Experiments on the Enterobacteriaceae using TSI, IMViC, Selective and Differential Media, MIO, LIA and Oxidase Test

Triple Sugar Iron Agar and Several Selective and Differential Media For Gram Negative Bacteria

MATERIALS

• Triple Sugar Iron (TSI) Slants in long tubes -- 3 per group of 2
• Eosin Methylene Blue (EMB) Plates -- 3 per person
• MacConkey (Mac) Plates -- 3 per person
• Salmonella and Shigella (SS) Plates -- 1 per person

CULTURES

• Escherichia coli slants
• Proteus vulgaris slants
• Salmonella enteriditis slants

Experiment 1

Triple Sugar Iron Agar Slants - (TSI Slants)

• Work in groups of 2
• Use three TSI tubes per group
• Inoculate ("stab the butt and streak the slant") each with
  • E. coli
  • P.vulgaris
  • S. enteriditis
• CAPS MUST BE LOOSE DURING INCUBATION
• After 48 hours incubation, observe for hydrogen sulfide production and carbohydrate fermentation.
• Look at an uninoculated TSI for comparison.
• Compare your results with those described in Leboffe and Pierce.

 

Experiment 2  

Selective and Differential Media used for Enterobacteriaceae

• Work individually
• Streak for isolation:
  • Inoculate S. enteriditis onto SS, EMB and Mac
  • Inoculate E. coli onto EMB and Mac
  • Inoculate either P. vulgaris or E. cloaca onto EMB and Mac
• Read about and compare your results with those described in Leboffe and Pierce.

MATERIALS

• Tryptone broth -- 2 per group
• MR-VP broth -- 2 per group
  • Note that Tryptone and MR-VP Broths look very similar - Please label immediately! NEVER put ANYTHING back in the materials racks if you're unsure of its' identity!
• Simmon's Citrate slants -- 2 per group
• Empty 13x100 mm test tubes -- 2 per group
• Kovac's reagent (dropper bottles) -- 5 per class
• Methyl Red (dropper bottles) -- 5 per class
• 5% alpha-napthol (dropper bottles) -- 5 per class
• 40% KOH (dropper bottles) -- 5 per class
• Plastic droppers or Pasteur pipettes -- 2 per group

CULTURES

• Escherichia coli slants
• Enterobacter cloacae slants

Experiment 3

• Work in groups of 2
• Use two tubes of each medium (Tryptone, MR-VP, Citrate) per group and inoculate one set each with:
  • E. coli
  • E. cloacae
• Remember to keep all caps LOOSE!!!
• Tryptone and MR-VP broths look alike, Please label your tubes immediately. (Never put ANYTHING back in the stock racks if you are unsure of its identity.)
• Incubate 48 hours and then perform and interpret the tests as follows:
  • Indole Test:
    • Add several drops of Kovac's Reagent to the Tryptone Broth. A positive test is indicated if the Kovac's Reagent (which floats on top) turns red. See Leboffe and Pierce.
  • Methyl Red - Voges Proskauer (MR-VP) Test
    • Using a pasteur pipet and an empty 13x100 mm test tube split the volume of the incubated MR-VP tube between the original tube and the 13x100 test tube. (Even though the new test tube and the pasteur pipet are not sterile, work aseptically! Why do you think that it is alright to work with nonsterile materials at this point in the test?)
    • Perform the MR test on the volume in the original tube by adding several drops of methyl red indicator. A cherry red color indicates a positive test.
    • Perform the VP test on the volume in the 13x100 test tube by adding in sequence:
      • 2 drops of Potassium Hydroxide
      • 2 drops of a-Napthol
      • a positive test is indicated if a red-brown color develops over a 15 minute incubation period.
    • Compare your results with those shown in Leboffe and Pierce.
  • Citrate
    • Read these results directly from the incubated slants. A blue color indicate a positive test. Compare your results with those shown in Leboffe and Pierce.

MATERIALS

• 2 LIA tubes per group of two

CULTURES

• Escherichia coli slants
• Enterobacter cloacae slants
• Proteus vulgaris slants

Experiment 4

• Work in groups of 2
• Inoclulate LIA tubes with the "stab the butt, streak the slant technique"
• Inoculate one tube with either E. coli or E. cloacae
• Inoculate the other tube with P. vulgaris
• THESE CAPS MUST BE TIGHT!
• Interpret the test reults for either lysine deaminase or lysine decarboxylase. Compare your results with those shown in Leboffe and Pierce.

MATERIALS

• 2 MIO deeps per group two

CULTURES

• Escherichia coli slants
• Enterobacter cloacae slants
• Proteus vulgaris slants
• Salmonella enteriditis

Experiment 5

• Work in groups of 2
• Inoculate one tube with P. vulgaris
• Inoculate one tube with either E. coli, E. cloacae, or S. enteriditis
• Inoculation is done by making one straight stab into (and out of) the deep, preferably with a needle
• CAPS MUST BE LOOSE
• Use this medium to determine ornithine decarboxylation. The medium may also be used to determine motility -- although that may be a bit difficult to judge. The indole test may also be performed on this medium, but that would duplicate the result you obtain from the tryptone broth above.
  • Read about the ornithine decarboxylation test in Leboffe and Pierce.

MATERIALS

• Filter paper
• Tetramethyl-p-phenylenediamine Reagent

CULTURES

• Escherichia coli plates
• Pseudomonas aeruginosa plates

Experiment 6

• Work in groups of two
• Use a wooden applicator stick to transfer some bacterial growth from the slant cultures. Rub it into a small spot onto a piece of filter paper. (Several different cultures can be tested on the same piece of filter paper.)
• Drop some Tetramethyl-p-phenylenediamine reagent onto the organism spots on the filter paper. An immediate (within ten seconds) appearance of a dark purple/blue color indicates a positive test.
• Read about this test in Leboffe and Pierce

©2008 by John M. Berestecky
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