Determination of the bacteriological quality of water is not a straightforward analysis. The testing for a specific pathogenic bacteria can often lead to erroneous conclusions. Analyses for pathogenic bacteria are difficult to perform. In general, data are not quantitatively reproducible. As an example, if Salmonella was found to be absent from a water sample, this does not exclude the possible presence of Shigella, Vibrio, or disease-producing viruses. The bacteriological quality of water is based on test procedures for nonpathogenic indicator organisms (principally the coliform group).
Coliform bacteria, typified by Escherichia coli and fecal streptococci (enterococci), reside in the intestinal tract of man. These are excreted in large numbers in the feces of humans and other warm-blooded animals. Typical concentrations average about 50,000,000 coliforms per gram. Untreated domestic wastewater generally contains more than 3,000,000 coliforms per 100 ml. Pathogenic bacteria and viruses causing enteric diseases originate from the same source (that is, fecal discharges of diseased persons). Consequently, water contaminated by fecal pollution is identified as being potentially dangerous by the presence of coliform bacteria.
Standards for drinking water specify that a water is safe provided that the test method does not reveal more than an average of one coliform organism per 100 ml. The number of pathogenic bacteria, such as Salmonella typhosa, in domestic wastewater is generally less than 1 per mil coliforms, and the average density of enteric viruses has been measured as a virus-tocoliform ratio of 1:100,000. The die-off rate of pathogenic bacteria is greater than the death rate of coliforms outside of the intestinal tract of animals. Consequently, upon exposure to treatment, a reduction in the number of pathogens relative to coliforms will occur. Water quality based on a standard of less than one coliform per 100 ml is statistically safe for human consumption. That is, there is a high improbability of ingesting any pathogens. This is an Environmental Protection Agency (EPA) standard applicable only to processed water where treatment includes chlorination. Coliform criteria for body-contact water use and recreational use have been established by most states. Upper limits of 200 fecal coliforms per 100 ml and 2,000 total coliforms per 100 ml have been established. These values are only guidelines since there is no positive epidemiological evidence that bathing beaches with higher coliform counts are associated with transmission of enteric diseases. Some experts feel that these standards may be too conservative from a standpoint of realistic public health risk. In recent years, no cases of enteric disease have been linked directly to recreational water use in this country. Coliform standards applied to water used for swimming are linked to water-associated diseases of the skin and respiratory passages rather than enteric diseases. This, naturally, is entirely different than the purpose of the coliform standard for drinking water, which is related to enteric disease transmission. Here tighter restrictions are imperative, since a water distribution system has the potential of mass transmission of pathogens in epidemic proportions.
Water sample collection techniques differ depending on the source being tested. The minimum number of water samples collected from a distribution system which are examined each month for coliforms is a function of the population. For example, the minimum number required for populations of 1,000 and 100,000 are 2 and 100, respectively. To ascertain compliance with the bacteriological requirements of drinking water standards, a certain number of positive tests must not be exceeded. When 10-ml standard portions are examined, not more than 10 percent in any month should be positive (that is, the upper limit of coliform density is an average of one per 100 ml).
Coliforms are defined as all aerobic and facultative anaerobic, nonsporeforming species. Gram-stain negative rods ferment lactose and produce gas within 48 h of incubation (at 35° C). The initial coliform analysis is the presumptive test which is based on gas production from lactose. In this test, 10ml portions of water samples are transferred into prepared fermentation tubes using sterile pipettes. The tubes contain lactose or lauryl tryptose, broth, and inverted vials. Inoculated tubes are placed in a warm-air incubator (at 35° C ± 0.5° C). Growth with the production of gas (the gas is identified by the presence of bubbles in the inverted vial) means a positive test. That is, it indicates that coliform bacteria may be present. A negative reaction, either no growth or growth without gas, excludes coliforms.
Such tests are employed to substantiate or refute the presence of coliforms in a positive presumptive test. In normal, potable water coliform testing, the test is confirmed using brilliant green bile broth. Occasionally, one may desire to run a completed test. This involves transferring a colony from an Endo (or EMB plate) to nutrient agar and into lactose broth. If gas is not produced in the lactose fermentation tube, the colony transferred did not contain coliforms and the test is negative. If gas is generated, a portion of growth on the nutrient agar is smeared onto a glass slide and prepared for observation under a microscope using the Gram-stain technique. If the bacteria are short rods, with no spores present, and the Gramstain is negative, the coliform group is present and the test is completed. If the culture Gram-stains positive (purple color), the completed test is negative. In examining surface water quality, an elevated-temperature coliform test is used to separate microorganisms of the coliform group into those of fecal and nonfecal sources. This approach is applicable to studies of stream pollution, raw-water sources, wastewater treatment systems, bathing waters, and general water quality monitoring. It is not recommended as a substitute for the coliform tests used in examination of potable waters.
The water analysis is incomplete unless the number of coliform bacteria present is determined as well. A multiple-tube fermentation technique can be used to enumerate positive presumptive, confirmed, and fecal coliform tests. Results of the tests are expressed in terms of the most probable number (MPN). That is, the count is based on a statistical analysis of sets of tubes in a series of serial dilutions. MPN is related to a sample volume of 100 ml. Thus, an MPN of 10 means 10 coliforms per 100 ml of water.
For MPN determination, sterile pipettes calibrated in 0.1-ml increments are used. Other equipment includes sterile screw-top dilution bottles containing 99 ml of water and a rack containing six sets of five lactose broth fermentation tubes. A sterile pipette is used to transfer 1.0-ml portions of the sample into each of five fermentation tubes. This is followed by dispensing 0.1 ml into a second set of five. For the next higher dilution (the third), only 0.01 ml of sample water is required. This small quantity is very difficult to pipette accurately, so 1.0 ml of sample is placed in a dilution bottle containing 99 ml of sterile water and mixed. The 1.0-ml portions containing 0.01 ml of the surface water sample are then pipetted into the third set of five tubes. The fourth set receives 0.1 ml from this same dilution bottle. The process is then carried one more step by transferring 1.0 ml from the first dilution bottle into 99 ml of water in the second for another hundredfold dilution. Portions from this dilution bottle are pipetted into the fifth and sixth tube sets. After incubation (48 h at 35'C), the tubes are examined for gas production and the number of positive reactions for each of the serial dilutions is recorded.
A final testing technique worth noting is the membrane filter method for coliform testing. This procedure involves passing a measured water sample through a membrane filter to remove the bacteria. The filter is then placed on a growth medium in a petri dish. The bacteria retained by the filter pad grow and establish a small colony. The number of coliforms present is established by counting the number of colonies and expressing this value in terms of number per 100 ml of water. This technique has been widely adopted for use in water quality monitoring studies, especially since it requires considerably less laboratory apparatus than the standard multiple-tubes technique. Also, this technique can be adapted to field studies. Equipment needed to perform the membrane filter coliform test includes filtration units, filter membranes, absorbent pads, forceps, and culture dishes. The common laboratory filtration unit consists of a funnel that fastens to a receptacle bearing a porous plate to support the filter membrane. The filterholding assembly can be constructed of glass, porcelain, or stainless steel. It is sterilized by boiling, autoclaving, or ultraviolet radiation. For filtration, the assembly is mounted on a side-arm filtering flask which is evacuated to draw the sample through the filter. For field use, a small hand-sized plunger pump or syringe is used to draw a sample of water through the small assembly holding the filter membrane.
Commercial filter membranes are normally 2-in diameter disks with pore openings of 0.45 (±0.02) R. This is small enough to retain microbial cells. Filters used in determining bacterial counts have a grid printed on the surface. To facilitate counting colonies, the filter membranes must be sterilized prior to use, either in a glass petri dish or wrapped in heavy paper. After sterilization, the pads are placed in culture dishes to absorb the nutrient media on which the membrane filter is placed. During the testing, filters are handled on the outer edges with forceps that are also sterilized before use.
Glass or disposable plastic culture dishes are used. If glass petri dishes are employed, a humid environment must be maintained during incubation. This prevents losses of media by evaporation (the dishes have loose-fitting covers). Disposable plastic dishes have tight-fitting lids which minimize the problem of dehydration. The size of the filtered sample is established by the anticipated bacterial density. An ideal quantity results in the growth of about 50 coliform colonies and not more than 200 colonies of all types. Often it may be difficult to anticipate the number of bacteria in a sample. Two or three volumes of the same sample must be tested. When the portion being filtered is less than 20 ml, a small amount of sterile dilution water is added to the funnel before filtration. This uniformly disperses the bacterial suspension over the entire surface of the filter. The filter-holding assembly is placed on a suction flask. A sterile filter is placed grid side up over the porous plate of the apparatus using sterile forceps. The funnel is then locked in place holding the membrane. Filtration is performed by passing the sample through the filter under partial vacuum. A culture dish is prepared by placing a sterile absorbent pad in the upper half of the dish and pipetting enough enrichment media on top to saturate the pad. M-Endo medium is used for the coliform group and M-FC for fecal coliforms. The filter is then removed from the filtration apparatus and placed directly on the pad in the dish. The cover is replaced and the culture is incubated (for 24 h at 35° C). For fecal coliforms, incubation is performed by placing the culture dishes in watertight plastic bags and submerging them in a water bath at 44.5° C. Coliform density is calculated in terms of coliforms per 100 ml by multiplying the colonies counted by 100 and dividing this value by the milliliters of the sample filtered.
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