Yeast Banking - #5 Frozen Yeasts - Eureka Brewing

Yeast banking #5 Frozen yeasts | Eureka Brewing
https://eurekabrewing.wordpress.com/2012/09/16/yeast-banking-5-fro...
Yeast banking #5 Frozen yeasts

Posted on September 16, 2012
Eureka, today is the last post in the series about yeast banking at home (or in a lab). Please
refer to the yeast basics page for links to all the other posts. The three previous methods
(agar plates, isotonic sodium chloride solutions and agar slants) work all at room
temperatures or colder. But not below 0C (32F) since the yeasts would probably die and
the media (agar and sodium chloride solution) would freeze. Storing your yeasts at colder
temperatures prevents some of the growth. If the yeasts do not grow during the storage
time, the chances are high to have the same exact strain after you revive them. If you store
your yeasts in a refrigerator your yeast can grow (even slowly) and might mutate and try to
adapt to the colder temperatures. The yeasts could therefore change and maybe lose
specific characteristics. This could lead to loss of flocculation or even loss of your most
loved aroma profile (banana or clove aroma in wheat yeasts for example). However, such a
conversion does not have to happen. It might. And this is why a lot of labs store their
microorganisms or cells at lower temperatures such as -80C (-112F). At this low
temperature no growth occurs. Even the whole metabolism of the cells arrest. The cells kind
of stops entirely. You can store your cells at this temperature for nearly forever.
I am not sure how many of you out there have a -80C freezer at home. Most of you might
have a freezer at around -20C (-4F). And you can store your yeasts at -20C as well. Just
dont use a freezer with thaw-cycles. The only disadvantage here is the metabolism of the
yeasts might still work and some changes could occur as well. In comparison to a storage at
room temperature or colder temperatures, far fewer changes can/do happen at -20C. And
this is why freezing your yeasts is as far as I know the only method to bank your yeasts over
a longer period of time (years) at home.
Description of the technique
As already described, this method here is about freezing your yeasts at -20C (-4F) or
lower/higher if you want. For this purpose you use a special media which consists of a
cryoprotectant (antifreezer) such as glycerin. Please dont use antifreezer you use for your
car. If you have your storage media ready you just add some yeasts to the media and put it
in your freezer and leave it there until you want to use the yeast for a future batch. Please
notice, this is about banking yeasts and not yeast storage. Only small amounts of yeasts will
be frozen here. Not pitchable amounts.
I would like to mention already, this is the most sophisticated method of all the four
described already (agar plates, isotonic sodium chloride solution and agar slants). I do not
recommend to go with this method if you havent tried one of the previous ones before. If
you are new to yeast banking try to bank your yeast with another technique than this one
before and get some experience. I recommend the isotonic yeast storage method for
beginners. If you manage to revive the yeasts without an infection you might step forward to
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this method. If infections occur regularly, try to find the source for the infection and work on
that. This method here does not work if you have troubles with your sterility and
cleanliness It just does not. In addition, the technique below is just one way to do it. I am
certain there are other ways to freeze your yeasts.
Material
Vial, tube or any other containment you
can heat sterilize to store your yeast in a
freezer. I use 1.5 mL reaction tubes for this
purpose (Fig 1). They are small and easy
to sterilize
Food grade glycerin. Glycerin solutions
work as well as long as the glycerin
concentration is above 60%. I use a 85%
food grade glycerin solution I bought at a
local pharmacy
Pressure cooker or any other source to
heat sterilize your tubes and the food
grade glycerin
Media. I guess dried malt extract solution
or even an isotonic sodium chloride
solution could work as well. I use a lab
media (called YPD) as a storage media.
Fig 1: Tube filled with storage media
And add some ascorbic acid as well. More
and yeast
about my storage media later on.

Sterile pipettes, micro pipette with sterile
tips or a sterile syringes. You need sterile devices to add the storage media to your
containments before freezing and get the yeast out of the containment for reviving. See
bank the yeast description below for further information.
Preparation
First, get the freshest, purest yeast you can get. This could be from a starter or from a fresh
yeast package or vial. This is very crucial. If you freeze unhealthy yeast you could risk to
either loose them entirely or have problems during reviving them. Or a different outcome of a
batch of beer (attenuation, taste, flocculation etc.). Please do not freeze or bank any
unhealthy yeasts. And dont expect the yeasts to come around during banking. If you have
problems during a fermentation (stuck or whatever) dont bank the yeast afterwards and
hope the yeasts will be fine. They probably wont.
What yeast sources could you use?
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1. Yeast starter
Get yeast directly from a fresh yeast starter. Wait until no
more growth occurs. Then mix up the whole yeast starter
to get the yeasts back into solution. Remove some of the
volume from the yeast starter and fill your pre-sterilized
containments. If you want to cool down the yeast starter
to let the yeasts settle to the bottom of the flask and
discard as much of the yeast starter as possible (and
only pitch the yeast sediment), remove the yeasts for
banking before cooling down the starter. Store the filled
containments for 48 h in a refrigerator. During this step
the yeast build up some important molecules they need
to survive and settle to the bottom (Fig 2). Remove as
much of the supernatant as possible (Fig 3). This can
mostly be done by just inverting the tubes, vials etc. The
yeast sediment at the bottom should stay at the bottom.
Just dont turn the tubes too fast and too slow. You now
should have a nice yeast sediment at the bottom (Fig 3).
The volume of the yeast sediment should be below 10%
of the volume of the containment. If it is a bit more or
less dont worry. However, discard some of the sediment
if it is more than 20% of the volume.
Then proceed with the steps described as bank the
yeast below.
Fig 2: Small yeast

starter with yeast at
2. Yeast package from manufacturer
bottom
Use yeast from the manufacturer directly. Make a small

yeast starter and add a few mL of yeast slurry from the
package or vial (Fig 2). I use glass tubes for this purpose which are filled with 4 mL of a malt
extract yeast starter media (10 g of dried malt extract dissolved in 100 mL of water) and
sterilized them in a pressure cooker.
Leave the starter at room temperature for 48 h. Then proceed with the steps described as
yeast starter above. If your yeast is very fresh, you might skip the whole starter-step and
bank the yeasts directly. Therefore fill your containments with the yeasts and let them settle
down in a refrigerator for 48 h then discard the supernatant. Then proceed with method
described as bank the yeast below.
3. Yeast sediment from fermenter
I would not recommend to directly bank yeasts from a slurry. At least wash them first to get
rid of trub and any dead cells and do a small yeast starter. Just harvest a small amount of
the sediment (like 100 mL) and wash them with sterile water for a few times until only the
viable cells remain. Discard as much of the supernatant as possible. Then make a small
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yeast starter (100 to 200 mL), add the washed yeast cells and leave the starter at room
temperature for 48 h. Then proceed with steps described as yeast starter above.
4. Yeast sediment from bottles
Procedure is similar to yeast package from manufacturer above. Make a small yeast starter
and add some of the bottle sediment. Leave the starter at room temperature for 48 h. Then
proceed with steps described as yeast starter above.
Bank the yeast
The yeast sediment you now have in your containments
should consist of very healthy and pure yeast cells (Fig
3). Now its time to add the storage media (see below)
and freeze the yeasts. I add about ten times the volume
of storage media for every volume of yeast. In my case I
have about 0.05 to 0.1 mL of yeast sediment (Fig 3). I
therefore add 0.5 mL of storage media. To add the
storage media you need a sterile device such as a
pipette, micro pipette with sterile tips or sterile syringes.
Please pre-sterilize the storage media in a pressure
cooker for 15 min if possible. Let the storage media cool
down to room temperature first before proceeding. Then
add the media and either gently shake the tubes, vials or
use the pipette, syringe, micro pipette for a thoroughly
mix. You are basically done. Just label your
Fig 3: Tube with yeast
containments very well and put them in your freezer.
sediment at bottom
Done!
Storage media:
1. Malt extract based (havent try this one): For 100 mL of storage media use 10 g of dried
malt extract, 50 g of glycerin and fill up to 100 mL with water. Add 0.1 g ascorbic acid (aka
vitamin C) if possible. The ascorbic acid helps to stabilize the membranes of the yeasts. If
you have a glycerin solution for example a 85% glycerin solution calculate the amount you
need as following: 50 g divided by percentage of solution (divided by 100). In this example
50 divided by 0.85 equals 58.8 g. You therefore have to add 58.8 g of your 85% glycerin
media. Sterilize the storage media in a pressure cooker for 15 min if possible.
2. YPD storage media (I use this one). The recipe for this YPD-media based storage media
is from the book Yeast: The Practical Guide to Beer Fermentation by C. White and J.
Zainasheff. For 100 mL you need: 5 g YPD bouillon, 50 g of glycerin and 0.1 g ascorbic acid.
Add up with water to 100 mL. Sterilize the storage media in a pressure cooker for 15 min if
possible.
Storage
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Put your containments in your freezer. Nothing to do more. I use a rack for my tubes to have
some organizing system (Fig 4).
Fig 4: Yeast library part 1
Reanimation
1. Make yourself a yeast starter. I recommend 100 mL for the first step. Therefore dissolve
10 g of dried malt extract in 100 mL of water, add some yeast nutrients if possible and
sterilize the starter for 15 min with a pressure cooker. Cool down the starter to room
temperature.
2. Get your tube, vial (or whatever containment you use for yeast banking) out of your
freezer and increase the temperature as fast as possible. I let the tubes warm up in my
hands. Then gently mix the yeast and storage media and add the whole content to your
yeast starter. I use a micro pipette for this step. Then wait a few days until signs of
fermentation arise (cloudiness, white foam, yeast sediment at bottom, bubbling etc.). Wait
until a yeast sediment formed at the bottom. You can either stir your yeast starter the whole
time or just leave it unstirred.
3. Prepare your next yeast starter. I normally do a 1 L stirred yeast starter as a second
starter here. Therefore dissolve 100 g of dried malt extract in 1000 mL of water and sterilize
it. Discard the supernatant from the first yeast starter and only transfer the yeast sediment to
your next 1 L yeast starter. I recommend to taste the supernatant (before discarding) to
check if the starter is okay. If the starter tastes bad probably an infection occurred. If the
yeast starter tastes good, congratulations!
4. Repeat the yeast starter steps until you have the amount of yeast you need. It is hard to
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tell how many yeast starters you need and what volume you should choose. There are way
too many different way on how to bank the yeasts. The only way to tell how many yeast cells
you have would be to count the cells (have a look at this post concerning this topic).
From my experience and with the amount of yeast I bank (about 0.1 mL as it can be seen in
Fig 3), I need a 100 mL yeast starter first, followed by a 1 L yeast starter, followed by
another 1 L yeast starter afterwards to have approximately 100E9 cells. This would be equal
to the amount of yeast you get in a Wyeasts Activator package or White Labs vial.
My experiences with this method
My procedure looks as following. As already mentioned, I use a YPD-based storage media
to bank the yeasts. And I use the tubes shown in Fig 3 for banking. After discarding the
supernatant after storing the tubes 48 h in the refrigerator, I add approximately 0.5 mL of the
storage media to the tubes with a micro pipette and a sterile tip (Fig 5).
Fig 5: Equipment for yeast banking. YPD-based storage media (left), yeast sediment in
tube (right) and micro pipette (1000 uL) with sterile tip
Then use the micro pipette to mix the yeast and the storage media. After that the tube look
like shown in Fig 1. I then put the tubes in a box (shown in Fig 4) and store them in my
freezer (at -20C).
What are the advantages and disadvantages for this method compared to the others?
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Advantage
Disadvantage
Long term storage method
Lot of equipment necessary (freezer, lab equipment etc.)
No maintenance work
Contaminations not visible
Does not require a lot of space
Cant store yeast mixtures, blends
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Rather complicated method
This is for sure one of the least labor intensive methods. And the only one to store your
yeast over a longer period of time. On the other hand, you do need some extra equipment
such as a freezer and some lab equipment (syringe or pipette or micro pipette,
containments, chemicals (ascorbic acid)). I think this method is only for the people really
interested in yeast banking. And I would not recommend to go with this method if you
havent tried one of the previous ones before. Sure the long-term storage seems very
advantageous. However, do not underestimate the time and equipment you need to prepare
the yeast for this banking method. On the other hand, your equipment has to be very clean
and mostly sterile.
As with other methods, it is not easily visible whether your yeast is infected or not by just
looking at your vial, tube etc. You will know after the first yeast starter. And you cant bank
yeast blends and other mixtures with this method as well. The ratio of the different
microorganisms will eventually change during the reviving. If you do want to store a blend
you might have to separate the blend before
For all of you still interested in freezing your yeasts, I would like to mention the book Yeast:
The Practical Guide to Beer Fermentation by C. White and J. Zainasheff again. In there are
further information on how to freeze your yeasts.
This is the end of the yeast banking posts. I hope I could give you some information about
the topic. Please feel free to comment and ask questions if something is not clear enough.
The next posts will be about some recipes, tasting notes and yeast hunting stories again.
Stay tuned!
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R E LATE D
Yeast banking - #3
Isotonic sodium chloride
Yeast banking #4 Agar

slants
Yeast banking - #1
Introduction
This entry was posted in Technique and tagged Agar plates, Banking, Lab, Technique,
Yeast, Yeast basic by eurekabrewing. Bookmark the permalink
[https://eurekabrewing.wordpress.com/2012/09/16/yeast-banking-5-frozen-yeasts/] .
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18 THOUGHTS ON YEAST BANKING #5 FROZEN YEASTS
JO
on September 19, 2012 at 10:36 am said:
This is a very good disciption!!
eurekabrewing
on September 19, 2012 at 11:20 am said:
Cheers! Glad you enjoyed it
Mike
on November 9, 2012 at 11:17 am said:
Extremely informative, as the other techniques and description. For me, its just pure
awesomeness
May I ask, what kind of pressure cooker are you using (model, brand?) and what is
your average sterilization time (20 min or more) ?
I know that most of european models (therefore mine too
) cant reach 15 psi (1
bar @ 121C).
eurekabrewing
on November 9, 2012 at 9:01 pm said:
Hi Mike, thank you very much for your comments. Always appreciate such
comments. I inherited my pressure cooker made by Kuhn Rikon from my
grandmother and it is a very standard pressure cooker for cooking potatoes
and such. I have no information about the model. It just comes with a standard pressure releasing valve. Thats it. No pressure gauge or anything
fancy. I therefore have no idea about the pressure in the pot during the sterilization process or the temperature. Concerning the time, I heat up the pot until it releases pressure. Just to get rid of the air inside and replace it by water
vapour. After releasing the pressure for some seconds, I decrease the temperature a bit to stop the valve from releasing further pressure. Then hold the
temperature for 10 to 15 min. If you want to sterilize bigger liquid volumes (>
500 mL) such as starter media etc. increase the time to 20 to 30 min. However, I sterilize my yeast starters (around 100- 200 mL) for 10 to 15 min and
never had any contaminations so far. After that simply turn off the heat and
let the whole pot cool down on the hot cooking plate. Hope I could give you
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some information here. I guess the best way would be to start with a defined
procedure and if it works, stick to it. Thats what I did here.
Cheers, Sam
mirogster
Thank you very much Sam! I really appreciate your time and effort,
dedicated to the implementation and placing the effects of your work,
in the form of this blog
Easily accessible to all interested sharing
means caring
After that, when I bought and learned that my pressure cooker, unfortunately, does not maintain 15 psi. I conducted a small research on
the subject
You may find yourself lucky, because apparently yours
Kuhn Rikon and maybe 2 other products (in EU), can produce and
maintain such pressure.
As I mentioned earlier, the majority of european pressure cookers
holds lower pressures & temperatures.
Of course, in US they got no such issues, all PC have as a standard
15 psi damn those lucky Yanks
What I did, was that, I just extended the time of test drive sterilization
for 45 min (~100 ml wort / agar medium and few petri dishes). The result was indeed very pleasing. After one week, of storing in ambient
temps, I didnt noticed any contamination
Once again, thanks for sharing your knowledge and the effects of
homebrewing /microbiologics experiments.
Cheers, and keep em coming!
eurekabrewing
Thanks for sharing your information as well. I havent put

many thoughts into pressure cookers so far
And I dont
know how important it is to reach a pressure of 15 psi. Sure

there is the golden rule of +1 bar (2 bar in total) and 121C
and somewhat keeping this temperature for 20 and 30 min.
Thats how it is done in most of the labs I have been working
so far. However, your test drive gave you satisfactory results.
And I manage to do the same with my pressure cooker without
measuring any temperatures or pressures.
Good luck with your yeast ranching. And I will keep writing further posts about yeast ranching and stuff for sure. Promised!
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Cheers, Sam
dbals (@dbals)
Thank you for such an inspiring and informative blog. Just started ranching myself
(3711, 1450, 3944 and 1768) and will be harvesting from several bottles soon!
Thank you for your time and effort,
Dan
eurekabrewing
You are welcome! Thanks for commenting and I hope some of my information can be helpful to you. Good luck with your yeast ranching. Cheers, Sam
G.Pascual
on February 4, 2013 at 10:05 pm said:
Hi Sam, Great work! I was wondering how is your frozen bank progressing. Have
you tested yeast viability after a couple of months stored in the freezer?
eurekabrewing
Hi, actually I have not measured any viabilities (yet). The only evidence that
all works is from all the successfully revived yeasts. Even Brettanomyces survive a freezing period (check out the post from 2nd of February 2013). Time
will tell how this all works on a larger timescale. Let me know if you have experience with a frozen yeast library over a longer period of time.
Thanks for commenting, cheers Sam
G.Pascual
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Ive started with a frozen library around half year ago. My very first
strains were successfully propagated a few times. Initially I took a
sample with the inoculation loop from the eppendorf tube kept in the
fridge and plate it. Then inoculated 10ml wort and propagated it up to
a liter that I use in my batchs. Recently I started inoculating the 10ml
tube directly from the bank (same result and less time) and only plate
to check viability or make new mother cultures. Well.. the thing is that
last week I plated that very first strain and I found few that colonies
grew and it took it more time than usual.. So.. my mother culture is dying after 6 months.. Unfortunatelly, I only have a frost free freezer
available right now (with thaw-cycles). I keep my tubes in a styrofoam
box with ice to amortiguate the temp variations.. but it doesnt seem to
be working that well.. So, Im moving to an isotonic backup until I get
a decent freezer for my yeast.. and Im starting to check viability on all
my strains.. and thats why I wanted to know your experience with it..
eurekabrewing
Thanks for sharing your experiences. Your procedure is very

similar to mine. I am sorry to hear that you observe a decrease of viability of your yeasts. I dont know if there might be
a connection to your freezer with thaw-cycles. Periodical thawing is for sure not great for the yeasts and stresses them a lot.
Still, there might be other reasons such as strain dependency
etc. The isotonic sodium chloride solution worked very well in
my opinion. A good choice for a backup.
Great blog of yours by the way. I can use my Spanish again
Cheers, Sam
Alyssa
on July 17, 2013 at 4:39 am said:
Hello! Excellent post even for those of us experienced in microbio. I work in a TB lab,
so this is all very familiar to me
I am wondering if there are growth log charts
available for different strains somewhere? I am used to being able to take an OD and
it makes me slightly uncomfortable to just wing it while doing upscales. Also, is there
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any practical way to get an OD reading without buying a spectrophotometer or using

a counting chamber? (I kind of hate counting chambers with a passionI blame all
the class labs Ive taken!)
eurekabrewing
on July 17, 2013 at 7:18 pm said:
Hi, I am not aware of any source concerning log charts. I imagine these are
strain dependent anyway. Concerning the OD. I estimate the cell counts from
the yeast volumes. To get the connection between volume and cell count I
use the yeast calculator from mrmalty (http://www.mrmalty.com
/calc/calc.html) and the re-pitching from slurry tab. This is a very easy way to
go. Cheers, Sam
david
on August 8, 2013 at 7:37 pm said:
Thank you. But I wish I had found this about 4 years ago. I got interested in yeast
ranching for the fun of it and just because I could (though I told the SO is was to
save money). Really it was about being able to have the yeast I wanted when I
wanted it and not have to depend on mailing liquid yeast in the middle of the
summer. It was a long road of mis-starts and mistakes. I pasted through the yeast
washing and storing slurry in bottles (very short term) and freezing with glycerin. I
finally found some information on sterile distilled water. Oh, by the way I dont have
any Microbiology background and it took weeks of googling to understand what
streaking was (as opposed to the running around naked of the late 60s and 70s) or
what a slant was. The last time I did any kind of chem lab work was about 40 years
ago! Now I am storing yeast in sterile distilled water. I had not seen anything concern
the .9% saline and osmotic pressure. It is time to re-master my stores and I will
definitely use the isotonic solution. I use the petri dish to isolate the original colony
and use the inoculation loop to pick up a few drops from the yeast bank to streak on
a slant. I will pull all of the colonies from the slant and pitch that to 150ml or so and
then step up to a full starter.
I am planning to experiment with pitching directly from the slant to a full starter. I
dont know of any down side except for the possible risk of contamination due to the
very low concentration of yeast in the 600-1000 ml starter.
Again, thank you for your research and for sharing it. I now have some confirmation
(and more information) that I stumbled onto the right path.
David
22.06.2015 18:12
eurekabrewing
on August 9, 2013 at 7:13 pm said:
David, thanks for reading and your feedback. Always appreciated and keeps
me motivated to write up such methods and such.
Good luck with re-mastering your current bank. I hope everything goes according to plan. By the way, your way of doing it sounds very reasonable to
me. You did your research very well.
Directly pitching from slants to starters. I agree that contaminations are the
most important factor to consider. I do not feel comfortable pitching from
slants directly to >600 mL starters. Simply because of the risk of contaminations. I go with 100 200 mL starters first and then go from there. Simply because I can sterilize the 200 mL starters in my pressure cooker and kill everything. But I would not be surprised either if it works.
Cheers, Sam
Karl
on October 12, 2014 at 4:27 pm said:
Thank you for this post, this is way better than any of the forum posts I have
encountered so far!
Buying small quantities of YPD is very, very expensive.
There are some guides on making your own YPD. Almost all of them use lab
chemicals, which are equally expensive in small quantities for a private individual.
For yeast extract, a few suggest simply autoclaving dry yeast, which would seem like
a good way to make your own yeast extract. Dextrose/glucose is usually sold in food
stores. This leaves the peptone, again very expensive in small quantities.
The peptone is simply a nitrogen source in the form of partially hydrolysed protein.
Could you simply use protein powder form a training store as the nitrogen source?
I would love to see a guide on making a good YPD substitute. Sure you can use malt
extract, but I prefer the empiric approach and eliminating variables.
eurekabrewing
on October 27, 2014 at 3:29 pm said:
Cheers. Dont know if protein from a training store would work as a substitute
for peptone. Never tried that myself. And since my time is very limited lately, I
dont even care to look for MRS (or YPD) substitutes. Its cheaper to just use
the more expensive material if one considers the time one needs to find
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cheaper alternatives. I am therefore sorry that I cannot help you out here.
Better have a look at bkyeast.wordpress.com. Dmitri is looking for media alternatives.
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