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Biochemical Tests

Biochemical Tests

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Biochemical Tests

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  1. Biochemical Tests

  2. Fermentation • Glucose acts as an electron donor to one or more of its organic products • End products include alcohols, organic acids, and CO2 • Today we use two tests • Phenol red broth • Triple sugar iron (TSI)

  3. Phenol Red Broth • Contains: • Carbohydrate • Dextrose/glucose • Lactose • Sucrose • peptone • Phenol red (pH indicator) • Below 6.8 = yellow • 6.8-7.4 = red • Above 7.4 = pink • Durham tube – inverted to catch gas bubbles

  4. TSI • Similar to phenol red broth, but has a 3 sugars in one slant • Only contains a small amount of glucose (0.1%), so if the organism only ferments glucose, it will quickly run out and will resort to breaking down protein, which will increase pH and cause red coloration • Also tells if organism reduces sulfur • Black precipitate • Streak the surface and stab it (get to oxygen poor area)

  5. Starch Hydrolysis • Starch can be broken down into glucose if the proper enzymes are produced by bacteria • Iodine turns from yellow to black in the presence of starch • So if the bacteria produces starch hydrolyzing enzymes, there will be no starch present and iodine will not produce black color

  6. Casein Hydrolysis • Casein = milk protein • Casease (enzyme) breaks down casein into amino acids to be taken up by bacteria • Casease-positive bacteria will produce a clear zone

  7. Gelatin Hydrolysis • Gelatin is another protein • Gelatinase (enzyme) breaks down gelatin into amino acids to be taken up by bacteria • Gelatin liquifies when it’s broken down

  8. Lipid Hydrolysis • Enzyme lipase breaks down lipids • Spirit blue dye gets pulled toward bacteria when lipids are taken up

  9. Indole/H2S • SIM determines • Sulfur reduction (black precipitate) • Indole production from trypophan (red color with Kovac’s reagent) • Motility (radiating growth)

  10. Methyl Red-Vogues Proskauer (MR-VP) • Methyl red test determines mixed-acid fermentation • Addition of methyl red reagent gives red color for positive bacteria • Vogues Proskauer test determines if fermenting bacteria utilize the butylene glycol pathway • Addition of vogues proskauer reagent gives red color for positive bacteria • Because both result in red color, next week we need to pour ½ of each broth into a fresh culture tube

  11. Citrate Utilization • Determines if bacteria use citrate as carbon source • Contains bromthymol blue dye • Green at pH 6.9 • Blue at pH 7.6 • Produces alkaline products when citrate is used • So it turns BLUE