality control procedures for the M-FC test attempt to eliminate sources of error.
The following quality controls are required at a minimum:
Positive control Anything that is toxic in the materials used in the test will suppress their growth, giving false negative results. By testing the materials on a sample known to contain Fecal Coliform, the materials can be shown to perform as intended. Typically, either a diluted pure strain of E. coli or 1 – 2 mL of raw influent is used as a positive control. These sources should create the characteristic blue colonies filtered and incubated.
Sterility checks Any contamination from sources containing Fecal Coliform bacteria will give false positive results. Sterility checks verify that the materials used in the test are not contaminated with bacteria. Tryptic Soy Broth (TSB) is used as a media to demonstrate sterile conditions. If Petri dishes, membrane filters, absorbent pads and other materials used in the test are incubated with TSB but show no growth, the material are sterile. If growth occurs, the materials are contaminated and should not be used in the test.
Negative control In order to determine which bacteria are Fecal Coliform and which bacteria are not, the media that the bacteria are grown upon (broth) contains a chemical that is only taken up by Fecal Coliform, turning them blue. A negative control proves that the media will differentiate between Fecal Coliform and all other bacteria. Usually, an organism such as Enterobacter Aerogenes is used as a negative control. Colonies of Enterobacter
Aerogenes will grow on the membrane, but will be some color other than blue, usually tan.
Pre-blank A pre-blank is used to demonstrate that the filter funnel apparatus was properly sterilized before the test. Pre-blanks are just like samples, except that 100 mL of sterile buffered dilution water are filtered. Pre-blanks are filtered BEFORE any of the samples. No growth should ever be observed on the pre-blank. If growth occurs, the filter funnel sterilizing procedures should be improved.
After-blank An after-blank is used to demonstrate that the rinsing following filtering of the samples was adequate. After-blanks are just like pre-blanks, except that they are done AFTER the samples have been filtered. No growth should occur on the afterblanks. If growth occurs, sample carry over is occurring and the rinsing procedure following each sample filtration should be improved.