The Big Job

One of the downsides of maintaining an yeast library is the work involved over time. Among the most annoying of these would be the periodic re-slanting; I know that it's been recommended that this be done once every 4 months, but I usually do it once every 6 months as those Corning cell culture flasks are really good at keeping the medium moist and fresh. However I've decided to switch over to the more 'traditional' slants using tubes, due to their lower cost. Yes, Corning cell culture flasks are through-the-nose expensive.

So that's how today was spent! Re-slanting is a rather hefty operation that takes place over several days. I bought these nice 15ml polypropylene (PP) centrifuge tubes at about $0.30 each online. I chose PP over glass due to the lower cost, and PP can also withstand typical autoclave temperatures. Also, compared to my old Corning cell culture flasks, these PP tubes can be reused over and over again. They don't come sterile though, so they are first sterilised at the highest pressure cooker setting for 25 minutes. I wet the tubes, place their caps loosely on, stand them in a PP beaker and seal this loosely with aluminum foil. At the end of 25 minutes, the pressure cooker is allowed to cool slowly and the PP beaker is removed.

Now comes the medium. DME is reconstituted to a S.G. of 1.040 in hot water. A pinch of Fermaid-K is added to this medium and agar at a rate of 15g/L is dissolved in it. Now here comes a big juicy secret to getting agar to solidify. If you read enough homebrewing blogs, you'd see many lamenting how you can't possibly get a 1.040 medium to solidify adequately. The trouble with the traditional technique is that this medium is autoclaved immediately, resulting in layering of the agar at the bottom. In order to make a nice juicy agar at SG 1.040, the trick is to dissolve the agar first! The medium is first microwaved at the high setting for a total of 3 minutes - every 30 seconds, heating is stopped and the medium is stirred thoroughly. At the end of 3 minutes, you'll find that the medium would have achieved a nice, ropy consistency. I then transfer it to a PP beaker, cap it with foil and pressure cook for 25 minutes to sterilise it. I've repeated this experiment of pre-microwaving vs directly autoclaving, and the results are always consistent - pre-microwaving will allow the agar to set later on!

After 25 minutes, the pressure cooker is again allowed to cool slowly. The medium is allowed to cool to 50C before pouring. 6ml of medium is added to each 15ml PP centrifuge tube, and this is propped up by a single large satay stick to allow the slant to form. After the agar solidifies, the slants are left in room temperature for 4 days - this is a 'proofing' step for me. If any of the slants had been contaminated, stuff would grow on the slants during these 4 days.

And finally, the streaking. A nichrome loop is flamed using an alcohol burner to incinerate any contaminants. After a brief cooling, yeast is picked up from the original cultures and re-streaked into the PP tubes. The cap is placed loosely on for the next few days. After visible colonies have formed, the cap is tightened and taped using Parafilm to conserve moisture within. These slants are then placed back into the fridge at 4C. Done!