Many testing methods are available.
Here are the four common testing methods including two assays that can produce more rapid results:
Four common testing methods
The methodology for the detection and quantitation of microorganisms has traditionally been performed by using various forms of cell culture. Over the years, numerous culturing media have been employed that are enriched with specific concentrations of nutrient broths mixed with agar for support and grown in individual dishes/plates. The microorganisms are allowed to grow for specific durations of time (hours to days), and the direct number of colonies are counted per milliliter of fluid (CPM) or through viable cell counting to determine cell proliferation using colony forming units per milliliter of sample (CFU/mL).
In recent years, much attention has been given to the use of chromogenic media for use in plating microorganisms. This application combines the use of synthetic substrates for the detection of enzymatic microbial activities with colorimetric reactions, allowing for more specific detection of species or sub-species, often with only visual analysis. Cell culturing requires a proficient knowledge of scientific technique and requires designated lab space for laminar flow hoods, orbital shakers, incubators, and other necessary equipment. Moreover, microbiologists who are experts in these techniques are reticent to accept some of the newer methodologies, so some debate exists in cannabis and pathogen detection as to whether cell culture is indeed the “gold standard” of microbiology. Moreover, since culturing is a manual and time consuming process, the need for more rapid results has pushed the development of other assays, namely ELISA and qPCR.
ELISA (enzyme-linked immunoassay) kits are enzyme-linked immunosorbent assays designed for both the rapid detection and quantitation of target proteins, antibodies, and other soluble targets of interest including cytokines, growth factors, and signaling molecules, as well as transcription factors and post-transitional gene regulators (RNAi). ELISAs are highly sensitive, with a low limit of detection, typically in the range of 0.1 ng to 0.01 ng. Their use for detecting microorganisms involves using a heat-inactivated bacterial strain or a target protein at known concentration for positive control and allows users to automate their sampling and produce far more replicates for statistical derivation in a typical 96-well format using a multi-plate reader and minimal additional equipment needs. Additionally, the reactions do not require a high degree of technical expertise, making them more suitable for use in the cannabis space.
PCR, and now the use of qPCR, has allowed for the detection and quantitation of genes found with less than 10 copies in each sample. The combined use of fluorophores also allows a researcher to quantify five genes simultaneously, with one as a reference or “housekeeping” gene. This ensures that the DNA amplified occurs in known relative concentrations to a DNA baseline, thereby minimizing human error. qPCR is slowly becoming the “go to” technology for operators in the cannabis space for microorganism detection and quantitation by virtue of such rapid generation of results and diversity of application. Despite having a reputation of being error prone, numerous studies have shown that qPCR-based microorganism detection protocols are faster and more sensitive than classical culturing/plating techniques for staph aureus, candida and E. coli (Paule et al., 2009) (Sankari et al., 2019) (Sreshtha et al., 2019). Moreover, it can be automated and routinely performed with a modest amount of equipment and technical expertise.
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