YIC: Isolation, and Making a Starter

*This is part two of the Yeast from the Iron City (YIC) series. For sampling, media, and plating information check out the first post, Yeast for the Iron City: Sampling and Plating

Figure 1. Mixed culture from several fruit. (picture looks hazy because of condensation on lid)

Now that we have a culture growing wild on the agar plates that looks something like figure 1, its time to move on to the next step, Isolation. One of the best isolation techniques to use is what's known as a 4-quadrant streak (Figure 2). This does take a bit of practice to get perfect, but is pretty easy and very effective at isolating colonies of microorganisms. The best tool to use is the inoculation loop. If you have a nichrome loop (non-disposable metal loop) you will need to run the wire and loop through a flame until it is red hot to sterilize it. Sterile plastic disposable loops are available, but you would need 4 per plate and who wants to buy something to throw it away after a single use?

Figure 2. 4- Quadrant Streak Technique. (Numbered as steps)

It may look like a 5 year old with the ability to color in the lines can do this,but as I said earlier, this technique takes a little practice to get good isolation. I'll give you the steps with a nichrome loop because that is what we used. If you are using plastic, one, I'm disappointed, and two, whenever the nichrome loop gets sterilized, toss yours in the garbage (or you can save them and autoclave them I suppose if they are autoclave-safe) and get out a new one. 

First identify a colony you suspect to be yeast that is mostly free of other colonies touching it. Typically, the yeast colonies I have seen will be opaque white/ off white with clean edges (Figure 3). The stuff you don't want to touch is either fuzzy (fungi), has a very slimy shiny appearance (typically bacteria) or is a color other than white/ off-white (Figure 1). Sterilize your loop in the method described earlier. Then wait about 10-15 seconds to let it cool (don't "shake it like a Polaroid picture" as they say to cool it faster, you will increase the chances of picking up microbes from the air). Take about half of the individual colony making sure not to touch any other colonies and streak onto the plate as in Figure 1-1. Sterilize your loop and let it cool. Make streak as in step two of figure 1. The key is to only cross over your initial streak a few times. Repeat these steps two more times with successively less crossing into the previous streak. 

Figure 3. Isolated Yeast Colonies

Let this stand at room temperature until you start to see growth. Theoretically, you will have diluted out the original streak to single individuals that will then form a single isolated colony in either the 3rd or 4th quadrant (Figure 4A). If you do Success! You can now take a sample (from a single colony) and look at it under magnification to tell whether or not you actually have yeast (Figure 4B).

Figure 4. A) Example of 4-Quadrant Isolation. B) Wild Yeast X400

At this point you can begin to build up a starter to use pitch. To avoid possible contamination, it is best to start with a relatively small starter and work up to the size you want to pitch with rather than just going straight from plate to a .5 L starter. This greater yeast to starter ratio will allow the yeast to out compete most other microbes that may have been introduced into the starter. Nothing left to do now but pitch and hope that you've caught a good one! The Saga continues in the next YIC post: Temperature-dependent Flavor Determination.