The Benefits and Pitfalls of Total Yeast and Mold Counts in Cannabis Labs

The standard for yeast and mold testing

For years, the benefits of total yeast and mold (TYM) testing have been widely debated in cannabis industry. There is evidence that quantitative polymerase chain reaction (qPCR) is the better option since all DNA is amplified in the extraction process, which will avoid missing certain yeast and molds that do not culture well in plating methods. Alternatively, other evidence indicates that false negatives and low quantitation/under estimation occur with qPCR tests. In which case, traditional plating methods may be the best choice.

So, regulators and laboratories are being forced to decide which method produces the most accurate results. However, the decisions tend to be made hastily and without extensive research to support the decision.

Our laboratory, Modern Canna, recently began looking at total yeast and mold methods closer, to better assess what the industry standard for this specific type of testing should be.

Throughout the investigation, we found several pieces of evidence that suggest, while there are benefits to total yeast mold testing in cannabis flower, there are also several pitfalls that may be leading labs to unintentionally report inaccurate results.

Which result is the correct answer?

One of the first questions that we hoped to answer was: which method is the most accurate and provides the most reproducible data? We began trying to find a certified reference material that could be used to determine which method matched the closest and was reproducible. During the process, we discovered that one of the only available reference materials for total mold in cannabis had two drastically different values (Part Number: FM-729, Lot Number: 220210rev1), one for each of the plating methods used. The certified activity is 152,000 colony forming units per gram (CFU/g) when Petrifilm Rapid Yeast/Mold Plates (P-RYM) are used, and 423,000 CFU/g when Sabouraud Dextrose (SabDex) Agar is the growth medium.

This fact alone poses an interesting question for the industry: which result is the correct answer? Or are both answers considered correct? In seeing this, the laboratory concluded that the next step to solving the mystery surrounding TYM was to run this analysis on cannabis samples using multiple different growth mediums and via qPCR to see how the results lined up with one another.

Comparison study of several growth mediums and qPCR analysis on cannabis samples

An initial study was conducted by analyzing ten cannabis flower samples for TYM using plating methods on various growth mediums (listed below) and qPCR technology.

• Compact Dry (CD)
• Petrifilm Rapid Yeast and Mold (P-RYM)
• Petrifilm Non-Rapid Yeast and Mold (P-NRYM)
• Potato Dextrose Agar (PDA)
• Potato Dextrose Agar with Chlortetracycline (PDAC)
• Dichloran Rose Bengal Chlorampenicol (DRBC)
• Sabouraud Dextrose (SabDex)

The results obtained from this experiment were inconclusive due to the large variations across the methods and the lack of reproducibility in results.

Therefore, the laboratory opted to have more control over the experiment by running two different studies. One that involved using a single sample and analyzing it five times at three different dilutions, and the second that looked at six different samples, processed in triplicate, (three of which were treated with radiation) at the same dilutions. This allowed the reproducibility of the methods to be measured when compared to itself and other methods. The experiment also involved documenting and counting colony growth at several time intervals (48, 72, 96, 120, and 144 hours), to determine incubation times for each type of plate used. True incubation times were established as the point when colonies no longer started appearing and instead just grew on the plates.

The conclusions obtained from the experiment suggest the following incubation times for the various growth mediums used:

The study revealed that while TYM numbers sometimes align from method to method, it is more common that the results will vary drastically, sometimes by more than 50%. This is one of the main pitfalls that cannabis methodologies face. Cannabis is a complex organic material that changes from sample to sample. Thus, the matrix plays a major role in the results being obtained. The initial studies conducted verified that more method development needs to be done to assess what assumptions can be made during the analysis regarding the matrices.

Moving forward – is there a need for a change?

While measuring the amount of yeast and mold is important for the cannabis industry, more emphasis needs to be placed on standardized methods for this analysis and a singular procedure should be selected. The variance seen from method to method indicates that while laboratories may not be intentionally passing failing products, this may occur depending on what technique they are using and how they are calculating their quantified results. Pitfalls, such as this one, can be avoided through more rigorous validation processes.

Throughout the investigation, the laboratory was able to confirm the previously determined benefits and pitfalls of various microbial testing techniques. In traditional plating methods, there is an indication that there is not enough selectivity and, as a result, bacterial colonies may grow on plates intended to detect TYM. To test this theory, we are in the process of having the colonies sequenced to determine how much bacterial growth is occurring. Additionally, our findings revealed that the typical dilutions performed in plating methods may cause data to be skewed. In most instances, calculated totals at each dilution were not duplicate numbers. Therefore, if an average of the three dilutions run is taken to determine the quantitative result, the true value may be increased or decreased depending on the skew seen.

Conversely, there are some concerns that qPCR may lead to false-negative results if there is too much contamination or if certain microbes are present. We are in the process of confirming if there is a threshold at which the contaminants present become too concentrated, thus skewing the qPCR data due to DNA saturation.

Additionally, it was determined that incubation time plays a pivotal role in the results obtained. During the experiment, it was noted that most of the agars used did not show complete colony growth in cannabis until at least 72 hours, even though some of the recommended incubation times are 48 hours. Therefore, if labs are processing samples based on recommended incubation times, TYM counts may be severely underestimated. This is another indication that the methodology used for TYM may be lacking and that further studies need to be conducted.

It is our lab’s suggestion that the industry consider moving away from including TYM as part of the standard cannabis testing panels and instead, test for additional yeast or mold species that commonly grow on cannabis and are dangerous to humans. Some of these microbes include Aspergillus species, Botrytis (bud rot), powdery mildew, Fusarium species (root rot), and other Penicillium species. One of the biggest pitfalls of TYM testing is that even if a sample contains less yeast and mold than the regulatory limit, there is still a chance that the microbes present could be extremely dangerous to humans. However, if speciation testing is not being performed to ensure that those microbes are not present, TYM analysis does not truly protect the consumer. It is important to note that some of these microbes do not culture well on some of the commonly used agars. As a result, labs may need to use molecular techniques, such as qPCR, to properly identify these contaminants.

There are clearly benefits and pitfalls to both plating and qPCR methodology. The quantitation of microbes can be extremely difficult based on the sample matrix and the homogeneity of the aliquot being used. While TYM analysis may be able to pinpoint when a grower should be concerned about the cleanliness of their processes, it may not be the best analysis to determine if products are safe for human consumption. The food and pharmaceutical industries have extensively researched how analyses such as TYM work for their products and ultimately, the cannabis industry needs to apply that same level of scrutiny to TYM in cannabis products.