Tag Archives: bacterial

Building An Integrated Pest Management Plan – Part 2

By Phil Gibson
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This is the second part of a series of articles designed to introduce an integrated pest management framework for cannabis cultivation facilities. To see Part One, click here. Part Three comes out next week and covers prioritization and preventative measures. Stay tuned for more!

This is Part 2: Pest Monitoring, Record Keeping, & Communications

Begin your pest identification process with a pest scouting document. You have already mapped out your facility with locations and potential access locations. For each of these pest types and room type assignments (mothers, clone, veg, flower), identify your employee scouts, their scouting methods, scouting frequency and the type of likely pest they are to search for and count.

Insect Types and Tracking Methods

Figure 1: Example Sticky Trap Scouting Map

Insect pest types include, but are not limited to, airborne flying or crawling insects, their various egg, lymph, larvae, pupal shells or immature forms. Look for trace remnants, plant damage or feces that let you know they are present in some form. If they are at the mature jumping or flying stage, this can be harder to count, but sticky traps distributed on an even basis around your rooms can make the counting process more consistent from survey to survey.

Note airflows in your rooms and fan locations so migrations can be predicted once an infestation is located.

Insects Can Be Everywhere – Crawlers & Fliers

Insects would like to be everywhere so they come in many types from the obvious flying and crawling types to root-zone microscopic, aquatic, fungal, bacterial or biofilm based. For those of you using soil or media, root-zone insects can be beneficial by digesting and breaking down organic matter into something useful for your plant’s roots (earthworms) or harmful by feeding directly on your plant roots and sucking the life out of your plants from out-of-sight below (nematodes, maggots).

Common pests in a cannabis environment include:

  • White flies – Oval shaped eggs on the underside of leaves, nymphs- oval crawlers that suck on the undersides of leaves, larger stage nymphs with pupae shells as they form wings and mature white flies.
  • Fungus gnats – Clear eggs deposited in overly wet soil or dead plant matter. Clear or white colored larvae in the soil or media, these worm-like critters go through multiple stages of molting as they grow, eventually pupating into brown cocoons and finally small black or dark flies with clear wings that flutter around your plants and suck on your leaves.
  • The dreaded spider mite – Clear, hard to see eggs on the underside of your leaves. These six-legged tiny moving bubbles begin the feeding as larva, add 2 legs in the intermediate and mature nymph stages and finally the oval shaped spider mites that every grower despises, adding their webs around the tops of your plants as their nurseries suck the life out of your flowers.

Insect Transfers of Bacterial Infections

Figure 2: The Dreaded Spider Mite

Many crawlers or fliers you may discover in your grow operation do not generate fungus or bacteria on their own. However, they do routinely pick these up along the feeding way and bring them into your shop. Sap-feeding insects like leafhoppers and aphids use their needle mouths to pierce your leaves to suck on the sap that is nourishing your greenery. These insects consume the fluids and transfer bacteria as they feed. Whiteflies fit into this category of leaf sucking bacteria carrying pests. These pests can make your healthy grow rooms look blotchy with color drained out of your canopy.

Obvious symptoms of these flying/hopping pests are sticky leaves, black fungus mold, or yellowing leaves that show up at the bottom of your plants and work their way upward as the infestation progresses. Leaf curling or plant wilting will be visible in the more advanced stages of these pests.

As if crawlers were not bad enough, invisible fungus and bacteria that get into your water supplies can be the worst challenges of any grow.

Water Sourced Bacteria

Baseline testing of your feed water is critical for any facility. This is true whether you are using surface water, well water or municipal water. Please see the water tutorials on the AEssenseGrows website for details on how to test your water sources and what to look for in the mineral content.

Regardless of your water source, bacteria can be present directly in your water supply, or it can be introduced from infected plant materials from one of your suppliers. Pythium, fusarium and the latest plague, hop latent viroid, are some of the most common threats that attack your plants from your water or soil sources. These can come from your wells, feed lines or plant materials.

Reverse osmosis (RO) is a typical method to clear water of most pathogens and bacteria using water that is pressed through filters with very small membrane apertures. These small openings usually stop impurities, salts and microorganisms. Of course, these systems come in many different types and they have to be maintained to keep their performance quality. Don’t take shortcuts on your RO system.

Once your water source is clean, strict hygiene procedures for tools, equipment and plumbing are the best way to minimize these threats to your plants downstream from your water source. These cleaning efforts are not a guarantee. Pests can still get into even the best facilities. Symptoms of these maladies vary, but root rot, stunted growth, wilting, discolored roots or leaves, and in some cases, the quick death of your plants is possible depending on the critter.

Use your scouting regimen and your data mapping to locate infestations before they expand and damage your facility. Isolate outbreaks and take appropriate measures to address the pests. We will give you suggestions on prioritization and preventative measures to take in the next chapter.

Figure 3: Example Pythium Brown Roots

Pythium is one of the most commonly harbored soil or water carried pests. When it is present and gets into your plants through cuts, natural openings, root surfaces or leaves on weakened plants, it can be devastating. In hydroponic systems, dirty looking brown roots evolve into full root rot if not addressed. Pythium is often the cause. In soil operations, pythium often shows up as wilting or yellowing patches on leaves.

Your lab testing partners are your friends when it comes to bacterial or fungal infections. Many diseases can resemble one another. It is not hard to misdiagnose environmental stress such as overheating or overwatering for a bacterial problem. Test results are necessary to accurately diagnose a problem.

Truly Airborne Molds & Mildews

Pythium and fusarium are not just present in water. They can also be airborne. Grey mold (botrytis) and powdery mildew are also common airborne pests. Proper humidity, air movement, air filtration and sterilization using HEPA (High-Efficiency Particulate Air) filters, activated carbon filters (also filter smells) and UV light sterilization can minimize these problems in your grow. Powdery mildew is the primary evil spore in this category. Airflow and regular cleaning to discourage fungal growth is the best way to limit these pests.

In conclusion, this week

Now that we have talked about identification (and clearly, this is not an exhaustive list), we will move into how to build in the cultural methods to prevent these problems from taking hold and ruining your business. In later chapters, we will dive into prioritization, treatment and control options for infestations, finally moving into control actions and emergency response.

Your integrated management response is how you pull all of this together and use your IPM procedures to increase your profitability. For the complete white paper on Integrated Pest Management Recommendations, download the document here.

Part three comes out next week and will delve into the world of Preventative Measures. Stay tuned for more!

Intimate Care Products with Cannabinoids Need More Safety Data

By Cindy Orser, PhD
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Cannabinoid products for intimate care use are now being sold in the unregulated cannabidiol (CBD) marketplace without proper evaluation of their impact on the vaginal microbiota or women’s health. Cannabinoids can exhibit complex and at times contra intuitive actions. The addition of CBD to these products is presumably for its anti-inflammatory pain-relieving qualities even though little is understood with regards to dosing and formulating. Moreover, in states where cannabis is legal, tetrahydrocannabinol (THC) along with other minor cannabinoids are also being added to intimate care products without purview of any federal agency. In general, the impact of vaginal products on vaginal microbiota is poorly understood. Ten years ago, Jespers et al. (2012) proposed to monitor lactobacilli indicator species of the vaginal microbiota in safety trials of intimate care products. Yet today, human safety data are not required prior to commercialization of intimate care products which are currently regulated like cosmetics except for lubricants which do require a 510K filing with the FDA.

The vaginal microbiota (VMB) of healthy women is dominated by Lactobacillus species, which exert important health-promoting effects to their host through the production of antimicrobial compounds, including hydrogen peroxide and lactic acid, to prevent invasive microbes from establishing in the vaginal epithelial mucosa (Pino et al. 2019). It was established almost 40 years ago by Speigel et al. (1983), that changes to the dominant Lactobacillus species, a process called dysbiosis, and overgrowth by diverse anaerobes can result in symptomatic conditions including bacterial vaginosis (BV), vaginal candidiasis, pelvic inflammation, and endometriosis (Taylor et al. 2013). The conditions resulting from dysbiosis are also linked to fertility problems, poor pregnancy outcomes, spontaneous miscarriages and preterm birth (Laniewski et al 2020).

An example of an infused intimate care product, the Foria Awaken Arousal Oil with CBD

Complications from dysbiosis of the reproductive tract can be serious in women wanting to become pregnant and in already pregnant women, including preterm premature rupture of membranes, spontaneous preterm labor and preterm birth (Ventolini et al. 2022). Reproductive tract microbiomes from idiopathic infertile women differ from fertile women’s VMB (Tomaiulol et al. 2020; Wee et al. 2018). Numerous studies have documented the vaginal community type microbiota associated with occurrence of BV around the world showing that HIV load is inversely proportional to lactobacilli species but positively correlated with BV (Sha et al 2005) as well as endometrial and ovarian cancer development (Walther- Antonio et al. 2016; Zhou et al. 2019).

Sexual lubricants often contain antimicrobial preservatives that have been shown to directly impact lactobacilli species in the cervicovaginal microbiome. The deterioration or absence of the lactobacilli-dominated vaginal mucosal biome through exposure to over-the-counter lubricants has been linked to increased incidence of BV (Brotman et al. 2010) and release of IL-8, a proinflammatory innate immunity mediator, produced by human epithelial cells to recruit leukocytes in response to infection, initiating an inflammatory response (Fashemietal.2013). The addition of under-researched cannabinoids to these products introduces the potential for further biological activity. Cannabinoids are widely reported to exhibit anti-microbial activity in vitro. The mechanism of CBD’s anti-microbial activity is thought to be due to its ability to intercalate into cytoplasmic membranes (Guard et al. 2022) and thereby modulate membrane vesicle (MV) release from bacterial cells which is associated with cell-to-cell communications (Kosgodage et al. 2019). Treatment of the gram-negative bacteria, E. coli, with CBD inhibited MV release and resulted in higher susceptibility to antibiotics but had minimal impact on gram- positive bacterial MV release. And CBD has recently been documented to inhibit the common human fungal pathogen, Candida albicans, from forming biofilms due to increased membrane permeability, reduced ATP levels, and modified cell walls (Feldman et al 2021).

In other reporting, the in vitro antimicrobial properties of CBD were demonstrated to have selective activity across a wide range of gram-positive bacteria, including several antibiotic resistant and anaerobic strains, with minimum inhibitory concentrations (MIC) in the low ppm range (Blaskovich et al. 2021). The conditions of a study impacted the observation of inhibition; for example, if human sera were present in the assay media, the antibacterial activity was drastically reduced. This has been attributed to CBD’s propensity to bind to non-specifically to proteins and thereby become unavailable (Tayo et al. 2018). Surprisingly, CBD does not exhibit broad antibacterial activity against Gram-negative species except against the human pathogens: Neisseria gonorrhoeae, N. meningitides, Moraxella catarrhalis, and Legionella pneumophila (Blaskovich et al. 2021). Bacteria do not develop resistance to CBD, but CBD is also non-systemic because of its high serum binding activity (Tayo et al. 2018).

The active ingredients in intimate care products can impact beneficial microorganisms but also deleterious ones. CBD has become a widespread, understudied active ingredient for women’s health. Today the molecular screening tools exist to conduct large scale epidemiological studies to further understanding of the consequences of dysbiosis and document the adverse effects on women’s reproductive health outcomes. Preventative treatments to reestablish dominant lactobacilli, in particular L. crispatus could have big impacts on not only women’s health but public health (Borgdorff et al. 2014).

As Ley R (2022) recently opined on the human microbiome, “there is much left to do.” Microbiomes are essential to the proper functioning of our bodies affecting social engagement, mental health, obesity, and disease states, and little is known about differences in microbiota across different groups of humans. More research is needed on the biological activity of cannabinoids as well as regulatory oversight to protect the health and safety of consumers.

References

  1. Blaskovich MAT, Kavanagh AM, Elliott AG, Zhang B, Ramu S, Amado M, Lowe GJ, Hinton AO, Thu Pham DM, Zuegg J, Beare N, Quach D, Sharp MD, Pogliano J, Rogers AP, Lyras D, Tan L, West NP, Crawford DW, Peterson ML, Callahan M, Thurn M (2021) The antimicrobial potential of cannabidiol. Commun Biol 4:7
  2. Borgdorff J, Tsivtsivadze E, Verhelst R, Marzorati M, Jurrrians S, Ndayisaba GF, Schuren FH, van de Wijgert J HHM (2014) Lactobacillus-dominateed cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women. The ISME J 8:1781-1793.
  3. Brotman RM, Ravel J, Cone RA, Zenilman JM. (2010) Rapid fluctuation of the vaginal microbiota measured by Gram stain analysis. Sex Transm Infect 86(4):297-302.
  4. Fashemi B, Delaney MA, Onderdonk AB, Fichorova RN (2013) Effects of feminine hygiene products on the vaginal mucosal biome. Microbial Eco in Health & Disease 24:19703-08.
  5. Feldman M, Sionov RV, Mechoulam R, Steinberg D (2021) Anti-biofilm activity of cannabidiol against Candida albicans. Microorganisms 9:441-457.
  6. Ilha EC, Scariot MC, Treml D, Pereira TP, Sant’Anna ES, Prudencio ES, Arisi ACM (2015) Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt. Ann Microbiol 66:597-606.
  7. Jespers V, Menten J, Smet H, Poradosu S, Abdellati S, Verhelst R, Hardy L, Buve A, Crucitti T (2012) Quantification of bacterial species of the vaginal microbiome in different groups of women, using nucleic acid amplification tests. BMC Microbiol 12:83.
  8. Kosgodage U et al. (2019) Cannabidiol is a novel modulato of bacterial membrane vesicles. http://doi.org/10.3389/fcimb.2019.00324.
  9. Laniewski P, Ilhan ZE, Herbst-Kralovetz MM (2020) The microbiome and gynaecological cancer development, prevention, and therapy. Nat Rev Urol 17(4):232-250.
  10. Laniewski P, Owen KA, Khnanisho M, Brotman RM, Herbst-Kralovetz MM (2021) Clinical and personal lubricants impact growth of vaginal Lactobacillus species and colonization of vaginal epithelial cells: an in vitro study. Sex Transm Dis 48(1):63-70.
  11. Ley R (2022) The human microbiome: there is much left to do. Nature p. 435
    Pino A, Bartolo E, Caggia C, Cianci A, Randazzo CL (2019) Detection of vaginal lactobacilli as probiotic
  12. candidates. Sci Rep 9:3355
  13. Sha BE, Zariffard MR, Wang QJ, Chen HY, Bremer J, Cohen MH, Spear GT (2005) Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis. J Infect Dis 191:25-32.
  14. Spiegel CA, Davick P, Totten PA, Chen KC, Eschenbach DA, Amsel R, Holmes KK (1983) Gardnerella vaginalis and anaerobic bacteria in the etioloty of bacterial (nonspeecific) vaginosis. Scand J Infect Dis Suppl 40:41- 46.
  15. Taylor BDP, Darville T, Haggerty CL (2013) Does bacterial vaginosis cause pelvic inflammatory disease? Sex Transm Dis 40:117-122.
  16. Tayo B. (2018) Exploration of the potential for plasma protein binding displacement and drug-drug interactions of valproate in combination with cannabidiol [abstract] Amer Epilepsy Soc Ann Mtg. New Orleans LA.
  17. Ventolini G, Vieira-Baptista P, DeSeta F, Verstraelen H, Lonneee-Hoffmann R, Leeev-Sagie A (2022) The vaginal microbiome: IV. The role of vaginal microbiome in reproduction and in gynecologic cancers. J Lower Genital Tract Dis 26(1):93-98.
  18. Walther-Antonio MRS, Chen J, Multinu F et al. (2016) Potential contribution of the uterine microbiome in the development of endometrial cancer. Genome Med 8:1-15.
  19. Zhou B, Sun C, Huang J et al. (2019) The biodiversity composition of microbiome in ovarian carcinoma patients. Sci Rep 9:1691.
Soapbox

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 tools,  https://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)

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.

Microbiology 101 Part One

By Kathy Knutson, Ph.D.
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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.

Part 1

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 petri dishes show sterilization effects of negative air ionization on a chamber aerosolized with Salmonella enteritidis. The left sample is untreated; the right, treated. Photo courtesy of USDA ARS & Ken Hammond

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.

Petri dish containing the fungus Aspergillus flavus. It produces carcinogenic aflatoxins, which can contaminate foods and cause an invasive fungal disease.
Photo courtesy of USDA ARS & Peggy Greb.

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.