Fecal Coliform samples must be collected and transported in specially prepared bottles that have been sterilized and contain a small amount of dechlorinating chemical (Sodium Thiosulfate). If the sample has been disinfected with chlorine and does not get completely dechlorinated, the chlorine will continue to disinfect the sample in the bottle on the way to the lab, yielding a false low result. During preparation, Sodium Thiosulfate is added to Fecal Coliform sample bottles as a dechlorinating agent, and then they are sterilized in an autoclave.
Care must be taken to draw the sample aliquot into the bottle without washing out the dechlorinating agent. Only grab samples are taken, and they should be drawn directly from the flow stream, (not collected with a sample dipper and then poured into the bottle).
Sterile sample bottles should only be opened once, at the time of sampling. The lid of the sample bottle should never be allowed to become contaminated. Samples that will take longer than 1 hour to analyze should be cooled to between 1º – 4º C on ice or in a refrigerator and then transported in a cooler on ice to the laboratory. The maximum holding time for Fecal Coliform samples that will be used for state or federal permit reporting purposes is 6 hours.
All materials used for the Fecal Coliform Membrane Procedure must be sterilized prior to running the test. Most labs purchase pre-sterilized Petri dishes, membranes, absorbent media pads and M-FC media. The equipment used to filter the samples is typically sterilized using an autoclave or ultra-violet light-sterilizing box. At the start of the test, the lab analyst disinfects the counter top where the test will be run using alcohol. Petri dishes are laid out for each sample volume to be filtered and absorbent pads are placed in the Petri dishes. Next, MFC media is poured onto the absorbent pad. Any excessive M-FC media on the absorbent pad is then discarded by pouring it out into a sink or trash can.
Using a set of tweezers that has been flame sterilized, a sterile membrane filter is placed upon a special filter funnel apparatus. Using a vacuum pump, a measured portion of sample is drawn through the membrane. A variety of sample volumes, ranging from dilutions that contain < 0.0001 mL of sample all the way up to 100 mL of sample, may be filtered through successive membranes. The sample volume range that is used is dependent upon the expected Fecal Coliform concentration.
Ideally, sample volumes or dilutions that yield between 20 and 60 colonies per plate should be selected. After the samples have filtered through the membrane, three successive 20 – 30 mL volumes of buffered dilution water are used to rinse the sides of the filter vessel and then drawn through the membrane filter. Next, the membrane is removed (using flame sterilized tweezers) and is carefully placed upon the absorbent pad containing M-FC media in the Petri dish. The Petri dish is then sealed. This procedure is repeated for all sample volumes to be filtered.
When all of the sample volumes have been filtered and placed into Petri dishes, they are collectively sealed in sterile, waterproof plastic bags and placed into a water bath incubator. Fecal Coliform samples are incubated at a very specific temperature: 44.5º C, +/- 0.2º C. Water bath incubators are generally used for this test because they can hold the temperature much more accurately than air incubators. After 24 hours of incubation, the Petri dishes are removed and the blue Fecal Coliform colonies are counted.