Germ Tube Test: Principle, Procedure, Results

A yeast has grown from a blood culture, and the team needs a fast answer: is this Candida albicans, which usually responds to first-line antifungals, or one of the other Candida species that are more often drug-resistant? Instead of waiting days for full identification, the technologist drops a colony into serum, warms it for a couple of hours, and looks. A slender tube grows straight out of the yeast cell with no pinch where it joins. That single feature, a germ tube with no constriction at its base, is enough to call it presumptively Candida albicans the same morning.

Germ tube test is a screening test for the presumptive identification of Candida albicans. Candida dubliniensis can also form germ tubes and Candida tropicalis can produce pseudohyphae that closely resemble germ tubes.

Figure: Germ Tube

The appearance of germ tubes is called the Reynolds-Braude phenomenon, named after Reynolds and Braude, who first reported it. This eponym is commonly used and frequently tested, so it is worth remembering: Reynolds-Braude phenomenon = germ tube formation by Candida albicans.

Buds and pseudo-hyphae 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.

Principle of Germ Tube Test

Germ tubes are short, non-septate outgrowths of germinating hyphae. They are about half the width and three to four times the length of the yeast cell they arise from. When Candida cells are incubated in serum at 37°C for 2 to 4 hours, Candida albicans and Candida dubliniensis produce these short, slender, tube-like structures. About 95 to 97% of C. albicans isolates form germ tubes under the right conditions.

Figure: Characteristics morphology of Candida albicans

A germ tube appears as a short lateral extension from the yeast cell and does not have a constriction (septum) where it meets the yeast cell or any constriction at the septum along the tube.

The formation of germ tubes is associated with increased synthesis of protein and ribonucleic acid. Various media like fetal bovine serum may be used as a substitute for human pooled serum.

Requirements

• Culture: Suspected Candida colonies from sabouraud dextrose agar (SDA).SDA is the best medium to isolate yeasts for germ tube production; sheep blood agar is an acceptable substitute
• Reagent: Serum (human, sheep, fetal bovine) or other commercially produced media for germ tube testing
• Others: Test tubes, loop or wooden applicator, Pasteur pipettes, slides, cover slips.

Figure: Candida albicans: Germ Tube Positive

Quality Control

The following yeasts should be included each time the test is performed.

1. Germ tube positive, C. albicans ATCC 14053
2. Germ tube negative, Candida tropicalis ATCC 66029

Procedure

1. Lightly touch a yeast colony with a wooden applicator stick.
2. Suspend the yeast cells in an appropriately labeled tube of fetal bovine serum (make a light suspension).
3. Incubate the tube for 2-3 hours in a 35 – 37°C incubator.
4. Place a drop of the suspension on a slide using a Pasteur pipette.
5. Place a coverslip over the suspension.
6. Examine the wet mount microscopically (at 40X) for the presence or absence of germ tubes.

Result and interpretation

Positive germ tube test: A short hyphal (filamentous) extension arising laterally from a yeast cell with no constriction at the point of origin is identified as C.albicans or C. dubliniensis. Some C. tropicalis isolates produce pseudohyphae that require careful observation to discriminate from those of C. albicans.

A minimum of about five germ tubes should be seen before calling the test positive, because occasional tubes may be produced by other species. Because both Candida albicans and Candida dubliniensis are germ-tube positive (and both also form chlamydospores), the germ tube test alone cannot separate them. The standard way to distinguish the two is growth at 45°C, at which C. albicans grows and C. dubliniensis does not. Some sources cite 42°C for this. CHROMagar color and molecular methods also separate them.

Figure: Difference between pseudohyphae and germ tube

Negative germ tube test: No hyphal extension arising from a yeast cell or a short hyphal extension with constriction at the point of origin. A constriction where the lateral extension meets the yeast cell is produced by pseudohyphae or budding cells.

Germ tube vs pseudohyphae

The most common mistake in reading this test is confusing a true germ tube with a pseudohypha or a bud. The whole test depends on telling them apart, and the deciding feature is the constriction at the base.

The single rule to remember: a germ tube has no constriction at its point of origin. If there is a pinch where the tube meets the yeast cell, it is a pseudohypha or a bud, not a germ tube. This is why the test is read by looking specifically at the base of each outgrowth, not just at the overall shape.

A practical safeguard: observe several germ tubes (a minimum of about five) before calling the test positive, because occasional species such as Candida tropicalis can produce pseudo-germ tubes that need careful discrimination, and these show a constriction at the base.

Limitations

1. Candida tropicalis may produce pseudo-germ tubes after 3 hrs of incubation but they show constriction at the point of origin.
2. Too heavy inoculum will inhibit germ tube formation.

How to Remember

Reynolds-Braude means germ tube. The eponym for germ tube formation by Candida albicans is the Reynolds-Braude phenomenon.

No pinch, positive. A true germ tube has no constriction where it leaves the yeast cell. A pinch at the base means pseudohypha, not germ tube.

Two are positive, so heat to separate. Both C. albicans and C. dubliniensis give germ tubes. Growth at 45°C separates them: albicans grows, dubliniensis does not.

Serum, 37°C, a couple of hours. The conditions for germ tube formation are serum, body temperature, and a short incubation. Longer incubation lets other species mimic the result.

Keep it light. A heavy inoculum inhibits germ tube formation, so make a light suspension.

Key exam facts

Where Students Get Confused

"A germ tube and a pseudohypha are the same." No. A germ tube has no constriction where it meets the yeast cell. A pseudohypha has a clear pinch at the junction. The base is where you look.

"A positive germ tube confirms Candida albicans." It is presumptive, not definitive. Candida dubliniensis is also germ-tube positive, so a positive result means C. albicans or C. dubliniensis.

"Germ tube positive rules out all other species." Not entirely. Rare pseudo-germ tubes from species such as Candida tropicalis can mimic the result, which is why several germ tubes should be seen and the base checked for constriction.

"You can leave it incubating longer to be sure." No. Longer incubation lets other species form pseudo-germ tubes and increases false positives. Read at 2 to 4 hours.

"Any serum and any inoculum will do." The inoculum must be light, because a heavy inoculum inhibits germ tube formation. Serum (human, sheep, rabbit, or fetal bovine) at 37°C is required.

Resumen en español

La prueba del tubo germinal (también llamada fenómeno de Reynolds-Braude) sirve para la identificación presuntiva de Candida albicans. Cuando las células de Candida se incuban en suero a 37°C durante 2 a 4 horas, Candida albicans y Candida dubliniensis forman una prolongación corta en forma de tubo llamada tubo germinal.

Un resultado positivo es un tubo que sale de la célula de levadura sin constricción (sin estrechamiento) en su base. Si hay un estrechamiento donde el tubo se une a la célula, se trata de una pseudohifa o una yema, no de un tubo germinal. Como tanto C. albicans como C. dubliniensis son positivas, se usa el crecimiento a 45°C para diferenciarlas: C. albicans crece y C. dubliniensis no. Un inóculo muy denso inhibe la formación del tubo germinal.

References and further readings

1. Sudbery, P. E. (2011). Growth of Candida albicans hyphae. Nature Reviews Microbiology, 9(10), 737–748.
2. Sudbery, P. E. (2001). The germ tubes of Candida albicans hyphae and pseudohyphae show different patterns of septin ring localization. Molecular Microbiology, 41(1), 19–31.
3. Leber, A. L. (Ed.). (2016). Clinical Microbiology Procedures Handbook (4th ed.). ASM Press. https://doi.org/10.1128/9781683670438.CMPH