# Culturing Yeast And Using Slants



## Hopsta (8/3/06)

I met up with fellow AHB member Airgead on the weekend, he lives local so we swapped a few yeasts. He told me about using slants to culture and store yeasts, this is new to me so i decided to do a bit of research and post some info on the subject for others that are interested.

I just have one question, one of the instructions is to flame the inoculation loop from the handle to the tip. Stick the tip into the Wyeast packet and swirl it in the liquid and smear the loop over the surface of the growth medium. Now im guessing that i am to flame the inoculation loop very quickly so as not to heat it up, because if it heats up wont it kill any yeast cells that it touches?

If anyone has any simplified methods or shortcuts in doing this let us know..... 

Anyway here is the info i found.........

- Hopsta

*Culturing Yeast and Using Slants - Part 1*
by Bill Bunning

This will be a three-part "Beer Geek Techneeks" on culturing yeast. Part 1 will consist of equipment and supplies needed as well as preparing your blank slants. Part 2 will deal with slanting yeast and preparing starters. Part 3 will deal with obtaining a pure culture from a suspect yeast source. 

Why culture yeasts? What you gain is indefinite (practically speaking) storage, assured maintenance of the original generation, and ease of sharing yeast with brewpals. Also, when it comes time to make up a starter to pitch into a batch of beer, you get 500 ml of active starter within 4 days every time. If you are doing slants, when someone sends you a sample of yeast, or you get one from some other means, you can make yourself a renewable lifetime supply from that sample. Finally, using a new package of yeast every time you brew is expensive. After your initial set-up costs, yeast cost per brew will plummet when you propagate your yeast this way. 

Minimum Equipment: 

1. A bunch of glass vials or test tubes which have caps that can: (a) withstand pressure cooker temperatures or boiling water temperatures; and ( B ) form a tight seal. 

2. An inoculation loop to transfer yeast. 

3. Some type of flaming mechanism for sterilization (an alcohol lamp or a butane pencil torch). 

4. Something to use for your starter vessel, like an old-style milk bottle, an Erlenmeyer flask (that's what I use), or other glass vessel that has a mouth to which you can affix a rubber-stopper and airlock. 

5. The other hardware you will already have if you brew beer: a scale, a big pot to boil in, kitchen stove, refrigerator, and spoons. Additionally, a pressure cooker is ideal for sterilizing solutions (that's what I use). 

Minimum Materials: 

1. Either agar or gelatin, to use as a growth medium. I use agar and it works great. 

2. A bag of dried malt extract. One bag will last you the rest of your life as far as keeping a full supply of yeast slants on hand is concerned. 

3. An additional item which is a terrific time saver is mini-yeast starters. These are 10 ml of sterile wort in 50 ml vials. Brewers Resource sells them, and they're really a must if you culture yeast. 

Preparing "Blank" Slants 

Bring 1 cup (about 250 ml) water to a boil. Remove from heat, add 2 tablespoons of dried malt extract, and stir till dissolved. Put back on the heat and boil for 5-10 minutes to ensure sterility. Remove from heat. Add 1 1/2 tablespoons agar (or a package of gelatin) into this "wort" and stir till completely dissolved. Pour this mixture into as many of your vials/test-tubes as you can; a small funnel is useful for this step. Fill the vials about 1/2 full - do not fill them all the way up. Screw the caps on. Now either boil the tubes for 30 minutes or place in a pressure cooker and "cook" for 15 minutes at 15 PSI. The pressure cooker will do a more complete job of sterilization. 

Remove from the heat, and have a couple homebrews while it cools off. When the vials have cooled enough to handle (still hot however), loosen the caps a bit and place them at an angle of about 40-45 degrees. When you do this, the surface of the still-liquid-but-cooling agar and malt extract will of course stay horizontal. Let the vials sit like this for 12 hours, after which time the agar and malt will be somewhat solid (still a bit soft and yielding--ideal for this purpose). After cooling, the surface of the medium is at an angle giving a greater surface area, hence the name "slant". These are now ready to be "inoculated" with cells of your favorite yeast. Store these in the refrigerator until you're ready to use them. 

Return to the Beer Geek Techneeks index 


--------------------------------------------------------------------------------

*Culturing Yeast and Using Slants - Part 2
by Bill Bunning*

This is part 2 of the three-part "Beer Geek Techneeks" on culturing yeast. We'll learn how to slant yeast and prepare starters. Part 1 dealt with equipment and supplies needed for yeast culturing as well as preparation of blank slants. Part 3 will deal with obtaining a pure culture from a suspect yeast source. 

Inoculating Blank Slants With Yeast 

So now you have a bunch of slants. At this point, the procedure depends on what the source is for your yeast to be cultured. You may be starting from another slant or a packet of Wyeast or Yeast Lab starter. I'll describe doing it from a packet of Wyeast, and then comment on variations used for other sources. 

The assumption here is that you are culturing from a pure source, like a Wyeast packet or a slant that someone sends you. If you are culturing, say, from a bottle of commercial bottle-conditioned beer, extra steps are required to isolate pure cultures (bottling strains are rarely pure). This is much more involved, and will be described in Part 3. A couple days prior to slanting the yeast, smack the Wyeast packet just as if you were going to prepare a starter. You can use the packet for a starter when your finished making your slant. When the packet is swelled, you should lay out your working area in an organized way to minimize having to get up and down, reach long distances for things, etc. Wash your hands thoroughly and begin. Have your slants, inoculation loop, flaming mechanism, and yeast packet ready to go. 

Shake the Wyeast pack well, and then open it using standard procedures. Flame the inoculation loop from the handle to the tip. Stick the tip into the Wyeast packet and swirl it in the liquid. Remove it from the packet and place it near your flame source, but not in it. The air is sterile within several inches of the flame. Open your slant, flame the opening, and smear the loop over the surface of the growth medium. Flame the opening and replace the cap. That's it. Use the remainder of the packet and make up a starter. Make sure you label the slant. 

When done, leave the slant out at room temperature for a week. Within a couple of days you will see a cloudy film on the slant surface, and a few days later it will develop into a milky white layer about a mm thick. You'll need to "burp" the slant every other day due to CO2 evolving from the yeast growing on the slant surface. No big deal - just bleed the gas out by cracking open the cap for a moment. After the week is over, store the slant in the fridge, where it will keep for at least 6 months in a perfectly viable condition. I've used them over 1 year old. 

From Slant To Slant 

When 6 months is nearly over, I reculture the strain by doing the above to another slant, but using an "old" slant as the source instead of a Wyeast packet. Otherwise the procedure is identical. Reculturing in this way does not increment the strain generation-number because the yeast have not made enough copies of themselves for mutation to occur. You can also culture yeast from a friend's slant this way. 

Making A Starter From A Slant 

Before starting, be sure to let your slant sit out for about an hour so it can slowly come up to room temp from fridge. The yeast will be stepped up in three steps: from slant to 10 ml, from 10 ml to 50-75 ml, and from 50 -75 ml to 500 ml. When making a lager I step up to 1000 ml (and it takes an extra day). Here's where the mini-yeast starters come in handy. They're 10 ml of sterile wort in a vial. If you don't have these, you can make mini-starters in test tubes ahead of time. Again, you'll need the inoculation loop, flaming mechanism, yeast slant, and starter. Flame the loop from handle to tip. Open the slant and flame the opening. Scrape some yeast on to the loop (try cooling the loop on some exposed growth medium first). Plunge the loop into your mini-starter and shake off the yeast. Remove the loop and replace the top. 

It will take about 2 days for this yeast to propagate. This is stepped up to 50 -75 ml and then to 500 ml. It only takes about 1 day for each of these steps. I use about 1 tbsp malt extract for the smaller volume and 4 tbsp for the larger volume when preparing my starters. After 4 days, you're ready to pitch. 

Return to the Beer Geek Techneeks index 


--------------------------------------------------------------------------------

*Culturing Yeast and Using Slants - Part 3
by Bill Bunning*

This is the final part of the three-part "Beer Geek Techneeks" on culturing yeast. We'll deal with obtaining a pure culture from a suspect yeast source. Part 1 dealt with equipment and supplies needed for yeast culturing as well as preparation of blank slants and part 2 dealt with how to slant yeast and prepare starters. 

Preparing Plates 

The first step is to prepare your petri dishes. You can either purchase plastic plates that you pour yourself or buy pre-poured plates. If you pour your own, prepare them when you make your normal slants; double your medium recipe and place the excess in a mason jar. Boil or pressure cook as described in Part 1. When it's cool enough to handle, pour the liquid into the plates (just a thin layer) quickly a leave to set. Store these wrapped (I use a zip-lock bag) in the fridge until ready to use. 

Slanting Suspect Yeast Sources 

If culturing from a bottled-beer source, make a small-volume starter and wait until it is fully active. WARNING! Many types of bottle-conditioned beer use either a different kind of yeast in the bottle than they fermented the beer with, or it is the same kind but has mutated, or any of several other possible complications. If you culture from a bottle of commercial beer, *taste* the small starter you make from it when inoculating your slants. If it tastes good (or at least, not bad) you are probably OK. But you should still test the yeast on a small, pilot batch of beer before committing your entire batch to it. Even when using a bottle of your own homebrew, things can happen, so I would recommend these cautions in that case as well. 

The steps here are similar to those used in slanting yeast in Part 2. You'll need the prepared plate, the inoculation loop, flaming mechanism, and the small starter you prepared above. Flame the loop from handle to tip. Place in the starter and swirl. Quickly remove the lid from the plate and streak the loop back and forth over one third of the plate. Flame the loop again and streak one third of the plate again overlapping the previous streak. Repeat this procedure one more time streaking the final third of the plate. Replace the lid and store at room temperature. This whole procedure should isolate individual cells of yeast by the third streak. You'll see individual colonies growing in the final area streaked several days later. These colonies are slanted the same as if you were going from slant to slant in Part 2. All the procedures remain the same. 

Maintaining Your Yeast Ranch 

In no time at all you'll have several different yeasts on slants; I've got over 20! (Editor's note: Bill's refrigerator must not have any beer, or food, in it). Make sure you label each slant with type and date. You'll want to keep track of dates so you'll know when to re-slant a given yeast. Good luck and happy ranching!

edit rogue smiley.


----------



## Steve Lacey (8/3/06)

> Now im guessing that i am to flame the inoculation loop very quickly so as not to heat it up, because if it heats up wont it kill any yeast cells that it touches?



Nah, you heat it up until it is red hot and glowing like a poker. At least the end part. You might not go that extreme near the handle. Remember, it quickly cools down in the air and then cools instantly when it contacts the liquid. You might kill a few million cells in that instant of contact, but there are a few billion left to carry on the good fight and stick to teh now cool loop.


----------



## Hopsta (8/3/06)

Cool thanks Steve, does anyone know of a good lab supply place i can get test tubes, test tube rack innoculation loop etc... from? 

I'll have a look on ebay.


----------



## Joel (8/3/06)

To anyone that does this, 

Can you possible post some pictures of the equipment described? I'm a very visual kind of guy and I need to see to believe...  

Also, I think some AHB guys did a bulk buy of test tubes last year. Perhaps one of them could let us know where to get the equipment from.

I can just see it now... (rollin', rollin', rollin'...) a yeast rancher with red hot innoculation loop in hand... (keep them wagons rollin')... endless herds of yeast spread out across an agar plain... (raw hiiiiiide!).


----------



## pint of lager (8/3/06)

OK, a few pointers and suggestions.

Please when posting stuff cut and pasted, include the link that it came from. Rtaher than posting whole articles, please post an excerpt or paragraph, and point people where to go for the rest of the article.

Have a browse through Sosman's brewiki source, I forget the link, but he includes it in his byline in every post. He has documented and included pictures of his voyage through yeast work. He also has jncluded many links to excellent yeast farming work.

In Australia we farm, in America, they ranch.

Have a read through the airlocked yeast thread. I am sure slants are covered there. As well as Scientific suppliers details.

When pressure cooking, never put a sealed container into it. Asking for blowups. Put the lid on, but make sure it is loose so that pressure can equalise. You also want glass testubes, or plastic that will handle 125 deg C.

There are no shortcuts or simplified methods.

The smaller the sample of yeast, the more chance of stuffups and infections. You want all transfers to be as aseptic as you can manage. If you do lots of reading and thinking about the process, there is less chance of problems, otherwise, every brew you do will be infected, and your money saving efforts will cost you heaps.

A pressurecooker is a yeast farmer's best friend.

Use powdered agar from either Asian foodstores or some healthfood stores.

Read on Craftbrewer.org, Graham Sander's article on sterile water storage. He includes an excellent description of using home equipment to achieve great results.


----------



## Airgead (9/3/06)

Hopsta said:


> Cool thanks Steve, does anyone know of a good lab supply place i can get test tubes, test tube rack innoculation loop etc... from?
> 
> I'll have a look on ebay.
> [post="113414"][/post]​



Hopsta

You are welcome to pop over any time and take a look at the process. I may well be making slants this weekend from the yeasts I got from you. They have been growing on plates since the weekend so I can isolate a pure colony. You will be pleased to know that I can't find any bacterial or fungal contamination in your samples.

I bought an innoculating loop from ebay (actually I bought 2 in case one broke). They are only about US$5 but the postage is a killer. Having seen one it would be easy to make out of some fine ss or nicrome wire.

I flame till red hot for about an inch. The loop cools on contact with the liquid so I just swirl it a bit to make sure I have moved it away from all the yeast cells that were toasted when the hot loop made contact. If I am picking up from a plate I cool the loop by pressing it into a blank area of the agar.

My tubes came from a bulk buy organised here. I can't remmber who we got them from but I seem to reemmber that we needed to buy 2000 or so. Livingstone has stuff that will work and they do sell in small lots (www.livingstone.com.au). I got my test tube racks and stuff from them. I'd give them a call and see what they have. You need something that is autoclavable. I use an old pressue cooker (ebay) as an autoclave. Works great.

As someone else said - there are no shortcuts. All it takes is one bacteria to land and your slant is stuffed as they will out-compete the yeast. Having said that its really not that hard if you are careful. If a klutz like me can manage it anyone can.

For those that want pictures, check Sosman's brewiki (brewiki.org). If there is anything specific you want to see, let me know and I'll try to take pictures while I'm slanting this weekend (warning - for digital shots I only have a crappy camera phone so the quality may not be great).

Cheers
Dave


----------



## Hopsta (9/3/06)

Cheers Airgead i'll check out the livingstone site.


----------



## Andyd (9/3/06)

I've bought lab gear for culturing from both Livingstone and ProSciTech (link below). Both were seriously good to deal with - my gear arrived at my door the next day!

http://www.proscitech.com.au/catalogue/home.asp

Andy


----------



## samhighley (4/4/09)

Hopsta said:


> Bring 1 cup (about 250 ml) water to a boil. Remove from heat, add 2 tablespoons of dried malt extract, and stir till dissolved. Put back on the heat and boil for 5-10 minutes to ensure sterility. Remove from heat. Add 1 1/2 tablespoons agar (or a package of gelatin) into this "wort" and stir till completely dissolved.



1-1/2 tablespoons of agar seems like *way* too much agar for this amount of water. The instructions on the powdered agar packet I picked up at the asian grocers recommends 25g per 3 litres of water, which equates to 1 gram per 120ml.

1-1/2 tablespoons of agar powder is about 10 grams, so I think it's a factor of 10 too much agar powder.

I want a nice solid growth medium that is stable at room temperatures, so I might try 2 grams per 120ml.


----------



## pdilley (6/4/09)

That Dave, spreading the joy of home lab work 

Reminds me, maybe I should have kept my Bacticinerator and brought it over and run it off the transformer, great for working with loops and no open flame hazard issues.







I also use X-acto style knifes for working with petri plates and slants. Especially when it comes time to slicing grown out growth medium into transfer cubes. So if you have a hard time sourcing a loop, hobby knives have been used successfully -- provided they are not those el cheapo Chinese ones sold at Bunnings that are plastic handles that will melt.

When working with plants from test tubes there was a gel growth medium that let you grow out in 3D, but I never used it much and don't recall the name anymore. For mycological work I grew out in 2D using Agar, usually in a P-D-A recipe and with some adjuncts to help fight unwanted organisms growing in it.

If you need a good knife source, try the hobby stores in the states. You can get full stainless steel shaft, rubber grip, safety twist down the bottom of the knife to lock and not up near the blade end to cut your fingers up, etc. $2 each

Buy a pack of 100 blades for $12

Back in the hobby days I'd cringe when hearing the locals here re-sharpening old blades because of the prices they were charged for new packs. At $2-3 a knife and $12 for 100 pack, you could do a group order and split the costs. Same for loops I suppose. I have not checked out eBay Hong Kong to see if there are any great China Factory -> Your Door in Oz deals going on in that area but thats another area.


----------



## Sammus (6/4/09)

Sammy said:


> 1-1/2 tablespoons of agar seems like *way* too much agar for this amount of water. The instructions on the powdered agar packet I picked up at the asian grocers recommends 25g per 3 litres of water, which equates to 1 gram per 120ml.
> 
> 1-1/2 tablespoons of agar powder is about 10 grams, so I think it's a factor of 10 too much agar powder.
> 
> I want a nice solid growth medium that is stable at room temperatures, so I might try 2 grams per 120ml.



That does seem like a lot. I like this guide, they recommend about 15g for a litre, but also state that different brands of agar will work differently. Last night I used 10g to about 700ml and i think it's perfect. I used to use 1g per 100ml before and it's ever so slightly too soft, I often cut it with my loop when I'm streaking. So I aim for a little over 1g/L.


----------



## samhighley (6/4/09)

Sammus said:


> Last night I used 10g to about 700ml and i think it's perfect. I used to use 1g per 100ml before and it's ever so slightly too soft, I often cut it with my loop when I'm streaking. So I aim for a little over 1g/L.



Presumably you meant 1g/100ml? I ended up using about 2g per 100ml and it's very solid, even sitting on the kitchen windowsill in the heat.


----------



## Sammus (6/4/09)

Sammy said:


> Presumably you meant 1g/100ml? I ended up using about 2g per 100ml and it's very solid, even sitting on the kitchen windowsill in the heat.



yep, that's what I meant 

Yeah heat shouldnt affect it too much. Agar is pretty weird compared to gelatin. Melts at about 95C then doesn't solidify till about 45C. Moreover, it will remelt and resolidify whenever you want it too, which is handy.


----------



## samhighley (7/4/09)

Sammus said:


> Yeah heat shouldnt affect it too much. Agar is pretty weird compared to gelatin. Melts at about 95C then doesn't solidify till about 45C. Moreover, it will remelt and resolidify whenever you want it too, which is handy.



And you can boil agar without affecting the setting properties, unlike gelatin which I believe denatures above about 90C.


----------



## Airgead (7/4/09)

Sammy said:


> Presumably you meant 1g/100ml? I ended up using about 2g per 100ml and it's very solid, even sitting on the kitchen windowsill in the heat.



My standard recipe is 1.5% agar. Sets up nicely.

If people are looking for loops and shipping is too much, a length of stainless wire and you could make one really easily.

Cheers
Dave


----------



## Sammus (7/4/09)

yeah, but if your ordering vials and whatnot from one of those lab supply sites, its usually only another $10 or so for a proper innoculation loop.


----------



## Airgead (7/4/09)

Sammus said:


> yeah, but if your ordering vials and whatnot from one of those lab supply sites, its usually only another $10 or so for a proper innoculation loop.


Yeah... if you can find one. Last time I checked all the pro lab supply places don't do the re-usable loops any more. all you can get are the plastic radiation sterilised ones. They are fine but they come in packs of 10 or so and once you open the pack you have to use them all straight away as you can't re-sterilise. Fine for a pro lab that uses heaps of them but on a homebrew scale that's a bit much.

Only place I found them was a mob on ebay selling biology supplies to schools.

Cheers
Dave


----------



## Sammus (7/4/09)

http://www.proscitech.com.au/cataloguex/on...p?page=t11#t067 - I got T067


----------



## Airgead (7/4/09)

Sammus said:


> http://www.proscitech.com.au/cataloguex/on...p?page=t11#t067 - I got T067



Well there you go then. I searched everywhere for one a couple of years ago and got nowhere. Good to see they are available.

Cheers
Dave


----------



## captaincleanoff (5/5/09)

i just used one of the slants I made to make a starter, for the first time.

I really struggled to pull off any yeast with the inoculation loop.. The agar was so hard i literally had to 'cut' it with the inoculation loop, which was quite hard as its floppy..

Managed to get a tiny bit out, stuck to a fair bit of agar..

Made up a 10ml starter, will step up to 100ml tomorrow if everything is ok..

Will a 'tiny' bit of yeast start 10ml?


----------



## Sammus (5/5/09)

Yeah, and that's basically what you're meant to do anyway.

The other method (which I do) is to make a fresh slant. Wait till that grows, then pour 10ml of wort into it and shake it up, leave it for a day or so, then add it to about 50ml, then 400ml on stirrer, then to 2L on stirrer.

You can then continue to use your old source (I have mine stored under distilled water, not on slants) to make new slants etc, as you can with your method.


----------



## Airgead (6/5/09)

I use the whole slant at a time method as well. I find its much easier and there is lesas chance of contamination as you are inoculating the starter with a much larger amount of yeast. You also don't run the risk of having your working slant contaminated (from being repeatedly opened and closed) and ending up with that contamination in all your starters.

When making slants from my masters (also stored under water) I usually make up 5 or 6 of each strain at a time as that's really not much more work than just making one. that way I can use a slant per brew with no hassle.

Cheers
Dave


----------



## white.grant (1/6/09)

I had my first go at making up some slants on the weekend. I goofed measuring up the ingredients so instead of 1040 wort I've got closer to 1020 and the growth medium looks very pale when compared to other photo's I have seen. Will my slants still work or should I start again?

cheers

grant


----------



## A3k (1/6/09)

Hi Grant,
I'm by no means a pro, but i'm pretty sure you'll still get growth. i wouldn't throw it away.


----------



## kabooby (1/6/09)

1020 will be fine. Still enough sugar there for the small amount of growth you need

Kabooby


----------



## white.grant (1/6/09)

A3k said:


> Hi Grant,
> I'm by no means a pro, but i'm pretty sure you'll still get growth. i wouldn't throw it away.






kabooby said:


> 1020 will be fine. Still enough sugar there for the small amount of growth you need
> 
> Kabooby



Thankyou gentleman. That's what I was hoping.

cheers

grant


----------



## Airgead (3/6/09)

kabooby said:


> 1020 will be fine. Still enough sugar there for the small amount of growth you need
> 
> Kabooby



A lot of professional growth media for yeast is way lower that 1040. Too much sugar in the growth media is a bad thing. What you want is just enough for it to grow to cover the surface then run out of food and go dormant. If there is too much sugar it won't go dormant and may not keep as well.

I've never measured the OG of my growth media but I use 1% malt which is probably going to give me something like 1004 given that my 1040 starters are about 10% malt.

Cheers
Dave


----------



## Fourstar (12/5/10)

Mods, is it possible to have this stickied?

I know alot of users would find this information useful, especially the wort/agar ratios and procedure as it is the prefered method over using gelatine.


----------

