Biochemical Test

Hi!

I was posted to the Anerobic(Quality control)on my 16th week.And we have been doing Biochemical test for the identification of Bacteria. The bacteria identification test done for the post is for E.coil

Biocehmical test is used for the identification the bacteria, by using the biochemical propaty of the bacteria. It is done using the following basic media.

Kliger iron agar

It uses the ablity to utilise carbohydrate fermentatively & to librate H2S.It contains one part glucose (0.1%) and 10 part lactose (0.1%).Phenol red is used as pH indicator.H2S formation is by the reaction of bacterium with sodiumthiosulplate with will form ferrous sulphide when H2S reacts with ferric ion.


Simmons citrate agar

Organism utilise citrate as their sole carbon source which is a intermediate metabolite in the kerb’s cycle. Media contain inorganic ammonium salt which the organisum use as a sole nitrogen source. The presence of organisum utilise the media a creats a alkaline environment that turns the bromthymol blue to a intense blue when pH reaches above 7.6.

Typtophan broth

The indole test determines the ability of an organism to produce indole from the degradation of the amino acid tryptophan in the broth. Tryptophan is hydrolysed by tryptophanase to produce three possible end products which is the Indole, pyruvic acid and ammonia.A bright red color product is produced when the indole is combined with aldehydes group.

Oxidation Fermentation (OF) basal Medium

The medium contains a high concentration of added carbohydrate to avoid the utilization of peptone by an aerobic organism as it would neutralize slight acidity produced by an oxidative organism.Bromthymol blue is used as a indicator.The agar permits the determination of motility and aids in the even distribution of any acid produced at the surface of the medium



Inoculation Method and reaction

Kliger iron agar

Stab to the bottom of the tube and streak the slant
Acidic butt and slant: Glucose and lactose fermentation (E.coil)
Acidic butt and alkaline slant:Glucose fermentation,Lactose nonfermentation
Alkaline butt and slant: Glucose and lectose non fermentation
Black media for H2S production.
Bubble when other gas is produced


Simmons citrate agar

Streak the slant.
+ve: Growth on the slant or deep blue colour observed. (E.coil)
-ve: No growth and no clour change (remains green)


Typtophan broth
Inoculate organisum by dissolving the colony in the broth.
After 24 hrs of incubation add 5 drops of kovac to the inner wall to see the pink interface.
+ve: Bright fuschia red interface with in secs.(E.coil)
-ve: No change

OF basal Medium
Stab the organisum stright to till the ¾ of meduim.
+ve: Medium becomes yellow indicating acid production. (E.coil)
-ve:Remains green.
Motality is observed when organisum grow away from the stright inoculum line.(E.coil)

By

Shameema :)