Germ Tube Test for Candida: Principle, Procedure, Results and Interpretation - microbeonline

Germ tube test is the confirmatory test for Candida albicans. Germ tube formation was first reported by Reynolds and Braude and hence the germ tube test is also known as Reynolds-Braude Phenomenon. This is a rapid method for identifying and differentiating C. albicans from other Candida spp.

Buds and pseudo-hyphae can be distinguished from germ tubes by the constricted attachment at the point of origin. Germ tubes don’t show constriction at the point of origin.

Principle of Germ Tube Test:
Germ tubes are short outgrowth, non-septate germinating hyphae. They are ½ the width and 3 – 4 times the length of the cell from which they arise. When cells of Candida are incubated in serum at 370C for 2-4 hours Candida albicans produce short, slender, tube like structures called germ tubes. Formation of germ tubes is associated with increased synthesis of protein and ribonucleic acid. Various media like fetal bovine serum may be used as a substitute to human pooled serum.

Requirements:

• Culture: Suspected Candida colonies from culture plate
• Reagent: Serum (human, sheep, fetal bovine)or other commercially produced media for germ tube testing
• Others: Test tubes, loop or wooden applicator, Pasteur pipettes, slides, cover slips

Procedure:

1. Aliquot 0.5 ml (12 drops) of serum or media in a test tube.
2. Make a light suspension of the suspect yeast colonies (by touching 1-2 large colonies or 3-4 smaller colonies with a sterile wooden applicator stick or loop) on serum.
3. Incubate the tube for 2-3 hours in a 35 – 37°C incubator.
4. Place a drop of the suspension on a slide using a Pasteur pipette and cover with a coverslip.
5. Examine the wet mount microscopically (at 40X) for production of germ tubes (long tube-like projections extending out from the yeast cells).

   

Result and interpretation:

• Positive Result: A short hyphal (filamentous) extension arising laterally from a yeast cell with no constriction at the point of origin. May be confirmed as C.albicans
• Negative Result: No hyphal extension arising from a yeast cell or a short hyphal extension with constriction at the point of origin.

Limitations:

1. Candida tropicalis may produce pseudo-germ tubes after 3 hrs of incubation but they show constriction at the point of origin.
2. Too heavy inoculum will inhibit germ tube formation.

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‏Source microbeonline.com