BIOCHEMICAL REACTIONS

1. BIOCHEMICAL REACTIONS By Dr. Emad AbdElhameed Morad Lecturer of Medical Microbiology and Immunology

2. Biochemical reactions Sugar fermentation: Composed of: Six tubes. Each tube contains peptone water + specific sugar in a concentration of 1%. The sugars are glucose, lactose, maltose, mannite, sucrose, salicin. Each tube contains also Andrade’s indicator which changes to pink if acid is produced. An inverted tube (Durham’s tube) is placed in the medium. Gas production is revealed by collection of bubbles at its apex.

3. Fermentation of glucose, maltose, mannite with acid only Fermentation of all sugars with acid + gas production Used to: Differentiate between bacteria according to their fermentative effect on different sugars and whether the fermentation is associated with gas production or not.

4. Indole test Principle: Demonstrates the ability of certain bacteria to decompose the amino acid tryptophan present in peptone water to indole. Indole is then tested for by adding few drops of Kovac’s reagent which gives a pink ring in the presence of indole. Procedure: The organism is inoculated in peptone water and after incubation at 37 degree for 24 hours, Kovac’s reagent is added. Interpretation: If a pink ring is produced, the organism is indole +Ve (E. coli). If a yellow ring is produced, the organsim is indole –Ve (Klebsiella).

6. Voges-Proskauer’s reaction (VP): Principle: Some bacteria ferment glucose with production of acetyl methyl carbinol. Procedure: Bacteria is grown in glucose phosphate peptone water for 48 hours. Then KOH is added to test for acetyl methyl carbinol formation. Interpretation: If an eosin pink color is produced, VP reaction is +Ve.

8. Methyl red test (MR): Principle: Detects the ability of some bacteria to produce large amounts of acid on fermentation of glucose, thus lowering the pH of the medium below 4. Procedure: The bacteria is grown in glucose phosphate peptone water. After incubation at 37 degree for 48 hours, few drops of methyl red indicator are added. Interpretation: A positive test gives bright red color. A negative test gives a yellow color.

10. Urease test: Principle: Some organisms produce urease enzyme. This enzyme splits urea with the release of ammonia. Ammonia causes alkalinity and increases pH of the medium. The phenol red indicator turns deep pink. Procedure: The bacteria is grown on a medium containing urea and phenol red indicator for 24 hours. Interpretation: Urease positive organisms such as proteus will turn the medium deep pink.

11. +Ve -Ve

12. Oxidase test: Principle: Some bacteria produce oxidase enzyme which reduces the oxidase reagent (tetramethyl-p-phenylene-diamine hydrochloride) to a deep purple color. Procedure: Done by picking up a portion of the colony tested and smearing it on a filter paper impregnated with oxidase reagent. Interpretation: The immediate development of a deep purple color indicates positive test. Examples: neisseria & pseudomonas & vibrios.

14. H2S test: Principle: Production of H2S from proteins. Procedure: Inoculate the organism on peptone water. After incubation, put lead acetate paper at the mouth of the tube. Interpretation: If the organism produces H2S, a black color is formed. Examples: proteus & Salmonella typhi.

15. Catalase test: Principle: Tests the ability of some bacteria to produce catalase enzyme. Procedure: Pick up the test colony on a platinum loop and immerse it in few drops of 3 % H2O2 (hydrogen peroxide). Interpretation: Rapid effervescence indicates oxygen production and a positive test. This test is used to differentiate between staphylococci (+Ve) and streptococci (-Ve).

16. Catalase +Ve

17. Slide method Coagulase test: Detects the coagulase bound to the organism. Homogenous suspension of the test organism is made in a drop of saline on a slide then mixed with a drop of undiluted human or rabbit plasma. Staphylococcus aureus clumps within 15 sec. because coagulase enzyme precipitates the fibrin in the plasma on the cell surface.

18. Tube method Detects the free coagulase. It is done by adding 5 drops of an overnight broth culture of the test organism to 0.5 ml of human or rabbit plasma diluted 1/10 in sterile saline. The tubes are incubated for 6-12 hours and inspected hourly for coagulation. Staphylococcus aureus is coagulase positive. Positive Negative

19. DNAase test: Principle: This test is used to identify Staph. aureus as it produces DNAase enzyme. Procedure: The tested organism is cultured on a medium which contains DNA. After overnight incubation, the colonies are tested for DNAase production by flooding the plate with weak HCL. The acid precipitates unhydrolyzed DNA. Interpretation: DNAase producing colonies (such as Staph. aureus) are surrounded by clear areas due to DNA hydrolysis.

20. DNAase +Ve

21. Analytical profile index (API): Principle: It is composed of a plastic strip with cupules containing dehydrated substances. Each cupule has a small hole at the top. Procedure: A saline suspension of the test organism is dropped in the cupules. The strip is covered with a lid and placed in a humidified plastic chamber and incubated at 37 degree for 24-48 hours. Interpretation: Biochemical profiles are determined by reading the color change and interpret according to the available charts. These are then converted to numerical codes which will be read from a profile index to identify the bacteria.

22. API

23. Automated bacterial identification systems: Principle: Examples: Vitek system, microscan, phoenix. These systems identify the organism and its antibiotic sensitivity by detecting color changes or turbidity in special plastic cards inoculated with the organism. Such cards are composed of tiny wells that contain substrates for detection of biochemical reactions and antibiotic sensitivity. Once the card has been inoculated and placed in the instrument, it will automatically perform all readings. Results are available within 4-6 hours.

24. Vitek card Vitek system

25. GOOD LUCK