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Germ Tube Test I. Principle: The Germ Tube Test is a screening procedure used to differentiate Candida albicans from other yeast. Approximately 95 - 97% of Candida albicans isolated develop germ tubes when incubated in a proteinaceous media. Germ tubes are short non-septate germinating hyphae. They are the width and 3 - 4 times the length of the cell from which they arise. The junction of the germ tube and cell is not constricted. Buds and pseudo-hyphae can be distinguished from germ tubes by the constricted attachment. II. Clinical Significance: N.A. III. Specimen: N.A. IV. Materials: 1. 10 x 75 mm tube (CardinalHealth #T1290-2) 2. Wooden applicator stick (CardinalHealth #A5000-1) 3. Glass microscope slide (Surgipath #220) 4. 22 x 22 mm coverslip (Surgipath #105) 5. Pasteur pipette (Cardinal Health #P5201-3) 6. Microscope Note: Our defacto standard for microscope field # is based on a field size defined by a fixed ocular diaphragm of 18 mm diameter. In order to obviate the need for scope-specific procedures, the scopes with larger field of view have been corrected to the standard by means of a custom reticle that has been installed in the right-side oculars. The reticle superimposes a thin circle over the visible field. 6. 35 - 37C incubator V. Reagents: Sheep serum (Colorado serum #CS1342) VI. Standardization: N.A. VII. Procedure: 1. Put 0.5 ml (12 drops) of sheep serum in a 10 x 75 mm tube. 2. Make a light suspension of the suspect yeast colonies by touching 1-2 large colonies or 3-4 smaller colonies with a sterile wooden applicator stick and then inoculating the sheep serum with the applicator stick. Note: Too large of an inoculum will inhibit germ tube formation.

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3. Incubate the tube for 2-3 hours in a 35 - 37C incubator. Warning: Do not over-incubate the tube. Candida tropicalis may produce pseudogerm tubes after 3 hours of incubation. 4. Place a drop of the suspension on a slide using a Pasteur pipette and coverslip. 5. Examine the wet mount microscopically for production of germ tubes (long tube-like projections extending out from the yeast cells).

VIII. Limitations: N.A. IX. Results Derivation: N.A. X. Expected Result(s) and/or Critical Value: N.A. XI. Quality Control: 1. Controls are run once per lot, batch or shipment. Record the results in the Lot Log. Refer to Labeling & Dating of Chemical and Biological Reagents operational policy for Lot Log instructions. A. Positive control: Candida albicans germ tube formation B. Negative control: Candida tropicalis or any other germ tube negative yeast, no germ tube formation 2. The Incidence Recording procedure is followed if quality control is unacceptable. XII. Reference: 1. Laboratory Methods in Medical Mycology, U.S. Department of Health, Education and Welfare, Center for Disease Control, Haley, Leanor D. and Callaway, Carey S., June 1978, pg. 119 2. Mycology Laboratory Procedure Manual, Mayo Clinic, Rochester, Minnesota, Roberts, Glenn D., 1979, pg. 117 3. Cumitech 11, Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology Laboratory, Sherris, John C., American Society for Microbiology, Washington, D.C., August 1980, pg. 10 4. Laboratory Procedures in Clinical Microbiology, Washington, John A., Mayo Clinic, Rochester, Minnesota, 1981, pg. 429-430 5. Medical Mycology, Kern, Martha, FA Davis Co., Philadelphia, 1985, pg. 142-143

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6. Bailey & Scotts Diagnostic Microbiology, Finegold, Sydney & Baron, Ellen Jo, CV Mosby Co., St. Louis, 1986, pg. 753

i. 5-14-85 C. Harms/E. Steiner ii. 9-18-87 B. Dunham (Revised: I., XI.) iii. March 2004 J. Wedig (Revised: XI.) iv. February 2006 J. Wedig (Revised: XI.1.B.)

Comprehensive Review: Pathologist: Technical Director:

Interim Review: June 2010 J. Wedig (Revised: IV.p/n) June 2011 C. Knapp (Revised: VII.3., 5.images added)

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