Chapter 12 Culture-Enriched Mycobiome Protocol for Amphibian Feces
12.2 Purpose:
To selectively enrich viable fungi from amphibian feces prior to DNA extraction and ITS barcoding.
12.3 Overview
This protocol enriches fungal taxa from frog feces using a short-term liquid incubation to increase fungal biomass and reduce bacterial load before DNA extraction. The resulting DNA can be used for ITS-based community profiling or culture isolation.
12.4 Materials
| Item | Notes |
|---|---|
| Fresh or frozen frog fecal pellets | Collect sterilely, store at −80 °C or in DNA/RNA Shield |
| Sterile PBS or 0.1% Tween-80 | For sample homogenization |
| Enrichment Medium | PDA- or SDA-based liquid medium |
| Antibiotics | Chloramphenicol (50 µg/mL), Streptomycin (100 µg/mL), Penicillin G (100 U/mL) |
| Sterile 50 mL tubes or flasks | For enrichment culture |
| Orbital shaker (80-100 rpm) | Gentle aeration for oxygenation |
| Centrifuge (≥5,000 × g) | To pellet cells/mycelia |
| DNA extraction kit | ZymoBIOMICS or Qiagen PowerSoil Pro |
12.5 Enrichment Medium (Generalist)
Per 1 L of medium:
| Component | Amount | Purpose |
|---|---|---|
| PDA broth powder (commercial) | 24 g | Nutrient base |
| Distilled water | 1 L | Solvent |
| pH | 6.0 – 6.5 | Adjust if necessary |
| Optional: antibiotics | Chloramphenicol (50 µg/mL), Streptomycin (100 µg/mL), Penicillin G (100 U/mL) |
Autoclave the base medium, cool below 50 °C, and aseptically add antibiotics from sterile stock solutions.
12.6 Inoculation
1. Homogenize 1 fecal pellet in 1 mL sterile PBS or 0.1% Tween-80.
2. Transfer 1 mL of suspension into 9 mL enrichment medium (1:10 ratio).
3. Include a no-sample control tube.
12.7 Incubation
| Parameter | Setting | Notes |
|---|---|---|
| Temperature | 25-28 °C | Optimal for gut fungi |
| Agitation | 80-100 rpm | Gentle shaking |
| Duration | 48-72 h | Check daily for growth |
- Cloudy medium or small hyphal fragments → fungal growth.
- Strong odor or heavy turbidity → bacterial contamination.
12.8 Harvesting the Enrichment
1. Centrifuge at 5,000 × g for 10 min.
2. Discard supernatant and wash pellet once in sterile PBS.
3. Proceed to DNA extraction or plate aliquots for culture isolation.
12.9 DNA Extraction
Extract DNA using a stool/soil DNA kit with bead-beating. Include blanks and medium-only controls. Quantify DNA with Qubit or Nanodrop.
12.10 ITS Barcoding / Amplicon Sequencing
| Step | Details |
|---|---|
| Target region | ITS2 (preferred) - Novogene |
| Analysis | QIIME2 + DADA2 + UNITE database |
12.11 Optional Modifications
| Goal | Modification |
|---|---|
| Broader diversity | Run SDA and YM broths in parallel |
| Zygomycete enrichment | Add 0.1 % casein hydrolysate; incubate microaerobically |
| Slow growers | Dilute medium 1:2 with water; incubate 5 days |
| Mixed-kingdom profiling | Extract DNA for both ITS2 and 16S rRNA amplicons |