- IndoleTest
- Culture and Reagent used:
- 24 hour Growth in Peptone Water
- p-dimethylamino benzaldehyde (Kovac’s reagent)
- Principle: Organism producing Tryptophanase enzyme that degrade (hydrolysis & deamination) the Tryptophan present in culture media & produce Indole as a metabolic end product. If p-dimethylamino benzaldehyde (Kovac’s reagent) is added in medium and if indole is present, it reacts with indole & form a Red ring at the top.
- Use : For identification of Gram negative bacilli, mainly Enterobacteriaceae
- Result : Positive – red surface layer
Negative – No red surface layer
- Organisms giving positive reaction
- E.coli, Vibrio cholera, Proteus vulgaris,
2. Citrate Test
- Culture and Reagent used:
- Simmon’s Citrate Medium
- Bacterial colony
- Principle: Organism using sodium citrate as a sole source of carbon & Ammonium phosphate as sole source of nitrogen present in the media produce NH3 (Ammonia) after utilization of Ammonium phosphate, which gives alkaline pH, at which Bromothymol blue pH indicator present in the medium gives Blue colour.
- Use : For identification of Gram negative bacilli, mainly Enterobacteriaceae
- Result : Positive – Blue colour of medium
Negative – No colour change of medium
No growth over streaking line.
- Organisms giving positive reaction
- Klebsiella, Salmonella paratyphi B, Providentia rettegerri, Enterobacter aerogenes, Pseudomonas aeruginosa.
3. Methyl Red Test (MR test)
- Culture and Reagent used:
- 48 hour growth of Bacteria in Glucose Phosphate broth
- 0.04% MR reagent
- Principle: This test detects acid production during fermentation of Glucose & maintenance of pH below 4.5 in old culture. Add 4-5 drops of 0.04% MR reagent in 48 hour incubated Glucose Phosphate Broth.
- Use : For identification of Gram negative bacilli, mainly Enterobacteriaceae
- Result : Positive – Red colour of medium
Negative – No colour change of medium
- Organisms giving positive reaction
- E.coli, Salmonella, Proteus
4. Voges Proskauer Test (VP test)
- Culture and Reagent used:
- 48 hour growth of Bacteria in Glucose Phosphate broth
- α- napthol (5%)
- 40% KOH
- Principle: This test detects production of Acetyl Methyl carbinol from Pyruvic acid produced from fermentative metabolism of glucose. In presence of Alkali (40% KOH) & atomespheric Oxygen, small amount of Acetyl Methyl carbinol (Acetoin) are oxidized into Diacetyl which react with peptone of broth & give red colour after adding α-napthol (5%) as a catalyst. 48 hour incubated Glucose Phosphate Broth is culture medium of this test.
- Use : For identification of Gram negative bacilli, mainly Enterobacteriaceae
- Result : Positive – Red colour of medium
Negative – No colour change of medium
- Organisms giving positive reaction
- Klebsiella, Hafnia, Serratia, Citrobacter
5.Triple Sugar Iron (TSI) Reaction
- Culture and Reagent used:
- TSI Slant
- Bacterial colony
- Principle :
- Peptone, Three sugar (Glucose, Sucrose & Lactose in concentration of 1:10:10) & Ferric Ammonium Citrate are present in this medium.
- After metabolism of peptone & sugar, bacteria produce alkali & acidic products respectively.
- Bacteria utilize sugar as 1st nutrient & peptones are utilized after that.
- Oxidative utilization of nutrients are faster than fermentative (in absence of O2) utilization of nutrients.
- Use : For identification of Gram negative bacilli, mainly Enterobacteriaceae
6.Tube Coagulase Test
- Culture and Reagent used:
- Peptone water growth of organism
- Plasma
- Principle: This test detect free coagulase enzyme produced by Staphylococcus aureus. In Human & rabbit plasma, Coagulase enzymes convert fibrinogen into fibrin clot.
- Use: For differntiation of coagulase positive staphylococcus aureus from coagulase negative staphylococcus epidermidis.
- Result : Positive – clotting of plasma
Negative – No clot formation
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