Under reporting of Aspergillus on Cannabis

In our most recent publication we demonstrate that Aspergillus appears to have poor viability on various culture mediums thus having 100-1000 fold under-estimate of CFUs on 3M TYM petrifilms compared to qPCR. This is likely due to Aspergillus forming heterogeneous macro-colonies in solution and thus single CFUs are actually derived from a clump of 100s to 1000s viable cells as show by de Bekker et al.

 

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McKernan et al. Figure 3.

Below we have plated Aspergillus stocks in solution and then performed serial dilutions on SabDex Agar plates and 3M rapid TYM petri-films. The stock solution generates qPCR signal that implied 1M CFU/gram and was diluted 1:10, 1:100, 1:1000. These correspond to 23.3 Cq ,26.6Cq, and 29.9 Cq. 10,000 CFU/g doesn’t show up on Agar and has 3-4 colonies on 3M. This is a result of heterogeneous macro colony formation in solution prior to seeding the Agar lawns and as a result the CFU counts for Aspergillus are orders of magnitude less sensitive than PCR.

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Taking these cultures of Aspergillus and placing them on a 40X microscope and hemocytometer after dilution into ddH20 before plating and one can see mycelium networks and heterogenous macro-colonies. These clustering of cells prior to plating destroy the quantitative accuracy of measuring CFU/g with culture based methods.aspergillus-macro_colony

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Examples of Heterogenous Macro Colonies from Bekker et al.

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Examples of Heterogeneous micro colonies from Veluw et al.

screen-shot-2016-11-21-at-8-56-49-amSummary

Dangerous Aspergillus species cannot be accurately quantitated with culture based methods and should not be used for cannabis safety testing.