Cultivation protocols for fungi?

Raghad Mouhamad

Advanced Protocol for Cultivation and Enumeration of Fungal Cells

1. Sample Collection and Preparation

Sample Source: Clinical, environmental, or culture stock samples.
Transport Medium: Use Amies medium or Sabouraud Dextrose Broth (SDB) to maintain fungal viability during transport.

2. Culture Media

Sabouraud Dextrose Agar (SDA): Standard medium for general fungal cultivation; contains 4% dextrose and 1% peptone, pH adjusted to 5.6.
Chromogenic Agar (e.g., CHROMagar Candida): Selective and differential medium for Candida species identification; C. auris colonies typically appear blue with a blue halo.
Potato Dextrose Agar (PDA): Commonly used for molds like Penicillium and Botrytis.
Sabouraud Salt Dulcitol (SSD) Broth: Recommended for selective enrichment of Candida auris.

3. Cultivation Steps

A. Candida auris Cultivation (CDC Recommended)

Enrichment in SSD Broth: Add 2 mL of SSD Broth into a 14 mL culture tube. Inoculate with 100 µL of sample. Loosely cap to allow gas exchange. Incubate at 40°C with shaking at 250 rpm for up to 5 days. Monitor daily for turbidity indicating growth.

Isolation on Chromogenic Agar: After 48 hours or upon visible growth, streak 10 µL from SSD Broth onto CHROMagar Candida plates. Incubate at 37°C for 24-48 hours. Identify C. auris colonies by their characteristic blue color with halo.

Colony Counting: Perform serial dilutions of the enriched culture. Plate 100 µL of appropriate dilutions on SDA or chromogenic agar. Incubate and count colony-forming units (CFU) to estimate fungal load.

Confirmation: Use molecular methods (PCR) or MALDI-TOF MS for species confirmation.

B. Cultivation of Candida albicans, Penicillium chrysogenum, and Botrytis cinerea

For yeasts (Candida albicans): Plate on SDA. Incubate at 30-37°C for 24-48 hours.
For molds (Penicillium, Botrytis): Plate on PDA. Incubate at 25°C for 5-7 days until colonies develop.
Colony Counting: Perform serial dilutions of fungal suspensions. Plate 100 µL aliquots on appropriate agar. Count CFUs after incubation.

4. Materials and Equipment

Culture tubes (14 mL) with breathable caps
Sabouraud Salt Dulcitol (SSD) Broth
Sabouraud Dextrose Agar (SDA) plates
Chromogenic agar plates (e.g., CHROMagar Candida)
Potato Dextrose Agar (PDA) plates
Sterile inoculating loops or pipettes (10 µL)
Incubators capable of 25°C, 30-37°C, and 40°C
Shaker incubator (for broth cultures)
Biosafety cabinet (BSL-2 recommended)
PCR or MALDI-TOF equipment for confirmation (optional)

5. Important Notes

Work in a biosafety cabinet (BSL-2) to prevent contamination and ensure safety.
Candida auris can be slow-growing; incubation up to 7 days may be necessary before ruling out growth.
Chromogenic media greatly facilitate differentiation between Candida species.
Combine culture-based counting with molecular methods for accurate identification.

6. References and Protocol Sources

CDC: Candida auris Isolation and Identification Protocol https://www.cdc.gov/candida-auris/hcp/laboratories/isolation-procedure.html
APHL: Candida auris Culture Method and Validation https://www.aphl.org/courses/Documents/2023/588-607-23%20slides.pdf
CDC: Detection of Candida auris Colonization https://www.cdc.gov/candida-auris/hcp/laboratories/detection-colonization.html
NCBI Bookshelf: Candida auris Overview https://www.ncbi.nlm.nih.gov/books/NBK563297/

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