A number of bacteria occur naturally in freshwater streams. Some are found living in the water and sediments as photosynthetic autotrophs or a saphrophytes living on dead matter. Others exist in or on other organisms as mutual symbiotes (providing some benefit to the host organisms in exchange for a place to live), commensuals (neither helping nor harming the host), or parasites (utilizing the host in a way that causes harm).

Certain bacteria that live in the intestinal tracts of animals are essential for the recovery of nutrients from digested food. Millions of these naturally occurring organisms are passed out of the body with fecal wastes. If pathogenic (disease-causing) organisms are present, they may be passed as well. When a stream is polluted by fecal material, pathogenic bacteria, viruses, and parasites may be introduced, posing a health hazard to those who come in contact with the water. Municipal and rural water supplies can transmit human diseases such as cholera (Vibrio cholerae), typhoid fever (Salmonella typhi), shigellosis (Shigella), salmonellosis (Salmonella), and gastroenteritis (Campylobacter jejuni, Escherichia coli, Giardia lamblia). The threat of such disease transmission becomes more serious as the population density increases and more sewage pollutes public water supplies, carrying with it human intestinal pathogens.

Rather than test water directly for pathogens, which can be difficult, expensive and even hazardous, researchers use indicator organisms to assess the possibility of fecal contamination. Fecal coliform bacteria, members of the family Enterobacteriacae, which include Escherichia coli , Citrobacter, Enterobacter and Klebsiella species, are often used as indicators. These gram negative bacilli (rod shaped bacteria) are found in the digestive tracts of all warm-blooded animals. Most are not pathogenic. However, because they are eliminated with feces, they are sometimes associated with pathogens such as Vibrio cholera bacteria or a form of Hepatitus virus that is found in the digestive tract. Total coliform bacteria counts are sometimes used to test for water contamination also. These organisms are less precise as fecal contamination indicators because many can live and reproduce in soil and water, without having a human host.

If high numbers of fecal coliform bacteria are found in a sample of stream water, one may conclude that there has been recent fecal contamination, although not necessarily human in origin. Other intestinal bacteria, such as streptococci or enterococci, may have a stronger correlation to human sewage, but no indicator has been identified that is exclusive to humans. The ratio of streptococci to fecal coliform was once thought to determine human versus animal fecal contamination. But, this is no longer though to be reliable because streptococci do not persist long in an open water environment, making it difficult to assess true concentrations. Enterococcal bacteria seem to be consistently associated with human sewage and subsequent diseases, but testing for these organisms involves a lengthy and complicated procedure.

Despite the fact that they can not be linked directly to contamination by human sewage, fecal coliform bacteria counts are often used to regulate surface waters for recreational use, shellfishing, and potability (ability to be safely consumed). Federal regulations stipulate maximun allowable numbers of these bacteria for various uses. If fecal coliform counts are high (over 200 colonies per 100 ml of water sample) in the river or stream, there is a greater chance that pathogenic organisms are also present. A person swimming in such water has a greater chance of getting sick from swallowing disease-causing organisms, or from pathogens entering the body through cuts in skin, the nose, mouth, or the ears. Diseases and illnesses such as typhoid fever, hepatitis, gastroenteritis, dysentery, and ear infections can be contracted in waters with high fecal coliform counts.

Cities and suburbs sometimes contribute human wastes to local rivers through their sewer systems. A sewer system is a network of underground pipes that carry wastewater. In a separate sewer system, sanitary wastes (from toilets, washers, and sinks) flow through sanitary sewers and are treated at the wastewater treatment plant. Storm sewers carry rain and snow melt from streets, and discharge untreated waters directly into rivers. Heavy rains and melting snow wash bird and pet wastes from sidewalks and streets into storm drains. In a combined sewer system, both sanitary waste and storm runoff are treated at a wastewater treatment plant. After a heavy rain, untreated or inadequately treated waste may be diverted into the river to avoid flooding the wastewater treatment plant. To avoid this problem, some cities have built retention basins to hold excess wastewater and prevent untreated waste from being discharged into rivers. Without retention basins, heavy rain conditions can result in high fecal coliform counts downstream from sewage discharge points. That is why it is important to note weather conditions on the days before a fecal coliform measurement.

1. Use a sterile container, of possible, to collect your water sample.
2. If sampling by hand, use gloves and hold the sample bottle or tube near the bottom. Face upstream and plunge the bottle (opening downward) below the water surface, then turn the bottle underwater into the current and away from you (make a sweeping "U" turn under the water). Be careful not to disturb the bottom sediment.
3. Leave a bit of air space at the top of the bottle by pouring out a little water, and replace the lid securely.
4. Avoid sampling the water surface because the surface film often contains greater numbers of fecal coliform bacteria than is represented in the rest of the stream or river. Also, avoid sampling the bottom sediments for the same reason, unless this is what you intend to analyze.
5. It is a good idea to collect several samples from any single location to minimize the variability that comes with sampling for bacteria.
6. If the purpose of your sampling is to test for suspected sources of fecal coliform contamination, then the samples should be taken just downstream from the source of contamination (like the mouth of a storm drain), and other samples should be taken upstream from this for comparison. If you are sampling downstream of a sewage treatment plant, take extra precautions to minimize direct contact with the water. Wash thoroughly with antibacterial soap after sampling. You may also want to check with the sewage treatment plant director to see if the sewage treatment effluent is sanitized (chlorinated or irradiated) before it is introduced to the river.
7. Put your water samples in a cooler on ice. Ideally, you should test your water samples within one hour of collection. If this is not possible, the samples bottles should be stored on ice until you get back to the lab, and then stored in a refrigerator. Samples need to be tested within 6 hours of collection to ensure adequate accuracy. After 6 hours, the probability of bacteria dying off or becoming too stressed to be cultured is greatly increased. If you will not be able to arrange to test your sample within 6 hours, you might want to consider having the instructor or another adult collect water on the morning before the experiment.

"Coliscan" is a type of commercially available bacterial growth media that contains a combination of color producing chemicals and nutrients that result in the growth of colonies of general coliform and fecal coliform bacteria in different colors. A test sample of water is added to the Coliscan media and general coliform bacteria will grow as pink-magenta colonies while E. coli (fecal coliform) will grow as purple colonies, and other bacterial types will grow as non-colored colonies. Many coliforms are normally found in soil and water and do not necessarily indicate the presence of fecal contamination, but E. coli is the primary bacterium in the mammalian (including humans) intestinal tract and its presence in food or water indicates fecal contamination. Therefore, E. coli is the coliform that is used as an indicator for fecal contamination.

The Coliscan media contains two color-producing substrates that are acted upon by the presence of the enzymes galactosidase and glucuronidase to produce pigments of different colors. General coliforms will produce the enzyme galactosidase (by fermenting lactose), and the colonies that grow in the medium will be pink in color. Fecal coliforms (E. coli) produce both galactosidase and glucuronidase and will grow as purple (or purple-blue) colonies in the medium. A count of the number of purple colonies will indicate the number of fecal coliforms per sample. The pink colonies indicate the total number of general coliforms per sample. The combined general coliform and fecal coliform number equals the total coliform number. Any non-colored colonies that grow in the medium are not coliforms, but may be other members of the family Enterobacteriacae.

Sterile collection container (sterile bottle or test tube) with water sample

Sterile pipets or sterile transfer pipets (dropper pipets)

Sterile petri dish

Coliscan Easygel (Micrology Laboratories)

Incubator set at 37° C

1. Label a petri dish with your name and the location of your water sample.
2. Wash your hands with antibacterial soap, then open a bottle of Coliscan Easygel.
3. Use a sterile pipet (or a sterile transfer pipet) to add between 1 and 5 ml of your water sample (the amount of water you add depends on the extent of fecal contamination you think is in the water).
4. Swirl the bottle to mix the water with the Coliscan Easygel.
5. Pour the mixture into a sterile petri dish. Gently swirl the dish to cover the bottom evenly.
6. Allow the petri dish to solidify for about 40 minutes.
7. Incubate the plate upside down (to minimize condensation on the agar surface) at 37° C.
8. Count colonies 24-48 hours later. See "Interpreting Coliscan pour plates" guide to assist you in determining which colonies are fecal coliforms.
9. Colonies should be analyzed further by bacterial staining and microscopy. Do simple stains and gram staining.

NOTE: You may want to include a negative control and a positive control in your experiment. Sterile water may be used for the negative control. Water from a toilet can be used as a positive control. Be sure to use reasonable precaution in collecting this sample!

To look at the total number of bacteria in your water sample, you may repeat this method using "Total Count" Easygel (instead of Coliscan Easygel). Decrease the amount of sample water that you add to the Total Count Easygel (0.1-1.0 ml should be sufficient). The resulting colonies that grow indicate the total number of colony-forming bacteria that are in your sample. These include both coliforms and non-coliforms. These colonies may be tested further, using microscopic staining.