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  Section: Biotechnology Methods » Microbiology
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Triple Sugar Iron Test

  The Microscopy
  The Bright Field Microscope
  Introduction to the Microscope and Comparison of Sizes and Shapes of Microorganisms
  Cell Size Measurements: Ocular and Stage Micrometers
  Measuring Depth
  Measuring Area
  Cell Count by Hemocytometer or Measuring Volume
  Measurement of Cell Organelles
  Use of Darkfield Illumination
  The Phase Contrast Microscope
  The Inverted Phase Microscope
  Aseptic Technique and Transfer of Microorganisms
  Control of Microorganisms by using Physical Agents
  Control of Microorganisms by using Disinfectants and Antiseptics
  Control of Microorganisms by using Antimicrobial Chemotherapy
  Isolation of Pure Cultures from a Mixed Population
  Bacterial Staining
  Direct Stain and Indirect Stain
  Gram Stain and Capsule Stain
  Endospore Staining and Bacterial Motility
  Enumeration of Microorganisms
  Biochemical Test for Identification of Bacteria
  Triple Sugar Iron Test
  Starch Hydrolysis Test (II Method)
  Gelatin Hydrolysis Test
  Catalase Test
  Oxidase Test
  IMVIC Test
  Extraction of Bacterial DNA
  Medically Significant Gram–Positive Cocci (GPC)
  Protozoans, Fungi, and Animal Parasites
  The Fungi, Part 1–The Yeasts
  Performance Objectives
  The Fungi, Part 2—The Molds
  Viruses: The Bacteriophages
  Serology, Part 1–Direct Serologic Testing
  Serology, Part 2–Indirect Serologic Testing

To detect whether the given test organism can ferment sugars and produce H2S by the triple sugar iron test.

Triple sugar iron agar is used for differentiation of members of Enterobacteriaceae, according to their ability to ferment lactose, sucrose, dextrose, and produce H2S. The medium contains phenyl red as a pH indicator. The indicator at different pH shows different colors in the medium. When oxidative decarboxylation of proteins take place, the H2S produced imparts a black precipitate resulting from the reaction between H2S and ferrous sulfate present in the medium (usually hydrogen, carbon dioxide). This is visible as bubbles in the medium or cracking of the medium.

The medium contains a small amount of dextrose as compared to lactose and sucrose, which results in the formation of very little amount of acid by dextrose fermentation. This is quickly oxidized by aerobic growth on the slant, which reverts back to the color of the slant to pink in absence of lactose and sucrose fermentation. Thus pink slant and yellow butt is dextrose fermentation.

Test cultures test tubes, conical flasks, glass rod, inoculation loop, and triple sugar iron agar.


  1. Prepare the TSI media and distribute among all the tubes.
  2. Sterilize the media in the test tubes in an autoclave for 15 minutes at 15 lb/sq inch.
  3. Cool the tubes after sterilization and prepare the slants in such a way that a small butt region remains in the bottom of the tube and an upper slant region.
  4. Allow this to solidify.
  5. Under aseptic conditions, using an inoculation loop, take the culture of the test organisms and pierce into the butt region and streak in the slant region.
  6. Incubate the tube for 24–48 hrs at 37°C and then record the observations.


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