The first step in the procedure is to prepare agar samples and set up the apparatus. The apparatus used in the Water lab at WKU is a water vacuum pump that is used to pull the water sample through filters. The next step is to disinfect the work area so as to eliminate the chance of outside contamination of the water sample. This is done using alcohol or another sterilization solution on the apparatus and any counter space that is to be used.
Now the test can begin in earnest. In order to avoid contamination, tweezers are used to handle the filter that will be used to collect the bacteria. These tweezers are dipped in ethanol and flamed to kill and residual bacteria from previous tests. The filter is now placed into the vacuum pump and a sterile funnel is placed over it to assist in pouring the sample. 100 ml of the sample water are now poured into the funnel and pulled through the filter by the vacuum pump. This 100 ml of sample water is followed by another 100 ml of sterile wash water that insures full recovery of any bacteria in the sample. The funnel is then removed and dried to be placed for 2 minute in an ultra-violet sterilizer for the next test sample. The tweezers are then resterilized and the filter is placed on an agar plate and inverted. The plate is then placed in an incubator for 24 hours (± 2hrs) at 35° C (± 0.5°C). A wet towel is included with the tray to make a moist heat for the colonies growth.
This process is repeated for each sample that has been gathered. In addition to the tests of the samples themselves, A precontrol sample is run before any of the real samples are analysed, and then a post control sample is run second to last in the days testing. These are the first samples checked when the test process is resumed after incubation.
After 24 hours of incubation, count the number of bacteria colonies that have grown in the agar plate. The filter is usually printed with a grid that assists in the bacteria count.
To avoid the misidentification of moisture droplets for colonies the lid if the plate is removed and held open so any water has a chance to evaporate. Then the number of colonies are counted. If there are less than 200 colonies then the exact number is recorded inthe data sheet. If there are more than 200 colonies but they are still recognisable as colonies then the plate is catagorised as "too numerous to count" or TNTC. Finally if the plate is one continous film of bacteria the plate is categorised as Confluent. Any number greater than 4 colonies results in a failure of the waters purity.
All of the above testing simply indicates the presence of bacteria. Now the verification process of total coliform bacteria test must be done. First the bacteria is recorded as either a Non sheen colony(Clear colony) or a Sheen colony (Green sheen to colony).
Then sample colonies are picked from the agar plate and placed into 3 test tubes containing solutions broths. The first tube contains Lauryl Tryptose broth (LT) and is a presumptive test solution. The second contains Brilliant Green Bile broth (BGB) and is a confirmed test. Finaly the third tube contains EC with MUG ans is another confirmed test. Smaller test tubes, completely filled with the broth, are inverted in the larger test tubes holding the sample to check for gas production. The tubes are then incubated under the same conditions as the agar plates. An additional 24 hours (for a total of 48) can be added to the incubation however if no results are postive after the first day of incubation.
Once the incubation has ocured the tubes are checked to varify the fecal coliform bacteria. The LT and BGB tubes are checked for trapped gas in the inverted tube. If gas production occurs in both tubes the test is positive. If only the BGB tube produces gas the test is positive. However if the LT tube is the only tube with positive gas production then bacteria is present but the total coliform test is inconclusive. In other words, there is bacteria in the sample, but it is not necessarially fecal in origin.
Finally the EC with MUG tube is checked. This test is diferent than the others in that it does not rely on gas production for verification, but the byproducts of a coliform colony instead. The tube is placed in blue light and checked for flourescence. If this occurs then the the tube contains byproducts of fecal bacteria giving a positive test result. If no flourescence occurs then the test is negative.