A glimpse into copper sulfate agar

Eureka, I would like to publish some preliminary results from my latest plating experiments. I am still interested in isolating Brettanomyces from different sources and still play around with different agar media to see what their impact is on the entire isolation process. The latest experiment I performed was a large scale bromocresol screening on different Saccharomyces yeasts to see whether bromocresol can be used to differentiate between Saccharomyces and Brettanoymces. My insight from this experiment: bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. In addition, some Saccharomyces strains grew as white colonies in presence of bromocresol green (possible false positive strains).

Yet another approach is to add copper sulfate to the agar media to inhibit the growth of domesticated yeasts [Yakobson, 2010, Taylor et al, 1984]. Wild yeasts therefore should be able to grow in presence of copper sulfate. I wanted to give this agar a go to see if it can be used to differentiate between domesticated Saccharomyces strains and wild yeasts (Brettanoymces in my case). I started by adding 0.6 g copper sulfate to 1 L of Sabouraud agar and streaked some strains on the plates. As controls, plain Sabouraud agar plates were used to test the viability of the strains (not all plates shown).

Fig 1: Saccharomyces yeasts on Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

The four domesticated Saccharomyces strains plated on plain Sabouraud agar showed a nice growth phenotype (Fig 1). Streaking the same strains on copper sulfate containing Sabouraud agar revealed that only one strain (WY1084 Irish Ale) was impaired in its growth (Fig 2). All the remaining Saccharomyces strains grew as normal. From this observation one can already conclude that the addition of copper sulfate to the agar media impaired only 25% of the domesticated Saccharomyces strains tested.

Fig 2: Saccharomyces yeasts on CuSo4-Sabouraud agar (1056 = Wyeast American Ale, 1084 = Wyeast Irish Ale, PtPtince = EBY049, Y05 = EBY050)

Plating Brettanomyces and isolated Saccharomyces strains on copper agar media revealed a growth phenotype for all tested Brettanomyces strains (Fig 3, 4). Only the Saccharomyces isolate (B04 green in Fig 3) and the bacteria strain (I10 in Fig 4) did not grow on copper sulfate agar. Since B04 green was isolated from a Gueuze, it can be argued that this particular strain might be a non-domesticated Saccharomyces strain. On the other hand, it might be a domesticated yeast strain concluding from the lacking growth on copper sulfate. Including the previous observation that only a small part of domesticated Saccharomyces strains were impaired in their growth makes it even harder to allocate the isolated yeast strain to domesticated or non-domesticated Saccharomyces.

Fig 3: Different yeasts on CuSO4-Sabouraud agar (B04 = EBY004 Brettanomyces, B04green = EBY041 Saccharomyces, B05 = EBY005 Brettanomyces, B02 = EBY002 Brettanomyces)

Fig 4: Different yeasts/bacteria on CuSO4-Sabouraud agar (B01 = EBY001 Brettanomyces, I10 = EBY024 Bacteria, I05 = EBY009 Brettanomyces, I11 = EBY013 Brettanomyces)

This small-scale experiment revealed that a copper sulfate addition to Sabouraud agar media does not impair most of the domesticated Saccharomyces strains tested. All the Brettanomyces strains tested in this experiment grew in presence of copper sulfate.

It seems to me that copper sulfate used at a concentration of 0.6 g per liter of Sabouraud agar media was not useful to differentiate non-domesticated from domesticated Saccharomyces yeasts. Simply because it could not inhibit the growth of most of the domesticated yeasts tested. As an outlook, one might increase the concentration of copper sulfate to levels where it impairs most of the domesticated Saccharomyces strains. Then test the Brettanomyces under the same conditions and see if they still grow or not. Maybe even change the Sabouraud agar to MYGP like published by Taylor et al. It is not clear to me yet if I even further investigate the use of copper sulfate.

References:

• Yakobson C. (2010) Brettanomyces project, http://www.brettanomycesproject.com/dissertation/analysis-of-culturability-on-various-media-agar/brettanomyces-identification-methods/
• Taylor GT, Marsh AS (1984) MYGP+Copper, A medium that detects both Saccharomyces and non-Saccharomyces wild yeasts in the presence of culture yeast, J. Inst. Brew., Vol 90, p 134-145

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About the morphology of colonies

Eureka, today’s post covers some general information about the morphology of bacteria, yeasts and other microorganism on agar plates and why it is important to know at least a bit about it to get the most information out of your agar platings.

Q: What do you mean by morphology of colonies?

The morphology of a colony describes how microorganisms appear on agar media such as Sabouraud, malt agar etc. Morphology just describes the colonies. If you streak some microorganisms on agar plates, they grown (if the media is appropriate for this particular organism) and form visible colonies. The colonies appear as spots like shown in Fig 1. It is important to remember that a colony are thousands to millions of microorganisms together, not a single microorganism cell. Ideally all the cells within a colony originated from one single cell at the beginning (clonal expansion). If single cells are closer together on the agar, the individual colonies overlap and no single colonies are visible (left-upper part in Fig 1). In this case, the concentration of the yeasts is just too high to observe individual colonies.

Fig 1: Brettanomyces bruxellensis on Sabouraud agar plate after 11 days

Fig 1 shows what you get if you streak some Brettanomyces yeast on Sabouraud agar. The roundish spots are the colonies (as you can see on the right side in Fig 1).

Q: How do you get single colonies?

To get an accurate description of a colony, single colonies are necessary. But how do you get single colonies in the first place? As mentioned above, if the individual cells after streaking are to close to each other, the colonies might overlap. To get single colonies one simply has to ensure a low concentration to prevent such colony-overlays. One example to do so is to dilute the cells directly on the plate itself by using a special streak technique called dilution streak or Z-streak (Fig 2). How this is done is shown in a video (YouTube) as well.

Fig 2: Dilution streak done with three streaks

Begin with a cell suspension. You might even use a yeast slurry in the first place. A first streak is done to get some cells on the plate (Fig 2, streak 1). One expects a lot of cells visible on the trajectory of the first streak and the individual colonies overlay each others. After the first streak, you sterilize your inoculation loop, let it cool down and collect some cells by passing the inoculation loop through the first streak for a second one. This time the concentration of cells is already lower because you only pick a subset of yeast cells. This process can be done for a second time to get three streaks in the end (Fig 2). The plate after a dilution streak might look like shown in Fig 1. Unfortunately, there are no colonies visible in the third streak anymore. Anyway, I hope you get the idea.

Single colonies are not only useful to describe their morphology but also to differentiate between different microorganisms. For instance, if you are interested in separating the Saccharomyces yeasts (brewer’s yeast) from Brettanomyces yeasts you can use the dilution streak and hopefully some colonies arise from single Saccharomyces colonies and others from Brettanomyces cells.

Q: Why is the morphology important?

Lets assume the morphology of a colony, representing one kind of microorganism (remember the concept of the single cell at the beginning), is unique for every microorganism there exists. The morphological description could therefore be used to identify the kind of microorganism on your agar plate. This is just an assumption because there are a lot of microorganisms which have similar morphologies. To summarize, the morphology of the colonies can be useful to identify the kind of microorganism you have on your plate. Lets go through some examples. Have a look at Fig 3.

Fig 3: Girardin bugs on Sabouraud agar plate

I assume it is obvious that there are different kinds of colonies and hence morphologies. There are differences in shapes, size and colors. To conclude, different morphologies originate from different microorganisms. I can give you even further information here. The white colonies (big ones and wavy) are yeast cells, the flat beige ones bacteria. The very small white colonies are another kind of bacteria. You see, the morphology can even be used to differ between yeasts and bacteria. That’s why agar media are very common in microbiology labs to identify different kinds of yeast/bacteria. One application here could be to test a beer for spoilage organisms such as Lactobacillus (beer turned sour). Plate some of the sour beer on a plate where Lactobacillus can grow and if colonies arise with a typical Lactobacillus morphology, you can be certain to have a Lactobacillus contamination in your beer. I will not get into further detail about the different media and strategies used to do these tricks. Just to give you an idea what the whole agar media method is capable of.

Fig 4: Water kefir on Sabouraud agar plate

Maybe an example to show that the colonies are not always circular. Some microorganisms tend to form large flat colonies as it can be seen in Fig 4. In this case, I plated some of my kefir culture on a Sabouraud agar plate. You can even observe some yellowish colonies. Colonies are not always white or beige either. Not only can you choose different kind of agar media but also add some dyes for further characterization. One such example is shown in Fig 5. In this case bromocresol green is added to differentiate between microorganisms that can grow as white colonies and such as green ones. The color differences suggest that there are at least two different kinds of microorganisms on the plate shown in Fig 5.

Fig 5: Jolly Pumpkin’s Madrugada Obscura dregs on bromocresol green Sabouraud agar

Q: How do you determine the morphology of a colony?

First you need a pure culture of the microorganism. This is important because the morphology can differ if other microorganisms are in the same colony. The morphology can even be different on other agar media. Lets assume you want to describe the morphology of a pure brewers yeast (Saccharomyces cerevisiae). The first thing to do is streaking the yeast on a suitable agar media with a dilution streak and incubate the plate until colonies arise like shown in Fig 6. Sabouraud is a typical agar media for Saccharomyces and other yeasts. Malt agar media works as well.

Fig 6: Wyeast’s 2112 California Lager on Sabouraud Agar plate

In case of Fig 6, I streaked some of Wyeast’s 2112 California Lager yeast on a plate to check the purity. Now what about the morphology? Lets take a single colony and describe the following characteristics: form, margin, elevation (shape of the colony from the side), size, texture, appearance, pigmentation, opacity. The following descriptions are just an example.

Fig 7: from: http://commons.wikimedia.org/wiki/File:Bacterial_colony_morphology.png#filelinks; (Adapted and redrawn from Seeley, HW & Vandemark, PJ (1962) Microbes In Action: A laboratory manual of microbiology. WH Freeman (San Francisco, London) by user Ewen)

One might describe the colonies shown in Fig 6 as following:

Margins: Entire
Form: Circular
Elevation: Convex
Surface: Smooth
Opacity: Not transparent, shiny
Color: Off-white

That is what you can expect when you streak a yeast colony on a Sabouraud plate. The morphology of Saccharomyces is very similar on malt agar. Maybe some of you observed that there are yet some other different colonies on the plate in Fig 6. There were some impurities in this yeast sample as expected in the first place.

Q: Is the morphology of a given microorganism always the same?

Unfortunately not. The morphology of colonies can depend on the type of agar media used, if oxygen is present, nutrients, vitality, pH-levels, incubation time, other microorganisms present… Just keep in mind that a morphology description is not universal. If you encounter a morphology description of a specific microorganism, always check the type of agar media used and the conditions how the plates were incubated.

Q: Is it possible to differentiate between Saccharomyces and Brettanomyces based on morphology?

One of the most simple tricks to differentiate between the two yeasts is the incubation time. Saccharomyces colonies arise relatively quickly (within few days). Brettanomyces grow much slower (days to weeks). The second trick is to use a microscope and have a look at the different colonies. A third one might be (haven’t tried that one yet) to inhibit the growth of Saccharomyces by adding some growth inhibiting substances. Differentiating those two yeasts based on morphology is not that easy in my opinion.

Q: Is it possible to differentiate between top and bottom fermenting yeasts or even yeast strains based on colony morphologies?

As far as I know and from my experiences, differentiating between bottom and top fermenting yeasts base on colony morphologies is not possible. And it is not possible as well to differentiate between different yeast strains as well. Although I encountered some different morphologies for wheat strains at one point. However, I would not do any strain differentiation based on morphologies.

I hope there were some useful information in this post to give you a better understanding of agar media cultivation. Agar media are a very powerful tool in microbiology and is also widely used in breweries to check for impurities in beer or water. Understanding the concept of colony morphologies is therefore very important to get the most information out of agar media cultivation. And know about some limitations of the method as well.

Saccharomyces bromocresol green screen

Eureka, I would like to share my latest plating results with you. You might know that I am very interested in isolating any kind of wild yeasts from commercial sour beers. The most difficult task in this whole isolation process is to differentiate normal Saccharomyces cerevisiae colonies from other yeast species such as Brettanomyces.

Previous studies to develop new kind of agar media to detect Brettanomyces in wine samples showed bromocresol green to be a useful indicator to detect acid producing Brettanomyces strains [Rodrigues et al., 2001; Couto et al., 2005, EP 1185686 A1]. In this case, bromocresol green acts as a pH-indicator and turns yellow in the presence of acid which is produced by some Brettanomyces species. The authors further added cycloheximide to the media to prevent any growth of Saccharomyces. Concluding from the previously cited publications an addition of cycloheximide to agar media should already be enough to differentiate between Saccharomyces and Brettanomyces colonies by simply inhibiting the growth of Saccharomyces. Further antibiotics could be added to prevent the growth of bacteria.

Other studies showed that bromocresol green alone can be used to differentiate between the two yeasts in absence of antibiotics [Yakobson, 2010]. In addition, bromocresol can diffuse into yeast colonies and form green colonies due to the accumulation of the dye [Yakobson, 2010]. However, some Brettanomyces strains seem to be able to form white colonies again. This has been shown in other experiments as well [Rodriguez, 2012; BKYeast, 2012]. Yakobson mentions that the dye gets actively metabolized by Brettanomyces and hence the white colonies again. Unfortunately, I could not find any source investigating how exactly the dye get metabolized. Yakobson further mentions that some Saccharomyces strains can form white colonies as well which would make it even more difficult to differentiate between Saccharomyces and Brettanomyces.

The aim of this study was to screen different Saccharomyces strains for their ability to form white colonies on bromocresol green containing agar.

Material

• Sabouraud agar 4% glucose, Art. X932.1, Roth
• Bromocresol green sodium salt, Art. KK18.1, Roth
• Saccharomyces strains from Wyeast and White Labs

19 different Saccharomyces strains including one Saccharomyces mixture (WY3056) were plated on Sabouraud agar containing bromocresol green. Bromocresol green was added as aqueous, sterilized solution to the sterilized Sabouraud agar until the agar turned blue. The plates were incubated at room temperature at a dark place until colonies were visible. A control was included (no yeast streaked) to observe any color changes of the agar due to environmental effects (photo bleaching, oxidation, decay etc). The following yeast strains were used for this screen.

Number	Product name
WY1010	American Wheat
WY1056	American Ale
WY1084	Irish Ale
WY1728	Scottish Ale
WY1762	Belgian Abbey II
WY2112	California Lager
WY2278	Czech Lager
WY2487	Helle Bock
WY3056	Bavarian Wheat Blend
WY3068	Weihenstephan
WY3333	German Wheat
WY3522	Belgian Ardennes
WY3638	Bavarian Wheat
WY3711	French Saison
WY3726	Farmhouse Ale
WY3864	Canadian/Belgian Ale
WY3942	Belgian Wheat
WY3944	Belgian Wit
WLP002	English Ale
Control	N/A

Results Part 1

Colonies were visible after four days of incubation (Fig 1-5). The control showed no colony formation and the agar showed no color change. The colors of the agar were compared with the control.

Fig 1: Yeasts on bromocresol agar after four days. Left: WY1762, Top: WY3522, Right: WY3864; Bottom: Control

Fig 2: Yeasts on bromocresol agar after four days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 3: Yeasts on bromocresol agar after four days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 4: Yeasts on bromocresol agar after four days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 5: Yeasts on bromocresol agar after four days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Some Saccharomyces strains were able to change the color of the agar from green-blue to yellow. Only two yeast strains, WY3333 German Wheat and WY3726 Farmhouse Ale, grew as white colonies on bromocresol green agar after four days. This already is proof that some strains indeed can grow as white colonies on bromocresol green. The plates were further incubated and after a total of twelve days, the color of the colonies were evaluated for a second time (Fig 6-10). Sorry for the bad quality of the pictures.

Fig 6: Yeasts on bromocresol agar after 12 days. Left: WY1762, Bottom: WY3522, Right: WY3864; Top: Control

Fig 7: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 8: Yeasts on bromocresol agar after 12 days. Left: WY2487, Top: WY2112, Right: WY2278; Bottom: WY1010

Fig 9: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 10: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

One could observe that some of the colonies now have white edges and a green centre. All these colonies were still counted as green colonies.

This time less yeast strains turned the agar to a yellow color because the control agar lost a lot of its blue color. The global decrease of the blue color in the agar might originate from diffusion of acids secreted by yeasts that turned the agar yellow. Or due to the diffusion of the dye into the colonies. Further on to the white yeast colonies. WY3333 German Wheat and WY3726 Farmhouse Ale still grew in white colonies. In addition, WY3864 Canadian/Belgian Ale and WLP002 English Ale now grew as white colonies as well. One might expect further yeast strains to form white colonies with a prolonged incubation time because a lot of the colonies already have white edges and a remaining green centre.

After 17 days of incubation, the colonies looked as shown below (Fig 11).

Fig 11: Yeasts on bromocresol agar after 17 days. Left/bottom: WY2487, Left/Top: WY2112, Right/Top: WY2278; Right/Bottom: WY1010

A lot of yeast colonies now turned into white colonies as expected (Fig 12). The color was now evaluated by looking at the entire colonies visible for a particular strain. If more than 50% of the colonies were white, the yeast was counted as white. Like the WY2278 Czech Lager shown in Fig 11. On the other hand, all the other yeasts shown in Fig 11 were counted as green like the WY3711 French Saison in Fig 12.

Fig 12:WY3711 French Saison colonies after 17 days of incubation

After 17 days of incubation, only seven out of the 19 screened yeasts still had green colonies. All the other ones turned white in the meantime. To put it in numbers. After 4 days 2/19, after 12 days 4/19 and after 17 days of incubation 12/19 yeast strains formed white colonies (Fig 13). This clearly shows a time dependency.

Fig 13: Yeast screen results on heavily stained bromocresol green agar

Results Part 2

The blue color in the agar plates (Fig 1-5) was quite heavy and to test whether a lower concentration of bromocresol green in the agar leads to the same results as discussed above, a second experiment was conducting by streaking the exact same yeast strains on some Sabouraud agar containing bromocresol green. This time a lower concentration of bromocresol green was used.

Quantification of the color after three days of incubation (Fig 14-18):

Fig 14: Yeasts on bromocresol agar after three days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 15: Yeasts on bromocresol agar after three days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 16: Yeasts on bromocresol agar after three days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 17: Yeasts on bromocresol agar after three days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 18: Yeasts on bromocresol agar after three days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Yet again some colonies grew as white colonies and others grew as green ones (Fig 19). Comparing the results with the one concluded from the first experiment, WY3726 Farmhouse Ale, WLP002 English and WY3864 Canadian/Belgian Ale showed white colonies. In contradiction with the first experiment are the color morphologies of WY3333 German Wheat, WY1728 Scottish Ale and WY3711 French Saison. WY3333 grew as white colonies in the first experiment and as green ones in the second one. On the other hand, WY3711 and WY1728 grew as white colonies in the second experiment.

Fig 19: Closer look at Fig 15

The colours were again determined after further incubation. Agar plates shown after 12 days of incubation (Fig 20-24).

Fig 20: Yeasts on bromocresol agar after 12 days. Left: WY3864, Top: Control, Right: WY1762; Bottom: WY3522

Fig 21: Yeasts on bromocresol agar after 12 days. Left: WY3942, Top: WY3944, Right: WY3726; Bottom: WY3711

Fig 22: Yeasts on bromocresol agar after 12 days. Left: WY2112, Top: WY2278, Right: WY1010; Bottom: WY2487

Fig 23: Yeasts on bromocresol agar after 12 days. Left: WY3638, Top: WY3068, Right: WY3333; Bottom: WY3056

Fig 24: Yeasts on bromocresol agar after 12 days. Left: WLP002, Top: WY1056, Right: WY1728; Bottom: WY1084

Twelve days of incubation and all the yeast strains have the same color like a few days ago. The plates were further incubated and a final color determination was conducted after 17 days (not shown).

The results of the second run are summarized in Fig 25. WY1010 American Wheat, WY1084 Irish Ale, WY1762 Belgian Abbey II, WY2112 California Lager, WY2278 Czech Lager, WY3068 Weihenstephan and WY3942 Belgian Wheat all had white colonies after 17 days (Fig 25). 13/19 yeast strains grew as white colonies after 17 days of incubation (Fig 25).

Fig 25: Yeast screen results on light-stained bromocresol green agar

Discussion

Comparing the two experiments, some strains such as WY1010 American Wheat, WY1728 Scottish Ale, WY2112 California Lager, WY3711 French Saison and WY3942 Belgian Wheat only grew in white colonies after 17 days on the light stained agar media and not the heavy stained one (Fig 26). WY3056 Bavarian Wheat Blend, WY3522 Belgian Ardennes and WY3638 Bavarian Wheat grew as white colonies on heavily stained agar but as green ones on lightly stained agar media (Fig 26). This might be an indicator that the bromocresol green concentration might influence the color change as well.

Fig 26: Differences between the two experiments

As a general trend, the different yeast strains seem to form white colonies after further incubation. However, two strains (WY3333 German Wheat and WY3726 Farmhouse Ale) grew on heavily stained agar as white colonies from very early on (Fig 13) and four additional ones on lightly stained agar (Fig 25). Yakobson states on his website that Wit yeasts can metabolize bromocresol green (http://www.brettanomycesproject.com/2009/03/wln-agar-medium/). In this screen the Wit strain from Wyeast (WY3944) did not grew as white colonies in both experiments (Fig 13, 25). Not even after 17 days of incubation.

Some words about the color of the agar media. Fig 1 to 5 are nice examples to show that the color of the bromocresol containing media changes its color from green to yellow. In both experiments, the color of the control agar turned to a yellow color as well. The plates were stored at a dark place to prevent any influence of light (photobleaching effects). The change in color might be due to secretion of acids (bromocresol changes color at lower pH to yellow), due to a take-up of the dye by the yeast cells like stated by Yakobson in case of Brettanomyces. Another possibility might be the stability of bromocresol green itself. If one imagines bromocresol green to be a relatively unstable molecule, the loss of the green color might be due to the depletion of the dye. Yakobson further mentions that Brettanomyces can even metabolize the dye and therefore grow as white colonies. All the cells not able to metabolize the dye remain as green colonies. Unfortunately, I could not find any evidence for this statement showing that Brettanomyces really metabolize the dye. Nor any evidence that Saccharomyces can do it. Maybe the cytoplasm of Brettanomyces cells have a lower pH and therefore turn the dye from green to yellow. There might even be some truth about this hypothesis since some Brettanomyces strains are known to secrete acetic acid under aerobic conditions. It is therefore not clear to me why/how the colonies turn from green to yellow.

I would like to discuss bromocresol green as a useful tool to differentiate between Brettanomyces and Saccharomyces. Although I did not show any Brettanomyces colonies here, the bromocresol screen strongly suggests that some Saccharomyces strains can grow as white colonies on bromocresol green containing agar media. This makes a differentiation already a bit harder. In addition, a majority of Saccharomyces yeast strains appear as white colonies after a longer incubation period. BKYeast came to the conclusion that differentiation based on bromocresol green might only be possible in a short time frame in mixed cultures (Saccharomyces and Brettanomyces grow on the same plate). The results from these experiments show that even in pure cultures, and in absence of Brettanomyces, a lot of the Saccharomyces strains tested turned from green to white within a short period of time. All these results strongly suggest that any differentiation solely based on bromocresol green might only be useful in a short period of time.

Summary

Bromocresol screen is a widely used differentiating dye to differentiate between Saccharomyces and Brettanomyces. Brettanomyces known for their capability to grow as white colonies while Saccharomyces grow as white ones. It has been reported that some Saccharomyces strains grow as white colonies as well and therefore making a differentiation more difficult [Yakobson, 2010]. Screening different Saccharomyces cerevisiae strains on bromocresol green containing Sabouraud agar revealed some strains capable of growing as white colonies from the very beginning where the majority of yeast strains grew as green ones. Therefore showing that indeed some yeast strains can grow as white colonies. After further incubation, the majority of the yeast strains turned from green to white coloured colonies. There seems to be a general trend for Saccharomyces cerevisiae strains to form white colonies after extended incubation times. However the reason for this observation is not clear at this point as well as the mechanism leading to the observed change in color. It can’t be excluded that different sources like instability of bromocresol green itself or any environmental factor lets the colonies turn from green to yellow.

Due to these observations, bromocresol green as a tool to differentiate between Brettanomyces (known to grow as white colonies) and Saccharomyces might only work within a small time frame. This has been previously observed by BKYeast as well.

Outlook

Brettanomyces bromocresol green screen similar to the one shown here for Saccharomyces. In addition, try to grow Brettanomyces anaerobically to test whether the colonies grow as white or green ones (acid theory mentioned in the discussion).

References:

• Couto J.A., Barbosa A. and Hogg T. (2005) A simple cultural method for the presumptive detection of the yeasts Brettanomyces/Dekkera in wines. Letters in Applied Microbiology, 41, 505-510
• BKYeast (2012) http://bkyeast.wordpress.com/2012/02/19/selective-media-part-i/
• EP 1185686 A1, Culture medium for detection of Dekkera and Brettanomyces, http://www.google.com/patents/EP1185686A1?cl=en
• Rodrigues N., Goncalves G., Pereira-da-Silva S., Malfeito-Ferreira M. and Loureiro V. (2001) Development and use of a new medium to detect yeasts
  of the genera Dekkera/Brettanomyces. Journal of Applied Microbiology, 90, 1-12
• Rodriguez J. (2012) Brooklyn Brewery Samples on New Wild Yeast Media, sciencebrewer.com
• Yakobson C. (2010) Brettanomyces project, http://www.brettanomycesproject.com/2009/03/wln-agar-medium/

I am open to any discussions and feedback concerning this experiment. Thank you for reading.

Freezing Brettanomyces

Eureka, another Brettanomyces post. This time about a feasibility study if you can freeze Brettanomyces like any other Saccharomyces strain. I would hereby like to discuss my latest results.

All started by preparing some Brettanomyces strains I either bought or isolated for cryo storage like described in a previous post of mine concerning freezing yeasts. I put the following Brettanomyces strains in my -20°C (-4°F) freezer in August/September 2012:

– Brettanomyces isolated from WY3191 Berliner Weisse blend
– Brettanomyces isolated from Girardin Gueuze
– Brettanomyces isolated from 3 Fonteinen Gueuze
– Brettanomyces isolated from Cantillon Kriek (3 strains)
– Brettanomyces isolated from Cantillon 2007 Lou Pepe Gueuze (2 strains)
– Brettanomyces bruxellensis (WY5526)
– Brettanomyces lambicus (WY5112)

Some isolates consisted of more than one strain which were separated during trial runs with bromocresol green (not published). All these different strains were frozen separately.

In mid November 2012, the Brettanomyces were taken out of the freezer and transferred into fresh YPD media. After two weeks some of the yeasts showed signs of growth such as turbid media and gas production. In the end all media showed signs of activity and formed off-white coloured sediments. Both yeasts isolated from the Cantillon beers even showed signs of pellicle formation (not shown). Although activity could be observed it still has to be evaluated if the activity originates from the yeasts and not any contamination. Due to lack of time the yeasts remained in the YPD media for nearly two months until further experiments could be conducted.

Micrographs

Some micrographs showing the yeasts from YPD liquid cultures before freezing and afterwards.

Fig 1: Brettanomyces from Cantillon Kriek before freezing

Typical elongated cell shape of Brettanomyces visible (Fig 1 and 2). Even some hyphae formation (Fig 2). Somehow the colonies in Fig 1 look smaller than the ones in Fig 2 although both pictures were taken with the exact same setup.

Fig 2: Brettanomyces from Cantillon Kriek after freezing

Fig 3: Brettanomyces from Cantillon Lou Pepe after freezing

Again some hyphae formation (Fig 3).

Concluding from the micrographs shown (Fig 1-3), Brettanomyces yeasts could be found in the YPD media after reviving them. Although Brettanomyces yeasts could be observed in the microscope observations still does not prove that the yeasts are viable. Liquid cultures were first streaked on some Sabouraud agar plates and incubated at room temperature until colonies were visible. Colonies were then picked from the Sabouraud plates and streaked on Sabouraud agar with an addition of bromocresol green.

Some yeasts had different morphologies like WY5526 B. bruxellensis on bromocresol green containing agar media (Fig 4). Some colonies grew as green, others as white ones. For the next agar platings, each a white and green colony were picked.

Fig 4: WY5526 B. bruxellensis on bromocresol green agar

One strain of Cantillon’s Kriek and the strain isolated from a 3 Fonteinen Gueuze grew in different morphologies (white and green colored colonies) as well and were treated separately for the next agar platings. Please further notice that WY5112 Brettanomyces lambicus was not streaked on Sabouraud Bromocresol green due to a mold contamination on the first Sabouraud plate. However, typical colonies of B. lambicus could be observed (not shown).

Agar plate results

All the revived Brettanomyces strains formed colonies on Sabouraud Bromocresol green agar (Fig 5-7).

Fig 5: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Kriek_green colony (B04_green); Top: Cantillon Gueuze 2007 (B05); Right: WY5526 B. bruxellensis colony 1; Bottom: WY5526 B. bruxellensis colony 2

All the yeasts grew as white colonies expect the one known to grow as green colonies (B04_green) (Fig 5). In the case of WY5526 B. bruxellensis, the two picked colonies from Fig 4 showed the same morphology again. Both grew as white colonies (Fig 5).

Fig 6: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Kriek (B04_2); Top: Cantillon Kriek (B04_1); Right: Girardin (B01); Bottom: Cantillon Geuze 2007 (B05 dark_1)

The two colonies that grew differently on the first bromocresol green agar from the Cantillon’s Kriek isolate grew again as white colonies (Fig 6).

Fig 7: Brettanomyces on Sabouraud agar after six days of incubation. Left: Cantillon Geuze 2007 (B05 dark_2); Top: Newly isolated Brett (nothing to do with this experiment); Right: 3 Fonteinen (B02_2); Bottom: 3 Fonteinen (B02_1)

The same is true for the different morphologies from a 3 Fonteinen isolate (Fig 6, 7). The Brettanomyces strain(s) isolated from WY3191 Berliner Weisse blend formed colonies as well (not shown).

In all cases, the bromocresol agar media turned from a blue color to yellow indicating the secretion of acid. Some plates even had a strong acetic acid smell. An un-streaked bromocresol agar media was included as a control and the color remained blue throughout the whole experiments (not shown).

Summary/ Conclusion of agar platings

It could be shown that all the frozen Brettanomyces strains formed colonies on Sabouraud agar. Some of the isolated yeasts grew in different forms (white and green colonies) but such a differentiation could not be observed after a second streak on agar media. This is not the case for the yeast strain isolated from Cantillon’s Kriek (B04 green) which grew in green colonies on every plating.

The differentiation between Brettanomyces and Saccharomyces based on bromocresol green and its issues will be covered in a future post. One could already observe in these platings that some of the yeast colonies grew as green colonies in a first run but grew as white colonies in a second run again.

Micrographs of the colonies

At last some micrographs of the colonies.

Fig 8: WY5226 Brettanomyces bruxellensis (5526_1)

Fig 9: WY5226 Brettanomyces bruxellensis (5526_2)

The two different samples of WY5226 B. bruxellensis look very similar (Fig 8, 9). The differences in color appearance on the bromocresol green might be due to some issues of bromocresol green as an indicator for Brettanomyces and wild yeasts. As mentioned already, more about that in a future post.

Fig 10: Brettanomyces from Girardin Gueuze (B01)

Typical Brettanomyces cells visible in the Girardin isolate (Fig 10).

Fig 11: Brettanomyces from 3 Fonteinen’s OudeGueuze (B02_1)

Fig 12: Brettanomyces from 3 Fonteinen’s OudeGueuze (B02_2)

Both colonies from 3 Fonteinen isolate seem to be Brettanomyces (Fig 11, 12). Hard to tell based on the morphology of the cells if the two samples are the same or not.

Fig 13: Brettanomyces from Cantillon’s Kriek (B04_1)

Not a lot of elongated cells were visible in B04_1 (Fig 13) like in B04_2 (Fig 14). Maybe these two samples are not the same strain of Brettanomyces. Maybe B04_1 is not a Brettanomyces strain. Further studies are necessary.

Fig 14: Brettanomyces from Cantillon’s Kriek (B04_2)

Fig 15: Brettanomyces(?) from Cantillon’s Kriek (B04_green)

Some elongated cells visible in B04_green (Fig 15). Yet a lot of the cells looked like Saccharomyces cerevisiae. Might be a mixture of S. cerevisiae and Brettanomyces.

Fig 16: Brettanomyces from Cantillon’s Kriek (B04_white_1)

Lots of hyphae visible in one of the isolates from Cantillon’s Kriek (Fig 16). Very typical for Brettanomyces.

Fig 17: Brettanomyces from Cantillon’s Kriek (B04_white_2)

On the other hand, in the second sample of B04_white not that many Brettanomyces cells visible form hyphae as shown in Fig 16 (Fig 17). Yet again, maybe these two samples are not the same strain of Brettanomyces. Further studies necessary.

Fig 18: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05)

Lots of elongated, boat-shaped cells in Cantillon’s Lou Pepe isolate B05 visible (Fig 18).

Fig 19: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05_dark_1)

The second strain from Cantillon’s Lou Pepe looks different from the first one shown in Fig 18 (Fig 19). Hard to tell if the second sample B05_dark_2 is another strain than B05_dark_1 or not.

Fig 20: Brettanomyces from Cantillon’s Lou Pepe 07 Gueuze (B05_dark_2)

Summary/ Conclusion of micrographs

Brettanomyces were visible in most of the micrographs shown above. However the shape of Brettanomyces can differ significantly. The experiment strongly suggest that it is possible to freeze Brettanomyces and successfully revive them. In addition, it could be shown that some of the frozen samples might contain further strains of yeasts. Additional experiments are necessary to further look into this possibility.

Unfortunately, the yeast isolated as Brettanomyces from WY3191 Berliner Blend looked very similar as Saccharomyces cerevisiae cells (not shown). It might be possible that the yeast isolated from the blend wasn’t a Brettanomyces strain in the first place. But beside the S. cerevisiae colonies were some smaller colonies visible as well (Fig 21).

Fig 21: Isolated cells from WY3191 Berliner Weisse blend

The cells shown in Fig 21 are no yeast cells. Theses cells look like Lactobacillus. Interestingly, these bacteria cells were in the freezer as well (therefore possible to freeze Lactobacillus like yeast cells as well) and they grew on bromocresol green containing agar.

Enough with the experimental part. I am very happy about theses results. Took me some time to do all the platings, micrographs but I have the feeling it was all worth the efforts. At least now I know that I can easily freeze all my Brettanomyces strains I have (well over 20) without any worrying. The only thing to keep in mind here is that theses yeast might take some additional time for reviving than normal S. cerevisiae strains. Some preliminary results even suggest that it is possible to freeze Lactobacillus like you would any yeast cells.

The next post will be about another big yeast experiment. Stay tuned and thanks for commenting!

Isolating the bugs from Cantillon Gueuze 2007

Fig 1: Cantillon’s Lou Pepe 2007 Gueuze

Eureka, this is another post concerning wild yeast isolation from a commercial beer. Today’s beer is Cantillon’s Lou Pepe 2007 Gueuze. I got a bottle of this particular Gueuze a year ago and stored it for another year in my cellar. By the end of June 2012, I finally got the opportunity to open the bottle and taste it.

Before heading into the tasting notes, let me give you some background information about the beer. The label on the bottle says: “Our Lou Pepe beers are all exceptional products. We only use the finest lambic to make these beers. The Lou Pepe Gueuze is a blending of only 2 years old lambics. Beer with tasteevolution. Best before 12/2029” (Fig 2). It comes in 0.75 L bottles and 5 ABV. Bottled on the 12th of October in 2009.

Smell: Very funky and a lot of horse blanket, leather and barnyard

Taste: Very light sourness, pretty dry on the palate, grainy. Some lemon and wood notes as well. Rather nice sourness (no vinegar). Subtle notes of funkiness.

Appearance: Pours in a golden-yellow color, clear and a pretty nice white head. Not very long lasting head though. Very fizzy. Looks like a champagne

Mouthfeel: Light body, average carbonation, dry and astringent aftertaste. Some bitterness is there as well

Overall: Not bad and very easy drinkable. Not a very sour and complex Gueuze compared to others. However, a good example for the style. My rating: 80/100. I expected this beer to be more complex and less astringent.

Fig 2: Bottle description

I then streaked some of the bottle’s sediment on some Sabouraud agar plates and left the plates at room temperature for approximately three weeks until colonies were visible. I could observe two different kinds of colonies (Fig 3).

Fig 3: Cantillon’s Lou Pepe 2007 sediments on Sabouraud agar

The most colonies were similar to the whitish colonies marked as 2 in Fig 3. And there were some darker colonies (light beige) marked as 1 in Fig 3. Nearly two years after bottling the Gueuze there are still some living organisms in the bottle. The morphology of these colonies is very similar to other Brettanomyces I isolated before. I expect these colonies (marked 2) to be Brettanomyces. On the other hand, I have no clue what the microorganisms in colony 1 could be since the color is very different from Brettanomyces or Saccharomyces colonies. Maybe the micrographs give me further information? Next step was to do some microscopy observations of the two different colonies. Lets begin with the colonies marked 2 in Fig 3.

Fig 4: Micrograph of colony 2 (see Fig 3)

Fig 5: Micrograph of colony 2 (see Fig 3)

To me, the colonies shown in Fig 4 and 5 look like a kind of wild yeast. At least no Saccharomyces cerevisiae for sure. Or any other kind of bacteria due to the size of these cells. I expect these cells to be Brettanomyces due to the elongated shape of the cells and other characteristics. The cells could be Kloeckera apiculata, Pichia membranaefaciens or Hansenula… The list is not complete here. Further investigations are necessary. What about the other colonies?

Fig 6: Micrograph of colony 1 (see Fig 3)

Fig 7: Micrograph of colony 1 (see Fig 3)

First of all, the cells shown in Fig 6 and 7 look very different from the ones shown in Fig 4 and 5. These cells here are mostly circular and look very similar to Saccharomyces cerevisiae cells. Aggregation of Saccharomyces cerevisiae as it can be seen on the upper left corner in Fig 7 can be observed in wheat yeast samples as well. For me theses cells look like typical Saccharomyces cerevisiae cells although some cells seem to have a more elongated form as well. I will have to do further investigations to get more information about the cells in colony 1. Wouldn’t it be cool to have isolated some Saccharomyces cerevisiae yeast cells from an old Gueuze from Cantillon?

To summarize, I could isolate two different kinds of yeasts from a Cantillon Gueuze bottled in 2009. I have a strong feeling the cells from colony 2 belong to the specie of Brettanomyces. This is just a feeling. The strains go into my library as B05 (colony 2) and Y03 (colony 1). Further investigations are necessary to differentiate the two different strains. Cool stuff. The only verified conclusion here is: It is possible to isolate some yeasts from a Gueuze that is nearly two years in the bottle. Stay tuned for further yeast related posts!

Yeast banking – #5 Frozen yeasts

Eureka, today is the last post in the series about yeast banking at home (or in a lab). Please refer to the yeast basics page for links to all the other posts. The three previous methods (agar plates, isotonic sodium chloride solutions and agar slants) work all at room temperatures or colder. But not below 0°C (32°F) since the yeasts would probably die and the media (agar and sodium chloride solution) would freeze. Storing your yeasts at colder temperatures prevents some of the growth. If the yeasts do not grow during the storage time, the chances are high to have the same exact strain after you revive them. If you store your yeasts in a refrigerator your yeast can grow (even slowly) and might mutate and try to adapt to the colder temperatures. The yeasts could therefore change and maybe lose specific characteristics. This could lead to loss of flocculation or even loss of your most loved aroma profile (banana or clove aroma in wheat yeasts for example). However, such a conversion does not have to happen. It might. And this is why a lot of labs store their microorganisms or cells at lower temperatures such as -80°C (-112°F). At this low temperature no growth occurs. Even the whole metabolism of the cells arrest. The cells kind of stops entirely. You can store your cells at this temperature for nearly forever.

I am not sure how many of you out there have a -80°C freezer at home. Most of you might have a freezer at around -20°C (-4°F). And you can store your yeasts at -20°C as well. Just don’t use a freezer with thaw-cycles. The only disadvantage here is the metabolism of the yeasts might still work and some changes could occur as well. In comparison to a storage at room temperature or colder temperatures, far fewer changes can/do happen at -20°C. And this is why freezing your yeasts is as far as I know the only method to bank your yeasts over a longer period of time (years) at home.

Description of the technique

As already described, this method here is about freezing your yeasts at -20°C (-4°F) or lower/higher if you want. For this purpose you use a special media which consists of a cryoprotectant (antifreezer) such as glycerin. Please don’t use antifreezer you use for your car. If you have your storage media ready you just add some yeasts to the media and put it in your freezer and leave it there until you want to use the yeast for a future batch. Please notice, this is about banking yeasts and not yeast storage. Only small amounts of yeasts will be frozen here. Not pitchable amounts.

I would like to mention already, this is the most sophisticated method of all the four described already (agar plates, isotonic sodium chloride solution and agar slants). I do not recommend to go with this method if you haven’t tried one of the previous ones before. If you are new to yeast banking try to bank your yeast with another technique than this one before and get some experience. I recommend the isotonic yeast storage method for beginners. If you manage to revive the yeasts without an infection you might step forward to this method. If infections occur regularly, try to find the source for the infection and work on that. This method here does not work if you have troubles with your sterility and cleanliness… It just does not. In addition, the technique below is just one way to do it. I am certain there are other ways to freeze your yeasts.

Material

Fig 1: Tube filled with storage media and yeast

– Vial, tube or any other containment you can heat sterilize to store your yeast in a freezer. I use 1.5 mL reaction tubes for this purpose (Fig 1). They are small and easy to sterilize

– Food grade glycerin. Glycerin solutions work as well as long as the glycerin concentration is above 60%. I use a 85% food grade glycerin solution I bought at a local pharmacy

– Pressure cooker or any other source to heat sterilize your tubes and the food grade glycerin

– Media. I guess dried malt extract solution or even an isotonic sodium chloride solution could work as well. I use a lab media (called YPD) as a storage media. And add some ascorbic acid as well. More about my storage media later on.

– Sterile pipettes, micro pipette with sterile tips or a sterile syringes. You need sterile devices to add the storage media to your containments before freezing and get the yeast out of the containment for reviving. See “bank the yeast” description below for further information.

Preparation

First, get the freshest, purest yeast you can get. This could be from a starter or from a fresh yeast package or vial. This is very crucial. If you freeze unhealthy yeast you could risk to either loose them entirely or have problems during reviving them. Or a different outcome of a batch of beer (attenuation, taste, flocculation etc.). Please do not freeze or bank any unhealthy yeasts. And don’t expect the yeasts to come around during banking. If you have problems during a fermentation (stuck or whatever) don’t bank the yeast afterwards and hope the yeasts will be fine. They probably won’t.

What yeast sources could you use?

Fig 2: Small yeast starter with yeast at bottom

1. Yeast starter

Get yeast directly from a fresh yeast starter. Wait until no more growth occurs. Then mix up the whole yeast starter to get the yeasts back into solution. Remove some of the volume from the yeast starter and fill your pre-sterilized containments. If you want to cool down the yeast starter to let the yeasts settle to the bottom of the flask and discard as much of the yeast starter as possible (and only pitch the yeast sediment), remove the yeasts for banking before cooling down the starter. Store the filled containments for 48 h in a refrigerator. During this step the yeast build up some important molecules they need to survive and settle to the bottom (Fig 2). Remove as much of the supernatant as possible (Fig 3). This can mostly be done by just inverting the tubes, vials etc. The yeast sediment at the bottom should stay at the bottom. Just don’t turn the tubes too fast and too slow. You now should have a nice yeast sediment at the bottom (Fig 3). The volume of the yeast sediment should be below 10% of the volume of the containment. If it is a bit more or less don’t worry. However, discard some of the sediment if it is more than 20% of the volume.

Then proceed with the steps described as “bank the yeast” below.

2. Yeast package from manufacturer

Use yeast from the manufacturer directly. Make a small yeast starter and add a few mL of yeast slurry from the package or vial (Fig 2). I use glass tubes for this purpose which are filled with 4 mL of a malt extract yeast starter media (10 g of dried malt extract dissolved in 100 mL of water) and sterilized them in a pressure cooker.

Leave the starter at room temperature for 48 h. Then proceed with the steps described as “yeast starter” above. If your yeast is very fresh, you might skip the whole starter-step and bank the yeasts directly. Therefore fill your containments with the yeasts and let them settle down in a refrigerator for 48 h then discard the supernatant. Then proceed with method described as “bank the yeast” below.

3. Yeast sediment from fermenter

I would not recommend to directly bank yeasts from a slurry. At least wash them first to get rid of trub and any dead cells and do a small yeast starter. Just harvest a small amount of the sediment (like 100 mL) and wash them with sterile water for a few times until only the viable cells remain. Discard as much of the supernatant as possible. Then make a small yeast starter (100 to 200 mL), add the washed yeast cells and leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

4. Yeast sediment from bottles

Procedure is similar to “yeast package from manufacturer” above. Make a small yeast starter and add some of the bottle sediment. Leave the starter at room temperature for 48 h. Then proceed with steps described as “yeast starter” above.

Bank the yeast

Fig 3: Tube with yeast sediment at bottom

The yeast sediment you now have in your containments should consist of very healthy and pure yeast cells (Fig 3). Now its time to add the storage media (see below) and freeze the yeasts. I add about ten times the volume of storage media for every volume of yeast. In my case I have about 0.05 to 0.1 mL of yeast sediment (Fig 3). I therefore add 0.5 mL of storage media. To add the storage media you need a sterile device such as a pipette, micro pipette with sterile tips or sterile syringes. Please pre-sterilize the storage media in a pressure cooker for 15 min if possible. Let the storage media cool down to room temperature first before proceeding. Then add the media and either gently shake the tubes, vials or use the pipette, syringe, micro pipette for a thoroughly mix. You are basically done. Just label your containments very well and put them in your freezer. Done!

Storage media:

1. Malt extract based (haven’t try this one): For 100 mL of storage media use 10 g of dried malt extract, 50 g of glycerin and fill up to 100 mL with water. Add 0.1 g ascorbic acid (aka vitamin C) if possible. The ascorbic acid helps to stabilize the membranes of the yeasts. If you have a glycerin solution for example a 85% glycerin solution calculate the amount you need as following: 50 g divided by percentage of solution (divided by 100). In this example 50 divided by 0.85 equals 58.8 g. You therefore have to add 58.8 g of your 85% glycerin media. Sterilize the storage media in a pressure cooker for 15 min if possible.

2. YPD storage media (I use this one). The recipe for this YPD-media based storage media is from the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff. For 100 mL you need: 5 g YPD bouillon, 50 g of glycerin and 0.1 g ascorbic acid. Add up with water to 100 mL. Sterilize the storage media in a pressure cooker for 15 min if possible.

Storage

Put your containments in your freezer. Nothing to do more. I use a rack for my tubes to have some organizing system (Fig 4).

Fig 4: Yeast library part 1

Reanimation

1. Make yourself a yeast starter. I recommend 100 mL for the first step. Therefore dissolve 10 g of dried malt extract in 100 mL of water, add some yeast nutrients if possible and sterilize the starter for 15 min with a pressure cooker. Cool down the starter to room temperature.

2. Get your tube, vial (or whatever containment you use for yeast banking) out of your freezer and increase the temperature as fast as possible. I let the tubes warm up in my hands. Then gently mix the yeast and storage media and add the whole content to your yeast starter. I use a micro pipette for this step. Then wait a few days until signs of fermentation arise (cloudiness, white foam, yeast sediment at bottom, bubbling etc.). Wait until a yeast sediment formed at the bottom. You can either stir your yeast starter the whole time or just leave it unstirred.

3. Prepare your next yeast starter. I normally do a 1 L stirred yeast starter as a second starter here. Therefore dissolve 100 g of dried malt extract in 1000 mL of water and sterilize it. Discard the supernatant from the first yeast starter and only transfer the yeast sediment to your next 1 L yeast starter. I recommend to taste the supernatant (before discarding) to check if the starter is okay. If the starter tastes bad probably an infection occurred. If the yeast starter tastes good, congratulations!

4. Repeat the yeast starter steps until you have the amount of yeast you need. It is hard to tell how many yeast starters you need and what volume you should choose. There are way too many different way on how to bank the yeasts. The only way to tell how many yeast cells you have would be to count the cells (have a look at this post concerning this topic).

From my experience and with the amount of yeast I bank (about 0.1 mL as it can be seen in Fig 3), I need a 100 mL yeast starter first, followed by a 1 L yeast starter, followed by another 1 L yeast starter afterwards to have approximately 100E9 cells. This would be equal to the amount of yeast you get in a Wyeast’s Activator package or White Labs vial.

My experiences with this method

My procedure looks as following. As already mentioned, I use a YPD-based storage media to bank the yeasts. And I use the tubes shown in Fig 3 for banking. After discarding the supernatant after storing the tubes 48 h in the refrigerator, I add approximately 0.5 mL of the storage media to the tubes with a micro pipette and a sterile tip (Fig 5).

Fig 5: Equipment for yeast banking. YPD-based storage media (left), yeast sediment in tube (right) and micro pipette (1000 uL) with sterile tip

Then use the micro pipette to mix the yeast and the storage media. After that the tube look like shown in Fig 1. I then put the tubes in a box (shown in Fig 4) and store them in my freezer (at -20°C).

What are the advantages and disadvantages for this method compared to the others?

Advantage	Disadvantage
Long term storage method	Lot of equipment necessary (freezer, lab equipment etc.)
No maintenance work	Contaminations not visible
Does not require a lot of space	Can’t store yeast mixtures, blends
	Rather complicated method

This is for sure one of the least labor intensive methods. And the only one to store your yeast over a longer period of time. On the other hand, you do need some extra equipment such as a freezer and some lab equipment (syringe or pipette or micro pipette, containments, chemicals (ascorbic acid)). I think this method is only for the people really interested in yeast banking. And I would not recommend to go with this method if you haven’t tried one of the previous ones before. Sure the long-term storage seems very advantageous. However, do not underestimate the time and equipment you need to prepare the yeast for this banking method. On the other hand, your equipment has to be very clean and mostly sterile.

As with other methods, it is not easily visible whether your yeast is infected or not by just looking at your vial, tube etc. You will know after the first yeast starter. And you can’t bank yeast blends and other mixtures with this method as well. The ratio of the different microorganisms will eventually change during the reviving. If you do want to store a blend you might have to separate the blend before…

For all of you still interested in freezing your yeasts, I would like to mention the book “Yeast: The Practical Guide to Beer Fermentation” by C. White and J. Zainasheff again. In there are further information on how to freeze your yeasts.

This is the end of the yeast banking posts. I hope I could give you some information about the topic. Please feel free to comment and ask questions if something is not clear enough. The next posts will be about some recipes, tasting notes and yeast hunting stories again. Stay tuned!