Biochemical reaction

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1. Biochemical Reaction Dr Avinanda Biswas Tutor of Microbiology

2. Biochemical Identification Based on the type of colony morphology and Gram staining appearance observed in culture smear , the appropriate biochemical tests are employed. Initially, catalase and oxidase tests are done on all types of colonies grown on media . For gram-negative bacilli: The following are the common biochemical tests done routinely , abbreviated as ‘ICUT’ : Indole test Citrate utilization test Urea hydrolysis test Triple sugar iron test For gram-positive cocci : The useful biochemical tests are as follows : Coagulase test ( for Staphylococcal aureus ) CAMP test ( Christine – Atkins – Munch – Peterson) for group B Streptococcus . Bile esculin hydrolysis test (for Enterococcus) Inulin fermentation test and bile solubility test ( for pneumococcus) Antimicrobial susceptibility tests are done for bacterial identification are as follows : Optochin susceptibility test – done to differentiate pneumococcus (sensitive) from viridans streptococci. Bacitracin susceptibility test – done to differentiate group A ( sensitive) from group B (resistant) Streptococcus .

3. Catalase Test When a colony of any catalase producing bacteria is mixed with a drop of hydrogen peroxide (3 % H2O2) placed on a slide , effervescence or bubble appear due to breakdown of H2O2 by catalase to produce oxygen . Catalase test is primarily used to differentiate between staphylococcus (catalase positive) from Staphylococcus ( catalase negative) . It is also positive for members of the families Enterobacteriaceae , Vibrionaceae , Pseudomonas etc .

4. Oxidase Test It detects the presence of cytochrome oxidase enzyme in bacteria , which catalyzes the oxidation of reduced cytochrome by atmospheric oxygen . When a filter paper strip or disk , soaked in oxidase reagent is smeared with a bacterial colony producing cytochrome oxidase enzyme , the smeared are turns deep purple within 10 seconds due to oxidation of the dye to form a purple – colored compound indophenol blue . Examples : Oxidase positive (deep purple) : Pseudomonas , Vibrio , Neisseria , Bacillus , Haemophillus etc . Oxidase negative ( no color change ) : members of family Enterobacteriaceae , Acinetobacter etc .

5. Oxidase test

6. Indole Test Inoculate the test organism into tryptophane broth Incubate at 37°C for 24 hours After incubation interval, add 1 ml Kovacs reagent which contain 4 (p) – dimethylamino benzaldehyde, shake the tube gently and read immediately Method

7. Contd. Result: A bright pink color in the top layer indicates the presence of indole The absence of color means that indole was not produced i.e. indole is negative . Significance: Indole Positive : A red-colored ring is formed near the surface of the broth . Example : Escherichia coli , Proteus vulgaris , Vibrio cholerae etc . Indole Negative : Yellow – colored ring is formed near the surface of the broth , e.g., Pseudomonas , Salmonella etc .

8. Positive for Escherichia coli Negative for Klebsiella pneumoniae Indole Test

9. Citrate Utilization Test It detects the ability of a few bacteria to utilize citrate as the sole source of carbon for their growth , with production of alkaline metabolic products . Test is performed on Simmon’s citrate medium. Citrate utilizing bacteria produce growth and a color change , i.e., original green color changes to blue . Citrate test is positive for Klebsiella , Pneumonia , Citrobacter , Enterobacter etc . The test is negative for Escherichia coli , Shigella etc .

10. Principle: Citrate Na 2 CO 3 Alkaline,↑pH Bromothymol blue CO2 + Na + H2O P y r u vate Simmone’s Citrate media Contains Citrate as a sole of C source Positive test CITRATE TEST

11. Positive Negative Citrate Utilization Test

12. Urea Hydrolysis Test Urease producing bacteria can split urea present in the medium to produce ammonia that makes the medium alkaline . Test is done on Christensen’s urea medium , which contains phenol red indicator that changes to pink color alkaline medium Urease test is positive for : Klebsiella pneumoniae, Proteus species , helicobacter pylori etc. Urea test is negative for : Escherichae coli , Shigella , Salmonella etc .

13. Urea Hydrolysis Test Negative Positive

14. Triple Sugar Iron (TSI) Test TSI contains Enzymatic Digest of Casein, Enzymatic Digest of Animal Tissue, and Yeast Enriched Peptone provide the nitrogen, carbon, and vitamins required for organism growth. Three different types of sugars Glucose (1 part) Lactose (10 part) Sucrose (10 part) Phenol red (acidic: Yellow) TSI dispensed in tubes with equal butt & slant

15. Contd. Principle – To determine the ability of an organism to attack a specific carbohydrate incorporated into a basal growth medium, with or without the production of gas, along with the determination of possible hydrogen sulphide production. When the carbohydrates are fermented, acid production is detected by the Phenol Red pH indicator. Sodium Thiosulfate is reduced to hydrogen sulfide , and hydrogen sulfide reacts with an iron salt yielding the typical black iron sulfide. Ferric Ammonium Citrate is the hydrogen sulfide (H2S) indicator . Sodium Chloride maintains the osmotic balance of the medium. Agar is the solidifying agent

16. Contd. Method: Inoculate TSI medium with an organism by inoculating needle by stabbing the butt and streaking the slant Incubate at 37°C for 24 hours

17. R eac t io n o n TS I Result Example Butt colo r Slan t colo r H 2 S Red Red Negative Alk/Alk/- (No a c tio n o n s uga r s ) N o n fe r m ente r e.g. Pseudomonas Yellow Red Negative A/Alk/- (G lu c o s e fe r m ente d without H 2 S) LNF e.g . Shigell a Yellow Red Po s iti v e bla c k i n butt A/Alk/+ (G lu c o s e fe r m ente d w it h H 2 S ) LNF e.g . Sal m onell a & Proteus Yellow Yellow Negative A/A/- ( th r e e s uga rs a re fermented) LF e.g . E . c oli , Klebsiella, Ente r oba c te r R es ul t

18. Triple Sugar Iron Test

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