Biochemical and microbiological evaluation of the water samples collected from different areas of district

Contamination of drinking water sources mainly due to microorganisms is the major problem in many areas of Pakistan. Pakistan is also facing the problem of contamination of drinking water which greatly affects human health and quality of life. The most important component of human beings for living is water. Therefore, it is important to analyze drinking water quality mostly in developing countries as the local people are mostly unaware of the water pollution. In this study, twenty three samples of water were analyzed during a 3-month period from the well and lake water supplies of different areas of Kohat and Mohamand Agency. The bacteriological evaluation was done and several tests were performed such as Total Plate Count, Coliform, Feacal coliform, Escherichia coli (E. coli) and Biochemical test. In this study, thirteen samples were in the normal range and 10 samples were out of safety ranges fixed by World Health Organization (WHO). The water which was not fit for drinking can be a consistent risk of the infectious diseases and continuous assessment and purification strategies should be developed in these areas to reduce the microbial contamination. The proper training by the local public authorities is required to educate the local community about water pollution, their causes and preventive measures in order to improve the health status of the people in the regions.


INTRODUCTION
Water is the most important component for living things and to support the life processes.Around 75% of the human body weight is composed of water.Without this it is not possible to maintain life on this planet.The quantity of water present on earth is approximately 1.4 trillion cubic meters

Water Sample Collection
Sample collection was performed from February to April (2013) and sampling was done randomly from different areas of Kohat and Mohamand Agency.Around 23 samples were collected from different dug wells, and lakes.The sampling method was according to standard method given by APHA (2005).Samples were collected aseptically in 250 ml sterilized autoclavable glass bottles in the laboratory.

Bacteriological Parameters:
The experiments for bacteriological analysis of drinking water were performed at the laboratories of Pakistan council of scientific and industrial research (PCSIR) complex Peshawar.

Total Plate Count:
Total plate count is determined by standard method.Serial dilution was prepared and aliquots of 1 ml were added to each replica petri dishes.
Plate count agar was added to every petri dish for total plate count and incubated at 35°C for 48 hours.After incubation, colonies were formed and then counted by colony counter and results were expressed as CFU/ml.

Total Coliform Bacteria:
Multiple tube fermentation technique was used to determine the total coliforms bacteria.One ml from previously prepared serial dilution was incubated in 3 duplicate tubes containing 10 ml of Lactose Broth with reversed Durham tubes and kept warm at 35°C for 24 and 48 hours.Tubes were observed for confirmation of gas formation at the end of 24 hours incubation.Gas making was calculated by gas dislocation in the reversed vial and bubble formation also occurred when the tubes was shaken smoothly.Unenthusiastic tubes were reincubated for extra 24 hours and observed for a second time for gas making.A positive tube with gas creation and turbidity was subcultured into BGB (Brilliant Green bile broth).All BGB tubes were kept warm at 35°C for 48 hours and checked for gas making.
Total Coliform was calculated from MPN table per 100 ml.

Total Fecal Coliform Bacteria
Tubes having 10 ml E.C broth with reversed Durham tubes were kept warm by means of 3 mm loop from the presumptive fermentation tubes viewing gas and keep warm 44.5°C for 24 hours and observed for gas making.

Isolation of E.Coli
EMB Agar was used for determination of E. coli.
All tubes of E. coli broth showing gas were subcultured by streaking on EMB Agar plates and incubated at 35°C for 24 hours.Positive plates contained typical colonies with metallic sheen were inoculated on PCA (plate count agar) slants for morphological and biochemical tests.After 24 hours incubation, the typical colonies were confirmed by biochemical tests and also by kits (E. coli O157:H7 latex test reagent kit Pro Lab.Canada) (31).

Results and Discussion
This study was conducted for the identification of Seven out of ten samples collected from Kohat were determined to be unfit for drinking due to the presence of E. coli as given in Table 1.Three out of 13 samples collected from Mohamand Agency were identified to be unfit for drinking purposes (Table 2).The result revealed that 10 (43.5 %) out of 23 samples have microbial contamination and unfit for drinking.The bacteriological analysis revealed that Total Plate Count (TPC) in the sample collected from Naveed Hotel (867 CFU/ml) was higher among all the samples, while that of Kohat Board (162 CFU/ml) was the lowest.The TPC values of all the samples are given in the mentioned tables where the samples have the TPC

Conclusion
It can be concluded from the study that the spring and dug well waters were mostly pure and free of contamination, however when these waters go through pipelines the water becomes contaminated.
The reason is the unsafe routes and leakage in the pipelines and should be properly managed to reduce contamination.The lake water was highly contaminated as it provides an open space for waste water, industrial solid waste and gutter waste Due to increase in environmental pollution, water resources are contaminated, which is a big hazard to life.This hazard is by regular discharge of industrial wastes and is a major problem in Pakistan [4].The main sources of drinking water in Pakistan are tube wells and dug wells.Water gets polluted when its condition or composition or both are changed.The major sources of pollution in natural water are discharge of domestic and industrial wastes from urban, rural and industrial areas.Kohat and Mohamand agency are important areas of Khyber Pukhtoonkhwa (KPK); therefore process of development is also fast.The population of these areas is constantly increasing.The increase in population give rise to many serious problems, especially increase in environmental pollution.As a result the sources of water are being polluted constantly [5].Drinking water must be free from turbidity, color and odor as well as from objectionable taste which have different inauspicious physiological effects [6-8].Fecal coliform bacteria include Total coliform and E. coli are the important microbiological parameters of water quality.Water contamination indicates the presence of E. coli either by fecal material of humans or other warm-blooded animals.According to U. S. Environmental Protection Agency (EPA), E. coli is a good indicator of fecal pollution than fecal coliform to evaluate fresh water quality [9-11].Contaminated water has direct and indirect effect; direct effect is on human health while indirect effect is through consumption of foodstuffs being irrigated with sewage and industrial effluents.World Health Organization estimated that around 80% of the population is deprived of pure drinking water in developing countries and every year around 5 million people die as a result of illness causes due to unsafe drinking water.The major pollutants present in water are mainly heavy metals, microorganisms, fertilizers and other toxic organic compounds [12].According to World Health Organization estimation every year about 500 million diarrhea cases take place in children usually less than five years in Asia, Africa and Latin America.The diseases like typhoid fever, bacillary dysentery, infectious hepatitis and other enteric infections are commonly transferred through contaminated water.Apart from this many other diseases are also transmitted through polluted water.Cancer is also caused by the assembly of certain materials carried by water to human organs [13-16].Escherichia coli is a unicellular microorganism, associated with faecal contamination of water.In human faeces, 95% of coliforms are Escherichia coli [17-20].For determination of water contamination and its standards the total aerobic plate count (TAC) is used as an indicator [21].TAC is a subgroup of E.coli used for the indication of faecal pollution of contaminated water [22].The use of bottled water is being increasing from the past 30 years worldwide [23].The consumer use bottle water mainly due to safety and potential health benefits because bottled water is much safer than tape water [24, 25].In Pakistan, rural populations are still deprived of clean drinking water and in urban areas contamination of drinking water is due to leakage in distribution systems [26-30].The aim of the study was to investigate the microbial contamination in the water samples collected from different areas of Kohat and Mohmand agency.This study will help to specify the areas where water is contaminated and purification strategies should be applied accordingly by the local authorities.
bacteria in drinking water samples from different areas of Kohat and Mohamand Agency.In total, 23 samples were collected from the above mentioned areas and checked for the presence of bacterial contamination.The Total Plate Count, Total Coliform, Fecal Coliform, E. coli and Biochemical tests were used for the bacterial identification.
within safety ranges of WHO was considered as fit for drinking and vice versa.Based on all the data and the given limits of WHO, all the water samples were considered as either fit or unfit for the drinking.The results revealed that the samples collected from lake water were highly contaminated and this could be due to the open access of the lake water to all the available wastes of those areas and no proper sanitation.The lake water contamination is not only alarming for the animals using this water for drinking but also for humans who are using these lakes for swimming in the summer season of Pakistan.In this study, the water belonging to different hotels of the mentioned areas were found highly contaminated and this is a question mark for the local authorities to adopt preventive measures and advise the hotels administration to improve their sanitation and water storage system in order to provide safe and pure water for drinking and washing.These hotels are mostly located in the surroundings of the Kohat University where mostly the young generation and specifically students are doing their lunch/dinner.This will have long lasting effect on the health of the local community.The spring water was mostly found safe and free of contamination.Mostly these springs are available in the areas have less population and much better environment, however when the spring waters comes in the pipelines and provided to local community, it becomes contaminated.According to previous literature, 90% of rural drinking water supplies system studied were found contaminated with Coliforms [31, 32].The methods used in this study were also previously used to investigate the microbial content of different waters in developing countries.Even some indicators were used to indicate surface contamination of drinking water supplies [33-35].