Thermotolerant (Faecal) Coliforms
Total Page：16
File Type：pdf, Size：1020Kb
Water Quality Monitoring - A Practical Guide to the Design and Implementation of Freshwater Quality Studies and Monitoring Programmes Edited by Jamie Bartram and Richard Ballance Published on behalf of United Nations Environment Programme and the World Health Organization © 1996 UNEP/WHO ISBN 0 419 22320 7 (Hbk) 0 419 21730 4 (Pbk) Chapter 10 - MICROBIOLOGICAL ANALYSES This chapter was prepared by J. Bartram and S. Pedley The discharge of wastes from municipal sewers is one of the most important water quality issues world-wide. It is of particular significance to sources of drinking-water. Municipal sewage contains human faeces and water contaminated with these effluents may contain pathogenic (disease-causing) organisms and, consequently, may be hazardous to human health if used as drinking-water or in food preparation. Faecal contamination of water is routinely detected by microbiological analysis. It is impractical to attempt the routine isolation of pathogens because they are present in relatively small numbers compared with other types of micro-organism. Moreover, there are many types of pathogen and each requires a unique microbiological isolation technique. The approach that has been adopted is to analyse for indicator organisms that inhabit the gut in large numbers and are excreted in human faeces. The presence of these indicator organisms in water is evidence of faecal contamination and, therefore, of a risk that pathogens are present. If indicator organisms are present in large numbers, the contamination is considered to be recent and/or severe. Bacteria in water are, in general, not present individually, but as clumps or in association with particulate matter. When enumerating bacteria in water it is not the number of individual bacteria present which are counted, but the number of clumps of bacteria or the particles and their associated bacteria. Each clump or particle may have many bacteria associated with it. 10.1 Characteristics of indicator organisms Total coliforms The term “total coliforms” refers to a large group of Gram-negative, rod-shaped bacteria that share several characteristics. The group includes thermotolerant coliforms and bacteria of faecal origin, as well as some bacteria that may be isolated from environmental sources. Thus the presence of total coliforms may or may not indicate faecal contamination. In extreme cases, a high count for the total coliform group may be associated with a low, or even zero, count for thermotolerant coliforms. Such a result would not necessarily indicate the presence of faecal contamination. It might be caused by entry of soil or organic matter into the water or by conditions suitable for the growth of other types of coliform. In the laboratory total coliforms are grown in or on a medium containing lactose, at a temperature of 35 or 37 °C. They are provisionally identified by the production of acid and gas from the fermentation of lactose. Thermotolerant (faecal) coliforms The term “faecal coliform” has been used in water microbiology to denote coliform organisms which grow at 44 or 44.5 C and ferment lactose to produce acid and gas. In practice, some organisms with these characteristics may not be of faecal origin and the term “thermotolerant coliform” is, therefore, more correct and is becoming more commonly used. Nevertheless, the presence of thermotolerant coliforms nearly always indicates faecal contamination. Usually, more than 95 per cent of thermotolerant coliforms isolated from water are the gut organism Escherichia coli, the presence of which is definitive proof of faecal contamination. As a result, it is often unnecessary to undertake further testing to confirm the specific presence of E. coli. In the laboratory thermotolerant coliforms are grown on media containing lactose, at a temperature of 44 or 44.5 °C. They are provisionally identified by the production of acid and gas from the fermentation of lactose. Nutrient-rich environments may encourage the growth or persistence of some species of thermotolerant coliform other than E. coli. This possibility should be considered when, for example, an unusually high result is obtained from water that was thought to be relatively clean. In such a case, the advice of a microbiology laboratory should be sought for the determination of the more specific indicator, E. coli. Faecal streptococci The presence of faecal streptococci is evidence of faecal contamination. Faecal streptococci tend to persist longer in the environment than thermotolerant or total coliforms and are highly resistant to drying. It is, therefore, possible to isolate faecal streptococci from water that contains few or no thermotolerant coliforms as, for example, when the source of contamination is distant in either time or space from the sampling point. Faecal streptococci grow in or on a medium containing sodium azide, at a temperature of 37-44 °C. They are usually detected by the reduction of a dye (generally a tetrazolium-containing compound) or the hydrolysis of aesculin. Routine methods may give “false positives” and additional confirmatory tests may be required. Table 10.1 Comparison of methods for analysis of coliform bacteria Multiple fermentation tube technique Membrane filter technique Slower: requires 48 hours for a positive More rapid: quantitative results in or presumptive positive about 18 hours More labour-intensive Less labour-intensive Requires more culture medium Requires less culture medium Requires more glassware Requires less glassware More sensitive Less sensitive Result obtained indirectly by statistical approximation Results obtained directly by colony count (high (low precision) precision) Not readily adaptable for use in the field Readily adapted for use in the field Applicable to all types of water Not applicable to turbid waters Consumables readily available in most countries Cost of consumables is high in many countries May give better recovery of stressed or damaged organisms in some circumstances Heterotrophic plate count The heterotrophic plate count includes all of the micro-organisms that are capable of growing in or on a nutrient-rich solid agar medium. Two incubation temperatures and times are used: 37 °C for 24 hours to encourage the growth of bacteria of mammalian origin, and 22 °C for 72 hours to enumerate bacteria that are derived principally from environmental sources. The main value of colony counts lies in comparing the results of repeated samples from the same source. If levels increase substantially from normal values, there may be cause for concern. 10.2 Selecting a bacteriological analytical technique Two techniques are commonly used to detect the presence of coliforms in water. The first of these is called the “multiple fermentation tube” or “most probable number” technique. In this method measured portions of a water sample are placed in test-tubes containing a culture medium. The tubes are then incubated for a standard time at a standard temperature. In the second technique, a measured volume of sample is passed through a fine filter that retains bacteria. The filter is then placed on culture medium and incubated. This is called the “membrane filter” technique. Features of the two techniques are compared in Table 10.1. 10.3 Multiple fermentation tube technique The technique has been used for the analysis of drinking-water for many years with satisfactory results. It is the only procedure that can be used if water samples are very turbid or if semi-solids such as sediments or sludges are to be analysed. The procedure followed is fundamental to bacteriological analyses and the test is used in many countries. It is customary to report the results of the multiple fermentation tube test for coliforms as a most probable number (MPN) index. This is an index of the number of coliform bacteria that, more probably than any other number, would give the results shown by the test. It is not a count of the actual number of indicator bacteria present in the sample. Principle Separate analyses are usually conducted on five portions of each of three serial dilutions of a water sample. The individual portions are used to inoculate tubes of culture medium that are then incubated at a standard temperature for a standard period of time. The presence of coliforms is indicated by turbidity in the culture medium, by a pH change and/or by the presence of gas. The MPN index is determined by comparing the pattern of positive results (the number of tubes showing growth at each dilution) with statistical tables. The tabulated value is reported as MPN per 100 ml of sample. There are a number of variants to the multiple fermentation tube technique. The most common procedure is to process five aliquots of water from each of three consecutive 10-fold dilutions; for example, five aliquots of the sample itself, five of a 1/10 dilution of the sample and five of a 1/100 dilution. Aliquots may be 1-ml volumes, each added to 10 ml of single- strength culture medium, or 10-ml volumes, each added to 10 ml of double-strength medium. Results are compared with values such as those given in Table 10.5 (see later). The use of one of the following variants of the technique may help to reduce the cost of analysis: • A smaller number of tubes is incubated at each dilution, for example three instead of five. A different table must then be used for the MPN determination (see Table 10.6 later). Some precision is lost, but using 9 tubes instead of 15 saves materials, space in the incubator, and the analyst’s time. • For samples of drinking-water, one tube with 50 ml of sample and five tubes with 10 ml of sample are inoculated and incubated. The results are compared with the values such as those given in Table 10.7 (see later) to obtain the MPN. 10.3.1 Culture media and buffered dilution water Each part of the test requires a different type of medium.