Pouring Plates and Making Slants

Plates are the bread and butter of a yeast rancher. They are quite easy to make and don’t require a lot of technical skill, but even so, one needs to have some basic skills and seeing how it’s done helps a lot. Recently I’ve been getting emails from a few people looking to get into building a home lab and start ranching so I thought a plate making tutorial would be helpful. And while we’re at it, why not also show you how to make slants?

First, you need the plates. They are easy to find online for a very decent price. Personally I prefer buying sterile plastic ones on eBay (which is where I buy almost all my stuff anyway) because I buy in packs of 500, which last me about 2-3 years. Now that I have my UV set up in the hood, mold contamination is almost entirely gone, though not completely because this is still an apartment setup, so I pour about two-three times less plates and imagine this new box of plates I just bought will last me many years. DO NOT WORK DIRECTLY UNDER UV. IT WILL BURN YOUR EYES AND SKIN. ALWAYS HAVE A SCREEN BETWEEN YOUR EYES AND UV AND IF YOU WORK IN THE HOOD WITH YOUR HANDS (OR OTHER BODY PARTS) TURN IT OFF TO AVOID INJURY! You can also get borosilicate glass plates which are reusable. They are more expensive, but the benefit is that you can use them indefinitely. I still prefer the plastic ones because they’re already sterile so I don’t need to sterilize them and there is just a lot less headache associated with them because sterilizing in a pressure cooker is always wet so glass plates will always be wet and just prone to infections.

 

Second, you need an agar recipe and the ingredients. You can find every agar recipe imaginable online and ingredients for the basic ones are easy to get. Check out my recipes and give them a try.

Third, put the ingredients together and cook in a pressure cooker at 15psi for 15-30 minutes, allow to cool to about 95˚F (35˚C) or thereabout, aka “kind of warm to the touch”, and pour into the plates. About 10-15mL is needed to cover a 100mm plate. One thing you can do is put your bottle into some water with crushed ice to rapidly cool it.

You can do this both with small and larger batches of agar.

If you choose to do so, make sure you only keep it in there for a few seconds at a time and swirl it constantly. Once it sets, it sets, and you’re in trouble because melting it again is going to be worse than just making it anew. In labs people usually leave the lids ajar and  let them sit and cool (typically in the hood with UV blasting at them) to avoid building up condensation inside the plate, but since this is a home lab, I’d recommend just closing the lids and letting the agar set. If you decided to go with the rapid-ice-cool method and catch your temperature just right, your plates will set within seconds. If you decided to let your agar passively cool until it’s well warm or just poured it hot, it could take up to a few minutes.

If you decided to make a slant, it’s much the same thing as pouring a plate except you pour it into a tube. After pouring just tilt the tubes until you get desired angle and leave it like that to set. For doing it at home, I’ve constructed this high-tech device and used it together with an old plate to slant my tubes.

High-tech science at its best!

I like my slants to be really slanted

If you want to make a tube for stab cultures, just pour it and let it set without slanting. The use for such tubes will be discussed in one of the later posts for those of you unfamiliar with them.

A tube ready for stab culture

Sure this isn’t the perfect tube for making slants, but it gets the job done. Notice how the agar comes almost to the edge of the thread.

Here it is viewed from the side

Another example of a slant

Here is a bit of a next level plate pouring which is rarely used, but I needed to make this. Put your plates on something to keep them angled.

Pour one agar and let it set at an angle. That way you’ll have more of it on one side and less on the other.

After it sets, pour another agar over it and let it set at an even level. What this will do is give you plates with two types of agar on them, but one part of the plate will have more of one and other will have more of the other with a more or less even gradient of the two in between. In this case the first level was some 123 IBU IIPA wort followed by another agar. That way on one end of the plate I have a lot of hop compounds, while on the other one I have much less. These plates will be used to help me pick the most hop resistant Lactobacillus strains.

Fourth. If you want to use your plates right away go ahead and do so, but if you want to store them, you’ll need to dry them. Obviously you should not dry them completely to the point where your agar almost ceases to exist – just enough to get rid of the condensation inside. My preferred method for drying is just letting them sit at room temperature for about 3-7 days, depending on when condensation disappears.

Remember how I mentioned that plastic cracks under UV in one of my previous posts? Well this is what my hood screen looks like now.

When the condensation inside is gone, I give them another day, then put them into a ziplock bag or the plastic sleeve in which the sterile plastic plates come and put into the fridge. Some of them will eventually get infected because no matter how much you try, there are mold spores everywhere and with humidity inside the bag of plates, some of them inevitably find their way into your plates. That’s OK though. If you do it right, it’ll be months before you see that happen. While drying I also like to let them sit under UV for a bit every day or every other day and before using them I also subject them to UV treatment for an hour or so just to make sure. You don’t need to do UV if you don’t have it, but in my experience it reduced streaking contamination down to zero unless there is mold in the actual sample.

Here are some more pictures of plate making:

How do you deal with bubbles while pouring plates? Here is some Milk Lactose Litmus Agar with some undissolved Litmus crystals in it. Sure it doesn’t look appetizing, but it works just the same and the dissolved Litmus is just enough to turn bright red when your yeast or bacteria produce acid! Adjusting pH with some base to around neutral will give you more purple-blue looking plates, but I forgot to do it this time. In any case, the colonies and agar get really noticeably red if there is acid production.

So what you do is take your trusty blow torch (mini blow torch in my case), set it to low oxygen flame by blocking the air or low flame setting, and go over the agar surface with the flame. Low oxygen/low flame because that way you’re less likely to melt and/or set your plate on fire.

Problem solved!

Some MYPG agar poured into plates and the rest of it ready to get its dose of Bromocresol Green. I like to keep my BG in 70% rubbing alcohol just so I don’t have to worry about sterility.

Add a few drops. Looks like a blooming swirling flower when you do it.
Mix gently to uniformity and pour.

Solidified MYPG plate

Solidified MYPG + Bromocresol Green plate

There you go! It’s pretty easy and from start to finish shouldn’t take you over an hour. I’ve heard of homebrewers who mix their agars, plate them, and then cook in the pressure cooker, and believe me, it’s not the way to go. This method is much simpler and much more reliable. Also, the more paranoid you are about sanitation and sterility, the better your plates and resulting yeast stocks will be.

Hope this helped!

Sending Out Cantillon Iris (2007) Brettanomyces C2

My fellow yeast mates!
The time has come to send you Cantillon Iris (2007) Brettanomyces C2 isolate.
Let me give you a brief description of this strain based on my own impressions and the feedback I got from around a dozen people who got back to me.

My impressions for C2:
Finished at 1.001. As I opened the carboy, the smell of wild strawberries erupted and filled up quite a radius around it. Very delicate and pleasant. As the beer was agitated by racking into the bottling bucket, strawberries were replaced by the signature bittersweet herbal-woody funk that all of the Iris isolates have. After allowing the gravity sample to stand and breathe for a while, the funk was replaced by fruit and pineapple. Given more time to stand, the strawberries came back. The gravity sample was dry, lightly tart with the signature Iris isolate funk. After allowing it to stand, strawberries, pineapple, apricot and mango came into the flavor as the funk recedes. The beer is lightly hazy and much more transparent than C1 portion was at the time of bottling. Hop bitterness is also not as pronounced as it was in C1 portion. Official tasting will be done in a couple months because I want to taste all 3 strains side by side.

Impressions from other people:
It was described as strawberries, bubblegum, green apple, pear, apricot, peach, wet sock/good cheese and slight vinegar. Strawberry, peach, apricot and pineapple were the most common descriptions. Finishes dry. Seems to be a good strain to use with fruity hops. Check out Kyler’s post about a beer he made with C2/C3 mix and Meridian Hops (http://haydtsbrewing.com/2013/02/13/100-brettanomyces-beer/)

As before, should your beer turn out horrible or poisonous because of this strain you agree that it’s not my fault and you are using it at your own risk. Also by using this strain you agree to give me feedback about it and possibly even send me a bottle of the resulting beer.

I don’t know exactly how many vials are available because I haven’t aliquoted the stock yet, but feel free to email me asking for it and I’ll try my best to accommodate as many people as I can. If you get a vial and plan to use it as the primary strain, make sure you pitch at least twice the amount of yeast that you would with normal brewer’s yeast.

ALL GONE! THANKS FOR PARTICIPATING!

Email me at bkyeast@gmail.com if you’re interested in acquiring a vial. As before, it’s $10 for the shipping and all that other good stuff. If you have some cool and unique strains of yeast or some cool beers, feel free to let me know and we could work out a trade.

Cheers!

Message for brewers in NYC or very close to it, i.e. Anthony, Simon, William, Aaron, Derek etc. Email me too and let’s arrange a meetup where we could taste some homebrew and swap yeast.

Part Three of the Dry Yeast Series: Temperature Differences

Part One of the Dry Yeast Series: Introduction

Part Two of the Dry Yeast Series: Rehydration

Part Three of the Dry Yeast Series: Temperature Differences

My latest post about dry yeast rehydration raised a rather unexpected wave of interest with surprising number of links from various websites and forums I didn’t even know existed. In about a week it became the most viewed post after Selective Medium. One of the readers (Luke) raised an interesting point regarding temperature difference between the yeast and the wort they’re pitched into. While I haven’t come across any studies of effects of this on flavor profiles, I found some whispers that pitching “hot” yeast into cool wort causes portion of the population to die. So I decided to do another mini study with dry yeast and whether or not this is true.

The experiments were designed as follows:

– Danstar Nottingham dry yeast were rehydrated in water at 32˚C (90˚F) for 20 minutes. Viability was assessed with Trypan Blue for all samples in the study.

– Samples of the resulting culture were placed into wort at various temperatures at dilution of 1:11 (0.1mL yeast into 1mL of wort) and incubated at those temperatures for 20 minutes, followed by viability assessment. Results summarized in Figure 1.

– After samples were taken, the culture was placed at 4˚C (39˚F) to passively cool until yeast began precipitating and culture started clearing up at the top (approx. 40 minutes).

– Viability of the resulting slow cooled culture was assessed, samples of it were placed into worts at various temperatures and treated as described above to observe the effects on yeast in a scenario of a washed yeast cake (since that’s done with water and stored in water) pitched straight into beer right out of the fridge. Results summarized in Figure 2.

Results:

Figure 1. Temperatures in ˚C correspond to following in ˚F:
90 | 0 (90 to 90) | 13 (90 to 77) | 29 (90 to 61) | 51 (90 to 39)

As seen in Figure 1, rehydration at 32˚C (90˚F) resulted in ~96% viability, which is consistent (though better) with the results of the previous study, validating it. Placing the yeast into isothermal wort results in retention of viability (~97%, higher due to random sampling). Rapid cooling from 32˚C to 25˚C and 16˚C (90˚F to 77˚F and 61˚F, respectively) resulted in ~20% viability loss in both cases, with statistically insignificant difference between the two. Rapid cooling from 32˚C to 4˚C (90˚F to 39˚F) resulted in ~40% viability loss.

Figure 2. Temperatures in ˚C correspond to following in ˚F:
39 | 0 (39 to 39) | 22 (39 to 61) | 38 (39 to 77) | 51 (39 to 90)

Results in Figure 2 show retention of ~96% viability in the culture that’s been passively cooled from 32˚C to 4˚C (90˚F to 39˚F). Transferring yeast to isothermal cold wort resulted in a significant viability loss of ~50%. Transferring yeast to wort at 16˚C and 25˚C (61˚F and 77˚F) resulted in ~30% viability loss, with statistically insignificant difference between the two. Transferring yeast to wort at 32˚C (90˚F) resulted in the most significant viability loss of ~70%.

Overall, both series largely imitate each other with major difference in initial isothermal transfer.

Discussion:

It looks like dry yeast really dislike the cold. In the previous post I showed that cold rehydration both in water and wort is detrimental to yeast health and this appears to support that notion. Transition from water to wort seems to be more detrimental in the cold rather than warm conditions. Percentage of cell death appears to decrease as the receiving wort gets warmer, ending with full viability potential at 32˚C (90˚F). As seen from gradual cooling of the yeast from initial rehydration temperature all the way down to 4˚C (39˚F), which retains full measure of viability, gradual cooling is a lot less detrimental than rapid. Can you pitch “hot” rehydrated yeast into your low-60s wort? Sure! Even with the loss of ~20% of cells, you’ll still probably have enough yeast there to ferment an average brew just fine. However, these data suggest that gradual cooling prior to pitching would be better. So if you’re willing to take that extra step of enjoying a pint or cleaning your gear as you let the yeast cool before pitching, I say “do it!” I think there are two factors playing here: 1 – temperature difference, and 2 – water to wort transition. Which is more important? Can’t say. Most likely it’s a combination of both. On one hand, we see that viability doesn’t change at all when you transfer from water to wort at the high end of the temperature series, but then when the same is done at the lowest end the effects are profound. Mid-range temperature difference is not all that horrifying, while large difference has profound effect in both cases. Perhaps the yeast dislike cold wort density and its dynamics. I don’t think it’s the weak cell walls because by that time they’ve had ample time to rebuild and utilize resources they have from dry preservation. Probably just general change of environment shock exacerbated by sudden low or high temperature that’s responsible for these results.

Effects of gradual cooling as well as equilibration with wort prior to pitching will be studied in the next part of the dry yeast series. Stay tuned.

Personal Notes:

– I originally planned to do the graduate cooling and equilibration study together with this, but as my time is limited and I just found out I have to present my research progress in 4 days due to sudden schedule change, I decided to put up these results now and finish the second part later.

– If anyone is interested in repeating these experiments in their home labs and validating or disputing my findings, please do. That way we can discuss our data and come up with more serious and credible results that would benefit the brewing community.

Sending Out Cantillon Iris (2007) Brettanomyces C1

Hello, my yeast mates!

The time has come for another yeast mail out!

This time it’s the Cantillon Iris (2007) Brettanomyces C1 strain.

Some information about this strain:

Ferments relatively fast to around 1.010 after which it slows down to a crawl. In my experience it fermented from 1.052 to 1.001 in 3.5 months resulting in 97% attenuation. It forms a pellicle very quickly. Pellicle is bright white and thick with large bubbles, but after a while it thins out and becomes patchy. The smell and flavor profile based on my experience as well as of those who got it from the October 2012 yeast mail is that it’s a pleasant strain which can be used as the primary strain. One of the people took first place in a competition with a beer that was 100% C1 fermented.

My impressions were as follows: Incredibly sweet smelling, strong fruity smell with apples, pears, mangos, honeydew, lots of honey, elderflowers, just general non-specific flowers and fruits, wood, hay, Bretty funk. Easily one of the best smelling beers I’ve made. Very dry and lightly tart with a hint of lemon. Since Brett thins out the beer and makes hops stand out like mad I’ll have to wait a few more months for an official tasting until the 1oz Callypso bittering hops fade enough for a Saison to not taste like it’s 1000 IBUs.

Other people who tried it and got back to me reported it being generally fruity and lightly tart with mostly apples, pears, lemons, wood and Brett funk.

As before, should your beer turn out horrible or poisonous because of this strain it’s not my fault and you are using it at your own risk. Also by using this strain you agree to give me feedback about it and possibly even send me a bottle of the resulting beer.

There are 23 vials available with around 25 billion cells each. If you get a vial and plan to use it as the primary strain, make sure you pitch at least twice the amount of yeast that you would with normal brewer’s yeast.

ALL OUT FOLKS! THANKS FOR PARTICIPATING! MORE YEAST COMING SOON!

Email me at bkyeast@gmail.com if you’re interested in acquiring a vial. As before, it’s $10 for the shipping and all that other good stuff. If you have some cool and unique strains of yeast or some cool beers, feel free to let me know and we could work out a trade.

Cheers!

P.S.

I will try to release a strain every month from now on so if you don’t get it this time there will be several other unique strains in the future.

More on Yeast Rehydration

The issue of dry yeast rehydration has been following me around since about the time I first wrote about it. A conversation here and there sparked up, often resulting in lengthy discussions on the topic of water vs wort and temperatures. Some took my suggestions and ideas readily, backed up with some info from a scientist who worked on these, while others were more skeptical. In either case it seems that people who tried rehydration in warm water preferred it to sprinkling straight into wort. The hardest issue for people to understand seems to be the matter of yeast viability. I find it very hard to explain that making a starter with dry yeast is pointless because osmotic pressure would kill a large portion of the initial culture and the time would be simply spent on rebuilding it to its original numbers. It doesn’t seem like it’s a very difficult concept to grasp, but the issue persists. Rather than debating and trying to back it up with simple scientific logic, I decided to conduct a little study to address the questions of temperature as well as sugar and hop compound concentration in dry yeast rehydration.

The original study was supposed to just be a temperature series in water, but right when I was about to start, it occurred to me that repeating the same series with different worts would also be interesting. The experiment consisted of three series:

Water – just plain sterile water.

Starter – a simple standard starter made with 100g DME per liter of water. OG ~1.040.

High OG/IBU – some wort I had saved in my freezer from an IPA I brewed some time ago. OG of 1.068 and 123 IBU.

The idea was to test the concept of temperature and osmotic pressure as well as assess the effects of high hop compound concentration on ensuing yeast viability after rehydration. For that purpose I used a packet of Danstar Nottingham that’s been sitting in my fridge for a while. It’s safe to assume that the viability of that culture is not at its optimum, but it’s what I had on hand and its response should be comparable to that of a fresh pack.

Experimental design was simple and straightforward:

Incubate yeast for 15-20 minutes in each medium at following temperatures: 35C (95F), 32C (89.6F), 25C (77F), 16C (61F) and 4C (39F). Stain with trypan blue and count dead and living cells. For each data point, between 120 and 1500 cells were counted, totaling in around 5000 cells. Let me tell you it wasn’t at all fun. In cases where cell viability was so low that counting would have been a waste of time (see example below), I assigned values of 1 and 5% based on qualitative observation.

An example of a sample with such low viability that counting was useless. Red arrow = dead cell. Green arrow = live cell.

The results were not completely unexpected. As expected, cell viability was much higher in yeast rehydrated in water rather than in wort. As the temperature decreased so did the viability, though more rapidly than I would have expected. This could be attributed to the age of the packet, but getting 85% viability in water suggests that they are still pretty healthy. What’s interesting is that starter wort largely imitates the water curve, but with significantly lower live cell counts, but high OG/IBU wort does not. It seems that in those conditions yeast get killed very quickly in warmer temperatures and they survive better in slightly cooler conditions. At around 17C (63F), which is a normal pitching temperature for most beers, the starter and high OG/IBU wort intersect and are almost the same as the temperature decreases. This suggests that when it comes to sprinkling dry yeast straight into wort it makes no difference if it’s a small or a big beer. Osmotic pressure will kill them just the same.

Final verdict: Warm water is better for rehydrating yeast than cool water, starter or pitching straight into beer.

I suppose the viability curves would be a bit higher if the pack was fresher and they vary a little from batch to batch, so I constructed a theoretical viability curve chart based on these findings, but with an increase of 10-15% so as to not surpass  100% viability and decrease the sudden drop below 30C.

I hope this little study has been helpful to you since some of you have been asking me to do a temperature series for a few months now. I also hope that this cleared up some doubts and concerns that you had regarding yeast rehydration.

Note: Don’t forget to equilibrate the rehydrated yeast prior to pitching into wort as a large temperature differential may kill or mutate a considerable portion of the yeast. Allow the rehydrated yeast to passively cool to temperature of your wort, or add small portions of the wort to the yeast until the temperatures are very close.

As always, comments, discussion, critique are always welcomed.

Cheers!