Microbiology Lab Exam 2

Last update by bubabe12 on 10/24/2009
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Hydrolysis Reactions-

Tryptophan Hydrolysis


Also known as "Indole" test.

Some bacteria can hydrolyze the amino acid tryptophan into pyruvate and indole.

The organism is inoculated into tryptone broth and allowed to incubate 24-48 hours at optimum temp.

Kovac's reagent is added to the tube after incubation.

A positive result= bright red ring at the surface of the broth

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  • bubabe12

    Durham sugar fermentation tubes
    Fermentation reactions are usually performed with glucose, sucrose and lactose although any available carb may be used.

    The test is done in phenol red indicator broth with appropriate sugar added.

    The tubes are incubated 24-72 hours.

    Acid production is indicated by a bright yellow color change. Gas production is indicated by gas bubbles in the durham tube.

    Some bacteria may grow in the broth without acid production and these are acid negative.

    Some bacteria will produce alkaline pH usually seen as pink or mauve color in the broth.
  • bubabe12

    Methyl Red Test
    Also known as mixed acid fermentation

    Some strains of bacteria will produce a mixture of acids during fermentation which may be detected by the methyl red test.

    The unknown is inoculated into MR-VP broth and is incubated at optimum temp for 24 hours.

    5 drops of methyl red indicator are added to the tube.

    A positive result= red/pink color change in the medium
  • bubabe12

    Catalase Production

    A test for the detection of the catalase enzyme.

    A drop of 3% hydrogen peroxide is placed on the bacterial colony.

    Postive result= production of bubbles
  • bubabe12
    Misc. Tests--

    Triple Sugar Iron Agar (TSI)

    A medium which contains 3 sugars (glucose, sucrose and lactose) with iron salts.

    Used to detect acid, gas production and hydrogen sulfide.

    The slant is inoculated with unknown using the streak and stab method.

    The tube is read 24-72 hours at the slant and butt for reactions.

    Results: acid production= yellow
    gas= bubbles or split in agar
    hydrogen sulfide= black color in agar

  • bubabe12
    Misc Tests--

    Citrate Utilization

    Done on Simmon's Citrate agar.

    The unknown is streaked onto the citrate slant.

    Bacteria which can use citrate as a sole carbon source will change the color of the medium from green to bright blue.

  • bubabe12

    Misc Tests--

    Litmus Milk Reactions

    Shows several reactions.

    The unknown is inoculated into litmus milk (10% skim milk + litmus pH indicator) and allowed to incubate at optimum temp for 5-7 days.

    Reactions: Fermentation of sugars= pink color
    Alkaline reaction= black or deep purple
    Peptonization (fat and proteinhydrolysis)
    =translucent appearance
    Reduction= loss of O2 causes bone white color
    Coagulation= curd or clump formation due (denaturation of protein in milk)
    Ropiness= incoluating loop passed through milk is followed by liquid strings or ropes
  • bubabe12
    Specialized Tests--

    Bile esculin agar
    Useful for identification of the intestinal bacteria, which can grow in bile and hydrolyze esculin.

    Positive reaction= agar becomes darkened
  • bubabe12
    Specialized Tests--

    Blood agar (hemolysis reactions)
    5% sheep erythrocytes in agar base. Used to test for hemolysis reactions important in identifying Streptococcus species.

    Three types of hemolysis can be seen:
    Beta: complete clearing of blood agar
    Alpha:cloudy/greenish opaque area
    Gamma: no apparent hemolysis
  • bubabe12
    Specialized Tests--

    Butanediol Fermentation
    (Voges-Proskauer Test)
    Test for detection of 2,3 butanediol, which is unique to Serratia, Enterobacter and Bacillus.)

    Incubated 24 hours at optimum temp in MR-VP broth.

    15 drops reagent A and 5 drops of reagent B added to 1ml of culture w/ constant mixing.

    Postive test= red color change after 5-15 minutes
  • bubabe12
    Specialized Tests--

    DNASE Agar
    Some organisms produce an enzyme which will hydrolyze DNA incorporated into an agar base.

    5N HCL added after incubation.

    Positive result= appearance of halo around colonies

    Available as agar plate.
  • bubabe12
    Specialized Tests--

    Eosin Methylene Blue Agar (EMB)

    A selective and differential medium for gram (-) bacteria.

    Colony color is noted for recognition of bacterial strains:
    E. coli= metallic green colonies
    Enterobacter= mucoid colonies with dark centers (fish eye)
    Proteus= thin, purple, filmy, spreading colonies
    Serratia= dark purple colonies
    Pseudomonas= pink/lavender colonies

Microbiology Lab Exam 2
Microbiology Lab Exam 2
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