Water is one of the most essential resources for human. Drinking water should be maintained under a certain level of quality standard to prevent health hazards because there are potentially harmful pathogens in drinking water. Natural water resources can be contaminated with human or animal waste which is consisting with lot of pathogenic microorganisms. Additionally there is natural mineral water resources presence with harmless bacteria which does not affect individual’s health .Therefore natural water is difficult to find without any contamination of microorganism. In Sri Lanka the demand and consumption for drinking water has been rapidly increasing. The major cause is difficult to find pure water naturally. People rely on drinking water in market assuming it to be free of pathogen. On the other hand, bottled drinking water may not always pure as it mentioned. There can be trace amount of disease causing pathogen which can affect consumer’s health.(Sasikaran et al,2012).
Table 2.1 Percentages of water borne pathogen.(Saati and Faidah,2012)
1.1 Regulation standard for drinking water in sri lanka
Natural mineral water is a good source of drinking water, also available in market as bottled drinking water. The quality of the bottled drinking water is highly questionable in current days in Sri Lanka. Out of 168 of brands of bottled drinking water only 15 have received SLS certification.(Perera,2003).SLS regulations are ensure the quality of the product. Ministry of health regulated the quality and condition of the bottled drinking water under food Act.No 26 of 1980. There are standards to identify two different types of bottled water, natural mineral water and bottled drinking water. Natural mineral water included none of chemicals. Natural water consists with harmless microorganisms. Purified bottled drinking water is free of microorganisms due to prevention treatments for growth of microorganism, Such as chlorination, UV radiation, High heat and filtration. (Madarasinghe, 2005). There are only less than 100 of bottle water brands registered in ministry of health and SLS certification. Most of the bottle water brands are not certified with the SLS. Regardless of the registration some products are available in market as purified water. Sri Lankan police arrested a man refilling the used plastic bottles with well water and sell it as purified water in medawachchiya.(Panagoda,2013)
1.2 Waterborne pathogens.
There are pathogens which are eliminates with feces capable of causing waterborne diseases. Bacteria, viruses and protozoan are causes for waterborne diseases. In developing countries waterborne diseases from bacterial origin are highly prevalent. Severe form of bacteria can initiate a disease with the low concentration. Bacterial pathogens were found in water distributing system where they can survive and multifaceted. Additionally pathogens found in drinking water are capable of causing severe infections, Such as P.aeruginosa , Aeromonas spp. Helicobacter pylori ,legionella which are able to cause acute gastroenteritis. There are high risk of infants, older age people, pregnant mothers and immunocompromised patients getting disease easily. (Leclerc,2002).
Figure 1.2.1 Bacteria presence in water .(Hayat,2013)
Coliform bacteria contaminated water via fecal matter. These types of bacteria are indicators for pathogenic bacteria in water.(Oram,2014). Waterborne bacteria can cause severe diseases such as, typhoid fever, basila dysentery, Diarrhea, shigellosis. The respective causative pathogen are Salmonella ,Shigella,Vibriosp,. Most of the causative bacteria are gram negative and spore forming. There is correlation between the concentration of the Coliform in water sample and pathogenic gastrointestinal bacteria. (Cabral, 2010).
Heterotopic bacteria are an indication for water is contaminated with fecal matter. Escherichia coli is one of the heterotrophic bacteria.. Furthermore heterotrophic bacteria does not give any evidence whether bacteria is pathogenic or harmless. The pathogenic bacteria are found in water sources are, Psuedomonas, Legionella and Pneumophila. (Deininige and Lee,2003). Polymerase chain reaction (PCR) amplification identify E. coli bacteria coliforms in water. PCR detect the trace amount of Coliform bacterial DNA contaminated in water.(Oram,2012)
The harmful pathogenic microorganisms which are living in water can increase the bacterial population during storage. Sallmonella spp, pseudomonas spp. are some of the pathogenic bacteria survive and reproduce in water. There are several regulation mechanisms to ensure the quality of drinking water. European community directive has established that there should not be any Coliform such as,E coli, Enterococcus. spp. Pseudomonas aeruginosa and parasite in 250 mL of bottled water. According to the world health organization there should be zero choliforms in drinking water. Using qualitative PCR can detect the total bacteria in water.(Ahmed et al,2013)
1.2 Methods of detection bacteria.
Polymerase chain reaction provides rapid and accurate amplified DNA. It is a novel technique in determination and quantification of DNA. The PCR technique facilitates to diagnose genetic, infectious diseases. Additionally also it facilitate the understanding of causative pathogen. (Ishmael and Stellato, 2008).
1.4 Bacterial cell wall differences and gram staining.
The bacterial cell wall is a multilayered structure which protect outside forces. According to the cell wall differences bacteria are dividing in to two groups, Gram negative and gram positive bacteria. Gram negative bacteria are bearing thin peptidoglycan cell wall surrounded by outer membrane.. Besides gram positive bacteria lack of outer membrane but covered with thick layers of peptidoglycan.. The grams stain procedures based on the reaction of bacterial cell wall with particular stain. These specific cell wall structures are adapted according to the environmental effect. Escherichia coli is a gram negative enteric bacteria lives in mammalian gut. It needs to exclude affect of chemical reaction such as bile salt which acts as detergent and capable to disrupt the cell. This specific environmental adaptative structures differs the bacteria as gram negative and gram positive. (Silhavy, Kahne and Walker,2010). The gram positive bacteria remain with the initial crystal violet stain where as gram negative bacteria decolorized by carbol fusion or safranin stains. Chemical components and cellular structures of bacteria respond differently to stain reaction. Gram positive bacterial cell walls are less permeable and thick. Gram negative cell walls are composed with thin peptidoglycan layer overlying with lipid protein bilayer which is called as outer membrane. Gram negative bacteria does not retain with stain. It disrupted by decolonization after apply alcohol. Beside gram positive bacteria retain the stain.( Beveridge, 2001)
1.5 Bacteria culturing.
Culturing is primary diagnostic tool to detect infectious microorganism. Under controlled laboratory condition, microorganism allowed to reproduce and multiply.. The advantage of culturing bacteria is from the low concentration can gain number of colonies. The agar is use as a media for culture. Escherichia coli is convenient to culture because of rapid growth and less nutritional demand. Although these small bacterial cells are small it needs less glucose concentration for produce energy source. ( Lodish, 2010)