Slanting yeast

I was recently talking to a Pro-Brewer about slanting and he told me that I wanted to make "stabs" not "slants". He stated that slants were only for something that you were going to use right away, and that you should make "stabs" for anything that you wanted to store over time. He said that I should be stabbing my loop down into the agar 3-4 time for each "stab". I notice that this is the technique that Sac suggests in the original posts, but a lot of the photos I have seen in the thread seem to just be growing on the surface.

stillbrewin said:

I was recently talking to a Pro-Brewer about slanting and he told me that I wanted to make "stabs" not "slants". He stated that slants were only for something that you were going to use right away, and that you should make "stabs" for anything that you wanted to store over time. He said that I should be stabbing my loop down into the agar 3-4 time for each "stab". I notice that this is the technique that Sac suggests in the original posts, but a lot of the photos I have seen in the thread seem to just be growing on the surface.

Click to expand...

correct me if I'm wrong. Slant refers to the agar being in an angle (slanted) giving more surface area to work with. that doesn't prevent you from stabbing into the media which is what you want to do when inoculating with a yeast strain.

You should slant the tube to give the agar more exposed surface area AND stab the loop into the agar in 3 or 4 different places. So you want to do both. The process works GREAT - One smackpack of yeast and I have 40 vials of usable product!

slanting like a mad man and saving money, thanks sacch

I have used your tutorial and it is excellent! I have a question - when making a starter from a slant you prescribe the following:

"With the sanitized small funnel, add a bit of wort to the vial. Re-cap, shake, and dump into a sanitized 250mL flask. Repeat to get all the yeasties out."

I did this and it worked fine but why can't you just use a thin blade of some sort (sterilized, of course) and just scoop the yeast off the top and flick it into the flask? It shouldn't matter if you get a bit of the agar jell with it, should it? Seems like this would be a lot less futzing about.

Just wonderin'...

I guess you could. I add some wort into the vial 3 or so times and it works well enough. Sometimes half the agar goes into the starter but it doesn't hurt (and is something to watch to ensure you have circulation with H Strain Yeast (Pilsner Urquell - it doesn't flocculate much to show you the stir bar is behaving)...

gkeusch said:

I have used your tutorial and it is excellent! I have a question - when making a starter from a slant you prescribe the following:

"With the sanitized small funnel, add a bit of wort to the vial. Re-cap, shake, and dump into a sanitized 250mL flask. Repeat to get all the yeasties out."

I did this and it worked fine but why can't you just use a thin blade of some sort (sterilized, of course) and just scoop the yeast off the top and flick it into the flask? It shouldn't matter if you get a bit of the agar jell with it, should it? Seems like this would be a lot less futzing about.

Just wonderin'...

Click to expand...

an inoculation loop is the best tool when selecting indiviual colonies, but with a inocculated slant what sacch reccomends is the most efficient. less time exposed less time to mess something up, not sure what "futzing" is but the method you have describe seem way more "futz"

Damned agar won't solidify.

I made two batches of media for slants this weekend. I boil for a minute or two and stir well to make sure everything is melted and in solution. I've been boiling in my microwave, so maybe that's the problem. Anyway, after that it all goes into the vials and then the pressure cooker for 15 minutes at 15psi.

• 200ml water
• 10g DME
• 4g agar agar.
• 2 drops red food coloring

Did it twice since it didn't solidify the first time. I even put them in the fridge after a day, slants and plates. Still liquid. I bought the agar agar a local oriental market, looked new and normal in a box and pouch. I'm going to double the amount and try again tonight. Geez, what a disappointment. Labor day weekend failure.

This is the agar agar powder I'm using. Anyone know why this would be? I guess I'll just double the amount tonight.

[edit] DON'T USE THIS STUFF. TOO MUCH SUGAR, NOT ENOUGH AGAR.

I'll bet doubling does it. I successful with 4-5% agar, by weight. That seems to be my "balance point.

ETA: you can probably just add the agar, stir, and reprocess without having to mix up a whole new batch.

UPDATE: Ok, I tried 15 grams in 200ml water and it solidified. I also took the time to look at the ingredients on the box and it lists ingredients as: Sugar, agar agar. So, it's mostly sugar. Tastes like it, too. I guess I'll look for some pure stuff, but I'm going forward with this for now. Not ideal.

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

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).

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)

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.

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...

edecambra said:

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.

Click to expand...

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?

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.

JUKES

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

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

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.

I brought my mixture to a slight boil for a few minutes. I have had no problems with the mixture.

Being cheap and not wanting to buy new yeast every time plus the fact that I'm hands on and like doing extra stuff for my beer I'm onboard for this. I don't like washing yeast and I've failed at freezing the stuff probably because my freezer doesn't maintain temps so I think this is it for me. Got the pressure cooker cooling now as I've just read through about every post on here. Wish I had done this before because I would have added some food coloring and made sure my caps were autoclavable. Guess we'll find out soon enough.

Well the caps survived 15PSI so I guess they are good! I used four grams of agar agar I found at the chinese grocery store; kind of long noodle like stuff. Can't read anything on it because it's all in another language but it did say 100% agar agar and something about it being vegetarian.

In the original post it says store in coldest part of the refrigerator, is that the freezer or actual refrigerator

Dukeman9988 said:

In the original post it says store in coldest part of the refrigerator, is that the freezer or actual refrigerator

Click to expand...

Refrigerator. Typically the bottom where you have slightly colder temps. You can't freeze yeast unless you use glycerin.

After buying 4 dead packs of Wyeast from the LHBS this is now on my Christmas list. Thanks for the great write up and the pictures.

I'm still trying to wrap my head around how to calculate starter sizes when growing up from slant. When starting from the slant, the culture will have X million or billion cells. But without a way of knowing what X is how do we calculate growth during the process?

If I start with a slant of WLP001 and go culture -> 20ml sterile wort (intermittent shaking for 24hr) -> 200ml sterile wort on stir plate -> 1L starter on plate -> ???

How can I estimate cell count so I know how far I need to step up for the given style (ale/lager) and gravity of the wort short of actual cell counting with a microscope and hemocytometer?

Jukas said:

I'm still trying to wrap my head around how to calculate starter sizes when growing up from slant. When starting from the slant, the culture will have X million or billion cells. But without a way of knowing what X is how do we calculate growth during the process?

If I start with a slant of WLP001 and go culture -> 20ml sterile wort (intermittent shaking for 24hr) -> 200ml sterile wort on stir plate -> 1L starter on plate -> ???

How can I estimate cell count so I know how far I need to step up for the given style (ale/lager) and gravity of the wort short of actual cell counting with a microscope and hemocytometer?

Click to expand...

Use yeastcalc.com and once you get to step three, reset the top value to show the cells you ended up with in step 3.

You can also estimate your cell count based on a 1-4.5 scale where the number represents the billions of cells per mL. A 1 would be a thin slurry, like cloudy wort, and a 4.5 would be a very thick, sticky compact yeast.

gkeusch said:

Thanks for the explanation - I'm sure I would have learned that the hard way.
While I have your attention, do I need to use an erlenmeyer type flask on a stir plate, or will a standard beaker (or even a canning jar) work OK?

Click to expand...

I'm sure you already got this answered.. But maybe for future subscribers... Anything with a flat bottom will work. Beakers or mason jars can do starters, but I do. Prefer an Erlenmeyer flask.

Got my pressure cooker for Christmas, and just placed my Cynmar order.. WOOHOO!!

Roadliner said:

I'm sure you already got this answered.. But maybe for future subscribers... Anything with a flat bottom will work. Beakers or mason jars can do starters, but I do. Prefer an Erlenmeyer flask.

Click to expand...

1 gallon flat bottomed jugs would work great and they are cheaper than 4000ml erlenmeyer's. Finding them is a bit of a pain. I may have to buy a few from labelpeelers (4.47 each) and test them as the bottom seems fairly flat on them in the pictures.

1 gallon jugs of Carlo rosse wine also work, they are 11 or 12 bucks but you do get some booze at least.

Thanks for the write-up! Was emailing links to my wife for months before Christmas to make sure she got me everything! Made my slants, couldn't get petri dishes, but found that wide-bottom canning jars work well too.
So here's my question...
I want to culture a bottle of Anchorage Brewing Co White IPA. It's brewed with ale yeast, then aged in wood with Brett. The beer has a strong Brett character, and I want to get the Brett out. What can I do to get the Brett, or am I going to have to culture this up as a mixed Sacro/Brett starter.

Thanks again for this awesome write up! I slanted some American Ale and British Ale II Wyeast a few months ago and just broke out the first slant and tossed it into a cup of my canned yeast starter (which by the way is also awesome to have around). It's been sitting on there for almost 36 hours and looks nice and milky so my question which may have been already answered but I'm struggling to find it scrolling through these pages:

After that initial step should the yeast hypothetically be somewhere near the 100 billion cells that come in a normal package? And then you just step up from there to the appropriate amount? That's kind of what I was getting but I'm not sure if you came out and said it. Sorry if I'm missing something.

Excellent information here. Any thoughts on using smaller vials? I already have some tall skinny 5ml vials, and it seems they should work the same way and would just maintain a smaller sample. Would the yeast have enough food for storage or would they not last as long?

zazbnf said:

Excellent information here. Any thoughts on using smaller vials? I already have some tall skinny 5ml vials, and it seems they should work the same way and would just maintain a smaller sample. Would the yeast have enough food for storage or would they not last as long?

Click to expand...

If the slurry is pourable into those vials you'd be somewhere between 1-2 billion cells per ml. So at best I think you'd have 10 billion cells, but more than likely 5-7. You could just do more of them, add like 10 of them to your starter I guess.

I just did some testing using water on my 5ml vials and to get a slant that doesn't cover the bottom so they won't push up, I can use 1 ml of the agar solution in each tube. I just need to buy some agar now to give them a shot.

My plan would be to build up a starter from 1 vial so I may need to add a smaller step to account for my smaller starting volume.

Has anyone tried and succeeded in slanting Brett? I see no reason it won't work but wanted to ask.

Has anyone tried the "water immersion" technique from the "yeast" book? Page 198.

peter78 said:

Has anyone tried the "water immersion" technique from the "yeast" book? Page 198.

Click to expand...

I haven't. I'm interested in doing it. Was a centrifuge needed to do this?