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).
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:
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.
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.
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.
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
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.
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.
J. Weissenbach. The rise of genomics. Comptes Rendu Biologies, 339 (7-8), 231-239 (2016).
R. Koch. 1882. Die Aetiologie der Tuberculose. Berliner Klinische Wochenschrift, 19, 221-230 (1882).
W. Wade. Unculturable bacteria—the uncharacterized organisms that cause oral infections. Journal of the Royal Society of Medicine, 95(2), 91-93 (2002).
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).
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).
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).
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 tools, https://f1000research.com/articles/10-624 (2021).
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)
In this “Leaders in Cannabis Testing” series of articles, Green interviews cannabis testing laboratories and technology providers that are bringing unique perspectives to the industry. Particular attention is focused on how these businesses integrate innovative practices and technologies to navigate a rapidly changing landscape of regulatory constraints and B2B demand.
PathogenDx is an Arizona-based provider of microbial testing technologies. Since their inception in 2014, they have broadened their reach to 26 states in the US. In addition to cannabis product testing, PathogenDx also provides technologies for food safety testing, environmental testing and recently started offering human diagnostics testing to support COVID-19 response efforts.
We interviewed Milan Patel, CEO and co-founder of PathogenDx. Milan founded PathogenDx as a spin-off from one of his investments in a clinical diagnostics company testing for genetic markers in transplant organs. Prior to PathogenDx, Milan worked in finance and marketing at Intel and later served as CFO at Acentia (now Maximus Federal).
Aaron Green: What’s the history of PathogenDx?
Milan Patel: PathogenDx was effectively a spin-off of a clinical diagnostics company that my partner Dr. Mike Hogan, the inventor of the technology, had founded when he was a professor at the University of Arizona, but previously at Baylor Medical College back in 2002. I had invested in the company back then and I had realized that his technology had a broad and wide sweeping impact for testing – not just for pathogens in cannabis specifically, but also for pathogens in food, agriculture, water and even human diagnostics. In the last 14 months, this became very personal for every single person on the planet having been impacted by SARS-CoV-2, the viral pathogen causing Covid-19. The genesis of the company was just this, that human health, food and agricultural supply, and the environment has and will continue to be targeted by bacterial, fungal and viral pathogens impacting the safety and health of each human on the planet.
We founded PathogenDx and we pivoted the company from its original human organ transplant genetics market scope into the bigger markets; we felt the original focus was too niche for a technology with this much potential. We licensed the technology, and we repurposed it into primarily cannabis. We felt that achieving commercial success and use in the hands of cannabis testing labs at the state level where cannabis was first regulated was the most logical next step. Ultimately, our goal was and is to move into markets that are approved at the federal regulatory side of the spectrum, and that is where we are now.
Green: What year was that?
Green: So, PathogenDx started in cannabis testing?
Patel: Yes, we started in cannabis testing. We now have over 100 labs that are using the technology. There is a specific need in cannabis when you’re looking at contamination or infection.
In the case of contamination on cannabis, you must look for bacterial and fungal organisms that make it unsafe, such as E. coli, or Salmonella or Aspergillus pathogens. We’re familiar with recent issues like the romaine lettuce foodborne illness outbreaks at Chipotle. In the case of fungal organisms such as Aspergillus, if you smoke or consume contaminated cannabis, it could have a huge impact on your health. Cannabis regulators realized that to ensure public health and safety there was more than just one pathogen – there were half a dozen of these bugs, at a minimum, that could be harmful to you.
The beauty of our technology, using a Microarray is that we can do what is called a multiplex test, which means you’re able to test for all bacterial and fungal pathogens in a single test, as opposed to the old “Adam Smith” model, which tests each pathogen on a one-by-one basis. The traditional approach is costly, time consuming and cumbersome. Cannabis is such a high value crop and producers need to get the answer quickly. Our tests can give a result in six hours on the same day, as opposed to the two or three days that it takes for these other approved methods on the market.
Green: What is your business model? Is there equipment in addition to consumables?
Patel: Our business model is the classic razor blade model. What that means is we sell equipment as well as the consumables – the testing kits themselves.
The PathogenDx technology uses standard, off-the-shelf lab equipment that you can find anywhere. We didn’t want to make the equipment proprietary so that a lab has to buy a specific OEM branded product. They can use almost any equipment that’s available commercially. We wanted to make sure that labs are only paying a fraction of the cost to get our equipment, as opposed to using other vendors. Secondly, the platform is open-ended, meaning it’s highly flexible to work with the volumes that different cannabis labs see daily, from high to low.
One equipment set can process many different types of testing kits. There are kits for regulated testing required by states, as well as required environmental contamination.
Green: Do you provide any in-house or reference lab testing?
Patel: We do. We have a CLIA lab for clinical testing. We did this about a year ago when we started doing COVID testing.
We don’t do any kind of in-house reference testing for cannabis, though we do use specific reference materials or standards from Emerald Scientific, for example, or from NCI. Our platform is all externally third-party reference lab tested whether it’s validated by our external cannabis lab customers or an independent lab. We want our customers to make sure that the actual test works in their own hands, in their own facility by their own people, as opposed to just shrugging our shoulders and saying, “hey, we’ve done it ourselves, believe us.” That’s the difference.
Green: Can you explain the difference between qPCR and endpoint PCR?
Patel: The difference between PathogenDx’s Microarray is it uses endpoint PCR versus qPCR (quantitative real time PCR). Effectively, our test doesn’t need to be enriched. Endpoint PCR delivers a higher level of accuracy, because when it goes to amplify that target DNA, whether it’s E. coli, Salmonella or Aspergillus pieces, it uses all the primer reagent to its endpoint. So, it amplifies every single piece of an E. Coli (for example) in that sample until the primer is fully consumed. In the case of qPCR, it basically reaches a threshold and then the reaction stops. That’s the difference which results in a much greater level of accuracy. This provides almost 10 times greater sensitivity to identify the pathogen in that sample.
The second thing is that we have separated out how the amplified sample hybridizes to the probe. In the case of our assay, we have a microarray with a well in it and we printed the actual probe that has the sequence of E. coli in there, now driving 100% specificity. Whereas in the qPCR, the reaction is not only amplifying, but it’s also basically working with the probe. So, in that way, we have a higher level of efficiency in terms of specificity. You get a definite answer exactly in terms of the organism you’re looking for.
In terms of an analogy, let’s take a zip code for example which has the extra four digits at the end of it. In the case of endpoint PCR, we have nine digits. We have our primer probes which represent the standard five digits of a zip code, and the physical location of the probe itself in the well which serves as the extra four digits of that zip code. The analyte must match both primary and secondary parts of the nine-digit zip code for it to lock in, like a key and a lock. And that’s the way our technology works in a nutshell.
Endpoint PCR is completely different. It drives higher levels of accuracy and specificity while reducing the turnaround time compared to qPCR – down to six hours from sample to result. In qPCR, you must enrich the sample for 24 to 48 hours, depending on bacteria or fungus, and then amplification and PCR analysis can be done in one to three hours. The accuracies and the turnaround times are the major differences between the endpoint PCR and qPCR.
Green: If I understand correctly, it’s a printed microarray in the well plate?
Patel: That’s correct. It’s a 96-well plate, and in each well, you’ve now printed all the probes for all targets in a single well. So, you’re not running more than one well per target, or per organism like you are for qPCR. You’re running just one well for all organisms. With our well plates, you’re consuming fewer wells and our patented foil-cover, you only use the wells you need. The unused wells in the well plate can be used in future tests, saving on costs and labor.
Green: Do you have any other differentiating IP?
Patel: The multiplex is the core IP. The way we process the raw sample, whether it’s flower or non-flower, without the need for enrichment is another part of the core IP. We do triplicate probes in each well for E. Coli, triplicate probes for Salmonella, etc., so there are three probes per targeted organism in each of the wells. We’re triple checking that you’re definitively identifying that bug at the end of the day. This is the cornerstone of our technology.
We were just approved by the State of New York, and the New York Department of Health has 13 different organisms for testing on cannabis. Think about it: one of the most rigorous testing requirements at a state level – maybe even at a federal level – and we just got approved for that. If you had to do 13 organisms separately, whether it’s plate culture or qPCR, it would become super expensive and very difficult. It would break the very backs of every testing lab to do that. That’s where the multiplexing becomes tremendously valuable because what you’re doing is leveraging the ability to do everything as a single test and single reaction.
Green: You mentioned New York. What other geographies are you active in?
Patel: We’re active in 26 different states including the major cannabis players: Florida, Nevada, California, Arizona, Michigan, New York, Oklahoma, Colorado and Washington – and we’re also in Canada. We’re currently working to enter other markets, but it all comes down to navigating the regulatory process and getting approval.
We’re not active currently in other international markets yet. We’re currently going through the AOAC approval process for our technology and I’m happy to say that we’re close to getting that in the next couple of months. Beyond that, I think we’ll scale more internationally.
I am delighted to say that we also got FDA EUA federal level authorization of our technology which drives significant credibility and confidence for the use of the technology. About a year ago, we made a conscious choice to make this technology federally acceptable by going into the COVID testing market. We got the FDA EUA back on April 20, ironically. That vote of confidence by the FDA means that our technology is capable of human testing. That has helped to create some runway in terms of getting federalized with both the FDA and the USDA, and certification by AOAC for our different tests.
Green: Was that COVID-19 EUA for clinical diagnostics or surveillance?
Patel: It was for clinical diagnostics, so it’s an actual human diagnostic test.
Green: Last couple of questions here. Once you find something as a cannabis operator, whether its bacteria or fungus, what can you do?
Patel: There are many services that are tied into our ecosystem. For example, we work with Willow Industries, who does remediation.
There’s been a lot of criticism around DNA based technology. It doesn’t matter if it’s qPCR or endpoint PCR. They say, “well, you’re also including dead organisms, dead DNA.” We do have a component of separating live versus dead DNA with a biomechanical process, using an enzyme that we’ve created, and it’s available commercially. Labs can test for whether a pathogen is living or dead and, in many cases, when they find it, they can partner with remediation companies to help address the issue at the grower level.
Another product we offer is an EnviroX test, which is an environmental test of air and surfaces. These have 50 pathogens in a single well. Think about this: these are all the bad actors that typically grow where soil is – the human pathogens, plant pathogens, powdery mildew, Botrytis, Fusarium – these are very problematic for the thousands of growers out there. The idea is to help them with screening technology before samples are pulled off the canopy and go to a regulated lab. We can help the growers isolate where that contamination is in that facility, then the remediation companies can come in, and help them save their crop and avoid economic losses.
Green: What are you most interested in learning about?
Patel: I would prefer that the cannabis industry not go through the same mistakes other industries have gone through. Cannabis started as a cottage industry. It’s obviously doubled every year, and as it gets scaled, the big corporations come in. Sophistication, standards, maturity all help in legitimacy of a business and image of an industry. At the end of the day, we have an opportunity to learn from other industries to really leapfrog and not have to go through the same mistakes. That’s one of the things that’s important to me. I’m very passionate about it.
One thing that I’ll leave you with is this: we’re dealing with more bugs in cannabis than the food industry. The food industry is only dealing with two to four bugs and look at the number of recalls they are navigating – and this is a multi-billion-dollar industry. Cannabis is still a fraction of that and we’re dealing with more bugs. We want to look ahead and avoid these recalls. How do you avoid some of the challenges around antimicrobial resistance and antibiotic resistance? We don’t want to be going down that road if we can avoid it and that’s sort of a personal mission for myself and the company.
Cannabis itself is so powerful, both medicinally as well as recreationally, and it can be beneficial for both consumers and industry image if we do the right things, and avoid future disasters, like the vaping crisis we went through 18 months ago because of bad GMPs. We must learn from those industries. We’re trying to make it better for the right reasons and that’s what’s important to me.
Green: Okay, great. That concludes the interview. Thank you, Milan.
Patel: Thank you for allowing me to share my thoughts and your time, Aaron.
As the cannabis market matures and the value chain becomes modernized, it’s important to address product safety in a comprehensive way. In other areas of manufacturing, Hazard Analysis & Critical Control Points (HACCP) has been the standard for reducing hazards both for employees and for the products themselves. A Critical Control Point (CCP) is any spot from conception to consumption where a loss of control can potentially result in risk (Unnevehr, 1996). In the food realm, HACCP has been used to drive quality enhancements since the 1980s (Cichy, 1982).
In a nutshell, HACCP seeks to help identify where a problem may enter a product or environment and how that problem may be addressed before it escalates. In cannabis, these hazards include many of the same problems that food products have: specifically molds, yeasts, and pathogenic bacteria (Listeria, E. coli, etc.). While the current industry standard is to test products at the end stage for these contaminants, this late-stage pass/fail regimen leads to huge lots of destroyed product and a risk for consumer distrust (Yamashiro, 2019). HACCP, therefore, should be applied at every stage of the production process.
Pathogen Environmental Monitoring (PEM) is a tool that can be used to identify CCPs in a cannabis cultivation or processing facility. The main goal of a PEM program is to find a contaminant before it reaches a surface that touches the product or the product itself. PEM is conducted using a pre-moistened swab or a sponge to collect a sample from the cannabis environment. The swab can then be sent to a lab for microbial testing. Keys to an effective PEM are:
1. Start with a broad stroke – When the FDA comes to a facility suspected of producing pathogen-laced food products, they conduct what is known as a Swab-a-thon. A Swab-a-thon is a top to bottom collection of samples, usually totaling 100 or more. Similarly, preemptively swabbing should be the first step in any PEM—swab everything to see what exists as a baseline.
2. Map your scene – identify on a map of your facility the following:
Flow of air and people (where do air and people enter and where do they go?
Identifying the above zones will help deepen your understanding of where contaminants may come into contact with cannabis and how they may migrate from a Non-CCS to a CCS.
3. Plan and execute:
Based on the results of mapping, and Swab-a-thon, identify where and when you will be collecting samples on a consistent and repeatable basis. Emphasis should be placed on areas that are deemed a risk based on 1) and 2). Samples should be collected at random in all zones to ensure comprehensive screening.
4. Remediate and modify:
If you get a positive result during PEM, don’t panic—pathogens are ubiquitous.
Remediate any trouble spots with deep cleaning, remediation devices or other protocols.
Re-test areas that were positive for pathogens to ensure remediation is successful.
Revisit and modify the plan at least once a year and each time a new piece of equipment is added or production flow is otherwise changed.
The steps above are a good starting point for a grower or processor to begin a PEM. Remember that this is not a one-size-fits-all approach to safety; each facility has its own unique set of hazards and control points.
Comprehensive guides for PEM can be found at the links below, many of the concepts can be applied to cannabis production.
You’ve survived seasons of cannabis cultivations, bringing in quality plants in spite of mold, mites, drought and other challenges that had to be conquered. Extraction methods are sometimes challenging, but you are proud to have a cannabinoid extract that can be added into your own products for sale. Edibles are just waiting to be infused with the cannabinoids, for consumers demanding brownies, gummies, tinctures and almost any food and beverage imaginable. You’ve been through the fire, and now the rest is easy peasy, right?
Actually, producing edibles may not be so seamless as you think. Just as in the rest of the food industry, food safety practices have to be considered when you’re producing edibles for public consumption, regardless of the THC, CBD, terpene or cannabinoid profile. Once you’ve acquired the extract (a “food grade ingredient”) containing the active compounds, there are three types of hazards that could still contribute to foodborne illness from your final product if you’re not careful- Biological, Chemical and Physical.
Biological hazards include pathogenic bacteria, viruses, mold, mildew (and the toxins that they can produce) that can come in ingredients naturally or contaminate foods from an outside source. Chemical hazards are often present in the kitchen environment, including detergents, floor cleaners, disinfectants and caustic chemicals, which can be harmful if ingested- they are not destroyed through cooking. Physical objects abound in food production facilities, including plastic bits, metal fragments from equipment, staples or twist ties from ingredient packages, and personal objects (e.g., buttons, jewelry, hair, nails.)
There are three main safety precautions that can help control these hazards during all the stages of food production, from receiving ingredients to packaging your final products:
1. Avoid Cross Contamination
Prevent biological, chemical or physical hazards from coming into contact with foods
Keep equipment, utensils and work surfaces clean and sanitized.
Prevent raw foods (as they usually carry bacteria) from coming into contact with “Ready-to-eat” foods (foods that will not be cooked further before consuming).
Keep chemicals away from food areas.
2. Personal Hygiene
Don’t work around foods if you’re sick with fever, vomiting or diarrhea. These could be signs of contagious illness and can contaminate foods or other staff, and contribute to an outbreak.
Do not handle ready-to-eat foods with bare hands, but use a barrier such as utensils, tissues or gloves when handling final products such as pastries or candies.
Wash hands and change gloves when soiled or contaminated.
Wear hair restraints and clean uniforms, and remove jewelry from hands and arms.
3. Time & Temperature control
Prevent bacterial growth in perishable foods such as eggs, dairy, meats, chicken (TCS “Time and Temperature Control for Safety” foods according to the FDA Model Food Code) by keeping cold foods cold and hot foods hot.
Refrigerate TCS foods at 41˚ F or below, and cook TCS foods to proper internal temperatures to kill bacteria to safe levels, per state regulations for retail food establishments.
If TCS foods have been exposed to room temperature for longer than four hours (Temperature Danger Zone 41˚ F – 135˚ F,) these foods should be discarded, as bacteria could have grown to dangerous levels during this time.
As cannabis companies strive for acceptance and legalization on a federal level, adopting these food safety practices and staff training is a major step in the right direction, on par with standards maintained by the rest of the retail food industry. The only difference is your one specially extracted cannabinoid ingredient that separates you from the rest of the crowd… with safe and healthy edibles for all.
With legalization rapidly increasing across states, the cannabis market is exploding. And with estimates of sales in the billions, it’s no surprise that greenhouses and grow rooms are emerging everywhere. As growers and extracting facilities continue to expand one important consideration that most tend to underestimate, is how flooring can impact both their production and product. Bare concrete is often a popular choice in cannabis facilities, as there are typically very minimal costs−if any at all−associated with preparing it for use. However, concrete floors can pose unique challenges when left untreated, which could inadvertently create unforeseen problems and unexpected costs.
Understanding the Risks of Bare Concrete Flooring
Whether a facility is growing or extracting, the proper flooring can play a critical role in helping maintain optimal safety and sanitation standards, while simultaneously contributing to production. That’s why its important for growers and extractors to know and understand the potential risks associated with bare concrete.
Concrete is porous: While concrete is a solid material, people may forget that it is porous. Unfortunately, these pores can absorb liquids and harbor small particles that spill on the floor. They create perfect hiding places for bacteria and other pathogens to proliferate. Pathogens can then contaminate product within the facility, causing a halt on production, and/or a potential product recall. This can incur unexpected costs associated with shutdown time and loss of product.
Concrete can be damp: When in a facility with an untreated concrete floor, at times the slab can feel slightly wet or damp to touch. This is due to moisture within the concrete that can eventually work its way up to the surface of the slab. When this happens, items that are placed on top of the floor can be damaged by trapped moisture above the slab and below the object. When this happens, if a product is not protected properly, it can be damaged.
Concrete is dark and unreflective: An untreated concrete slab can often make a room feel dark and it does not reflect lighting within the room. This can result in the need for extra lights and electricity to properly grow cannabis.
Concrete lacks texture: When working in areas where water and other liquids can fall to the ground and accumulate, flooring with traction can play a key role in helping aid against slip and fall incidents. Untreated concrete typically does not provide sufficient texture and can become very slippery when wet.
The Benefits of Bare Concrete Flooring
While the previously mentioned risks can be associated with bare concrete flooring, there is an upside to the situation! Concrete is the perfect substrate for adding a coating that is built to withstand the industry’s demands.
With the application of a fluid-applied or resinous floor coating, the risks of bare concrete flooring can be mitigated. There are a variety of resin and fluid-based coating systems that can be applied, such as:
Epoxy and Urethane Systems
Urethane Mortar Systems
Decorative Quartz Systems
Decorative Flake Systems
These durable coatings have numerous benefits and can offer:
Protection against the proliferation bacteria and other pathogens: Unlike porous concrete, a smooth and virtually seamless floor coating eliminates the little crevices where pathogens can grow. This in turn helps aid against the growth of bacteria, keeping hygiene standards at the forefront and grow rooms in full operations.
Protection against moisture damage: As moisture within the concrete can move upward to the surface of the slab, there are moisture mitigation coating systems, that keep it trapped below the surface, thus helping toprotect items placed on the floor.
Brighter spaces and light reflection: Installing a floor coating that is light in color, such as white or light gray, can help brighten any space. The benefits of this are twofold: First, it can help with visibility, helping employees navigate the space safely. Secondly, light reflectivity of the flooring improves lighting efficiency, resulting in fewer light fixtures and smaller electric costs.
Texture options to help aid against slip and fall incidents: Floor coating systems can offer a variety of texture options−from light grit to heavy grit−depending on how much accumulated water and foot traffic the area receives. Without additional texture in wet areas, slip and fall incidents and injuries are inevitable.
A wide range of colors and decorative systems: These coating systems can be designed to match the aesthetics of the building or corporate colors. Some manufacturers even offer color matching upon request. When it comes to colors, the options are virtually endless.
Choosing the Right Flooring: Considering Bare Concrete
Choosing the right flooring for a cannabis greenhouse or processing facility requires important consideration as every grow room and greenhouse is different. Bare concrete is a popular flooring option for manufacturing and processing facilities across industries, however, as discussed, it can pose unique challenges due to its innate nature. That said, by taking the right steps to ensure that the concrete substrate is properly sealed, it can then be an effective and hygienic flooring option, offering high durability and a longer life cycle.
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.”
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.
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:
Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
Drop sample in solution. Swab should already be in solution
Transfer 1ml of solution into 1.5ml tube
Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
Conduct Loci enhancement PCR of sample for 1 hour
Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
Conduct wash cycle for 15 minutes
Dry and image the slide in imager
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.
EVIO Labs Florida received their ISO 17025:2005 accreditation in February of 2018. Last week, EVIO Labs Florida announced via a press release that they completed their ISO 17025:2017 accreditation and received a certification from AOAC International. The accreditation helped them to further expand their testing scope to shelf life and stability testing, the ability to detect harmful bacteria and calculate degradation in samples.
The certification that they received from AOAC helps verify their ability to conduct accurate and fair 3rd party testing, meeting Florida’s requirements for the market. Back when the laboratory first started in 2017, there were no requirements for lab testing cannabis products under Florida’s regulations.
Upon expanding to their Gainesville location in November last year and getting accredited to ISO 17025:2017 last week, EVIO Labs Florida expects the new location to be compliant and operational by April 2019, in preparation for the state’s new regulations. “Our team has worked diligently to maintain our stance as the Gold Standard in Cannabis Testing,” says Chris Martinez, co-founder and president of EVIO Lab Florida. “The ability to obtain the recent ISO 17025:2017 and AOAC certification is a testament to our dedication in maintaining public safety and product integrity in an ever-growing industry.”
Martinez is also presenting during the 2ndAnnual Cannabis Labs Virtual Conference on April 2, where he will discuss how EVIO Labs Florida began as a laboratory and how they were able to expand to a second location and grow their market presence in Florida. Click here to register for his talk.
The cannabis industry is probably more informed about patients and consumers of their products than the general food industry. In addition to routine illness and stress in the population, cannabis consumers are fighting cancer, HIV/AIDS and other immune disorders. Consumers who are already ill are immunocompromised. Transplant recipients purposely have their immune system suppressed in the process of a successful transplant. These consumers have pre-existing conditions where the immune system is weakened. If the immunocompromised consumer is exposed to viral or bacterial pathogens through cannabis products, the consumer is more likely to suffer from a viral infection or foodborne illness as a secondary illness to the primary illness. In the case of consumers with weakened immune systems, it could literally kill them.Bacteria, yeast, and mold are present in all environments.
The cannabis industry shoulders great responsibility in both the medical and adult use markets. In addition to avoiding chemical hazards and determining the potency of the product, the cannabis industry must manufacture products safe for consumption. There are three ways to control pathogens and ensure a safe product: prevent them from entering, kill them and control their growth.
Prevent microorganisms from getting in
Think about everything that is outdoors that will physically come in a door to your facility. Control the quality of ingredients, packaging, equipment lubricants, cleaning agents and sanitizers. Monitor employee hygiene. Next, you control everything within your walls: employees, materials, supplies, equipment and the environment. You control receiving, employee entrance, storage, manufacturing, packaging and distribution. At every step in the process, your job is to prevent the transfer of pathogens into the product from these sources.
The combination of raw materials to manufacture your product is likely to include naturally occurring pathogens. Traditional heat methods like roasting and baking will kill most pathogens. Remember, sterility is not the goal. The concern is that a manufacturer uses heat to achieve organoleptic qualities like color and texture, but the combination of time and temperature may not achieve safety. It is only with a validated process that safety is confirmed. If we model safety after what is required of food manufacturers by the Food and Drug Administration, validation of processes that control pathogens is required. In addition to traditional heat methods, non-thermal methods for control of pathogens includes irradiation and high pressure processing and are appropriate for highly priced goods, e.g. juice. Killing is achieved in the manufacturing environment and on processing equipment surfaces after cleaning and by sanitizing.
If you have done everything reasonable to stop microorganisms from getting in the product and you have a validated step to kill pathogens, you may still have spoilage microorganisms in the product. It is important that all pathogens have been eliminated. Examples of pathogens include Salmonella, pathogenic Escherichia coli, also called Shiga toxin-producing E. coli (STEC) and Listeria monocytogenes. These three common pathogens are easily destroyed by proper heat methods. Despite steps taken to kill pathogens, it is theoretically possible a pathogen is reintroduced after the kill step and before packaging is sealed at very low numbers in the product. Doctors do not know how many cells are required for a consumer to get ill, and the immunocompromised consumer is more susceptible to illness. Lab methods for the three pathogens mentioned are designed to detect very low cell numbers. Packaging and control of growth factors will stop pathogens from growing in the product, if present.
Control the growth of microorganisms
These growth factors will control the growth of pathogens, and you can use the factors to control spoilage microbes as well. To grow, microbes need the same things we do: a comfortable temperature, water, nutrients (food), oxygen, and a comfortable level of acid. In the lab, we want to find the pathogen, so we optimize these factors for growth. When you control growth in your product, one hurdle may be enough to stop growth; sometimes multiple hurdles are needed in combination. Bacteria, yeast, and mold are present in all environments. They are at the bottom of the ocean under pressure. They are in hot springs at the temperature of boiling water. The diversity is immense. Luckily, we can focus on the growth factors for human pathogens, like Salmonella, pathogenic E. coli, and Listeria monocytogenes.
Temperature. Human pathogens prefer to grow at the temperature of the human body. In manufacture, keep the time a product is in the range of 40oF to 140oF as short as possible. You control pathogens when your product is at very hot or very cold temperatures. Once the product cools after a kill step in manufacturing, it is critical to not reintroduce a pathogen from the environment or personnel. Clean equipment and packaging play key roles in preventing re-contamination of the product.
Water. At high temperatures as in baking or roasting, there is killing, but there is also the removal of water. In the drying process that is not at high temperature, water is removed to stop the growth of mold. This one hurdle is all that is needed. Even before mold is controlled, bacterial and yeast growth will stop. Many cannabis candies are safe, because water is not available for pathogen growth. Packaging is key to keep moisture out of the product.
Nutrients. In general, nutrients are going to be available for pathogen growth and cannot be controlled. In most products nutrients cannot be removed, however, recipes can be adjusted. Recipes for processed food add preservatives to control growth. In cannabis as in many plants, there may be natural compounds which act as preservatives.
Oxygen. With the great diversity of bacteria, there are bacteria that require the same oxygen we breathe, and mold only grows in oxygen. There are bacteria that only grow in the absence of oxygen, e.g. the bacteria responsible for botulism. And then there are the bacteria and yeast in between, growing with or without oxygen. Unfortunately, most human pathogens will grow with or without oxygen, but slowly without oxygen. The latter describes the growth of Salmonella, E. coli, and Listeria. While a package seals out air, the growth is very slow. Once a package is opened and the product is exposed to air, growth accelerates.
Acid. Fermented or acidified products have a higher level of acid than non-acid products; the acid acts as a natural preservative. The more acid, the more growth is inhibited. Generally, acid is a hurdle to growth, however and because of diversity, some bacteria prefer acid, like probiotics which are non-pathogenic. Some pathogens, like E. coli, have been found to grow in low acid foods, e.g. juice, even though the preference is for non-acidic environments.
I have been studying microorganisms for over 35 years, and the elusive critters still fascinate me! Here in Microbiology 101, I write about the foundation of knowledge on which all microbiologists build. You may have a general interest in microbiology or have concerns in your operation. By understanding microbiology, you understand the diversity of microorganisms, their source, control of microorganisms and their importance.
The term microbiology covers every living being we cannot see with the naked eye. The smallest microbe is a virus. Next in size are the bacteria, then yeast and mold cells, and the largest microbes are the protozoans. The tiny structure of a virus may be important in the plant pathology of cannabis, but will not grow in concentrates or infused products. A virus is not living, until it storms the gate of a living cell and overtakes the functions within the cell. Viruses are the number one cause of foodborne illness, with the number one virus called Norovirus. Think stomach flu. Think illness on cruise ships. Viruses are a food service problem and can be prevented by requiring employees to report sickness, have good personal hygiene including good hand washing, and, as appropriate, wear gloves. Following Good Manufacturing Practices (GMPs) is critical in preventing the transfer of viruses to a product where the consumer can be infected.
The largest microbial cell is the protozoan. They are of concern in natural water sources, but like viruses, will not grow in cannabis products. Control water quality through GMPs, and you control protozoans. Viruses and protozoans will not be further discussed here. Bacteria, yeast and mold are the focus of further discussion. As a food microbiologist, my typical application of this information is in the manufacturing of food. Because Microbiology 101 is a general article on microbiology, you can apply the information to growing, harvesting, drying, manufacture of infused products and dispensing.
It is not possible to have sterile products. Even the canning process of high temperature for an extended time allows the survival of resistant bacterial spores. Astronauts take dehydrated food into space, and soldiers receive MREs; both still contain microbes. Sterility is never the goal. So, what is normal? Even with the highest standards, it is normal to have microbes in your products. Your goal is to eliminate illness-causing microorganisms, i.e. pathogens. Along the way, you will decrease spoilage microbes too, making a product with higher quality.
Yeast and mold were discussed on CIJ in a previous article, Total Yeast & Mold Count: What Cultivators & Business Owners Need to Know. Fuzzy mold seen on the top of food left in the refrigerator too long is a quality issue, not a safety issue. Mold growth is a problem on damaged cannabis plants or cuttings and may produce mycotoxin, a toxic chemical hazard. Following Good Agricultural Practices (GAPs) will control mold growth. Once the plant is properly dried, mold will not grow and produce toxin. Proper growing, handling and drying prevents mycotoxins. Like mold, growth of yeast is a quality issue, not a safety issue. As yeast grow, they produce acid, alcohol and carbon dioxide gas. While these fermentation products are unwanted, they are not injurious. I am aware that some states require cannabis-infused products to be alcohol-free, but that is not a safety issue discussed here.
What are the sources of microorganisms?
People. Employees who harvest cannabis may transfer microorganisms to the plant. Later, employees may be the source of microbes at the steps of trimming, drying, transfer or portioning, extract processing, infused product manufacture and packaging.
Ingredients, Supplies and Materials. Anything you purchase may be a source of microorganisms. Procure quality merchandise. Remember the saying, “you get what you pay for.”
Environment. Starting with the outdoors, microbes come from wind, soil, pests, bird droppings and water. When plants are harvested outdoors or indoors, microbes come from the tools and bins. Maintain clean growing and harvesting tools in good working condition to minimize contamination with microbes. For any processing, microbes come from air currents, use of water, and all surfaces in the processing environment from dripping overhead pipes to floor drains and everything in between.
In Part 2 I will continue to discuss the diversity of microorganisms, and future articles will cover Hazard Analysis and Critical Control Points (HACCP) and food safety in more detail. What concerns do you have at each step of operations? Are you confident in your employees and their handling of the product? As each state works to ensure public health, cannabis-infused products will receive the same, if not more, scrutiny as non-cannabis food and beverages. With an understanding and control of pathogens, you can focus on providing your customers with your highest quality product.
Editor’s note: This article should serve as a foundation of knowledge for yeast and mold in cannabis. Beginning in January 2018, we will publish a series of articles focused entirely on yeast and mold, discussing topics such as TYMC testing, preventing yeast and mold in cultivation and treatment methods to reduce yeast and mold.
Cannabis stakeholders, including cultivators, extractors, brokers, distributors and consumers, have been active in the shadows for decades. With the legalization of recreational adult use in several states, and more on the way, safety of the distributed product is one of the main concerns for regulators and the public. Currently, Colorado1, Nevada and Canada2 require total yeast and mold count (TYMC) compliance testing to evaluate whether or not cannabis is safe for human consumption. As the cannabis industry matures, it is likely that TYMC or other stringent testing for yeast and mold will be adopted in the increasingly regulated medical and recreational markets.
The goal of this article is to provide general information on yeast and mold, and to explain why TYMC is an important indicator in determining cannabis safety.
Yeast & Mold
Yeast and mold are members of the fungi family. Fungus, widespread in nature, can be found in the air, water, soil, vegetation and in decaying matter. The types of fungus found in different geographic regions vary based upon humidity, soil and other environmental conditions. In general, fungi can grow in a wide range of pH environments and temperatures, and can survive in harsh conditions that bacteria cannot. They are not able to produce their own food like plants, and survive by breaking down material from their surroundings into nutrients. Mold cannot thrive in an environment with limited oxygen, while yeast is able to grow with or without oxygen. Most molds, if grown for a long enough period, can be detected visually, while yeast growth is usually detected by off-flavor and fermentation.
Due to their versatility, it is rare to find a place or surface that is naturally free of fungi or their spores. Damp conditions, poor air quality and darker areas are inviting environments for yeast and mold growth.
Cannabis plants are grown in both indoor and outdoor conditions. Plants grown outdoors are exposed to wider ranges and larger populations of fungal species compared to indoor plants. However, factors such as improper watering, the type of soil and fertilizer and poor air circulation can all increase the chance of mold growth in indoor environments. Moreover, secondary contamination is a prevalent risk from human handling during harvest and trimming for both indoor and outdoor-grown cannabis. If humidity and temperature levels of drying and curing rooms are not carefully controlled, the final product could also easily develop fungi or their growth by-product.
What is TYMC?
TYMC, or total yeast and mold count, is the number of colony forming units present per gram of product (CFU/g). A colony forming unit is the scientific means of counting and reporting the population of live bacteria or yeast and mold in a product. To determine the count, the cannabis sample is plated on a petri dish which is then incubated at a specific temperature for three to five days. During this time, the yeast and mold present will grow and reproduce. Each colony, which represents an individual or a group of yeast and mold, produces one spot on the petri dish. Each spot is considered one colony forming unit.
Why is TYMC Measured?
TYMC is an indicator of the overall cleanliness of the product’s life cycle: growing environment, processing conditions, material handling and storage facilities. Mold by itself is not considered “bad,” but having a high mold count, as measured by TYMC, is alarming and could be detrimental to both consumers and cultivators.
The vast majority of mold and yeast present in the environment are indeed harmless, and even useful to humans. Some fungi are used commercially in production of fermented food, industrial alcohol, biodegradation of waste material and the production of antibiotics and enzymes, such as penicillin and proteases. However, certain fungi cause food spoilage and the production of mycotoxin, a fungal growth by-product that is toxic to humans and animals. Humans absorb mycotoxins through inhalation, skin contact and ingestion. Unfortunately, mycotoxins are very stable and withstand both freezing and cooking temperatures. One way to reduce mycotoxin levels in a product is to have a low TYMC.
Yeast and mold have been found to be prevalent in cannabis in both current and previous case studies. In a 2017 UC Davis study, 20 marijuana samples obtained from Northern California dispensaries were found to contain several yeast and mold species, including Cryptococcus, Mucor, Aspergillus fumigatus, Aspergillus niger, and Aspergillus flavus.3 The same results were reported in 1983, when marijuana samples collected from 14 cannabis smokers were analyzed. All of the above mold species in the 2017 study were present in 13 out of 14 marijuana samples.4
Aspergillus species niger, flavus, and fumigatus are known for aflatoxin production, a type of dangerous mycotoxin that can be lethal.5 Once a patient smokes and/or ingests cannabis with mold, the toxins and/or spores can thrive inside the lungs and body.6, 7 There are documented fatalities and complications in immunocompromised patients smoking cannabis with mold, including patients with HIV and other autoimmune diseases, as well as the elderly.8, 9, 10, 11
For this reason, regulations exist to limit the allowable TYMC counts for purposes of protecting consumer safety. At the time of writing this article, the acceptable limit for TYMC in cannabis plant material in Colorado, Nevada and Canada is ≤10,000 CFU/g. Washington state requires a mycotoxin test.12 California is looking into testing for specific Aspergillus species as a part of their requirement. As the cannabis industry continues to grow and advance, it is likely that additional states will adopt some form of TYMC testing into their regulatory testing requirements.
Centre for Disease control and prevention. 2004 Outbreak of Aflatoxin Poisoning – Eastern and central provinces, Kenya, Jan – July 2004. Morbidity and mortality weekly report.. Sep 3, 2004: 53(34): 790-793
Cescon DW, Page AV, Richardson S, Moore MJ, Boerner S, Gold WL. 2008. Invasive pulmonary Aspergillosis associated with marijuana use in a man with colorectal cancer. Diagnosis in Oncology. 26(13): 2214-2215.
Szyper-Kravits M, Lang R, Manor Y, Lahav M. 2001 Early invasive pulmonary aspergillosis in a leukemia patient linked to aspergillus contaminated marijuana smoking. Leukemia Lymphoma 42(6): 1433 – 1437.
Verweii PE, Kerremans JJ, Voss A, F.G. Meis M. 2000. Fungal contamination of Tobacco and Marijuana. JAMA 2000 284(22): 2875.
Ruchlemer R, Amit-Kohn M, Raveh D, Hanus L. 2015. Inhaled medicinal cannabis and the immunocompromised patient. Support Care Cancer. 23(3):819-822.
McPartland JM, Pruitt PL. 1997. Medical Marijuana and its use by the immunocompromised. Alternative Therapies in Health and Medicine. 3 (3): 39-45.
Hamadeh R, Ardehali A, Locksley RM, York MK. 1983. Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient. Chest. 94(2): 432-433.
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