Carbohydrate Differentiation Discs for Identification of Microorganisgms

By: Jvo Siegrist, AnalytiX Volume 6 Article 2

Differentiate and identify bacteria based on their carbohydrate fermentation profile in liquid, solid and semi-solid growth media

By Jvo Siegrist, Product Manager Microbiology …

A microorganism’s carbohydrate fermentation profile is often characteristic and can facilitate its identification. The differences arise because of the varying types and activity of enzymes that target specific carbohydrates1, 2. As an example, the different carbohydrates metabolized by Salmonella typhimurium ATCC 14028 are shown in Figure 1.

Figure 1 ......... Salmonella typhimurium ATCC 14028

 

The presence of carbohydrate-utilizing enzymes can be detected visually via gas (CO2) production or as a pHinduced color change from the production of acid.

The Sigma-Aldrich analytical microbiology product line includes Carbohydrate Differentiation Discs to detect microorganisms based on their ability to ferment twentyone different carbohydrates. The discs are effective yet economical, and work in any sugar-free medium – solid, liquid or semi-solid. The detailed instructions supplied with the discs lists the recommended media

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Using Discs with Liquid and Semi-solid Media

Liquid or semi-solid media are dispensed in 5 mL amounts in test tubes and sterilized. A single Carbohydrate Differentiation Disc and the inoculum, which is the sample to be analyzed for the presence of the microorganism, are aseptically added to each tube. With liquid medium, CO2 gas produced during fermentation is collected in an inverted Durham‘s tube while any acid produced changes the color of the medium. Figure 2 shows an example of some developed tubes indicating the presence (or absence) of the target microorganism based on the ability to ferment the specific sugars on the discs. The discs are visible at the bottom of the tubes.

Figure 2 ......... Test row with Carbohydrate Differentiation Discs for carbohydrate fermentation abilities in liquid media. The discs can be seen at the bottom of the tubes.


In semi-solid medium, the disc and inoculum are pushed into the medium just below the surface. The inoculumfree medium at the bottom of the tube serves as the control, while fermentation can be detected at the upper level. Any CO2 gas produced is trapped and seen as bubbles in the semi-solid media.

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Using Discs with Solid Media

By using solid media, it is possible to detect the fermentation of a number of carbohydrates on the same plate. Sterile plates containing the agar medium of choice are surface-seeded with the test organism. Carbohydrate Differentiation Discs are pressed gently onto the surface of the plate at sufficient distance (2 cm) from each other. The plate is incubated 36 ± 1.0°C for 18 - 48 hours and results are recorded at 18 - 24 hours and again at 48 hours. The results should be monitored frequently since reversal of fermentation can occur. On agar plates fermentation is visualized by change color of pH-indicator around the discs.

 

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References

  1. Bergey‘s Manual of Systematic Bacteriology, 1984, Vol. 1, Williams and Wilkins, Baltimore.
  2. Bergey‘s Manual of Determinative Bacteriology, 1994, 9th ed., Williams and Wilkins, Baltimore, MD, USA. (Available from Sigma-Aldrich, Cat. No. B0411)