No Gold Standard: The Problems with Plating Total Yeast and Mold

As part of an AOAC Emergency response validation (ERV) in the State of Michigan, the Medicinal Genomics team investigated the impact media selection has on using plating to test total yeast and mold on cannabis, using three different media:

  • Potato Dextrose Agar (PDA)
  • PDA with chloramphenicol 
  • Dichloran Rose Bengal with chloramphenicol (DRBC)

The study found that media containing antibiotics significantly impacts the diversity of microbes that grow, and as a result, the CFU values vary by orders of magnitude. Antibiotic selections are often utilized to reduce background bacteria but many of these antibiotics (chloramphenicol) inhibit the growth of the most pathogenic fungi found on cannabis (Fusarium, Pythium, and Aspergillus). These results should be considered as state regulatory agencies set standards for cannabis microbial testing.

Lack of Diversity 

The team isolated colonies from each media type and whole genome sequenced to identify the types of microbes that grow in each media selection. PDA with chloramphenicol demonstrated the highest diversity and the highest concordance with whole genome sequencing. DRBC plating demonstrated the lowest diversity. The team expected this result because DRBC contains three different selection agents (Dichloran, Rose Bengal, and Chloramphenicol). While this limits bacterial contamination it also limits yeast and mold growth.

The figure below uses Simpson’s diversity index to display the diversity of each media. Simpson’s diversity index takes into account the number of species present, as well as the relative abundance of each species and assigns a value between 0 and 1. A diversity index of 1 represents infinite diversity where 0 reflects no diversity.

To further demonstrate this effect, the team tested colonies from each plate type using Total Yeast and Mold and Total Aerobic Count qPCR assays to correlate these CFUs with fungi and bacterial species. 

qPCR testing identified that DRBC undercounts fungi while PDA overcounts CFUs due to bacterial growth without selection.

Finally, the team ordered organisms from ATCC and plated as pure monocultures on the three different plating mediums to confirm the differential growth on each media. Fewer colonies are consistently found on DRBC, which is consistent with the other findings.

Other Issues with Plating 

The study also highlighted two significant limitations of culture plating methods. 

First, plating has a very limited dynamic range. Since it is difficult to count colonies when more than 100 colonies are present on a plate, lab techs typically perform multiple dilutions to understand the full range of CFU counts one may encounter with a test that is attempting to quantitate 10,000 CFUs/gram. This results in multiplying diluted CFUs by 10, 100 and even 1,000-fold to back estimate the total CFU count. In this scenario, a single colony can swing the CFU count from passing to failing (9 colonies x 1,000 fold dilution vs 10 colonies at 1,000 fold dilution). Quantitative PCR has a linear dynamic range over 5-6 orders of magnitude and no such multiplication is required. Thus, qPCR provides a more accurate itemization of actual CFUs counts.

Second, all plating media studied, even DRBC, which uses 3 different forms of selection, had bacterial contamination and each level of selection reduced fungal CFU counts. Conversely, in-vitro inclusion and exclusion testing with ITS3 qPCR on ATCC sourced organisms demonstrated over 96% inclusion (50 yeast and mold) and zero bacterial cross-reactivity (30 bacteria).

Regulatory Implications

Despite the fact that the FDA references both PDA and DRBC in its Bacteriological Analytical Manual (FDA-BAM), some states exclusively consider DRBC for ease of colony visualization. This study showed that DRBC selection reduces bacterial growth more than the other media types tested, but it does so at the expense of fungi, limiting fungal growth 5-fold in the process. This has important implications for chloramphenicol-sensitive cannabis endophytes like Aspergillus, Pythium, and Fusarium

Based on these results, states who exclusively consider DRBC should also implement species-specific testing for pathogenic organisms to complement a partial yeast and mold test offered from a single selection-based media. PCR-based techniques can capture more organisms than DRBC alone because the qPCR uses no selection.

Read the PDF version of the preprint study “Whole genome sequencing of colonies derived from cannabis flowers and the impact of media selection on benchmarking total yeast and mold detection tools

To review additional data the Medicinal Genomics team has generated to compare different plating types, visit the “Benchmarking qPCR to Culture: PDA vs DRBC” page.