New Video! Casting Agar Plates
The next video in my “your home yeast lab made easy” video series is finally complete. This video covers the preparation of agar plates for yeast and bacterial culture. The video covers two media preparation for propagating yeast and brewhouse bacteria, proper plate handling, sterilization, casting and growth characteristics. Additional media recipes can be found below the fold…
Additional Media Recipes:
Recipes are given in percentages or gravity units to make scaling to desired size easy. You need 25-30 ml of media per 10cm/100mm (standard sized) petri dish,
Wort-Agar (in-video):
- 2-4% by weight dry malt extract (2-4g/100ml)
- 2% agar by weight (2g/100ml)
- tap water (or other brewing-compatible water)
- Optional: yeast nutrient
- Optional but recommended: Fermcap S
Dissolve DME, yeast nutrient and Fermcap S into the water; once compeltely dissolved slowly add the agar while stirring, being careful to avoid lump. Steralize for 15min in a pressure cooker at maximum pressure, or by simmering for 5 minutes. Cool to 60-65C/140-145F and pour into sterile plastic or glass petri dishes.
Wort-Agar (from wort):
Collect or prepare wort at a gravity of 1.020 to 1.040. Dilute to 1.002-1.004 (1:10 dilution of 1.020 to 1.040 wort). Add Firmcap S (optional) and 2% agar by weight to the diluted wort. Sterilize and cast as described above/in the video.
Hopped Wort-Agar (from wort):
To reduce the growth of brewhouse bacteria such as lactobacillus a wort with a final (i.e. post-dilution) hop alpha acid content of at least 10IBU can be prepared. Dilute some collected wort to 1.020, or or prepare 1.020 wort from DME. Boil sufficient high alpha-acid hops in the wort to achieve at least 50 IBU (preferably 75-100IBU) of bitterness. Dilute 1:5 (gravity of ~1.004, min 10 IBUs) and prepare as with “Wort-Agar (from wort)”, above.
Potato-Dextrose Agar (PDA, in-video):
Grate 50g of potato, with skin, into a small sauce pot. Add 100ml of water and bring to a simmer for 30 minutes. Strain the potato extract through a fine-meshed wire strainer – if clearer plates are required additional filtration through cheesecloth or a coffee filter can also be performed – warning: this will be extremely slow and is generally unnecessary. Next, dissolve 2%/weight (2g/100ml) of dextrose (corn sugar) or another fermentable sugar like table sugar (sucrose) into the potato extract. Then slowly stir in 2%/weight agar, being careful to avoid agar lumps. Steralize and cast as described above/in the video.
Hopped Potato-Dextrose Agar:
To get hopped PDA, pre-boil the hops in the water later used to boil the potatoes, aiming for 10-20IBU. Then prepare the plates as per PDA (above), using the hopped PDA.
Other Media Additives:
There are many other things that can be added to these medias – especially PDA – to enhance the growth of specific organisms, or alternatively, to allow for identification, or to achieve other purposes. I have not worked extensively with these, as I rely on genetic methods and antibiotics (some of which are described below); options not available to most brewers. However, several others – notably, Sam over at Eureka Brewing and Dmetri at BKYeast, have experimented with a few options. A few ideas (and links, where relevant) to try:
- pH indicator such as litmus. This will help identify acid-producing organisms such as Brettanomyces yeast and lactic-acid bacteria [BKY]
- Bromocresol green: This dye is selectively degraded by Brettanomyces but not Saccharomyces, allowing for identification of these yeast species. [Eureka | BKY]
- Cycloheximide: an antibiotic, available from some scientific supply companies, which selectively kills Saccharomyces, but not Brettanomyces or brewhouse bacteria. Make a 100X solution (10mg/ml), and dilute 1:1000 into agar immediately before casting. DO NOT presurecook/boil this reagent. CAUTION: This is a potential carcinogen and is highly toxic, so wear appropriate safety equipment and avoid exposure.
- Pen/Strep (100X): This is a mixture of penicillin and streptomycin, antibiotics which when combined will prevent the growth most (all?) beer-contaminating bacteria. Very useful for separating Brett/Sacc from wild/sour fermentation. Dilute 1:100 into media immediately before casting plates – do not boil/pressurecook this reagent.
The above represent a summary of reagents that are available to some/most brewers that I have seen work either first hand, or via the blog posts of others, A few other options that are out there, but I can offer no direct/indirect experience with are:
- Addition of 75g/L table salt (sodium chloride). This should result in a medium which allows for the growth of halotolerant lactobacilli, which would include Pediococcus and Lactobacillus. Be sure to use kosher, pickling, or otherwise non-iodated salt only.
- Replacement of dextrose with lactose in PDA media. This should create a medium which allows for the growth of all Lactobacilllus species, a some species of Pediococcus, and some strains of Brettanomyces. No Saccharomyces should not grow on this medium.
- Copper sulfate. When added at a rate of 0.6g/l this should impair the growth of non-Brettanomyces yeasts.
Commercial Medias:
There are a number of bacterial and fungal medias available from commercial sources – YPD (yeast peptone dextrose) being a commonly used media for yeasts and de Man, Rogosa and Sharpe (MRS) a commonly used medium for lactobacilli. While excellent medias, these tend to be expensive and offer only minor advantages in terms of ease-of-preparation and growth quality.
But more to the point, PDA is broadly recognized as the preferred medium for the growth of food-relevant fungi and lactic acid bacteria – indeed, it is a medium recommended by the FDA and by numerous food-inspection agencies for enumerating fungi and lactic acid bacteria in food and cosmetics. So save yourself some green and use what the pro’s use – potato-sugar-water!
Bryan first thanks for the videos, I have two questions.
What is the recommended temperature to grow (ALES and Lagers) yeast, for agar plates?
How many grams of yeast nutrient is enough for 100 ml of wort agar(DME)?
Most ale yeast can be grown up to 37C, with maximum growth seen at that Temperature. That said, I’ve had a few strains struggle or die at that temperature, so I typically use either room temperature (which is slower) or 34C for ale yeasts.
Lagers will die at temperatures over 28C, so I typically grow them at room temperature (20C).
As for yeast nutrient, it depends on what you’re adding. For DAP, I use 1/8th of a teaspoon per liter for liquid medium. For a more complete nutrient (Fermaid, servomyces, etc) I use 1/4 tsp. That said, nutrient isn’t strictly necessary for agar plates, and I generally don’t bother adding it. The growth advantage is minimal.
How much litmus would you recommend adding to a PDA/Lactose Agar mix? Trying to find ratios and lots of places recommend litmus but not how much to add
Thanks!
0.1% (w/v) – e.g. 1 gram per litre.
Great video and thanks for all the info.
What about pouring agar half way up the plate, THEN put in the oven for sterilization?
Skips the potentially infectious pour stage.
Would this not work and why?
Thank you again.
For plastic plates that won’t work as they will melt. For glass you can steralize the medium in the plate. There are two issues though – the first is that the agar can settle, leaving you a layer of highly dense gel below a layer of liquid medium, and the second is that condensation tends to form in the plates, diluting the agar.
In the end, its faster/easier to pour into pre-steralized plates. And with practice (and good technique), contamination during pouring is essentially a non-issue.
If you are getting contamination under these conditions you likely have a contamination somewhere in your hood that is being blown onto your samples. I'd recommend cleaning the hood thoroughly.
Bioshop Canada may send it to a home address, but I am not certain.
https://www.bioshopcanada.com/secure/detail.asp?Pin=PST999
Love your blog. Even with the use of a flow hood and autoclaving I'm haveing a small bit of contamination, viewed at 1000x. A few rods and some very tiny guys that move somewhat.
I'm interested in trying pen-strep. Is this available in Canada to the public?
See my recipe for potato dextrose agar, which can be used instead
liquid malt extract, potato, oats, yeast extract + sugar
Is there a way to substitute the dry malt extract with something which can be found at any house or quite in general?
No, starch will not work. The potato is providing a lot of things other than starch; various vitamins and nutrients, plus a nitrogen source. Pure starch will lack all of that (flour, presumably, will not – but I haven't tested that option).
I you want to use starch in place of potato you will need to replace all of those nutrients – using a complete yeast nutrient (e.g. servomyces) may work, but I've not tested it so I cannot say for certain.
This series has been a great source for setting up my home lab so firstly I just want to say thanks. Now for a question regarding the potato. I came across potato starch in the store, not potato flour. I was wondering whether this couldn't replace the whole cooking of the potato. Question is how much would you substitute for the 50g of raw potato. Also help with filtration.