Lab 24: Gram Negative Rods

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