Friday, June 3, 2011

Laboratory Exercise No 8. Biochemical Reactions of Bacteria

OBJECTIVES:  
     1.     To differentiate bacteria by biochemical reaction.
  1. To demonstrate enzymatic action of bacteria
  2. To emphasize importance of biochemical reactions in the evaluation and identification of bacteria.    
METHODOLOGY
  1. Triple Sugar Iron Agar (TSI). Inoculate a TSI slant/butt with a 24h culture of bacterium to be investigated by streaking the sloped surface and centrally stabbing the butt. With platinum loop and needle, respectively.Incubate for 48h at 37C. Read results.
  2. Sulfur Indole Motility (SIM). Inoculate a SIM butt with a 24h culture of bacterium to be investigated by stabbing the butt. Incubate for 18-24h at 37C. Read results for motility and hydrogen sulfide production. For indole test, cover the medium with 1ml ether, gently shake to allow the ether to seep into the medium. Allow the ether to resurface before adding 6 drops of Ehrlich reagents. Read results.
  3. Simmon’s citrate (SC) Inoculate an SC slant with a 24h culture of bacterium to be investigated by streaking the slant. Incubate for 18-24h and read results.
  4. Methyl Red—Vogues Proskauer Test (MR-VP). Inoculate 2 MR-VP tubes with 24h culture of bacterium to be investigated, one tube for the Methyl Red test and the other for the VP test. Incubate up to 4 days at 37C. For MR add 5 drops of methyl red indicator. Observe results. For the VP add 2-3 drops of alpha-napthol and 2-3 drops of 40% KOH. Read results.
  5. Nitrate reduction. Inoculate nitrate broth with 24h culture of bacterium to be investigated. Incubate for 48h. Add 5 drops of sulfanilic acid and 5 drops of alpha-napthylamine. Read results.
  6. Catalase test. Take a block of agar from a 24h culture of bacterium under investigation. Add 2-3 drops hydrogen peroxide. Observe for effervescence.
RESULTS AND DISCUSSION
  1. Tabulate your results and discuss
  2. Compare your results with Bergey’s Manual for Determinative Bacteriology. Try identifying your bacteria by their biochemical reactions.
  3. Discuss advantages and disadvantages in using biochemical reactions in identifying bacteria over other methods
 REFERENCES:


Laboratory Exercise No. 8

                                       Biochemical Reactions of Bacteria

Name:
Date Performed
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RESULTS


                                      


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