Microdase Test – Principle, Procedure, Uses and Interpretation

Microdase Disk is a reagent-impregnated disk recommended for use in qualitative procedures to aid in the differentiation of Staphylococcus from Micrococcus by the detection of the oxidase enzyme.

The oxidase method was originally described by Kovacs in 1956 as a method of differentiating gram-negative bacilli. However, in 1981, Faller and Schleifer modified Kovacs’ oxidase reagent by utilizing a tetramethyl-pphenylenediamine (TMPD) in dimethyl sulfoxide (DMSO).This is referred to as the modified oxidase test.

Objective

To differentiate gram-positive, catalase-positive cocci

Principle

The microdase test is a rapid method to differentiate Staphylococcus from Micrococcus spp. by detection of the enzyme oxidase. For the detection of oxidase enzyme a filter paper circular disks impregnated with tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide (DMSO) are used. The modified oxidase reagent is prepared as 1% (w/v) tetramethyl-p-phenylenediamine in certified grade dimethyl sulfoxide. DMSO provides solubility and stability against auto-oxidation and also aids in the permeability of cells to the reagent. In the presence of atmospheric oxygen, the oxidase enzyme reacts with the oxidase reagent and cytochrome C to form the colored compound, indophenol indicated as blue or purplish blue coloration on the disc after the introduction of bacterial colony on the disc.

All micrococci possess cytochrome c, whereas most staphylococci, with few exceptions, lack this type of cytochrome. The oxidase reagent substantiates the presence of type c cytochrome.

Media:

Oxidase Disc: Filter paper disks impregnated with tetramethyl-p-phenylenediamine dihydrochloride (oxidase reagent) in dimethyl sulfoxide (DMSO).

Method

Testing should be performed on aerobic, catalase-positive, gram-positive cocci.

  1. Using forceps, place the disk in an empty petri dish or on a clean glass slide.
  2. Using a wooden applicator stick, rub a small amount of several colonies of an 18- to 24-hour pure culture grown on blood agar onto a small area of the microdase disk.
    Note: Do not rehydrate the disk before use.
  3. Incubate at room temperature for 2 minutes.
  4. Examine for a blue color development.

Expected results

Positive: Development of blue to purple-blue color within 2 minutes time

Negative: No color change (white to gray color remains).

Uses

This test is used to differentiate Micrococci from Staphylococci. (Micrococci should yield a positive result. Staphylococci should yield a negative result, with the exception of Staphylococcus sciuri, S. lentus and S. vitulinus.

Limitations

  • Staphylococci should yield a negative color change, except for S. sciuri, S. lentus, and S. vitulus.
  • Bacteria from a culture grown on Blood agar for 24 – 36 hours must be used. Cultures which are too young or too old may give inaccurate results.
  • Modified oxidase test (microdase) is recommended for Gram-positive, catalase-positive cocci, only.
  • Macrococcus species and Kocuria kristinae are also known to possess cytochrome
  • Microdase is not designed for routine testing for oxidase activity in organisms other than Staphylococcus and Micrococcus.

References

  1. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  2. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/IFU21132.pdf
  3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC273944/
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC272470/

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