R.N.BHABU VIGNESHM.Sc. MICROBIOLOGY

Principle

This test demonstrates the ability of certain bacteria to decompose the amino acid TRYPTOPHANE to indole, which accumulates in the medium. Indole production is tested by adding Para dimethyl amino benzaldehyde

Biochemical mechanismTryptophane tryptophanase Indole

Tryptophane is converted to indole when the organism has the enzyme tryptophanase , this can be identified by adding Kovac’s reagent.

Requirements Sterile test tubes. Positive control . (E. coli )Negative control. (Klebsiella sp).Test organism.Peptone water.Kovac’s reagent.

Medium Peptone [containing sufficient amount of

tryptophane ]. Sodium chloride. Distilled water. pH- 7.4.

Kovac’s reagent Amyl or iso amyl alcohol. Para dimethyl amino benzaldehyde. concentrated Hydrochloric Acid. Preparation of Reagent: Dissolve aldehyde in the alcohol

and slowly add the acid. Store in refrigerator and shake gently before use.

Quality controlPositive control- E. coli.Negative control- Klebsiella sp.

Procedure Inoculate the test organisms in the

tubes containing peptone water.Add positive control and negative

control in the tubes containing peptone water separately.

Incubate for 48 hrs at 37oC.Add 0.5 ml of Kovac’s reagent and

shake gently .

Interpretation A fuschin red color ring appears

after adding Kovac’s reagent indicates positive result.

No color change after adding Kovac’s reagent indicates negative result .

Other method 1) Ehrilich method :Here Ehrilich reagent is used. Ehrilich reagent Para dimethyl amino benzaldehyde.Ethyl alcohol.Concentrated hydrochloric acid.

Procedure Inoculate peptone water with 1 drop of

a 24 hours broth culture.Incubate at 35oC in ambient air for 48

hours .Add 1 ml of xylene to the culture.Shake vigorously to extract the indole

and allow to stand until the xylene forms a layer on top of the aqueous phase.

Add 0.5 ml of Ehrilich reagent along the sides of tube.

2) Filter paper method Para dimethyl amino cinnamaldehyde is

coated. Rapid development of blue color

indicates a positive result. Most indole positive organisms turn blue within 30 seconds.

Precautions Bacterial inoculum should not be selected

from Mackonkey agar because color of lactose fermenting colonies on this medium can interfere with test interpretation.

Examples Indole positive bacteriaE. coli .Proteus vulgaris. Morgenalla morganii.Providencia sp.

Indole negative bacteria K. pneumonia.S. typhi.S. paratyphi.Enterobacter sp.Proteus mirabilis .

PrincipleThis test to demonstrate the ability of an

organisms to utilize citrate as the sole carbon and inorganic ammonium salts as its only nitrogen source. Koser’s liquid citrate medium or Simmons citrate agar may be used.

Requirements Sterile test tubes. Positive control (Klebsiella

pneumonia).Negative control (E.coli).Test organism.Simmons citrate agar.

Medium Koser’s medium Sodium chloride Magnesium sulphate Ammonium dihydrogen phosphate Sodium citrate Distilled water pH of 6.8

Simmons citrate agar It is modification of Koser’s

medium with agar and indicator Bromothymol blue

Quality controlPositive control- Klebsialla

pneumoniaNegative control- E.coli.

Procedure Inoculate Simmons citrate agar

slightly on the slant by touching tip of a needle to a colony that is 18- 24 hours old.

Incubate at 35oC for upto 7 days.

Interpretation The development of blue color

indicates the positive result , that is utilization of citrate and the medium becomes alkaline.

Examples Citrate test positive bacteriaKlebsiella pneumonia.Enterobacter sp.Proteus sp.Providencia sp.

Citrate test negative bacteria E. coli.S. typhi.Shigella sp.Morgenalla morganii.

Principle This is a test for the ability of the

organism to utilize urea by the action of enzyme urease . The occurrence of this enzyme can be tested by growing the organisms in the presence of urea and testing for alkali [NH3] production by means of suitable pH indicator.

An alternative method is to test for the production of ammonia from urea by means of Nesselar’s reagent.

Biochemical mechanism

Urea +water urease carbon dioxide+ water+

ammonia

Quality controlPositive control- Proteus sp.Negative control- E. coli.

Requirements Sterile test tubes. Positive control (Proteus sp).Negative control (E. coli).Test organism.Christensen’s medium.

Medium Christensen’s medium:Peptone .Sodium chloride.Dipotassium hydrogen phosphate.Agar.Distilled water .Glucose 10% solution. Urea 20% solution. Phenol red indicator. pH of 6.8- 6.9

Preparation of mediumSterlisation of this medium is done

by filtration or Urea should be added in the medium after sterlising in autoclave when temperature comes down to 45°C.

Procedure Inoculate heavily over the entire slope

surface and incubate at 37°C.Examine after 4 hours and overnight

incubation, no tubes being reported until after 4 days.

Urease positive cultures change the color of the indicator to purple.

Examples Urease positive organisms:Proteus vulgaris.Proteus mirabilis.Morgenalla morganii.

Urease negative organisms:E. coli.Salmonella typhi.Shigella spp.Vibrio cholera.

Other methods 1) Elek’s test:Urea Potassium hydrogen phosphate 0.2

mol/ litre.Sodium hydroxide, 0.2mol/ litre.Distilled water , ammonia free. pH of 7.2.

Urea broth Urea.Monopotassium phosphateDisodium phosphate.Yeast extract.Phenol red.Distilled water.

Urea broth for MycobacteriaPeptone.Dextrose.Sodium chloride.Potassium phosphate mono basic.Urea.Phenol red.Tween 80.Distilled water.

Rapid Urease test – It is used to detect Helicobacter pylori.