hunterlab

Recipes 

I just made my slants tonight there setting right now. I used agar agar flakes from whole foods and seems to be working well. I have a question after I pulled the tubes from the cooker there was a powder looking matter in the bottom of the tube. I made sure I mixed and melted the flakes and dme all together. I'm sure it won't matter just wonder why it separated?

Dan

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passedpawn

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OK, don't use that stuff in the picture above. It's total crap and never worked out. I found 100% pure agar agar powder at a whole foods place and it worked great (too great, I need to cut back on how much I use).

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Jukas

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Since i niw have a pressure cooker I finally put together an order from cynmar to get the ball rolling in slanting yeast. I current use washed yeast and storing 20+ jars of washed yeast even in baby food jars is a pain. I had three questions I didn't see answered in this thread I'm hoping someone can chime in on....

1) when going to build up a starter from a slant how/when do you figure out initial cell count approximations to use with a pitching calculator? Currently I can take the ml of yeast I have washed and get a viability % by age to get a baseline of how many cells I'm starting with. Then I can figure out if I need two or three stepped starters (500ml, 1L, 2L). How do you do do with a slant? I currently have 250ml, 500ml, 1L and 2L flasks.

2). I see reference to plating prior to slanting, or slant to plate to isolate good colonies, but I'm unsure how you would go about differentiating good colonies from mutated colonies or worse, wild yeast/undesirable bacteria?

3) I have a couple yeasts washed that are either a) "1st gen" bottle harvest (where I bottle harvested and split the yeast, pitching some and saving othe rest) or b) made up a starter from a smack pack and then couldn't brew so I split up the yeast and saved it for later. Is it possible to safely revive these washed yeasts to slant? Some I can easily replace, but several are bottle harvested that will be hard for me to get a fresh source of (I.e Bells)

edecambra

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If you wanted, you could get some plates, and make them up with your slant tubes. Streak out a culture on a plate and use that to isolate some colonies. If you don't have White's Yeast book, you should get it there is some great info on culturing.

commander flatus

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Great thread guys. Here's what I did - a little different...

Pressure canned jars with 40 cc of 30% glycerol solution.
Make a starter on the stir plate.
When that's done, sterile transfer 40 cc of concentrated slurry to make 80 cc of 15% glycerol/yeast solution.
Suck that up in a 60cc syringe (sterile), attach a needle and put 10-12 cc each in sterile red-top phlebotomy tubes.
All that medical junk can be found on fleabay. I got expired supplies from work (I'm a doctah).

Picture attached...

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Jukas

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I'm guessing this is an answer to my question #2 & 3? I've ordered the yeast book by Jamil Z & Chris White and should have it in a couple days so I'll read up on culturing & plating.

How do you estimate cell count to figure what size starters you'll need? I know I won't get an accurate count short of doing an actual cell count with a microscope but an estimate would be groovy. Is there a saturation point (billion cells/ml wort) that can be used to figure out an approximate cell count off the inital starter done on a stir plate?

Jukas

Recipes 

So I made my first slant media last night. 400ml water to 35g of DME to 2.5g Agar taken from Sacc's second post. I heated the mixture in a pan for 10 minutes or so to make sure all the agar was disolved and mixed in, but did not boil it. When pulling vials out of the pressure cooker, I noticed mine seems to have a ton of hot break material that I didn't see in anyone elses slant. It's kind of hard to photograph but you can see it in the upper part of the slant below.



I assume this isn't a problem?

Next time I plan to either have some sterile baby food jars to store leftover agar/wort mixture in or cut the amount in half as adding about 8ml to each vial left me discarding almost half the mixture.

hunterlab

Recipes 

JUKES

I noticed the same thing. It hasn't affected my yeast that I slanted so should be good to go.

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Jukas

Recipes 

Looks like I'm going to have to redo all the agar medium. It solidified just fine, but has started to reliquify. Seems like 2.5g of Agar may not have been enough

Jukas

Recipes 

To add to the above post. I redid my slant medium last night with 400ml water, 35g DME and 6g of Agar. I think I'm having problems getting the agar fully disolved before it goes into the vials. It looks like it's disolved, but half the slants never set properly and when I examined one of the ones that did the medium was so firm it would have been impossible to stab with an inoculation loop.

Next time I think I'll try the same amount of agar, but bringing the mixture to at least 190F on the stove and making sure it's completely mixed before adding to the vials.