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Subject: Course No: Submitted To: Submitted On: Seat No:

Environmental Microbiology and Public Health MIC-509 Dr.Tanveer Abbas 13th.Dec.2011 B0819075




1ndicators of Microbial and Chemical Water Quality


Indicator organisms are types of bacteria used to detect and estimate the level of fecal contamination of water. Water quality is the physical, chemical and biological characteristics of water. Current guidelines in the three water-related areas (drinking water, wastewater and recreational water) assess quality, in microbiological terms, by measuring indicator organisms. Traditionally, indicator micro-organisms have been used to suggest the presence of pathogens. 1.1.1 Fecal pollution of water Fecal pollution of water from a health point of view is the contamination of water with disease causing organisms (pathogens) that may inhabit the gastrointestinal tract of mammals, but with particular attention to human fecal sources as the most relevant source of human illnesses globally. Ingestion of water contaminated with feces is responsible for a variety of diseases important to humans via what is known as the fecal-oral route of transmission. Food, air, soil, and all types of surfaces can also be important in the transmission of fecal pathogens, and thereby implicated in disease outbreaks. Most fecal microorganisms, however, are not pathogenic. Indeed, some are considered beneficial to the host as they can out-compete pathogens for space and nutrients, complement the biochemical potential of the host‟s gastrointestinal tract, and help in the development of the host immune system. Nonetheless, animal feces can also carry a number of important frank and opportunistic pathogens, capable of inflicting debilitating illnesses and, in some cases, death.[1] 1.1.2 Criteria for Indicator Organisms The US EPA lists the following criteria for an organism to be an ideal indicator of fecal contamination:      The organism should be present whenever enteric pathogens are present. The organism should be useful for all types of water. The organism should have a longer survival time than the hardiest enteric pathogen. The organism should not grow in water. The organism should be found in warm-blooded animals‟ intestines.

None of the types of indicator organisms that are currently in use fit all of these criteria perfectly, however, when cost is considered, use of indicators becomes necessary. [2] 1.1.3 Indicator and Index Micro-Organisms of Public Health Concern There is no direct correlation between numbers of any indicator and enteric pathogens. To eliminate the ambiguity in the term „microbial indicator‟, the following three groups are now recognized:

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1ndicators of Microbial and Chemical Water Quality Process Indicator A group of organisms that demonstrates the efficacy of a process Fecal Indicator: A group of organisms that indicates the presence of fecal contamination such as E. coli Index and Model Organisms A group/or species indicative of human pathogen. 1.1.4 Fecal Coliforms as Index of Water Pollution It is almost impossible to isolate from water the organisms responsible for water-borne diseases so water quality bacteriologist have adopted the practices for certain indicator bacteria normally found in the human intestinal tract. Few organisms are present and they do not multiply in water. The only safe method to prevent waterborne disease is to condemn fecal polluted water as being unfit for human use, as it may contain harmful organisms. Fecal pollution can be determined by examination of water for colon bacilli (E. coli). E. coli is abundant in feces and not found outside intestinal tract in nature. The E. coli in water indicates the presence of pathogenic microorganisms in water, which may be responsible for a number of water-borne diseases. Hence, E. coli is known as indicator organism. Water also contains bacteria that resemble E. coli but may or may not be of fecal origin. These bacteria also ferment lactose with formation of gas like E. coli. The other indicator organisms are Enterococci including Streptococcus faecalis. 1.1.5 Significance of Fecal Coliforms The group of coliform bacteria as an indicator of other pathogenic micro-organisms, specifically organisms of fecal origin, has had much emphasis in all countries. This is due primarily to the fact that the coliform bacteria groups meets many of the criteria for a suitable indicator organism, and are thus a sensitive indicator of fecal pollution:       They are abundant in feces approximately billions/g of feces. They can grow in water under certain conditions. They are generally found only in fecally polluted waters They are easily detected by simple laboratory tests Can be detected in low concentrations in water The number of indicator bacteria seems to be correlated with the extent of contamination.

It is important to remember, however, that not all coliforms originate from human feces as they can originate from other mammalian species or from other environmental sources (e.g., bird droppings). When coliforms are discharged to the aquatic environment they will tend to die at a rate which depends, amongst other things, on the temperature and turbidity of the water and the depth to which solar radiation penetrates. Therefore, it is not safe to conclude that the lack of coliforms in a water means that it has not been subject to fecal pollution. [3] It is necessary to be familiar with a number of terms are as follows:

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1.1.6 Total Coliforms and Fecal Coliforms Coliforms Coliform is not a taxonomic classification, rather it is a group of Gram-negative, facultative anaerobic rod-shaped non-spore forming bacteria that ferment lactose to produce acid and gas within 48 h at 35°C. Coliform bacteria are a commonly used bacterial indicator of sanitary quality of foods and water. Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of warm-blooded animals. By and large, coliforms are represented by four or five genera of the family Enterobacteriaceae. Typical genera include:          Salmonella Shigella Proteus Citrobacter Enterobacter Hafnia Klebsiella Serratia Escherichia (Fecal coliform)

Coliform bacteria include genera that originate in feces (e.g. Escherichia) as well as genera not of fecal origin in normal conditions (e.g. Enterobacter, Klebsiella, Salmonella, Citrobacter). The assay is intended to be an indicator of fecal contamination; more specifically of E. coli which is an indicator microorganism for other pathogens that may be present in feces. Fecal Coliforms Fecal coliform, first defined based on the works of Eijkman. They are facultativelyanaerobic, rod-shaped, gram-negative, non-sporulating bacteria capable of growth in the presence of bile salts or similar surface agents, are oxidase negative, that grow and ferment lactose at elevated incubation temperature at 44.5 to 45.5°C, hence also referred to as thermotolerant coliforms. The fecal coliform group consists mostly of E. coli but some other enterics such as Klebsiella can also ferment lactose at these temperatures and therefore, be considered as fecal coliforms. Escherichia coli (E. coli), a rod-shaped member of the coliform group, can be distinguished from most other coliforms by its ability to ferment lactose at 45.5°C in the fecal coliform test, and by its growth and color reaction on certain types of culture media. When cultured on an EMB plate, a positive result for E. coli is metallic green colonies on a dark purple media. Unlike the general coliform group, E. coli are almost exclusively of fecal origin and their presence is thus an effective confirmation of fecal contamination. Rarely causing problems, it has theretofore been used as a marker to indicate the contamination of water or food with human feces, and hence the potential dangers that might accompany the presence of human

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feces in our food and water supply. Some strains of E. coli can cause serious illness in humans. Discovery Escherichia coli, (fig I-1) originally known as Bacterium coli commune, was identified in 1885 by the German pediatrician, Theodor Escherich. In 1892, Shardinger proposed the use of E. coli as an indicator of fecal contamination. This was based on the premise that E. coli is abundant in human and animal feces and not usually found in other niches. Hence, the presence of E. coli in food or water became accepted as indicative of recent fecal contamination.

Fig 1-1

Escherichia coli

Distribution E. coli is widely distributed in the intestine of humans and warm-blooded animals and is the predominant facultative anaerobe in the bowel and part of the essential intestinal flora that maintains the physiology of the healthy host. E. coli is a member of the family Enterobacteriaceae, which includes many genera, including known pathogens such as Salmonella, Shigella, and Yersinia. Although most strains of E. coli are not regarded as pathogens, they can be opportunistic pathogens that cause infections in immunocompromised hosts. There are also pathogenic strains of E. coli that when ingested, causes gastrointestinal illness in healthy humans. In practice not all such coliforms are fecal in origin although most (> 95%) are Escherichia coli (E. coli). This bacterium is a particular member of the fecal coliform group of bacteria; this organism in water indicates the presence of fecal contamination. E. coli reside in human intestinal tracts. They are excreted in large numbers in feces, averaging about 50 million per gram. Whereas untreated domestic wastewater generally contains 5 to 10 million coliforms per 100 ml. Perhaps the biggest drawback to using coliforms as indicators is that they can grow in water under certain conditions. [4][5][6]

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1.1.7 Fecal Streptococci This group of bacteria includes several species or varieties of streptococci and the normal habitat of these bacteria is the intestines of humans and animals. Examples include Streptococcus faecalis which represents bacteria of humans. So Escherichia coli (E. coli) and enterococci are used as indicators of microbial fecal pollution with reference to the water quality standards. 1.1.8 Waterborne Gastroenteritis Caused By E. coli (Diarrheagenic E. coli) Foodborne gastroenteritis occurs from the consumption of food (and water) contaminated with pathogenic E. coli. The Recognized Virulent Groups Based on disease syndromes and characteristics, and also on their effect on certain cell cultures and serological groupings, five virulence groups of E. coli are recognized which are as follows;       Enteroaggregative (EAggEC) Enterohemorrhagic (EHEC) Enteroinvasive (EIEC) Enteropathogenic (EPEC)
Enterotoxigenic (ETEC)






















Table 1-1The Recognized Virulent Groups of E. coli

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1.1.9 Detection and Enumeration The examination of water for the presence, types, and numbers of microorganisms and/or their products is basic to food microbiology. In spite of the importance of this, none of the methods in common use permits the determination of exact numbers of microorganisms in a in water. Although some methods of analysis are better than others, every method has certain inherent limitations associated with its use. The four basic methods employed for “total” numbers are as follows:  Standard plate counts (SPC) or aerobic plate counts (APC) for viable cells or colony forming units (cfu).  The most probable numbers (MPN) method as a statistical determination of viable cells.  Dye reduction techniques to estimate numbers of viable cells that possess reducing capacities.  Direct microscopic counts (DMC) for both viable and nonviable cells. A large number of methods have been developed for the detection and enumeration of E. coli and coliforms. Almost all the methods used to detect E. coli, total coliforms or fecal coliforms are enumeration methods that are based on lactose fermentation. [14] Some commonly used methods are as follows;  Conventional Method for coliforms, fecal coliforms and E. coli o The Most Probable Number (MPN) method o Solid medium method o Membrane Filtration (MF) Method  Fast Detections Using Chromogenic Substances  Application of Antibodies o IMS/Culture and Other Rapid Culture-Based Methods o Gene Sequence-Based Methods 1.1.10 Water Quality Standards for Bacteria Drinking Water Standards

World Health Organization Guidelines for Drinking Water Quality state that as an indicator organism Escherichia coli provides conclusive evidence of recent fecal pollution and should not be present in water meant for human consumption. In the U.S., the EPA total coliform rule states that a water system is out of compliance if more than 5% of its monthly water samples contain coliforms. Recreational Standards

Early studies showed that individuals who swam in waters with geometric mean coliform densities above 2300/100 mL for three days had higher illness rates. In the 1960‟s, these numbers were converted to fecal coliform concentrations assuming 18% of total coliforms were fecal. Consequently, the National Technical Advisory Committee (NTAC) in the US recommended the following standard for recreational waters in 1968: 10 % of total samples during any 30-day period should not exceed 400 fecal coliforms/100 mL or a log mean of
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200/100 mL (based on a minimum of 5 samples taken over not more than a 30-day period). Despite criticism, the US Environmental Protection Agency (EPA) recommended this criterion again in 1976, however, numerous studies were initiated by the EPA in the 1970‟s and 80‟s to overcome the weaknesses of the earlier studies. In 1986, the EPA revised its bacteriological ambient water quality criteria recommendations to include E. coli and enterococci. [7] 1.2 CHEMICAL POLLUTION INDICATOR Common chemical water quality pollution indicators include;        Dissolved Oxygen Ph Toxic Organic Compounds Biochemical Oxygen Demand (BOD) Chemical 0xygen Demand (COD) Heavy Metals Nutrients

1.2.1 Dissolved Oxygen The amount of molecular oxygen dissolved in water is an important measure of habitat availability for aquatic organisms. Low levels of oxygen result from the introduction of organic waste pollution which increases the rate of eutrophication and decreases the suitability for aquatic animal life. Sources include: agricultural runoff, urban runoff, and wastewater treatment plants. 1.2.2 pH Because humans and aquatic organisms are dependent on water with pH levels within a range near neutral, pH is a crucial water quality indicator. The pH test, one of the most common and easily performed water quality tests, measures the concentration of hydrogen ions, which then allows us to infer the strength of the acid or base. Pollution from burning fossil fuels increases the amounts of sulfur and nitrogen oxides introduced into the water, thereby increasing the overall acidity. 1.2.3 Toxic Organic Compounds There are many chemicals that have the capacity to travel throughout a waterway like pesticides including herbicides and insecticides. They have deleterious effects on human health if consumed so proper testing is necessary to ensure the standard quality of water. 1.2.4 Biochemical Oxygen Demand (BOD) Biochemical oxygen demand or B.O.D. is the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material present in a given water sample at certain temperature over a specific time period. BOD can be used as a gauge of the effectiveness of water treatment plants. It is listed as a conventional pollutant in the U.S. Clean Water Act.
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1ndicators of Microbial and Chemical Water Quality

1.2.5 Chemical 0xygen Demand (COD) The chemical oxygen demand (COD) test is commonly used to indirectly measure the amount of organic compounds in water. Most applications of COD determine the amount of organic pollutants found in surface water (e.g. lakes and rivers) or wastewater, making COD a useful measure of water quality. 1.2.6 Heavy Metals Industrial effluents are major sources of heavy metals, and aquatic environments are extremely sensitive to even the smallest concentrations of these materials. Serious abnormalities have been reported in many aquatic organisms. Arsenic and mercury are two common examples of heavy metals, but other similar substances and compounds can also have significant effects on an aquatic community. 1.2.7 Nutrients Additional nutrients, such as phosphorus and nitrogen, are added to streams by many avenues, but primarily through human sewage, animal waste, fertilizers and erosion. This area of water quality monitoring is greatly affected by both urban and agricultural human practices. Although these chemical variables are useful to monitor impacts, they only provide a shortterm picture of water quality at a sampled site and each can only represent a portion of a complete assessment. A couple disadvantages of using only chemical indicators include:  Effects of certain isolated chemicals in a laboratory setting can be dramatically different than its interactive effects with other variables  Chemical testing is extremely expensive and labor intensive and not practical for monitoring non-point pollution sites such as urban and agricultural run-off Chemical testing is much more applicable for point source pollution where industrial contamination is suspected. [8]

[1][2][3] Indicators of microbial water quality by Nicholas J. Ashbolt, Willie O.K. Grabow and Mario Snozzi [4] Fundamental Food Microbiology by Bibek Ray, 3rd Edition CRC Press. [5] Modern Food Microbiology by James M. Jay, Martin J. Loessner and David A. Golden 7th Edition, Food Science Text Series. [6] Coliforms and Fecal Coliforms, Wikipedia. [7] [8] Water Quality Indicators)

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