Practical # 2

Simmons Citrate Test

(Biochemical test)

Principle:
This test detects the ability of a few bacteria to utilize citrate as the source of carbon and
inorganic ammonium salt as the source of nitrogen for their growth, with the production of the
alkaline metabolic products. Bacteria that can grow on the medium produce an enzyme citrate
permease capable of converting Citrate into pyruvate. Pyruvate enters the metabolic cycle for the
production of energy.

Composition of Media:
Composition
Sodium Chloride 5.0 gm
Sodium Citrate (dehydrate) 2.0 gm
Ammonium Dihydrogen Phosphate 1.0 gm
Dipotassium Phosphate 1.0 gm
Magnesium Sulfate (dehydrate) 0.2 gm
Bromothymol Blue 0.08 gm
Agar 15.0 gm
Procedure:
 We took test tubes with Simmons citrate agar in slant position.
 We bacterial colony of test organism (Escherichia coli).
 Then we sterilized the loop needle.
 Then we picked up a minimum amount of bacteria and transferred it into Simmons citrate
agar.
 Then we inoculated the media in zig zag portion.
 Then we incubated the culture media at 37°C for 24 hours.

 After 24 hours incubation we added pH indicators such as bromo

methyl blue green-blue above pH 7.6.

Result Interpretation:
Dark blue color appeared on the surface of the slant indicates result is
positive. (Green color turns into blue color)
Green color appeared means no color change indicates result is negative.
(No color change)
Our test organism (Escherichia coli) tested negative for Simmons citrate test (no color
change was observed).
 Practical # 5
Most Probable Number (MPN)
MPN:
It is commonly used techniques for the detection of total coliform contamination of
water. It provides an approximate count of viable bacteria and can detect any type of coliform
genera and other coliform bacteria too.

Steps of MPN Test:

1. Presumptive Test
Procedure:
 First, we prepared MacConkey Lactose Broth media in test tube with Durham’s tube and
then autoclaved tube.
 We took three sets of test tubes containing five tubes in each set with 10ml of media.
 Using sterile pipettes, we transferred 10ml of water to each of the first set of tubes, then
transferred 1ml of water sample (Escherichia coli) to second set of tubes
and then transferred 0.1ml (100µl) to remaining tubes which are of third
set.
 After that we incubated tubes at 37℃ for 24 to 48 hours.
 After incubation, observed the color change and gas production in
Durham’s tube and in media.
Results:
The formation of gas in Durham’s tube and media color change within 24
to 48 hours is positive test for fecal coliform bacteria.

2. Confirmatory Test
Some microorganisms other than coliform also produce acid and gas from lactose
fermentation. In order to confirm the presence of coliform a confirmatory test is done.
Inoculation of lactose-broth
 We took a loop full of suspension from positive presumptive test tube and inoculated in
Lactose broth.
 Incubated at 37℃ for 24 hours and inspected gas production.
Inoculation in Media Slants
 First, we took a loop full of suspension from positive presumptive test tube and inoculated it
on agar slant.
 Incubated agar slant at 37℃ for 24 hours.
 We examined the colonies by staining (i.e. Red Pink Colonies of E. coli).
Inoculation in Tryptone Water
 We inoculated the colony in tryptone water and incubated for 24 hours.
 Added Kovac’s reagent and we observed for ring formation.

Results:
 Formation of gas in Lactose broth is indication for Positive test.
 Staining of Colonies on slant as Red Pink Colonies of E. coli is positive test.
 Ring formation in Tryptone water is indication of Positive test.
3. Completed Test
Procedure:
 We streaked the inoculum from positive confirmatory test
tube on Eosin Methylene Blue (EMB) or Endo Agar and
incubated at 37℃ for 24 hours.
 Observed the color change.
 Maintained temperature at 44.5℃ to indicate presence of
thermotolerant E. coli.
Results:
Coliforms produced Greenish Metallic color that is
Indication for Positive test.

ADVANTAGES & DISADVANTAGES

Advantages of MPN:
 Ease of interpretation either by observation or gas production.
 Effective method for analyzing highly turbid sample such as sediments, sludge and mud etc.
Disadvantages of MPN:
 Laborious and expensive in terms of material, glassware and incubator space.
 Poor accuracy and precision associated with MPN usually means that it is example of
classical microbiology.