Tempest in a Pipette: What a Typo in an Illinois Regulation Reveals About the Insanity of Cannabis Testing Regulations

Before medical and adult-use cannabis were legalized in Illinois—10 and 5 years ago, respectively—the state’s regulators established a colony-forming unit per gram (CFU/g) limit for Total Yeast and Mold (TYM) testing. The state’s adopted microbial testing rules follow the American Herbal Pharmacopeia’s Cannabis Inflorescence monograph (2013), which set fungal limits for orally consumed botanical products and other cannabis products, such as flower (see footnote in Table 1) at 10,000 CFU/g.

Table 1: American Herbal Pharmacopeia’s Cannabis Inflorescence monograph (2013) Table 9

However, due to a transcription error, this limit was mistakenly lowered tenfold to 1,000 CFU/g instead of 10,000 CFU/g (see Table 2). As a result, Illinois adopted some of the strictest TYM limits in the country, making it difficult for flower samples to meet the standard, likely leading to the use of remediation techniques, which can introduce secondary safety risks, such as endotoxins.

Table 2: Illinois - Title 8: Agriculture and animals Chapter I: Illinois Department of Agriculture subchapter v: licenses and regulations.

Normally, a minor typographical mistake of this nature—albeit with significant consequences—would have been swiftly and easily corrected. However, that did not occur. Instead, these erroneous regulations, which were off by an order of magnitude, went into effect and have since led to testing failures and remediation of hundreds of millions of dollars' worth of cannabis. We suspect that some growers have begun preemptively remediating their crops before testing, hoping to meet the TYM standard, despite the fact that this process often compromises the quality of their product. The continued failure to address this mistake is nothing short of perplexing. For those readers whose microbiology is a bit rusty, here is the significance of the Colony Forming Unit provided by Google:

A colony-forming unit (CFU) per unit (g or mL) is a measure of the number of viable cells in a sample, such as bacteria or fungi. CFUs can be used to estimate the number of microbial cells that are able to multiply in a controlled environment.

In the context of counting microorganisms, such as molds in cannabis flowers, CFU/g assays are short-term quantitative tests that measure a cell's potential to form a colony. The principle behind “plating” assays is that a single microbial cell will grow on a semi-solid nutrient medium to form a visible colony with distinct characteristics, such as size, color, and morphology. CFUs are determined by performing serial 10-fold dilutions of an aqueous sample, spreading aliquots from three successive dilutions onto triplicate agar plates, incubating them under specific conditions, and counting the colonies on each plate. One of the dilutions will typically yield an ideal colony count on the triplicate plates, usually between 100-200 CFUs. To calculate CFU/g, the weight of the test sample, the volume of medium used to desorb microorganisms, the sample volume for dilution, and the dilution that produced the average number of colonies on the plates are all factored in. Ideally, CFU/g limits should be a key step in identifying the presence of potentially harmful pathogenic microorganisms—like bacteria, yeasts, and molds—that could pose health risks to cannabis consumers, particularly those who are immunocompromised and prefer smoking their cannabis. If this method were flawless, it would be easy for everyone to agree on an acceptable yeast and or mold cell count per gram and move forward. Unfortunately, that’s not the case. This issue is far more complex than just one state's failure to transcribe regulations accurately.

Why use CFUs at all?
CFUs are employed in various applications, including:

• Clinical settings: CFU-Hill assays are used as a marker for disease.
• Probiotics: The required CFU count depends on the intended purpose. Most studies suggest that 1-10 billion CFUs are sufficient for general health, but higher doses may be needed for certain conditions.
• Microbial Cannabis Testing: CFUs have been adopted as acceptance criteria for “clean” cannabis

Historically, CFU/g is determined by using a single medium, incubated at a specific temperature for a set period. Unfortunately, this method doesn't account for the fact that not all microorganisms of a particular type will grow on that medium under those conditions. In fact, there is no single medium capable of cultivating all the microbes in a cannabis flower sample, whether it’s total aerobic bacteria, total yeast and mold, Bile-Tolerant Gram-Negative bacteria (BTGN—around 60 genera), or pathogenic strains of E. coli.

I am not the first, nor the only, microbiologist to raise this concern. Numerous peer-reviewed articles have highlighted the limited ability of most microorganisms to grow on any given agar medium. These studies often estimate the percentage of microorganisms that can grow on a "universal" medium for specific sample types, such as human oral microbes, within specific incubation conditions. Yet these conditions only allow for the growth of a fraction of the microorganisms present in the sample.

Incubation temperature also isn’t universally applicable. Some species struggle at the prescribed temperature—either growing too quickly, too slowly or not at all—depending on whether it’s too hot or too cold for their optimal growth.

In addition, there are behaviors that plating regulations ignore entirely. For example, a colony may form from more than one cell. Certain molds and bacteria tend to clump together, forming a single colony that results in an undercount. Other microbes can only grow in the presence of specific metabolites secreted by neighboring species. If these nutrients aren’t available, the microbe won’t form a colony, even if it is present in the sample.

The most ironic aspect, given all these inconsistencies and the potential for errors, is that some states mandate plating as the only allowable method for enumerating certain microorganisms like TYM. However, an AOAC-approved PTM using qPCR is available for TYM enumeration, which provides a more comprehensive approach by detecting all cells and removing DNA from dead cells before quantifying the DNA of live cells. Unfortunately, some states still prohibit this method, despite its AOAC approval. To me, this represents a far more significant mistake than a simple transcription error—it’s a failure to embrace the most accurate technology available, ultimately undermining the goal of testing: to provide the most reliable results.

About the author

Sherman Hom, Ph.D., Director of Regulatory Affairs or Medicinal Genomics Corp.

Sherman Hom, Ph.D. is the Director of Regulatory Affairs for Medicinal Genomics Corp. Prior to joining MGC, Dr. Hom was a Research Scientist 1 in New Jersey’s Division of Public Health and Environmental Laboratories (PHEL), where, as a Project Manager, he coordinated all PHEL’s pre-analytical activities supporting its SARS-CoV-2 testing. Prior to that, he was the Project Manager for creating the state’s Cannabis Testing Lab and subsequently for validating Microbial Testing of Cannabis products. He has also been a professor of microbiology, a laboratory manager, a senior research scientist, a writer, and an inventor. He was a Postdoctoral Fellow in Molecular Genetics at The Johns Hopkins University, holds a Ph.D. in Microbiology from the University of California at Davis, and a B.A. in Biology from UC San Diego.