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Introduction*
Pathogen contamination also can be indicated by fecal streptococci and enterococci. Methods for their detection and enumeration include multiple-tube dilution and membrane lter procedures. Methods for differentiating coliforms are included. Generally, differentiation is of limited value in assessing drinking water quality because the mere presence of coliform bacteriaparticularly thermotolerant coliforms or E. colirenders the water potentially unsatisfactory and unsafe. However, identication may conrm the validity of coliform results or show how a distribution system is colonized. Differentiation also yields valuable information about the possible source of water pollution especially its remotenessbecause nonfecal coliforms may be expected to survive longer than thermotolerant coliforms in water (an unfavorable environment). Procedures for thermotolerant coliforms and E. coli include a 24-h, multiple-tube test using A-1 medium; a rapid (7-h) method; and chromogenic substrate coliform tests. Genetic testing also is becoming available. Unlike other coliforms, those found in the guts and feces of warm-blooded animals generally include organisms capable of producing gas and/or acid from lactose in a suitable culture medium at 44.5 0.2C. Both the multiple-tube dilution technique and the membrane lter procedure have been modied to incorporate incubation in conrmatory tests at 44.5C so analysts can estimate the density of thermotolerant organisms. Heterotrophic plate counts may be done via pour plate, spread plate, membrane lter, or multiwell-enzyme substrate methods. The methods provide an approximation of viable bacteria that may yield useful information about water quality and indicate the signicance of coliform test results. They may be used to judge the efciency of various treatment processes, application as an in-plant control test, and to check water quality in the laboratorys reagent-grade water system or in distribution systems (by indicating microbial colonization and sediment buildup in slow-ow sections and dead ends). Procedures for isolating certain pathogenic bacteria and protozoa are presented. These procedures are tedious, complicated, and followed by laboratory personnel with related expertise. Likewise, tentative procedures for enteric viruses are included, but their routine use is not advocated. However, these methods are useful in investigating waterborne diseases, characterizing watersheds, or determining groundwater quality. Pollution in tidal estuaries and other bodies of saline water has raised concerns about whether existing bacteriological techniques need to be modied to analyze such waters effectively. However, the methods used to analyze fresh waters generally are also satisfactory when testing saline waters. Methods for examining waters in swimming pools and other bathing places are included. The standard plate count procedures for thermotolerant coliforms and streptococci are identical to those used for other waters. Procedures for Staphylococcus and Pseudomonas aeruginosa organisms commonly associated with the skin or upper respiratory tractalso are included, as are procedures for aquatic fungi and actinomycetes. Sections on rapid coliform tests and on recovering stressed organisms are included. The quality control section has been expanded because of increased interest in this topic.
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The following sections describe procedures for examining a water samples microbiological content. These methods were developed primarily to permit rapid examination of water samples, so analysts often assume they only apply to routine examinations. However, they are suitable for both compliance monitoring and research studies of ambient, drinking, waste, and marine waters. The methods are used by many laboratories and are the best techniques currently available, but to be effective, their limitations must be thoroughly understood and appropriate quality control practices must be followed. Likewise, all techniques should be investigated to establish their specicity, improve their procedural details, and expand their uses in analyzing various types of water. Keep in mind that routine bacteriological analyses do not provide a complete water quality picture. Always consider bacteriological results in light of information available about sanitary conditions in and around the sample source. Water quality evaluations based on test results of one sample from a given source are inadequate. When possible, base water quality evaluations on the results of a series of samples collected and analyzed over a known, protracted period. Also, experience has shown that when un-iced samples are mailed, noticeable changes may occur in the types or numbers of bacteria (even if shipping time is brief). So, samples should be refrigerated during transportationparticularly when ambient air temperature exceeds 13Cand minimal time should elapse between collection and analysis. One of the principal indicators of a waters suitability for domestic, industrial, or other uses is the coliform group of bacteria, as herein dened. Experience has established the signicance of coliform densityin particular, that of Escherichia coli or thermotolerant coliforms (previously called fecal coliforms) as a water quality criterion, and the groups cultural reactions and characteristics have been studied extensively. Tests for detecting and enumerating indicator organisms and pathogens are presented, including two for coliforms: the membrane lter technique and the multiple-tube fermentation test. The membrane lter technique involves direct plating to detect and estimate coliform densities. Procedural modicationsparticularly the culture mediumhave made its results comparable to those of the multiple-tube fermentation procedure. Although the membrane lter technique has limited applications, it is equivalent when used appropriately. Either procedure can be used to evaluate water quality or treatment process effectiveness. Coliform density can be reported as a most probable number (MPN) index or membrane lter count per 100 mL. It is customary to report results of the multiple-tube fermentation procedure as MPN, which is not an actual enumeration but rather an index of the number of coliform bacteria most likely to give the results produced during testing. In contrast, the membrane lter procedure and other direct plating methods enable analysts to actually count coliform colonies. Not all sample types require a quantitative assessment of coliform bacteria, so qualitative, presenceabsence tests are included.
* Approved by Standard Methods Committee, 2008. Joint Task Group: Margo E. Hunt.