Germ tube test

Table of Contents

Introduction

The germ tube test for identifying yeasts is probably the most important and easiest test to perform. The germ tube test offers one of the quickest approaches to presumptive identification of C. albicans and C. dubliniensis. The yeasts recovered from clinical specimens are approximately 75 % C. albicans and germ tube tests usually provide adequate identification within a few hours of this organism.

Germ tubes occur as early hyphal-like extensions of yeast cells formed at the point of origin from the yeast cell, without a constriction. Another Candida species, C. dubliniensis, has been shown to also produce true germ tubes. Although C. dubliniensis is infrequently encountered, supplemental biochemical or morphologic testing may be needed to differentiate it from C. albicans. C. tropicalis produces what has been termed “pseudo-germ tubes,” which are constricted from the yeast cell at the base or point of the germ tube. Unless this is recognized and the lab has developed the ability to differentiate between true germ tubes and pseudogerm tubes, C. tropicalis isolates will be misidentified as C. albicans.

The standard procedure requires serum or plasma, for example fetal bovine serum. Expired fresh-frozen plasma from the blood bank is useful in this test and can almost indefinitely be stored at 4 ° C. Many other liquid media (e.g. BHI, soy broth trypticase, nutrient broth) have been widely used as an alternative. The substratum is inoculated, then incubated for 3 hours at 37 ° C. Care must be taken not to incubate the test after 3 hours, since other species can form germ tubes with extended incubation.

Procedure

  • Lightly touch a yeast colony with a wooden applicator stick.
  • Suspend the yeast cells in an appropriately labeled tube of fetal bovine serum.
  • Incubate the tubes at 35° to 37°C for no longer than 3 hours.
  • Place a drop of the suspension on a microscope slide.
  • Place a coverslip over the suspension.
  • Examine under high power for the presence or absence of germ tubes.

Result

A germ line emerges from the yeast cell as a small lateral branch, which has no constriction (septum) where it reaches the yeast cell. Pseudohyphae or budding cells form a constriction where the lateral extension reaches the yeast cell. Before calling the isolate positive a minimum of 5 germ tubes should be observed.

Germ tube production by Candida albicans. A positive germ tube base won’t have constrictions.
Candida tropicalis shows constriction at the base of the germ tube, called pseudo-germ tube.

True germ tubes at their bases lack constriction, where they attach to the mother cell. If there is a constriction at the base of a germ tunnel, so the yeast is not a animal. Such constricted germ tubes, called pseudo–germ tubes, are more characteristic of C. tropicalis. C. dubliniensis is differentiated from C. albicans by its inability to grow at 42°C

References:

  • Linne & Ringsurd Clinical Laboratory Sience 6th Edition
  • Textbook of Diagnostic Microbiology 5th Edition, Saunders Elsevier
  • Clinical Microbiology Procedures Handbook 2nd Edition, ASM Press
  • Bailey & Scott’s Diagnostic Microbiology 13th Edition, Elsevier
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