Selective Media Part I

Beer microflora is one of the most important factors in brewing. It’s no secret for any brewer that if you brew a batch, slit it into 100 portions and ferment with 30 different yeast stains at different temperatures and pitching cell densities you’ll end up with 100 different beers. Yeast and bacteria in beer impart their unique flavor profiles to the product which are subject to varying fermentation conditions. While for beginning homebrewers working with yeast goes about as far as opening a packet or a smack-pack and pouring it into the wort, a more advanced beer lover will start to crop the yeast, collect yeast cakes, wash, and eventually become a bit of a “homebrewer microbiologist.”

One of the biggest challenges homebrewer microbiologists, especially those who dabble in wild beers, face is yeast and bacterial strain differentiation. This problem goes far beyond the scope of making starters and maintaining a frozen yeast bank or slants, that at least at this stage of homebrewer culture, are considered to be about as fancy microbiological techniques as can be achieved in your kitchen. Some brave souls venture as far as reculturing yeast from bottle dregs by pouring them into starters and hoping they do not get infected which works with varying degrees of success. And only few who are crazy enough and have some real scientific background go as far as making plates, using various media, know-how and techniques to separate and differentiate different bugs.

Such people include myself, Jason at Brew Science, and Sam at Eureka Brewing. There are of course others, but I haven’t had the pleasure of communicating with them. We, the scientifically inclined brewers, however, have a flaw in that we tend to deviate from the truly “homemade” and cheat a little by utilizing lab equipment at our disposal which, of course, is way out of reach for the average brewer.

With the recent explosion and popularization of wild beers homebrewers are spearheading the funk movement and often produce beers far superior to commercial examples due to their ability to experiment with small batches. In such conditions it is becoming increasingly more important for us to be able to distinguish between the different bugs we use. While it is all but impossible to distinguish between Saccharomyces cerevisiae from Sierra Nevada and Chimay in home setting, it is possible to differentiate between Saccharomyces, Brettanomyces and bacterial species.

Over the course of the last few months I’ve been trying to come up with some homebrewer friendly media that could grant one the ability to tell various organisms apart without too much trouble or expense. Perhaps the most unexpected result of this endeavor was the fact that these things actually work to a certain extent. While I’ve had my share of failures I feel like there is enough good information gathered by this time to share and be helpful to others. So without further ado, here it is…

The format will be the following: Name of the medium, its recipe and preparation, what it looks like, and my observations of various bugs on it. Then I’ll write some thoughts on how to improve it or what I did wrong.

Let’s start with malt based media.

Malt Extract/Wort Agar

Composition per liter:

Dilute Wort ………………… 1L

Agar ……………………………15-20g

pH 5.2 ± 0.2 at 25°C

Note: If using Malt Extract, use 20g DME in 1 liter of water. The gravity of the wort or DME solution should be around 1.002 otherwise you’ll have problems with agar consistency.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure and 121°C AKA cook in pressure cooker. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of yeast and bacteria commonly associated with brewing.

Appearance: Clear and dark-golden in color.

Observations: Pretty much everything grows on these plates. Can’t tell the difference between Saccharomyces and Brettanomyces. Bacteria can be distinguished by the different color colonies they form. This medium is not effective for differentiation, but it quite good and cheap for isolating colonies and maintaining your yeast ranch.

Bromocresol Green and Universal Indicator Addition: By adding BG and UI you may change the properties of this agar. First let’s talk about stability of the indicators. I tried both pre and post “autoclaving” and found that BG is thermostable and keep working while UI degrades with temperature. If added post autoclaving it doesn’t seem to do a very good job doing anything either. I will no longer try it in any of my media. Plates are bluish-green. I wrote before about my observation that BG seems to have detrimental effect on Lactobaillus growth and this is once more confirmed on these plates. In other words – Lactobacillus doesn’t grow on plates with BG. After digging around I was able to find that BG is a member of the triphenylmethane dye family which is lethal to most gram + organisms. So this explains the effect. As for Saccharomyces vs Brettanomyces differentiation, there seems to be a relatively short timeframe when this medium is effective. Within the first week or two you can observe a difference in coloration, but after that all colonies look the same in a mixed culture. In that window, Brett seems to form blue colonies which Sacch will form green colonies. Afterwards everything is greenish-white and medium loses coloration. All in all dye addition does not make it a very effective differential medium. My guess is that there needs to be an additional carbon source to facilitate better dye breakdown. I’ll have to reseed the cells as well as try the same recipe with some sugar addition to reach a more definite conclusion.

Failure Notes: I bet when you read that you have to dilute wort to 1.002 most of you thought that I’m an idiot and don’t know what I’m talking about. Believe me I tried using more concentrated wort and more DME to make a nice and juicy agar and every time I did that I failed. Unless you’re willing to spend a ton of agar on a juicy malt plate, the best you’ll end up with is something with a consistency of apple sauce.

Malt with Bromocresol Green Agar. Mix of Saccharomyces, Brett, and Pediococcus. 3 colony morphologies.

On to my personal favorite – the Potato Based Medium! I talked about Potato Dextrose/Sucrose Plates and the following is continuation of the “sugar switching” trend.

Potato Lactose Agar with Universal Indicator

Composition per liter:

Potatoes ………………………… 300.0g

Lactose …………………………. 10.0g

Agar …………………………….. 15.0g

Preparation of Medium: Dice potatoes and place in 500.0mL of boiling water for 30 min. Strain through cheesecloth. Adjust volume to 1.0L with distilled/deionized water. Mix thoroughly. Add agar and lactose. Gently heat and bring to boiling. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure @ 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of numerous fungi and Lactic bacteria.

Appearance: Clear and bright-golden yellow in color.

Observations: The main purpose for making this medium was seeing how lactic acid bacteria grow, since BG is toxic to lactobacillus, I think it’s obvious why it’s not used. This turned out to be far more useful than just looking at Lactobacillus and has quickly became my favorite medium. As controls I used WY Lactobacillus delbrueckii, WY Brettanomyces B, and WY Biere de Garde.

Lactobacillus grows readily, forming large whitish-yellow umbonate colonies.

Brettanomyces B took about two weeks to grow tiny round white colonies. Not really surprising because Brett B reportedly does not ferment lactose so it had to live off of whatever I got out of boiling potatoes.

-Biere de Garde yeast landed me with more questions than answers. It is supposedly the Fantome strain known for it’s super attenuative nature, and I can see why it has such reputation. I made a few beers with it and they all ferment quite dry. Now the most interesting part. The colonies grew as well and as fast as the Lactobacillus colonies on the other side of the same plate, forming medium sized white colonies! I have plated some mixed cultures on these plates (from commercial beers that I’ll describe in later posts) and in each case Saccharomyces grew very slowly. I can’t draw conclusions yet, but right now the data points in direction that this particular yeast strain can in fact ferment lactose. Weird? You bet!

Pediococcus forms round raised bright yellow colonies.

Brettanomyces L isolated from WY Old Ale Blend readily grows brownish brain-shaped colonies.

Brettanomyces ??? isolated from WY Berliner Blend forms same colonies and its counterpart from Old Ale Blend.

Overall this seems like a very useful medium for homebrew bug differentiation. It allows you to clearly pick out Lactobacillus and Pediococcus, differentiate between Brett B and its brothers, and it retards Saccharomyces growth enough to allow just about everything else to grow before they start forming decent sized colonies.

Failure Notes: As described before, UI didn’t seem to survive the thermal treatment and adding it afterwards doesn’t seem to be one bit useful. I was hoping that the acid production would become apparent with this indicator, but alas. What I’ll do instead next time is use litmus. That’s right, plain good old fashioned litmus that is all but gone from memory by now. You can still find the powder on eBay for a decent price. It is thermostable, non toxic and gives excellent acid-base differentiation.

Potato Lactose UI Agar. Mix of Saccharomyces, Brettanomyces and Pediococcus. 3 colony morphologies seen.

Potato Lactose Agar. Lactobacillus and WY BdG yeast. Weird? YES!!!

Potato Sucrose Agar with Bromocresol Green

Composition per liter:

Potatoes ………………………… 300.0g

Sucrose ………………………….. 10.0g

Agar ………………………………. 15.0g

Bromocresol Green …………Enough to turn the medium bluish-green (~0.5 – 1mL of 1% solution)

Preparation of Medium: Dice potatoes and place in 500.0mL of boiling water for 30 min. Strain through cheesecloth. Adjust volume to 1.0L with distilled/deionized water. Mix thoroughly. Add agar, sucrose and BG. Gently heat and bring to boiling. Mix thoroughly. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure @ 121°C. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of numerous fungi.

Appearance: Clear and bluish-green in color.

Observations: As stated previously gram + bacteria (Lactobacillus and Pediococcus) don’t grow  while yeast does just fine. In agreement with previous observations, Brettanomyces break down BG, forming whiter and brain shaped colonies while Saccharomyces stay bluish-green. Medium loses color and becomes colorless with Brett present by about 4-7 days. All colonies become greenish-white after about 2 weeks regardless of species. Seems like an easy way to tell between wild and usual yeast.

Potato Sucrose with Bromcresol Green. WY Brettanomyces B. Strangely, two colony morphologies are seen. Further investigation required.

Continuing the Lactic bacteria growth trend is Milk Lactose Agar.

Milk Lactose Agar

Composition per 500mL:

Milk (Whole) …………………. 25mL

Lactose ………………………….. 10g

Agar ……………………………… 10g

pH 7.0 ± 0.2 at 25°C

Note: Also added about 2g of meat tenderizer powder and let sit prior to autoclaving in hopes of imitating something resembling casein digest since the powder contains a protease and salt. See failure notes.

Preparation of Medium: Add components to distilled/deionized water and bring volume to 500mL. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure and 121°C AKA cook in pressure cooker. Let cool and add Universal Indicator. Pour into sterile Petri dishes or leave in tubes.

Use: For the cultivation and maintenance of microorganisms from dairy and water samples.

Appearance: Pink, chunky with white flakes, non-transparent.

Observations: This came quite handy for the initial stages of my yogurt and raw wort bug hunt. The project is still at the initial stages, but that’s for later posts. Can’t really tell you much about it right now because I haven’t had the time to plate any controls on these plates yet, but preliminary results from yogurts and stuff seem to show that Lactic acid bacteria grow very well and some form bright yellow colonies while others form colonies that look like pellicles. Further analysis is required. From a single mixed culture from a commercial beer that contains Sacch, Brett, Lacto and possibly 2 or 3 more bugs I can tell that they behave similarly to Potato Lactose agar and Brett or one of the other bugs in the dregs breaks down UI turning the plates from pink to white. Seems like a good medium for lactic acid bacteria so far because non bacterial species grow real slow.

Failure Notes: OK so perhaps trying to proteolyse milk wasn’t the best idea and resulted in white chunks spotting the plates most likely caused by breakdown of milk. Also 25ml of milk may have been too much because plates turned out soft. Not too soft as to be unusable, but still too soft. Next time I’ll not play around with tenderizer powder and use less milk. I’m also going to add litmus to part of the preparation, making a classical litmus milk medium that’s been used since about right after discovery of lactic bacteria.

Milk Lactose Agar. 3 of the organisms isolated from raw wort. As you can see the colonies are different and one of the samples is contaminated with another organism.

Milk Lactose Agar. Streaked a mix from commercial dregs. So far looks like Lactobacillus is taking over everything, but other colonies also visible. You can't really see it very well, but the agar color changes where the colonies grew.

To conclude this article I’ll talk about the most advanced home-made agar I made thus far. Recently I got my hands on about 50mL of 1% Brilliant Green solution in EtOH (ready available in any and every Eastern European pharmacy, known as Zelyonka or Solutio Viridis nitentis spirituosa 1%). It has been widely used as a topical antiseptic due to the fact that it kills all gram + and most gram – bacteria (of course as with everything in biology, there are exceptions). This makes it a perfect candidate to use for selection because it’ll give the homebrewer ability to kill off all bacteria from the dregs leaving only the yeast which can be differentiated by colony morphology, lactose fermentation etc. While the original recipe uses tryptone and yeast extract, which simple mortals can’t really get for cheap, I tweaked it to use what is available, and chose lactose as the main carbon source in hopes of slanting the selectivity toward Brettanomyces spp. In almost every grocery you can find bouillon cubes which are for most part hydrolyzed meat and yeast extracts. For this experiment I chose Herb Ox Beef Bouillon Cubes (~3.6 g each, contain hydrolyzed beef and yeast extract – good nitrogen, carbohydrate, mineral, and vitamin source, salt – electrolyte source) and Rapunzel Vegan Vegetable Bouillon with no salt (~8g cubes, contains yeast extract and various vegetable extracts).

Home-Made Brilliant Green Agar

Composition per 500mL:

Beef Cube ……………………………… 1

Vegetable Cube ……………………… 1/2

Lactose ………………………………… 10g

Sucrose ………………………………… 5g

1% Brilliant Green Solution ………. 0.6mL

Meat Tenderizer …………………….. 2g

Agar …………………………………… 10g

pH 7.0 ± 0.2 at 25°C

Preparation of Medium: Add components to distilled/deionized water and bring volume to 500mL. Mix thoroughly and incubate at room temperature for 20 minutes to allow further proteolysis. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure and 121°C AKA cook in pressure cooker. Pour into sterile Petri dishes or leave in tubes.

Use: For selective cultivation and maintenance of Salmonella and non-bacterial species.

Appearance: Clear, green with tiny specks – vegetable pieces from the bouillon cubes. Smells like soup!

Observations: As with Milk Agar I haven’t had the time yet to work properly with this one. Preliminary results show that it is indeed quite effective in killing off bacteria. Some bacterial colonies are able to survive in cases where I streak so much of them that they’re probably able to live above the actual medium, but the growth is still extremely limited. Yeast form green colonies.

Bromocresol Green Addition: By adding BG the properties of this agar change slightly. BG gives agar a bluish color. So far I haven’t plated pure Brett, but I would expect it to break it down as with any other medium. It did come in handy as a pH indicator. Some of the organisms I pulled from yogurts and raw wort seem to survive Brilliant Green and Bromocresol Green and are acid producers as indicated by the medium turning yellow. Right now this medium seems like a good choice for initial dregs plating since it kills off bacteria leaving you with various Sacch and Brett species. Further analysis with control strains is requires and will ensue in upcoming weeks.

Failure Notes: The only problems I have with these plates are that there are vegetable pieces in it and it’s fatty. You can actually see a thin oil/fat layer on the surface. I’m sure vegetable oils in the bouillon cubes are the source. It may not actually be such a bad thing. As for veggie pieces I’ll just try to filter the medium better next time. Back when I first prepared it my hand pump vacuum setup wasn’t good enough to filter the oily soup so I ended up just doing it as is. However this week I managed to adapt my filtering flask to a vacuum cleaner and that should improve my filtration capacity quite a lot.

Brilliant Green Agar with Bromocresol Green. Same 3 raw wort organisms as shown before on Milk plates. You can see the colony morphologies as well as medium color changes.

So there is it guys. I think the progress thus far is pretty good given the fact that I’m busy up to my ears with school, lab and a few other things I can manage to sneak into my life. I would love to keep experimenting, but time and money are not in such abundant supply. I hope this information will be useful to you and as always feel free to leave feedback, suggestions, questions, requests and so on. Stay tuned for more posts about media and bug isolation from commercial dregs as well as yogurts and raw wort.

Cheers!

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35 thoughts on “Selective Media Part I

  1. Wow, thank you for this. As one of these “homebrewer microbiologists” I greatly appreciate anything that can bridge the gap between what we do and what real microbiologists do. Obviously it’s impossible to close the gap when one involves at the least years of schooling and a dissertation but any amount of information beyond what typical homebrew resources offers is both helpful and incredibly interesting. There is a big gap there that blogs like yours are filling in.

    I do have some questions:

    – With the potato lactose agar, am I understanding correctly that lacto/pedio differentiation would be possible even without the UI addition?

    – Can you assume, at least in the context of brewing yeasts, that an organism that can grow using a particular sugar can also ferment it?

    Thanks for your time.

    • Hey Claudio!
      To answer your questions. Sure, they’ll grow absolutely fine without UI. In fact, UI got degraded as far as I can tell and is thus was utterly useless.
      I’d say it’s a pretty safe bet that an organism can ferment the sugar it can grow on or at least metabolize it if not convert to alcohol (or lactic acid in case of lactic fermentation). If I’m wrong, please someone correct me.
      I’m excited about making litmus supplemented agars to see if the organisms really are acidogenic.
      I just finished plating a bunch of samples… ooooh, it’s tiring hahaha.
      Cheers!

  2. Eureka, well there is a lot of great information here. Thank you very much for your streaking and your explanations. Really great stuff! And the plates are very beautiful with all the different colours. Wrote myself a short summary of your results to plan my next experiments. Thanks again for all your work, really appreciate it.

    Cheers!
    Samuel

  3. This was an awesome read. good job going through all of this. I would love a simple way to test what I have growing in my casks… but I’m not even gonna bother trying to do actual gram stains (college was awhile ago) any ideas?

    • To be honest gram stain is very quick and easy to do and will allow you to at least tell gram+/- and to see them better under microscope. You can try plating a sample and picking colonies, but that may not tell you very much without a microscope.

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  6. I managed to buy both brilliant green solution and bromocresol green. I want to make some of the brilliant green media, but I’ve been reluctant since I have no filtration equipment. Do you think I could replace the boullion cubes and tenderizer with some commercial yeast nutrient?

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  9. Are you growing your plates anerobically in CO2? Lactobacillus and Pediococcus both grow in media with Bromocreosol green added in my tests. I use HLP and differential agar, the indicator is used to achieve a light green or turquoise color.

      • No, it”s the fact that you aren’t growing Lactobacillus and Pediococcus anerobically. These bacteria cannot generate energy aerobically. They are not killed by oxygen, but they synthesis energy via fermentation.

      • Thanks for telling me. Much appreciated.
        I toyed with the idea of maybe incubating them in a ziplock bag with a piece of dry ice to provide CO2, but never got around to doing that.

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  12. Hi Dmitri, with the malt extract/wort agar you say the gravity needs to be 1.002 but you also talk about 20g in 1L of water which works out between 1.007-1.008. I used the 20g in the first batch I made and had no problems with it setting but I thought it’d be worth clarifying.

    • It’s more of an estimate really. My point was to make it very light wort so that it would set easily and you’d have no problems plating.
      If it works out for you, then great. I just sometimes have problems with it setting if I make the agar OG too high.
      That’s all 🙂

  13. Thanks so much for this writeup! I’m planning on plating and isolating Saccharomyces from any and all ‘infections’. (I’ve yet to get into funky brews, intentionally). I have a large collection of washed yeast in mason jars in my fridge that I’d like to revive and store properly. Hence why I’d like to plate and isolate the yeast.
    I intend to mix up your malt extract agar recipe. Have you had any recent success with Brilliant/Bromocresol Green additions to assist in isolation? If so, how much do you add to the agar? Or have you found a better mix for yeast isolation?
    Also, am I on a fools errand with trying to isolate yeast without a microscope?

    • Hey Mark!
      No, you’re not on a fool’s errand. You can isolate yeast without microscope just fine. You just won’t be able to see individual cells, but if you’re streaking your yeast cakes etc, chances are that you’ll end up picking a yeast colony about 99.999% of the time. If one or two look different from the rest, then obviously they are something else. Probably bacteria or some wild yeast.
      Brilliant green will kill off most bacteria while Bromcresol green will be able to distinguish between yeast that can break it up (usually Brettanomyces) and those that can’t (usually Saccharomyces). I have noticed, however, that saison strains don’t behave the same as, say, English strains or even other Belgians when it comes to Bromcresol green. I can’t distinguish if they’re metabolizing it or it just changes color due to pH change, but from what I’ve seen Saison strains appear discolored, similar to Brett rather than regular brewer yeast.
      I make something like 10% Bromcresol green solution in alcohol (or maybe 1%, I don’t remember but it makes no difference anyway) and then add it right before pouring plates to final concentration of about 0.02g per Liter of agar.

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  16. Great write up!! What agar powder do you use? I’ve been trying to distinguish the difference between what is called “beer agar” powder vs “potato agar” powder. Is there a difference? Which do you recommend?

    • I just use “agar agar” or just plain “agar”.
      In my experience when there is something preceding the word “agar” it usually means a mix of ingredients just like you see in my recipes here.
      I get mine from eBay and it’s pretty cheap. I’d recommend getting just plain agar with markings something like this:
      Agar, Powder
      (Ash 2-4.5%)
      Sigma Cat. # A7002
      At least that’s what I usually get.

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  19. This might sound a bit extreme to some people but I was wondering if you have any suggestions for alternatives to the mediums you use or how to make those mediums from scratch. I’m getting to the point where I grow almost all my own food, make my own apple cider vinegar from orchard apples and lacto ferment vegetables from garden. I’m having trouble isolating a good wild yeast strain for ethanol fermentation from scratch. Seems most of them aren’t very alcohol tolerant and halt at about 3 or 4 % ABV. I don’t need tons of varieties (woud be nice though) of yeast. I just want to know how to isolate a yeast strain from my own local fruit that has high enough alcohol tolerance that I can make a strong wine to drink and be strong enough that I can distill it into hard liquor to drink or use as a disinfectant for wound care when shit hits the fan. I know I’m a bit extreme but if you have any answers for me it would be appreciated.

    P.S. As an example I have lots of potatoes for medium and lots of maple syrup/sugar from my black maple trees ( and sugar beets for molasses) as a sugar source but don’t know what I could use in place of agar agar.

    • You think I could just harvest gelatin from bone broth? I mean like letting it cool skimming it from the top and seperate from the fat then strain? Then cook the gelatin again and dry it? Would that be a replacement for agar ?

  20. Hey, thanks for the great write-up!

    I am busy trying to find a recipe for a medium I can use for QC in my brewery. Ideally something that can relatively easily ID lactic acid bacteria. HLP medium is recommended but very expensive to get shipped down here to South Africa. Any ideas?

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