Germ tubes are short  outgrowth, non-septate germinating tube-like structures called pseudohyphae produced by Candida albicans when incubated at a temperature of 35-37°C for 2-4 hours in pooled human serum. The use of human serum as a germ tube induction medium has raised questions about standardization, reproducibility, and safety.There are various media, including fetal bovine serum that have been evaluated as substitutes for human serum in performing the germ tube test.

Principle of Germ tube test

• The formation of germ tubes is associated with increased synthesis of protein and ribonucleic acid(RNA) by Candida albican.
• Germ Tube Solution contains tryptic soy broth and fetal bovine serum, essential nutrients for protein synthesis but human serum can still be used since it also contains proteins.

Germ tube solution reagents

Tryptic Soy Broth	20.0 g
Fetal Bovine Sera	..Q.S
Demineralized Water	1000.0 ml

OR

You can simple use Human serum

Materials required

• Inoculating loop
• Incubators
• Inoculum
• Quality control organisms
• Test tubes
• Microscope slides
• Coverslips
• Disposable pipettes
• Sterile demineralized water.

Germ tube test procedure

• If using the germ tube reagent,Rehydrate Germ Tube Solution with sterile demineralized water according to the volume size indicated on the vial label.
• Now aliquot 0.5 ml of solution into clean test tubes, one tube for each test isolate and control.
• Make a dilute suspension of the test isolate or control from a single colony.
• Touch the tip of a disposable pipette to the colony and emulsify in the solution.

Do not heavily inoculate the solution because excessive inoculum causes a significant decrease in the percentage of cells forming germ tubes.

• Positive and negative controls should be tested simultaneously.
• Incubate aerobically at 35-37°C for 2-4 hours.
• Examine microscopically under high (40 X) magnification for the presence of germ tubes.

Procedure using Human serum

• To about 2 to 3 ml of Human serum in a test tube
• Make a dilute suspension of the test isolate or control from a single colony.
• Touch the tip of a disposable pipette to the colony and emulsify in the serum.
• Do not heavily inoculate the solution because excessive inoculum causes a significant decrease in the percentage of cells forming germ tubes.
• Incubate aerobically at 35-37°C for 2-4 hours.
• Examine microscopically under high (40 X) magnification for the presence of germ tubes.

Result and Interpretation

Positive Test	A short hyphal (filamentous) extension arising laterally from a yeast cell, with no constriction at the point of origin
Negative Test	No hyphal (filamentous) extension arising from a yeast cell or a short hyphal extension constricted at the point of origin

Quality control

CONTROL	INCUBATION	RESULTS
Candida albicans	Aerobic for 2-4 h at temperature 35-37°C	Positive
Candida glabrata	Aerobic for 2-4 h at temperature 35-37°C	Negative