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Brettanomyces Brewing
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01/28/09 07:18 AM  
Making slants
Hey Al (or anyone with the know how)

when making slants do you normally use agar mixed with wort, or can/should I use YPD and peptone or some other more standard lab mix?

If you have a standard recipe that you'd post I'd be much appreciative.

I'm wondering how best to keep strains in longer term storage and I've read that media that is not enriched for maltose may effect fermentation properties down the road.



Al B
01/28/09 09:52 AM  
Re: Making slants

This is the way to go:

YM Media 500mL

Malt extract 1.5 g

Dextrose 5 g

Yeast nutrient 2.5 g

Agar 7.5 g

A small amount of DME covers any maltose defiency issues. You may substitute all DME. I transfer cultures once/yr with no issues in viability or fermentability.

01/28/09 10:10 AM  
Re: Making slants
Thanks Al

so 500 ml is the total media volume? No additional water?

also, for yeast nutrient would you use something like fermax or is peptone sufficient?



Al B
01/28/09 10:20 AM  
Re: Making slants

500 ml of water - if you have an autoclave ....and I think you do.....boil the ingrediates to dissolve the agar in 500ml water, then autoclave.

I use WY nutrients, but Fermax will do. Peptone, while excellent as a source of amino acids, lacks enough vitamins. Its a good idea to have a vitamin-based medium for storage as well as for propagation.

01/28/09 10:26 AM  
Re: Making slants

Coolio. Yeah I have an autoclave. I got a little confused here. You mean this as a recipe for

500ml of YM?

or do you make up the YM, then add the other components and another 500ml of water.

I'm thinking the former but just want to be sure.

thanks again.


Al B
01/28/09 10:34 AM  
Re: Making slants
Its a recipe (modified YM agar - due to the additon of some DME)- 500mL total. Its the former, you don't add more water (there won't be THAT much evaporation!) Sorry for the confusion, I had just cut & pasted that for convenience.

PS - after inoculating the slants, don't forget to keep the caps loose for O2. After sufficient growth on the slant then cap tighly for refrigeration (colder the better)......good luck!

01/28/09 11:22 AM  
Re: Making slants
Colder the better...

I have a -20 and a -80 but I'm thinking those are both too cold without glycerin.

4 deg. C?

Al B
01/28/09 11:30 AM  
Re: Making slants
Egads. I meant refrigerated temps (4C is fine).

01/28/09 12:33 PM  
Re: Making slants

01/29/09 11:33 AM  
Re: Making slants
While basically Al has laid it all out simply and correct I use to look at this site also


good info


01/30/09 11:15 AM  
Re: Making slants
Thanks Chad and Al

Now I have a friend at work telling me I should just grow up a saturated colony in a small volume of wort and then add 15-20% glycerol, flash freeze in liquid nitrogen and store in my -80.

Is this the better plan?


Al B
01/30/09 11:56 AM  
Re: Making slants
That's for really long storage. You can do that as a back-up. It's really up to you and what you find easiest.
02/01/09 12:17 PM  
Re: Making slants
Hey Al

What about when culturing up brett.. is the inclusion of CaCO3 vital? Does the acid production need to be neutralized or do you find its fine without?


Al B
02/02/09 07:36 AM  
Re: Making slants

Despite literature & ATCC media recommendations for the inclusion of CO3 in the media, I have found that Brett isolated from beer do not require buffering for culturing. It may be optimum to add it, but it isn't necessary.

02/02/09 10:33 AM  
Re: Making slants
Ditto--not that it's recommended, but I've revived colonies from 9 month old unbuffered petri dishes without any trouble.
02/02/09 11:41 AM  
Re: Making slants
I went to make up petri dishes and slants in the lab today and decided to hold off as I didn't have "yeast nutrient".

What is ideal for Brettanomyces?

Liddil from the biohazard lambic brewers site says wort agar with CaCO3 made to 1.040..

You listed your reagents above... Will using spray malt make up for the yeast nutrient and maltose? What exactly is Yeast nutrient. I have all the other reagents including Yeast Extract, Malt Extract, Glucose, CaCO3 if I want, agar.. what should I do about the "Yeast Nutrient" as we don't have that. Will Servomyces (from WLP) work the same or will peptone suffice? or am I putting way too much consideration into this?

02/02/09 11:45 AM  
Re: Making slants
...and what about autoclaving the yeast nutrient? Are any of the vitamins and amino acids vulnerable to denaturing due to the temps?
Al B
02/02/09 12:04 PM  
Re: Making slants
Spray malt or Dried malt extract (DME) will suffice. It usually supplies most of the nutrients. However, for propagation (growth), a considerable amount of amino acids, vitamins, etc. are optimum for building new cells.

Yeast nutrients are a generic term here in US. Depending on the brand, it is basically Yeast extract, peptone, essential minerals such as zinc, etc. The yeast extract is an excellent source of vitamins and peptone for amino acids. For the above recipe, I would use about 1g peptone and 1.5g yeast extract in place of the nutrient. Servomyces will do the job, but it is more expensive - save that for high gravity beers.

Provided that you don't overheat the media, vitamins and amino acids will be bioavailable (Autoclave 15 minutes/liter). Obviously stuff like enzymes denature much more quickly.

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