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General Category => Yeast and Fermentation => Topic started by: pete b on June 03, 2015, 07:21:48 am

Title: Keeping Yeast Slurry Long Term
Post by: pete b on June 03, 2015, 07:21:48 am
I have not brewed since April and cannot brew anytime soon. I have a large kitchen renovation/addition project that unfortunately could not happen until spring when I had to get the garden in etc. and then my girlfriends brother died suddenly last week which of course brought emotional trauma and a lot of practical things we need to do to help the family.
I have a lot of yeast slurrys which I normally use successfully as is from the fridge within 3-6 weeks. I have a couple question about using and storing months old slurry. Should I feed them kind of like sourdough starter (but with wort) from time to time? I won't be trying to use them as is, I'll make a starter when I get to them: what's the best method in this case? Use a couple tablespoons and step up? Use all of it (maybe 300 ml) and make directly into a pitchable sized starter? Any other advice?
Title: Re: Keeping Yeast Slurry Long Term
Post by: brewinhard on June 03, 2015, 07:23:47 am
How much yeast approximately do you have saved in each container?  Are these slurries saved from the primary fermenter of previous batches?
Title: Re: Keeping Yeast Slurry Long Term
Post by: a10t2 on June 03, 2015, 09:28:59 am
You can store yeast under beer in a sealed container for years as long as your sanitation is good, although at this scale, if you have access to fresh yeast it's probably worth the $7.

Ideally you'd want the beer in question to be 2-3% ABV like from a starter. Don't feed the cultures - it's a contamination point and the whole idea of long-term storage is to keep the cells dormant. When you want to use the yeast just put a couple tablespoons in a starter. Unless it's a large (more than ~2 L) starter I wouldn't worry about stepping up. Some yeast nutrient in the starter can't hurt either.
Title: Re: Keeping Yeast Slurry Long Term
Post by: Joe Sr. on June 03, 2015, 11:22:27 am
I agree with Sean.  I store my yeast in exactly the fashion you are describing and regularly use slurries that are months old.  When they get beyond a yearI will typically toss them but more because that means its a strain I'm not using much than because I worry about sanitation or viability.

Make a starter.  Use a small amount and use some yeast nutrient.

I've found this approach works much better than trying to wake up the entire slurry.

There is no need to feed the yeast during storage and I would not recommend doing that.
Title: Re: Keeping Yeast Slurry Long Term
Post by: pete b on June 03, 2015, 11:27:21 am
How much yeast approximately do you have saved in each container?  Are these slurries saved from the primary fermenter of previous batches?
About 300m and yes.
Title: Re: Keeping Yeast Slurry Long Term
Post by: pete b on June 03, 2015, 11:33:35 am
You can store yeast under beer in a sealed container for years as long as your sanitation is good, although at this scale, if you have access to fresh yeast it's probably worth the $7.

I save yeast so I have a variety around as much if not more than to save money. I don't live near a lhbs.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 03, 2015, 11:58:13 am
I have been experimenting with using the double-drop technique to start slurries that are older than 5 weeks from the date that the batch was pitched.

Here's my process:

1.) Pitch the entire slurry (100 to 150ml of solids) into starter wort
2.) Separate the liquid fraction from the sediment when signs of low krausen appear
3.) Pitch the separated liquid fraction at high krausen 

This technique allows me to capture a large number of viable cells from the slurry while discarding the non-viable cells and all of the remaining break and organic matter.  I have yet to try it with a really old (> 6 months) slurry because it is just as easy to grow a new culture from slant.  A really old culture is going to have lysed cells in it.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 03, 2015, 12:13:53 pm
I save yeast so I have a variety around as much if not more than to save money. I don't live near a lhbs.

If I were you, I would look into maintaining a small bank of cultures on slant.  Aseptic technique is relatively easy to learn.

I started plating and slanting yeast back in 1993, so that I could have easy access to a variety of yeast strains as well as brewery strains that were not available via the home brew trade. 
Title: Re: Keeping Yeast Slurry Long Term
Post by: pete b on June 04, 2015, 04:40:30 am
I have been experimenting with using the double-drop technique to start slurries that are older than 5 weeks from the date that the batch was pitched.

Here's my process:

1.) Pitch the entire slurry (100 to 150ml of solids) into starter wort
2.) Separate the liquid fraction from the sediment when signs of low krausen appear
3.) Pitch the separated liquid fraction at high krausen 

This technique allows me to capture a large number of viable cells from the slurry while discarding the non-viable cells and all of the remaining break and organic matter.  I have yet to try it with a really old (> 6 months) slurry because it is just as easy to grow a new culture from slant.  A really old culture is going to have lysed cells in it.
how much wort? 1 litre? This seems sound.
Title: Re: Keeping Yeast Slurry Long Term
Post by: pete b on June 04, 2015, 04:46:38 am
Mark, I have been interested in doing this since some of your early posts showing some of your collection. Right now I I'm in a phase of life where I need to carve out some time because I'm a bit too busy. I can picture doing this in a few years.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 04, 2015, 07:16:42 am
how much wort? 1 litre? This seems sound.

I use 1 liter of 10% w/v (1.040) wort and my "shaken, not stirred" technique with a 5L media bottle; however, a 1-gallon glass jug will work.

Note: For those who live in the UK, a 1-gallon American jug is what you refer to as a 1-gallon demijohn.  A British jug is called a "pitcher" in the U.S.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 04, 2015, 08:05:36 am
Mark, I have been interested in doing this since some of your early posts showing some of your collection. Right now I I'm in a phase of life where I need to carve out some time because I'm a bit too busy. I can picture doing this in a few years.

I had to quit brewing for several years due to demands on my time, so I know what you are up against.

With that said, once you get setup and start maintaining your own bank on slant, you will kick yourself for not doing it sooner.  The basic skills necessary to prepare absolutely sterile media and plate/slant yeast are high school-level biology.   

I was basically in the same situation that you are today when I first started to maintain a yeast bank.  At that point in time, anything other canned kits, questionable dry yeast, and oxidized hops was considered to be exotic.  White Labs did not exist, and the Wyeast catalog could be enumerated using two hands.  Many Wyeast strains were difficult to obtain due to the perishable nature of liquid yeast and the relative immaturity of the market.   

With that said, things changed rapidly after 1993.  However, the impetus for that change was not the AHA.  The driving force behind the rapid technological advancement within the hobby was a magazine called Brewing Techniques (compare Zymurgy articles from the same period with those published in Brewing Techniques, and you will see what I mean).  I did not bother to join the AHA during my first pass through the hobby because I did not care for Zymurgy (I still only scan the magazine). While the quality of the articles in Zymurgy has improved greatly over the years, it's still more of an entry level/brewing lifestyle magazine than a true nuts and bolts small-scale brewing magazine. The community needs a modern version of Brewing Techniques.  Neither BYO nor Zymurgy fills the void that was created when the publisher of Brewing Techniques shuttered its doors.  In my humble opinion, the BA/AHA should consider offering a subscription above and beyond the cost of membership technical journal much in the way that the Amateur Radio Relay League (ARRL) offers QEX (the ARRL's lifestyle magazine is called QST).  I would write for such a journal.
Title: Re: Keeping Yeast Slurry Long Term
Post by: a10t2 on June 04, 2015, 11:08:19 am
I save yeast so I have a variety around as much if not more than to save money. I don't live near a lhbs.

I'm in the same boat. 250 miles round-trip to my "local" homebrew shop. I tend to mail-order new cultures once every year or two, in either the spring or fall when shipping temperatures are reasonable.
Title: Re: Keeping Yeast Slurry Long Term
Post by: erockrph on June 04, 2015, 11:14:26 am
I save yeast so I have a variety around as much if not more than to save money. I don't live near a lhbs.

I'm in the same boat. 250 miles round-trip to my "local" homebrew shop. I tend to mail-order new cultures once every year or two, in either the spring or fall when shipping temperatures are reasonable.
My LHBS is open hours that generally conflict with my work/family schedule, so I'm in the same boat. I keep a stash of dry yeast on hand for short-notice brews, and try to brew a couple of successive brews when I use liquid yeast. For strains I like but can't get a hold of easily, I keep a few mason jars of the yeast cake from a previous batch and store them in the fridge. I've had good luck with most strains just growing up a starter from these.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 04, 2015, 11:23:29 am
I'm in the same boat. 250 miles round-trip to my "local" homebrew shop. I tend to mail-order new cultures once every year or two, in either the spring or fall when shipping temperatures are reasonable.

Two hundred and fifty miles to the nearest home brew supply store?  Man, that's hardcore.

I maintained several cultures in my original bank for over a decade.  I kick myself for allowing a few of the cultures in that bank to expire.  For example, I had the two major strains in real Ringwood isolated on different slants.  I kept Brewtek CL-170 and Brewtek CL-660 alive for most of the time that that bank was in existence much in the same way that Denny kept Brewtek CL-50 alive long enough to convince Wyeast to package it as Wyeast 1450.  I kept Brewtek CL-210 alive for a long time as well.
Title: Re: Keeping Yeast Slurry Long Term
Post by: Wort-H.O.G. on June 05, 2015, 11:10:48 am
I have been experimenting with using the double-drop technique to start slurries that are older than 5 weeks from the date that the batch was pitched.

Here's my process:

1.) Pitch the entire slurry (100 to 150ml of solids) into starter wort
2.) Separate the liquid fraction from the sediment when signs of low krausen appear
3.) Pitch the separated liquid fraction at high krausen 

This technique allows me to capture a large number of viable cells from the slurry while discarding the non-viable cells and all of the remaining break and organic matter. I have yet to try it with a really old (> 6 months) slurry because it is just as easy to grow a new culture from slant.
  Mark- can you explain what it means to do this^^^^^^ grow from slant. thanks
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 05, 2015, 03:51:21 pm
Growing a new culture from slant involves taking a small amount of yeast from the surface of an agar slant with a laboratory device known as a loop and using the yeast to inoculate a small volume of autoclaved (pressure cooked) wort (the operation is performed using what is known as aseptic transfer technique).  The inoculated wort is incubated for a day or two depending on strain and slant age before being stepped up in volume.   Many breweries that maintain their own cultures use this procedure when they need to grow a new culture.

Here’s what yeast looks like when grown on the surface of agar solidified wort:

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/CCyeast1_zpsdc754fa7.jpg)


Here’s what I inoculate with yeast scraped from a slant:

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/MediaBottle_zpsdff03f83.jpg)

The 5% w/v (1.020) wort on the 100ml media bottle has been autoclaved; hence, it is absolutely sterile.  An aseptic transfer is a sterile transfer.

By the way, I have switched to using an agar solidified laboratory medium called MYGP since shooting the photograph shown above.  MYGP stands for Malt extract, Yeast extract, Glucose, and Peptone.  While I do not have enough data at this point, MYGP does appear to increase the length of time that a culture remains viable on slant.


Title: Re: Keeping Yeast Slurry Long Term
Post by: pete b on June 06, 2015, 05:39:18 am
How are you getting it on the slant?
Title: Re: Keeping Yeast Slurry Long Term
Post by: quattlebaum on June 06, 2015, 06:36:38 am
how much wort? 1 litre? This seems sound.

I use 1 liter of 10% w/v (1.040) wort and my "shaken, not stirred" technique with a 5L media bottle; however, a 1-gallon glass jug will work.

Note: For those who live in the UK, a 1-gallon American jug is what you refer to as a 1-gallon demijohn.  A British jug is called a "pitcher" in the U.S.

Do you have any suggestions on where i/WE can get Media bottles for a reasonable $. It seems that i can only ever find bulk cases for $$$. 
Title: Re: Keeping Yeast Slurry Long Term
Post by: narcout on June 06, 2015, 09:18:45 am
If you have a copy of Yeast, it covers the process of creating slants. 
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 06, 2015, 10:57:36 am
Do you have any suggestions on where i/WE can get Media bottles for a reasonable $. It seems that i can only ever find bulk cases for $$$.

If you find a cheap source for media bottles, please let me know.  I believe that I paid close to $100.00 for ten 100ml media bottles with shipping.  I paid $80.00 for my 5L media bottle, and it was NOS surplus.  I would avoid purchasing used media bottles because one does not know what was previously stored in the bottle.

I used 4oz baby food jars for my first-level starters for a very long time.  The liner on a baby food jar will withstand being processed in a pressure cooker for 15 minutes.  It's just a pain in the backside to get the lids to vacuum seal after removing them from a pressure cooker.  The threads are formed in the rubber liner when the jar and lid cools.  The liner returns to its original state when pressure cooked, which means that one has to keep screwing the lid down periodically while the jar and its contents are cooling to ensure that the jar vacuum seals.

With that said, there are a couple of companies that manufacture aftermarket replacement lids for baby food jars.  Baby food jars are used in plant tissue culture. 

These caps are made of polypropylene; therefore, they will hold up to autoclaving.  You need to make sure that you order the non-vented caps.

http://phytotechlab.com/index.php/equipment/culture-vessels/culture-vessel-closures/jar-closures/closure-i-phyto-i-cap-trade-baby-food-jar-closure.html
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 06, 2015, 11:01:48 am
Here's a photo that I shot of the last batch of first-level media that I made using baby food jars.  The jars used here are 2.5oz jars, which I do not recommend.

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/22mlSterileStarters_zps5c1db602.jpg)
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 06, 2015, 12:13:32 pm
How are you getting it on the slant?

Yeast is transferred to a slant by streaking cells taken from another pure culture source onto the surface of the slant using a nichrome loop.  Contrary to what White Labs and Wyeast claim, their cultures are not 100% pure (no mass-produced culture is 100% pure 100% of the time), which means that one should plate liquid cultures for single colonies.  Plating for singles is a standard laboratory procedure when transferring a liquid culture to slant.  The cells are spread out on the plate using a one of several different streaking patterns.

Here's a video that demonstrates a three-sector streak:

https://www.youtube.com/watch?v=Ay2hhujTuvg

The technique shown in the video is used to isolate yeast in addition to bacteria.  The technique was originally developed to isolate bacteria, and was later adopted for isolating yeast.

Here's a plate that I streaked with a culture from Scottish and Newcastle's Tyneside Brewery:

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/SandNYeast_zpsc0067d33.jpg)

The well-isolated round colonies in the lower right-hand corner of the photo shown above are all the offspring of single yeast cells; therefore, each colony is a single-strain pure culture.  That's what we want to transfer to a blank slant using aseptic technique. Yeast will cover the entire surface (or at least most of it) of the slant after it has been streaked and incubated.  From this point forward, we should not have to re-plate the culture.  We only have to perform periodic sub-culturing to keep it alive.  Sub-culturing is process by which yeast is taken from one slant and used to inoculate (streak) another slant using aseptic technique. 

My goal is to be able to piggyback a starter propagation event on top of a sub-culturing event, which means that I attempt to use my cultures in a steady rotation, so that I do not have to perform sub-culturing-only events.  Each culture needs to be sub-cultured to a new slant at least once every year.  The more that I can piggyback making a starter on top of a slant-to-slant sub-culture the better.   How frequently one brews will determine the number of cultures that one can maintain through normal use without resorting to pure sub-culturing events.   I had over forty cultures in my first bank.  I was sub-culturing all of the time.  I am attempting to limit this bank to a dozen cultures.  I usually keep two copies (two slants of each strain).  I currently prepare twenty slants and six plates every time I make solid media, which means that I need at least 44 cultures tubes (I have multiples of this number at my disposal).


Title: Re: Keeping Yeast Slurry Long Term
Post by: quattlebaum on June 06, 2015, 05:22:16 pm
Do you have any suggestions on where i/WE can get Media bottles for a reasonable $. It seems that i can only ever find bulk cases for $$$.

If you find a cheap source for media bottles, please let me know.  I believe that I paid close to $100.00 for ten 100ml media bottles with shipping.  I paid $80.00 for my 5L media bottle, and it was NOS surplus.  I would avoid purchasing used media bottles because one does not know what was previously stored in the bottle.

I used 4oz baby food jars for my first-level starters for a very long time.  The liner on a baby food jar will withstand being processed in a pressure cooker for 15 minutes.  It's just a pain in the backside to get the lids to vacuum seal after removing them from a pressure cooker.  The threads are formed in the rubber liner when the jar and lid cools.  The liner returns to its original state when pressure cooked, which means that one has to keep screwing the lid down periodically while the jar and its contents are cooling to ensure that the jar vacuum seals.

With that said, there are a couple of companies that manufacture aftermarket replacement lids for baby food jars.  Baby food jars are used in plant tissue culture. 

These caps are made of polypropylene; therefore, they will hold up to autoclaving.  You need to make sure that you order the non-vented caps.

http://phytotechlab.com/index.php/equipment/culture-vessels/culture-vessel-closures/jar-closures/closure-i-phyto-i-cap-trade-baby-food-jar-closure.html
What's your thoughts on the nalgene square Lexan bottles for media 4oz?  I know Pyrex is the way to go but these are plentiful in my are for cheep.
Title: Re: Keeping Yeast Slurry Long Term
Post by: mchrispen on June 06, 2015, 06:24:53 pm
Mark, seriously, consider writing a book, digest - or at least a blog. Your insight in really valuable. Not only on the application side, but also the history.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 06, 2015, 09:53:26 pm
What's your thoughts on the nalgene square Lexan bottles for media 4oz?  I know Pyrex is the way to go but these are plentiful in my are for cheep.

Borosilicate glass is the only way to go when culturing yeast.  Quality borosilicate glassware will last a very long time when used in a home lab. I used the first batch of glassware that I purchased for over ten years before selling it.

With respect to culture tubes, only purchase true culture tubes.  A lot of brewers, including Kai, use vials.  Vials were designed for collecting samples.  Culture tubes were designed for culturing microbes.  A culture tube looks like a test tube with a screw-on cap.  A culture tube has a deep cap that was designed for culturing microbes.

There are two types of culture tubes being sold today. The first type is known as a reusable culture tube.  A reusable culture tube is manufactured using thick borosilicate glass (borosilicate glass is also known as Pyrex or Kimax glass).  A reusable culture tube will have a cap that is made from phenolic resin impregnated wood fiber (an early thermoset plastic known as bakelite).  Disposable (single-use) culture tubes are made from thinner borosilicate glass and have polypropylene caps.

It is not easy for the layman to be able to distinguish between a reusable culture tube and a disposable culture.  The culture tube on the left in the photo shown below is a Corning 9825 reusable culture tube.  The culture tube on the right is a generic disposable culture tube that one can find on eBay for a reasonable price. 

(http://i699.photobucket.com/albums/vv356/tonestack/Brewing/SCCT1_zps2cc12802.jpg)

A re-usable culture tube costs many times that of a disposable (single use) culture tube.  eBay sellers take advantage of unknowing buyers to charge a premium price for disposable culture tubes.   When purchasing culture tubes, look for Corning numbers 9825 and 9826 or Kimble Chase numbers 45066 or 45066A.  I am partial to Corning lab glassware.  Corning is the Mercedes of lab glassware.  Corning owns the Pyrex brand name.  A Corning purchase is buy once, cry once purchase.   I have a Corning 4980-500 500ml Erlenmeyer flask that is 22 years old.


Title: Re: Keeping Yeast Slurry Long Term
Post by: quattlebaum on June 07, 2015, 06:01:57 am
Much appreciated Mark. It seems all my hobbies are $$$.  Glad there hobbies :)
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 07, 2015, 10:45:54 am
Assembling a home lab does not have to be ridiculously expensive.  One just needs to purchase wisely.  Biotech is a volatile industry. Biotechs and liquidators that feed on the carcasses of dead Biotechs routinely dump surplus NOS labware on eBay.  The trick is being in a position to purchase when the good stuff is listed for a decent price.
Title: Re: Keeping Yeast Slurry Long Term
Post by: a10t2 on June 07, 2015, 11:28:46 am
If you have a decent-sized university nearby, you also have an auctioneer or surplus store or something along those lines that has a ton of cheap used glassware.
Title: Re: Keeping Yeast Slurry Long Term
Post by: narcout on June 07, 2015, 12:18:49 pm
If you have Amazon Prime, you can get 100ml Pryex media bottles for $12.50 each (including shipping).

Right now on Ebay, someone is selling two boxes (20 bottles - new) for $130 (including shipping) which is only $6.50 per bottle.
Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 07, 2015, 01:00:53 pm
Corning 100ml media bottles can be had for $10.27 each on Amazon as well, but one has to spend $35.00 to get free shipping.

With that said, anyone who is thinking about purchasing media bottles should think about purchasing a minimum of 10 (which is a case when ordering 100ml media bottles).  It does make sense to prepare sterile starter media one bottle at a time.   I purchased my media bottles from Capitol Scientific.  I believe that I paid about $15.00 in shipping.  The Corning  number for 100ml media bottles is 1395-100.  The 1395 part of the number is the type of glassware, which is media bottle in this case.  The 100 is the volume of this type of glassware.

http://www.capitolscientific.com/Corning-1395-100-PYREX-100mL-Round-Media-Bottles-with-GL45-Screw-Cap-Graduated


Title: Re: Keeping Yeast Slurry Long Term
Post by: S. cerevisiae on June 07, 2015, 01:03:25 pm
This case price with shipping is better than the Capitol Scientific case price without shipping.  That's about as cheap as one is going to find new 1395-100 media bottles.

http://www.amazon.com/PYREX-100mL-Round-Storage-Bottles/dp/B004XR5WAO/ref=sr_1_3?ie=UTF8&qid=1433703704&sr=8-3&keywords=1395-100
Title: Re: Keeping Yeast Slurry Long Term
Post by: MoreHops on June 12, 2021, 01:37:42 pm
This case price with shipping is better than the Capitol Scientific case price without shipping.  That's about as cheap as one is going to find new 1395-100 media bottles.

http://www.amazon.com/PYREX-100mL-Round-Storage-Bottles/dp/B004XR5WAO/ref=sr_1_3?ie=UTF8&qid=1433703704&sr=8-3&keywords=1395-100

Just clicked on this link and found the 250 ml bottles selling for $36 for a case of 10 - someone jump on whatever is left (might only be 1 case remaining). 
Title: Re: Keeping Yeast Slurry Long Term
Post by: MoreHops on June 12, 2021, 01:43:52 pm
This case price with shipping is better than the Capitol Scientific case price without shipping.  That's about as cheap as one is going to find new 1395-100 media bottles.

http://www.amazon.com/PYREX-100mL-Round-Storage-Bottles/dp/B004XR5WAO/ref=sr_1_3?ie=UTF8&qid=1433703704&sr=8-3&keywords=1395-100

Just clicked on this link and found the 250 ml bottles selling for $36 for a case of 10 - someone jump on whatever is left (might only be 1 case remaining).

Looks like this is unfortunately back to the normal price now