Slanting yeast

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Sterile technique is critical. Of the two books I listed, I most highly recommend Rajotte's as it is thorough but brief. Assuming you also have access to a pressure cooker with a gauge, you should have no problem with contamination. The selection of an isolated colony ensures that you have a pure culture derived from a single cell.

I'll have to get that book. I do have a pressure cooker, I use it to make my slants and also to can my starter wort, so sterile technique isn't an issue.

I was more concerned the fact that I can't pressure cook the petri dishes like I can the slants. Also about airborne stuff when I plate the yeast, then again when I transfer it to the slant.

Finally, last time I tried to make plates, I failed and didn't end up with any single cell colonies, just thick smears of yeast. I did use small petri dishes for that though, now I have some larger ones.

I have a bunch of empty slants I need to make an agar medium for though, so I will definitely fill some petri dishes as well and give it a try.
 
Here are my 1st couple of slants. The yeast looks nice and white, but for some reason my slants are accumulating fluid at the bottom of the vials. Even the newer ones I have made with more agar are doing this. When putting the yeast in the vial, I poured out any liquid that had accumulated by condensation, so I'm not sure what is going on and whether it's a problem.

2013-10-16143451.jpg

Why are you using vials? The proper tool for the job is a piece of lab glassware known as a screw-cap culture tube. A screw-cap culture tube looks like a test tube with a screw-on cap that has an autoclavable rubber liner. What kind of liner does that vial have?

Here's a photo that I took a few months ago when I made plates and slants:

slants_zpsd8559e74.jpg


Additionally, you should dump the Asian food store-grade agar. It's junk. Ideally, you should use lab-grade powdered agar. eBay seller solared2011 is selling 100 grams of lab-grade powered agar for $12.99 + $0.86 shipping (http://www.ebay.com/itm/100-Grams-0...326?pt=LH_DefaultDomain_0&hash=item20ce3e94ee), which is a very good deal because 100 grams of lab-grade agar will make 6.67 liters of malt agar. That's enough media to last an active amateur brewer ten years because 250ml of malt agar media will make twenty 20x125mm culture tube-based slants and six 100x10mm plates. If you must use non-lab-grade agar, then look for agar flakes in a health food store. Agar flakes, while not as pure as lab-grade powered agar, work very well.

Lab-grade agar is so pure that one only needs to 1.5% agar weight/volume when making malt agar media. A simple recipe for malt agar is 10% light dry malt extract (DME), 1.5% agar, and 0.1% yeast extract (yeast energizer also works). Putting those ratios into practice, in order to make one liter of malt agar, we need to use 100 grams of DME, 15 grams of agar, and 1 gram of yeast nutrient. I make up 250mls of malt agar media at a time, so I use 25 grams of DME, 3.5 grams of powdered agar, and 1/8th teaspoon of yeast extract.

With that said, it looks like your media is breaking down. How much agar did you use? How long did you autoclave your media? One only needs to autoclave media for 15 minutes at 100C/212F @ 15 PSI. While a gauge is nice to have, any quality pressure cooker will hit this temperature and pressure without a gauge. I have been using the same T-FAL/SEB 3125 stainless pressure cooker to sterilize media at home for over twenty years without single infection that could be traced back to the pressure cooker.

Finally, the proper tool to use when inoculating slants is a nichrome loop. A decent nichrome loop can be had for a few dollars. A nichrome loop is sterlized by heating it until it is red hot. Also, ALL LIQUID YEAST CULTURES should be plated for "singles" before transferring well isolated colonies to slants. Contrary to what you may believe, packages of Wyeast yeast and preforms of White Labs yeast are not 100% pure yeast. Plating for singles and using well isolated colonies that exhibit good morphology to inoculate your slants using aseptic technique will ensure that nothing else grows on your slants.
 
Why are you using vials? The proper tool for the job is a piece of lab glassware known as a screw-cap culture tube. A screw-cap culture tube looks like a test tube with a screw-on cap that has an autoclavable rubber liner. What kind of liner does that vial have?

Here's a photo that I took a few months ago when I made plates and slants:

slants_zpsd8559e74.jpg


Additionally, you should dump the Asian food store-grade agar. It's junk. Ideally, you should use lab-grade powdered agar. eBay seller solared2011 is selling 100 grams of lab-grade powered agar for $12.99 + $0.86 shipping (http://www.ebay.com/itm/100-Grams-0...326?pt=LH_DefaultDomain_0&hash=item20ce3e94ee), which is a very good deal because 100 grams of lab-grade agar will make 6.67 liters of malt agar. That's enough media to last an active amateur brewer ten years because 250ml of malt agar media will make twenty 20x125mm culture tube-based slants and six 100x10mm plates. If you must use non-lab-grade agar, then look for agar flakes in a health food store. Agar flakes, while not as pure as lab-grade powered agar, work very well.

Lab-grade agar is so pure that one only needs to 1.5% agar weight/volume when making malt agar media. A simple recipe for malt agar is 10% light dry malt extract (DME), 1.5% agar, and 0.1% yeast extract (yeast energizer also works). Putting those ratios into practice, in order to make one liter of malt agar, we need to use 100 grams of DME, 15 grams of agar, and 1 gram of yeast nutrient. I make up 250mls of malt agar media at a time, so I use 25 grams of DME, 3.5 grams of powdered agar, and 1/8th teaspoon of yeast extract.

With that said, it looks like your media is breaking down. How much agar did you use? How long did you autoclave your media? One only needs to autoclave media for 15 minutes at 100C/212F @ 15 PSI. While a gauge is nice to have, any quality pressure cooker will hit this temperature and pressure without a gauge. I have been using the same T-FAL/SEB 3125 stainless pressure cooker to sterilize media at home for over twenty years without single infection that could be traced back to the pressure cooker.

Finally, the proper tool to use when inoculating slants is a nichrome loop. A decent nichrome loop can be had for a few dollars. A nichrome loop is sterlized by heating it until it is red hot. Also, ALL LIQUID YEAST CULTURES should be plated for "singles" before transferring well isolated colonies to slants. Contrary to what you may believe, packages of Wyeast yeast and preforms of White Labs yeast are not 100% pure yeast. Plating for singles and using well isolated colonies that exhibit good morphology to inoculate your slants using aseptic technique will ensure that nothing else grows on your slants.

Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!
 
DWhitwell said:
Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!

Yes... And thank you for your contribution as well.
 
Whew! That is a whole lot of words to tell us that the way the original poster described his slanting technique has led us all astray! I'm glad I didn't read this first, or I would have never started slanting!

You can look at it that way, or you can look at my posting as a clarification of the process. There is a lot of rigor involved in maintaining a healthy yeast bank. One needs to ensure that the only microflora on one's slants are the reference cultures, and the only way to ensure that level of purity is to plate every liquid culture before transferring it to one or more slants. All commercial yeast cultures contain low levels of contamination. It is not economically feasible for the yeast manufacturers to ship lab-grade cultures at home brew trade price points, but that's exactly what one needs to plate if one wants to bank a culture for an indefinite amount of time.

Here's a plate that I made from a culture that I grew from a bottle of commercial bottle-conditioned beer:

PlatedYeast_zps10c1ab8c.jpg


The plated culture shown above was clean enough to use, but I wanted to ensure that I was banking the reference culture and not any wild microflora that made it into the bottle during bottling. That's how one ends up with mold and/or bacteria laden slants. The well-isolated colonies in the red rectangle are each the offspring of a single yeast cell; therefore, they are single-cell isolates (a.k.a. pure cultures). The colonies all demonstrate good morphology, that is, they are round, dome shaped, and creamy white, which makes them good candidates for transfer to slants. One can be assured that the slants containing these colonies will remain clean if one practices good aseptic transfer technique when performing plate to slant, slant to slant, and slant to starter transfers.
 
I've got bubbles in all of my slants... probably from pouring/agitation. Any reason I can't just crack the caps and pop them all back in the pressure cooker for another 15min?
 
What is the amount of DME needed and what is the cost of bringing a slant to a White Labs vial size? Trying to convince myself to buy the equipment :)
 
you won't be able to slant in a white labs vial (the caps are not able to be put into a pressure cooker). I'm sure they have a more sophisticated way of sterilizing them than with steam under pressure. The proper vials are very cheap - about $1 each and re-usable.
 
Thanks Fred. My question is, what is the cost in agar, dme, yeast nutrient etc.? I'm trying to justify spending ~$160 in upfront costs and once I have all the equipment, how much a vial will cost me if I make it at home as oppose of buying a new vial every time I brew?
 
Hmmm. Mathematical problem. Lets see 35 grams of DME plus 2.5 grams of Agar yields an average of 27 vials. One vial will grow via starter one fresh White Labs yeast vial worth $8.00.

35 grams of DME = $0.39 (3 lbs Pilsen DME = $15.34 on Amazon shipped = 1361 grams)

2.5 grams of Agar = $0.28 (5 oz = $15.95 on Amazon shipped = 142 grams)

Total materials for 27 vials of $8.00 yeast ($216.00) is $0.67. Add some time and electricity in there of course but you are looking at a savings of 99.69% or roughly $215 of $216. Good Deal!
 
I have been looking into doing this also but haven't found a lot of info on it. Once I get home ill have to start doing some research. This is really good for those seasonal strains etc
 
Thanks Fred. My question is, what is the cost in agar, dme, yeast nutrient etc.? I'm trying to justify spending ~$160 in upfront costs and once I have all the equipment, how much a vial will cost me if I make it at home as oppose of buying a new vial every time I brew?

Here's how I justified it:

A vial of yeast costs $7. It costs nowhere near $7 in raw materials (DME, agar, yeast nutrient) to produce a starter for a batch of beer.

Therefore, eventually, I will break even on it. I don't particularly care when, I just know that I've helped reduce a recurring cost and also the overall price per batch.
 
The most difficult issue with this process is planning ahead. You will need to plan every step to enure you have a good starter for brew day. So those days you say, "Lets brew!" May be limited. Other then that this is the way to go!

I love this thread:ban:
 
As mentioned earlier, the proper tool for the job is called a screw-cap culture tube. I know that it's just a minor difference in terminology; however, it pays to know the proper terminology when ordering from a supply house. If one asks for vials when placing an order, one will receive vials. Most vials are made from soda lime glass and come with paper or foam liners. Neither of these liners is a good investment, and soda lime glass is not designed to withstand multiple trips through an autoclave.

As mentioned earlier in this thread, a screw-cap culture tube looks like a test tube with a screw-on cap (i.e., culture tubes have round bottoms). If at all possible, one wants to purchase reusable screw-cap culture tubes with reusable phenolic caps and rubber liners. Polypropylene caps will work, but they are considered to be single-use caps. The reason why culture tubes that ship with polypropylene caps are cheap is because they are designed to be thrown away after a single use.

20mm x 125mm Screw-Cap Culture Tubes

SCCT1_zps2cc12802.jpg


The culture tubes shown above look similar, but the one of the left is a Corning 9825-20 20mm x 125mm reusable culture tube with a reusable phenolic cap. The culture tube on the right is a disposable culture tube with a polypropylene cap.

Culture Tube Caps (exterior surface)

SCCT2_zps66d74738.jpg


The cap on the left in the photo shown above is phenolic. Notice how it is less shiny than the polypropylene cap on the right. That's because the material from which phenolic caps are made is combination of cellulose fiber and phenolic resin. It is a thermoset material that does not melt.


Culture Tube Caps (interior surface)

SCCT3_zpscc4dc509.jpg


The phenolic cap on the left in the photo shown above is much thicker than the polypropylene cap on the right. Phenolic caps do not flex.

There is a cost difference between reusable culture tubes and disposable culture tubes. Quality reusable culture tubes usually cost between $2.00 and $2.50 each in quantity. Culture tubes that costs less than $2.00 each are usually made to be thrown away after a single use. My preferred culture tubes are Corning (Pyrex) 9825 and Kimble-Chase (Kimax) 45066. Corning 9826 culture tubes are also very nice, but there is no need to pay extra for a PTFE surfaced liner. One can often find good deals on NOS and used culture tubes on eBay. However, I would refrain from purchasing NOS or used culture tubes that do not come with caps. It's difficult to find reusable caps in less than quantity 192. One needs to remember that the keyword here is "reusable," as there are disposable phenolic caps.


Answering Peter78's question, yes, the investment is worth it if one looks at yeast management as a sub-hobby within the brewing hobby. I have brewed with cultured yeast since my fourth batch of beer. My first home lab easily paid for itself. The key to building a self-supporting home laboratory is to purchase wisely and only purchase what one is going to use. It is easy to get carried away with labware; therefore, I recommend starting small and adding additional labware as needed. The minimum setup that one needs to culture yeast is a pressure cooker that is capable of at least 13 psi (15 psi shortens the amount of time that media must be pressure cooked), twenty culture tubes, a nichrome loop, and an alcohol lamp (a.k.a. a spirit lamp). A large-capacity pressure canner is nice to have, but a 6-quart pressure cooker will handle most culturing media needs. In fact, I have been using the same 6-quart stainless steel pressure cooker for over twenty years. While I currently use 100ml Corning media bottles for canning 40ml absolutely sterile starters (used when propagating yeast from slant), I used 4oz baby food jars for years (use jars that contained fruit or vegetables). The lids on baby food jars will reseal after autoclaving. However, they are only good for a couple of passes through a pressure cooker. The glue residue that is left on a baby food jar after delabeling can be removed with "Goo Gone" or mineral spirits.

I recommend using pre-poured disposable plates until one becomes comfortable with making sterile media. Making plates correctly is considered to be an advanced skill. Contrary to what one will see on YouTube, properly made plates are never placed in an autoclave, as autoclaving plates results in too much condensation forming on the inside of the lids. Glass plates are usually made by dry sterilizing the petri dishes in the oven, autoclaving the media in a separate container, and pouring the plates hot after the media has cooled to between 120F/49C and 140F/60C. Another thing to consider is that quality 100mm borosilicate plates such as Corning 3160-100s cost around $7.00 each plus shipping. Plates are only used when adding new cultures to one's bank (as I mentioned earlier in this thread, one should plate for "singles" before adding a culture to one's bank to ensure purity); therefore, glass plates are a luxury.

If anyone is wondering why he/she should learn how to plate and slant, here's a photo of a new culture that I am adding to my bank:

SandNYeast_zpsc0067d33.jpg


The culture on the plate shown above is Scottish and Newcastle's Tyneside culture. Each of the well-isolated round colonies is composed entirely of the offspring of a single yeast cell. Single-cell isolation is how we purify a culture.

Learning how to plate and slant yeast means that one is freed from the selection constraints imposed by the major yeast suppliers. The culture shown above is not available from Wyeast or White Labs. None of the cultures in my bank are directly available from Wyeast or White Labs because they are either brewery cultures or cultures that I acquired from culture collections.

Here's my current collection:

MyCurrentBank1_zps31b27281.jpg
 
I took a slant out of the fridge that was made about 3 months ago. I've noticed a marked increase in lag time going from the slant to the 150 ml starter. When the slants were fresh, the step 1 starter was ready in about 24 hours. Now it takes 48 hours because the starter doesn't kick off for about 24 hours.

I don't care about the increase in time, but I'm worried about yeast health and the impact this might have on my beer. Any thoughts?
 
I am using 17 month old slants and the beer tastes just fine. Like you noticed the starter takes 3 days to even begin growing even at the 150ml level.... But once you wake up the yeast they take off like normal. I'm sure there is an increased chance of mutation from older slants, knock on wood... Or maybe not?
 
I have used cultures that were two years old. The key to propagating yeast from old slants is to use a small volume of autoclaved wort.

I do not know how you are inoculating your first-level starters, but 150mls is fairly large for a first-level starter, especially if the wort is not autoclaved and one is working with an older slant. Ideally, one should start with 10mls of absolutely sterile wort when propagating yeast from a slant, especially a very old slant. I started out using 10mls of autoclaved wort, but have managed to push the volume up to 40mls of autoclaved wort over the years. Using "canned" 40ml starters takes some of the sting out of starting with a smaller volume of wort.


Canned 40ml Starters in Media Bottles


MediaBottles_zpseed0bf41.jpg


The media bottle in the bag is from an older batch. I placed it in a bag as a way of indicating that it needs to be used first.




Canned 22ml Starters in 2.5oz Baby Food Jars

22mlSterileStarters_zps5c1db602.jpg


The canned starters shown above are the last batch that I made in baby food jars. The wort is dark because I sent the media through the pressure cooker twice due to my inability to get the jars to vacuum seal after the first trip through the pressure cooker. Four ounce baby food jars are better for canning small amounts of wort because there is more headspace in which to form a vacuum.
 
I am slanting exactly as shown beginning on page 1 of this thread - same equipment. I guess I am mixing a little luck in with a healthy dose of PFM starting at 150 ml. I then step to 1000ml, 3000ml and 5000ml (one step per 24 hours) followed by a cold crash and decant.

PFM (Pure F%$#@'in Magic) is a highly viable method for completing arduous tasks in an efficient manner as taught in the U.S. Navy...
 
I am slanting exactly as shown beginning on page 1 of this thread - same equipment. I guess I am mixing a little luck in with a healthy dose of PFM starting at 150 ml. I then step to 1000ml, 3000ml and 5000ml (one step per 24 hours) followed by a cold crash and decant.

PFM (Pure F%$#@'in Magic) is a highly viable method for completing arduous tasks in an efficient manner as taught in the U.S. Navy...


I haven't heard that acronym in over thirty years. :rockin:

What is your batch size? Are you using a stir plate? I have been able to seriously reduce the size of my starters since moving to a stir plate.
 
So... Zymurgist, would those be a good option for slants? Will they be big enough?

http://www.cynmar.com/ProductDetail/11520774_Glass-Culture-Tubes-Wscrew-Cap-16x150mm-20ml-10pk

Besides being questionable quality (all quality tubes carry a brand marker), the diameter of that tube is not as much of a problem as is the length. Ideally, one wants to limit tube length to 100mm with 16mm diameter tubes and 125mm with 20mm diameter tubes (I would love to find high-quality 20m x 100mm culture tubes). It is difficult to inoculate a tube with a loop when the tube length exceeds 125ml unless one uses more media than is really necessary. Ideally, one doesn't want much more than the wire portion of one's nichrome inoculation loop inside of the tube.

With that said, it looks like Midland Scientific offers a few Corning 9825 and 9826 sizes in singles. The best size tube that they offer in singles is the 9826-16, which is a 16mm x 100mm culture tube (I used to use 16mm x100mm tubes exclusively). Midland Scientific wants $2.04 each for these tubes, which is a very good price for a 9826-16 in quantity 1. The 9826 is a step up from the 9825 in that the reusable phenolic caps have PTFE faced rubber liners. The 9826 is Corning's top of the line reusable culture tube.

http://www.midlandsci.com/default.aspx?page=item+detail&itemcode=CORNING+9826-16

Midland Scientific also offers the Kimble-Chase equivalent of the Corning 9825-20 in singles for $2.35 each. The Kimble-Chase number for this tube is 45066-20125.

www.midlandsci.com/default.aspx?page=item+detail&itemcode=KIMBLE+45066-20125
 
So... Zymurgist, would those be a good option for slants? Will they be big enough?

http://www.cynmar.com/ProductDetail/11520774_Glass-Culture-Tubes-Wscrew-Cap-16x150mm-20ml-10pk

I agree with EarlyAmateurZymurgist that those would be too long. However, you definitely do not need to buy the best money can buy. I have been using the Cynmar 16ml vials recommended by Kai Troester on his wiki page on slanting. I find them to be a great size and relatively inexpensive. Actually, the last batch of vials I purchased I got off of ebay. Same size and no issues with "autoclavability".
 
Given how much great information, and motivation, I have received from this thread I thought I would add something back in:

I struggled quite a bit trying to figure out the "best" way (for me) to step up the slant to a full starter. I couldn't come to terms with not knowing, or at least being able to estimate, how much yeast I was dealing with. Kai Troester's page helped a bit and there are number of pages with non-microscope methods of estimating cell counts (such as yeast volume) but ultimately, after the 3rd or 4th re-reading of the Maltose Falcons excellent page on yeast propagation, I arrived at a methodology that I felt comfortable with.

In that article Dr. MB Raines shows that the yeast content of a typical slant in 10ml of wort will produce about .8 billion cells in ~2 days. She also recommends fewer step-ups (or at least shows that a lot of steps aren't necessary).

Given this, I can 10ml of wort in the same vials I use for the slants. The first step for making a starter is to pour the contents of a vial into a slant vial. Swish it about until all of the yeast is suspended in the wort. I then pour this back into the empty vial. I let this sit for 48 hours with an occasional shake. From there I use a yeast calculator starting, with .8billion cells, to determine the next steps. This usually leads me to a step resulting in ~100b cells (to put me where a typical commercial yeast vial is; this is about 230-250ml of wort). From there I step up to where I need to be.

So far this approach has worked quite well for me. The estimated cell amounts may be totally wrong but at least I have a number to put on it :D
 
What size stir bar will fit well in a 250 ml flask? I use stir starter plate, if that matters.
 
If one wants an almost free method of slanting yeast, 2.5 and 4 oz baby food jars provide a huge amount of surface area on which to grow yeast. I used whatever I could get my hands on for culturing for the first six months that I cultured yeast. It was very difficult for consumers to purchase quality lab glassware in 1993, and baby food jars did the job. I also used two and four dram vials during that period.

With that said, I discovered that Fungi Perfecti sold screw-cap culture tubes in less than lab-size quantities in the fall of 1993 and never looked back. Using proper culture tubes made the job so much easier than using vials because the deeper caps made handling and slanting the tubes while the media was cooling so much easier. The deeper cap also provided more protection during incubation. I paid about the same amount that Fungi Perfecti charges today for my first two dozen culture tubes. That set of culture tubes went through my pressure cooker at least once every six months for ten years without losing seal (that's 10 cents per use). The tubes were perfectly functional when I sold all of my gear in 2004 after having not brewed for a year. To the best of my knowledge, they are still in use today.

One thing that one needs to keep in mind when purchasing lab glassware is that there is a difference between being rated as being autoclavable and being rated as being able to withstand multiple trips through an autoclave (a.k.a. reusable). The $1.00 tube that I displayed earlier is rated as being autoclavable. However, it is not rated as being able to withstand multiple trips through an autoclave without losing integrity. On tubes with caps that are rated as autoclavable, but not reusable, the adhesive that holds the liner in place breaks down after a couple of uses. If the adhesive doesn't break down, the liner itself breaks down.


In closing, as MichaelBrock mentioned, good deals can be had on eBay. I acquired 106 surplus 9825-20 20mm x 125mm culture tubes for $30.00 with shipping when I decided to re-enter the hobby. The only hitch was that the caps were for 13mm diameter tubes. I wound up having to purchase 192 Corning No. 9999 caps at a total cost including shipping of around $80.00 because that's the smallest packaging that Corning offers and it was cheaper than purchasing single caps. I am still happy with the deal because $1.04 beats $2.00+, but I would be lying if I said that I would have not preferred to pay under $0.30 per tube. The best deal that I have gotten on glassware was $20.00 plus shipping for 48 NOS 9825-16 16mm x 100mm culture tubes. The tubes and caps are truly NOS, not surplus. They have the nice etched marking area.
 
By the way, here's a link to one of the better videos that demonstrates how to perform aseptic transfer in a home environment. The guy in the video is using a bunsen burner, but I prefer to use a denatured alcohol-fueled spirit lamp (it's just a personal preference). He also dips the loop in an alcohol-filled culture tube between inoculations because makes it easier to sterilize the shaft. Flaming that much of the shaft between inoculations is necessary because of the length of the culture tubes that he is using. Anything that enters the tube must be sterile. The shaft barely enters the tube when using 100mm or shorter tubes.

 
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In that article Dr. MB Raines shows that the yeast content of a typical slant in 10ml of wort will produce about .8 billion cells in ~2 days. She also recommends fewer step-ups (or at least shows that a lot of steps aren't necessary).

Maribeth is definitely the godmother of yeast culturing within the amateur brewing community. She brought yeast culturing to masses with her yeast culturing kit and pamphlet in the nineties.

A large proper subset of the cultures in my old bank came from Maribeth. I sent a Ringwood culture that I isolated in early 1994 to Jeff Mellem to give to Maribeth. I am not sure if that culture made it into Brewtek's line-up, but it was passed around the community throughout 1994 and 1995.

Without Maribeth, there would be no Denny's Favorite 50. She offered that culture on slant as Brewtek CL-50 California Pub Brewery Ale. That's where Denny Conn first obtained it. I suspect that many of the cultures that found their way into the Wyeast and White Labs catalogs came from Maribeth and the Maltose Falcons' culture collection.
 
I agree with EarlyAmateurZymurgist that those would be too long. However, you definitely do not need to buy the best money can buy. I have been using the Cynmar 16ml vials recommended by Kai Troester on his wiki page on slanting. I find them to be a great size and relatively inexpensive. Actually, the last batch of vials I purchased I got off of ebay. Same size and no issues with "autoclavability".

The glass vials the OP suggests, has anybody had any problems with the caps after a few autoclaves?

By the way, Happy New Year everybody!
 
I just read Kai's wiki page. From personal experience, I can tell you that sterilizing media filled petri dishes in an autoclave (pressure cooker) is a guaranteed way to make plates with a high failure rate due to excessive condensation. Unlike slants, plates are not sealed off from the elements. Parafilm and/or tape helps, but a large amount of condensation almost always leads to mold. There's almost no way to get rid of that much extra condensation without compromising plate sterility. Contrary to what Kai claims, that much condensation does not magically disappear. Plus, plates need to be relatively dry when streaked.

The generally accepted protocol for making glass plates is to dry sterilize the petri dishes in the oven at 350F/177C for 90 minutes before allowing them to cool 150F/66C (the sterilization period is shortened at higher temperatures and lengthened at lower temperatures). The media is autoclaved in a covered separate container and allowed to cool to between 120F/49C and 140F/60C before removing the petri dishes from the oven and pouring the plates hot in order to minimize condensation. It takes a little practice to be able to lift one side of the lid, pour enough media to cover the bottom of the plate, and lower the lid in one smooth motion; however, anyone who is willing to put forth the effort can master the technique.

Here's what properly poured plates look like:

plates_zpsfb5c4940.jpg


The plates shown above were poured hot. One can clearly see that there is only a light film of condensation on the lids. This film will evaporate during the "proofing" period (I usually hold my plates at room temperature for two to three days to ensure that they are sterile). After this proofing period has been completed, the plates are sealed with Parafilm and stored upside down in plastic bags.

Here's one of the plates shown above put into action:

PlatedYeast_zps10c1ab8c.jpg


By the way, I made plates the way Kai makes them until Maribeth Raines told me how they are poured in a lab. A modified version of the procedure that I outlined above is used to make plates using pre-sterilized plastic petri dishes. With pre-sterilized plastic plates, the plates can be poured with the petri dishes cold or warm.


Happy New Year!
 
I'm not sure I get the purpose behind plating. If you have a pure culture to start with why not just slant it in a vial and move on? Is the concept of plating to see what grows and "choose" which colony to replicate based on visual observation of said colony on the plate?
 
Plating is a quality control measure. Despite the claims made by the major yeast manufacturers, anyone who has plated enough commercial yeast on selective media knows that home brew trade yeast cultures are not 100% pure 100% of the time. It is impossible to produce and package 100% pure lab-grade cultures 100% of the time at home brew trade price points (that's why Siebel charges so much for their cultures). Almost all manufacturers have an acceptable level of contamination that is maintained via statistical quality control (at least the dry yeast manufacturers are honest about it). Given enough time, many commercial cultures that are slanted directly without going through isolation will show signs of infection. The plating process ensures that the culture is free from wild microflora because only well-isolated colonies that exhibit good morphology (physical characteristics) are transferred to slant.

If you want to see how clean your cultures are, leave them at room temperature for two weeks. A sterile slant that was inoculated from a plate using good aseptic technique will remain free from infection.
 
I've really enjoyed this thread and have posted several questions, and have received several responses. I won't rehash my previous questions other than to say I've followed the directions implicitly and produced 12 blank slants which were being stored in a sterilized ziplock bag with lids tight and electrical tape sealing. The unused slants were made about 2 months ago. Upon examining them today I noticed one slant was completely corrupted by green mold. A couple of others had a light film of mold. Any ideas what went wrong? I actually pressure cooked the mixture filled vials a little longer than the directions implied. Where was the mistake? Should I assume the good looking slants are still okay? If they were supposedly sterile they should have lasted indefinitely, even if they hadn't been inoculated yet. I don't see how the slants could have been contaminated during the brief moments of lifting them out of the pressure cooker, tightening the lids, and wrapping them with the tape. Thank you!
 
Obviously it is hard to say what went wrong. How old are the slants you are using? The only thing I can think of that I do differently is I don't keep the slants all in a bag together where moisture can build up. That said it shouldn't make a difference if the caps are tight....
 
I've really enjoyed this thread and have posted several questions, and have received several responses. I won't rehash my previous questions other than to say I've followed the directions implicitly and produced 12 blank slants which were being stored in a sterilized ziplock bag with lids tight and electrical tape sealing. The unused slants were made about 2 months ago. Upon examining them today I noticed one slant was completely corrupted by green mold. A couple of others had a light film of mold. Any ideas what went wrong? I actually pressure cooked the mixture filled vials a little longer than the directions implied. Where was the mistake? Should I assume the good looking slants are still okay? If they were supposedly sterile they should have lasted indefinitely, even if they hadn't been inoculated yet. I don't see how the slants could have been contaminated during the brief moments of lifting them out of the pressure cooker, tightening the lids, and wrapping them with the tape. Thank you!

If it's not getting contaminated post-processing, then the spoilage bacteria/mold had to be there in the beginning, so, just throwing this out there - if your pressure cooker isn't getting up to 15 pounds, it would not be rendering your media sterile. If you don't have a gauge, or the gauge is inaccurate, that could be the problem.

Let us know what you find out!
 
I attempted my first slanting and it seems like everything went ok. I used the agar agar powder found at the asian markets and on my first attempt I used 6 grams to 400ml of wort, and when I put them to slant they never gelatinize. On the second attempt I used 12 grams of agar agar powder in 400 ml of wort. They did gelatinize this time, however there is still some liquid left in the tubes. I have them sitting at room temp for now sealed to make sure the autoclaving killed everything. One week in and no growth.
 
I just made a whole batch of blank slants yesterday. I used 35g DME, 2.5g of Agar powder, and 400 ml of water, as indicated in the OP (or close to it). They all set up just fine. In fact, I ran two batches of slants through the pressure cooker, and by the time the first set had cooled, the leftover wort in the beaker had already gelatinized. I had to heat it back up to get it to where I could pour it into a vial.
 

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