Fecal coliform bacteria is found in sewage. However, Fecal Coliform is a bacteria found in the feces of warm-blooded animals. Animals that include: cattle and other farm animals, wild animals, domestic animals, water fowl, and HUMANS.
* BEFORE YOU START, run a dummy sample (e.g. water from any local stream) to TEST THE THERMOMETER on your incubator. If you follow the directions and your colonies come out reddish, your incubator is too cold (the thermometer is reading too high), and you aren't distinguishing your fecal coliforms from other coliforms. If your filter comes out a dull gray, your incubator is too hot (your thermometer is reading too low), and you killed all your bacteria. A couple of degrees of error makes a huge difference. We have found serious problems with thermometers in the past. Check yours!
1. Plastic petri dishes, culture media, absorbent pads, and filters are presterilized, packaged, and ready to use. Sterilize everything else (i.e. your Millipore filter assembly and pipettes) using an autoclave or a pressure cooker at 15 pounds of pressure for 30 minutes. Teachers should do this before going to the river. Assemble the filtration systems, with rubber stoppers covering three of the four holes in the cover on top of the unit, as well as one of the two holes in the bottom half of the unit.
2. Take a representative sample of water from moving (not stagnant) water. Rinse your sample bottle out several times in the river, so that any bacteria in your sample actually come from the river and that the concentration of bacteria in the sample water is the same as that in the river. Don't take a sample from the water surface, since the concentration of these bacteria at the surface is abnormally high, and your reading won't be accurate. For the same reasons, you should avoid sediments from the bottom in sampling the river for fecal coliform.
3. Sanitize your forceps by dipping them in alcohol and burning off the alcohol in a flame. If your petri dish does not contain an absorbent pad already, place a pad into the petri dish using the sanitized forceps. Don't touch the pad with your fingers!
4. Remove the top from the ampule of bacterial food broth and drain the broth onto the pad. Be careful not to let bacteria from the air (or anywhere else) settle on the pad! Replace the top on the petri dish, and put it aside for a few minutes.
5. Sanitize your forceps again as in step 3. Take a filter out of its sterile wrapping, using the forceps. The wrapping includes the filter and its backing. The filter is the thing with the grid on it. You don't need the backing! Be sure that your forceps are cool when you remove the filter, or it will melt. Quickly (don't expose the filter to bacteria in the air, soil, or your body) unscrew the filter system and put the filter on top of the white membrane. Be sure that (1) the grid side is up, (2) the filter is centered on the membrane, and (3) the filter is flat and unwrinkled. Screw the filter system back together.
6. Using a sterile pipette, add a small amount of sterile water to the filter system through the unstoppered hole in the top. Swirl it around a bit. Using either a sterile pipette or a pipette that has been thoroughly rinsed in the river, add 10 ml of water to the filtration system through the unstoppered hole in the top. You must know exactly how much river water you add, so be careful.
7. Put the filter system on a level surface. With the suction pump attached to the unstoppered hole in the unit's bottom, draw the sample through the filter, swirling to minimize the bacteria adhering to the upper funnel. Continue until the filter appears dry.
8. Sanitize the forceps again. Unscrew the filter. Quickly remove the filter with the forceps. Open the petri dish with the absorbent pad and nutrient broth and place the filter on the pad. Again, insure that the grid side is up! Label the petri dish with a pencil or ball-point pen (felt-tips don't work so well) as to site and volume of water used to make the sample.
9. As quickly as you can (preferably within 30 minutes), transfer the petri dish to an incubator at 44.5 degrees C (plus or minus 1/2 degree). Place the petri dish in the incubator upside down! This will let condensation water, which forms on the bottom surface, to settle on the top of the petri dish and not on the filter. Incubate for 24 hours. Count the bacterial colonies on the filter. You can use a magnifying glass if you want to, but almost all should be visible with your unaided eye. Each bluish spot is a fecal coliform colony (derived from a single bacterium initially in the water). Cream-colored or gray colonies are non-fecal coliform and should not be counted.
10. Convert your count to "bacterial colonies per 100 ml." If you used a 10-ml water sample, multiply the number of colonies you counted by 10; if you used a water sample of some other size, use the following formula: Bacteria (per 100 ml)= 100 x Colonies (counted)/ Water Sample (size in ml)
How does your Fecal Coliform level compare with standards?
A. Other organisms, such as bacteria and viruses are likely to be present
B. The opportunity exists for other waterborne diseases to occur:
C. Swimming is inadvisable at this site
D. The quality of the river is impaired, as is the quality of the aquatic life
E. The river may smell
How old and how near is the sewage plant?
Are there any new housing developments in the area?
Are the storm sewers connected to the sanitary sewers?
Have there been sewer overflows nearby?
Are there dairy farms, pig farms or feedlots nearby?
Is the sewage plant large enough?
Is the city government aware of the problem?
Has there been a dry spell followed by a heavy rainfall recently?
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