Singco, Marvin John J.

Microbiology-Mycology

Germ Tube Test- Principle, Procedure, Results, Interpretation and Limitations

Germ Tube Test is a screening test which is used to differentiate Candida albicansfrom other yeast. Germ
tube (GT) formation was first reported by Reynolds and Braude in 1956. When Candida is grown in
human or sheep serum at 37C for 3 hours, they forms a germ tubes, which can be detected with a wet
KOH films as filamentous outgrowth extending from yeast cells. It is positive for Candida
albicans and Candida dubliniensis. Approximately 95 97% of Candida albicans isolated develop germ
tubes when incubated in a proteinaceous media.

Principle of Germ Tube Test

Formation of germ tube is associated with increased synthesis of protein and ribonucleic acid. Germ Tube
solutions contains tryptic soy broth and fetal bovine serum, essential nutrients for protein synthesis. It is
lyophilized for stability. Germ tube is one of the virulence factors of Candida albicans. This is a rapid test
for the presumptive identification of C. albicans.

Procedure of Germ Tube Test

1. Put 0.5 ml of sheep or human serum into a small tube.

Note: Fetal bovine serum can also be used instead of human serum.
2. Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum.
Note: Too large of an inoculum will inhibit germ tube formation.
3. Incubated the tube at 37C for 2 to 4 hours.
4. Transfer a drop of the serum to a slide for examination.
5. Coverslip and examine microscopically under low and high power objectives.

Results and Interpretation of Germ Tube Test

Singco, Marvin John J. Microbiology-Mycology

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.

Quality Control in Germ Tube Test

Positive Control: C. albicans (ATCC 10231)

Negative Control: C. tropicalis (ATCC 13803), C. glabrata (ATCC 2001)

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.

Slide culture for Fungi

Materials Required:

Culture:

7-10 day old fungal culture

Media:

Sabouraud agar

Preparation of Sabouraud agar (pH-5.6)

Sabouraud agar supplemented with aureomycin*

Peptone-10g/liter
Dextrose-40g/liter
Agar-15g/liter
*Aseptically add aureomycin ,10g per ml, to the sterile, molten and cooled medium
 Singco, Marvin John J. Microbiology-Mycology

Equipments:

Sterile Petri dish

Filter paper (9cm diameter)
U-shaped glass rod
Microscope slides and coverslips (Sterile)
Sabourauds plate with mixed culture of fungi
Sterile Sabourauds agar plate
Lactophenol cotton blue stain
Glass capillary tube
Scalpel
Inoculating needle
Sterile distilled water
95% ethanol
Forceps

Procedure:

A) Slide Culture Preparation

Aseptically, with a pair of forceps, place a sheet of sterile filter paper in a Petri dish.
Place a sterile U-shaped glass rod on the filter paper. (Rod can be sterilized by flaming, if held
by forceps.)
Pour enough sterile water (about 4 ml) on filter paper to completely moisten it.
With forceps, place a sterile slide on the U-shaped rod
Gently flame a scalpel to sterilize, and cut a 5 mm square block of the
medium from the plate of Sabourauds agar or Emmons medium.
Pick up the block of agar by inserting the scalpel and carefully transfer this block aseptically to
the centre of the slide.
Inoculate four sides of the agar square with spores or mycelial fragments of the fungus to be
examined. Be sure to flame and cool the loop prior to picking up spores.
Aseptically, place a sterile cover glass on the upper surface of the agar cube.
Place the cover on the Petri dish and incubate at room temperature for 48 hours.
After 48 hours, examine the slide under low power. If growth has occurred there will be
growth of hyphae and production of spores. If growth is inadequate and spores are not
evident, allow the mold to grow for another 2448 hours before making the stained slides.

B) Application of Stain

Place a drop of lactophenol cotton blue stain on a clean microscope slide.

Remove the cover glass from the slide culture and discard the block of agar.
Add a drop of 95% ethanol to the hyphae on the cover glass. As soon as most of the alcohol has
evaporated place the cover glass, mold side down, on the drop of lactophenol cotton blue
stain on the slide. Examine the slide under microscope
 Singco, Marvin John J. Microbiology-Mycology

Advantages of slide culture:

It is a rapid method of preparing fungal colonies for examination and identification.

Permits fungi to be studied virtually in situ with as little disturbance as possible
Fungi are identified mostly by close examination of its morphology and the characteristics it
possess. In slide cultures, we are growing the fungi directly on the slide on a thin film of agar. By
doing this, there is no need to remove a portion of the fungus from a culture plate and transfer it
to the slide. So there is less chance for the features that are key to identification, notably the
spore-bearing structures, to be damaged.