1. IN THE NAME OF ALLAH ALMIGHTY THE MOST COMPASSIONATE THE MERCIFUL

2. Biochemical Tests for Identification of Bacteria

3. Biochemical tests: These tests are performed for identification of bacteria. All of these tests depend upon production of certain enzymes by the bacteria. 1.Catalase test This test is used to differentiate those bacteria that produce the enzyme catalase, such as staphylococci, from non-catalase producing bacteria such as streptococci. This enzyme converts hydrogen peroxide into water and oxygen.

4. Principle: Catalase producing bacteria will produce O 2 when mixed with H 2 O 2.Methods: 1.Slide Test 2.Tube Test Slide Method: Take a drop of 3% H 2 O 2 on a glass slide. Mix it with a small quantity of test bacteria. 2 H 2 O 2 -------------> 2 H 2 O and O 2 (Bubbles)

5. Results: Active bubbling............ Positive catalase test. No bubbles.............. Negative catalase test.

6. Caution: Performing the test on a slide is not recommended because of the risk of contamination from active bubbling. When the rapid slide technique is used, the hydrogen peroxide solution should be added to the organism suspension after placing the slide in a petridish. The dish should then be covered immediately, and the preparation observed for bubbling through the lid.

7. Tube Method Pour 2–3 ml of the hydrogen peroxide solution into a test tube. Using a sterile wooden stick or a glass rod (not a nichrome wire loop), remove several colonies of the test organism and immerse in the hydrogen peroxide solution. Important: Care must be taken when testing an organism cultured on a medium containing blood because catalase is present in red cells & a false positive reaction may occur. Look for immediate bubbling.

8. Results Active bubbling............ Positive catalase test. No bubbles.............. Negative catalase test.

9. 2- Citrate Utilization Test: This test is one of several techniques used occasionally to assist in the identification of Enterobacteria. Principle: The test is based on ability of an organism to use citrate as its only source of carbon. Citrate method using Simmon’s citrate agar (Green color) Prepare slopes of the medium. Using a sterile straight wire, first streak the slope with a saline suspension of the test organism and then stab the butt. Incubate at 35 0 C for 48 hours. Look for a bright blue color in the medium.

10. e.g. Enterobacter and Klebsiella are citrate positive while E. coli is negative. Results Bright blue...... Positive citrate test. No change in color..... Negative citrate test.

11. 3- Coagulase Test: This test is used to identify Staph. aureus, which produces coagulase enzyme. Principle: Coagulase causes plasma to clot by converting fibrinogen to fibrin. Two types of coagulase are produced by most strains of Staph. aureus: Free coagulase which converts fibrinogen to fibrin by activating a coagulase-reacting factor present in plasma. Free coagulase is detected by clotting in the tube test. Bound coagulase(clumping factor) which converts fibrinogen directly to fibrin without requiring a coagulase reacting factor. It can be detected by the clumping of bacterial cells in the rapid slide test.

12. A tube test must always be performed when the result of a slide test is not clear, or when the slide test is negative and Staphylococcus has been isolated from a serious infection. Requirements Anticoagulated human plasma or rabbit plasma ( by EDTA, oxalate or heparin). The plasma should be allowed to warm to room temperature before being used. Do not use citrated plasma because citrate-utilizing bacteria e.g. enterococci & Pseudomonas may cause clotting of the plasma (in tube test).

13. 1- Slide test method (detects bound coagulase): Place a drop of distilled water on each end of a slide or on two separate slides. Emulsify a colony of the test organism in each of the drops to make two thick suspensions. Add a loopful (not more) of plasma to one of the suspensions, and mix gently. Look for clumping of the organisms within 10 seconds. Results Clumping within 10 sec...... Staph. aureus. No clumping within 10 sec..... No bound Coagulase.

14. 2- Tube test method (detects free coagulase): Label two small test tubes one as: Test organism (18–24 h broth culture) and the other as: Control (sterile broth). Pipette 0.2 ml of plasma into each tube. Add 0.8 ml of the test broth culture to “Test” tube. Add 0.8 ml of sterile broth to “Control” After mixing gently, incubate the tubes at 35–37 0 C for 6-12 hours and examine hourly. If the test is still negative, leave the tube at room temperature overnight and examine again. Note: When looking for clotting, tilt each tube gently.

15. Results  Clotting of tube contents........... Staph. aureus.  No clotting ………………. Not Staph. aureus.

17. 4- Oxidase Test: The oxidase test is used to assist in the identification of Pseudomonas, Neisseria, Vibrio, Brucella, and Pasteurella species, all of which produce the enzyme cytochrome oxidase. Principle When the organism is oxidase-producing, the phenylenediamine (oxidase reagent) will be oxidized to a deep purple color.

18. Requirements: 1.Oxidase reagent strips. 2.Stick or glass rod. Method using an oxidase reagent strip: Moisten the strip with a drop of sterile water. Using a piece of stick or glass rod (not an oxidized wire loop) remove a colony of the test organism and rub it on the strip. Look for a deep purple color within 20 seconds. Results Deep purple color........ positive oxidase test

20. 5- Urease Test: Proteus strains are strong urease producers. Principle: The test organism is cultured in a medium which contains urea and the indicator phenol red. When the strain is urease producing, the enzyme will break down the urea to give ammonia and carbon dioxide. With the release of ammonia, the medium becomes alkaline as shown by a change in colour of the indicator to pink-red.

21. Method Inoculate the media with the tested organism. Incubate at 37°C for 18 –24 hours. Results: Change of the color into pink red ……………. Urease producing “Proteus” No change of the color …………… non urease producing organism.

22. The tube on the left is a positive reaction; the tube in the middle is a negative reaction and the tube on the right in an un-inoculated control.

23. 6- Indole production test Testing for indole production is important in the identification of enterobacteria.Principle: To determine the ability of an organism to produce enzyme tryptophanase that splits amino acid tryptophan into indole. Indole production is detected by adding Kovac’s reagent to the test solution Kovac’s reagent which reacts with the indole to produce a red colored compound.

24. SIM agar method (Sulfide-Indole-Motility): This agar is used for detection of the organism’s: 1. H 2 S production. 2. Indole production. 3. Motility. Inoculate test organism two-thirds into the medium by stabbing. Incubate at 37°C for 18 –24 hours. Examine tubes after incubation for motility and H 2 S production. Add 3-4 drops of Kovac’s Reagent after determining motility and H 2 S production. Record as indole positive if a pink or red color ring appear, or as indole negative if there is no color change.

25. Results Motility is indicated by turbidity of the medium or growth extending from inoculating stab line. H 2 S production is shown by a blackening along the stab line. Indole production is seen as the production of a red color ring after the addition of Kovac’s Reagent.

27. Escherichia coli in SIM Medium Motile, indole positive and hydrogen sulfide negative Proteus mirabilis in SIM Medium Motile, indole negative and hydrogen sulfide positive

28. 7- Triple Sugar Iron Test (TSI) In this test we use TSI agar slants (slope media). Principle: Triple Sugar Iron (TSI) agar slants differentiate bacteria on their ability to ferment glucose, lactose, and/or sucrose and on their ability to reduce sulfur to hydrogen sulfide gas (H 2 S). This test is used to differentiate among the members of Enterobacteriaceae i.e. E. coli, Salmonella, Shigella, Klebsiella, Enterobacter etc.

29. Composition Of TSI agar: 1. 0.1% glucose. 2. 1% lactose. 3. 1% sucrose. 4. Ferrous sulfate for detection of H 2 S production. 5. Phenol red as indicator that changes yellow in acidic media. 6. Beef & yeast extract. 7. Low conc. of agar. The media is poured in test tubes in the form of slants & deep butt. The color of the media is red

33. 8- Bile solubility test Principle This helps to differentiate Strep. pneumoniae, which is soluble in bile and bile salts, from other alpha haemolytic streptococci (viridans streptococci) which are insoluble.Requirements Sodium deoxycholate reagent. Sterile physiological saline.

34. Method Emulsify several colonies of the test organism in a tube containing 2 ml sterile physiological saline, to give a turbid suspension. Divide the organism suspension between two tubes. To one tube, add 2 drops of the sodium deoxycholate reagent and mix. To the other tube (negative control), add 2 drops of sterile distilled water and mix. Leave both tubes for 10–15 minutes at 35–37 0 C. Look for a clearing of turbidity in the tube containing the sodium deoxycholate.

35. Results Clearing of turbidity................Strept. Pneumoniae. No clearing of turbidity... ……. is not Strept. Pneumoniae.

36. 9- DNA-ase test This test is used to help in the identification of Staph. aureus which produces deoxyribonuclease (DNAase) enzymes. The DNA-ase test is particularly useful when plasma is not available to perform a coagulase test or when the results of a coagulase test are difficult to interpret. Principle Deoxyribonuclease hydrolyzes deoxyribonucleic acid (DNA). The test organism is cultured on a medium which contains DNA. After overnight incubation, the colonies are tested for DNA-ase production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates unhydrolyzed DNA. DNA-ase-producing colonies are therefore surrounded by clear areas due to DNA hydrolysis.

37. Requirements DNA-ase agar. Hydrochloric acid solution 1 mol/l (1N).Method Using a sterile loop or swab, inoculate the test and control organisms. Incubate the plate at 35–37 0 C overnight. Cover the surface of the plate with 1 mol/l hydrochloric acid solution. Look for clearing around the colonies within 5 minutes of adding the acid.

38. Results Clearing around the colonies.......... DNA-ase positive strain Staphylococcus aureus. No clearing ………… Negative DNA-ase Staphylococcus epidermidis.