Plating Yeast & Selecting Colonies

[jester5120]

I've seen several articles on this site about the proper way to slant yeast but haven't found one that shows the process of streaking yeast onto a plate and selecting individual colonies. This is the method I use. It's not flawless and it certainly has room for improvement but it's cheap and works well for me.
What you'll need to do this:

• yeast source (bottle dregs, old washed yeast)
• Petri dishes (ideally ones made of tempered glass that can be boiled but I used sterile disposable ones)
• agar
• DME
• inoculationloop (I use a paper clip because it's practically free andI'veseen others use it online)
• aluminum foil
• small pot or flask

The first thing you'll need to do is create the growth medium. I use the same recipe that Saccharomycesuses in his slanting tutorial: https://www.homebrewtalk.com/f163/slanting-yeast-133103/
It is 35g DME, 400ml water, 2.5g agar
When I first did this I used those exact numbers and filled 8 plates. That left me with way too much growth medium. So either fill more plates or cut back on the recipe.
Mix all the ingredients for the growth medium into a 1L flask and get it boiling.

While the medium is cooking away, lay out some sterile aluminum foil to set the plates on for when you pour them.

Once the growth medium has boiled long enough to sterilize it, take off the heat and pour right from the flask and into the Petri dishes. The hot liquid helps to sterilize them again, just in case. They need time to cool and solidify after they're poured. This is where the highest risk for infection is, so what I do is keep a steady updraft going right next to them so anything floating in the air doesn't have a chance to land. This was achieved by putting the plates on one side of my stove, turning the burners on on the opposite side of the stove, and also turning on the exhaust fan.

After the plates have cooled and solidified you'll need to streak the yeast onto the plate. Give them some extra time if you're not sure. If they're still too hot you'll kill the yeast and if they're just a little watery you'll make a mess. Illustrated below in my awesome Microsoft Paint drawing is the pattern that you want to use when streaking the plate.

When you streak the plate all you're doing is trying to spread out the yeast so when it begins to grow you can identify a healthy, single, uniform yeast colony to eventually start some delicious beer from. This is where you'll need your yeast source (mine is some rinsed Pacman yeast that was on its last leg in a mason jar) and your paper clip. Straighten your paper clip out. You'll need to sterilize your paper clip somehow. I've found it's easiest to just crank the gas burners up and heat it up that way.

After its good and hot let it cool back down so you don't kill the yeast. Then you want to stick your freshly sterilized paper clip into your yeast source. Make sure to keep the paper clip fairly dry after you bring it back out of the yeast, I give it a little shake or tap otherwise the plate will get watery and messy. There will be plenty of yeast left on it, don't worry.

After your paper clip has some lovely yeast on it you need to streak the plate by rubbing the paper clip across the growth medium in the pattern illustrated above.

Now you need to put the lid on the plate, seal it up either in aZiplocbag or plastic wrap, put it in a dry place between 60-70 degrees F and wait. After a a day or two you'll start to see yeast growth.

From this point you can either put the individual colonies into a small starter and begin to step it up to a pitchable volume (I do steps of 10ml, 100ml, 1L) or you can slant the yeast and save it for later. To get them off the plate use the same method as before for handling the yeast. Sterilize the paper clip, let it cool, scrape the colonies off the plate, and move them onto their next step. I circled some of the ideal colonies that you would want to select.

And following the Saccharomyces' guide to slanting, I put several of those colonies into slants for later use.

 

[savannahbrew]

the streak plate pattern you drew is close to being right but is a bit off. Google streaking or streak plates and it will show you. For example draw one squiggly line in one quadrant of a dish. Then flame the loop. Then drag the sterilized loop through your streak pattern on the plate and squiggle another quadrant. repeat this and it will thin your colonies out by the third and fourth quadrant. This will make colony selection much easier. Great article though this is a good way for people to learn how to do this stuff and save their favorite strains.

 

[jester5120]

yeah i've seen the regular way to streak plates. I have the yeast book and read through braukaisers info online. I never noticed a difference in outcome but i suppose if i were doing it all the time i'd see the advantages of it. Thank you for pointing that out though, i'll try to edit it later when i figure out how.

 

[Fionnbharr]

@savannahbrew I second this / your method.
The focus should be on minimum transfer to the plate, and then culling individual cells from the streaks into new areas of the plate.

 

[mkingston2]

How do you identify the colonies as "ideal"? What makes the isolated growth better than the growth that occurs where inoculation was more continuous in the streaks?

 

[jester5120]

the ideal colonies are round, uniform in shape, and not above average size compared to the other visible colonies. From what i've read in the yeast book and from braukaisers tutorial this is primarily to make sure the cell aren't mutated. The reason you don't want growth from the streak is because you're trying to select a single healthy colony to start from. Selecting from the streak wouldn't guarantee that you are getting a single colony and would make it very difficult to select a non-mutated cell. That is my understanding, there may be more reasons though.

 

[jaapie]

You want to select one colony because you want something that is genetically uniform. If you pull through a streak you will get different cells that started each its own colony and fused together into a lawn of cells. If you want to stock something you do not want that because there is a chance that you have a mixed population. However, if you grow say a starter its not such a huge deal.
Personally, what I do is pick a colony, streak it on a fresh plate, and from that plate on then i do not really care anymore. If the cells look ok and uniform (no weird stuff) then its all the same organism.

 

[mheitt]

Forgive my ignorance, but what's the advantage of plating, selecting and slanting over just taking a small portion of a fresh starter and growing from there? I have done the later and it's very simple: I buy a vial of yeast, make a starter and I only pitch about 4/5 of the starter. I let the yeast settle and decant the wort, then I pour about 5ml of the remaining 1/5 of the starter into sterile tubes and top off with some glycerin and either store in the fridge or even in the freezer (the glycerin protects the yeast cell walls). My way is easy, but probably more prone to contamination. Any other advantages to your way?

 

[jester5120]

the reasons i'm aware of, and i'm sure there are more knowledgeable people on this topic that could add more info, are that, as you said, it can help keep out contamination but also eliminates mutations in the cells. by selecting one single colony you can see that it is healthy and growing uniformly which is an indicator that it is not mutated. a mutation or contamination would change the character of the original yeast. I only plate the yeast out after i've repitched it for 4-5 generations and am worried about it getting funny.

 

[HBCBrewmaster]

I have successfully stored yeast for over a year in a pure sugar solution. 90 grams water 10 grams white table sugar.
put 20 mils in a autoclaved testtube and inocculate with 1 loop of a clean known to be pure yeast source, like a fresh vial of whitlabs. so far 100 percent success with reanimation after 1 year storage. I got this method from Pierre Rajots book. http://www.amazon.com/First-Steps-Yeast-Culture-Part/dp/2921327171

 

[microbebrew]

Thanks for posting this article. I have a microbiology background and I can clarify a few things for you. The agar recipe looks good.
I can't tell if you did or not from the photo, but you definitely want to cover the flask with aluminum foil. When the media has boiled (all of the particulate should be dissolved), let it cool to ~50C (122F) before pouring the plates. A good rule of thumb is to cool the media until you can hold the bottle by the base, where the media is, and not burn yourself. Keep the foil on the flask during cooling, and swirl the flask occasionally to keep everything mixed up. Try to not agitate the media too much to prevent bubbles from forming.
Your plates (glass or disposable) can comfortably hold ~35 mL of media. You can see in your photo a ridge along the inside of the plate. That's the max fill line. With your recipe of ~400 mL of media, you should be able to get 10-12 plates, depending on how thick you pour. As soon as a plate is poured, put the lid on! This eliminates the need for the plates to dry next to an updraft and prevents contamination. Always, always, always put the lid on the plates! Let the plates dry and solidify for several hours, but overnight would be best. Once the plates have solidified completely, you're ready to streak. Store any unused plates in a Ziploc bag in the fridge. This will prevent contamination and will help your plates last longer without drying out.
For streaking the plates, the paperclip is perfect! Heating it over the burner will work great. To cool your loop, you don't want to just let it stand at room temperature, and you especially don't want to wave it around to cool it off. Any contaminants in the air can stick to it and will be transferred to your plate. A better option is to heat the loop and then touch it against the agar in your petri plate. Touch it near the rim of the plate and it will hiss for a second. Flip the loop over and repeat. Don't stab into the agar, just set the loop on top. This will quickly cool it against a sterile surface.

Next, pick up the yeast as you have indicated. There was a comment that mentioned how to perform a three-phase streak of a plate. That will dilute out the cells on the plate and help you get individual colonies, which are what you want. Incubate your plates at room temperature for ~5 days.
The whole purpose of doing this is indeed to get isolated colonies, which grew from a single cell. It's impossible to tell by looking at a plate if some of the colonies are mutants. The best way to reduce the risk of mutation is to streak a plate with your fresh new vial of yeast, not yeast that's been used in several subsequent batches of beer. The yeast is more likely to pick up mutations growing in broth culture than on plates. If you're repitching yeast for 4 or 5 batches of beer, I would be worried that it's already funny, and probably very different than the yeast you started with.

 

[medusa1066]

Great writeup! Good comments too! If you notice that there is too much condensation on the lids of the petri dishes, incubate them UPSIDE DOWN and handle them upside down. This way the condensation won't drip onto your colonies and wash growth across your plate.

 

[jester5120]

Awesome info! thanks for the clarification and corrections

 

[microbebrew]

Good call, Medusa. I forgot to include that.

 

[dozer5454]

Love the write up. I'm just staring to get into slanting and this info is just what I needed.

 

[microbebrew]

@jester5120 You're welcome! Happy to help.

 

[MitchyBourbon]

Great write up! The only things I would add / include are that when your not actively playing you should keep your plates upside down. Part of the goal is to grow several isolated colonies where the colonies are started from one cell. Last, when starting a propagation from a plate it is beneficial to select several well formed isolated colonies because it does provide for some genetic diversity. If you plated from a fresh vial of yeast and select only well formed isolated colonies you can be confident that they are of the same organism.

 

[peterlonz]

The article & comments provide a very practical & easily understood procedure with reasons for each step.
Genuinely excellent!

 

[pithy]

Whu-whu-whuutt is all this hullabaloo bout yeast!!!! Read many years ago in an Aust brewing mag not to use dried yeast but culture live yeast from a conditioned comm bottle , put in frig, top up each brew from yeast dregs, add to new brew!!!! did this for years using same bottle!!! Some of best beer I drank!!- Then came "x-perts!!!) saying oh no- cant do that- the yeast become unstable!!! Well pilgrims, an article in New Scientist disproves that!!! Yeast actually improve the longer they are used!! heard of evolution!!!? And how bout natural wine from yeast on grape skins- bootiful!!! And as to yeast, the best yeast for beer & cider is bakers yeast!! Yes that,s right- works quick & ferments right out!!! Having said all that, I recently was given a carton of Pale Ale from Little creatures brewing in WAust- tasted just like Avon Ale which was made during prohibition in Aust 2nd WW- a powerful bitter brew, using treacle, molasses, brown sugar, double hops, double malt. Anyway, for some reason or other, I made a yeast starter from the dregs of a bottle, & put in my next brew(Coopers Cerveza)- WOW! Best beer I have drunk for a looonnnggg time!!! since then bottle in frig/dregs trick- added to each new brew!! working great!!! This yeast forms a thick white layer at bottom of fermenter- unlike 99% of other yeasts!!! And showing NO sign of "weakening!!!" Guess I'll keep on doing this for ages to come!!! Cheers- glug-glug, glug!!

 

[mike191]

Point to the New Scientist article so we can read it please!

 

[jester5120]

@pithy the whole idea here is to keep the yeast from evolving and use as close to the original culture as possible. there are certain characteristics we want from our yeast and if the yeast evolve those characteristics do to. i respect your opinion but disagree with almost everything you have said

 

[Baloo]

The streaking method savannahbrew posted provides a higher number of single cell colonies with each plate so you need fewer plates to get the requisite number of single cell colonies.
Maybe I spent too many hours streaking plates as a workaday Molecular Biologist right out of college, but this seems like a lot of work for little gain. I wholeheartedly agree that propagating your own yeast saves time and money over buying new for each brew. This, however, seems like a lot of extra work compared to picking up a fresh batch from Wyeast when your working batch is past the point of prudent propagation.
Lastly, what test do you use to confirm the single cell colonies you choose to slant were formed from your desired strain rather than a mutant or contaminant?

 

[FLJohnson]

As stated, the intent is to propagate a colony that has arisen from a single yeast cell. However, no one has provided a means of knowing with any certainty if the colony one selects has actually come from a single yeast cell. Yeast tend to stick together, so there is a high likelihood that a colony has NOT come from a single cell unless you have set the conditions during the streaking such that there is a high probability that each colony comes from a single cell. Just because you have a nice round colony says nothing about how many cells the colony arose from.
I believe the standard way of performing the initial streaking is to create a very dilute solution of the yeast such that the initial streaking results in MANY single colonies of uniform size. Occasional large colonies are likely to be have arisen from multiple cells that were stuck together during the streaking. A colony resembling many others in size (from a dilute solution) is much more likely to have arisen from a single cell. Also, it would be important to not select a small colony if it is not representative of many other colonies of the same size. I believe this could be the colony from a petite mutant. (Isn't that where the name "petite mutant" comes from, ie. the colony size?) The key to getting a colony from a single cell is to create the dilute solution before streaking, so that one gets LOTS of colonies of the same size.
I would love to hear comments on this from a microbiologist who really knows yeast, how to create dispersions of single cells, and how mutants behave.
(Incidentally, mutation does not equal "evolution". Mutation is a loss of genetic information, and "evolution" would require the gain of new, meaningful genetic information.)

 

[microbebrew]

The simplest solution to the mutation concern is to start your plates from a fresh, brand new yeast culture. It's not possible to visually pick out a single colony with no mutations, at least mutations we care about. The problem is that unless the colony looks different (different color, not shiny, rough looking, etc.) you can't really SEE a mutation. We can, however, taste the mutation. The only way to make sure the isolated colony that you pick has the characteristics you want would be to brew up a batch of beer made from a starter of that isolated colony. If the beer has characteristics you like, you win! If not, you can always pick a different colony and try again.
If you really want to get into the weeds, the spontaneous mutation rate of S. cerevisiae is ~9E-7. In a nice dense yeast culture, there should be about 1E8 cell forming units (CFU) / mL. So, in 1 mL of culture, you pick up about 90 spontaneous mutations. When you streak to a plate, especially using the paperclip method described above, you're probably putting about 10 microliters (0.01 mL) onto the plate and then diluting it out with the three phase streak. So, you're putting about a million (1E6) CFU onto the plate and then diluting it out. In the first streak, you can't see isolated colonies. When you get down to the secondary or tertiary streaks, you can usually see nice isolated colonies. Of the million CFU you put on the plate, maybe 1 will have picked up a spontaneous mutation. That 1 mutation might have killed the cell, so you won't see it. Also likely is that the one mutation won't make any difference to the flavor of your beer. It COULD, but it's not likely.
In the sense of the "evolution" of the yeast, there's some truth to it. Essentially, some yeast cells will do better than others in particular styles of beer. Those yeast cells are indeed mutants. HOWEVER, mutation can be the loss or change of genetic information. When you recover yeast after fermentation, the majority of the cells will likely be mutants that performed better under the particular conditions of your beer. Maybe the pH was a little low and one of your yeast cells had a spontaneous mutation that allowed it to perform a little better under acidic conditions. Those will be the cells that are most prominent in the trub after fermentation. If you're using those cells in another beer, the pH might be a little higher than usual, and the yeast won't be as well adapted and may not perform as well as you would like.
Using the same yeast for several subsequent generations of beer can absolutely work and I'm sure can produce delicious beer. Pithy, this is a great discussion about how to properly and safely plate yeast. And some genetics. It's not a place for insults and foul language. If you don't want to plate your yeast, no problem. Some of us do.

 

[FLJohnson]

Thanks for the reply, microbebrew. When I plate, unless I start with a dilute solution--I am usually just replating from a slant--I usually get several individual colonies, but they aren't the same size. I conclude that these colonies came from small clumps of cells, not individual cells. I usually just pick one of these up to slant, but I know that I am increasing the probability that I'm picking up a colony containing an undesirable mutant. As I said before, it seems that the best way would to start with a dilute solution from which one can obtain many uniformly sized colonies, increasing the probability that when I pick a colony from all of these similarly looking colonies, that I'll pick one that is not from a mutated cell.
How are petite mutants identified?

 

[dharalson]

My two cents on the subject.
The purpose of streaking to reduce the odds of selecting a "mutant" to near zero. If my math is right based on the comments by microbrew the odds of a mutant are about 1 in a million. So after you have "spread" out the yeast cells the odds of any individual cell colony being started from a mutant are 1 in million. So unless you are REALLY unluck the one to three colonies you select will be "pure".
What I have been doing for several years is once a year I will start with a fresh commerical yeast (Wyeast or White Labs) and streak onto a petri dish. I will collect about 10 or so of these colonies and store these in distilled water that has be sterilized in a autoclave (pressure cooker). I use a 10 ml vial with about 5 mil of water.
When it comes time to brew I will take a drop of the distilled water (shake it up first to disperse the yeast cells) in a Slant (see Slant below). Let the yeast on the Slant grow for a 3-4 days and I will have a nice crop of yeast. I try to scrap as much of these colonies as I can in to a starter batch and build up the numbers as any starter. I have used progressively larger starters and I have pitch a million or so cells into 600ml and watched it grow to the 150-200 billion cells needed.
A Slant is similar to a petri dish; same media in a vial. The Agar culture media is allowed to cool with the vial on a "slant" so that the surface of the media "fills" the bottom and is a the edge near the top.
I would be interested in the "microbiologist's" comments on this method.
As far as "dry yeast" or using a fresh "liquid yeast" for each batch. That is probably much less expensive than culturing your own. But its a hobby ! I can do it any way I want !

 

[beerchemist]

@pithy,
Your way does indeed evolve the yeast. Every time you re-use the dregs, you're selecting for 1) flocculence and 2) tolerance to whatever conditions you use in your brewing (temperature, pH, etc.). Your beer will change over time, but not necessarily in a bad way. The yeast will adapt to the conditions you use when you brew.
The point of streaking and picking a single colony is to ensure reproducibility. Every batch will be made from a clonal (i.e. genetically identical) colony, so if you do everything else the same way, you will get the exact same beer. I wouldn't necessarily worry about mutations on your plates, since there's no selective pressure, but in your fermenter, fluctuations in temperature, pH, and whatever else you're adding to your brew might change things over time. I'm content to let Fermentis take care of that for me and spend the extra couple bucks per batch. YMMV.
If you want to get deep into the genetics of it, commercial brewing strains have evolved in a very weird way, including extra copies of chromosomes that contain fermentation-related genes (go figure):
http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001287
Also, for the OP, your media ratios are a bit off. I'd go with 20-30 g/L DME and 15-20 g/L agar. With the amount of agar you're using, your plates might come out a little on the squishy side.

 

[DocG308]

My 2 pennies to add to microbebrew:
The vast majority of yeast mutations are due to recombination of the chromosomes. These recombinations are for increased survivability in a given media. The "best" way to ensure you don't have mutations you don't want on the plate is to make the agar as close to your wort conditions as possible. We use drug resistance genes to isolate coding regions of DNA for use in genome research by adding the drug to the agar, making sure only the yeast/bacteria with the drug resistance and genome of interest survive.

 

[Vigo_Carpathian]

The morphology on that plate looks homogeneous throughout. What differentiates those colonies over from the rest? I don't see a point to this because during growth, the healthy yeast cells will out-compete and dominate the tank anyway. If there was a difference in morphology, that's one thing, but in this case....
Plates are good for resistance studies, plasmid retention studies, contamination monitoring, and stability monitoring, not so much for health determination. This all seems like a lot of trouble for very little benefit.

 

[KeyWestBrewing]

This has been an interesting read. Loving the conversation, you all are putting a lot of knowledge out on the table.

 

[mirogster]

Gentlemen, with great pleasure, I'd like to point you at certain excellent blog. Over there you'll find almost all techniques and writeups, how to isolate, store, slant and brew from those yeast colonies. For some it's well known site,BUT didn't showed up earlier in above dispute.
It's not mine, therefore I'm not trying to spam here . I've learned TONS of useful & interesting things from there. I really, REALLY recommend to have a look!
So, after slightly long introduction, here it is:
http://eurekabrewing.wordpress.com/yeast-cultivation/

 

[Micr0beBrew]

Someone stole my screen name AND doesn't know what they are talking about up above...

 

[ashenafi]

my question here.i'm on the way going to do my project my project title is isolation of yeast from cane molasses for ethanol fermentation.and i need simple methodology.pleases