SummaryThe coliform bacteria group have been identified as the Klebsiella, Escherichia coli, Citrobacter, Enterobacter, and Serratia. Of these, the most frequently viewed are the Klebsiella group (found in higher concentrations in hospital patients and is abundant in the environment) and the E.
coli. Procedures have been devised through which the detection of microorganisms that indicate faecal contamination can be detected. These microorganisms are usually the coliform bacteria, though other types of bacteria that do not fit the coliform description are also taken as indicators of faecal contamination. The United Kingdom law stipulates that these procedures must be carried out on water made available to the public, but the laws have had to take into consideration the limitations of statistical methods of measuring the amount of coliform bacteria in a sample.
IntroductionThe presence of certain micro-organisms has usually been used to infer the presence of pathogens, yet it is now known that many possibilities exist in which each might exist in the absence of the other (Trest et al., 2004; A. Leclercq, et al., 2002).
The term “microbial indicator” has therefore become ambiguous, and to eliminate this ambiguity, more specific names have been given to different types of micro-organisms. General or process microbial indicators refer to those that “demonstrate the efficacy of a process” (Ashbolt, Grabow, and Snozzi, 2001). Faecal indicators point toward the existence of faecal contamination, and index/model organisms indicate the presence of pathogens (2001).BackgroundIt was Von Fritsch who in 1880 first attributed to Klebsiella pneumoniae and K.
rhinoscleromatis the property of being organisms generally found in human faeces. Just a few years later began the routine examination of water in London for bacterial contaminants. In 1885, Escherich provided a description of the Bacillus coli, later renamed for him as Escherichia coli (E. coli), and not long after this Percy and Grace Frankland suggested that organisms normally found in sewage ought to be identified so that better methods of detecting pollution could be adopted.
The enumeration of B. Coli using the Wurtz method of directly plating water samples was used by bacteriologists. The concept that acid would arise from the lactose was used to diagnose the presence of these coli. The idea of coliform bacteria (or those bacteria that take the form of the B.
coli) was present in Britain as early as 1901, although colony counting was not officially introduced until the first Report 71 (Ashbolt, et al. 2001). It was known, even then, that though B. Coli was predominantly faecal in origin, other coliforms could not be definitively said to be so.
PropertiesThe coliform bacilli group is made up of the Escherichia and Klebsiella mentioned before, along with Citrobacter, Enterobacter, and Serratia (Guentzel). Collectively, they are described as having the following properties: they are non-spore forming, gram- and oxidase-negative, rod-shaped, anaerobic, and are able to effect the fermentation of lactose to gas and acid using ?-galactosidase. This occurs at around 34-38°C and within 24 to 48 hours. These bacteria can also exist in a variety of substrates (Ashbolt, et al.
, 2001; Reynolds, 2003; Prieto, et al. 2001; A. Leclercq, et al. 2002).
EscherichiaSome coliforms are thermotolerant, and these form acid and gas from lactose at approximately 44.5°C within 22-26 hours. They are also termed faecal coliforms because of the role they play as faecal indicators. Escherichia coli are such coliforms that thrive in warmer environments, and therefore are called thermophilic.
They utilise tryptophan toward the production of indole. Their definition has recently come to include their ability to produce ?-glucuronidase. These coliforms are the most indicative of faecal contamination from warm blooded animals (Ashbolt, et al., 2001).
KlebsiellaeThe genus Klebsiella contains large, non-motile bacteria that produce a heat stable enterotoxin, which contribute to their pathogenicity (“Klebsiella,” 1995). Klebsiella resides as a saprophyte in the intestines of 5-38% of humans and animals, although it is also found in the nasopharynx and is abundant in the environment. They are (usually) rod-shaped and encapsulated, gram-negative, non-motile, and are of the family Enterobacteriaceae. Most produce gas from lactose at 44.
5°C and more than half grow at 10°C, though none of the pneumoniae type do so. They “usually develop prominent capsules composed of complex acidic polysaccharides” (Podschun and Ullmann, 1998). Citrobacter is gram-negative and flagellated and is also a part of the Enterobacteriaeae cloacae complex (Hoffmann and Roggenkamp, 2003).Methods of EnumerationThere are three major methods used to enumerate coliforms in a water sample.
One is known as the multiple tube fermentation method which gives an analysis of the Most Probable Number of coliforms present in the testing sample. The membrane filter (MF) method is used to number the total coliform colonies that form from a sample placed on the membrane, and the defined substrate method uses a medium that targets the specific microbacteria that one wishes to detect. Most Probable Number (MPN) MethodIn executing the MPN method, a water sample is diluted ten-fold in a series and inoculated in 1-ml aliquots into a medium. After a period of incubation (approximately 24 hours), the samples are examined for colony growth.
Growth is theoretically probable if one organism is present in each sample. The greater the number of samples done from each dilution, the better the statistical accuracy becomes. When the incubation period ends, patterns are noted regarding growth or non-growth of the bacteria. Optimal colony count for best results should range from 30 to 300.
The number of samples from each dilution that shows growth is recorded and the number pattern is compared with an MPN table, which gives the most probable number of the bacteria one can expect to find per unit of the substance tested (Lindquist, 2003).The MPN, though simple and traditionally considered superior to the Membrane Filtration Method (Lin, 1976), has several drawbacks. It is time consuming, as it takes approximately 48 hours just to get a presumptive reading. After this a complete test is required, which is also in itself time consuming, requiring an additional 24 to 72 hours for a result.
Even then, the species of the coliform is never identified and no members of the faecal group of coliforms are distinguished from the entire coliform group. (Ashbolt, et al. 2001; Edberg, et al. 1988; Health Canada, 2003).
The UK Environment Agency states that “it is important to realise that the MPN is only an estimate, based on statistical probabilities and that the actual number may lie within a range of values” (2002, p. 39).Membrane Filtration MethodIn the first part of the process, a sample of the appropriate volume passes through the filtration membrane. (Standard volume varies according to the type of water being tested).
The membrane must have a small enough pore-size to retain the bacteria while allowing the fluid to pass through. The standard amount of time for filtration is five minutes. Then on a Petri dish or plate containing a pad saturated with the appropriately selected medium (designed to maximise the growth of coliform), the membrane is placed and incubated for 22-24 hours. The medium used is one enriched with lactose, and the temperature for incubation is approximately 34.
5C to 35.5C. Plates are inverted for the duration of the incubation period. Bacteria will grow on the on surface of the medium.
After this period, the colonies are counted and recorded as number of colony forming units (CFU’s) per 100 ml. (Chevalier and Craddock, 2000).In order get an accurate estimate, it is best to get a colony count of between 20 and 80, but the higher the number, the greater the statistical accuracy. However, a higher number than 200 would probably be too tedious.
The coliform density can be computed thus:Number of colonies counted x 100 /ml of sample filteredOne of the limitations of this method is the inability of the membranes to detect gas production. The result of this drawback is that in some cases, E. coli or other related coliforms have gone undetected because it has failed to react in the principal ways that are best detected by this test (Lindquist, 2003; Chevalier and Craddock, 2000).The Defined Substrate MethodThe defined substrate method is based on past methods calculated to analyse microbacteria on the basis of the enzymes that constitute them.
The method utilises a substrate that is hydrolysable and that is defined for the targeted microbe. The technique is defined by its simplicity, as water is merely added to a pre-fabricated formula that comes in powder form, and incubation immediately follows. Colour changes occur by the actions of the targeted microbacteria. This method is convenient because of the speed with which results can be obtained, and also because the fewer number of steps makes it resistant to errors, (Edberg, et al.
1988).Other Faecal Indicating BacteriaSulphite-reducing clostridia (SRC) form spores are gram positive, non-motile, and anaerobic. Clostridium perfringens are SRC’s that because the fermentation of lactose, inisitol, and sucrose with the effect of producing gas. They cause milk to coagulate, cause the hydrolysation of gelatin and reduce nitrate.
C. perfringens are the clostridian indicator of faecal contamination; not all SRC’s are faecal indicators. Advantages of clostridia are that the three main detecting methods are able to detect these bacteria and their persistence in the environment makes them easy to detect (Sobsey, 2001; Ashbolt, et al., 2001; USFDA, 2005).
Bifidobacteria are anaerobic and gram-positive, but most are catalase- and indole-negative. Approximately 17 species have been discovered and most are found in the intestines of humans, pigs, and sheep. These have been proposed as indicators of faecal contamination, and methods have been developed to detect them. However, the fact that they do not survive well in the environment limits their usefulness as a faecal indicator.
In addition, the medium developed to detect them has not proven to be sufficiently selective to produce very conclusive results (Sobsey, 2001; Ashbolt, et al., 2001).Faecal streptococci (FS) are gram-positive and catalase negative whose medium is selective. They can thrive in bile aesculin agar at temperatures around 45°C, and the genera to which they belong are Enterococcus and Streptococcus.
Most FS can produce growth at a pH of 9.6 and in 6.5% NaCl, and nearly all can withstand temperatures as high as 60°C for about thirty minutes. They can grow aerobically and can hydrolyse “4-methlumbelliferyl-?-D-glucoside […] in the presence of thallium acetate, nalidixic acid and 2, 3, 5-triphenyltetrazolium chloride” (Ashbolt, et al.
, 2001).Government LegislationIt has been a requirement of UK law since 1847 that wholesome drinking water be supplied to the public, and “quality criteria for the protection of aquatic ecosystems are now being based on an ecological quality index” (Enderlein, et al., 1997). The 1989 Water Act, the Water Supply (Water Quality) Regulation and the 1991 Water Resource Acts define wholesome as “clear, palatable and safe” (Emmerson, 2001) and specified the requirements for amounts of substances legally allowed in water supply.
Corresponding legislation in the form of the Surface Waters Regulation of 1994 have set standards for the water found naturally in rivers, streams, and other part of the ecosystem. This requires that water providers test for the common faecal indicators, such as E. Coli and Klebsiella, as well as such “surrogate markers” as Clostridium Perfringens. The detection of E.
Coli in “any one sample is an infringement of the legislation” concerning coliform, which stipulates that 95% of any samples taken (when fewer than 50) should be free from coliforms (2001).In order to deal with inaccuracies regarding the statistical records, laws have allowed that “laboratory techniques should have a detection limit that is preferably, one order of magnitude lower than the water quality objective for the substance in question” (Enderlein, et al., 1997). It is often the case that the objectives regarding water quality have a margin of error already built in, so that should water quality actually fall below that margin, the public would remain relatively safe (1997).
ConclusionThe coliform most indicative of faecal contamination is the E. Coli group, although included in the coliform group are Klebsiella, Citrobacter, Enterobacter, and Serratia. These are most generally used as indicators of contamination in water. Along with these, the Clostridia, bifidobacteria, streptococcus and enterococcus families have been taken as evidence of faecal contamination.
The tests generally used to indicate the presence of these bacteria are the Most Probable Number (MDN) test, the Membrane Filtration (MF) Test, and the Defined Substrate Method. Though they are very good at detecting the presence of bacteria, they have drawbacks, though, as the first two are not particularly selective of the bacteria they do detect, and (especially the MDN) are time consuming. The Defined Substrate Method is quite expedient, and is a simple test that gives fewer chances for human error. The United Kingdom has mandated that these tests be performed on water designated for public use, although allowances have had to be made for statistical errors.
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