Slanting yeast

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There has been plenty of discussion of yeast slanting in various threads, and I know there are other slanters here on the forums, but I have not seen a definitive thread which walks through the process step by step. So, last time I prepared some slants I took some photos and decided to post a tutorial here.

Although it is an advanced technique, yeast slanting has some advantages. You can share yeast strains easily by exchanging slants. Each yeast culture you buy can be used to make 25 batches or so without re-using yeast. You can save platinum/seasonal yeast strains for use year-round. You can harvest yeast from a brew buddy's starter to add to your library. Or if you go to the trouble of bottle harvesting yeast you can save it for future use. As long as you are very careful with sanitation while handling yeast slants, the risk of a contaminated batch is very low -- especially if you compare this technique to re-using yeast from prior fermentations.

I didn't make up all this stuff myself. There are some good resources out there on the web, so be sure to check them out as well before you get started. To name a few good ones:

Making Plates Slants - German Brewing Techniques
Yeast/Culturing - Brewiki
Culturing Yeast and Using Slants
Yeast Propagation and Maintenance - Principles and Practices
 
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You will need some equipment:

  • A pressure cooker, 8 quarts or larger
  • A small pan (unless you are a cave man, I'm sure you already have one)
  • Filtered or bottled water
  • Dry malt extract
  • Vials. I use the 24mL vials from Cynmar, stock no. 115-27910.
  • Beakers. I purchased the set from Cynmar, stock no. 115-20035.
    An extra 500mL and/or 1000mL beaker would be handy as well depending on
    the size of your pressure cooker.
  • Large paper clips, half straightened
  • Agar agar. You can find this in an Asian market or in the Asian section of
    a large grocery. If you can't find agar agar you can use plain gelatin.
  • 250mL flask
  • 1L and/or 2L flask
  • Stir plate
  • Small and medium size stir bars
  • A digital scale
  • A measuring cup
  • A small funnel suitable for filling your vials
  • Aluminum foil
  • Electrical tape
  • Scissors
  • A small box such as a shoe box
  • Sanitizer
  • Candle
  • Lighter
  • Half-pint canning jars for canning sterile starter wort (optional)

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Measure out 35g of DME and about 400mL of water. Combine in the pan along with half a bar of agar agar (2.5 grams) and heat gently while stirring until the agar agar and DME are fully dissolved. It isn't necessary to boil it, it gets boiled later in the pressure cooker.

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Pour the mixture into the 500mL beaker and fill each vial one third to one half full using the funnel (it's hot, so you may want to use gloves). You should have enough wort to fill 25-30 vials. I will use 5-6 vials per yeast strain, so this is enough for five strains. As you can see I did this on a baking sheet so I would have easier cleanup for spillage.

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Cap each vial and loosen the cap one half turn. Place the vials inside the large beakers so they are snug -- you don't want them to tip over in the pressure cooker. Use empty vials to fill any empty space in the beakers. It's not a bad idea to keep some empty sterile vials on hand for collecting some yeast from a buddy's yeast starter which you can take home and slant later (I'll get to that topic in a bit). Make sure the cap is loose on the empties too. You don't want them to explode.

Cover the beakers with foil and place them in the pressure cooker. Add about 1L of water, or enough to get at least 1" in the bottom. You definitely do NOT want to end up running your pressure cooker dry or your equipment will be toast.

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Gently bring the pressure cooker up to temperature. You can start on high but once the water starts boiling I recommend backing off on the heat so you don't overpressurize it by accident. Once the cooker hits 12psi set a timer for 15 minutes.
 
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After 15 minutes at 12psi, carefully slide the pressure cooker off the hot burner and set a timer for one hour to allow it to cool. Do not move it, open the valve, or use a water bath -- if you do, you will cause the vials to boil over as the pressure drops too rapidly. We don't want that to happen! Let it cool for one hour.

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After the pressure cooker has cooled for one hour carefully open the cooker. With clean and sanitized hands, on a clean and sanitized counter top, carefully remove the glass vials, screw them shut, and tape the lids with electrical tape so they won't dry out. Place them in a cardboard box (such as a shoebox) and prop it at a 45 degree angle (so they slant!) for 24 hours or until they set. Then you can store them in zip bags in a dark, cool closet until ready for use. They are sterile so they should last indefinitely.

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Do this when you have fresh yeast on hand, and are ready to make a starter for a batch.

Clean and sanitize your work area as well as your hands. A spray bottle of sanitizer works great for this. Shut off the A/C, close windows, turn off fans. The goal is to eliminate any drafts. Setup your candle and light it. The candle is a flame source to sterilize the paper clip. It does double duty by providing a hot updraft in your work area so dust doesn't (hopefully) settle into your open slants as you work.

Fill the 50mL beaker with sanitizer (I use Star San because it keeps for about a month, any no-rinse sanitizer will do, as long as it is chlorine free). Place the paper clip inside the beaker to hold it. Remove the tape from 5-6 vials, make sure the lids are tight, and dip them in sanitizer. Sit them to the side of your work area. Setup a beaker large enough to hold the yeast package as you work near the flame. Open your yeast package and place it in the beaker so it won't fall over. Place a piece of sanitized foil next to the flame to hold your caps as you work. Now you are ready to slant.

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Grab a slant, unscrew the cap, and place it on the foil (open side down). Keeping the slant near the flame as you work, grab the paper clip and remove it from the sanitizer. Run it through the flame a few times to sterilize it, and dip it back in the sanitizer to cool it off.

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Now dip the clip into the yeast package, and then into the slant 4-5 times. Drop the clip back into the sanitizer to free up your hand, and screw the cap on. Sit the vial to the side. Repeat for the remaining vials.

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When you are finished slanting make your starter with the yeast package as usual.

To incubate, unscrew each cap a half turn (don't forget to do this!!) and place the vials in a sanitized beaker. Cover it with aluminum foil and place in a warm, dark place for 7-10 days.

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After the slants have incubated, screw the caps shut (don't forget clean sanitized hands again!), and re-seal them with electrical tape. Examine each slant carefully. There should be white streaks of yeast throughout the slant. If there is any discoloration, wash and store the vial for re-use -- I have only had one contaminated slant so far, and it was obviously green with mold so I tossed it. Store the slants in the coldest part of your refrigerator. Don't forget to label the vials! I use masking tape and a marker, and label each one with the strain code and name. It is a good idea to date them also if you think you will be keeping your slants for a long time.

When the yeast turns brown, it is time to re-slant. Most yeast will keep for over a year on slants.
 
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This process takes 2-3 days with a stir plate. If you don't have a stir plate (craziness!) I would allow two days for each step. At a minimum you will need pure O2 from a stone if you don't have a stir plate, given the large number of generations the yeast will have to reproduce to grow from 1M cells to 100B+ cells.

On the left is a 'blank' slant. On the right is a slant which has been incubated for nine days. Notice the creamy white yeast all over the slant. This is what a healthy slant looks like:

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Remove the slant from the refrigerator and leave it out for a few hours to warm to room temperature so you don't temperature shock the yeast. I like to use pre-canned wort for the first step because it's sterile, but other folks have done OK with just boiled DME. You need 1 cup of wort either way, so if you are using DME the vial can warm up while you make your wort and let it cool.

Remove the tape from the vial, make sure the lid is tight, and drop the vial in sanitizer before opening. 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. Drop in your stir bar (if it isn't already in the flask), and add the remaining wort to the flask.

Cover the flask with sanitized aluminum foil and let it ride on the stir plate for 24-36 hours. When it's ready to step up you will know because the wort will look like chocolate milk rather than wort -- that's healthy yeast cells in there!

Being careful not to drop in the stir bar, decant your small starter into a 1L or 2L flask depending on batch size and gravity. Add enough fresh wort for the volume of starter required. 24 more hours on the stir plate and it will be ready to pitch, or you can refrigerate/decant after 48 hours if you wish. I pitch the whole thing into the fermenter right off of the plate. Typical lag time is about 2-3 hours!
 
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Harvesting from a starter

You can harvest yeast from a brew buddy's starter using a stainless steel baster and an empty sterile vile (told ya those come in handy!). These can be had for $10 from Amazon.com, I didn't have any luck at the local kitchen stores. Clean and sanitize the baster like you have never cleaned anything before, or nuke it in the pressure cooker before use... the risk of contamination is very real! Using the baster, suck up a little bit of the yeast cake from the bottom of the finished yeast starter, and put it in a sterile vial. Cap and tape up the vial for transport home. You can then inoculate slants with it. This procedure is not recommended if the yeast is not first generation due to the risk of contamination -- you want to harvest yeast that came from a pure culture so you have a pure culture every time you build up yeast from the slant.

Re-slanting

This is easy. When you use your last slant (or next to last?), or the yeast get old, just dip your paper clip into the slant and innoculate fresh slants. Then use the parent slant to build up a starter. It really is that easy.

You will know when it's time to re-slant your yeast if it gets old, as the yeast will turn the color of putty as they use up their glycogen reserves. This can take 6-18 months depending on the yeast strain and the conditions in your refrigerator. If the yeast reaches the color of peanut butter, it is dead and the slant should be discarded.
 

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Thanks Sacc. Awsome! This is going to be my next home brewing endeavor.

Prost!:mug:
 

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What is the recommended size of pressure cooker?
 
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What is the recommended size of pressure cooker?
As big as you can find. The bigger the better especially if you plan to pressure can wort. Mine is the Presto 8 quart and it is very small for the job, though I already had it when I started. I have heard you can find them at thrift stores and garage sales quite frequently.
 

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Grab a slant, unscrew the cap, and place it on the foil (open side down). Keeping the slant near the flame as you work, grab the paper clip and remove it from the sanitizer. Run it through the flame a few times to sterilize it, and dip it back in the sanitizer to cool it off. Now dip the clip into the yeast package, and then into the slant 4-5 times. Drop the clip back into the sanitizer to free up your hand, and screw the cap on. Sit the vial to the side. Repeat for the remaining vials.
When you dip the paper clip into the slant, are you jabbing it into the agar? or rubbing it along the slanted surface?
 

ChillyP

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Finally been waiting to a tutorial. And Subscribe.
 

jason.mundy

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- Vials. I use the 24mL vials from Cynmar, stock no. 115-27910.
- Beakers. I purchased the set from Cynmar, stock no. 115-20035.
Good post! This is motivating me to slant my yeast.

Thanks for the part numbers and the reference to www.cynmar.com.

I have a lot of this stuff already. I may be able to do a few equipment substitutions.

Pyrex beaker could be replaced with a pyrex measuring cup.

A candle could be replaced with a propane torch I bought to sweat copper fittings.
 
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There is enough yeast that a 250mL starter is actively fermenting on the stir plate after 24 hours. Assuming a growth rate of one generation every two hours, and a maximum yeast concentration of 80M cells per mL, there would have to be at least 5M cells in the slant. So a conservative estimate would be somewhere around 1M ~ 10M cells... but it's just a guess.

This is the best online resource IMO regarding yeast cell concentration and growth rates in wort:

http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices

If you read, you will see why I use a stir plate AND sterile wort for the first step; the method he gives you can use non-sterile wort safely but it requires adding 10mL of wort (which you could add directly to the slant vial) and wait 2-3 days before pitching the 250mL starter. Since I use sterile wort and am very very careful with my sanitation in the first step, I go straight from the slant into 250mL without any problems.
 

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Just a note for those planning to do this with equipment from Cynmar: read their shipping info carefully to see if what you're buying will need special shipping, since their system will not tell you in advance.
Growth media (like agar plates) must be shipped separately and/or overnight, which costs extra. See my thread on ordering from them for details.
 

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does the amount of medium in the tube relate to how long the culture will survive? i mean can the yeast pull nutrients out of the agar media or only off the surface? why not use a more radical angle to form the agar so you have a greater colony in there?
 
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does the amount of medium in the tube relate to how long the culture will survive? i mean can the yeast pull nutrients out of the agar media or only off the surface? why not use a more radical angle to form the agar so you have a greater colony in there?
The yeast begin to die off when their glycogen reserves are depleted; they cannot replenish those without going through a whole adaptive - growth - fermentation - flocculation cycle.

If you use long test tubes for your slants, you will get more yeast growth because of the larger surface area. This will help when you go to build up the slant since you would have more yeast. It wouldn't make it last more than 6-12 months though.
 
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One of the resources I researched suggested the tape so the caps don't come loose in storage potentially drying out the slants. It's cheap insurance against losing a strain in my library I suppose. I may skip wrapping the unused slants next time around since it's a bit of a PITA to tape up 20 slants at once. :)

Canning the wort is simple, just fill jars about 80% full with 1.040 wort (you can use leftover wort from brewing diluted to 1.040 or just mix up DME and hot water to dissolve). Put on a fresh canning lid and loosely screw on the ring. Load the jars into a pressure cooker with about 1" of water in the bottom, heat to 12 PSI, and hold for 15 minutes before allowing the pressure cooker to cool for about 45 minutes. Remove the jars making sure the lids are tight, and ensure that the jars do vacuum seal properly. I had a few jars that didn't seal last batch which I stuck in the fridge and re-boiled before using for starters.
 

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I've used a simular process in the past. One problem I keep having, is after inoculating the slants and the yeast start to grow they produce a lot of C02. I vent the tubes but am always worried about contamination. When activity starts to die down (less gas to vent) I place the tubes in the fridge. But they still ferment a bit, and when I go to use the slant, I get a large puff of C02 and the agar actually starts to bubble up from the base of the tube and push its self slowly out of the tube.

Do you vent your culture tubes?

Have you ever had this problem?

Maybe I just used the wrong mix of agar/wort.
 
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No I incubate the tubes in a sanitized beaker covered with foil with the lids slightly loose so they can offgas. After 10-14 days I tighten them up and into the fridge. I have had one vial give off a slight puff of CO2 so far when opening it but it was very slight.

Yeast/bacteria/mold can only get in through dust which can't get through the threads under the cap, but moisture and CO2 can escape.
 

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i tested that theory, i sterilized (15 psi for 30 min and cooldown time) 500ml wort. left the flask for a week with an shimmed, inverted beaker.. to leave plenty of gap for airflow. no mold or anything visibly colonized it. so i agree. airborn bad guys cant fly up.
 

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There is enough yeast that a 250mL starter is actively fermenting on the stir plate after 24 hours. Assuming a growth rate of one generation every two hours, and a maximum yeast concentration of 80M cells per mL, there would have to be at least 5M cells in the slant. So a conservative estimate would be somewhere around 1M ~ 10M cells... but it's just a guess.

This is the best online resource IMO regarding yeast cell concentration and growth rates in wort:

http://www.maltosefalcons.com/tech/yeast-propagation-and-maintenance-principles-and-practices

If you read, you will see why I use a stir plate AND sterile wort for the first step; the method he gives you can use non-sterile wort safely but it requires adding 10mL of wort (which you could add directly to the slant vial) and wait 2-3 days before pitching the 250mL starter. Since I use sterile wort and am very very careful with my sanitation in the first step, I go straight from the slant into 250mL without any problems.

I'm just getting started in slanting, but I've been going from slant to ~200mL for the first step, then from there to ~1000mL, then higher if necessary. It's working fine so far.

Also, 'he is a 'she'. The author of that Maltose Falcons article is Maribeth Raines. ;)


Here's a question i don't think I've seen an answer to...What makes a slant 'go bad'?
 

TimWeber

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A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.

Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.
 

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To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?
 

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i got 12 ml vials . a little small. do you think i could work with those?
 
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A slant can go bad if it dries out, or if it becomes infected with mold, fungus, wild yeast, ect.
I have only had one slant 'go bad', it had visible green mold growth from a single spore that managed to sneak into the slant vial while I had the cap off.

Another question, what do you do about the condensation that forms inside the slant tubes while setting? With the petri dishes it's fine, you just turn them upside down. With the slants, I store them upside down but then when I go to innoculate, a few drops of water leak around the cap. I blow torch this away, but just wondering how you handle it.
I haven't been that concerned about it, doesn't seem to effect anything.

To me this looks like an alternative to washing yeast and keeping it in 200ml jars but slants take up less space - a typical test tube stand could easily keep ten strains. Are there any other advantages to slanting as opposed to washing ?
Washing yeast the viability drops off quickly so you have to re-use the yeast relatively soon, or re-wash and propagate the yeast prior to pitching. I have also had a few infection problems with washed yeast, whereas building up from a slant everything is sterile (you are always working with yeast which came from a pure culture rather than from a fermenter).

i got 12 ml vials . a little small. do you think i could work with those?
Most folks use the 12ml vials for yeast banking. I went with the bigger ones because I think they are easier to handle. :)
 

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thanks for all the information Saccharomyces .:mug:
 
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