Germ Tube Test For Candida: Principle, Procedure And Results Interpretation

Introduction

germ tube test is a diagnostic test in which a sample of fungal spores are suspended in animal serum and examined by microscopy for the detection of any germ tubes. It is particularly indicated for colonies of white or cream color on fungal culture, where a positive germ tube test is strongly indicative of 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.

Principle

This procedure is used for presumptive identification of Candida species and is also known as germ tube test. The culture of Candida species is treated with normal human pooled serum or sheep or rabbit or fetal calf serum and incubated at 37°C for 2-4 hours. A drop of suspension is examined on the slide under microscope. The germ tubes are seen as long as tube like projections extending from the yeast cells.There is no constriction at the point of attachment to the yeast cell as seen in case of Pseudohypahe.

The demonstration of the germ tube is known as Reynolds-Braude phenomenon.This is a rapid method for identifying and differentiating C. albicans from other Candida sppBuds and pseudohyphae 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.

Germ tube is a short outgrowth, non-septate germinating hypha. It is ½ the width and 3 – 4 times the length of the cell from which it arises. When Candida species are incubated in serum at 37°C for 2-4 hours and producing short, slender, tube like structures called germ tube. Formation of this germ tube is associated with increased synthesis of protein and ribonucleic acid and observed in Candida albicans.

Requirements For Germ Tube Test

  • Test tube (12Χ75 mm)
  • Inoculating loop/ sterile bamboo stick
  • 24 hours old culture of suspected fungal colony to be tested
  • Incubator
  • Clean and grease free slide and cover slip
  • Droppers
  • Microscope
  • Human pooled serum

Quality Control For Germ Tube Test

  • Positive control (PC): Candida albicans
  • Negative control (NC): Candida parasilosis

Procedure

  1. Take four test tubes and label as uninoculated (UN), negative control (NC), positive control (PC) and test (T).
  2. Add 0.5 ml of serum to each of the test tube.
  3. Take half of a single colony to be tested by using a sterile loop , and mix with serum in the test tube ‘T’.
  4. Similarly, take half of C. albicans single colony by using a sterile loop , and mix it with serum in the test tube PC.
  5. Similarly, take half of C. parasilosis single colony by using a sterile loop , and mix it with serum in the test tube NC.
  6. Leave uninoculated tube (UN) without any disturbance.
  7. Now incubate  all the tubes at 37°C for 2 hours.
  8. Place one drop of suspension from tube tubes Un, NC, PC and T onto  different slides and place cover slips over the drops.
  9. Examine the slide under low power (10X) and finally  high power (40X) objective.

Observations And Results Interpretation of Germ Tube Test

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. tropicalisC. glabrata and other yeasts.

3. Uninoculated (UN) tube: Lacking yeast cells.

4. Negative Control (NC) tube: Absence of germ tube formation.

5. Positive Control(PC): Presence of germ tubes.

Differences between germ tube and pseudohyphae

Germ Tube   Pseudohyphae
No constriction at the site of attachment .Constriction at the site of attachment
Non-septate with parallel sides.Septate  and not necessarily with parallel sides.

Limitations of Germ Tube Test

  1. C. tropicalis may form early pseudohyphae which may be falsely interpreted as germ tubes.
  2. The yeast formerly named Candida stellatoidea also produces germ tubes; however, it has been combined with C. albicans and no longer exists as separate species.
  3. This test is only part of the overall scheme for identification of yeasts. Further testing is required for definite identification.

Further References

  1. C.J. Alexopolous, Charles W. Mims, M. Blackwell, Introductory Mycology, 4th ed. (John Wiley and Sons, Hoboken NJ, 2004).
  2. Hoppe J, Frey P. Evaluation of six commercial tests and the germ-tube test for presumptive identification of Candida albicans. Eur J Clin Microbiol Infect Dis. 1999;18:188-91.
  3. Pfaller MA, Barry A, Bille J, Brown S, Ellis D, Meis JF, et al. Quality control limits for voriconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol. 2004;42:1716-8.
  4. Romeo O, Criseo G. Morphological, biochemical and molecular characterisation of the first Italian Candida africana isolate. Mycoses. 2008;52:454-7.
  5. Brown S, Traczewski M. Quality control limits for posaconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue. J Clin Microbiol. 2007;45:222-3.
  6. Hoppe J, Frey P. Evaluation of six commercial tests and the germ-tube test for presumptive identification of Candida albicans. Eur J Clin Microbiol Infect Dis. 1999;18:188-91.