INTRODUCTION :-
Water is very essential to life. The greatest microbial risk are associated with ingestion of water that is contaminated with human or animal feces. The most bacterial diseases transmitted through water - cholera, typhoid fever & bacillary dysentery. Microbiological water analysis is mainly based on fecal indicators bacteria i.e coliforms. Routine basic microbiological analysis of drinking water is carried out by detecting the presence of Escherichia coli by culture method.
Detection of coliforms, to determine the quality of water can be carried out by performing following test.
1) Presumptive test
2) Confirmed test &
3) Completed test
Water is very essential to life. The greatest microbial risk are associated with ingestion of water that is contaminated with human or animal feces. The most bacterial diseases transmitted through water - cholera, typhoid fever & bacillary dysentery. Microbiological water analysis is mainly based on fecal indicators bacteria i.e coliforms. Routine basic microbiological analysis of drinking water is carried out by detecting the presence of Escherichia coli by culture method.
Detection of coliforms, to determine the quality of water can be carried out by performing following test.
1) Presumptive test
2) Confirmed test &
3) Completed test
1) Presumptive test
Requirements:-
- sterile Mackonkey's lactose broth (double strength-5 tube of 10 ml each and signal strength-3 tube of 5 ml each with the Durham's tube)
- sterile 10 ml pipettes - 5 no.
- sterile 1 ml pipettes - 2 no.
- water sample
Requirements:-
- sterile Mackonkey's lactose broth (double strength-5 tube of 10 ml each and signal strength-3 tube of 5 ml each with the Durham's tube)
- sterile 10 ml pipettes - 5 no.
- sterile 1 ml pipettes - 2 no.
- water sample
Principle :-
It is based on the principle that coliform group if present in water will ferment lactose to produce acid and gas within 24 to 48 hours. Production of acid is indicated by pH indicator dye & gas is collected in Durham's vial.
Composition of MacConkey's broth:-
Peptone - 20.0 g
Lactose - 10.0 g
NaCl - 0.5 g
Bile salt - 5.0 g
Neutral red - 30.0 g
Distilled water -1 liter
pH - 7.4
Procedure :-
1) shake the water sample vigorously to ensure uniform distribution of organisms.
It is based on the principle that coliform group if present in water will ferment lactose to produce acid and gas within 24 to 48 hours. Production of acid is indicated by pH indicator dye & gas is collected in Durham's vial.
Composition of MacConkey's broth:-
Peptone - 20.0 g
Lactose - 10.0 g
NaCl - 0.5 g
Bile salt - 5.0 g
Neutral red - 30.0 g
Distilled water -1 liter
pH - 7.4
Procedure :-
1) shake the water sample vigorously to ensure uniform distribution of organisms.
2) with sterile graduated pipette inoculate water sample as follow
a) 5 MacConkey's broth double strength with 10 ml water sample
b) 1 MacConkey's broth single strength with 1.0 ml water sample
c) 1 MacConkey's broth single strength with 0.1 ml water
d) 1 MacConkey's broth single strength serve as a CONTROL tube.
a) 5 MacConkey's broth double strength with 10 ml water sample
b) 1 MacConkey's broth single strength with 1.0 ml water sample
c) 1 MacConkey's broth single strength with 0.1 ml water
d) 1 MacConkey's broth single strength serve as a CONTROL tube.
3) incubate all tube at 37°c for 24 hours.
4) next day examine the tubes for the presence of acid and gas production. Reincubate if no acid & gas formed for another 24 hours.
2) Confirmed test
Requirement :-
- positive presumptive tube
- sterile BGLB tube
- sterile EMB agar medium
Requirement :-
- positive presumptive tube
- sterile BGLB tube
- sterile EMB agar medium
Mechanism :-
Sometimes false positive presumptive test may also result because of a type of association known as synergism. In this association joint action of two organisms on carbohydrate result into production of gas. For that specific concentration of brilliant green dye is incorpereted in medium which inhibit the growth of Gram +ve organisms.
Composition - BGLB
Peptone - 10.0 gm
Lactose - 10.0 gm
Bile salt - 20.0 gm
Brilliant green - 0.0133 gm
Distilled water - 1 L
pH - 7.2
Procedure :-
1) from positive MLBB tube inoculate 1 loopful in BGLB tube and incubate at 37°c for 24 hours .
2) if coliforms present than BGLB medium get turbid. From +ve BGLB streak one loopful on EMB agar media anf incubate platd at 37°c for 24 hours.
3) next day observed the typical colonies on EMB agar media.
Sometimes false positive presumptive test may also result because of a type of association known as synergism. In this association joint action of two organisms on carbohydrate result into production of gas. For that specific concentration of brilliant green dye is incorpereted in medium which inhibit the growth of Gram +ve organisms.
Composition - BGLB
Peptone - 10.0 gm
Lactose - 10.0 gm
Bile salt - 20.0 gm
Brilliant green - 0.0133 gm
Distilled water - 1 L
pH - 7.2
Procedure :-
1) from positive MLBB tube inoculate 1 loopful in BGLB tube and incubate at 37°c for 24 hours .
2) if coliforms present than BGLB medium get turbid. From +ve BGLB streak one loopful on EMB agar media anf incubate platd at 37°c for 24 hours.
3) next day observed the typical colonies on EMB agar media.
3) Completed test
Requirement :-
- EMB agar plate with typical greenish colony
- sterile lactose broth tube
- sterile N- agar slant
- steile distiled water phials
Procedure :-
1) select the typical colony on EMB agar and prepare suspension.
2) one loopful of suspension inoculate into lactose broth and one loopful streak on slant. Incubate both at 37°c for 24 hours.
3) next day obeserved acid and gas production in lactose broth and perform Gram staininv from pure growth on N.agar slant.
