Water Quality Analysis: Examination of Coliforms and Indicators PDF

Pure water does not exist in nature, regardless of how much companies that sell bottled water want us to believe this. Water is polluted by a variety of substances, both chemical and microbiological. The chemical pollutants in water are of great environmental importance. Historically, however, most...

Pure water does not exist in nature, regardless of how much companies that sell bottled water want us to believe this. Water is polluted by a variety of substances, both chemical and microbiological. The chemical pollutants in water are of great environmental importance. Historically, however, most of our concern about water purity has been related to transmission of disease by microbial pollution. Diseases such as dysentery, typhoid fever, and cholera are intestinal infections spread principally by the fecal-oral route through contaminated water sources. Additionally, as observed in recent disease outbreaks, if contaminated water is  sed for irrigation purposes, the produce itself may become contaminated, which may result in human and animal disease. Therefore, detection of microorganisms present in water has become a routine procedure performed by both scientists and water purification companies. It would be too lengthy to identify each organism present in a contaminated water sample, and it would simply not be practical to examine the water sample for pathogens only. When pathogens are present in water, their numbers are generally low and they would probably be missed during the identification process. Also, by the time individual pathogens are detected, it may be too late to prevent them from being spread. It is practical, however, to examine water for the presence of only one or two organisms. The presence of these organisms indicates that pathogens may also be present. Because many diseases transmit- ted by water are caused by organisms found in human and/or animal feces, scientists examine water for the presence of Escherichia coli, an organism present in the intestinal tracts of animals and humans. Therefore, E. coli is an indicator organism, one whose presence in a sample suggests the presence of other, potentially pathogenic fecal organisms. E.coli is the most commonly used indicator organism, but any of the coliform bacteria, which are members of the family Enterobacteriaceae, can be used as indicator organisms. Coliforms are aerobic or facultatively anaerobic, gram-negative, nonsporing rods that ferment lactose with acid and gas formation within 24-48 hours. Genera of coliforms include Enterobacter, Klebsiella, Citrobacter, and Escherichia. The presence of a significant number of coliforms in a water sample indicates fecal contamination has occurred and that the water may not be safe for recreation or drinking. Established public health standards specify that the maximum number of coliforms in each t 100 er. wa ml sample of water depends on the intended use of the water. For example, the standard set for drinking water is a limit of one coliform per 100 ml of water and the action limit for drinking water, the limit at which action must be taken, is four coliforms per 100 ml of water. Water used for recreation (water sports), for irrigation, and for discharge into a bay or river has higher standard sets and action limits than those for drinking water. I I One method for determining the number of coliforms in a set volume of water is the mem- brane filter technique. The membrane filter method is often used for sampling water because it gives results in a short period of time, because large volumes of water can be processed at one time, and because the results are highly reproducible. In this technique, water is passed through a filtering apparatus, and a nitrocellulose acetate or polycarbonate filter retains particles, including bacteria, which are larger than 0.45 micrometers. The filter is then removed and transferred to a sterile Petri plate containing a pad saturated with a liquid or agar-based medium. During incubation, nutrients from the pad diffuse through the filter and are metabolized by the bacteria retained on the filter. Each bacterium trapped on the filter grows into a separate colony. Therefore, by counting colonies and calculating the CFU/ml, one can deter- mine the number of bacteria present in the water sample. There are several different types of nutrient pads available, each of which is a selective and differential medium. Depending on the type of pad used, desired colonies will develop distinctive appearances. The medium usually employed for coliform detection ism-Endo medium, which is both a selective and differential medium. Sodium lauryl sulfate and sodium desoxycholate are included to inhibit the growth of gram-positive organisms, while lactose serves to differentiate between coliforms and noncoliforms. Basic fuchsin serves as the pH indicator and sodium sulfite is added to decolorize the basic fuchsin. Lactose- fermenting bacteria will exhibit a red color with a metallic sheen. The red color results when bacteria ferment lactose And produce acetaldehyde which reacts with sodium sulfite and basic fuchsin. The metallic results from the interaction of basic fuchsin with aldehydes produced when bacteria rapidly ferment lactose. If a heavy inoculum is used, production of the metallic sheen will be suppressed. Colonies of non-lactose-fermenters will remain the color of the medium. M- Endo medium is recommended by the American Public Health Association (APHA) for the enumeration of coliforms in water, wastewater, and foods. Another medium that is often incorporated into nutrient pads is KF Streptococcus agar, which detects fecal streptococci. These organisms are present in the intestinal tracts of humans and animals and are also indicative of fecal contamination. If both fecal coliforms and fecal streptococci are present in a water sample, officials can be reasonably certain that fecal contamination of the water has occurred. By using the FC/FS ratio (number of fecal coliforms divided by the number of fecal streptococci), public health officials can determine if the contamination is of animal or human origin. Human fecal contamination results in a higher fecal coliform count than fecal streptococci count, while fecal contamination from animal sources results in a higher fecal streptococci count than fecal coliform count. A second technique for determining the number of coliforms in a set volume of water is to perform the spread plate technique with diluted samples of the water and m-Endo agar plates. The spread plate technique is a third method for achieving isolated colonies. In this procedure a small volume of a broth culture of microbes is spread over the surface of an agar medium. The broth culture may have to be diluted significantly prior to preparing the spread plate. A sterile, L-shaped glass rod known as a glass spreader or a hockey stick is used to spread this volume over the surface of the agar. This rod is sterilized by dipping it in a beaker of 95% ethanol and then passing it through a Bunsen burner flame. Once the flame has burned out, the hockey stick can be used to spread the culture over the medium. During the spreading procedure, it is helpful to rotate the plate so that the volume can be spread completely over the surface of the medium. The hockey stick is then returned to the beaker of ethanol. As with the streak plate, one bacterium gives rise to millions of bacteria which are then visible as a discrete colony. A single colony can then be picked and grown in broth to generate a large volume of pure culture. In addition to being a method for obtaining isolated colonies, the spread plate technique can also be used to estimate the total number of bacteria in a broth culture. The formula for this calculation is identical to that used for the pour plate in Exercise 14. As was mentioned previously, keep in mind that this number may represent only a fraction of the total microbes in a culture. The medium used as well as the incubation temperature may only select for certain populations of microbes. In today's experiment, each group will calculate the number of coliforms/ml from a water sample collected by the group. Students will then compare the coliforms/ml_found in water samples collected by each group in the section. The classic example of a PSE is the discharge of water from a wastewater treatment plant.