Tag Archives: TYMC

March 1, 2018

Colorado vs. California: Two Different Approaches to Mold Testing in Cannabis

By Swetha Kaul, PhD

Across the country, there is a patchwork of regulatory requirements that vary from state to state. Regulations focus on limiting microbial impurities (such as mold) present in cannabis in order for consumers to receive a safe product. When cultivators in Colorado and Nevada submit their cannabis product to laboratories for testing, they are striving to meet total yeast and mold count (TYMC) requirements.In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

TYMC refers to the number of colony forming units present per gram (CFU/g) of cannabis material tested. CFU is a method of quantifying and reporting the amount of live yeast or mold present in the cannabis material being tested. This number is determined by plating the sample, which involves spreading the sample evenly in a container like a petri dish, followed by an incubation period, which provides the ideal conditions for yeast and mold to grow and multiply. If the yeast and mold cells are efficiently distributed on a plate, it is assumed that each live cell will give rise to a single colony. Each colony produces a visible spot on the plate and this represents a single CFU. Counting the numbers of CFU gives an accurate estimate on the number of viable cells in the sample.

The plate count methodology for TYMC is standardized and widely accepted in a variety of industries including the food, cosmetic and pharmaceutical industries. The FDA has published guidelines that specify limits on total yeast and mold counts ranging from 10 to 100,000 CFU/g. In cannabis testing, a TYMC count of 10,000 is commonly used. TYMC is also approved by the AOAC for testing a variety of products, such as food and cosmetics, for yeast and mold. It is a fairly easy technique to perform requiring minimal training, and the overall cost tends to be relatively low. It can be utilized to differentiate between dead and live cells, since only viable living cells produce colonies.

Petri dish containing the fungus Aspergillus flavus.
Photo courtesy of USDA ARS & Peggy Greb.

There is a 24 to 48-hour incubation period associated with TYMC and this impedes speed of testing. Depending on the microbial levels in a sample, additional dilution of a cannabis sample being tested may be required in order to count the cells accurately. TYMC is not species-specific, allowing this method to cover a broad range of yeast and molds, including those that are not considered harmful. Studies conducted on cannabis products have identified several harmful species of yeast and mold, including Cryptococcus, Mucor, Aspergillus, Penicillium and Botrytis Cinerea. Non-pathogenic molds have also been shown to be a source of allergic hypersensitivity reactions.The ability of TYMC to detect only viable living cells from such a broad range of yeast and mold species may be considered an advantage in the newly emerging cannabis industry.

After California voted to legalize recreational marijuana, state regulatory agencies began exploring different cannabis testing methods to implement in order to ensure clean cannabis for the large influx of consumers.

Unlike Colorado, California is considering a different route and the recently released emergency regulations require testing for specific species of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus). While Aspergillus can also be cultured and plated, it is difficult to differentiate morphological characteristics of each species on a plate and the risk of misidentification is high. Therefore, positive identification would require the use of DNA-based methods such as polymerase chain reaction testing, also known as PCR. PCR is a molecular biology technique that can detect species-specific strains of mold that are considered harmful through the amplification and analysis of DNA sequences present in cannabis. The standard PCR testing method can be divided into four steps:

The double stranded DNA in the cannabis sample is denatured by heat. This refers to splitting the double strand into single strands.
Primers, which are short single-stranded DNA sequences, are added to align with the corresponding section of the DNA. These primers can be directly or indirectly labeled with fluorescence.
DNA polymerase is introduced to extend the sequence, which results in two copies of the original double stranded DNA. DNA polymerases are enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA.
Once the double stranded DNA is created, the intensity of the resulting fluorescence signal can uncover the presence of specific species of harmful Aspergillus mold, such as fumigatus.

These steps can be repeated several times to amplify a very small amount of DNA in a sample. The primers will only bind to the corresponding sequence of DNA that matches that primer and this allows PCR to be very specific.

PCR testing is used in a wide variety of applications
Photo courtesy of USDA ARS & Peggy Greb.

PCR is a very sensitive and selective method with many applications. However, the instrumentation utilized can be very expensive, which would increase the overall cost of a compliance test. The high sensitivity of the method for the target DNA means that there are possibilities for a false positive. This has implications in the cannabis industry where samples that test positive for yeast and mold may need to go through a remediation process to kill the microbial impurities. These remediated samples may still fail a PCR-based microbial test due to the presence of the DNA. Another issue with the high selectivity of this method is that other species of potentially harmful yeast and mold would not even be detected. PCR is a technique that requires skill and training to perform and this, in turn, adds to the high overall cost of the test.

Both TYMC and PCR have associated advantages and disadvantages and it is important to take into account the cost, speed, selectivity, and sensitivity of each method. The differences between the two methodologies would lead to a large disparity in testing standards amongst labs in different states. In a nascent industry, it is prudent for state regulators to reference specific testing methodologies so that an industry standard can be established.

February 20, 2018

An Insider’s View: How Labs Conduct Cannabis Mold Testing

By Swetha Kaul, PhD

2 Comments

As both recreational and medical cannabis legalization continues to progress across the country, each state is tasked with developing regulatory requirements to ensure that customers and patients receive clean cannabis for consumption. This requires cannabis to undergo laboratory testing that analyzes the presence of microbial impurities including yeast and mold.

Some states, such as Colorado, Nevada, Maine, Illinois and Massachusetts use total yeast and mold count testing (TYMC) and set a maximum yeast and mold count threshold that cultivators must fall below. Other states, such as California, require the detection of species-specific strains of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus), which requires analyzing the DNA of a cannabis sample through polymerase chain reaction testing, also known as PCR.

Differences in state regulations can lead to different microbiological techniques implemented for testing.Before diving in further, it is important to understand the scientific approach. Laboratory testing requirements for cannabis can be separated into two categories: analytical chemistry methods and microbiological methods.

Analytical chemistry is the science of qualitatively and quantitatively determining the chemical components of a substance, and usually consists of some kind of separation followed by detection. Analytical methods are used to uncover the potency of cannabis, analyze the terpene profile and to detect the presence of pesticides, chemical residues, residuals solvents, heavy metals and mycotoxins. Analytical testing methods are performed first before proceeding to microbiological methods.

Microbiological methods dive deeper into cannabis at a cellular level to uncover microbial impurities such as yeast, mold and bacteria. The techniques utilized in microbiological methods are very different from traditional analytical chemistry methods in both the way they are performed and target of the analysis. Differences in state regulations can lead to different microbiological techniques implemented for testing. There are a variety of cell and molecular biology techniques that can be used for detecting microbial impurities, but most can be separated into two categories:

Methods to determine total microbial cell numbers, which typically utilizes cell culture, which involves growing cells in favorable conditions and plating, spreading the sample evenly in a container like a petri dish. The total yeast and mold count (TYMC) test follows this method.
Molecular methods intended to detect specific species of mold, such as harmful aspergillus mold strains, which typically involves testing for the presence of unique DNA sequences such as Polymerase Chain Reaction (PCR).

Among states that have legalized some form of cannabis use and put forth regulations, there appears to be a broad consensus that the laboratories should test for potency (cannabinoids concentration), pesticides (or chemical residues) and residual solvents at a minimum. On the other hand, microbial testing requirements, particularly for mold, appear to vary greatly from state to state. Oregon requires random testing for mold and mildew without any details on test type. In Colorado, Nevada, Maine, Illinois and Massachusetts, regulations explicitly state the use of TYMC for the detection of mold. In California, the recently released emergency regulations require testing for specific species of Aspergillus mold (A. fumigatus, A. flavus, A. niger and A. terreus), which are difficult to differentiate on a plate and would require a DNA-based approach. Since there are differences in costs associated and data produced by these methods, this issue will impact product costs for cultivators, which will affect cannabis prices for consumers.

January 30, 2018

10 Treatment Methods to Reduce Mold in Cannabis

By Ketch DeGabrielle

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As the operations manager at Los Sueños Farms, the largest outdoor cannabis farm in the country, I was tasked with the challenge of finding a yeast and mold remediation treatment method that would ensure safe and healthy cannabis for all of our customers while complying with stringent regulations.

While outdoor cannabis is not inherently moldy, outdoor farms are vulnerable to changing weather conditions. Wind transports spores, which can cause mold. Each spore is a colony forming unit if plated at a lab, even if not germinated in the final product. In other words, perfectly good cannabis can easily fail microbial testing with the presence of benign spores.

Fun Fact: one square centimeter of mold can produce over 2,065,000,000 spores .

If all of those landed on cannabis it would be enough to cause over 450 pounds of cannabis to fail testing, even if those spores remained ungerminated.

Photo credit: Steep Hill- a petri dish of mold growth from tested cannabis

It should also be known that almost every food item purchased in a store goes through some type of remediation method to be considered safe for sale. Cannabis is finally becoming a legitimized industry and we will see regulations that make cannabis production look more like food production each year.

Regulations in Colorado (as well as Nevada and Canada) require cannabis to have a total yeast and mold count (TYMC) of ≤ 10,000 colony forming units per gram. We needed a TYMC treatment method that was safe, reliable, efficient and suitable for a large-scale operation. Our main problem was the presence of fungal spores, not living, growing mold.

Below is a short list of the pros and cons of each treatment method I compiled after two years of research:

Autoclave: This is the same technology used to sterilize tattoo needles and medical equipment. Autoclave uses heat and pressure to kill living things. While extremely effective, readily available and fiscally reasonable, this method is time-consuming and cannot treat large batches. It also utilizes moisture, which increases mold risk. The final product may experience decarboxylation and a change in color, taste and smell.

Dry Heat: Placing cannabis in dry heat is a very inexpensive method that is effective at reducing mold and yeast. However, it totally ruins product unless you plan to extract it.

Gamma Ray Radiation: By applying gamma ray radiation, microbial growth is reduced in plants without affecting potency. This is a very effective, fast and scalable method that doesn’t cause terpene loss or decarboxylation. However, it uses ionizing radiation that can create new chemical compounds not present before, some of which can be cancer-causing. The Department of Homeland Security will never allow U.S. cannabis farmers to use this method, as it relies on a radioactive isotope to create the gamma rays.

Gas Treatment: (Ozone, Propylene Oxide, Ethylene Oxide, Sulfur Dioxide) Treatment with gas is inexpensive, readily available and treats the entire product. Gas treatment is time consuming and must be handled carefully, as all of these gases are toxic to humans. Ozone is challenging to scale while PPO, EO and SO2 are very scalable. Gases require special facilities to apply and it’s important to note that gases such as PPO and EO are carcinogenic. These methods introduce chemicals to cannabis and can affect the end product by reducing terpenes, aroma and flavor.

Hydrogen Peroxide: Spraying cannabis plants with a hydrogen peroxide mixture can reduce yeast and mold. However, moisture is increased, which can cause otherwise benign spores to germinate. This method only treats the surface level of the plant and is not an effective remediation treatment. It also causes extreme oxidation, burning the cannabis and removing terpenes.

Microwave: This method is readily available for small-scale use and is non-chemical based and non-ionizing. However, it causes uneven heating, burning product, which is damaging to terpenes and greatly reduces quality. This method can also result in a loss of moisture. Microwave treatment is difficult to scale and is not optimal for large cultivators.

Radio Frequency: This method is organic, non-toxic, non-ionizing and non-chemical based. It is also scalable and effective; treatment time is very fast and it treats the entire product at once. There is no decarboxylation or potency loss with radio frequency treatment. Minimal moisture loss and terpene loss may result. This method has been proven by a decade of use in the food industry and will probably become the standard in large-scale treatment facilities.

Steam Treatment: Water vapor treatment is effective in other industries, scalable, organic and readily available. This method wets cannabis, introducing further mold risk, and only treats the product surface. It also uses heat, which can cause decarboxylation, and takes a long time to implement. This is not an effective method to reduce TYMC in cannabis, even though it works very well for other agricultural products

extraction equipment — Extraction can be an effective form of remediating contaminated cannabis

Extraction: Using supercritical gas such as butane, heptane, carbon dioxide or hexane in the cannabis extraction process is the only method of remediation approved by the Colorado Marijuana Enforcement Division and is guaranteed to kill almost everything. It’s also readily available and easy to access. However, this time-consuming method will change your final product into a concentrate instead of flower and usually constitutes a high profit loss.

UV Light: This is an inexpensive and readily available method that is limited in efficacy. UV light is only effective on certain organisms and does not work well for killing mold spores. It also only kills what the light is touching, unless ozone is captured from photolysis of oxygen near the UV lamp. It is time consuming and very difficult to scale.

After exhaustively testing and researching all treatment methods, we settled on radio frequency treatment as the best option. APEX, a radio frequency treatment machine created by Ziel, allowed us to treat 100 pounds of cannabis in an hour – a critical factor when harvesting 36,000 plants during the October harvest.

January 17, 2018

10 Ways to Reduce Mold in Your Grow

By Ketch DeGabrielle

3 Comments

Regardless of whether your grow is indoor or in a greenhouse, mold is a factor that all cultivators must consider.

After weeks of careful tending, pruning and watering to encourage a strong harvest, all cultivators are looking to sell their crop for the highest market value. A high mold presence, measured through a total yeast and mold count (TYMC), can cause a change of plans by decreasing crop value. But it doesn’t have to.

There are simple steps that any cultivator can take that will greatly eliminate the risk of mold in a grow. Below are some basic best practices to incorporate into your operation to reduce contaminants and mold growth:

Isolate dirty tasks. If you are cleaning pots, filling pots or scrubbing trimming scissors, keep these and other dirty tasks away from grow and process areas. Dirty tasks can contaminate the grow area and encourage mold growth. Set up a “dirty room” that does not share heating, ventilation and air conditioning with clean areas.
Compartmentalize the grow space. Mold can launch spores at speeds up to 55 miles per hour up to eight feet away without any air current. For this reason, if mold growth begins, it can become a huge problem very quickly. Isolate or remove a problem as soon as it is discovered- better to toss a plant than to risk your crop.
No drinks or food allowed. Any drinks or food, with the exception of water, are completely off limits in a grow space. If one of your employees drops a soda on the ground, the sugars in the soda provide food for mold and yeast to grow. You’d be surprised how much damage a capful of soda or the crust of a sandwich can do.
Empty all trash daily. Limiting contaminants in turn limits the potential for issues. This is an easy way to keep your grow clean and sterile.
Axe the brooms. While a broom may seem like the perfect way to clean the floor, it is one of the fastest ways to stir up dirt, dust, spores and contaminants, and spread them everywhere. Replace your brooms with hepa filter backpack vacuums, but be sure that they are always emptied outside at the end of the work day.
No standing water or high humidity. Mold needs water to grow, therefore standing water or high humidity levels gives mold the sustenance to sporulate. Pests also proliferate with water. Remove standing water and keep the humidity level as low as possible without detriment to your plants.
Require coveralls for all employees. Your employee may love his favorite jean jacket, but the odds are that it hasn’t been cleaned in months and is covered with mold spores. Clean clothing for your staff is a must. Provide coveralls that are washed at least once a week if not daily.
Keep things clean. A clean and organized grow area will have a huge impact on mold growth. Clean pots with oxidate, mop floors with oxidate every week, keep the areas in front of air returns clean and clutter-free, and clean floor drains regularly. The entire grow and everything in it should be scrubbed top to bottom after each harvest.
Keep it cool. Keep curing areas cool and storage areas cold where possible. The ideal temperature for a curing area is roughly 60 degrees and under 32 degrees for a storage area. Just like food, the lower the temperature, the better it keeps. High temperature increases all molecular and biological activity, which causes things to deteriorate faster than at cooler temperatures. However, curing temperature is a function of water activity more than anything.
Be Careful With Beneficials. Beneficial insects certainly have their place in the grow environment. However, if you have a problem with mold on only a small percentage of plants, any insect can act as a carrier for spores and exacerbate the problem. By the same token, pests spread mold more effectively than beneficials because they produce rapidly, where beneficials die if there aren’t pests for them to eat. It is best to use beneficials early in the cycle and only when necessary.