Biochemical Tests

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Biochemical Tests
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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)

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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

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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)

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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

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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

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Gelatin Hydrolysis

• Gelatin is another protein
• Gelatinase (enzyme) breaks down gelatin into
  amino acids to be taken up by bacteria
• Gelatin liquifies when its broken down

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Lipid Hydrolysis

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

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Indole/H2S

• SIM determines
• Sulfur reduction (black precipitate)
• Indole production from trypophan (red color with
  Kovacs reagent)
• Motility (radiating growth)

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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

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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