Apr 9, 2021

Case Study #5: Fungal Inoculation

Test Method Background and Study Timeline

ASTM International, formerly the American Society for Testing and Materials (ASTM), is an internationally recognized organization that develops and publishes product and testing standards. ASTM G21 is a qualitative test method designed to assess the ability of synthetic materials to resist fungal attack. The method is typically conducted over a 28-day period where treated materials are inoculated with a pooled suspension of fungal spores, incubated, then compared to untreated controls at intervals. The untreated controls serve as references for fungal resistance. A diverse array of fungal species are used in this method, so it is considered to be a good indicator of fungal resistance in a variety of environments.

Products Included In This Study


Test Substance and Microorganisms

test microorganism selected for the Nycote fungal resistance test

1. Aspergillus brasiliensis 9642

This fungi is a conidiophore, or a sexual spore generating aerobic fungus. A. brasiliensis, formerly listed as a strain of A. niger, is related to other Aspergillus species in that they produce spores highly resistant to chemical and environmental conditions. A. brasiliensis is commonly used as a benchmark fungus for antimicrobial fungicides and preservatives used in pharmaceutical and personal care products.

2. Talaromyces pinophilus 11797

This fungi is a faculative tonophile, meaning it can survive and grow in extreme conditions, such as arid or high pH environments. Growth in these conditions is uncharacteristic of other mold fungi. This species is known to utilize molecular components from several materials as a food source, namely cotton and paper products. T. pinophilus, formerly listed as a strain of Penicillium funiculosum, is associated with rotting fruit and seeds, and is commonly used in fungus resistance of articles.

3. Chaetomium globosum 6205

This fungi is a saprotrophic species that is normally found in soil, air, and plant debris. C. globosum thrives in cellulose rich areas, including seeds, textiles, straw, and sacking. Typically prevalent in homes with water damaged areas, C. globosum is one of the more prevalent fungi encountered in household environments. This prevalence makes C. globosum a commonly used model for fungus resistance testing.

4. Aureobasidium pullulans 15233

This fungi is a ubiquitous saprotroph, meaning it can be found in a multitude of environments and process nutrients by extracellular digestion of dead or decayed organic matter. A. pullulans has been known to cause pneumonitis (humidifier lung) over extended periods of exposure. This fungi is often employed in fungal resistance testing due to it’s ability to prevail in numerous environments and to metabolize organic matter.

5. Trichoderma virens 9645

This fungi is typically prevalent in soil and decayed wood. T. virens is often used in the agriculture industry as it is beneficial to crop production and plant metabolism because of its ability to produce antibiotics and parasitize other fungi. Due to its widely accepted use, it is a recommended microorganism for fungi resistance testing of adhesives and lumber.



Diagram of Nycote procedures to conduct fungal resistance testing

Summary of the Procedure

  • The test fungi are prepared individually, prior to the test, by growth on agar.
  • Suspensions of fungi are standardized by dilution in a buffered saline solution, and then pooled into a single suspension.
  • Test and control substances are aseptically placed on agar plates.
  • The pooled suspension of fungi is then applied to the surface of test and control substances using a sprayer. Sufficient spray is applied to wet the surface of the test substance.
  • Inoculated test and control substances are placed in a sealed, humid environment and incubated for the predetermined contact times.
  • At the conclusion of each contact time, visual assessments of each sample are made, noting the percentage of fungal growth on the inoculated surfaces of both test and control substances.
  • Based on the percentage of growth observed, a numerical score is assigned to each substance.



Criteria for Scientific Defensibility of an ASTM G21 Study

  1. The average number of viable spores of each fungal species shall be approximately 1 x 106 spores.
  2. Greater than 85% growth observed on inoculated untreated control substances after 14 days of incubation.



Study Modifications

No further modifications were made to the method for this study.

Study Notes

No additional notes were made for this study.

Study Photographs — Day 28

Negative controls of the study show no fungal growth

No fungal growth was observed on the inoculated mineral salt agar replicates used in this study.


Positive controls show heavy fungal growth observed on both inoculated filter paper

Heavy fungal growth was observed on both inoculated filter paper replicates.



Study Photographs — Day 28

The photograph below shows the three replicates (1-3, Left to Right ) of the Nycote 99 Ecoshield, sample on day 28 of incubation after inoculation with pooled fungal spores. No fungal growth was observed on the test surfaces of the three replicates.

Nycote 99 Ecoshield on day 28 of the fungal resistance Nycote case study


Study Photographs — Day 28

The photograph below shows the three replicates (1-3, Left to Right ) of the Nycote 7-11 sample on day 28 of incubation after inoculation with pooled fungal spores. No fungal growth was observed on the test surfaces of the three replicates.

Nycote 7-11 sample on day 28 of Nycote case study



Control Results:

Neutralizer: N/A
Growth Confirmation: Pure
Media Sterilty: Sterile


No calculations are made for this study.

Scoring Results:

Observations of growth on test substances is rated by the method according to the following system:

Graphs displaying evaluation criteria and results of Nycote case study

About Nycote

Nycote coating and resin products provide an excellent barrier from all degradative environments and can increase safety and part life-span with complete encapsulation technology. Please contact sales@nycote.com or call +1 (818) 764-9498.

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