1. Biochemical Activities of Microorganisms Part (2)

2. Indole. Methyl Red. Voges Proskauer. Citrate. IMViC.UreaseMotility. Triple Sugar Iron. Kligler Iron Agar. TSI & KIA.

3. Principle. Indole is a component of the amino acid “Tryptophan”. Some bacteria have an enzyme (Tryptophanase). Function: Breaking Tryptophan, releasing indole. The presence of indole reflects the presence of enzyme. Media. Tryptophan broth. Peptone broth. Reagent. Kovac’s reagent.

5. Inoculate the test tubes of Tryptophan broth with a loopful of bacterial growth. Incubate tubes at 35º-37ºC for 24 hours. After incubation period, add 5 drops of kovac’s reagent. Read the results immediately.

7. Purpose. To determine the ability of bacteria to produce ACIDS as final products from glucose fermentation. Media. MR/VP broth. Reagent. Methyl Red indicator.

8. Inoculate the test tubes of MR/VP broth with a loopful of bacterial growth. Incubate tubes at 35-37ºC for 48 hours. After incubation period, add 4 drops of Methyl Red reagent. Read the results immediately.

10. Purpose. To determine the ability of bacteria to produce ACETOIN as final products from glucose fermentation. Media. MR/VP broth. Reagent. Alpha Naphthol. 40% KOH

11. Inoculate the test tubes of MR/VP broth with a loopful of bacterial growth. Incubate tubes at 35-37ºC for 48 hours. After incubation period, add 6 drops of Alpha Naphthol & 2 drops of 40% KOH (IN THIS ORDER) Loosen the cap of the tube and put it in a slant position for 15 to 45 minutes Read the results after 15 minutes If positive – report as positive. If negative – keep it for another 30 minutes

13. What happens when alpha naphthol and 40% KOH are added to the test tube? Acetoin, if present, is oxidized to diacetyl, in the presence of oxygen and alkaline conditions. Alpha naphthol catalyzes the reaction between diacetyl and peptone to produce a complex of pink colored compound.

14. Purpose. To determine if the organism is capable of utilizing Citrate as the ONLY source of carbon. Principle. Bacteria can break the conjugate salt of citrate into organic acids and CO2. CO 2 Combines with sodium from the conjugate salt to form sodium carbonate (a basic compound). Media. Simmon’s citrate. Reagent. Bromothymol blue pH indicator (incorporated in the medium).

15. Inoculate the slant of test tube with an organism (Fish tail). Incubate the tube for 1-2 days at 35-37ºC. After incubation, read the results immediately.

17. Purpose. To determine the presence of urease enzyme Principle. Urea may be broken down by Urease into CO2 and ammonia. Ammonia turns the medium alkaline (pH>7). Media. Urea media broth or slant. Reagent. Phenol red pH indicator (incorporated in the medium).

18. Inoculate the tube medium with bacteria. If slant agar, do the fish tail. Incubate the tubes at 35-37ºC for 24 hours. After incubation, observe color change and read results.

20. Why do they move? Toward beneficial things like O2 and nutrients. Away from deleterious chemicals and toxins like O2 What is the structure they use? Most motile bacteria use Flagella. Spirochetes are helical bacteria that have an internal structure that is responsible for spiral rotation. How do they move? When flagella rotate counterclockwise they act like Propeller. As a result bacteria move through the liquid.

22. Purpose. To determine the ability of bacteria to move (Motile Vs Non-motile bacteria). Media. Semi-solid (less than 1% of Agar). Motility media. Procedure. With a sterile needle, inoculate bacteria within the media (single stab). Incubate for 24 hours at 35-37ºC. After incubation, read the results. Motile bacteria can move away from the original site of stabbing.

24. Purpose. Used to differentiate between bacteria in terms of: 1-Fermentation of Glucose, Lactose and/or Sucrose. Acids are the final products of fermentation. 2-Production of gas. 3-Production of hydrogen sulfide (H 2 S) Note. TSI & KIA differ in that: TSI agar contains glucose, lactose and sucrose. KIA contains only glucose and lactose.

25. Components of TSI include: 1% Lactose. 1% Sucrose. 0.1% Glucose. pH sensitive dye (Phenol red – pH indicator) Sodium thiosulfate. Ferrous sulfate / Ferric ammonium sulfate (H 2 S indicator)

26. If bacteria can ferment glucose but not lactose. Acid is enough to turn the butt yellow, but not the slant. If bacteria can ferment both glucose and lactose. Fermentation of glucose occurs first, resulting in a yellow butt. Lactose fermentation occurs next, resulting in a yellow slant.

27.  Where does H 2 S come from? ◦ H 2 S is a toxic gas that is produced by some bacteria form:  Breakdown of sulfur-containing amino acids, or  The use of sodium thiosulfate during anaerobic respiration (as in our experiment).  What is the reaction? ◦ When H 2 S is produced, it reacts with IRON in the medium.  Source of iron is Ferrous sulfate / Ferric ammonium sulfate. ◦ The result is a black precipitate, which is ferric sulfide (FeS).

28. What are the produced gases? H2 and CO2. Results. Detection by the presence of cracks or bubbles in the medium. Figure is shown later in slides.

29. Pay attention: Inoculation involves stabbing and fish-tail

30. Tube (1): A/A, G Tube (2): K/A Tube (3): K/A, H 2 S Tube (4): K/K 1234

32. Example Escherichia coli Klebsiella pneumonia Enterobacter aerogenes Salmonella typhi Proteus vulgaris Shigella sonnei Pseudomonas aeruginosa KIA A/A/G K/A, G, H 2 S K/A K/K IMViC + + - - - - + + - + + + - - - - - + Urease - + - - + - - Motility + - + + + - +

34. FINAL EXAM ALL LABORATORIES ARE REQUIRED 100 questions 80 Qs Theory 20 Qs form Laboratory STUDY SMARTER NOT HARDER