【Summary】 Although the cell sorter was an effective tool for isolation of not only eucaryocytes but yeast and bacteria, the same equipment was avoided to use all types of cells because of worries about contamination in the conventional cell sorter. The flow cytometry system of our company can be performed all because of a disposable type of microchips.
【Purpse】 Both analysis and sorting of yeast or bacteria were carried out using the flow cytometry system of our company.
The method of distinguishing “live or dead bacillus” and calculating bacteria quickly with culture medium were enforced by evaluation of the colony method until now.
However, since evaluating of the number of bacilli would be inaccurate if centrifuge processing made a mycelia cake condenses, it was examined whether evaluation of medicine screening could be performed with a culture medium.
【Method】 Syto-9 and PI were used for “live or dead” judgment for yeast or bacteria. Since PI cannot penetrate the cell membrane of live cells, live cells are dyed only by Syto-9. On the other hand, dead cells are dyed with Syto-9 and PI as double positive on analysis.
After heating yeast, nuclear dye was carried out and 100 dead cells and 50 live cells were sorted by On-chip Sort respectively, and they were cultivated with the culture dish after that.
Each concentration of Meropenem was mixed to the pseudomonas aeruginosa, and sampled for every time progress. The standard protocol of “LIVE/DEAD BacLight Bacterial Viability and Counting Kit” was improved, and the number of dead cell was counted by it.
【Result】 Since disposable microchip is used, there is no contamination of the main part of equipment from yeast or bacteria.
Although the colony was not observed from the fraction of dead cells in sorting of yeast, 40 colonies were observed in fraction of the live cell.
It was also possible to have judged the antibacterial activity of medicine simply for a short time by measuring bacterial “live-and-dead” judgment using FCM.
Furthermore, since whole-quantity measurement was possible, the absolute quantity of the cell in equivalent solution was able to be measured.
The judgment of “live and dead” of yeast using On-chip Sort.
The judgment of “live and dead” of bacteria (pseudomonas aeruginosa) using On-chip Sort