General Microbiology Laboratory Biochemical Test biology and biotechnology
General Microbiology Laboratory Biochemical Test biology and biotechnology department 1
Exercise 9 Coagulase Test By: Mahmoud W El-Hindi 2
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” from other coagulase negative Staphylococcus sp “non-pathogenic “. By: Mahmoud W El-Hindi 3
Cont.
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 5
Cont. 2 - Free Coagulase (“clotting factor”): This factor is extracellular and reacts with a substance in plasma “serum factor” factor (also referred to as coagulase-reacting factor “CRF”). By: Mahmoud W El-Hindi 6
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 7
SIGNIFICANCE This test is a confirmatory test for the differentiation of the pathogenic Staphylococcus aureus from the nonpathogenic Staphylococcus epidermidis By: Mahmoud W El-Hindi 8
Staphylococcus aureus By: Mahmoud W El-Hindi 9
Staphylococcus epidermidis By: Mahmoud W El-Hindi 10
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 11
Cont.
Result of slide test By: Mahmoud W El-Hindi 13
Cont. By: Mahmoud W El-Hindi 14
Cont. Tube test: 1. Place 0. 5 m. L of rabbit plasma in a test tube. 2. Place 0. 5 m. L 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 15
Result of Tube Test The Tube test more reliable from slide test. By: Mahmoud W El-Hindi 16
Cont. By: Mahmoud W El-Hindi 17
Cont. By: Mahmoud W El-Hindi 18
Limitations of the procedure: The slide test should be read very quickly, as false positives can occur. The slide test should not performed with organisms taken from high-salt media such as Mannitol Salt Agar, as the salt content can create false positives. The tube test is more reliable than the slide test.
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 Enterobacteriaceae. By: Mahmoud W El-Hindi among 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 p. H 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 p. H (6. 9) and deep blue above p. H 7. 6 Koser's citrate broth is another medium used to test for citrate utilization. Growth is evidence of a positive reaction. By: Mahmoud W El-Hindi 23
Cont. By: Mahmoud W El-Hindi 24
Cont. By: Mahmoud W El-Hindi 25
Cont. Brom thymol blue Test Tube Blue By: Mahmoud W El-Hindi 26
Control POSITIVE CONTROL : Enterobacter aerogenes Klebsilla By: Mahmoud W El-Hindi NEGATIVE CONTROL E. coli 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 o. C for 48 hours. 3. Examine for growth. Is there a change the indicator dye in the agar? By: Mahmoud W El-Hindi in 28
Results By: Mahmoud W El-Hindi 29
Cont. By: Mahmoud W El-Hindi 30
Cont. By: Mahmoud W El-Hindi 31
Cont. By: Mahmoud W El-Hindi 32
Cont. By: Mahmoud W El-Hindi 33
Exercise 11 Nitrate Reaction Test By: Mahmoud W El-Hindi 34
Principle Organisms that possess the enzyme, nitrate reductase reduces nitrate to 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 40
Cont. By: Mahmoud W El-Hindi 41
Cont. By: Mahmoud W El-Hindi 42
Cont. By: Mahmoud W El-Hindi 43
Cont. By: Mahmoud W El-Hindi 44
Good Luck
- Slides: 45