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Baums
07/18/07 10:34 AM  
Brett plates
Brett creates a "zone of clearing" on plates that are made with CaCO3 in the media. The acid produced by the brett reacts with CaCO3 around the colony, changing the agar from opaque to clear.

Does anyone know whether using 1% vs 2% CaCO3, or pouring thick versus thin plates, makes this effect more noticable? I am not seeing much evidence of clearing even around the colonies on a CaCO3-laden plate that I streaked directly from a tube of WLP claussenii--but it's only been 10 days of incubation so far.

I am interested in this because I want to be able to confirm that colonies from a stored slurry I have are really brett. Is there another easy way to do this? Put a drop of baking soda solution on a colony and look for fizzing?

Al B
07/18/07 11:38 AM  
Re: Brett plates
Baums,

I don't think 1% vs 2% would have a huge effect since the colonies are on the surface, and much of the undissolved CaCO3 probably settles out. There are other agars more selective, but are expensive. Then again, perhaps more sugar in the agar you're using is needed to create more acid.

Its possible to differentiate Bretts from Sacchromyces under the microscope based on cell shape, although both exhibit multi-planor budding.

I think the easiest thing to do is inoculate a starter, oxygenate, and taste over a few weeks. Observe for a pellicle. Take a pH reading.

If still in doubt, I could take a wack at it.......

Al

Baums
07/20/07 10:17 AM  
Re: Brett plates
You are probably right--just building up a starter should be enough for me to tell. I just wish there was a more immediate way to ID a colony without selective media or a good microscope.

By the way, what kind of microscope settings are good for looking at bretts? I have a cheap microscope laying around that might be good enough.

There are pictures of the "zone of clearing" on liddil's website, I think, and I actually did see them on a plate of WY b. lambicus--but not until it had incubated for quite a while. I took a colony off that plate and I noticed that the whole area under the colony (the entire depth of the agar) had become clear.

My claussenii plate also seems to have what look like popped or exploded bubbles at a few spots. I don't think these are contaminants--they look more like physical disruptions to the colonies. Has anyone seen something like that before?

Al B
07/20/07 10:31 AM  
Re: Brett plates
100x is best w/ the microscope.

<< don't think these are contaminants--they look more like physical disruptions to the colonies>>

I haven't seen that, but suppose that was from CO2 expelled.

One thing - what agar are you using?

With a medium with extra dextrose, I notice more distinct morphology traits distinguishing different species more clearly - which can help identify "other" yeasts.

Baums
07/20/07 12:52 PM  
Re: Brett plates
I am using 3% DME, 1.5% agar, and either 1 or 2% CaCO3. Maybe I'll replace some DME with dextrose.
Al B
07/20/07 01:07 PM  
Re: Brett plates
You can keep it at 3% DME and add an additional 3% dextrose.

Baums
08/01/07 10:30 AM  
Re: Brett plates
So I have been messing around with claussenii, and although I can get colonies to grow on agar with no trouble, when I transfer a few colonies to 10 mL of room-temp 1.040 wort (no CaCO3), I see no signs of growth in 3 days (unlike with WY b. lambicus). I will wait longer, but in this scenario (first propagation into wort) longer waits make me start to worry a little about infection.

Does anyone have a proven procedure for getting BC from agar colonies into exponential growth in liquid media?

Al B
08/04/07 08:31 PM  
Re: Brett plates
The yeast was probably in a lag phase (as opposed to log phase) - should get going in a few more days.

How's it doing now?

Baums
08/06/07 11:19 AM  
Re: Brett plates
Still no sign of life... very strange. As I said before the lambicus grows really fast for me when propagated from plate to liquid.

The claussenii plate I streaked from was a few weeks old and stored at room temp. Could the brett have lost viability that quickly?

I guess the next idea is to streak another plate and then try to catch the colonies while they are still growing fast on the plate. Any other thoughts?

Al B
08/07/07 05:35 PM  
Re: Brett plates
These things happen sometimes. Colonies of bugs (yeast or bacteria) can lose viability - or population diminished so much that its a matter of luck to get a few survivors.

Plates with agar will lose water faster than test tubes for example. Room temp vs. refrige. temps. - many other variables can contribute.

Have a back up plate in the refrige for these occurrances - same thing happened to me with the PAcman yeast stored in old wort at refrige. - piffed

 
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