GERM TUBE TEST

GERM TUBE TEST

AIM

     To identify and differentiate Candida albicans from other Yeasts by Germ Tube Test or Reynold’s Brande Phenomena.

PRINCIPLE   

     Germ Tube Test is the Confirmatory test which is used to differentiate Candida albicans from other yeast. Germ tube formation was first reported by Reynolds and Braude in 1956 so it is also called as Reynold’s - Brande Phenomenon. Germ tubes are short outgrowth, non-septate germinating hyphae. They are half the width and 3 – 4 times the length of the cell from which they arise. When Candida albicans is grown in human or sheep serum at 37 °C for 3 hrs, they forms a Germ tubes, which can be detected with a Wet films as filamentous outgrowth extending from yeast cells. It is positive for Candida albicans and Candida dubliniensis (produce Germ tube after 3 hrs). Approximately 95 – 97 % of Candida albicans isolated develop Germ tubes when incubated in a proteinaceous media. Formation of Germ tube is associated with increased synthesis of Protein and Ribonucleic acid. Germ tube is one of the virulence factors of Candida albicans. This is a rapid test for the presumptive identification of Candida albicans.

MATERIALS REQUIRED

• Sheep serum or Human serum
• Test tube
• Wooden applicator stick
• Microscopic Glass slide
• Cover slip
• Pasteur Pipette
• Micropipette

PROCEDURE

a)     Add about 0.5 ml of sheep serum or pooled human serum in a test tube.

b)   With a sterile wooden applicator stick, lightly touch a yeast colony and then place the stick into the serum. Too large of an inoculum will inhibit germ tube formation.

c)     Incubated the tube at 37 °C for 2 to 4 hours.

d)     After incubation, place a drop of suspension on a clean microscopic slide and coverslip it.

e)     Examine the Wet mount microscopically for production of Germ tubes.

OBSERVATION AND RESULTS

• Positive Test: A short hyphal (filamentous) extension arising laterally from a yeast cell, with no constriction at the point of origin. Germ tube is half the width and 3 to 4 times the length of the yeast cell and there is no presence of nucleus. Examples: Candida albicans and Candida dubliniensis.
• Negative Test: No hyphal (filamentous) extension arising from a yeast cell or a short hyphal extension constricted at the point of origin. Examples: C. tropicalis, C. glabrata and other yeasts.

Figure – 1: Germ tube test