Biochemical Test By: Mahmoud W El-Hindi 2013

1. General Microbiology Laboratory Biochemical Test By: Mahmoud W El-Hindi 2013

2. Exercise 9 Coagulase Test By: Mahmoud W El-Hindi

3. Principle • Coagulase is a protein enzyme having a prothrombin-like activity capable of converting fibrinogen to fibrin, which results in formation of a visible clot. • The coagulase test is intended for use in differentiation of Stapylococcus. Aureus “pathogenic” fromother coagulase negative Staphylococcus sp “non-pathogenic “. By: Mahmoud W El-Hindi

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5. Coagulase is present in two forms: 1. Bound Coagulase ("clumping factor"): • This factor is found on the surface of cell walls, and converts fibrinogen to fibrin directly, with no involvement of plasma factors, and is not inhibited by antibodies to free coagulase. • The slide coagulase test detects “bound coagulase “. By: Mahmoud W El-Hindi

6. Cont. 2- Free Coagulase (“clotting factor”): • This factor is extracellular and reacts with a substance in plasma “serum factor” (also referred to as coagulase-reacting factor “CRF”). By: Mahmoud W El-Hindi

7. Cont. • Free coagulase reacts with CRF to form a coagulase-CRF complex. • This complex acts indirectly to convert fibrinogen to fibrin, forming a clot. • The tube coagulase test detects ‘bound’ and ‘free’ coagulase. By: Mahmoud W El-Hindi

8. SIGNIFICANCE • This test is a confirmatory test for the differentiation of the pathogenic Staphylococcus aureusfrom the non-pathogenic Staphylococcus epidermidis By: Mahmoud W El-Hindi

9. Staphylococcus aureus By: Mahmoud W El-Hindi

10. Staphylococcus epidermidis By: Mahmoud W El-Hindi

11. PROCEDURES Slide Test: • 1- Place a drop of coagulase plasma (rabbit plasma) on a clean, dry glass slide. • 2- Place a drop of distilled water or saline next to the drop of plasma as your negative control. • 3- With a loop, emulsify an amount of the isolated colony being tested in each drop, inoculating the water or saline first. Try to create a smooth suspension. By: Mahmoud W El-Hindi

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13. Result of slide test By: Mahmoud W El-Hindi

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15. Cont. Tube test: • 1. Place 0.5 mL of rabbit plasma in a test tube. • 2. Place 0.5 mL of distilled water in a test tube. • 3. Add one inoculating loopful of the organism being tested to each tube. By: Mahmoud W El-Hindi

16. Result of Tube Test The Tube test more reliable from slide test . By: Mahmoud W El-Hindi

17. Cont. By: Mahmoud W El-Hindi

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19. Exercise 10 Citrate Utilization By: Mahmoud W El-Hindi

20. Principle • Some bacteria may be able to use organic compounds other than sugars as their sole source of carbon. • The ability to metabolize citrate for example is useful for differentiating among Enterobacteriaceae. By: Mahmoud W El-Hindi

21. Cont. • Simmons Citrate agar is a medium containing citrate as the sole carbon source and ammonium salts as the sole nitrogen source. • Organisms that metabolize citrate utilize the ammonium salts releasing ammonia and increasing the pH of the medium. By: Mahmoud W El-Hindi

22. Cont. • Brom thymol blue is present in the medium as the indicator dye. • It is green at neutral pH and deep blue above pH 7.6 • Koser's citrate brothis another medium used to test for citrate utilization. • Growth is evidence of a positive reaction. By: Mahmoud W El-Hindi

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25. Cont. Bromthymol blue Test Tube Blue By: Mahmoud W El-Hindi

26. Control • POSITIVE CONTROL : Enterobacter aerogenes Klebsilla • NEGATIVE CONTROL E. coli By: Mahmoud W El-Hindi

27. PROCEDURE • 1. Using a sterile inoculating needle, streak one organism over the surface of the agar slant, then stab the butt. Repeat with the second organism. • 2. Incubate the tubes at 37 oC for 48 hours. • 3. Examine for growth. Is there a change in the indicator dye in the agar? By: Mahmoud W El-Hindi

28. Results By: Mahmoud W El-Hindi

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33. Exercise 11 Nitrate Reaction Test By: Mahmoud W El-Hindi

34. Principle • Organisms that possess the enzyme, nitrate reductase reduces nitrateto nitrite. • Nitrite combines with an acidified substrate naphthylamine to form red colored end product. • If the organism has further reduced nitrite to nitrogen gas, the test for nitrate will yield negative (colorless) results. By: Mahmoud W El-Hindi

35. Cont. • An additional test for the presence of unreacted nitrate must be performed to validate such colorless results. • Metallic zinc catalyzes the reduction of nitrate to nitrite, thus, with the addition of zinc, a negative test will yield a red color, indicating the presence of unreacted nitrate. By: Mahmoud W El-Hindi

36. By: Mahmoud W El-Hindi

37. SIGNIFICANCE • Most Gram-negative bacilli are positive for nitrate reduction and negative for Nitrite reduction. • This test is used in the identification of only few gram negative bacilli such as Moraxella and Acinetobacter, some strains of Pseudomonas and Vibrio and Flavobacterium. By: Mahmoud W El-Hindi

38. PROCEDURES • 1- Inoculate a nitrate broth with the test organism. • 2- Incubate at 37 C for 24 hours. • 3- Add 5 drops of reagent A (Sulfanilic acid) and 5 drops of reagent B (naphthylamine) to the broth. By: Mahmoud W El-Hindi

39. RESULTS • Positive: A red color develops in 1-2 minutes. • Negative: Colorless NB: IF NEGATIVE Add a pinch of zinc dust to the tube • Positive: Colorless • Negative: Red color By: Mahmoud W El-Hindi

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43. Cont. By: Mahmoud W El-Hindi

44. Good Luck