M. A. El-Farrash

Water is the most important substance in our daily life. Safe drinking water is essential to humans and other life forms, as water is important to the mechanics of biological metabolisms in the body. Drinking water should be pure and free of contaminants to ensure proper health and wellness. Drinking Water

Water is made fit for drinking by filtration, distillation, or by a range of other methods. Poor water quality and bad sanitation are deadly; about five million deaths a year are caused by polluted drinking water. WHO (2010) estimated that safe water could prevent 1.4 million child deaths from diarrhea each year. Drinking Water

The contamination of drinking water sources with microbial pathogens in an on-going problem. More than three million people die every year from water-related disease and 43% of water- related deaths are due to diarrhea. The majority of diseases are infectious in nature caused by bacteria, fungi, viruses and parasites. Unsafe Drinking Water

Water is unsafe for human consumption when it contains pathogenic microorganisms. Pathogenic microorganisms may include: 1. Bacteria : Salmonella typhi, Vibrio cholerae, Shigella, and 2.Viruses: Poliovirus, Rotavirus, Norovirus or Hepatitis A virus and 3.Protozoa: Giardia lamblia or Cryptosporidium parvum. Unsafe Drinking Water

Outbreaks of disease attributable to drinking water still occur even in developed countries and can lead to serious acute, chronic, or sometimes fatal health consequences, particularly in sensitive and immunocompromized populations. In the last 10 years, there were 764 outbreaks associated with drinking water, resulting in 575,457 cases of illness and 79 deaths in USA. Unsafe Drinking Water

Bacteria Disease Caused Escherichia coli Gastroenteritis Leptospira spp. Leptospirosis Salmonella typhi Typhoid fever Salmonella spp. Salmonellosis Shigella spp. Shigellosis (bacillary dysentery) Vibrio cholerae Cholera Unsafe Drinking Water

Viruses Disease Caused Enteroviruses Gastroenteritis (poliovirus) Hepatitis A virus Infectious hepatitis Norwalk agent Gastroenteritis Rotavirus Gastroenteritis Unsafe Drinking Water

Protozoa Disease Caused Balantidium coli Balantidiasis Cryptosporidium parvum Cryptosporidiosis Entamoeba histolytica Amebiasis Giardia lamblia Giardiasis Unsafe Drinking Water

Indicators of unsafe water Measuring all of these pathogens routinely for determining presence or absence or acceptable concentration in drinking water is not possible. Methods are not available to recover and measure some of them. Available methods for others are technically demanding, slow to produce results and their costs are high.

Indicators of fecal pollution of water Alternative approach: measure something other than a pathogen that is indicative of contamination, predicts pathogen presence and estimates human health risks. Those are called indicators of fecal pollution of water and they are measured routinely and frequently.

The criteria for an organism to be an ideal indicator of fecal contamination: 1. The organism should be present whenever enteric pathogens are present 2. The organism should be useful for all types of water 3. The organism should have a longer survival time than the hardiest enteric pathogen 4. The organism should not grow in water 5. The organism should be found in warm-blooded animals’ intestines. Indicators of fecal pollution of water

Selected bacteria should be: 1.Commonly present in intestines of warm blooded animals in high numbers 2.Easy to measure 3. Applicable to all types of water (and other relevant samples). 4. Present in feces, sewage and fecally contaminated samples when pathogens are present; numbers correlate with amount of fecal contamination; outnumber pathogens.

5. No "aftergrowth" or "regrowth" in the environment 6. Survive/persist > than or = to pathogens. 7. Easily detected/quantified by simple lab tests in a short time. 8. Constant characteristics 9. Harmless to humans and other animals 10. Numbers in water are associated with risks of enteric illness in consumers.

E. coli E. coli is the most numerous coliform in human and animal intestine and is derived exclusively from these sources. It does not survive long in water. It is therefore the best indicator of recent human or animal faecal pollution. It is presence in water in high counts indicates a heavy or recent pollution, and in low counts indicates a slight or more remote one.

Other coliform bacilli Other coliform bacilli, e.g. klebsiellae and citrobacters, are much less abundant in faeces than E. coli. They also survive longer in water than E. coli. Their presence in water may therefore indicate either contamination from soil or contamination with faecal material at a time remote enough to have allowed E. coli to die out.

The presence of any coliform bacilli in chlorinated water indicates either a failure of the chlorination process or a contamination after chlorination, and the fault should at once be investigated and corrected. The presence of E. coli that can grow at 44 o C “thermotolarent” in water is a strong indication of recent sewage or faecal contamination. Thermotolarent E. coli

Faecal Streptococci Faecal streptococci are less abundant in faeces than E. coli and are often more abundant in animal than human faeces. Faecal streptococci survive longer in water than E. coli and are more resistant to chlorination than the coliforms. Their presence with coliforms, confirms faecal pollution of the water.

Clostridium perfringens Clostridium perfringens is present in faeces in even smaller number than the streptococci and is the least sensitive indicator of faecal pollution. But it is spores may survive chlorination and persist in water much longer than the other indicator bacteria, so that it is finding in the absence of the latter implies an intermittent or remote pollution.

It is necessary not only to attempt to detect the presence of the indicator bacteria, but also to count them, for the greater their number, the greater the danger of infection from the supply. Indicators of fecal pollution of water

Separate counts are made of presumptive coliform bacteria and confirmed E. coli bacteria. If the results of these tests are difficult to interpret, counts are also made of faecal streptococci and C. perfringens. Indicators of fecal pollution of water

Collection of water samples

Glass bottles used for water sampling should have a capacity of at least 200 ml. They should be fitted with ground glass stoppers or screw caps. Silicon rubber liners, that will withstand repeated sterilization by autoclaving, should be used inside screw caps. After being sterilized the bottle should not be opened before the sample is collected.

When collecting the sample, exercise extreme care to avoid contaminating it with bacteria from the environment. Flame the mouths of taps and hydrants and allow water to run to waste for 3-5 min before running it into the bottle. Collection of tap-water samples

When sampling from streams or lakes, open the bottle at a depth of about 20-30cm with it is mouth facing the current and ensure that water entering the bottle has not been in contact with the hand. Sample wells with weighted bottles. Collect at least 100ml in each bottle. Collection of other water samples

Collection from an river

Collection from an open well

Minimum sampling frequencies for drinking water in the distribution system Population served Samples to be taken monthly 1.Less than sample sample / 5000 population 3.More than sample / population, plus 10 additional samples

Un-chlorinated water supplies before distribution Population served Maximum interval 1. Less than One month Two weeks Four days

Multiple tube method In this method a measured sub-sample (10ml) is diluted with 100ml of sterile fluid Mac Conkey’s medium and an aliquot of 10ml is then decanted into each of ten tubes. The remaining 10ml is then diluted again and the process repeated. At the end 50 tubes covering the dilution range of 1:10 through to 1: Bacteriological Examination

The tubes are then incubated at 37 o or 44 o C for 24 hrs and the number of tubes with growth in is counted for each dilution. Statistical tables are used to derive the bacterial numbers in the original sample. This method can be enhanced by including a tiny inverted tube in each sample tube to catch any gas produced. The production of gas at 44 o C is a strong indication of the presence of fecal E. coli. Multiple tube method

Plate count The laboratory procedure involves making serial dilutions of the sample (1:10, 1:100, 1:1000 etc.) in sterile water and culture on nutrient agar in a dish that is sealed and incubated. Typical media include Plate count agar for a general count or MacConkey agar to count Gram-negative bacteria such as E. coli.

The plates showing an average between 30 and 300 colonies of the target bacterium are counted. Fewer than 30 colonies makes the interpretation statistically unsound whilst greater than 300 colonies often results in overlapping colonies and imprecision in the count. To ensure that several dilutions are normally cultured. Plate count

The total number of colonies is referred to as the Total Viable Count (TVC). The unit of measurement is cfu/ml (or colony forming units per ml) and relates to the original sample. For calculation of cfu/ml : the counted number of colonies is multiplied by the dilution used. Plate count

Membrane Filtration Method A membrane filter of the appropriate pore size is placed in a filter holder, and the sample is filtered by suction pump. In the process microorganisms in the test sample are retained on the filter surface by the screening action of the membrane filter. Growth inhibitors can be removed by flushing the holder with sterile water following filtration.

Afterwards, the membrane filter is placed on a culture medium and incubated. Nutrients and metabolites are exchanged through the pore system of the membrane filter. Colonies, which have developed on the membrane filter surface during incubation, are counted and related to the sample volume. Membrane Filtration Method

Results from routine samples Coliform / 100ml E. coli / 100ml 1- Excellent Satisfactory Intermediate Unsatisfactory 10 or more Quality of drinking water samples