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
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.