Sunday, 15 May 2016

Water Analysis : To Characterize the coli-forms

AIM:  To characterize the coli-forms.
Coliform is a group of bacteria which is aerobic and facultative anaerobic, gram-negative, nonspore-foaming, rod shaped bacteria which ferment lactose with gas formation within 48 hours at 35°C.  This definition includes the following bacteria:  Escherichia coli, E. Aurescens, E. freundii, E. Intermedia;  Aerobacter, Aerogenes, A. Cloacae. 
These bacteria are found in the intestines of warm blooded animals and therefore are present in sewage, on and in soils, surface waters and vegetation.  The total coliform group has been used for sometime as an "indicator organism".  An indicator organism by itself is considered to cause no diseases in man or animals, but its presence usually indicates the presence of pathogenic or disease-causing organisms.  By measuring the number of total coliform present in a sample a judgment can be made as to the water's usability for a given purpose. 
However, there are problems with using the total coliform group as an indicator and this has resulted in an analyses for only those bacteria of the coliform group that are of fecal origin. 
Water which receives animally & human which form a primary source of water borne disease. Direct testing procedures capable of detecting & quantifying the full spectrum of pathogens & identifying their source is time consuming process because of their variable occurrence, survival rate & origin; hence an indicator system has been identified as a best method for evaluating the microbiological quality of water.
A fecal coli form test is considered as most reliable test available for detesting  the presence of contamination of an intesting origin, the coli form group comprises all aerobic & facuttative an aerobic, gram –ve  non spirulating, rod shaped bacteria that ferment lactase with gas format within 48hrs at 35 ˚c.
 The discharge of wastewater from municipal sewages is one of the most important sources of coliform in drinking water. Municipal sewage contains human feces & water contaminated with these effluents may contain pathogenic organisms & consequently, may be hazardous to human health if used as drinking water. In rural areas, open detection in the field, bathing & wasting of cloths, etc are some of the common sources of coliform contamination. Fecal contamination of water is routinely detected by microbiological analysis. Pathogenic microorganisms are transmitted by water include bacteria, viruses & protozoa. The intestinal troet of men contains countless rod shaped bacteria known as colliforms. Each person discharges from 100 to 400 billion coliforms per day in addition to other kind of bacteria.
Coliform bacterial densities can be determined by either multiple tube fermentation technique (MPN) or by membrane filter procedure. The multiple tube formation technique providing the most probable no. is an indirect countira technique reluing on statistical interpretation of growth (gas) or no growth (no gas) observation in the inoculated tubes. The test is conducted in three steps such as
1.      Presumptive test.
2.      Confirmatory test
3.      Complete test.
Generally for routine examination, the first two phases are conducted. In the membrane filter procedure, the water sample is passed through sterile membrane filter & the filters are incubated on the culture media. For the quantitative analysis of coliform the complete test is carried out.
            For microbiological analysis water samples have to be collected in sterilized bottle directly from the source leaving air space at the tap. Contamination from other sources like bucket tip of the tap, etc have to be avoided, the tap can be allowed to run for 2 to 3 min. before collection. Microbiological analysis has to be carried out immediately after collection. Sample can be stored below 10˚c for the may time of 6 hrs.
Test for coliforms with MPN
  •  Single strength of sactose bile growth (10 ml)- 10 test tubes. 
  • Double strength of sactase bile growth (10 ml)-  5 test tubes
  • Water sample.
  • Sterile pipettes.
  • Mac Cankey broth.
-    Distribute 10ml single strength lactase broth each to test tubes.
-     Add 10ml of double strength lactase broth in each of test tubes.
-     Introduce the Durhalm’s wial in all test tubes & sterilize them at 121˚c for 15 min.
-     Inoculate 10ml of water sample to all double strength lactose broth tubes.(DSLB)
- Inoculate 1ml water sample to each of 5 SSLB tubes & 0.1ml of water sample to remaining 5 SSLB tubes.
-   Incubate all the tubes at 37˚c for 24hrs.
-          If gas is formed in 24hrs indicate +ve test. If no gas is formed in 24 hrs, incubate for next 24 hrs. If no gas is formed even after 48 hrs of incubation test is –ve.
-          Count the no. of +ve tubes in each dilution.
-          Determine the MPN by Mc Cardy’s table.
Detection of Water Born Coliform and Fecal Coliforms with Coliscan Easy Gel
This new process for coliform and fecal coliform testing does not require an incubator or water bath.
  1. Use a sterile calibrated dropper to collect a 1 ml water sample and deposit the sample into bottle containing liquid coliscan medium (this procedure may be done in the field and the coliscan-water mix can be kept on ice until returning to the lab).
  2. Pour the coliscan water mix into a pre-treated petri dish and swirl to cover entire bottom of petri dish.
  3. Place the petri dish containing the coliscan-water mix in a warm place and incubate for 24-48 hours (this is best done in a place such as an incubator which holds the temperature in a range of 850-950' F).
  4. Count the red colonies in the petri dish as coliforms and the purple colonies as fecal coliforms (E. coli). (White or blue-green colonies should be noted, but they are not classified as coliforms or fecal coliforms).
  5. Perform gram staining of the coloformms.

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