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Lab 24: Gram Negative Rods

Lab 24: Gram Negative Rods. Biochemical tests. identification of gram negative rods IMViC series of tests Indole MR VP Citrate. Indole Test. Determine if the organism can produce the enzyme tryptophanase which cleaves tryptophan, generating indole Add 4-5 drops of Kovacs reagent.

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Lab 24: Gram Negative Rods

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  1. Lab 24: Gram Negative Rods

  2. Biochemical tests • identification of gram negative rods • IMViC series of tests • Indole • MR • VP • Citrate

  3. Indole Test • Determine if the organism can produce the enzyme tryptophanase which cleaves tryptophan, generating indole • Add 4-5 drops of Kovacs reagent

  4. MR test • Determine if the organsim can produce acid end products from glucose fermentation • Add 4-5 drops of methyl red (pH indicator)

  5. VP test • Determine if the organism can produce alkaline and non-acid end products from fermentation of glucose. • Add __ drops of VP-A, __ drops of VP-B and incubate at 37oC for 30 minutes.

  6. Citrate test • Determine if the organism can metabolize citrate as the sole carbon source • pH indicator is brom thymol blue

  7. Sugar Fermentation Broths • Determine if the organism can ferment the sugar (glucose, lactose, or sucrose) • pH indicator is phenol red • Turns yellow in acidic conditions • Turns hot pink in alkaline conditions • Look for gas production in the durham tube

  8. Phenol red fermetation broths

  9. Urea Test • Determine if the organism can produce the enzyme urease which cleaves urea generating ammonia • pH indictor is phenol red

  10. Phenylalanine Slants • Tests for the presence of the enzyme phenylalanine deaminase • If produce enzyme, organism will break down phenylalanine to phenylpyruvic acid • When you add ferric chloride, slant turns green

  11. Motility Deeps • Used to determine if the organism produces flagella • Look at the tube in all dimensions • If you see a “fanning” of growth, or lack of a stab line, then it is motile

  12. Catalase Test • Use the TSA plate, find an isolated colony to test • Determine if the organsim produces the enzyme catalase which breaks down hydrogen peroxide generating oxygen and water • Add few drops of hydrogen peroxide and look for bubbles

  13. Oxidase • Test for the presence of enzyme cytochrome oxidase • Use sterile swab to pick up a colony and add a few drops of “oxidase reagent” to swab

  14. QuickTime™ and a decompressor are needed to see this picture. MacConkey Agar • Selective and differential agar • Selects for gram negative rods • Differentiates lactose fermenters from non-fermenters

  15. XLD plates • Selective and differential agar (selects for gram negative rods, also helps differentiate Salmonella from Shigella) • Organisms which ferment lactose, sucrose, or xylose will generate yellow colonies on agar • Organisms which decarboxylate lysine will raise the pH and colony will appear red • Salmonella will appear as red colonies with black precipitate (produce hydrogen sulfide) • Shigella will not ferment or produce hydrogen sulfide

  16. XLD plates • Salmonella are black colonies • Other gram negatives are yellow to orange colonies

  17. Hektoen Enteric Plates • Selective and differential agar (selects for gram negative rods, differentiates between those that ferment lactose and/or sucrose) • Contains a high concentration of bile salts to inhibit gram positive • Organisms that ferment produce yellow colonies, those that don’t ferment generate blue-green colonies

  18. HE plates • Organisms that ferment produce yellow to orange colonies, those that don’t ferment generate blue-green colonies

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