Microbiological Analysis of Potable Drinking Water

Drinking water sources can be accidently contaminated by human fecal pollution. But release of small quantity of fecal matter does not cause serious health problems because it is highly diluted within the natural water bodies. If water bodies are once fecal polluted, it contains pathogens of fecal origins.  This pathogen may include protozoan cysts, nematodes larvae, nematode eggs, viruses, bacteria and fungi. But it is difficult to test for each and every above organism so test for indicator organisms such as tests for E coli is done. It is an intestinal microorganism that lives in human and most animal bodies. E coli releases through the fecal matter into aquatic water bodies. They do not die as soon as they release from the human body. But they cannot survive for many days in the water due to low nutrient availability. It is assumed that fecal origin pathogens also have similar duration of survival within aquatic water bodies. According to WHO, drinking water cannot contain at least one E coli. If drinking water contain at least one E coli, it is not suitable for human consumption.

                      Testing for E coli is cost effective and time saving. If E coli are present, it indicated that there is high probability of presence of fecal origin pathogens. But even if direct testing for E coli is cost because electricity has to be applied to inoculate the microorganisms in the water sample at high temperatures  in order to test for E coli. So initially testing for coliform is done. Coliform is a group of bacteria that are capable of fermentation lactose and producing acids and gas. Coliforms are gram negative and rod shaped bacteria. E coli are also one of the members of coliform. If the result of coliform test is negative, there is no need of further testing. Therefore water can be released for human consumption.

                Testing for E coli is done in three steps such as presumptive test, coliform test and conform test. In presumptive test, MaConkey broth is used the culture media. MaConkey broth media acts as the selective and differentiate medium for isolation of gram negative lactose fermentative bacteria.  It is comprised of lactose as the carbon source, peptone as the nitrogen and other nutrient source, bile salt, pH indicator and NaCl. Bile broth is added to suppress the growth of gram positive bacteria. Bromo cresol purple or neutral red is added as the pH indicator to observe to acid production of bacteria. If Bromo cresol purple is added, it changes its color as yellow due to the acid production. If neutral red is added as the pH indicator, it changes it color as dark red due to the acid production. NaCl is added to t maintain the osmotic balance of the medium.

                  Here five test tubes are prepared with 9 mL of double strength MaConkey broth and 1ml of water sample to prepare 10◦ series. Then another five test tubes are prepared with 9mL of single strength MaConkey broth and 1ml of water sample to prepare 10^-1 series. 1mL of water sample is taken into another test tube containing 9mL of peptone to dilute water to 10^-1. Then from that test tube 1mL is added to five test tubes filled with 9mL of single strength MaConkey broth to prepare 10^-2 series. These test tube series are inoculated at 37◦C for 24-48 hours. Color change due to the acid production and gas collected in the Durham tube are taken as the positive result of this test.

If the result of presumptive test is negative, there is no need of further testing.

 It indicated that there is no coliform that means there are no E coli. If results are positive, it indicates the presence of coliform. So further testing is needed to confirm whether presence or absence of coli.

                       The test tubes those shows positive results are used to inoculate in the conform test. Two methods are available such as BGLB broth or EMBA. BGLB broth means brilliant green lactose bile broth. Here lactose also is added as carbon source for bacterial fermentation. Brilliant green dye suppress the growth some selective gram negative bacteria. Bile suppresses the growth of gram positive bacteria. One set of BGLB broth containing test tube are inoculated at 37◦ C for total coliform. Total coliform contain both environmental coliform and fecal coliform. Another set of BGLB broth containing test tube are inoculated at 44◦ C for fecal coliform. Gas production in the Durham tube is taken as the positive results in this test.

Plate containing EMBA agar is used to conform the presence of E coli. Inoculum from the test tubes that shows positive results are taken into plates for inoculate at 37◦C for 24-48 hours. EMBA means Eosin Methylene Blue Agar. Eosin Methylene Blue dyes suppresses the growth of gram positive bacteria. Agar is added as the solidifying agent. Lactose is added as the carbon source for bacterial fermentation. It gives characteristic green color with metallic sheen if strong acid producers are present such as E coli. It gives pink colored colonies if weak acid producers are present such as Enterobactor. Non lactose fermentative does not give any color.

 If results from conform test is negative, there is no fecal coliform. So water can be released for human consumption. But if results are positive, further testing is needed because In addition to E coli, Enterobactor also shows positive results for confirm test.

                          Then complete test is done to confirm exactly presence of E coli. IMViC tests are done. IMViC means Indol Methylene red VP Citrate tests. E coli give positive results for Indol and Methylene test. In Indol test, only E coli is able to digeste tryptone because only E coli tryptonase enzyme to form Indol ring. As Indol ring cannot be observed due to the colorless, knovas reagent is added. If Indol ring is present, it appears in red in color. Enterobactor gives positive results for VP and Citrate tests. If the results of complete test are also positive, it indicated that presence of E coli. So water cannot be released for human consumption due to the possibility of presence of fecal origin pathogens.