Yeasts (Disk Diffusion) Assay: Introduction, Test Requirements, Procedure, Result-Interpretation, and Keynotes

Introduction

Disk diffusion assay is a standardized method used for testing antifungal susceptibility of yeasts, especially Candida spp. It evaluates the inhibitory effect of antifungal agents on yeast growth by measuring the zone of inhibition around drug-impregnated disks placed on inoculated agar. This method is recommended by CLSI M44-A2 and EUCAST guidelines for routine antifungal susceptibility testing.

Test Requirements

1. Culture Media
   • Mueller–Hinton agar supplemented with 2% glucose and 0.5 µg/mL methylene blue (MHA-GMB).
   • Sterile saline or broth for preparing the inoculum.
2. Yeast Suspension
   • Pure yeast colonies (18–24 h old on SDA or CHROMagar).
   • Standardized inoculum adjusted to 0.5 McFarland (≈ 1 × 10⁶ to 5 × 10⁶ CFU/mL).
3. Antifungal Disks
   • Fluconazole (25 µg), Voriconazole (1 µg), Itraconazole (8 µg), Ketoconazole, Amphotericin B (10 µg), Echinocandins (where available).
4. Other Essentials
   • Sterile cotton swabs.
   • Forceps/automatic disk dispenser.
   • Incubator (35 ± 2 °C).

Procedure

1. Inoculum Preparation
   • Suspend yeast colonies in sterile saline.
   • Adjust turbidity to 0.5 McFarland standard.
2. Inoculation
   • Dip a sterile swab into the suspension.
   • Evenly streak over the surface of the MHA-GMB agar plate in three directions to ensure uniform lawn growth.
3. Application of Disks
   • Place antifungal disks on the agar surface using sterile forceps.
   • Ensure proper spacing (≥24 mm apart).
4. Incubation
   • Incubate plates at 35 ± 2 °C for 20–24 h (extended up to 48 h for slow growers).

Result Interpretation

• Measure the zone of inhibition in millimeters around each antifungal disk.
• Compare results with CLSI/EUCAST interpretive breakpoints: Example (CLSI M44-A2 for Candida spp.):
  • Fluconazole (25 µg)
    • Susceptible (S): ≥19 mmSusceptible-Dose Dependent (SDD): 15–18 mmResistant (R): ≤14 mm
    Voriconazole (1 µg)
    • S: ≥17 mmSDD: 14–16 mmR: ≤13 mm
  (Breakpoints vary depending on drug and yeast species.)

Keynotes

• Disk diffusion is a simple, low-cost method suitable for routine laboratory use.
• Standardized medium (MHA-GMB) is critical for reproducibility.
• Always compare results with clinical breakpoints for specific species.
• Use control strains (Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258) for quality control.
• Though less precise than broth microdilution (gold standard), disk diffusion provides rapid and clinically useful guidance for antifungal therapy.

Further Readings

• CLSI (Clinical and Laboratory Standards Institute). Reference Method for Antifungal Disk Diffusion Susceptibility Testing of Yeasts; Approved Guideline—Second Edition (CLSI Document M44-A2). Wayne, PA: CLSI; 2009.
• EUCAST (European Committee on Antimicrobial Susceptibility Testing). EUCAST Definitive Document E.DEF 7.3.2: Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. 2022.
• Larone, D.H. Medically Important Fungi: A Guide to Identification. 6th Edition. ASM Press, 2018.
• Murray, P.R., Rosenthal, K.S., & Pfaller, M.A. Medical Microbiology. 9th Edition. Elsevier, 2020.
• Pfaller, M.A., & Diekema, D.J. (2012). “Progress in antifungal susceptibility testing of Candida spp. by use of Clinical and Laboratory Standards Institute broth microdilution methods, 2010–2012.” Journal of Clinical Microbiology, 50(9), 2846–2856.
• CLSI & EUCAST Joint Recommendations (2018). “Breakpoints and methods for antifungal susceptibility testing of yeasts.” Clinical Microbiology Reviews, 31(4): e00002-17.