Soil Extraction Plating for the Detection of Aspergillus terreus from Environmental Samples

Soil Extraction Plating for the Detection of Aspergillus terreus from Environmental Samples

Authors and Affiliations

Jan Schobert¹, Paul Illmer², Roya Vahedi-Shahandashti¹*, Cornelia Lass-Flörl¹*

¹ ISHAM Working Group of Aspergillus terreus, ECMM Excellence Center of Mycology, Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Austria
² Institute of Microbiology, University of Innsbruck, Austria

Abstract

This protocol describes a selective soil extraction plating method for the detection and isolation of Aspergillus terreus from environmental soil samples. Sieved soil is suspended in a saline–surfactant spore buffer (0.9% NaCl, 0.1% Tween 20) to release fungal propagules, followed by plating of the supernatant onto Sabouraud agar supplemented with chloramphenicol, with or without amphotericin B. Differential incubation at 37 °C and 50 °C enhances selectivity for thermotolerant A. terreus. Suspected colonies are subcultured on oatmeal agar and identified by microscopy and sequencing.

Background

Aspergillus terreus is a clinically relevant filamentous fungus and an important environmental mold with intrinsic reduced susceptibility to amphotericin B. Soil is considered one of its main ecological reservoirs. Recovery of A. terreus from environmental samples is complicated by the heterogeneous distribution of fungal propagules and by the presence of abundant competing microorganisms.

The present protocol is based on soil extraction plating and selective cultivation. Soil particles are first homogenized and fungal propagules are released into suspension using a saline-Tween buffer. After a short sedimentation step, aliquots of the supernatant are inoculated onto selective media. Chloramphenicol suppresses bacterial growth, amphotericin B provides additional selectivity, and incubation at 50 °C exploits the thermotolerance of A. terreus. Colonies suspected to represent A. terreus are purified on oatmeal agar and further identified by morphology and sequencing.

Materials and Reagents

Table 1. Sabouraud Agar Composition (per 1 L)

Table 2. Oatmeal Agar Composition (per 500 mL)

Media Preparation

Sabouraud Agar (SAB)

30 g Sabouraud 2% dextrose bouillon and 20 g agar are dissolved in 1000 mL deionized water in a Schott bottle. The medium is autoclaved and cooled to approximately 45 °C before supplementation (Table 1).

Chloramphenicol Stock Solution

Prepare a stock solution by dissolving 0.5 g chloramphenicol in 10 mL ethanol (50 mg/mL). Add 1 mL of this stock per 1 L medium to obtain a final concentration of 50 mg/L.

Amphotericin B Supplementation

Using a stock solution of 3200 mg/L amphotericin B, add 156.5 µL per 1 L of medium already containing chloramphenicol to achieve the desired final concentration.

Plate Pouring

Pour approximately 20 mL of medium per Petri dish under sterile conditions.

Oatmeal Agar

Grind 15 g oatmeal using a mortar and pestle. Add to 7.5 g agar in a flask and mix with 500 mL deionized water. Autoclave, cool to ~45 °C, and pour into Petri dishes (~20 mL per plate) (Table 2).

Procedure

1. Soil Sampling

Using a disinfected shovel, collect approximately 10 g of soil from the top 5–10 cm and transfer into a sterile 50 mL Falcon tube.

2. Sieving

Remove plant material, stones, and debris by passing soil through a sterile 2 mm mesh sieve.

3. Subsampling

Weigh two independent 2 g portions of soil into sterile 15 mL Falcon tubes.

4. Spore Extraction

Add 5 mL of sterile spore buffer (0.9% NaCl + 0.1% Tween 20) to each tube. Perform all steps under aseptic conditions.

5. Homogenization

Vortex samples for 1 minute to release fungal propagules.

6. Sedimentation

Allow tubes to stand for 1 minute to sediment larger particles.

7. Plating

Plate 100 µL of the supernatant from each replicate onto:

• 3 × SAB + CAP plates

• 1 × SAB + CAP + AmB plate

Total: 4 plates per replicate.

8. Incubation

Incubate plates for 48–72 hours:

• All plates at 37 °C

• One SAB + CAP plate at 50 °C

9. Screening

Examine plates macroscopically for colonies consistent with Aspergillus terreus.

10. Subculture and Identification

Transfer suspected colonies onto oatmeal agar. Confirm identity by:

• Morphology (macro- and microscopic)

• Molecular methods (sequencing)

Notes

• Replicate subsampling improves detection due to heterogeneous distribution in soil.

• The Tween-containing buffer enhances spore release.

• Short sedimentation reduces debris carryover.

• Chloramphenicol suppresses bacterial growth.

• Amphotericin B increases selectivity.

• Incubation at 50 °C is critical for selecting thermotolerant A. terreus.

Applications

• Environmental detection of Aspergillus terreus

• Isolation of environmental strains

• Epidemiological and ecological studies

• Downstream susceptibility testing and sequencing

Acknowledgments

Adapted from: 
Schobert J., Illmer P., Vahedi-Shahandashti R., Lass-Flörl C.

Conflict of Interest

The authors declare no conflict of interest.