[S. cerevisiae]

Is anyone interested in acquiring resuable culture tubes for making slants?  I have been sitting on three shelf packs of new Kimble-Chase 45066A-13100 13x100mm (a little more than 1/2" in diameter by just short of 4" in length) culture tubes (72 tubes per shelf pack).  The 45066A line of culture tubes are Kimble-Chase's top of the line reusable glass culture tubes.  They have phenolic caps with PTFE (teflon)-coated rubber liners.  The pricing for these culture tubes is all over the place, but the lowest price is around a dollar and half each in quantity 288 (usually more in smaller quantities).  The size is conducive to maintaining a nice size bank in a small amount of space.  These tubes are much nicer than the culture tubes that one can acquire via the home brew trade.  The culture tubes that More Beer sells are disposable borosolicate tubes.  One can tell a disposable tube from a reusable tube by the cap material (the glass is also usually thinner on a disposable tube).  Disposable tubes have caps that are made from polypropylene instead of phenolic resin.  Phenolic caps are thermoset, which means that they will not melt or deform once cured. 

[RPIScotty]

What would your price per tube be Mark?

[S. cerevisiae]

I really do not know if I am going to sell the tubes at this point.  I just put the post out there to see if there is any interest.  However, I do not see myself parting with them for much less than $1.50 each.  That's what it would cost me to replace the tubes, and I would have purchase 288 tubes in order to approach that price.  The advantage here would be the ability to acquire a small lot of high quality tubes at a large lot price point. I have never seen these tubes sold in less than pack size (72 tubes).

[RPIScotty]

I would be interested in 8 if enough interest gets created.

Is your slanting method documented here on the forum? I was thinking maybe your "Say No..." post.

[Phil_M]

I'd be interested as well, I've been (slowly) acquiring things to start slanting. I'd be good for $20-$25 worth.

[S. cerevisiae]

I am not looking simply for a way to get rid of these culture tubes.  I am looking to increase the number of amateur brewers who culture yeast.  If I all wanted to do was sell the tubes, I would list them on eBay.

I would like part these tubes out in two dozen lots to brewers who are serious about learning how to isolate yeast and maintain a yeast bank.  Maintaining a yeast bank is a hobby unto itself that requires an investment in time.  Anyone who is solely looking to get into yeast culturing as a way to save money is going to quickly tire of the commitment that is required to maintain a healthy yeast bank.

Why am I limiting the lots to two dozen?   Well,  two dozen is really the smallest number of tubes that someone who is seriously interested in culturing should own.  Yeast strains should be banked two tubes per strain or culture.  One wants to have a back up in case a slant goes south, and one needs to have blank slants available for periodic subculturing because slanting is not a "set and forget it" way to maintain yeast.   Two dozen culture tubes will allow a brewer to maintain up to about eight cultures, but six is more usual.  It is difficult to prepare media for less than a dozen slants because one is only going to use about 3 to 4 milliliters of media per tubes.  The tubes only hold 8ml, and one only fills the tubes 1/3rd to 40% full.  I use a straight tip irrigation syringe to fill my tubes.  This type of tip is known as a catheter tip.



I suggest heating the media up in a 2 to 4 cup glass measuring cup in a microwave.  One only needs to heat the media until the agar melts.  The pressure cooker will take care of rendering the media absolutely sterile.

Simple Solid Media

pale DME - 5% w/v 
agar flakes or powder - 1.5% w/v


Solid MYGP

pale DME - 0.3% w/v
yeast extract - 0.3% w/v
soy-based peptone - 0.5% w/v
dextrose - 1.0% w/v
agar power - 1.5% w/v


One last thing, one should not transfer a liquid culture directly to slant.  A liquid culture is plated for "singles" using a technique known as streaking.  Well-isolated single colonies are transferred to slant.

Southern Tier's yeast strain in a 60mm x 15mm petri dish



The well-isolated colonies (technically colony forming units, or CFUs) in the red box are each the offspring of a single yeast cell; hence, they are pure cultures.


Scottish and Newcastle's strain in 100mm x 10mm petri dish



The colonies in the lower right hand quadrant are the candidates for transfer to slant.

There are several videos on YouTube that demonstrate how to streak plates and perform aseptic transfers over a flame source.  I suggest starting out with a flint glass alcohol lamp. 

I like this style the best (http://prolabscientific.com/Flint-Glass-Alcohol-Lamps-p-18335.html)



Even a small Bunsen burner produces an intense flame that may be too much for someone who is just starting out.  I would practice sterilizing the loop to get a feel for how it is performed before attempting a transfer.  Like learning to ride a bicycle or roller skate, there is a learning curve with aseptic technique.  However, just like learning how to ride a bicycle or roller skate, it is a set movements that one's hands remember even after long layoffs.

With the above said, while I am not looking to to make money on this venture, I am also not looking to lose  money either.  I need to look into shipping because I would like to find a way to bring the total cost down to around $35.00 including shipping.

[RPIScotty]

Sounds good Mark. Keep us posted. Great info.

What is it about those isolated parts of the colonies above that make them ideal?


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[S. cerevisiae]

What is it about those isolated parts of the colonies above that make them ideal?

A well isolated colony is almost guaranteed to be a pure culture.  Once transferred to a slant, an isolated colony becomes what is known as an isolate.  A lot of brewers have used W-34/70.  W-34/70 is the 70th isolate of strain number 34.  When I plate a culture, I usually transfer one colony per slant.  I usually pick the slant that has the most robust growth to use for further propagation.  The use cycle is as follows

1) Pick the slant that has the most robust growth
2) Subculture two new slants from this slant
3) Inoculate 20 to 40mls of autoclaved wort from the slant
4) Discard the slant that was subcultured and used to make the starter (i.e., the other old slant for this culture can be discarded at this point, or one can wait until the subcultured slants proof before discarding it)
5) Incubate the slants (caps loose to allow for offgassing) and the 20 to 40ml starter

The key is to always propagate the healthiest slant.  There is no need to propagate both slants in a pair (that move would result in an ever growing number of slants  for each culture).

[RPIScotty]

Yet another thread to bookmark. Thanks again Mark.


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[Phil_M]

My initial amount I'd be willing to spend was based on not understanding just how many tubes I'd need.

I'd be willing to spend more, and will be following this thread closely.

[S. cerevisiae]

Phil, you have to look at this cost as the beginning of the cost of culturing.  You will still need an inoculation loop, a flame source, a pressure cooker that is capable of generating at least 13 PSI above normal atmospheric pressure (13 PSI requires 25 minutes of autoclaving whereas 15 PSI shortens the processing time to 15 minutes), and plates.  I recommend starting out with pre-poured plates.  Making plates correctly is more difficult than making slants, and you going to have your hands full with mastering the basics.

I guess what I am attempting to do here for a select group of brewers is what Maribeth Raines and Jeff Mellem did for many more brewers back in the 90s without having to set up a company to do so.  I do not want to be in the yeast or yeast culturing business, but I welcome anyone who wants to give it a shot.   

The beauty of learning how to plate and slant is that one is no longer limited to the cultures that one can acquire through the home brewing trade.  For example, if you are lucky enough to capture a wild culture that you like, you can plate the culture, select individual colonies, and test to see which ones you like.  A wild culture on a plate is going to be potpourri of colors and textures because it is more than one strain and/or type of microflora.   You can also use selective media.

[Phil_M]

Yep, I've been shopping around/pricing things out for a while. Back in the spring I thought I'd go ahead and pull the trigger on much of the gear, then you recommended not culturing over the summer here in MD. Now that the weather has started to cool off I've been thinking about culturing again, and your post seemed well timed.

On that note, any advice on pressure cookers? It seems most household ones *should* reach the desired pressures, but many are aluminum. I'm assuming eventually fatigue would be a (scary) concern.

[erockrph]

The beauty of learning how to plate and slant is that one is no longer limited to the cultures that one can acquire through the home brewing trade.  For example, if you are lucky enough to capture a wild culture that you like, you can plate the culture, select individual colonies, and test to see which ones you like.  A wild culture on a plate is going to be potpourri of colors and textures because it is more than one strain and/or type of microflora.   You can also use selective media.

This is the part of yeast wrangling that I'd be interested in dipping my toes into. There are a few mixed-culture beers that I'd love to be able to isolate the Brett strain(s) from (Orval & Girardin in particular).

Mark, any recommendations on where to get pre-poured plates?

[RPIScotty]

Some quick research on Amazon shows ~$45 additional investment for flame source, 3 loops and a pack of 10 pre-poured plates.


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[S. cerevisiae]

Mark, any recommendations on where to get pre-poured plates?

Here is a company that is selling pre-poured malt agar plates in 60mm and 90mm diameter dishes.  I personally prefer 90mm or 100mm diameter dishes.  They are easier to streak for singles.

https://www.emlab.com/app/store/Store.po?event=showItem&item=2

The way that Kai shows people how to make plates on his web site is a no-no.  Plates should never be sterilized in an autoclave.  That is a recipe for plates that contain so much condensation that contamination is going to be an ever present threat.  No one who has ever worked in a lab pours plates that way, which why Kai lost a huge amount of credibility with me, that, and the fact that he uses the wrong piece of lab glassware for slants.  A screw cap culture tube has a deep-skirted cap for a reason.

A reusable borosilicate glass culture tube on the left with a disposable borosilicate culture tube on the right (the differences are not obvious to the untrained eye)




Glass dishes are dry sterilized in an oven at 350F for 90 minutes, and allowed to cool to 150F while the media is being autoclaved in a separate covered container.  I hold my Petri dishes at 150F after the sterilization step to reduce the amount of condensation that forms when the plates are poured.  The media is removed from the autoclave and allowed to cool to between 120F and 140F before I remove the plates from the oven.  I usually light an alcohol lamp to create an updraft where I am pouring.  The pouring process involves lifting one side of the cover up high enough to be able pour enough media to cover the bottom of the dish to a depth of about 1/8th of an inch, and then quickly placing the cover back down on the dish.  It takes a little practice to be able to perform the technique as one fluid movement, but anyone who is willing to put forth the effort can learn it.   The beauty of this technique is that it works for presterlized plastic dishes as well.  Presterilized plastic dishes eliminate the dish sterilization step.

Kai's plates



Did you notice the large water droplets?  Those droplets are a contamination event in the making because a plate is stored upside down, turned over for streaking, incubated upside down, and turned back over inspection. The seal between the cover and the bottom of the dish is not air tight.  Anything that gets between the cover and the bottom while the plate is upside down can end up on the surface of the media via condensation on the lid when the plate is turned over.
 
A set of plates that I poured using the technique outlined above



Did you notice that there is only a thin film of condensation?  This condensation will disappear during the proofing step.

Now, those who have small children can save quite a bit of money on small media bottles for first-level autoclaved starter media by using delabled 4oz baby food jars (Goo Gone will soften the glue).  Baby food jars are designed to be autoclaved. I used 4oz baby food jars for a long time.  The only downside to using baby food jars is that the threads are formed in the rubber liner when the jar and lid cool.  The threads disappear when the jar is processed in a pressure cooker, so one has to periodically keep screwing the lids down as the jars cool.

First-level starters in 2.5oz baby food jars (this size is too small)