Your Home Yeast Lab Made Easy - Making Agar Plates
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- เผยแพร่เมื่อ 15 ต.ค. 2024
- In this video I demonstrate how to prepare, sterilize and cast two different (but cheap!) types of agar plates, both of which are suitable for growing brew-house yeast and bacteria.
The recipes for the media used in this video, as well as the recipes for some derivations of these media, can be found in the associated post over at my blog:
suigenerisbrewi...
Great content and one of the best and easiest forms of home brewed agar plates i've seen yet.
Keep up the great work.
Cheers
Outstanding how-to videos! I'm getting into yeast ranching for my homebrewing, and I have no laboratory background. This helps so much! Thanks!
Glad they are of use. Good luck with your yeast ranch.
Thank you for your excellent video. I found it extremely interesting and informative.
Glad you enjoyed!
Brilliant video thanks. I am hooked and will be a devotee for many months.
Ah this brings back memories of my home lab days back in the 80s. It was great having access to the professional lab grade bottles of dried PDA and MEA powder but when that wasn't available I just made it from scratch just like was shown here. (I was not cultivating yeasts at the time but rather isolating various mushroom mycelia).
How did you store fungi? I'm trying to grow P. microspora
I learned a lot from your videos, I hope to see more videos about beer yeast, thanks, greetings from Venezuela
What a great video! Will this agar process work on fungus grow?
Yep. Most fungi will grow on the potato dextrose agar.
@@SuiGenerisBrewing I was boiling my agar for an Hour and still got contaminated, inside the agar itself, some white spots starting to appear.. is it essential to use Distilled water to keep it cleaner? I used tap water instead and poured inside still air box
@marksabota4056 still air boxes aren't the greatest for limiting contamination. I'd suggest pouring with a budsen burner or alcohol lamp, like shown in the video. Tap water is usually fine.
@@SuiGenerisBrewing yes I was surprised you haven't used air box, no gloves, nothing haha and me trying to battle this thing so much with iso alcohol all over. Thanks for reply. I will give it a shot again!
Did you sterilized your glass container prior agar boiling??
@@marksabota4056 no, as it would be sanitized as the solution in it boiled.
Great video. I like this little series you are doing on home made yeast lab. Cheers and thanks for doing it!
I read that yeast for brewing should be cultured on malt extract to avoid changes in their preference for sugar sources. If true, the MEA media should be used even if that results in weaker growth. Is that your experience?
In theory yeast can downregulate the transporters they use to import maltose if grown on simple sugars, which would then lead to a longer lag phase one pitched. That said, I've not seen a meaningful effect when I use PDA, YPD, or other media that use simple sugars like glucose or sucrose.
Appreciate your video. I notice you do not add and "dextrose" to the DME & Agar mix. All other recipes I have seen use some form of sugar, even the ones done by laboratories.
DME contains a mixture of sugars (including dextrose), protein/nitrogen and micronutrients, so it can be used "straight" as a culture medium. Commercial medium tends to be made of more basic components, e.g. yeast extract for micronutrients, peptone for protein, etc, so an exogenous source of sugar has to be added.
You can add sugar to DME based mixes, but it doesn't really give you anything that you get from straight DME.
Thank you for clearing that up for me....EXCELLENT! Much appreciated!
I liked your video the second I saw that potato slice weighing 50g. Bravo!
another great video. One question. When you were demonstrating making the potato dextrose plates, you didn't say how much dextrose you added or if you did, I didn't hear it. How much was it? I'd like to try this method. Also, would you recommend making several plates to have ready at any given time or just make a few at a time?
I forgot that bit - I've added an annotation to provide that info, but the long and short is you want 2% by weight dextrose and 2% by weight agar. That would be 2g of each if preparing 100 ml of PDA.
Thank you for your video! I have a question. What yeast is the one you've mentioned at 12:57? Koka-chair?
Kloeckera, which is also called Hanseniasporia
@@SuiGenerisBrewing Thank you so much!
Very useful and instructive ! Thank you so much !
Greetings from France.
Thanks for the very informative ideas you shared!
very informative. I would like to know if we can put the plates under a temperature of around 30'C (India) ? This temperature would be just the room temperature without the incubator.
30C would work for most ale yeasts, as well as brettanomyces and brewing bacteria (lactobacillus, pediococcus). However, some lager strains may die at those temperatures.
Thank you for all the useful infos! How long will the plates be good for? Should I use parafilm and store in the fridge?
Store them in a sealed bag (e.g. ziplock/zipper-sealed) with as much air pushed out as possible. They are good for a few months stored like that.
One issue you may find is condensation on the stored plates. The best way to avoid this is to place the plates in a warm oven (e.g. with the light on) for a few hours after you cast them (with lids on). This will dry the gel slightly and reduce condensation.
Always store plates with the gel-side up (e.g. lid-down). This way condensation that forms on the gel will drip onto the lid, rather than the reverse (which can carry contaminants onto the gel)
@@SuiGenerisBrewing wow, thanks for the quick reply! I wish the professors at my uni were that fast... I'm really excited about yeast ranching, I did my first streak plate to purify a yeast from an infected batch, I hope I've picked the right colony:)
Excellent video! It's a perfect how-to video on agar plates. I have a question: Have you ever measured the specific gravity of the potato extract or somehow figured out how much sugar/fermentables it is contributing? When I pour agar plates, I usually use DME at about 7-10%, yielding an SG of about 1.030 to 1.040. At this amount of sugar, after streaking my yeast out, the plate grows to completion in less than a week. I was wondering why you make your DME agar plates at such a low percentage of DME? Thanks again for such a great video!
I've not measured the SG of the potato-dextrose; its quite viscous as a liquid as the . bulk of the extract is gelatanized starches. From the yeast perspective those starches are not fermentables - indeed, without the addition of dextrose (or another sugar) yeast grow minimally on the potato extract. The potato is there to provide nutrients to the yeast, not energy. Nutritionally speaking, PDA has ~50x the amino acids, metals and other components yeast need for growth than does wort-based media.
As for DME, the rational for the lower-gravity is two-fold:
Firstly, malt-based agar is a well established microbiology medium, and the 1.002-1.006 range (i.e. 2-6% w/v) is generally considered optimal; keep in mind this is not liquid media, and the yeast are exposed directly to air. Higher gravities under these circumstances place a lot of osmotic stress on yeast, which can limit the growth of many strains, increase the risk of mutation and other stress-related defects, and even prevent some strains/species from even growing. There is a higher-gravity version of wort-agar, which comes in around 1.024, but this is used to preferentially select for osmotically tolerant moulds over their less-tolerant yeast cousins.
Secondly, higher gravity = wasted money/materials. Your yeast are growing on the top of the plate, not throughout it, meaning the yeast are feeding off the entire depth of the agar beneath them. As such there is a tremendous amount for them to eat, even given the lower gravity.
As for growth success, I'd point out my plates grew to near-completion in less than a week at well below the temperature we'd normally prefer to grow yeast at (16C vs. 20-28C). If I grow them at work (where room temp is actually "room temp" - i.e. 21C/72F) growth is generally complete in 48 hours; if I use an incubator at 28C growth is complete overnight.
Sui Generis Brewing Thanks for the quick response! I will definitely lower my DME next time I make plates. Does the same logic hold when making slants? That is currently my preferred method of storage. Do you use slants for long term storage, or are you set up for glycerol stocks in microcentrifuge tubes?
+Brian McConeghy Same rules apply for slants. I'm planning video's on slants and at-home freezing, but no promises as to when those will actually get done.
@@SuiGenerisBrewing FYI. For my home "lab", I am using an Instant Pot Duo Evo as my incubator. The Duo has a Sous Vide function, and the temperature control is very good after it settles down. I notice temperature overshoot initially and then a very stable temperature (monitored with lid off and a Kegland RAPT thermometer). The plate is contained in a stainless steel steamer, bain-marie style. It's the kind with no holes and a lid, which can float and keep condensation drips off the plate. I will run a pressure cook cycle after use as a safety precaution.
I have learned a lot from your videos.Exselant. Please can you tell me the name of the drops you used . I just cannot make out what you call this. Thanks Richard
+Richard uk They are fermcap-s, and can be purchased from most homebrew supply shops.
I'm confused at 4:18.... Wart, wort? That was not in the ingredients anywhere, I don't have that :-\
Also, what is the alcohol lamp for? It looks to be just sitting around in the video...?
Wort, which is unfermented beer. You can make it from malt, or dry malt powder.
The alcohol lams use is explained in a separate video, but the short version is that it relates an updraft of air which will carry contaminants away from your samples- so long as you work close enough to it, and move slowly to prevent drafts.
Question, could you use the PDA without Agar to grow yeast in a starter or pre-starter (ie, taking from a slant, putting in Potato-dextrose, then adding to a starter)?
Probably, but I don't know how much growth you'd get compared to DME. PDA is a fairly low in protein, which you need to get good cell yields from a starter.
@@SuiGenerisBrewing Ah, ok! Will experiment and return!
Really enjoy your videos! Any tips as to how we should wash glass plates?
Just dishsoap, water and a bit of elbow-grease. You can use a dishwasher as well, but make sure the plates wont get banged around too much.
Very awesome stuff, thank you Bryan!
Very nice videos.
Do contaminants not gather on the tinfoil cap while it is off on the counter?
Ah, I just saw the Aseptic Techniques video.
Thank you for your great video. Are the plastic dishes Polystyrene and are they 100mm X 15mm?
Can you re-use the polystyrene dishes?
I noted that the dishes you placed in the oven were made of glass.
I use both glass and polystyrene; glass can be reused (following autoclaving/pressure cooking), polystyrene is generally considered single-use...that said, I know a few brewers who remove the gel, wash the polystyrene plates and then soak the plates in starsan overnight before casting new gels in the plate.
And yes, my plates (glass and plastic) are 100mm x 15mm
I was just about to buy some pitri dishes and I have a choice of 3 vents or single vented or non vented. Please could you tell me the correct pitri dishes?
Thank you for your reply.
It doesn't really matter - the "vents" are nothing more than a tiny bump of plastic on the lid that creates an air gap. I generally buy whichever are cheapest; yeast don't really care.
How much agar powder should we add, is it ok to use unpowdered form of China grass
+Vahin Mohan 2% by weight (i.e. 2 grams per 100 ml wort). The unpowdered stuff is fine, but you need to slowly melt it on the stove before boiling/pressurecooking the media
If we can both for tissue culture ?
Thank you very much for putting this together. I am starting to plate yeast and have found these very useful. I have an excellent source of many different beers/yeasts/bacteria from my homebrew club to work with and can't wait to get my yeast bank up and running!
Just tried to make some plates on Sunday night. Must have become contaminated somehow because now they all have a thin clear/white slim growing across the entire surface of all 4 plates. I even used the pressure cooker to sterilize the agar solution. I guess I'll try again soon...
Sounds like mold, which usually comes in through the air; if you didn't before, pour near a flame (bunsen burner, alcohol lamp, etc) in as a draft-free area as you can find. Lift the lids the minimal amount you need to pour the plate, and cover them as soon as the plate is poured.
How much sugar and agar did you use for the 2nd experiment?
2% (weight/volume) of each. E.g. 2g agar + 2g sugar per 100 ml
I really appreciate your videos. However I have a question.. When freezing the yeast at what ratio do you maix slurry/ glycerol and is 80% glycerol sufficient? I just tried freezing some yeast that I propagated from un sulfured sultanas and figs and mixed slurr at about 50%/50% with 80% glycerol. It's not really freezing solid in my domestic freezer though.
+rimmersbryggeri
80% is excessive. For lab-freezers (-80C) we generally use 15-20% glycerol. For home freezers (typically -10C to -20C), 30-50% glycerol is generally best. They should not freeze solid in your freezer, but rather should be slushie-like. For freezing you generally want to let the yeast settle completely, decant as much liquid as you can, a then suspend the yeast slurry in at least 2-volumes of freezing solution (i.e. at least 2X the volume of slurry; if you had 20ml of slurry I'd use at least 40ml of freezing solution). I would recommend 1.020 wort + 30 glycerol for a home freezing solution.
Sui Generis Brewing
Thanks we will see what happens I did about 50% slurry to 50% Glycerol (80%). Lets just hope it survives.
Hi, In where Im living there is no Dry Malt Extract (DME) in the market. What do you suggest to use instead of DME to make agar or liquid culture (LC) for growing mycelium that could have the same or even better results than DME? Thank you
You could buy a commercial media like YPD (yeast peptone dextrose). Another option is homemade potato-dextrose-agar, which I discuss in this video.
The recipe for PDA can be found on my blog as well: suigenerisbrewing.com/index.php/2015/03/18/new-video-casting-agar-plates/
@@SuiGenerisBrewing can i use Dextrose Monohydrate ?
@@amintaleghani2110 yes, or glucose, or even sucrose (table sugar).
Nice Video...
What is the liquid you add at 5:50 ?.....
Fermcap-S, which is used to prevent boil-overs. Not required, but it makes it a lot easier to boil the agar. Without it, you have to be very careful that the agar solution doesn't boil over.
If you cannot find it in your area you can add a quarter of a crushed anti-gas tablet - but make sure the tablet is one that contains "Simethicone"; the anti-gas tablets that contain "Alpha-galactosidase" will not work.
In reference to the " hop the PDA"..... What purpose would this serve?
It will inhibit lactobacillus growth
@@SuiGenerisBrewing so how would you perform this correctly?
@@628Amos just add a gram of hops per 100ml, pressurecook for 15 mon, then strain to remove the hops
This is GREAT information :-). Thanks
Thanks for the detailed explanations. I have a question though: "Since Hydrogen peroxide is produced by the human body, it is a natural anti-septic and its commercial form is used to clean wounds to prevent infection. So, can I use HYDROGEN PER OXIDE instead of STAR SAN, which is not on the German market?".
3% hydrogen peroxide can be used, but you need to rinse with water afterwards as peroxide is quite toxic to yeast and can lead to stress and off-flavours, even in small amounts. There is a German homebrewing community (hobbybrauer.de) who may have suggestions for alternative products that may take starsans place.
Thank you very much for the swift answer. You are very kind and you know your staff right to the smallest detail, so please keep up with good work.
Is there a reason you don't sterilize the dish's (wrapped) and some test tubes for slants in the pressure cooker while already filled?
Its not practical to do it with plates, for two reasons. The first is that you will get an inordinate amount of condensation via that method, the second being that any slight tipping of the plates can spill the agar. And, of course, plastic plates cannot be autoclaved/pressure treated. In the (real-world) lab, plates are almost always poured on the bench, as per the video. I have a separate video on slants, and in that video the slants are autoclaved/pressure-cooked after filling, and then angled afterwards to cool/solidify as a slant.
TY
Thank you again. Wonderful presentation.
If I want to make 100ml of Agar. Is it necessary to put 2-4g of DME? Or can I just add 2g of simple Agar? I don't know what DME is or what it means?
DME = dry malt extract; its the 'food' for the yeast, so if you don't add it the yeast wont grow. If you lack DME, you can instead add 2g of agar to 100ml of 1.004 to 1.008 gravity wort.
But I've seen other lab videos and nobody mentioned DME before. I bought agar from a Lab and they didn't mentioned that either. Can I buy DME from store too?
If you do not need to sterilise your Agar-wort solution, then why do you need to sterilise your vials, plates, mineral oil, starter wort? That could save me from buying an pressure-cooker. Or is it because you can see contaminations on your plate right away? Confused
You can achieve near-sterility by boiling, which 99% of the time is sufficient. A pressure cooker simply guarantees that your materials start out truely sterile.
So you don't need one, and indeed, I banked by first 80 or so yeast without one. However, a pressure cooker offers more certainty that your cultures will remain pure.
Sui Generis Brewing thanks a lot. I’ll keep that in mind.
Slowcooker is on the “to buy” list 😉
@@martijnblokzijl1350 FWIW we use ours for cooking all the time. Its got a lot of good uses outside of canning/brewing
Sir can we make banana culture like mushroom culture or pda
I've not used banana for yeast, but it may work similar to pda.
excellent, i learn a lot , this is so informative
Glad you found it useful!
i want to know what's contient in potatos extract? pleas
Potatoes and water:
1) Finely dice 100g of potatoes
2) Add diced potatoes to 0.5L of water
3) Simmer for 20-30 min
4) Separate liquid (potato extract) from remaining potato solids
The potato extract can be used immediately, or frozen for future use. To prepare potato-dextrose agar (PDA) plates:
1) Mix 100 ml of potato extract with 2 g of dextrose (corn sugar) and 2 g of agar
2) Heat until agar is dissolved
3) Autoclave/pressure cook for 15 min to steralize
4) Pour agar plates as per my video
100 ml of agar should provide you with 3-4 100mm diameter agar plates.
Sorry, small mistake. Add 100g of potato per 1L of water.
Thank you I just finished but I have no d-glucose so I just use regular sugar, and I have no pressure cooker. I should buy one in a week. Thank you dude
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Is a great video for revision too!
I have 20% agar and 80% msltodextrin mixture. Can I use it or should I buy 100% agar?
I would suggest getting agar, as the high amount of Maltodextrin may osmotically stress the yeast
@@SuiGenerisBrewing Thank you!
Can I use steam rice pot to steam instead using pressure cooker ?
I have no idea. Does it cook under pressure?
Pda formula? Liqued formula?
Aspergin?
All those details can be found in the link at the bottom of the video description.
thank you for the video
how much agar to the potato mix and how much corn sugar
2% ny weight of each. E.g. for 100 ml you'd use 2 g agar and 2 g corn sugar
this is in addition to the grated potato it is just starchy water plus dry malt yeast 2-3 gr. and agar2 gr. I just did some agar but it did not do as much as I thought it should have so I wanted to do something different. Thanks
If I don`t have alcohol lamp , Can I use candles or gas lamp ?
I have a video on making an alcohol lamp; super cheap, and requires only a drill. A candle lacks the power; a gas lamp is not safe to use for this purpose.
yes , it is under pressure.
Then it may be OK. Ideally the pressure should be around 1 atm (~15 PSI) of pressure.
I've read to never put an erlenmeyer flask on an electric stove element.
It is fine so long as the flask has liquid in it. You cannot heat an empty flask on an electric element, but you can on a flame.
My country doesn't have dried malt extract
Really? How about liquid - that would work as well (although I'd wait to add it until after the inversion step). If malt extracts, in general, are unavailable you can try other protein sources - but be sure to look for protein sources that don't have fat or lipids. Options would include skim milk powder, protein powder (for body building) and yeast nutrient (the type made of broken-up yeast).
Sui Generis Brewing you mean protein can i use soy protein drink
So long as it has a low fat content. Fats will interfere with the formation of melanoidins and can produce unpleasant side-products. If using something like a soy drink, I would add it after the inversion of the sugar is complete.
It should work, so long as it has a low (or zero) fat content. Fats produce unpleasant flavours when heated like this, and may interfere with the formation of the melanoidins. If using this kind of beverage, I would add it after the inversion step of the process.
Sui Generis Brewing ok I don't have dried milt extract what can i use to substitute that
is this psilocybe mushrooms?
Are you?
yah know what would have been nice... you pick up the brown bottle and hold the name of it in your hand instead of showing what it was...
6
?
50g on da nose!