During the month of April and beginning of May, I worked to standardize batch culture propagation methods for maximizing cell growth. I had previously finished observing colony morphology and growth characteristics on various mediums and chose MYPG and WLN agar as my two mediums of choice. With eight strains of Brettanomyces to use throughout the research and needing to propagate each strain in duplicate (in order to replicate the experiments) I had to use multiple orbital incubators and tied up a lot of the University lab’s resources. The following is the conditions which I found to produce the highest concentration of cells which were physically ready to start fermentation with high vitality and viability.
- Started with single colonies from MYPG agar, inoculated into 100 ml of wort in a 250 ml flask.
- All flask pre-filled with the wort solution were autoclaved with a sterile foam bung and aluminum foil loosely around the top.
- Initial propagation from single colonies was allowed to go for 7 days.
- The initial propagations were then decanted aseptically into an additional 500 ml of wort in a 1.5 liter flask.
- This second propagation step went 8 days before it was used for pitching into fermentations.
- All propagations occurred in orbital incubators rotating at 80 rpm.
- Temperature was set at 28°C in the incubator.
- Semi-aerobic conditions were present as a result of using a foam bung in the conical flask tops with aluminum foil covering.
A previous post or two mentions the propagation methods as they were going and some variations in the length of the initial propagation from the colonies. In the end the info above is what I used throughout the rest of the study to propagate yeast cells. Check out the following link to read more about the Propagation and Batch Culture Methods used throughout the dissertation.
Thanks for the great information.
How long did the second propagation last before decline or pitched?
Thank you,
B-Dub
With the second propagations I was growing the yeasts for generally 7 days. I also had propagations that went 8 days and 12 days. I found I had very little change in viability when I went even twelve days (always above 91%). The cell counts either stayed about the same or declined slightly from day 7 to day 12 with all the strains, except for the A-15 matted and A-15 glossy strains which kept growing to larger cells counts.