Study Notes on Identification of Gram-Negative Rods

Introduction

• The focus of these notes is on testing and identification of gram-negative rods in a laboratory setting.

Overview of Flowcharts for Gram-Negative Rods

• There are four flowcharts in the lab manual (section 9-1) that facilitate the identification of gram-negative rods.
• The identification of which flowchart to use is determined by conducting the MBIC battery test.
• Tests included in the MBIC battery:
  • Indole test
  • Methyl Red test
  • Voges-Proskauer test
  • Citrate test

Understanding the Unknowns

• There are both easier and more difficult unknowns associated with gram-negative rods based on chance.
• Gram positive cocci generally have shorter flowcharts compared to gram positive rods and gram negative rods which have longer flowcharts.
• It is suggested that students may learn more from unknowns that require performing more tests.

The MBIC Battery Tests

• The MBIC battery is essential for identifying different species of gram-negative rods.

• The battery includes:

  • Indole Test:

  • Uses SIM (Sulfide-Indole-Motility) agar.

  • Procedure: Inoculate with an inoculation needle into solid media.

  • Test Components:

    • Sulfur reduction (H2S production): indicated by black precipitate.
    • Indole production from tryptophan.
    • Motility: evidenced by growth outside the stab line. A stable stab is required to avoid false motility results.

  • Methyl Red Test:

  • Accompanied by Voges-Proskauer (these two tests share the same MRVP broth).

  • Purpose: To identify mixed acid fermentation of glucose.

  • Procedure: Inoculate MRVP broth, incubate, then split and add methyl red reagent to one portion and Voges-Proskauer reagent to the other.

  • Methyl red will indicate a positive result for acid production; the result indicates the ability of the organism to perform mixed-acid fermentation directly linked to some species of Enterobacteriaceae.

  • Voges-Proskauer Test:

  • Checks for the production of 2,3-butanediol from glucose fermentation.

  • Important as not all gram-negative rods can perform this fermentation.

  • Citrate Test:

  • Assesses whether an organism can utilize citrate as its sole carbon source.

  • Uses a slant medium; growth or a color change to blue indicates a positive result.

  • Procedure: Inoculate a citrate agar slant using a stab and zigzag streak technique for proper aeration and visibility of growth.

  • Growth alone indicates citrate utilization; a color change indicates ammonia production, which is also considered.

Decarboxylase Test

• The presence of decarboxylase enzymes is assessed with specific broths (e.g., ornithine broth).
• Procedure: Inoculation with an inoculation loop followed by a layer of mineral oil to create an anaerobic environment is crucial.
  • About one quarter inch of mineral oil is required; forgetting to add the mineral oil necessitates restarting the test.
• This test requires a longer incubation time of 96 hours to yield results, contrasting with other tests that generally require only 48 hours.

Practical Application

• Each pair of students will work with either Escherichia coli or Salmonella typhimurium, inoculating four types of tests (SIM Agar, MRVP broth, citrate slant, decarboxylase broth) based on structured guidelines provided by the instructor.
• Accurate labeling and tracking of each student’s unique unknown number is crucial for successful identification.