Automated image analysis with ImageJ of yeast colony forming units from cannabis flowers

Abstract

Currently, in the state of Colorado and all other states within the United States of America with legalized marijuana programs, testing is required for bacteria, yeast, and mold on marijuana products. The Code of Colorado Regulations, 1 CCR 212-1, considers a passing result when a 1 g sample contains <10⁴ colony forming units (CFU) for the total yeast and mold count (TYMC). These measurements are usually obtained by manually counting colonies on petri-dishes or 3 M™ Petrifilms™, which is a time consuming and user subjective process. Therefore, an automated counting method utilizing ImageJ has been developed for CFU analysis of TYMC on Petrifilms. The performance of this colony counting method was demonstrated by comparing manual and automated counts from marijuana flower samples containing spikes of Candida albicans as well as samples that tested positive for the presence of yeast and mold. Fifteen images of Petrifilms showing various concentrations of colonies were studied by fifteen users at two institutions using both the automated and manual counting methods. All counts from the automated ImageJ procedure were within 12% of those obtained manually. In twelve out of fifteen Petrifilms, the average count of the automated method was statistically similar to the manual counts. The statistical differences of the other three samples were observed to be random and caused by user errors. The automated counting method could be used to quickly count numbers that are as high as 400 CFUs, reducing time of analysis with improved documentation because the images and the electronic colony counts can be saved on a computer or cloud for long term storage and data access.

Keywords: Automation of CFU counting; Candida albicans; Colony forming units of yeast and molds; ImageJ; Manual CFU counting; Petrifilms.