Isolating Yeast from a Honduran Vineyard

I'm currently taking some of the last basket-weaving classes required to finish up my undergraduate degree in Microbiology. One of those classes offered a short trip to an agricultural school in Honduras to assist with harvesting their vineyard and subsequent wine making. Having just read through the Bootleg Biology website; I was inspired to make an entirely different project out of it.

I set out to isolate a wild yeast strain from the grape skins in the vineyard we were working in. In case you weren't aware, the white powdery film on the fruit in grocery stores isn't some pesticide or preservative - it's yeast. They float around in the air outside until they eventually encounter something sweet. If that something happens to be a fruit, then they wait there for the skin to eventually break open so they can nom on the sugary juice. You might ask, "Why not just isolate a wild yeast from a local vineyard? Or one of those fruit farms that lets you go pick blueberries in the summer?" Rest assured, I will. But at the time it happened to be December, and I was going to a vineyard in Honduras anyway.

So I brought several sterile individually wrapped cotton swabs along with me to Honduras. Once in the vineyard I swabbed several grape clusters, and carefully broke the tips of the swabs off to drop them back in their little sterile packages and wrap them up.

Pre-Enrichment

Once back home, the real fun began. I filled three old White Labs yeast vials with diluted starter wort (1.015-1.020ish) and inoculated each with two cotton swabs. The idea here is to pre-enrich your culture by creating a healthy environment for the yeast and hoping they out-compete whatever else is on that swab. It's important to remember to practice aseptic technique here too, even though you're working with an environmental sample. I would hate to have gone through all this trouble to isolate California Ale yeast because I didn't sanitize my vials. Throughout this project, I used the highest proof vodka I could find in a big cheap jug at the liquor store as my sanitizer. Frequently using it to sanitize little vials made it more convenient than mixing up some Star-San. I kept my kettle full of boiling water in case I felt like I needed to rinse whatever I had just soaked in vodka, but it's not so bad to skip that at this step. A few drops of ethanol diluted into your culture can only help to select for alcohol resistant microbes such as yeast. Every couple days I loosened the caps on the vials just enough to let gas escape. Once the little vials stopped producing gas I transferred them to flasks and fed them some more starter wort.

Appearance and aroma will tell you what's growing in your cultures. Each of the three cultures I had started were growing different things, which is exactly why it's important to have more than one attempt going. One very quickly grew an enormous bright orange fungal glob (similar to a kombucha scoby) and was disposed of quicker than I could take a picture of it. Of the other two, one was producing lots of CO2 and had that characteristic bready yeast aroma. The other was not producing any gas and smelled very strongly of vinegar. I kept feeding the culture with the yeast to continue raising the alcohol concentration until I could get a sample into the lab and plate it.

Isolation Plating

There are some great resources out there with instructions for various plating techniques that I won't really go into. As an undergraduate doing personal work in the university lab I am subject to using whatever plates are left over from other various undergraduate classes. I have in past used YPGA plates with good results, but this time PCA (Plate Count Agar) is what was available and it worked fine too. So I streaked two PCA plates and two PCA + CAM (antibiotic) plates.

Yeast grew on all four plates, all with the same colony morphology, thin creamy white colonies with a dense brighter nipple in the center. Some yellow bacteria also grew on the plates without antibiotics. To make pitchable yeast from a plate, I selected a colony and used it to inoculate a tube of diluted starter wort much the same way I built up the cotton swab cultures, but with even greater care towards sanitation since this is going into beer now. 

Microscopy

Under the scope, these yeast cells are elongated and show bipolar budding which lead me to jump to the conclusion that this was a strain of Brettanomyces. I was stoked! But after the culture had started to ferment it had a very strange smell. The weird aroma persisted for weeks and weeks, like wet cheez-its and onions. I cracked open my dusty Mycology textbook and found out there are lots of yeasts out there that have bipolar budding. Considering on all four plates I streaked the yeast colonies were of the same morphology, I concluded there probably wasn't a yeast in that culture that is suitable for brewing. But this was a really fun project and it got me very excited to try again and find something useful!