#3 Agar plates (#1056, #3787)

Eureka, this is the third post in the pentade about some agar plates. The other two posts about dregs and Lactobacillus can be found here. Today, some pictures of brewers yeast Saccaromyces cerevisiae. I plated Wyeast’s #1056 American Ale and #3787 Trappist High Gravity yeasts a while ago on Sabouraud agar and now its time to have a look at it.

#1056 American Ale

#1056 American Ale yeast on Sabouraud agar after three days of incubation.

Could observe just one sort of colonies after three days of incubation: Off-white color, even, circular, glossy, convex, 2 mm diameter. Typical yeast morphology.

Microscopy picture #1056 American Ale yeast colony

And the microscopy observation confirms that the colonies are indeed yeast cells. This is a very typical picture of the slightly oval yeast cells. But let me just add some further information that can be derived from the picture. Not only is it possible to identify these cells as yeasts, but it is further possible to identify this particular yeast strain to be top fermenting. Top fermenting yeasts tend to stick together, bottom fermenting ones don’t (or less). Unfortunately, I do not have a picture of bottom fermenting yeast cells available right now to show the difference.

#3787 Trappist High Gravity from 2010

I have a sample of this yeast strain in my yeast library since 2010 and I originally isolated this strain from an agar plate. As I was not sure if the yeast was really pure (had no microscope yet), I plated it and hoped to get an answer.

#3787 Trappist High Gravity yeast on Sabouraud agar after three days of incubation.

And there were just one sort of colonies, so far so good: Off-white color, serrated, irregular, glossy, flat, 2 mm diameter. Some look like snow flakes. Well, this is a bit strange. I expected the colonies to look like those in the picture of #3787 below. The colonies look very similar to those from the 3 Fonteinen dregs.

And the microscopy analysis identified the colonies to be bacteria not yeasts. I have no picture of the microscopy sample as it is really difficult to get a good picture due to the really small size of bacteria. This means that my #3787 from 2010 was indeed not pure.

#3787 Trappist High Gravity from 2011

I brewed a batch of Belgian style Ale in 2011 and as I was not really sure about the quality of the yeast strain in the yeast library (see above), I got myself a fresh culture of #3787 Trappist High Gravity to be sure.

#3787 Trappist High Gravity yeast on Sabouraud agar after three days of incubation.

Just one sort of colonies on the agar: Off-white color, even, circular, glossy, convex, 2 mm diameter. Typical yeast morphology again.

Microscopy picture #3787 Trappist High Gravity yeast colony

And again the typical yeast cells in the microscopy sample. I observed something interesting as well. Look at the colony in the middle of the left side, the one which looks like a club. I already observed a cell like this before in the BFM dregs analysis a while ago. See Fig5 in this thread. This would mean that there could be some yeasts in the dregs as well. And this is of no surprise to me as I took the dregs out of a fermenter and there could certainly be yeast cells in the bottom of the fermenter.

So far so good. Because I had some cool results in the past two posts and not in this one yet, I kind of tried to shoot a cool picture of yeasts. I already mentioned, my microscope and camera are not the best ones. But they do their job. One thing that is really difficult is to look at the samples with the highest magnification of 2000x using the immersion oil objective. And to take a photo of such a magnification is even harder. But I did my best to get the following picture.

Microscopy picture #3787 Trappist High Gravity yeast colony

I think it is a quite good picture. And you can even see the club-formed cell as well. I like this picture very much.

Well, that’s all about Saccaromyces cerevisiae so far. I hope some of you enjoyed the pictures as I did and maybe learned something new. If someone noticed that I did not mentioned anything about the Heidelberger Kellerhefe, although mentioned in the first post, well, the yeast seems to have finally given up on me. I could not observe any colonies, even after further incubation…

The next post about agar will be about Wyeast’s #3763 Roeselare Blend culture(s). And there will be some cool results, I promise. And I just decided to post some pictures of top and bottom fermenting yeasts in the future as well to show the differences. Feel free to comment below anytime. I really appreciate it.

#2 Agar plates (Water kefir, Lactobacillus)

Eureka, this is the second post in the pentade about some agar plates. The first post was about different dregs from commercial sour beers. This post is about a water kefir culture and a Lactobacillus starter. I made a Lactobacillus starter with sterile apple juice and dumped some acidified malt in there for a future Berliner Weisse. Made the starter a few days ago and it smells really sour. Lets get into the results.

Water kefir:

Plated my first water kefir on a Sabouraud plate to see what’s in there. I could observe only two kinds of colonies after three days of incubation:

Water kefir bugs on Sabouraud

– Off-white color, even, circular, glossy, convex, 2 mm diameter.

– White color, wavy, irregular, not glossy, flat, 10 mm diameter.

Since I had no idea what these colonies might be, I took a sample of each colony and did a microscopy analysis.

Microscopy picture Water kefir circular colony

And now the really fun part begins: The circular colonies on the agar are no bacteria due to the size of the bugs. These must be some sort of yeasts. Maybe even more than one kind. But a kind of yeast that exists in a slender rather than in a circular form like brewers yeast (aka Saccaromyces). I have no idea what sort of yeast I have here.

Microscopy picture Water kefir wavy colony

And the wavy colonies look even more inhomogeneous than the circular colonies. But these are yeast cells as well due to the size. There are the slender ones again like in the picture above, and a kind of roundish smaller one as well. I have no idea what I got myself here. I expected to find some Lactobacillus bacteria on the plates since bacteria grow much faster than yeasts in general. But I could not find any. And no Lactobacillus on the microscopy samples as well. I guess that Sabouraud agar does not support the growth of Lactobacillus at all.

Comparing the two pictures gives me the impression that there are more than one different kind of yeasts. These results were very unexpected to me and I am quite fascinated what I got myself here.

Lactobacillus starter:

And for the starter: No sign of colony formation at all. Even after further incubation. This might prove that Sabouraud agar is indeed not a media to cultivate Lactobacillus bacteria. Unfortunately I do not have a microscopy picture of the Lactobacillus starter to show the bacteria.

Summary

What did I learn? First, Sabouraud might be unsuitable for cultivating Lactobacillus strains. Second of all, there are some sort of yeasts in the water kefir culture I could not identify further. Lets go back to the results from the first post about the dregs where I could observe only bacteria. The microscopy showed bacteria, Bacillus to be exactly. And I expected that the bacteria might be Lactobacillus or Acetobacter. As these results here showed that Lactobacillus might not grow on Sabouraud, I guess that the bacteria from the dregs must be Acetobacter then.

Did some research about the bugs in water kefir. Some sources state, that there are Candida strains or Kloeckera apiculata in there. I compared my pictures with published pictures of both yeasts and they looked very different to me.

I plated the different colonies on fresh Saboraud plates and will do further investigations.

If there is someone out there who can identify any of the bugs in the pictures, please comment below. And if someone has any ideas how/what to do next with the colonies, please let me know as well.

The next, third post about agar plates will be about some normal Saccaromyces strains.

#1 Agar plates (BFM, 3 Fonteinen, Girardin)

Eureka, after some recipe posting now a post that is right down my alley. I did some agar pouring, streaking and finally some microscopy analysis of different colonies. So this is the first post out of five about bugs. Today, pictures of bugs from commercial sour beers.

Plated bugs

01/30/2012: I plated the following bugs on Sabouraud plates: BFM La Torpille dregs, 3 Fonteinen dregs, Girardin dregs, Water Kefir liquid, Lactobacillus from a apple juice starter for a future Berliner Weisse, my Heidelberger Kellerbier yeast, #1056 from a starter for an IPA batch, Flanders Red, Kombucha liquid, #3787 Trappist yeast from 2011 and 2010 and I put the last plate outdoors for about six hours to collect some wild yeasts.

The plates were stored at room temperature for approximately three days until colonies appeared. The following pictures were taken after three days of inoculation.

I can already tell, that I did not collect any kind of wild yeast with my last plate, just some beautiful molds…

BFM La Torpille dregs:

I dumped some La Torpille dregs in a blended Belgian style beer (#39 Another Reason to Live) a while ago and poured myself a sample from the fermenter to taste and streak.

BFM bugs on Sabouraud agar

Well, I could observe just one kind of colonies: Off-white color, even, circular, glossy, convex, 2 mm diameter.

I guess it is normal brewers yeast due to the size, morphology and time the colonies needed to grow. If this is a yeast, then it must be #3522 Belgian Ardennes or/and #3787 Trappist High Gravity.

Microscopy picture BFM colony

And the microscopy picture above proves my guess. These colonies are cells of Saccaromyces cerevisiae. Unfortunately not Brettanomyces as hoped… Will use a different media next time to inhibit the growth of brewers yeasts.

3 Fonteinen dregs:

Isolated bugs from dregs of a 3 Fonteinen Oude Gueuze and streaked them as well to see what I got. The wort I used for isolating the bugs smelled very acidic and was kind of sticky. The stickiness might come from the work of Pediococcus.

3 Fonteinen bugs on Sabouraud agar

And I could observe just one kind of colonies again: Off-white, wavy, irregular, not glossy, 5 mm diameter. I was already aware that these might be something different than brewers yeast because they tend to grow in circular colonies and not wavy-ones.

Microscopy picture 3 Fonteinen colony

And the microscopy revealed that these colonies are in fact bacteria. Some sort of Bacillus. My guess is that these are maybe Acetobacter due to the acidic smell of the wort. I have to apologize for the poor quality of the pictures, it is quite tricky to get some pictures with my camera of bacteria due to their very small size. Well, no yeasts or Brettanomyces.

Girardin Gueuze dregs:

Girardin bugs on Sabouraud agar

And at last, some bugs from a Girardin Gueuze. The starter was sticky, like the 3 Fonteinen one as well, but the acidic note was subtle. Once again, just one kind of colonies:

Off-white color, even, circular, glossy, flat, 2 mm diameter. Darker than colonies from BFM dregs.

These colonies were circular again but had a different color than the ones from the BFM dregs (which were Saccaromyces cerevisiae). Maybe Brettanomyces this time?

Microscopy picture Girardin colony

And once again, these colonies are bacteria not yeasts. And they look very similar to the ones from the 3 Fonteinen colonies.

Although I could not detect any colonies of Brettanomyces so far, I learned, that I have to use a different kind of agar to prevent the growth of Saccaromyces and bacteria to give the Brettanomyces a chance. And I already have a different media in mind…

That’s all so far about the bugs from the dregs. Next post about agar plates will be about the water kefir culture and Lactobacillus. And the third one about some common yeasts, the fourth about the #3763 Roeselare Blend from Wyeast and the last one about Kombucha cultures. I can already tell that some of the results were quite unexpected and really interesting and to finish: spoiler alert: I could isolate some Brettanomyces!

About Sabouraud agar plates

Eureka, I guess this might be an interesting post for some of you. I will mention some details about my technique about agar plates. But I will not get into much details, just a brief overview. I learned the techniques in the lab, so the following techniques are very basic microbiological methods.

Label of the Sabouraud bottle

About the media, I normally use Sabouraud agar. This is a media for growing yeasts and some other bacteria. The agar consists of the following components [g L^-1] (when used in a concentration of 65 g of the powder to 1 Liter of water):

10 g Meat- and casein protein, 40 g glucose, 15 g agar, pH around 5.6 +/- 0.2 (according to the label). Produced by Carl Roth GmbH, Karlsruhe, Art. Nr. X932.1, Sabouraud – 4% – Glucose Agar, Ph.Eur. f.d. Mikrobiologie, Agarmedium C.

Why Sabouraud? I first tried to make my own malt agar with dried malt extract and agar but the firmness of the agar turned out to be very inconsistent. And I used Sabouraud before in the lab for culturing yeasts.

Procedure:

Schott bottle with agar ready to sterilize

It begins by adding 26 g of the Sabouraud mixture to 400 mL of tap water. I use a 500 mL Schott bottle and a pressure cooking pot and boil the whole thing for approximately 15 minutes to sterilize the media. I then let it cool down in the pot until the pressure is back to normal, take the hot bottle out and let it cool.

Greiner petri dish 94 mm

When the agar is approximately 50°C (122°F), I pour about 20 mL of the media in a petri dish (I use 94 mm Greiner plastic petri dishes with vents) and let it cool down. 400 mL should be enough for 20 plates but I always have less than that. Thats all about the procedure to get a petri dish with agar ready to plate some bugs.

Agar is ready

I plate with an inoculating loop which I heat up in a gas burner until it is red, let it cool down until the red color vanishes and stick the loop into the agar to cool it down. I then pick up a colony, some liquid or whatever I want to plate and plate it. I normally use a dilution streak technique to have single colonies later on. This makes it easier to determine the morphology of a colony and distinguish between different microorganisms (see picture below).

And I do not have a fancy flow bench. I just use a piece of glass as a base which I can clean with some alcohol. The most important part, in a flow bench as well, is to work fast. Just open the dish for a very short time for streaking and close it again. This technique works for me, the plates with molds and other contaminations are very limited.

Wyeast's #3787 Trappist High Gravity on Sabouraud

After a few days of incubation at room temperature, the plate might look as the on the left. As already mentioned, the advantage of the dilution streak is that you get single colonies. A colony is a single spot. This is easy to observe on the picture on the left. The single colonies are the ones on the upper right corner. A typical yeast colony looks like (in my experience): Circular, white- to off-white color, even, convex, a bit glossy and a diameter of about 2 mm. About the terminology:

• Circular: Defines the form of the colony. Colonies might as well have an irregular shape.
• Even: Defines the margins of the colony. Some colonies might be wavy, filamentous etc.
• Convex: Describes the elevation of the colony. Some colonies are flat, some convex, hemispheric, raised etc.

And thats how I prepare my Sabouraud plates and use them for growing and screening for different yeasts and some bacteria.