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How Do You Know You’re Right? qPCR vs. Plating

By Dr. Sherman Hom
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Cannabis testing to detect microbial contamination is complicated. It may not be rocket science, but it is life science, which means it’s a moving target, or at least, it should be, as we acquire more and more information about how the world we live in works. We are lucky to be able to carry out that examination in ever increasing detail. For instance, the science of genomics1 was born over 80 years ago, and just twenty years ago, genetics was still a black box. We’ve made tremendous progress since those early days, but we still have a long way to go, to be sure.

Much of that progress is due to our ability to build more accurate tools, a technological ladder, if you will, that raises our awareness, expertise, and knowledge to new levels. When a new process or technology appears, we compare it against accepted practice to create a new paradigm and make the necessary adjustments. But people have to be willing to change. In the cannabis industry, rapid change is a constant, first because that is the nature of a nascent industry, and second because in the absence of some universal and unimpeachable standard, it’s difficult to know who’s right. Especially when the old, reliable reference method (i.e. plating, which is basically growing microorganisms on the surface of a nutritional medium) is deeply flawed in its application to cannabis testing vs. molecular methods (i.e., quantitative polymerase chain reaction, or qPCR for short).

Dr. Sherman Hom, Director of Regulatory Affairs at Medicinal Genomics

Plating systems have been used faithfully for close to 130 years in the food industry, and has performed reasonably well.2 But cannabis isn’t food and can’t be tested as if it were. In fact, plating methods have a host of major disadvantages that only show up when they’re used to detect cannabis pathogens. They are, in no particular order:

  1. A single plating system can’t enumerate a group of microorganisms and/or detect specific bacterial and fungal pathogens. This is further complicated by the fact that better than 98% of the microbes in the world do not form colonies.3 And there is no ONE UNIVERSAL bacterial or fungal SELECTIVE agar plate that will allow the growth of all bacteria or all fungal strains. For example, the 5 genus species of fungal strains implicated in powderly mildew DO NOT plate at all.
  2. Cannabinoids, which can represent 10-30% of a cannabis flower’s weight, have been shown to have antibacterial activity.4 Antibiotics inhibit the growth of bacteria and in some cases kill it altogether. Salmonella species & shiga toxin producing coli (STEC) bacteria, in particular, are very sensitive to antibiotics, which leads to either a false negative result or lower total counts on plates vs. qPCR methods.
  3. Plating methods cannot detect bacterial and fungal endophytes that live a part or all of their life cycle inside a cannabis plant.5,6 Examples of endophytes are the Aspergillus pathogens (A. flavus, A. fumigatus, A. niger, and A. terreus). Methods to break open the plant cells to access these endophytes to prepare them for plating methods also lyse these microbial cells, thereby killing endophytic cells in the process. That’s why these endophytes will never form colonies, which leads to either false negative results or lower total counts on plates vs. qPCR methods.
  4. Selective plating media for molds, such as Dichloran Rose-Bengal Chloramphenicol (DRBC) actually reduces mold growth—especially Aspergillus—by as much as 5-fold.This delivers false negative results for this dangerous human pathogen. In other words, although the DRBC medium is typically used to reduce bacteria; it comes at the cost of missing 5-fold more yeast and molds than Potato Dextrose Agar (PDA) + Chloramphenicol or molecular methods. These observations were derived from study results of the AOAC emergency response validation.7
  5. Finally, we’ve recently identified four bacterial species, which are human pathogens associated with cannabis that do not grow at the plating system incubation temperature typically used.8 They are Aeromonas hydrophila, Pantoea agglomerans, Yersinia enterocolitica, and Rahnella aquatilis. This lowers total counts on plates qPCR methods.

So why is plating still so popular? Better yet, why is it still the recommended method for many state regulators? Beats me. But I can hazard a couple of guesses.

A yeast and mold plate test

First, research on cannabis has been restricted for the better part of the last 70 years, and it’s impossible to construct a body of scientific knowledge by keeping everyone in the dark. Ten years ago, as one of the first government-employed scientists to study cannabis, I was tapped to start the first cannabis testing lab at the New Jersey Dept. of Health and we had to build it from ground zero. Nobody knew anything about cannabis then.

Second, because of a shortage of cannabis-trained experts, members of many regulatory bodies come from the food industry—where they’ve used plating almost exclusively. So, when it comes time to draft cannabis microbial testing regulations, plating is the default method. After all, it worked for them before and they’re comfortable with recommending it for their state’s cannabis regulations.

Finally, there’s a certain amount of discomfort in not being right. Going into this completely new area—remember, the legal cannabis industry didn’t even exist 10 years ago—we human beings like to have a little certainty to fall back on. The trouble is, falling back on what we did before stifles badly needed progress. This is a case where, if you’re comfortable with your old methods and you’re sure of your results, you’re probably wrong.

So let’s accept the fact that we’re all in this uncharted territory together. We don’t yet know everything about cannabis we need to know, but we do know some things, and we already have some pretty good tools, based on real science, that happen to work really well. Let’s use them to help light our way.


References

  1. J. Weissenbach. The rise of genomics. Comptes Rendu Biologies, 339 (7-8), 231-239 (2016).
  2. R. Koch. 1882. Die Aetiologie der Tuberculose.  Berliner Klinische Wochenschrift, 19, 221-230 (1882)
  3. W. Wade. Unculturable bacteria—the uncharacterized organisms that cause oral infections. Journal of the Royal Society of Medicine, 95(2), 91-93 (2002).
  4. J.A. Karas, L.J.M. Wong, O.K.A. Paulin, A. C. Mazeh, M.H. Hussein, J. Li, and T. Vekov. Antibiotics, 9(7), 406 (2020).
  5. M. Taghinasab and S. Jabaji, Cannabis microbiome and the role of endophytes in modulating the production of secondary metabolites: an overview. Microorganisms 2020, 8, 355, 1-16 (2020).
  6. P. Kusari, S. Kusari, M. Spiteller and O. Kayser, Endophytic fungi harbored in Cannabis sativa L.: diversity and potential as biocontrol agents against host plant-specific phytopathogens. Fungal Diversity 60, 137–151 (2013).
  7. K. McKernan, Y. Helbert, L. Kane, N. Houde, L. Zhang, S. McLaughlin, Whole genome sequencing of colonies derived from cannabis flowers & the impact of media selection on benchmarking total yeast & mold detection toolshttps://f1000research.com/articles/10-624 (2021).
  8. K. McKernan, Y. Helbert, L. Kane, L. Zhang, N. Houde, A. Bennett, J. Silva, H. Ebling, and S. McLaughlin, Pathogenic Enterobacteriaceae require multiple culture temperatures for detection in Cannabis sativa L. OSF Preprints, https://osf.io/j3msk/, (2022)

Beyond Compliance: Understanding and Combating Contamination

By Jill Ellsworth MS, RDN, Tess Eidem, Ph.D.
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As an emerging field in cannabis, contaminant testing remains a gray area for many businesses. The vast differences in state-by-state regulations, along with the frequent changes of previously established rules make testing a difficult, time-consuming process. But at its core, the science and reasoning behind why we test cannabis is very clear – consumer safety and quality assurance are key factors in any legal, consumer market. The implications of federal legalization make cannabis testing even more important to the future of the cannabis supply chain. Understanding the types of contaminants, their sources and how to prevent them is essential to avoiding failures, recalls and risking consumer safety.

When talking about cannabis contaminant testing there are four groups of contaminants: pesticides, heavy metals, foreign materials and microbes. The microbes found on cannabis include plant pathogens, post-harvest spoiling microbes, allergens, toxin release and human pathogens. While all of these can be lurking on the surface of cannabis, the specific types that are tested for in each state vary widely. Understanding the full scope of contaminants and looking beyond state-specific compliance requirements, cultivators will be able to prevent these detrimental risks and prepare their business for the future.

Environmental controls are essential to monitor and regulate temperature and humidity

Beyond just the health of the plant, both medical patients and adult use consumers can be adversely affected by microbial contaminants. To immunocompromised patients, Aspergillus can be life-threatening and both adult use and medical consumers are susceptible to allergic reactions to moldy flower. But Aspergillus is just one of the many contaminants that are invisible to the human eye and can live on the plant’s surface. Several states have intensive testing regulations when it comes to the full breadth of possible harmful contaminants. Nevada, for example, has strict microbial testing requirements and, in addition to Aspergillus, the state tests for Salmonella, STEC, Enterobacteriaceae, coliforms and total yeast and mold. Over 15 states test for total yeast and mold and the thresholds vary from allowing less than 100,000 colony forming units to allowing less than 1,000 colony forming units. These microbes are not uncommon appearances on cannabis – in fact, they are ever-present – so understanding them as a whole, beyond regulatory standards is a certain way to future-proof a business. With such vast differences in accepted levels of contamination per state, the best preparation for the future and regulations coming down the pipeline is understanding contamination, addressing it at its source and harvesting disease-free cannabis.

The risk of contamination is present at every stage of the cultivation process and encompasses agricultural practices, manufacturing processes and their intersection. From cultivation to manufacturing, there are factors that can introduce contamination throughout the supply chain. A quality control infrastructure should be employed in a facility and checkpoints within the process to ensure aseptic operations.

Microbial monitoring methods can include frequent/consistent testing

Cultivators should test their raw materials, including growing substrates and nutrient water to ensure it is free of microbial contamination. Air quality plays an important role in the cultivation and post-harvest processes, especially with mold contamination. Environmental controls are essential to monitor and regulate temperature and humidity and ensure unwanted microbes cannot thrive and decrease the value of the product or make it unsafe for worker handling or consumers. Developing SOPs to validate contact surfaces are clean, using proper PPE and optimizing worker flow can all help to prevent cross-contamination and are part of larger quality assurance measures to prevent microbes from spreading across cultivars and harvests.

Methods of microbial examination include air quality surveillance, ATP surface and water monitoring, raw materials testing, and species identification. Keeping control of the environment that product is coming into contact with and employing best practices throughout will minimize the amount of contamination that is present before testing. The solution to avoiding worst case scenarios following an aseptic, quality controlled process is utilizing a safe, post-harvest kill-step, much like the methods used in the food and beverage industries with the oversight of the FDA.

The goal of the grower should be to grow clean and stay clean throughout the shelf life of the product. In order to do this, it is essential to understand the critical control points within the cultivation and post-harvest processes and implement proper kill-steps. However, if a product is heavily bio-burdened, there are methods to recover contaminated product including decontamination, remediation and destroying the product. These measures come with their own strengths and weaknesses and cannot replace the quality assurance programs developed by the manufacturer.

Connecticut Seeks to Change Microbial Testing Regulations

As of now, there are only two cannabis testing labs in Connecticut. Last year, regulators in the state approved a request from AltaSci Labs to raise the testing limits for yeast and mold at their lab from 10,000 colony forming units per gram (cfu/g) up to 1 million. The other lab, Northeast Laboratories, has kept their limits at 10,000 cfu/g.

Connecticut state flag

According to CTInsider.com, that request was approved privately and unannounced and patients were notified via email of the change. Ginny Monk at CTInsider says patients enrolled in Connecticut’s medical cannabis program have been outspoken over safety concerns, a lack of transparency and little voice in the decision-making process.

Connecticut has a small medical cannabis market with roughly 54,000 patients in the program and they are in the midst of readying the launch of their adult-use market.

A yeast and mold test showing colony forming units

Following public outcry regarding the change at the recent Social Equity Council meeting, state regulators have proposed a change to microbial testing regulations. The new rule will set the limit at 100,000 cfu/g for yeast and mold and requires testing for specific forms of Aspergillus, a more harmful type of mold.

Kaitlyn Kraddelt, spokeswoman for Connecticut’s Department of Consumer Protection, the agency in charge of testing regulations for the state’s cannabis program, told CTInsider.com that they involved several microbiologists to develop the new rule. “These new standards, which were drafted in consultation with several microbiologists, will prohibit specific types of yeast and mold in cannabis flower that may cause injury when inhaled and allow 10^5 cfu/g of colony forming units that have no demonstrated injurious impact on human health,” says Krasselt.

The rule change is now undergoing a public comment period, after which the Attorney General’s office will get a review period. If approved, it’ll head to the legislature, where a committee has 45 days to act on it.

Detecting Microbial Contamination in Cannabis

By Mike Clark
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Increasing cannabis use across the US has come with increased scrutiny of its health effects. Regulators and healthcare providers are not just concerned about the direct effects of inhaling or consuming cannabinoids, however, but also about another health risk: microbial contamination in cannabis products. Like any other crop, cannabis is susceptible to contamination by harmful pathogens at several points throughout the supply chain, from cultivation and harvesting to distribution. Many state regulators have set limits for microbial populations in cannabis products. Consequently, testing labs must adopt efficient screening protocols to help companies remain compliant and keep their customers safe.

Some of the pathogens common to cannabis flower include Aspergillus fungus species such as A. flavus, A. fumigatus, A. niger and A. terreus. Cannabis might also harbor harmful E. coli and Salmonella species, including Shiga toxin-producing E. coli (STEC). Regulations vary by state, but most have set specific thresholds for how many colony forming units (CFUs) of particular species can be present in a sellable product.

The gold standard method for detecting microbes is running cultures.

Growers and testing labs need to develop a streamlined approach to remain viable. Current methods, including running cultures on every sample, can be expensive and time-consuming, but by introducing a PCR-based screening step first, which identifies the presence of microbial DNA – and therefore the potential for contamination – laboratories can reduce the number of cultures they need to run, saving money and time.

The Risk of Aspergillus Contamination

Contamination from Aspergillus species can bring harm to cannabis growers and their customers. The state of Michigan is currently undergoing the largest cannabis recall in its history from Aspergillus contamination.

If contamination grows out of control, the pathogen can damage the cannabis plant itself and lead to financial losses. Aspergillus can also cause serious illness in consumers, especially those that are immunocompromised. If an immunocompromised person inhales Aspergillus, they can develop aspergillosis, a lung condition with a poor prognosis.

A Two-Step Screening Process

The gold standard method for detecting microbes is running cultures. This technique takes weeks to deliver results and can yield inaccurate CFU counts, making it difficult for growers to satisfy regulators and create a safe product in a timely manner. The use of polymerase chain reaction (PCR) can greatly shorten the time to results and increase sensitivity by determining whether the sample has target DNA.

Using PCR can be expensive, particularly to screen for multiple species at the same time, but a qPCR-based Aspergillus detection assay could lead to significant cost savings. Since the average presumptive positive rate for Aspergillus contamination is low (between 5-10%), this assay can be used to negatively screen large volumes of cannabis samples. It serves as an optional tool to further speciate only those samples that screened positive to comply with state regulations.

Conclusion

Overall, screening protocols have become a necessary part of cannabis production, and to reduce costs, testing labs must optimize methods to become as efficient as possible. With tools such as PCR technology and a method that allows for initial mass screening followed by speciation only when necessary, laboratories can release more samples faster with fewer unnecessary analyses and more success for cannabis producers in the marketplace.

Cannabis Recalls: Lessons Learned After Three Years of Canadian Legalization

By Steven Burton
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Three years ago, Canada became one of the first countries in the world to legalize and regulate cannabis. We’ve covered various aspects of cannabis regulation since, but now with a few years of data readily available, it’s time to step back and assess: what can we learn from three years of cannabis recalls in the world’s largest legal market?

Labelling Errors are the Leading Cause of Canadian Cannabis Recalls

Our analysis of Health Canada’s data revealed a clear leader: most cannabis recalls since legalization in October 2018 have been due to labelling and packaging errors. In fact, over three quarters of total cannabis recalls were issued for this reason, covering more than 140,000 units of recalled product.

The most common source of labelling and packaging recalls in the cannabis industry (more than half) is inaccurate cannabinoid information. Peace Naturals Project’s recall of Spinach Blue Dream dried cannabis pre-rolls this year is a good example. Not only did the packaging incorrectly read that the product contained CBD, but the THC quantity listed was lower than the actual amount of THC in the product. The recall covered over 13,000 units from a single lot sold over 10 weeks.

In another example, a minor error made a huge impact. British Columbia-based We Grow BC Ltd. experienced this firsthand when it misplaced the decimal points in its cannabinoid content. The recalled products displayed the total THC and CBD values as 20.50 mg/g and 0.06 mg/g, respectively, when the products contained 205.0 mg/g and 0.6 mg/g.

Accurate potency details are not just crucial for compliance. For many customers, potency is a deciding factor when selecting a cannabis product, and this is especially important for medicinal users (including children), people who are sensitive to certain cannabinoids and consumers looking for non-psychoactive effects. In this case, at least six consumer complaints were submitted to Peace Naturals Project, the highest number for any cannabis recall in Canada.

Frequent, integrated lab testing, an effective and robust traceability system, smaller lot sizes during production and consistent quality checks could have helped Peace Naturals Project and We Grow BC limit the scope of their recall or avoid them altogether.

Pathogens are the #2 Cause of Cannabis Recalls in Canada

Pathogens are the second most common cause of recalls in Canada, claiming 18% of total cannabis recall incidents. And while that doesn’t sound like much compared to the recalls caused by labelling errors, it affects the highest volume of product recalled with over 360,000 units affected.

Canadian Cannabis Recalls – Total number of affected units and noted causes

A primary cause of allergens and microbiological contamination of cannabis products is yeast, mold and bacteria found on cannabis flower (chemical contaminants like pesticides can also be a major concern). Companies like Atlas Growers, Natural MedCo and Agro-Greens Natural Products have all learned this lesson through costly recalls.

These allergenic contaminants pose an obvious health risk, often leading to reactions such as wheezing, sneezing and itchy eyes. For people using cannabis for medical conditions and may be more susceptible to illness, pathogens can cause more serious health complications. Moreover, this type of cannabis recall not only drives significant cost since microbiological contamination of flower could easily affect several product batches processed in the same facility and/or trigger downstream recalls, but also affect consumer confidence for established cannabis brands.

Preventive control plan requirements for cannabis manufacturers mandate that holders of a license for processing that produce edible cannabis or cannabis extracts in Canada must identify and analyze the biological, chemical and physical hazards that present a risk of contamination to the cannabis or anything that would be used as an ingredient in the production of the edible cannabis or cannabis extract. Biological hazards can come from a number of sources, including:

  • Incoming ingredients, including raw materials
  • Cross-contamination in the processing or storage environment
  • Employees
  • Cannabis extract, edible cannabis and ingredient contact surfaces
  • Air
  • Water
  • Insects and rodents

To mitigate risks, addressing root causes with preventative measures and controls is essential. For instance, high humidity levels and honeydew secreted by insects are common causes of mold on cannabis flowers. Measures such as leaving a reasonable distance between plants, using climate-controlled areas to dry flowers, applying antifungal agents and conducting regular tests are necessary to combat such incidents.

control the room environment
Preventative measures and controls can save a business from extremely costly recalls.

Of course, placing all the necessary controls into action is not as simple as it may sound. Multiple facilities and a wide range of products in production mean more complexity for cannabis producers and processors. Any gaps in processing flower, extracts or edibles can result in an uncontrolled safety hazard that may lead to a costly cannabis recall.

These challenges are not just limited to cannabis growers. The food industry has been effectively mitigating the risk of biological hazards for decades with the help of food ERP solutions.

Avoid Recalls Altogether with Advanced ERP Technology

An effective preventative control plan with regular quality checks, internal audits and standardized testing is important to minimize the threats evident from Canada’s recall data. If these measures ever fail, real-time traceability systems play a pivotal role in the event of a cannabis recall by enabling manufacturers to trace back incidents to the exact point of contamination and identify affected products with surgical precision.

Instead of starting from zero, savvy cannabis industry leaders turn to the proven solutions from the food industry and take advantage of data-driven, automated systems that deliver the reliability and safety that the growing industry needs. From automated label generation to integrated lab testing to quality checks to precision traceability and advanced reporting, production and quality control systems are keys to success for the years ahead.

AOAC Approves Two New Microbiological Assays

By Cannabis Industry Journal Staff
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On August 11, PathogenDx announced that they received an AOAC Performance Tested Methods Certificate for their QuantX total yeast and mold test. Six days later, on August 17, Medicinal Genomics announced that AOAC approved their PathoSEEK 5-Color Aspergillus Multiplex Assays under the same AOAC Performance Tested Methods program.

Both assays are specifically designed with cannabis and hemp testing in mind and designed to expedite and simplify microbiological testing. PathogenDx’s QuantX quantifies the total amount of yeast and mold in a sample while also measuring against safety standards.

In addition to the total yeast and mold count test, PathogenDx has also introduced a 96-well plate, improved sample preparation and new data reporting with a custom reporting portal for compliance testing.

The Medicinal Genomics platform can detect four species, including A. flavus, A. fumigatus, A. niger, and A. terreus in both flower and infused edibles. The PathoSEEK microbial testing platform uses a PCR-based assay and provides an internal plant DNA control for every reaction.

This technique verifies the performance of the assay when detecting pathogens, allegedly minimizing false negative results commonly due to set up errors and experimental conditions.

AOAC International is a standards organization that works in the cannabis testing space through their CASP program to evaluate and approve standard testing methods for the industry.

Ask the Experts: Microbiological Contamination in Cannabis & What You Should Look for

By Cannabis Industry Journal Staff
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Testing cannabis and cannabis derived products for microbiological contamination should be a straightforward conversation for testing labs and producers. However, a patchwork of regulations and a wide variety of perspectives on what we should, or should not, be looking for has left much of the cannabis industry searching for reliable answers.

Organizations like the AOAC are taking the first crack at creating standardization in the field but there is still a long way to go. In this conversation, we would like to discuss the general requirements that almost all states share and where we see the industry headed as jurisdictions start to conform to the recommendations of national organizations like AOAC.

We sat down with Anna Klavins and Jessa Youngblood, two cannabis testing experts at Hardy Diagnostics, to get their thoughts on microbiology testing in the current state of the cannabis industry.

Q: What are the biggest challenges facing cannabis testing labs when it comes to microbiology?

The CompactDry Yeast and Mold Rapid plate provides fast results.

Anna Klavins & Jessa Youngblood: For microbiology testing, it comes down to a lack of standardization and approved methods for cannabis. In the US, cannabis regulation is written on a state-by-state level. As a result, the rules that govern every aspect of bringing these materials to market is as unique and varied as the jurisdiction writing them. When we are speaking specifically about microbiology, the question always comes back to yeast and mold testing. For some, the challenge will often be centered on the four main Aspergillus species of concern – A. terreus, A. niger, A. fumigatus, and A. flavus. For others, it will be the challenges of total count testing with yeast, mold, and bacteria. These issues become even more troublesome by the lack of recognized standard methodology. Typically, we expect the FDA, USP, or some other agency to provide the guidelines for industry – the rules that define what is safe for consumption. Without federal guidance, however, we are often in a situation where labs are required to figure out how to perform these tests on their own. This becomes a very real hurdle for many programs.

Q: Why is it important to use two different technologies to achieve confirmation?

Dichloran Rose Bengal Chloramphenicol (DRBC) Agar is recommended for the enumeration of yeasts and molds.

Klavins & Youngblood: The push for this approach was borne out of the discussions happening within the industry. Scientists and specialists from across disciplines started getting together and creating groups to start to hash out problems which had arisen due to a lack of standardization. In regards to cannabis testing, implementing a single method for obtaining microbiology results could be unreliable. When clients compared results across labs, the inconsistencies became even more problematic and began to erode trust in the industry. As groups discussed the best way to prove the efficacy of their testing protocol, it quickly became apparent that relying on a single testing method was going to be inadequate. When labs use two different technologies for microbiology testing, they are able to eliminate the likelihood of false positives or false negatives, whichever the case may be. In essence, the cannabis testing laboratories would be best off looking into algorithms of detecting organisms of interest. This is the type of laboratory testing modeled in other industries and these models are starting make their way into the cannabis testing space. This approach is common in many food and pharma applications and makes sense for the fledgling cannabis market as well.

About Anna Klavins

Anna Klavins earned a Molecular and Cellular Biology B.S. degree from Cal Poly San Luis Obispo while playing for the Cal Poly Division I NCAA women’s tennis team. Since joining Hardy Diagnostics in mid-2016, she has gained experience in FDA submissions [510(k)] for class II microbiology in vitro devices. She has worked on 15 projects which led to a microbiology device becoming FDA cleared. She has recently begun participating in the AOAC Performance Tested Methods program.

 

About Jessa Youngblood

Jessa Youngblood is the Food, Beverage and Cannabis Market Coordinator for Hardy Diagnostics. A specialist in the field of cannabis microbiology for regulatory compliance, she is seated with the AOAC CASP committee working on standard methods for microbiological testing in cannabis and hemp. She also sits on the NCIA Scientific Advisory Council as well as the ASTM Cannabis Council.

Content sponsored by Hardy Diagnostics.

Product Safety Hazards: Looking Beyond Food Safety in Cannabis

By Radojka Barycki
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I think that we need to start changing the terminology around the hazards associated with cannabis from food safety hazards to product safety hazards. These hazards have not only been associated with harmful effects for those that ingest cannabis infused products, but also for those that consume the cannabis products in other ways such as inhalation (vaping or smoking). So, when we refer to these hazards as food safety hazards, the immediate thought is edibles, which misleads cultivators, manufacturers and consumers to have a false sense of security around the safety of products that are consumed in other ways.

Food processing and sanitation
By standardizing and documenting safety procedures, manufacturers mitigate the risk of cannabis-specific concerns

There are several product safety hazards that have been associated with cannabis. These hazards can become a public health problem if not controlled as they could harm the consumer, regardless of the method of consumption.

Let’s take a look at the different types of hazards associated cannabis:

Biological Hazards refer to those microorganisms that can cause illness to the consumer of a product that contain them. They are not visible to the naked eye and are very dangerous when their metabolic by-products (toxins) are ingested or their spores are inhaled. The symptoms for illnesses caused by these microorganisms will vary. Consumers may experience gastrointestinal discomfort (vomiting, diarrhea), headaches, fever and other symptoms. The ingestion of these pathogens, allergens or their by-products may lead to death, if the illness is not treated on time or if the consumer of the product is immunocompromised. In addition, the inhalation of mold spores when smoking cannabis products, can lead to lung disease and death. Some of the biological hazards associated with cannabis are: Salmonella sp., E. coli, Clostridium botulinum, Aspergillus sp. and Penicillium sp.

Chemical Hazards refer to those chemicals that can be present in the plant or finished product due to human applications (pesticides), operational processes (extraction solvents and cleaning chemicals), soil properties (heavy metals), environmental contamination (radiological chemicals) or as a result of occurring naturally (mycotoxins and allergens). Consuming high concentrations of cleaning chemicals in a product can lead to a wide range of symptoms from mild rash, burning sensation in the oral-respiratory system, gastrointestinal discomfort or death. In addition, long term exposure to chemicals such as pesticides, heavy metals, radiological contaminants and mycotoxins may lead to the development of cancers.

Physical Hazards refer to those foreign materials that may be present in the plant or finished product. Foreign materials such as rocks, plastics or metals can cause harm to the consumer by chipping teeth or laceration of the mouth membranes (lips, inner cheeks, tong, esophagus, etc.) In the worst-case scenario, physical hazards may lead to choking, which can cause death due to asphyxiation.

These hazards can be prevented, eliminated or reduced to an acceptable level when foundational programs (Good Agricultural/Cultivation Practices, Good Manufacturing Practices, Allergen Management Program, Pest Control, etc.) are combined with a Food [Product] Safety Plan. These lead to a Food [Product] Safety Management System that is designed to keep consumers safe, regardless of the method of consumption.

Preventing Mold & Fungus in Cannabis with Data Analytics

By Leighton Wolffe
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Cannabis legalization has taken the United States by storm, with 33 states approved for medicinal cannabis use — 11 of which are also approved for recreational use for adults aged 21 and over. With new patients and consumers entering the market every day, it’s more important than ever for cannabis cultivators to establish more effective methods for mold and fungal prevention in their crops and to ensure consumer confidence in their brands.

Today, many cultivators address the risk of mold and fungus growth by testing crops for contaminants at the end stage of production. While this helps to catch some infected product before it reaches the market, this method is largely ineffective for mold and fungal prevention during the cultivation process. In fact, recent studies have shown an 80% failure rate in mold and fungal testing in Denver cannabis dispensaries. By relying on late-stage, pass/fail testing, cannabis entrepreneurs also expose themselves to increased risk of lost crops and profits.

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

However, emerging sensor technologies exist that can test plants during the grow process, significantly reducing the risks associated with cannabis cultivation while increasing the bottom line for commercial grow operations. By leveraging data from these monitoring sensors along with environmental automation systems that are integrated with data analytics platforms, cannabis professionals can take a proactive approach to achieve the ideal environmental conditions for their crops and prevent against mold and fungal infestation.

Common Causes for Bud Rot in Indoor Growing Systems 

Botrytis cinerea — commonly known as “bud rot” — is a pathogenic fungi species that creates a gray mold infection in cannabis plants. An air-borne contaminant, it is among the most prevalent diseases affecting marijuana crops today and can lead to significant damages, particularly when left untreated during post-harvest storage. Bud rot is one of the most difficult challenges cannabis entrepreneurs face: Once plants have been affected, only 2% can be expected to recover. This is because Botrytis cinerea can use multiple methods for attacking host plants, including using the plant’s natural defenses against it to continue infestation.

While difficult to contain, bud rot is very easy to spot. Plants affected with the fungus will begin yellowing, experience impaired growth, and develop gray fungus around its buds. Overall crop yield will be significantly reduced, leading to decreased profit for cannabis cultivators. The biggest contributing factors to a Botrytis cinerea infestation are as follows:

  • Humidity: Indoor grow facilities that maintain humidity levels in excess of 45% are breeding grounds for mold and fungus. These environments can become perfect conditions for mold and fungal growth.
  • Temperature: Bud rot typically thrives in environments where temperatures fall between 65- and 75-degrees Fahrenheit, which is why greenhouses and grow rooms are often the victim of such infestations.
  • Ventilation: Poor airflow is another contributing factor to Botrytis cinerea Without proper ventilation, excess moisture buildup will eventually result in mold and mildew growth.
  • Strain: Some marijuana strains are better equipped to fend off bud rot infection. In particular, sativa plants have a higher resistance to mold development than their C. indica and C. ruderalis cousins.

Controlling mold and fungal growth in commercial grow facilities is a top priority for cannabis cultivators. Not only detrimental to their profitability and crop yield, infected plants can pose serious health risks to consumers, especially for immunocompromised patients. Consuming cannabis products that have been compromised by bud rot or other mold and fungal infections can cause a wide range of medical concerns, including pneumonitis, bronchitis, and other pulmonary diseases. As a result, growers are required to dispose of all infected plants without the possibility to sell.

Bud rot isn’t the only culprit responsible for cannabis plant destruction. Powdery mildew, Fusarium, sooty molds, and Pythium all contribute to the challenges faced by cannabis professionals. In fact, a recent study conducted by Steep Hill Labs and University of California, Davis – Medical Center found that in 20 randomly-selected samples submitted for testing, all samples showed detectable levels of microbial contamination7. Many of these samples also contained significant pathogenic microorganism contamination. Without proper detection and prevention methods in place, these pesky plant-killers will only continue to terrorize the cannabis cultivation industry.

The Current Cannabis Cultivation Landscape 

The data is clear: Current practices for cannabis cultivation are insufficient for preventing against mold and fungal growth. Sterilization and pass/fail testing do not identify the root cause of harmful infestations in plants, therefore leaving cannabis professionals in the dark about how to better optimize their grow conditions for improved crop reliability and safety. In order to prevent against damages incurred from mold and fungal infestation, marijuana growers must be more diligent in their grow condition monitoring practices.

Many cannabis professionals rely on manual monitoring to identify environmental changes within their indoor grow facilities. While it’s important to collect data on your operation’s essential systems, doing so without the right tools can be time-consuming and ineffective. Manual monitoring often relies on past data and does not illustrate the relationship between different systems and their impact on environmental changes. The goal is to assemble data from all the grow systems and create correlations on actual bio-environmental conditions during the grow process to compare to yield results. This is only available when an information management platform is synthesizing data from all the systems within the grow facility and presenting meaningful information to the growers, facility operators and owners.

Especially as the cannabis industry is expected to grow exponentially in coming years, growers need more robust tools for tracking and manipulating environmental changes within their indoor growing systems.

Leveraging Building Automation Systems & Data Analytics in Cannabis Cultivation 

A powerful approach to prevent environmental conditions that are known to lead to mold and fungus growth exists in leveraging the data produced from your grow facility’s various automation systems. Most commercial cultivation facilities have multiple stand-alone and proprietary systems to control their indoor environment, making it difficult to not only collect all of this valuable data, but also to achieve the level of grow condition monitoring necessary for mold and fungal prevention.

With some data analytics platforms, such as GrowFit Analytics, data is collected across disparate systems that don’t normally communicate with one another, providing access to the key insights necessary for achieving environmental perfection with your cannabis crops. A viable solution collects vital grow facility system data and relevant bio-environmental monitoring data, and delivers this information in one, centralized software interface. The software then will apply analytic algorithms to develop key performance indicators (KPIs) while working to detect system anomalies, faults, and environmental fluctuations. The right analytics solution should also be customizable, allowing you to track the KPIs that are most important to your unique facility, and to achieve the vision of your chief grower. Ultimately, the software should serve up actionable insights that empower facility management and growers.

Sample data visualization dashboard from GrowFit Analytics showing real-time Temperature and Relative Humidity readings and indicating potential Mold Risk as defined by the Grower.

Collecting reliable data from different grow facility systems and environmental sensors can be a complex process and the information collected illustrates more than just what’s working right and what isn’t. By implementing an advanced data analytics solution, cannabis cultivation professionals can now be empowered to track minute details about their indoor grow facility, providing a safer, healthier environment for their crops and avoiding those environmental conditions that lead to mold and fungus altogether.

An ideal data analytics platform won’t simply collect data to be analyzed at a later date, and simple trending of sensor data is not enough. Information — especially in a commercial grow facility — is time-sensitive, which is why growers should select a system that offers real-time analytics capabilities. Some platforms offering real-time analytics utilize cloud computing, allowing for easy access from anywhere while also providing enhanced security to protect sensitive facility data. The most robust data analytics platforms provide detailed historical data for your entire crop’s lifecycle that provide a “digital recipe” to replicate successful crops, and fine-tune the process for continuous improvement.

Data analytics tools can also impact the bottom line by lowering operational costs. GrowFit Analytics, for example, was born out of a software solution designed to lower energy costs for large complex buildings like commercial grow facilities.

The data and insights provided can help identify opportunities for greater energy efficiency, which can lead to significant utility savings. Grow facilities operate 24 hours/day, with energy expenses representing one of the largest operational costs. With data analytics tools at their disposal, facility managers are armed with the information they need to improve system efficiency, increase energy savings, and improve profitability.

Eliminating Mold & Fungus from the Future of Cannabis Cultivation 

By focusing on grow condition monitoring using data analytics tools, cannabis professionals can effectively eliminate the risk of mold and fungus growth in their crops. Leading data analytics tools make tracking environmental changes simple and easy to manage, allowing cannabis professionals to take a proactive approach to mold and fungus prevention. As we look to the future of the cannabis cultivation industry, it’s paramount for professionals to explore the technological advancements available that can help them address their business’ most pressing challenges.

Rapid Pathogen Detection for the 21st Century: A Look at PathogenDx

By Aaron G. Biros
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In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.

The global food supply chain moves much faster today than it did in the late 19th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21st century pathogen detection solutions.”

Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”

Milan Patel, CEO of PathogenDx
Photo: Michael Chansley

He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”

While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.

The PathogenDx Microarray

Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”

When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”

The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:

  1. Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
  2. Drop sample in solution. Swab should already be in solution
  3. Vortex
  4. Transfer 1ml of solution into 1.5ml tube

    A look at how the sample is added to the microarray
  5. Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
  6. Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
  7. Conduct Loci enhancement PCR of sample for 1 hour
  8. Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
  9. Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
  10. Conduct wash cycle for 15 minutes
  11. Dry and image the slide in imager
  12. The imager will create a TIFF file where software will analyze and deliver results and a report

Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.

Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.

Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.

Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.