BIOCHEMICAL TEST 1

1. Biochemical tests-I

2. What are biochemical tests ? • Biochemical tests are the tests that are used for identification or confirmation of different bacterial species based on biochemical activities that they perform. • By their nature, Bacteria produce large volume of enzymes and it is these enzymes that allow for their identification via biochemical methods . • Each species of bacteria has specific metabolic needs and relies on different enzymes to fuel those unique needs.

3. BIOCHEMICAL REACTIONSBased on the type of organisms observed in culture smear the appropriate biochemical tests are employed- • Catalase • Coagulase • Oxidase • Bile esculin hydrolysis • Indole • Methyl Red (MR) • Voges-Proskauer (VP) • Urea hydrolysis • Citrate Utilization • Nitrate Reduction • Phenyl Pyruvic Acid (PPA) • Oxidation fermentation (OF) • Sugar fermentation • Decarboxylase • CAMP (Christie-Atkins-Munch-Peterson) • PYR • DNase • Triple sugar iron(TSI)

4. Catalase Test • All members of Micrococcaceae (Staphylococci and micrococci) are Catalase positive, Which differentiates them from Streptococcacea (Catalase negative) • It is also positive for members of family Enterobacteriaceae,Vibrionaceace,Pseudomonadaceac,etc • It catalyzes hydrogen peroxide into water and oxygen and produce effervescences .

5. Coagulase Test • This test is utilized to separate Staphylococcus aureusfrom different Staphylococcus species like S.epidermidis • It has two methods- • Slide method • Tube method • Coagulase causes plasma to clot by converting fibrinogen to fibrin. • Coagulase positive –Staphylococcus aureus • Coagulase negative-S.epidermidis, S.saprophyticus

6. Oxidase Test • It detects the presence of cytochrome oxidase enzyme in bacteria which catalyzes the oxidation of reduced cytochrome by atmospheric oxygen. • Procedure-a disc ,soaked in oxidase reagent (1%tetramethylpara-phenylenediamine dihydrochloride) is smeared with a bacterial colony producing cytochrome oxidase enzyme ,the smeared area turns deep purple within 10 seconds due to oxidation of dye to form iodophenol blue. • Example- Pseudomonas,Vibrio,Niesseriaetc.

7. Bile-esculin Hydrolysis test • This test is based on the hydrolysis of esculin into glucose and esculetin (6,7-dihydroxy-coumarin) by a bacteria that produce an enzyme esculinase. • Esculetin reacts with an iron salt (ferric citrate) in the medium to form a phenolic iron complex which produces a dark brown or black color. • This test differentiates between group D streptococci from other not group D streptococci. • Positive-Enterococcus faecalis • Negative-Streptococcus agalactiae or Streptococcus pyogenes

8. Indole test- • It detects the ability of certain bacteria to produce enzyme tryptophanase that breaks down amino acid tryptophan present in the medium into indole. • When Kovac’s reagent (para-dimethyl amino –benzaldehyde) is added to overnight incubated broth of a bacterial colony ,it complexes with indole to produce a cherry red colored ring near the surface of medium. • Indole positive-Escherichia coli, Proteus vulgaris etc. • Indole negative-Klebsiella pneumonia, Pseudomonas, Salmonellaetc.

9. Methyl Red (MR) Test • In glucose phosphate broth certain bacteria ferment glucose to produce stronger acids(lactic,acetic and formic) that maintains the pH below 4.4,which turns methyl red indicator from yellow to red color. • MR positive- Escherichia coli • MR negative- Klebsiella pneumonia.

10. Voges-Proskauer(VP) Test • This test is also done in glucose phosphate broth .certain bacteria produce acetoin (acetyl methyl carbinol)as chief end product of glucose fermentation. • In the presence of alkali(40%potassium hydroxide VP reagent-II ) and atmospheric oxygen ,acetoin is oxidized to diacetyl which reacts with α-naphthol (VP reagent-I) to give a red color. • VP positive- Klebsiella pneumonia,Enterobacter etc • VP negative- Escherichia coli,Shigella,etc.

11. Urease test • Urea test media contains 2% urea and phenol red as a pH indicator . • An increase in pH due to production of ammonia results in color change from yellow ( pH 6.8) to bright pink ( pH 8.2). • Urease positive- Proteus, Helicobacter, Klebsiellaetc.

12. Citrate Utilization test • It detects the ability of few bacteria to utilize citrate as a sole source of carbon for their growth. • This test is performed on a citrate containing medium ,such as- • Simmon’s citrate(solid) medium-original green color of bacteria changes to blue, bromothymol blue is used as an indicator. • Koser’s(liquid) medium-it becomes turbid . • Citrate positive- Klebsiella pneumonia, Citrobacter, Enterobacter etc. • Citrate negative- Escherichia coli, Shigellaetc.