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How to make solid culture medium plates for yeast culture

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If you would like to culture your own yeast, either from strains available commercially or from unpasteurized craft beer, then you can easily do this outside of the lab with a pressure cooker and a few supplies.  First, I encourage you to read my previous Instructables on how to use a pressure cooker to sterilize materials and the other on how to make an alcohol lamp for microbiology purposes: 

http://www.instructables.com/id/How-to-Sterilize-Autoclavable-Materials-at-Home-us/?ALLSTEPS

http://www.instructables.com/id/How-to-make-an-alcohol-lamp-for-home-microbiology-/?ALLSTEPS

If you haven't already, I also **highly** recommend the book 'Yeast' by Chris White and Jamil Zainasheff, especially if you don't have any laboratory experience.  
 
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Step 1: Obtain materials

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You will need several 100 mm borosillicate glass pyrex (lab-grade) petri dishes which can be purchased from Amazon, along with lab-grade pyrex Erlenmeyer flasks of appropriate size for the quantity of plates that you want to make.  Here I am using two pyrex petri dishes and two small Erlenmeyer flasks, since I am only making two plates and a small slant (50 mL).  If you want to also make slants, then you should purchase borosillicate glass 'slant tubes' with screw top.  Also, some other microbiological instruments like a spreader and inoculation loop could be helpful.  I made it at TechShop with some scrap metal, pliers, and a vise.  www.techshop.ws 

You will also need the raw materials that will make the solid culture medium.  You can obtain laboratory-grade materials from Amazon, or you can make your own 1.040 wort and use yeast nutrient and agar-agar to solidify the plates.  You can buy D-(+)-Maltose, Peptone, yeast extract, and agar technical (solidifying agent) on Amazon, believe it or not (autoclave the maltose separately to prevent caramelization of the sugar).  While this method produces solid growth medium that is perfect for keeping yeast in a laboratory setting, many folks don't want to shell out the dough for these materials, and prefer to make use of beer-making equipment and materials they might have at home already.  

The easy way to do this is to get agar-agar (solidifying agent similar to gelatin) online or at an Asian food store.  Then, make a simple 1.040 gravity wort using only brewer's 2-row base malt, add yeast nutrient and 20 grams of agar-agar into 1 liter of culture liquid (can be scaled down accordingly depending on your needs), and autoclave in an appropriately-sized Erlenmeyer flask (you don't want to exceed about 50% of the volume of the flask when autoclaving).  Personally, I don't have a pressure cooker capable of autoclaving my 2 liter Erlenmeyers upright and full of liquid, so I make small batches.  You can easily make up 1.040 wort and split into several batches (in mason jars) for autoclaving purposes, and these will keep in the fridge or basement as long as they are sealed.  Then, you can use this liquid for starters or 50-100 mL at a time for making solid culture medium plates such as we are discussing.  In this case, you would take 50 mL of this nice pre-autoclaved wort and add 1 gram of agar-agar to it, dissolve, and autoclave again in an Erlenmeyer flask in your pressure cooker.  

Step 2: Surface clean and autoclave your glass petri dishes

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I like to wrap my petri dishes (with lid) in aluminum foil, and also to pre-sterilize my Erlenmeyers just for storage purposes.  This covering allows you to maintain a sterile environment within the wrapping when you take it out of the pressure cooker, but won't prevent the steam from entering and sterilizing your equipment.  REMEMBER, you NEVER want to autoclave a container with a screw top lid on it.  Remove the screw top lid from your slant tube and wrap it separately in foil.  The mouths of Erlenmeyers and the slant tube can be covered in foil.  Autoclave these safely as per previous instruction (pressure can be 'quick-released' to speed up the process, ONLY if you are using 100% borosillicate glass materials), but be patient.  This is a preparation stage and can be done well in advance of actually making solid culture medium plates, if desired.  

Step 3: Drink a Michigan-made craft beer while you wait!

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While you wait patiently and attentively for your pressure cooker, why not enjoy a Michigan-made craft beer?  Choose one that is also un-filtered and un-pasteurized and you also will have plenty of dregs to work with later in the day for culturing yeast!  In my case, I chose to gently pour out a Dark Horse Crooked Tree IPA, leaving visible dregs in the glass, and covering the mouth with foil afterwards.  Don't put the bottle to your lips!  You will end up culturing some nasty bacteria instead of yeast, and end up with much more contamination than is practical.  For added protection you can pour yourself the beer with an alcohol lamp lit and close by.  

Step 4: Make wort, dissolve powders, and autoclave liquids

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I like to dissolve the powders before autoclaving, using very low heat in a small pot/pan before I autoclave it, but this is not strictly necessary.  I like to do it at home because I feel like there is a lower amount of thermal mixing going on in my pressure cooker than I get with the autoclave I use in my laboratory (there I just chuck the powder in to the flask and cover with liquid, hit the button and walk away).

Please remember that proper maintenance of your pressure cooker is essential.  Read up on the manual for your model and please follow all safety precautions indicated there.  A pressure cooker is not a medical sterilization device.  Spore test your autoclave after 10 hours of run time to ensure it is sterilizing effectively.  A 3M Attest biological indicator capsule is very easy to use.  Safety first, people.

Step 5: Setup a semblance of a sterile environment to pour your plates in

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Arrange your pre-sterilized and foil-wrapped petri dishes, slant materials (if applicable), some latex or nitrile gloves, and your alcohol lamp(s) on paper towel in an area free of disturbances (wife/husband/kids, pets, air drafts, etc).  You want an area of still air, preferably.  The area has to remain undisturbed for 15-30 minutes while the agar plates solidify (the corollary to this rule is that your agar will solidify in the Erlenmeyer flasks after autoclaving, if you don't pour the plates within a reasonable amount of time!). 

I like to keep the alcohol lamp(s) lit and between me and the plates I'm pouring.  This acts to catch any stray particulates emanating from my breath or circulating around the disturbed air around my person.  While the lid of the petri dish is off, you always want a lit alcohol lamp nearby creating an updraft which will give it protection from a majority of airborne contaminants which would love to encounter this rich growth medium.  

PLEASE NOTE: working in proximity to FLAME and FUEL (denatured alcohol) can be dangerous.  You are responsible for your own safety.  I recommend practicing the motions beforehand, without the lamps lit.  Good sterile technique can take years to master, so don't become discouraged.  I can't go over all of this since it would require much more than an Instructable, but I think we can reach a good level with common sense.  Sterilization is different than sanitization, and anything (upstream or downstream) that comes into contact with these plates will grow on it, whether it be bacteria, mold, non-saccharomyces yeast, or the desired brewer's S. cerevisiae yeast).  Just try to be cognizant of the fact that this is GROWTH media.  Any small amount of contamination will lead to growth of that contamination, so mistakes can be easily amplified (since we are actually attempting to amplify/grow organisms).  If at first you don't succeed, LEARN FROM YOUR MISTAKE, and then try again.  

Step 6: Pour plates/slant(s)

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You don't need a thick plate.  The yeast are only going to grow on the surface, and these aren't plates designed for storage for long term (this will come later).  The slant, however, should have a reasonable amount of medium in it, although it does need to be tipped on its side for solidifying, so plan accordingly.  This pouring process should be done before the agar solidifies in your erlenmeyer flask, so work at an appropriate and safe pace.  

When you are done, leave the lid slightly off of the plate.  This will allow the heat to escape as the agar solidifies, and you won't get a crazy amount of condensation containing contaminants on the inner surface of your petri dish lid.  ALWAYS have a lit alcohol lamp very close by while the lid is off, even slightly.  

For my slant, I like to put the foil lid back on while it is cooling, and recycle nearby foil from the plates into a makeshift holder, which allows you to set a custom angle for your slants while they solidify.  This means you can use any size slant tube that you might be able to find.  Keep the screw top lid wrapped in foil, and put it on later after the slant has cooled and solidified.  

Step 7: Cover and store plates

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In short order your plates/slants will have solidified.  You can 'jiggle' them slightly to make sure they are solidified before you put the lid on them.  I like to store them upside down so that the lids don't continue to accumulate condensation from the interior environment of the dish.

At home, I like to pour a small amount of denatured alcohol on a paper towel and wipe out the interior of a plastic sandwich bag, and store the plates unwrapped inside of that.  Once my slant cools completely I'll add the sterilized screw lid to that.  These can be stored at room temperature, or inside the fridge.  Each method has its own advantages (the cool temp inside the fridge will inhibit growth of any potential contaminants and prevent them from drying out as quickly, although storage at room temperature will fairly quickly alert you to the growth of any potential contaminants.  It's a give and take situation).  Before you use a plate for culturing purposes, you always want to bring it out of the fridge and let it come to room temperature before you plate the yeast dregs, giving the yeast a bit of a nice warm environment to grow on and preventing a sudden temperature shock.  

Step 8: Grow yeast!

An upcoming Instructable will go over the process of using these plates to make a pure culture of yeast from an unfiltered/unpasteurized (impure) craft beer, both on plates and slants.  It will also cover the propagation process to go from a small amount of yeast on solid growth medium plates up to a liter of liquid yeast culture, or more!     
Jelonek2 years ago
Nice work! Thank You.
Kiteman2 years ago
Yay, science!
SHOE000727 days ago

I am for a while tried to grow baker yeast on Nutrient agar which I bought from BOREAL SCIENCE but I just bought maltose from Prolab scientific and it is very expensive 15 dollars for 100 g. So Maltose is required to grow yeast. Would this work with baker yeast and other fungi?? This is because nutrient agar for a strange reason did not grow any yeast expect for pencillium italium instead. The spores in the air were naturally there in small amounts.

J.p.H6 months ago

This is a pretty awesome tutorial. I am going to try this, but a little differently. I think it would be better to use disposable petri dishes. You can get a 10-pack for like $5 and they are already sterile. Also, it's better to wrap your dishes with parafilm. They will get contaminated if you leave them unsealed in a bag. Plus, you want some gas exchange. After isolation on the petri you can then move to a culture slant.

mirogster2 years ago
1000 @°°@ it's me baby !!! Very interesting tutorial. Cheers!!