Beer Science Literature Review: Selecting and Generating Superior Yeasts for the Brewing Industry.

Some time ago I surfed the net for some beer science articles, but left it off due to just being too busy. Then, a recent email exchange with one of my readers regarding yeast breeding and selection rekindled my interest in research in the field of brewing. Upon digging around through the ever-expanding databases of online research articles, I was pleasantly surprised by the amount of science that’s being done in malting, hop and yeast research all directly aimed at brewing. There are journals specifically dedicated to beer science. Some of them are freely accessible while others require subscriptions. Luckily, I can get my hands on most of them.

Since reading even the free articles is not an easy task for those who don’t read scientific literature regularly, I hatched the following idea: I’m going to try reading a beer science article, digest it, and write a brief summary in every day terms for the benefit of the homebrewing community. I’m going to try to make it a regular thing if you, my readers, think it necessary and educational and I’m not just wasting my time and brain power.
Since the conversation that sparked this idea was about yeast selection and breeding, we’re going to review an article on the state of yeast selection and engineering in the brewing industry. The following article was published in Cerevisia in 2012.

Cerevisia
Belgian Journal of Brewing and Biotechnology
Journal of the Associations of Former Students of Belgian Schools of Brewing Sciences
Cerevisia provides a medium for the publication of both full-length articles and short communications on all aspects of malting and brewing, including biotechnology. The Journal will accept papers ranging from genetic or molecular biological aspects to those covering biochemical, chemical or bioprocess engineering aspects, provided that in each case the material is relevant to malting and brewing.
Editor-in-Chief: Jean-Marie Rock
Taken from http://www.journals.elsevier.com/cerevisia

The article is titled “Selecting and Generating Superior Yeasts for the Brewing Industry” and it’s by a group from Belgium. The tone is rather defensive and tries to convince the reader that genetically modified (GM) yeast is not from the devil, but is rather something that may become very useful and beneficial in the brewing world.
That’s about enough introduction, so let’s jump straight into it!

Title

Fermentation has been utilized for food and beverage production for well over 8000 years, and had historically been spontaneous. Such processes resulted in inconsistent results and it was not until 19th century that using single pure yeast for fermentation became a viable idea. While this approach gave much more consistent results, producers now faced a new problem – selecting yeast strain that carried the desirable characteristics. Tools to do that were unavailable until just a few decades ago and so yeast used in breweries and wineries today have more historic rather than scientific reasons. With advent of molecular biology and genetics it has become possible to select or even make yeast strains with “custom tailored” traits.

From the dawn of agriculture humans have been selectively breeding superior livestock and harvesting seed to use for planting resulting in an unnatural, but beneficial to us, evolution known as artificial selection. Its impact on the species subjected to it is very profound. Many modern crops yield harvests that are often over tenfold higher than their wild progenitors. Many such species, especially animals, have been selectively bred to an extent that they wouldn’t be able to survive in nature. One of the most striking examples is the Belgian Blue cattle that is so muscular that calves are too thick to pass through the birth channel and Caesarian section is required for every birth.
Due to differences in size between microbes and cattle, their selective breeding is far behind compared to the latter. While people bred animals for about 10,000 years, it was not until 1680s that we even became aware of existence of microbes thanks to microscopy pioneer Antoni van Leeuwenhoek. It took yet another two centuries for people to even start thinking about involvement of microbes in food production as well as diseases. Louis Pasteur was the first person to show the correlation between various microbes and the corresponding flaws they contributed to wine. Even after it was established that yeast are responsible for characteristics of fermented products, selective breeding was impossible because, unlike cattle, you can’t you take two yeast cells and breed them. Advent of technology that would allow us to do that took place only a few years ago and is still not widely available.
Genetic modification is a recent technique that has already found its way into improving microbes, crops and livestock. Without going into too much detail, genetic modification is very similar to selective breeding. A desired gene is inserted into the DNA of an organism and the resulting GM entity now carries it. Unlike selective breeding, though, the genes are not randomly recombined and genes are not restricted to those of species that can sexually reproduce. For example, cotton plants now carry a gene from a bacterium Bacillus thuringiensis, which produces a compound that’s lethal to parasites while being harmless to humans. This serves two purposes – higher yields of cotton and reduced pesticide use. 77% of soy, 26% of corn (which is by the way a product of millennia of selective breeding, original “corn” being about 2 inches in size) and 21% of canola worldwide are genetically modified.
Even though our understanding of molecular genetics is nowhere near as deep as some people would like to portray it, our techniques are constantly being refined, resulting in more precise and efficient ways of modifying the genome. The newborn field of Synthetic Biology has already shown that it’s possible to construct fully functional genome from scratch and “tailor-made” genes are no longer science fiction (Fig 1). ***Here I want to clarify that “tailor-made” genes does not mean genes that do what you want, i.e. turn sugar into hop alpha acids. It means that we can select a known gene from a library of sequences and insert it precisely where we want to. Activity of genes is very heavily influenced by where they are positioned on the genome as well as the environment. We’re not going to go into discussion about promoters, enhancers, chromatin states, non-coding RNAs and other players in mindblowingly complex world of epigenetics. An example of “tailor-made” gene would be inserting a gene encoding a fluorescent protein precisely after some other gene. That way they are both expressed if the original gene is active allowing us to track it. Example below.***

Fig1 GM Yeast

Fig. 1. Examples of experiments to improve industrially relevant phenotypes in yeast strains. (A) Genetically modified yeasts in which one of the most important genes for aroma production is fused to a gene which codes for a green fluorescent protein (GFP). This way, the enzyme that accounts for the production of aroma compounds lights up green, so we are able to study its activity and localization in the cell. (B) Genetic modification of a yeast strain to fine-tune the level of flocculation. 1: control, 2–9: gradual increase of flocculation.
Steensels, et al. Cerevisia, 2012


In recent decades there was a rush to create genetically modified organisms for industrial applications which resulted in wonderful inventions like the transgenic safflower seeds that accounts for majority of world’s pro-insulin production and saves millions of diabetics worldwide. There were successes in the food industry, but food scandals, mad cow disease, coupled to ill-informed population resulted in their disappearance and they were eventually forgotten. Despite the initial defeats, GM organisms are slowly taking over and about 10% of crops worldwide are modified.
In recent years several research teams have developed yeast strains that could potentially be very useful in brewing industry. There are now genetically modified strains of yeast that produce less diacetyl, that can ferment dextrins and starch, produce specific flavors etc, but brewers refuse to use them due to bad hype of GM organisms. These strains were often developed to meet specific industrial needs. Strains that can ferment dextrine and starch were developed for the purposes of creating low calorie beers because the resulting beers would have much less residual sugar. Despite commercial interest these beers were never commercialized due to pressure from protest groups and consumer fear of GM organisms. Food scandals and protests resulted in cessation of funding for development on GM bacteria and yeast for industrial and commercial use and the field dwindled to obscurity by late 90s.
Very recently food industry started recognizing benefits of GM yeast once again. One of the latest victories for GM was patenting of a yeast strain in the US that is able to break down certain undesirable aroma compounds in wine with few winemakers already using it in North America. With increased interest, money once more started flowing into yeast development and research teams are now working on making strains to meet specific needs in beer, wine, chocolate, biofuel and other industries. There are already strains that produce various fruity compounds, ferment quickly, ferment completely, produce high or low levels of alcohol, flocculate more or less, able to survive in high alcohol, or even genetically tagged to be distinguished from other strains easily. Though no GM yeasts can be used industrially at this time, it is anticipated to change in coming years and some breweries are already showing interest in such strains.
While we’re waiting for official permissions to use GM yeast, scientists are also working on selecting superior yeasts by means other than genetic modification. This is essentially done the same way it’s done with cattle, only on a much smaller scale. Individual yeast cells are picked, genetically screened to see what genes are up and down and thus determined which ones are better for this or other application, and are then crossed with other yeasts that possess some other desirable characteristics to result in an even more superior strain. Results of such breeding are already in use in our  daily lives. Bakers can now choose yeast strains from a whole array of strains each with different characteristics and profiles such as fermentation speed, dough rising and temperature tolerance. Since these strains were developed by selective breeding, they are not GM organisms and can therefore be freely used.
Paradoxically, the biggest roadblock on the road to using GM yeast in brewing is the beer itself. Since yeast contributes such a major portion of aroma and flavor to the beer, brewers are reluctant to change their house strain for something new. It may be somewhat ironic because the traditional brewing strains were mostly selected by trial and error based on the “whatever works better” criterion. That may be the case with old traditional breweries with many years of tradition and consistency behind them, but new breweries and new generation of brewers are less afraid of taking risks, experimenting and making scientifically informed decisions. There are already collaborations between researchers in Belgium and some Belgian breweries to perform large scale screenings to select and breed strains beneficial to brewing industry via non GM means. Some strains that were selected from such screenings are already in industrial use. This, of course is not only limited to beer and Saccharomyces. Other species of yeast that are of importance for food industry, for example chocolate production, are undergoing the same process.
Yeast are grown in variety of different conditions and their industrially relevant characteristics in these conditions are mapped (Fig 2).

Fig2 Map

Fig. 2. Graphical representation (heat map) of different characteristics of industrial yeast strains. Every row consists of data from a different yeast strain, every column is a different characteristic. “Yellow” is a low score, and “red” is a high score for this certain characteristic. The dendrogram on the left represents the genetic relatedness of the yeasts, based on an AFLP fingerprint exploiting transposon TY1 insertion site polymorphisms. The colour code on the top right indicates the origin of the yeast strains. This kind of analysis allows us to select yeasts with specific beneficial traits, for example to use in industry, or for breeding.
Steensels, et al. Cerevisia, 2012

Analysis of these maps allows for picking of strains with exactly the characteristics desired. Mating different strains is a powerful approach to select for traits like alcohol tolerance. Rather than mating two strains, researchers now utilize an approach called “genome shuffling” where several strains are mated at the same time. This results in millions of strains in the same flask (essentially each cell is its own strain that’s different from others) with a mosaic genome consisting of a mix of the parent strains. Repeating this several times results in progeny with complex traits. In the beginning the parental strains are irradiated to create mutations and thus more genetic diversity. They are then mixed and allowed to mate, after which the ones with some particular desirable trait (like alcohol tolerance) are selected out and further analyzed to assess their other characteristics. This is repeated several times to obtain superior strains. ***At this point I feel it’s necessary to point out that there is more to mating yeast than just taking WY1056 and WY3711, mixing them together and getting a hybrid strain (though you WILL get some hybrid cells, but they’ll be outnumbered 500 billion to one so you’ll never even know they’re there). As far as I know, sporulation (sexual reproduction) is induced so each strain creates spores that can then interbreed with other spores and create hybrid strains. Usually, like in a starter or your fermenter, yeast reproduce asexually by budding. So each cell in the culture is essentially a clone of every other cell so under such conditions they can’t interbreed because there is no breeding. It’s just making copies of yourself.***
Researchers recognize the importance of spontaneous fermentations and are working of creating superior bred blends of yeasts and bacteria to be used for processes like cocoa fermentation in chocolate making. They aim to mimic the complexity and characteristics of spontaneous fermentation while bringing the consistency and superiority of selectively bred microbial strains. ***I just can’t help but think of WY Lambic/Roeselare blends and the possibility of these custom made “superior spontaneous bug blends” that could be used in future brewing.***
With all these scientific advances in both genetic modification as well as selective microbial breeding, brewing and wine industry are still reluctant to try them. Other than government sanctions, tradition, bad publicity and consumer fears play important roles in preventing the use of “superior” microbes on industrial scale. While the GM taboo has almost completely disappeared in medical and agricultural fields, food industry has yet a long way to go. Should they adopt these new strains for the custom made ones? Benefits and dangers of using GM microbes in food are out of scope of that paper.

Some remarks and personal thoughts:

When I said “there are groups working” on this or that throughout this article, those groups often include the one who wrote the original paper. The authors are the same people who actually generated and bred those strains of yeast that are talked about here.

I couldn’t help but think of an article written by Derek at Bear Flavored Ales about the future of hops as I read and wrote this. He speculates that with all the new hop varieties that are becoming increasingly more fruity and specific in terms of which fruit character they impair we will soon see beers that taste like fruit beers without ever touching fruit. I believe that the combination of the new hop breeds and genetically modified yeast made so that they throw off high amounts of strawberry, or apple, or cherry, or pear, or watermelon, or apricot etc will indeed result in very fruity and interesting beers.

I also believe it’s only a matter of time before GM yeast are finally approved for wide use and they will flood the market and brewing industry. Personally I support it and don’t see any zombie apocalypses or Frankenstein coming out of it based on what little I know on the topic, but it’s not my place to tell you if they’re good or bad. If you believe they’re bad and we should only stick to the original “wild” or “feral” strains, that’s perfectly fine and it’s been done for thousands of years with great success. While most old world and traditional breweries will probably choose to maintain their old cultures and stay true to the quality and traditional flavors particular to those breweries, I think new breweries (especially in USA and Belgium where tradition is good, but novelty is better) will most likely embrace these and give us a myriad of exciting new flavors.

With advances in biology being what they are and an ever-growing number of scientifically educated brewers, I think we are standing at the very doorstep of a brewing revolution the likes of which were never seen in history. With increasing number of brewers embracing Brettanomyces, the yeast species that’s been selected against for as long as beer microbiology existed, and realizing what an amazing depth it can contribute to beer. With new hop breeds appearing every year, each with such an amazing and unique array of flavors and aromas. With increasing variety of malted grains at our disposal, some of which have been brought back from near extinction, adding to the malty complexity of the beverage we’re all so fond of. And now, with advances in biology being such that we can practically custom make yeast with properties that we want, I think just a couple decades from now we’ll think over a pint of “Lemon Garden With a Strawberry Twist Saison”, look back at this time and think how back in those days such beers were only a fantasy.

I claim no authorship of all or any parts of the original paper. The original work was done by and belongs to Jan Steensels, Tim Snoek, Esther Meersman, Martina Picca Nicolino, Elham Aslankoohi, Joaquin F. Christiaens, Rita Gemayel, Wim Meert, Aaron M. New, Ksenia Pougach, Veerle Saels, Elisa van der Zande, Karin Voordeckers, and Kevin J. Verstrepen with all due credit and recognition.

I ask my readers to write a comment or send me an email with their thought on whether or not the Beer Science Literature Review is a worthwhile project or not. Do you think doing these mini reviews would benefit the brewing community? Do you have suggestions or some topics that perhaps I could find some literature on? Please let me know. Personally I really enjoyed working on this post. I feel like it was really beneficial for me and expanded my knowledge and, hopefully, it did for you as well.

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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:

Decr

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.

Incr

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.

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.

20130311_014415186

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.

MesChart

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.

ProjChart

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!

 

Dyeing Yeast Cells: Life vs Death

I wanted to do this post for probably around a year now, but just was never quite able to sit down and do it. Surprisingly, now that I am at the busiest I’ve been at the lab, the inspiration finally struck me and I was up until past 2 am squinting into the microscope after hours of microscopy in the lab. Note: don’t do 8 hours of microscopy in one day or your eyes will hate you. By that time I was so tired that quality of the micrographs wasn’t even close to a top priority, so I’ll have to ask your forgiveness if some of the images are even worse than usual.

Anyway, what was it that was on my mind for so long? Vital staining.

There has been a resurgence of interest on the topic among the scientifically slanted homebrewing blogs and, as far as I can tell, everyone out there is using methylene blue to check their yeast viability. It’s not really surprising since MB has long been used as the standard vital stain in the brewing industry and thus adopted by many homebrewers. Another stain often utilized by a wider audience of fermentation and ethanol production industries is methylene violet, which seems to be pretty much the same as methylene blue except for the color. In the science world there are a number of ways to determine viability of the cells ranging from fluorescent dyes to apoptotic/necrotic marker antibodies etc., but such methods require expenses and preparations way out of homebrewing range. The gold standard for non-fluorescent methods, however, is trypan blue. First let’s take a look at some of them.

Methylene Blue – a blue dye that will stain the dead cells blue. Living cells also take in the dye, but the active enzymes within them will process (reduce) the dye and make it colorless. Problems with this stain include that some dead cells still have enough active enzymes left in them to reduce the dye, the dye may block some intracellular processes and result in failure to reduce it by living cells, below pH 4.6 the uptake of the dye increases so that many living cells also become blue, also it may stain stressed live cells with fractured membranes. In my search for more info I found some articles about using various microscopic techniques to tell between the false positive and false negative cells, all of which are beyond the homebrewing level. Despite all its shortcomings, methylene blue remains the standard viability stain most likely due to its availability.

Availability: easily available and inexpensive.

Trypan Blue – also a blue dye that stains dead cells blue. Unlike methylene blue, this stain’s action is based on cell membrane integrity. It is a negatively charged compound that can’t cross the membrane unless it has been damaged. Dead cells have ruptured membranes, allowing the dye to enter and stain them while living cells are very selective in respect to what goes in or out and trypan blue is not allowed to enter. This stain is not dependent on chemical reactions, gradients, reduction states etc., which makes it the gold standard for non-fluorescent vital dyes. It should be noted that it is toxic and cells exposed to it for too long will die and stain blue. Other two stains in this study will be compared to it to measure their effectiveness.

Availability: not easily available to homebrewers and expensive.

Safranin – a red-pink dye that is often used in histology and cytology to stain nuclei as well as the last stain in the gram staining procedure. Not many people know that it’s also often used as a vital dye in microbiology (often together with methylene blue) and stains dead cells red.

Availability: easily available and inexpensive.

In this post I’ll test these three stains to assess viability of a couple of yeast strains and see how well they correlate. Trypan blue will be used as the standard with methylene blue and safranin compared to it to assess staining accuracy. While it is common knowledge that, despite its faults, methylene blue can be used to check Saccharomyces viability with some degree of accuracy, there isn’t much about using it to look at Brettanomyces. With growing interest in Brettanomyces, both in homebrewing as well as industry, it is important to have a reliable means of assessing culture viability, especially since all-Brett beers require at least a twofold increased pitch compared to regular ales. Checking these stains with some Brettanomyces cultures was therefore the logical thing to do.

Here you can see some micrographs of the various stains in Brettanomyces and Saccharomyces. For this study I looked at 5 cultures:

WY 1275 Thames Valley – washed yeast cake from a primary that has been stored in the fridge for over 2 months. Very interesting how viable the cells are! According to Mr. Malty’s calculator, the viability of this slurry should have been 10%.

1275 TB 1 1275 Saf

A yeast sample I got from a local bakery – mixed culture with 2 or 3 yeast strains and some Lactobacillus floating around.

Bakery TB 1 Bakery MB Bakery Saf 1

Cantillon Iris C3 isolate – a bit of that yeast strain that’s been sitting at room temperature since the time I was sending out these cultures to you guys.

No good pictures, sorry

Cantillon Iris C2 isolate – sample pulled from an all-Brett mild that’s currently in primary. Micrographs say C1 on them, and that’s a typo. I made sure to take cells from the pellicle and it appears that some of them are dead, while the ones from the liquid are at near 100% viability (the last photo in this series). The total viability was calculated from a homogenized sample – meaning both liquid and pellicle mixed together.

C1 TB Long C1 MB C1 SafC1 TB

Cantillon Mamouche Dregs – straight dregs from a bottle of Mamouche that I left to sit at room temperature for 15 days before sampling to allow the organisms to wake up a little before trying to culture anything from it. The viability is so low that staining was essentially meaningless and so was not used to determine efficiency.

Mamouche MB Mamouche Saf

I found it especially curious how trypan blue and safranin can differentially stain cells in clumps while methylene blue has a bit more trouble differentiating between live and dead ones. It’s also curious that the “filamentous” forms of Brettanomyces that we often see in old cultures and dregs, but very seldom in active ones are stained as dead. It makes sense though. Perhaps that form is the final stage in the nutrient poor environment, which is why we see them in old cultures, and they eventually die afterwards. Another cool thing is that unlike methylene blue, trypan blue and safranin don’t stain living bacteria, which means it can be used to assess the viability of your lactic acid bacteria cultures. This is something that’ll have to be followed up on.

Mamouche TB cluster C1 MB Cluster Bakery Saf cluster

As a result of this little staining series, it appears that methylene blue and safranin are good for assessing Saccharomyces viability, but not Brettanomyces. While safranin seems to be more accurate for Brettanomyces than methylene blue, it still isn’t as good as trypan blue. At least it somewhat approximates the actual viability. The error in counts may be too great to actually be useful so the search for a reliable Brettanomyces stain that’s readily available still goes on. If any of you have had a different experience with these stains please let me know. It is entirely possible that my results do not reflect every Brettanomyces strain out there.

Chart

Total cells counted = 2248

There are a couple more stains I’m interested in looking at, but that’s for another post. Once again sorry for not posting more often, but I’ve just been so very busy that I can’t even brew. It looks like the research is getting into its rhythm and I should be able to manage my time better in the upcoming weeks. Hopefully soon enough the blog will be back to normal with timely posts.

How to Build a Yeast Ranch

First of all, I’d like to apologize for prolonged silence. Research is what it is and doesn’t leave me much time to write…

A lot of my readers are what one might call “advanced homebrewers” who are not afraid of experimentation and know their way around the mashtun and yeast cultures. Some are also fellow yeast ranchers and know the pains of proper yeast ranch maintenance. My recent move forced me to disassemble and reassemble my lab so I thought I’d make a post on how to set up a home yeast lab or at least the way I went about it. The fact that it was all in pieces gave me a chance to rethink a few things and change/add/remove certain aspects and a couple people inquired about setting up a home lab so here is how I did it.

I’d like to point out that this is how I set it up and how I want it to be based on my experience working with bacterial, insect and mammalian cultures for a few years in a research laboratory setting. There is no one way that a lab, especially a home-set one, should be so this is by no means dogmatic. Feedback and suggestions are welcome, as always. Also I’m not trying to promote this or that store. I’m simply telling you where I got my equipment.

All right, let’s get started. I didn’t know how to best organize this and in the end decided to go from the basic and most necessary towards luxurious and fancy while describing how I use them and where I got them. There will be no in-depth technique discussion in this article. Otherwise it’ll be 30 pages long and will take a month longer to write. At this point I should also emphasize that eBay is home yeast rancher’s best friend. The amounts of cell culturing equipment available there as well as the prices are just unbelievable. You’ll see examples of this throughout this article.

Basic “Blind” Yeast Ranching:

Things that would allow you to easily do basic yeast ranching such as making starters, saving yeast, washing yeast, etc. It’s “blind” because you cannot visualize the yeast cells or know what is growing in your culture.

Common Sense: The rarest of things. This is an absolute must. With it you’ll be able to utilize and adapt common household things for your yeast ranching needs, and without it you can have state of the art lab at your disposal and still manage to screw it up. Common sense includes things like making sure your equipment is clean, assuming that nothing is ever microbe-free unless you yourself made it so or it’s some certified sterile equipment like pipettes and test tubes, not breathing into your yeast flask or not using your mouth to start the syphon. Remember, there are microbes everywhere!

Sanitation and Aseptic Technique: This is not trivial and requires diligence and practice. Another absolute must. Read up on it, watch videos, remember your old microbiology course if you ever took one, ask people. It can be extremely frustrating when you suddenly have mold growing in your yeast or your started going sour (not the good kind) and so you must be very careful and paranoid about sanitation and aseptic technique while working with yeast.

Pressure Cooker: This may be a little surprising, but I maintain that a pressure cooker is a must for yeast ranching. A lot of people just boil flasks on their stoves or soak them in Star San etc., but it’s just never as good as a pressure cooker. It’s a stovetop autoclave that sterilizes your gear! Make a starter, pressure cook it and store on a shelf or in the fridge for weeks. When you need to make a starter, just take it and you’re good to go. Can’t really think of anything that would beat it short of installing a real autoclave in your house. Mine had to be bought new for around $80 since we didn’t have one. If you have one already or your grandma hasn’t used her old one in many years and it holds the pressure, there is no reason not to use it.

PressureCooker

Balance: To measure your DME when making starters. These also come in handy when you’re making other kinds of media including agar plates. Every brewer has these for hop measurements etc. anyway.

Glass Containers: Mason jars or other kinds of jars to make a starter in. There is absolutely no rule about having to make a starter in a flask, especially if you’re not making a stirred starter. Just make sure it’s not something made of thin glass as it may crack or break under pressure. After that just put some yeast slurry or a smack pack or however you make your starters and it’s good to go.

Pressure Sterilization vs. Tyndallization: Sure there is a way to sterilize your starter other than pressure-cooking it, but I still advocate it over any other method. First let’s familiarize ourselves with the process of tyndallization. It is a low-tech way to sterilize media and is basically heating or boiling the medium for 15-30 minutes, letting it cool and incubate overnight and repeat the boil for three consecutive days. This method relies on germination of heat-shocked spores to vegetative cells that can then be killed by boiling. Repetition of boiling and incubation is to make sure all spores germinate. This method is only effective for media that supports growth of spore-forming organisms and so is not particularly useful for sterilizing water for yeast washing for example. Pressure sterilization is a lot more effective in killing cells and spores and works well not only for medium, but water as well. As a precaution, it is wise to let your starter/medium incubate overnight and pressure cook it again the next day to kill the last of the spores that may have survived. It’s rare, but it happens that one round of pressure sterilization is not enough and I’ve seen mold growing in starters that have been sterilized only once after a couple of weeks on a shelf.

Intermediate “Blind” Yeast Ranching:

Advanced enough to make stirred starters and save yeast to use for later. 

Flasks: Flasks are made of heat resistant borosilicate glass that allows them to survive the thermal and pressure changes. They also have flat bottoms so that the stir bar can easily spin. There are many different kinds of flasks. Most people to prefer Erlenmeyer. They are very nice, have a wide flat bottom and are easy to handle. Besides those I also use flat-bottomed Florence flasks. I like those because their shape makes it easy to pour the yeast out while leaving the stir bar behind so that you don’t have to worry about dumping it into the wort. My go-to place for those is HomeScienceTools.com where they sell 1L flasks for around $10 and 2L ones for $14 plus have an excellent customer service. They also have a lot of smaller flasks that are handy for growing up your culture from a small amount of yeast and working your way up to a full volume starter.

Culture Bottles: Same thing as flasks, but with screw-on caps. Handy for growing yeast as well as yeast washing and storage. Also very handy for making agar when you get advanced enough to pour your own plates. Best place for those is eBay. Normally culture bottles are ridiculously expensive, just insanely expensive, just criminally expensive! However on eBay you can find them for something like $5-8 per 500mL bottle, which is a great bargain. Smaller ones, like 125mL, are very useful for storing washed yeast in pitchable portions that you can just take out of the fridge and pitch. They can also be used to grow your yeast enough to pitch into a starter. Those run at about $3 or less per 125mL bottle on eBay so that’s nice. I got my small ones in a fortunate occurrence when a neighboring lab was throwing out a whole box of them.

ShelfGlass

Flasks, culture bottles, and other stuff

Heavy Foil: Needed for covering the flask mouth when pressure cooking, storing and growing. It prevents microbes from getting into your wort while still allowing a complete gas exchange. Cost is negligible and you can find it in almost any store.

Stir Plates + Magnetic Stirrers: We all know how beneficial it is to have an agitated starter. Agitation allows for a very good gas exchange that keeps your yeast happier and lets them grow to higher numbers. The most common way to achieve that in homebrewer setting is through a stir plate and a magnetic stirrer. The principle is pretty straightforward – a magnet is attached to a rotor under the plate and there is a magnetic stir bar in your flask. When you turn on the plate, the rotor starts rotating and the magnetic field rotates with it, rotating the stirrer in your flask. Thus there is an agitation of your culture. There are a countless number of articles with pictures and videos about how you can make one out of a computer fan and a hard-drive magnet so check them out if you’re interested. What I did was just regularly check my best friend eBay and eventually came across some amazing deals. There are currently only two plates in my possession, but it’s perfectly sufficient for my needs. One plate was bought for $10 and other than having adjustable speed it also has a temperature control. Most of you know about stir plates with heating, but this one is a stir plate with cooling, which comes in handy if you want to maintain a certain temperature. The other one cost me $14 and included a set of 5 stir bars (which are around $3-5 each if you buy separately). Compared to ~$50-70 and up that homebrew stores and other science stores charge for these, I’d say those were pretty sweet deals and even doing it yourself can’t beat the price. That being said, stirred starters are a great thing that reduces the volume of starter you need to prepare and ferment out much faster than stationary ones and every yeast rancher should invest in at least one of these. Another good method is a shaker plate or a shaker incubator that work by swirling your whole flask rather than just the liquid inside it, but that’s out of reach for most people.

A little note: put your stir bar into the starter before you pressure-cook it. That way it also gets sterilized and you don’t have to worry about putting it in later. Don’t worry about it loosing its magnetic properties. They are made for such applications and will last a very long time.

RegStirrer

Plain stirrer

ColdStirrer

Stirrer with cooling

Intermediate “Visual” Yeast Ranching:

Now that you’re advanced enough you can start looking at your yeast and other organisms. 

Microscope: A must have for any hardcore rancher. A microscope allows you to actually see your yeast and bacteria. Sometimes you can tell the difference between yeast just by looking at the cells. For example in general Saccharomyces and Brettanomyces look very different, and bacteria are unmistakably different. This is very useful for seeing if you have a mixed culture or contamination. Of course sometimes you won’t see the invading cells because there are so many more of your native cells there especially if the “infection” is very low. Nonetheless, a microscope is an immensely powerful tool. Other than just looking at your little workers, it gives you the ability to count cells as has been described in the previous post. To see yeast you’ll need a microscope with 40X objective and a 10-15-20X eyepiece for 400-600-800X-magnification power. For looking at bacterial cells you’ll need at least 1000X magnification, which would require a 100X immersion oil objective with the aforementioned eyepieces. There are a number of different ones available online for a good price. Celestron offers good microscopes at affordable prices and they’re even cheaper on Amazon or eBay to the point of being able to get a usable one for under $100. What I like about those scopes is their sturdy build and good optics and I initially used one. Later on I upgraded to AmScope – 40X-2000X Vet Clinical Stereo Compound Microscope Model B400AB that I found on sale for just below $200 and couldn’t pass it by. 2 eyepieces make it much easier to work with. It has a nice construction, dimmable light, and is pretty nice overall. However the light source is rather poor – dim and yellow and optics at 100X objective aren’t as good as Celestron. It’s still very nice and usable and those flaws don’t impede my yeast ranching process in the least and I prefer this one to my original Celestron. AmScope has a lot of microscopes of different grades and prices so check them out. I’ve been meaning to buy a better light bulb for it and have a spare 100X objective that I bought on sale for around $60 to try out and see if it’s better, but can’t get to it yet. Whatever scope you choose to get, make sure the magnification is high enough and it has an electrical light source. It’s worth to spend a few bucks on that rather than trying to play with a mirror for your light.

Microscope with camera connected to my computer

Microscope with camera connected to my computer

Slides + Cover Glasses: To look at your yeast you need to make slides. The easiest way to do it is to put a drop of your culture onto a slide and drop a cover glass on it. HomeScienceTools has them at decent prices. I prefer the plastic cover glasses to the glass ones because of their durability. There is nothing more annoying and horrifying than dropping a piece of 0.1mm thick glass in your room and worrying whether or not you’re going to step on that sliver of glass.

Transfer Pipettes + Pipetters: How would you put that drop without contaminating your culture? Our best friend eBay offers a solution to that. You can buy a ton of sterile transfer pipettes of different volumes for very cheap. I bought 500 1mL ones for $20-30 and 500 of 3mL ones for around the same price. Basically they’re almost free and let you take samples and transfer samples without having to worry about sterilizing your pipette. Another option for transferring your samples between flasks or tubes would be serological pipettes. People on eBay sell them for down to 20% of what they normally cost. You need a pipetter for those though, which you can also find there. There are manual and bulb-action as well as motorized ones.

Sterile transfer pipettes

Sterile transfer pipettes

Serological pipettes

Serological pipettes

Test Tubes: Another must have for cell culture work, and again eBay saves us with best prices and that’s where I get them. Sterile 15 or 50mL tubes in racks for quite cheap. Perfect for starting cultures from colonies (not advanced enough yet!) or dregs as well as keeping yeast in them. Those of you who received the Cantillon Brett cultures know what I’m talking about. Glass ones are also very nice, but I prefer plastic. They come sterile and you just use them without worrying about sterilizing them first. If you really want to reuse them you can just wash and pressure-cook them and you’ll be fine. Even though the manufacturers usually say those tubes are not autoclavable, you can go ahead and cook them. I’ve grown insect cells without antibiotics that way for 15 months without a single instance of infection, which earned me the nickname of “cell whisperer” in the lab.

TubesInFridge

TubeRack2

SpareTubes

Hemocytometer: AKA counting chamber. Used for calculating culture density as described in the previous article. These things are so ridiculously expensive that even culture flasks look cheap in comparison. Luckily eBay sells cheap ones from China that take a few weeks to get to you, but for $20-30 it’s worth the wait.

Spray Bottle and Alcohol: You need that to sanitize your hands, flask mouth and whatever else you want. Bottles can be found in just about any store and alcohol is also not that hard to find.

 

Advanced Yeast Ranching:

Now that you’re an accomplished yeast rancher, you can start picking out individual yeast strains. 

Knowledge: You’d think that it’s a basic thing to have, but I don’t find it all that necessary until you reach a certain level. There really isn’t all that much thinking involved when you’re just growing from slurries, making starters, or even just looking at what you have in there under microscope. Best thing about knowledge is that it’s free. The Internet is full of free books and articles about yeast and microbiology in general. You could also audit a microbiology course at your local college. All you need to do is talk to the professor teaching it and I’ve never heard of one refusing to let anyone audit their class and some even take you in for the ride in the lab sections of the class where you can learn just about every technique you need to know for yeast ranching. Another option would be iTunes U – one of the most amazing things ever done by Apple. I’m sure most of you are familiar with the iTunes store where you buy music, but iTunes U is a part of the music store that is completely free and 100% aimed at education. It’s where professors upload their lectures in audio and sometimes even in video and anyone is free to download and reap the benefits. It’s like going to class without leaving your home. Just great stuff! You can find a lot of general biology, cell biology, microbiology, virology and just whatever classes you want there. I cited one in the previous post. It’s truly an amazing thing. You could also read books, of which there are several on microbiology in general as well as yeast specifically. Anyone can reach a relatively high level from the free recourses I just told you about in 6-12 months of casual learning. Knowledge and ability to think will take you to the next level.

Plates: aka Petri Dishes – bread and butter of yeast isolation. You need plates to pour agars into and grow your yeast or bacterial colonies. You have a choice between reusable glass ones and disposable plastic. I choose to go with plastic because I don’t have to sterilize them. You can find both kinds on eBay, and I usually go with a case of 500 for ~$60. Slants that enjoy certain popularity with yeast ranchers are the same thing as plates, but much smaller and with a screw cap that prevents them from drying, making them the preferred method for culture storage among homebrewers. Plates may be sealed with Parafilm (cheap on eBay, just make sure you know how to use it and get the lab stuff, not the tree wrapping one) that also prevents drying and keeps them for a very long time in a refrigerator.

UVPlates

Agar and Media: See my agar recipes. Agar is the medium you grow your cultures on as well as the solidifying agent used to make agars semi-solid. When you buy agar powder, make sure it’s purified and has low amount of ash in it. Generally people who sell it sell good stuff, but sometimes it’s very dirty and you can’t use it for making plates. Again you can get 100g for around $10-20 on eBay and that’s easily enough for over 100 plates. Other than the solidifying agent, your agar medium should contain sources of carbon and nitrogen at the very least plus selective agents and indicators. Handbook of Microbiological Media by Ronald M. Atlas is a great book with hundreds of media recipes and what they’re used for and how they should be prepared. I’d recommend getting it if you can find it for cheap. Internet is also a great resource for media recipes and if all fails, knowledge and common sense will guide you. For example you have a mixed culture of Saccharomyces and Brettanomyces and you want to isolate the Brett. You know that Brettanomyces bruxellensis is able to ferment lactose while the other one can’t. So you can quickly make an agar with lactose as a sole source of carbon and plate your mixture. Saccharomyces will starve and not grow as well as Brettanomyces, or not at all. Or you can use brilliant green in your agar to kill off bacteria in your culture and be able to pick out yeast colonies. Things like that can make your life much easier.

Poured plates cooling in the hood

Poured plates cooling in the hood

Same plates, but under UV light

Same plates, but under UV light

Alcohol Lamp: Used in plating your cultures onto the agar plates and run on high% alcohol, such as denatured alcohol that you can get at paint stores. Burning one creates a “hot air hood” that prevents bacteria and mold from falling into your agar as you’re plating because the resulting flux of hot air pulls the particles in the air upwards. It’s quite effective and is often used in laboratories, though mostly they use Bunsen burners, but it’s the same thing for our purposes. Takes some practice, but you’ll get there. The flame is also used to flame sterilize inoculating loop or cell spreader. HomeScienceTools sells them for a reasonable price.

Alcohol lamp with its fuel plus a little butane burner and an inoculating loop

Alcohol lamp with its fuel plus a little butane burner and an inoculating loop

Inoculating Loop: Used for streaking your cultures on agar. It’s basically a piece of wire with a loop on the end and a handle. The loop acts the same way as a soap bubble loop and is used to take a small liquid sample and streak across your medium to separate single cells that later grow into colonies. The loop is first heated over flame until red hot to sterilize. You can make your own, but I bought mine from HomeScienceTools.

Cell Spreader: Similar to inoculating loop, but rather than streaking a sample you use it to spread a drop of it evenly through the whole plate. There are a number of them available from single use disposable ones to nice glass ones. In either case none compare in price to just buying a glass rod and bending it with help of a propane or butane burner. HomeScienceTools sells glass rods that I use to make my spreader.

Glass rod and a cell spreader made from one just like it

Glass rod and a cell spreader made from one just like it

Stains: Stains are more geared towards bacteria rather than yeast, but you can use several to stain the nuclei and cytoplasm if you wish to see. Gram stain is quite useful for figuring out what bacteria you have in your culture. For example it would allow you to easily distinguish between Lactobacillus and Acetobacter that may be plaguing your beer. HomeScienceTools has a wide selection at reasonable prices as well as educational videos about staining procedures.

Reagents

Luxury Yeast Ranching:

These things are luxury. Completely unnecessary, but cool to play with and make your process faster and more effective

Scientific Friends: Having friends in science, such as students, professors, postdocs and technicians who enjoy your homebrew goes a very long way.

Incubator: You don’t really need a special temperature to grow your yeast and they’re perfectly happy at room temperature. In labs though, they’re usually incubated at ~30°C (86°F). In my recent attempt to isolate some aggressive strain of Lactobacillus (presumably L. brevis that can withstand hops and sour even hoppy beers) from Cascade Brewery dregs (thanks for all the samples guys!) I decided it was time to take the plunge and get me one because these bacteria prefer temperatures closer to that of our body and grow much better at 30-35°C (86-95°F). The incubator you see in the photo is a ~$400 piece of equipment what I got on eBay for $72. I just love when people sell things without knowing what they are and this person thought it was something else due to some notes that were taped to it so his description of the item was messed up, which reflected on the final price. I was watching several of these at the same time and others that were more beat up and older sold like hot cakes for $200-300-400 because people who sold them knew what they had. That’s a pretty good deal in my opinion. It’s almost new, no scratches, perfect interior, digital dial, great temperature control, basically I’m very happy with it. While Lactobacilli seem to like growing there and I may have already isolated that special sour strain, I wanted to see how the yeasts like it. I have to tell you, it’s amazing. Growth is probably 2-3 times as fast as in room temperature. Colonies appear on plates the next day after plating rather than 2-3 days and grow much faster. Liquid cultures also grow very well. Now I grow all my initial yeast samples and isolates in the incubator and it works great. I’m very happy so far.

Incubator

InsideIncubator

Hood: An enclosed, separate space where you work with cultures with a transparent “window” through which you see what you’re doing is a very nice thing to have, though not really necessary. It keeps the amount of particles falling into your work lower than if it was your kitchen table. Mine is pretty small and not as comfortable as I’d like it. It originally was a bookshelf that I modified into using as a hood. There is no laminal flow because I’m a grad student, not an oil magnate and can’t afford going that far. When we were moving I originally planned to adopt another shelf as my hood, which would have been deeper and wider, but in the end I decided to stay with the old one and just adapt to working in it plus adding a UV lamp and rewiring the daylight fixture inside so that it’s more compact and with a switch on the outside. It’s also positioned differently now so it’s more comfortable than before though I really wish it was deeper. If you plan to make one for yourself, I’d recommend making it about 2 feet deep, about 3-4 feet wide and 2-2.5 feet high. At least that would be perfectly sufficient for me.

Improved hood + UV lamp

Improved hood + UV lamp

UV Lamp: Unnecessary, but probably the best addition to my lab. Jason at Science Brewer recommended me getting one, but I didn’t at the time because I had to share my room and I was afraid the Plexiglas would crack. The lamp itself cost ~$80 on eBay. Installation was pretty straightforward – just drill a hole in the hood wall and poke it through. I turn it on about 10 minutes before working in it. When I pour plates I let them cool and solidify under the UV for an hour or two and the results are just stellar. I poured a lot of plates since installing it and not one had mold growing on it after incubation for a week. It’s amazing! My fear about Plexiglas cracking was correct though. You can’t really see it in pictures, but the area closest to the lamp has a spider web of cracks radiating from it. Eventually it’ll have to be changed, but it’s not a big deal. Also if you work in it with UV on, wear gloves and long sleeves or you’ll start to feel the burn after a while. I do that sometimes when I really want something to be as contaminant free as possible, and even though I hate wearing gloves when doing cell culture and almost never do because they get in the way, I wear them to protect myself from the UV. Another nifty feature was adapting a broken stainless steel dish rack as a movable tray inside the hood. As you can see in the photo, I can move it up close to the lamp and leave my plates there or tubes with medium, or flasks with water etc. to sterilize them just that little bit more. Works great so far.

Microscope Camera: Another luxury item. Lets you easily project your micro images onto your computer and analyze them quickly. You can get a cheap one for ~$50 on Amazon or from Celestron, but I’d advise against it. 2MP just doesn’t cut it. You’ll end up with the fuzzy pathetic pictures like what I have all over my blog that really don’t do justice to what’s really there. A decent camera would cost a couple hundred dollars and that’s out of my league, at least for now. Putting your regular camera or phone to the eyepiece and snapping a picture will give you a better quality image, but makes analysis such as measurements a lot more difficult because you can’t calibrate it very well. I also like being able to just snap the picture right into the computer and not have to transfer files back and forth.

Micropipettes: Allows you to measure out and transfer tiny volumes that you can’t do with transfer or serological pipettes. Perfect for when you need to use tiny amounts of reagents for your agar mixes such as antibiotics, cupper sulfate, p-coumaric acid etc. A workaround could be just making highly dilute solutions and using larger volumes, but there are errors associated with these methods. They are also very handy when you need to make serial dilutions of your culture for counting. I’ve been toying with the idea of getting some, but it’s just an idea so far. They cost a couple hundred dollars each, but you can get used ones on eBay for as low as $20. They are probably not calibrated, but you’re not planning to do research at your yeast ranch anyway.

Pipet-Aid Motorized Pipetter: Got on eBay for $24. Essentially a pump with a pipette nozzle. Very nifty for pipetting with the serological pipettes mentioned above. Plugs into the outlet and you control uptake and release with two buttons on the handle. You can use the manual or bulb pipetters instead, but your thumb will hate you for rolling the wheel up and down or having to squeeze the bulb every 100mL.

Darkfield Condenser: Very cool toy! Too bad it costs almost as much as the microscope itself. Basically it’s a light condenser for your microscope that changes the way light passes through your sample and makes things stand out as white on black background. It allows you to see detail much better in unstained samples. Worth checking out if you like looking at your cells in detail. I got mine from AmScope – Oil Darkfield Condenser For Compound Microscopes Model DK-OIL100. It’s a high power oil condenser and works very well with 100X objective. Just way cool! No real relevance to yeast ranching aside from being able to see more detail such as nuclei at high power.

Culture Fridge: You need to keep your cultures somewhere, right? People who lived here previously left their old fridge behind and I took it for my ranching needs. You can store your plates, cultures, yeast packs, bottles, hops, brewing herbs and spices and even grain in it.

Fridge

GlassInFridge

PlatesInFridge

FlaskInFridge

So this is how I setup my lab and what equipment I amassed over time. Procedures weren’t covered in this post because I didn’t want it too long and most people know them anyway. Recently a request came in to post the procedure for isolating yeast from bottles so I’ll try to do a step-by-step with photos in upcoming weeks. Please feel free to ask for “how-to” posts if you want me to clarify something or walk you through something. If you have some suggestions or comments, please feel free to make yourself heard and I’ll gladly discuss things with you.

To list the sites that helped me in setting up the lab and may be useful to you:

eBay

Amazon

Home Science Tools

American Science and Surplus

AmScope

Celestron Microscopes

All of them are great sources of all sorts of equipment and worth checking out.

Cheers!