Novel flow cytometry based analysis methods within aquaculture

Project summary

Aquaculture is meeting an increasing pathogenic pressure. Transportation systems, such as well boats, can cause the spread of pathogenic organisms across seas. Recirculating systems faces similar problems with maintaining a good microbial quality, free from pathogens. Current methods to identify infections rely on PCR detection of specific pathogens. However, the vitality of the identified species is not determined.

Flow cytometry has been used for decades to determine bacterial vitality by using fluorescent dyes, such as live/dead staining with SYTO9/PI, CFDA-AM, and so on. In this project, we wanted to explore and expand possibilities for bacterial analysis in the aquatic environment with a recently developed method: Bioorthogonal Amino Acid Tagging (BONCAT).

BONCAT is a click-chemistry reaction, where an analogous amino acid is incorporated into newly synthetized proteins, before being coupled to a fluorescent dye with a click reaction. The method allows to follow proteins synthesis at a time point and over time. Protein production requires intact DNA and informs about the general metabolic activity in cells. We adapted the method for flow cytometry to improve analysis time and combine it with SYBR Green, an established method for staining marine microbes DNA.