Bacteriological analysis of water – Most Probable Number (MPN) Technique

 Bacteriological analysis of water – Most Probable Number (MPN) Technique

AIM

                   To check the potability of the given Drinking water sample by Most Probable Number (MPN) Technique.  

 

PRINCIPLE

                   Washing receives human and animal waste from a primary source of water borne disease. Direct heating produces detecting and quantifying the full spectrum of pathogens and identifying their source are time consuming sources because of their variable accordance survival rate and origin. Hence, an indicator system has been identified as the best method for evaluating the microbiological quantify of water. Coliform especially faecal coliform test is considered as the most reliable test new available for detecting the presence of contamination of an intestinal origin. The coliform groups comprises in all aerobic and for cultivating anaerobic Gram negative non sporulating, rod shaped bacteria that ferment lactose with gas formulating within 48 hours at 35 °C coliform includes bacteria of most common internal organism. Escherichia coli less common intestinal organism Klebsiella pneumoniae and of organism rate association in intestine Enterobacter aerogenes. Other common organisms includes this class are Salmonella, Shigella, Streptococcus and Aeromonas.

 

                   Coliform bacterial densities can be determined by either Multiple tube fermentation technique (MPN Test) by Membrane filter (MF) procedure. The Multiple tube fermentation technique providing the Most portable number (MPN) is an Indirect count technique relying on statistical interpretation of growth (90s). No growth (no gas) observation in the inoculated tubes the test was conducted in three steps such as the Presumptive test, Confirmatory test and the Complete test. Microbiological analysis has to be carried out. Immediately after collection sample can be stored below 10 °C for a maximum time of 8 hours during transient and refrigeration afterwords storage time should in any care not exceed 30 hours.

 

MATERIALS REQUIRED

a)             Brilliant Green Lactose Broth

b)            Eosin Methylene Blue (EMB) agar

c)             Gram staining kit

d)            Erlenmeyer flask

e)             Pipettes

f)              Test tubes

g)            Durham’s tube

h)            Petridishes

i)              Glass L – rod

j)              Distilled water

k)            Microscope

l)              Immersion oil

PROCEDURE

I. Presumptive Test

1)    Distribute 10 ml Single strength lactose broth (SSLB) each to 6 test tubes and Double distilled strength broth (DSLB) to 3 test tubes.

2)    Introduce the Durham's tube and sterile them at a time.

3)    Inoculate the 0.1 ml and 1 ml of water sample to each of three SSLB tubes and 10 ml of water sample in three DSLB correspondingly.

 

4)    Incubate the tubes at 37 °C and examine the gas formation in Durham’s tubes at 24 hours (positive test) or not 48 hours (doubt full tests) proceed to confirmed.

 

II. Confirmative Test

1)    Take a loopful culture from the Lactose broth tube from the highest dilution that still showed positive test and streaks it on EMB agar plate.

2)    Incubate the plates at 37 °C for 24 hours.

3)    Observe for the typical Escherichia coli colonies showing greenish metallic sheen.

 

III. Complete Test

               Pick up a suspicious colony (Escherichia coli) from EMB agar plate, make a smear and conduct Gram staining.