Gelatinase Test and Bacterial Unknowns

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Sugar Fermentation Test

        Lab #20
• 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
                    HYDROLYTIC ENZYMES

             Gelatinase Test
Teams of 4
  Escherichia coli
  Staphylococcus aureus
  2 gelatin tubes

Put a heavy loopful of the
  organism all the way
  down to the bottom of the
                  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
  positive…the bacteria is able to digest gelatin.
               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
        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.
      Phenol Red Broth for Sugar
• 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
              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
S. Aureus
Alcaligenes faecalis
                BIOCHEMICAL TESTS

  Phenol Red Broth for Sugar
Reading results
Yellow color in the
 phenol red pH
 indicators shows acid
 hot pink shows
Gas production will
 show as a bubble in
 the durham tube           ALKALINE   ACID/GAS   ACID
            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
  1 – 2 Nutrient agar slants or 1 Nutrient
    Agar plate

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