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Biochemical Tests

Objectives. Tests to knowLactose fermentationIndole MotilityH2S production in SIMMethyl Red/Voges ProskauerCitrateOxidase test. Lactose Fermentation. MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as

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Biochemical Tests

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    1. Biochemical Tests

    2. Objectives Tests to know Lactose fermentation Indole Motility H2S production in SIM Methyl Red/Voges Proskauer Citrate Oxidase test

    3. Lactose Fermentation MacConkey Agar contains bile salts and crystal violet, both inhibitory to Gram-positive bacteria and selects Gram-negative bacteria, such as E. Coli. It also differentiates lactose-fermenting bacteria, such as E. Coli from non-lactose fermenting bacteria. Media and Reagent: MacConkey Agar and neutral red dye Method: Streak MAC plate and incubate at 37°C for 2 days. Expected results: Positive test: Lactose fermentation = Growth and color change to pink Negative test: No lactose fermentation = May or may not grow and no color change

    4. Enteric Gram-negative rods Lactose-fermenters (LFs) Escherichia coli Klebsiella pneumoniae Non-lactose fermenters (NLFs) Salmonella enterica Shigella spp. Proteus mirabilis Pseudomonas aeruginosa

    5. Results of Lactose Fermentation

    6. Indole Production Test The amino acid tryptophan can be broken down by enzyme tryptophanase to form indole, pyruvic acid and ammonia as end products. Tryptophanase differentiates indole-positive enterics, such as E. coli and P.vulgaris from indole-negative enterics, such as S. marcescens. Media and Reagents: SIM with tryptophan and Kovac’s reagent. Method: Inoculate medium and incubate at 37°C for 24-48 hours. After incubation, add five drops of Kovac’s reagent to the surface. Do not stir or shake the tube. Expected Results: Positive test : Kovac’s reagent combines with indole and turns the surface red. Negative test: No red color development

    7. Motility Test This is not a biochemical test, but it can distinguish bacteria. It determines presence of flagella. Media and reagent: Deep agar Method: Inoculate deep with a needle and incubate at 37°C for 24-48 hours. Expected results: Positive test: Growth spread away from the line of inoculation = motile Negative test: Growth only occurred at the line of inoculation = Non-motile

    8. Results for Motility Test

    9. Indole Production Purpose: The indole test is used to identify bacteria capable of producing indole using the enzyme tryptophanase. Kovac’s Reagent

    10. Results of H2S production

    11. Sulfur Reduction Purpose: This test is used to identify those bacteria capable of reducing sulfur.

    12. . SIM Tube Results A. Indole positive and hydrogen sulfide positive B. Hydrogen sulfide positive C. Indole positive and motility positive (note fuzzy growth away from stab line) D. Negative Control

    13. Methyl Red Test Methyl red test is used to identify enteric bacteria based on their pattern of glucose metabolism. If they use mixed acid pathway and produce acidic products, then they are called methyl-red-positive. If they use butylene glycol pathway and produce neutral end products, then they are called methyl-red-negative. Media and reagents: MR-VP medium and methyl red indicator Method: Inoculate broth and incubate at 37°C for 2-5 days. After incubation, transfer 2.5 ml of inoculate to another tube and add five drops of methyl red. Roll between the palms of hands to disperse methyl red. Expected results: Positive test: acids + methyl red = red solution Negative test: neutral end products + methyl red = yellow color

    14. Results for Methyl Red Test

    15. Methyl Red (MR) Test Purpose: Used to identify bacteria that produce stable acid end products by means of mixed acid fermentation of glucose

    16. Voges Proskauer Test It is used to identify enteric bacteria based on their pattern of glucose metabolism. The enterics that produce neutral end-products, such as acetoin are detected by VP test. Media and Reagent: MR-VP medium and Barritt’s Reagent A (contains alpha-naphthol) and Barritt’s Reagent B (contains KOH). Method: Inoculate medium and incubate at 37°C for 48 hours. After incubation, transfer 2.5 ml of inoculate to another tube and add six drops of Barritt’s Reagent A and two drops of Barritt’s Reagent B. Gently mix and let it sit for 10-15 minutes to allow time for color development. Expected results: Positive test: acetoin + alpha-naphthol + KOH = red color Negative test: alpha-naphthol +KOH = copper color

    17. Results for VP test

    18. Citrate Utilization Citrate is an organic molecule that can be utilized by bacteria that produce the enzyme citrase. Citrase is produced by some bacteria such as E. aerogenes but not by others like E. Coli Media and Reagent: Simmon’s Citrate Agar. It has citrase as the only carbon source and PH indicator bromothymol blue Method: Inoculate the slant and incubate at 37°C for 24-48 hours. Expected results: Positive test: Growth and color changes to blue Negative test: No growth and color remains green

    19. SIMMONS CITRATE Purpose: The citrate utilization test is used to determine the ability of an organism, using the enzyme citrase, to use citrate as its sole carbon source

    21. IMViC Reactions I M Vi C Escherichia coli + + – – Proteus vulgaris + + – – Klebsiella pneumoniae – – + + Enterobacter spp. – – + + Serratia marcescens – – + + Citrobacter freundii – + – + Citrobacter koseri + + – +

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