Testing Cannabis Plant Material for Microbial Analysis

Testing Cannabis Plant Material for Microbial Analysis

Testing Cannabis Plant Material for Microbial Analysis

Professor DeBacco

Research Article
McPartland, J. M., & McKernan, K. J. (2017). Contaminants of concern in cannabis: microbes, heavy metals and pesticides. In Cannabis sativa L.-Botany and Biotechnology (pp. 457-474). Springer, Cham.
https://www.researchgate.net/publication/318020615_Contaminants_of_Concern_in_Cannabis_Microbes_Heavy_Metals_and_Pesticides

Cannabis Diseases
At least 88 fungal species cause diseases in Cannabis as do eight pathovarieties of plant pathogenic bacteria.
The most threatening diseases of flowering tops are caused by three ubiquitous fungi—Botrytis cinerea (the cause of gray mold), Trichothecium roseum (white mildew or pink rot), and Alternaria alternata (brown blight).

Pathogen Threat to Humans
Phytopathogens cannot infect humans, except perhaps immunocompromised individuals.
Opportunistic infections by A. alternata have been reported in patients receiving chemotherapy, recent organ transplant patients, and people with AIDS.
Airborne conidia (spores) of B. cinerea and A. alternata cause mold allergies and asthma, particularly in greenhouse workers

Indoor and Outdoor Problem
Aspergillus and Penicillium spp. Predominated indoors, and Cladosporium spp. predominated outdoors.
Cladosporium may be an emerging problem; this fungus also infests hemp mills (McPartland 2003).
About 1% of cannabis supplies received by Harborside Medical Cannabis Dispensary in Oakland, California were returned to vendors because of unacceptable levels of Aspergillus contamination

American Herbal Pharmacopoeia (AHP) Protocols
The AHP’s protocols were based on tests for commodity food products issued by the EPA and the Food and Drug Administration, as well as assays for cannabis used in Holland
For orally consumed cannabis, the AHP recommended four tests:
(1) total yeast and mold count
(2) total coliforms
(3) Escherichia coli
(4) Salmonella spp.
In addition, they recommended immunochemical methods to screen for aflatoxins.
For products to be inhaled, more stringent tests were recommended:
(1) total yeast and mold count
(2) total aerobic count
(3) bile-tolerant gram-negative bacteria,
(4) E. coli and Salmonella spp., and aflatoxin assays
The AHP proposed specific limits in CFU/g counts, but emphasized that these values did not represent pass-fail criteria. Rather they were recommended levels when plants are cultivated and harvested under normal circumstances.

Colorado Testing
In 2015 Colorado updated its testing regimen:
(1) total yeast and mold count (limit less than 104 CFU/g)
(2) Salmonella (limit less than 1 CFU/g)
(3) Shiga-toxin producing E. coli (STEC, limit less than 1 CFU/g)
Colorado recommended testing for three species of Aspergillus:
A. flavus
A. fumigatus
A. niger
*Although this was never implemented.

Species Detection
Aspergillus is a large genus with 250 species, and separating three specific species from the others is not easy.
Due to the challenges associated with species-specific detection, Colorado changed their testing requirements again in 2016, to a 10,000 CFU/g total yeast and mold test, but left in place single CFU/g testing for E.coli and Salmonella spp.

qPCR Pros and Cons
To accelerate testing turn-around time, some laboratories now use quantitative polymerase chain reaction (qPCR) assays which detects DNA sequences in cannabis samples.
The drawback to qPCR is the method’s indifference to living or non-living DNA.
To accommodate this, an enrichment step is performed, where the cannabis samples are incubated overnight in TSB broth prior to qPCR detection.
Overnight growth in TSB ensures only live organisms are measured, but raises questions over preferential culture conditions for broader total yeast and mold tests.
To address this conundrum, some labs perform a qPCR on total yeast and molds, and positive results are confirmed with an additional test extracted 24 h later to ensure the signal from the pre-incubation test was from live organisms.

Gamma Radiation
Gamma radiation remains controversial and it may destroy terpenoids, and it does not destroy mycotoxins
Hazekamp (2016) evaluated the effects of 10,000 grays in four cultivars of THC or CBD-dominant Cannabis.
Levels of total THC and/or CBD were not altered by irradiation treatment in any of the cultivars tested, compared to controls.
Irradiation decreased four monoterpenoids:

Hazekamp compared these reductions to similar decreases arising from short term storage in a paper bag

Link to Lecture Slides: https://drive.google.com/file/d/12hXb9DoBu802ou3l2wVzolFOs1rowV4E/view?usp=sharing

*Due to the description character limit the full work cited for “Testing Cannabis Plant Material for Microbial Analysis” can be viewed at… https://drive.google.com/file/d/16SHfebj_xNW9zj2mU-fELF7RA-ei7D-a/view?usp=sharing