[Wort-H.O.G.]

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

[S. cerevisiae]

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:




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



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.

[pete b]

How are you getting it on the slant?

[quattlebaum]

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 $$$. 

[narcout]

If you have a copy of Yeast, it covers the process of creating slants. 

[S. cerevisiae]

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

[S. cerevisiae]

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.

[S. cerevisiae]

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:



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).

[quattlebaum]

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.

[mchrispen]

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.

[S. cerevisiae]

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. 



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.

[quattlebaum]

Much appreciated Mark. It seems all my hobbies are $$$.  Glad there hobbies

[S. cerevisiae]

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.

[a10t2]

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.

[narcout]

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.