Gelatinase Sugar Fermentation Test

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Gelatinase Sugar Fermentation Test

• Lab 20

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Experiment

• Gelatinases comprise a family of extracellular
  enzymes produced by some microorganisms to
  hydrolyze gelatin
• The smaller amino acids can then be taken up by
  the bacterial cell for metabolic purposes
• Bacteria is positive for the enzyme and will
  liquefy the media
• Bacteria does not secrete the enzyme and the
  media will remain hard
• E. coli
• S. aureus

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HYDROLYTIC ENZYMESGelatinase Test

• Teams of 4
• Organisms
• Escherichia coli
• Staphylococcus aureus
• Media
• 2 gelatin tubes
• Put a heavy loopful of the organism all the way
  down to the bottom of the tube

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Using Gelatin

• This media is used to test if bacteria can digest
  the protein gelatin. To digest gelatin, the
  bacteria must make an enzyme called gelatinase.
• To inoculate this media, use a transfer needle to
  stab the gelatin. After incubating the inoculated
  media for at least 48 hrs, transfer the tube into
  a refrigerator. The tube should be completely
  chilled prior to observation.
• If the media is solid after refrigeration then
  the test is negative (the bacteria did not digest
  gelatin). If the media is liquified even after
  refrigeration, then the test result is
  positivethe bacteria is able to digest gelatin.

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Example results

• Results -examples
• The 'Serratia marcescens' on the left is positive
  for gelatinase production, as evidenced by the
  liquidation of the media.  The 'Salmonella
  typhimurium' on the right is negative, as
  evidenced by the solidity of the media. 

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Sugar Fermentation

• Metabolic process by which organic molecules act
  as electron donors (become oxidized) and one or
  more of its organic products acts as a final
  electron acceptor.
• Bacteria use protein or carbohydrate as a carbon
  and energy source and catabolize carbohydrate and
  produce acid, the pH indicator changes color come
  produce gas as well as acid.

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Phenol Red Broth for Sugar Fermentation

• Lactose fermenters are organisms that split
  lactose into glucose and galactose and ferments
  the monosaccharides (hydrolysis reactions)
• Glucose fermenters produce pyruvate
  (glycolysis)and ferment pyruvate to products
  alchohol and CO2 and H.
• Phenol Red is an acid indicator Yellow below 6.8,
  Pink above 7.4, and red from 6.8 to 7.4.
• Durham tube is and indicator for gas production.
• Differentiation of Enterobacteraciae from other
  Gram rods

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Tests results

• Acid Gas - produces yellow bubble in the tube
• Acid no Gas- produces yellow with no bubble
• Red Broth control pH 7.3
• Degradation of peptone produces alkaline media
  with NH3 as the end product and will be
  pink/reddish
• E.Coli
• S. Aureus
• Alcaligenes faecalis

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BIOCHEMICAL TESTSPhenol Red Broth for Sugar
Fermentation

• Reading results
• Yellow color in the phenol red pH indicators
  shows acid hot pink shows alkaline
• Gas production will show as a bubble in the
  durham tube

ALKALINE
ACID/GAS
ACID
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UN Bacterial Unknowns

• Check your streak plates if you have isolated
  colonies, put them on nutrient slants if not,
  re-streak your unknown
• If you have growth on your slant
• -Gram Stain
• -Inoculate into Sugars and Gelatin
• Media
• 1 2 Nutrient agar slants or 1 Nutrient Agar
  plate