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Biochemical Tests

Biochemical Tests. 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). Phenol Red Broth. Contains: Carbohydrate Dextrose/glucose

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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

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