Using Agar Slants - In Pictures

Share your experiences of using brewing yeast.
Wolfy

Using Agar Slants - In Pictures

Post by Wolfy » Fri Aug 27, 2010 3:50 pm

In the set of pictures here, I outlined how I use build a starter from yeast saved on an agar slant.
And I outlined how to make agar slants here.
Between those two steps we need to inoculate our slants and use them to save and grow yeast.

Any yeast source can be used to inoculate the slant, a direct 'virgin' source such as a freshly opened Wyeast or Whitelabs pack is best, but the dregs of bottle conditioned beer, another (older) slant or even a glass of beer at the local if the beer has been cask conditioned can also be used.
Yeast stored on agar slants will remain viable for a few months - up to a year - after which they should be reinoculated.

Here is the typical setup I use when working to inoculate new yeast slants.
Bench surface, vials, everything else wiped down with a bleach solution, flame source, isopropyl (rubbing) alcohol, slants, inoculation loop and other thing in order and ready to use.
Image
In this case I'm inoculating the new slants from one I used to isolate individual colonies of yeast (see below).

Sterilise the inoculation loop, easily done with metal loops by heating them in a flame, plastic loops can be dipped/soaked in alcohol.
Use the loop to pickup the yeast sample, this can be done by dipping it into a Wyeast/Whitelabs pack, immersing it in yeasty-bottle-dregs or selecting a small colony of yeast from an agar slant or plate.
Gently rub the loop over the surface of the agar slope, being careful not to touch the sides of the tube.
There is not much to see when the slants have been freshly inoculated:
Image

Store the slants at room temperature (20-30DegC is good):
Image
I incubate my slants on the (cleaned with bleach solution) shelf above the TV, the lounge room is heated in winter and air conditioned in summer, so it's as good place as any.

After a few days the first signs of yeast growth on the agar surface:
Image

Another day or two and the yeast will be visible:
Image

Depending on temperature and conditions after 3-6 days the yeast should have covered most of the surface:
Image
There is a large amount of freshly grown viable yeast on the agar surface, which can be used to create a starter or the slant can be stored in the fridge.

Before storing the slant in the fridge, seal it so it stays air-tight and label it well.
The slants should be sealed to prevent contamination during storage, this is best done with Parafilm (sticky tape, electrical tape, masking tape tends not to stick so well, leaves sticky residue and does not fully seal the tubes, cling wrap may work if you can't get Parafilm).
Parafilm is a stretchy elastic 'tape' that will stick to itself often used in labs, however rather than paying (~$30 a roll), florist's Parafilm (~$2 a roll) works just as well:
Image
(The plastic tubes are frozen yeast samples not slants.)

To protect the slants from the various bugs and nasties in most fridges, I seal the slants in a zip-lock bag and put them into a plastic container which fits well on the bottom shelf of the refrigerator:
Image
When making slants for long-term yeast storage, it is always a good idea to make 2 or 3 for each yeast strain, that way if one sample gets contaminated or is otherwise unusable, the others should still be viable. Try also to store each of the slants in different zip-lock backs or even a different fridge if possible, to further reduce the risk of losing an entire strain.

Slants stored in the fridge should remain healthy and viable for a few months, after about 6 months there will be noticeable autolysis, but I usually re-slant my yeast samples after 1 year. It is a good idea to visually check stored slants every month or so, that way if there are any contamination or other problems they can be fixed in a timely manner.
Last edited by Wolfy on Wed Mar 02, 2011 8:11 pm, edited 1 time in total.

Wolfy

Re: Using Agar Slants - In Pictures

Post by Wolfy » Fri Aug 27, 2010 3:50 pm

Agar slants are best inoculated from a 'virgin' yeast source such as freshly opened Wyeast or Whitelabs pack.
However they can also be used for yeast sourced from bottle conditioned beer, a keg, or even a pub-beer that has been conditioned and served from the cask.
One way to do this is to simply immerse the inoculation loop in the yeasty-dregs and then coat the surface of the slant.
However,bottle/keg/cask/pub yeast samples are not always the most pristine so by isolating individual yeast cells, you can be sure there is no contamination when the sample is saved or stepped up into a starter.

Once the yeasty-dregs have been poured into a sanitized shot glass, you can see just how many cells the sample contains:
Image
In order to isolate individual yeast cells, an agar filled petri dishs are often used, however if you don't have a petri dish you can also use the a slant.
Dilute the yeasty-bottle dregs in about 70-90% boiled/sterilised water (the cling wrap covered shot glass in the picture above).
If you are lucky this will dilute the yeast so that when you dip the inoculation loop into diluted sample it will be easier to isolate single colonies.

Incubate the slant as normal, and after a few days it should look something like this:
Image
The two samples on the left are from the diluted mixture, and the tube on the right was made from the undiluted yeasty-dregs as shown in the first picture above.

After a few more days the individual yeast colonies (small round white blobs) should be noticeable and with a steady hand you can transfer one or two of those individual colonies to a new slant to be sure that only pure yeast is saved and there is no contamination from the bottle or source of your yeast (this is what I was doing in the first picture in the first post - above).
With the correct dilution the slants should look something like this, and the individual yeast cells/colonies can be easily distinguished:
Image
Last edited by Wolfy on Fri Aug 27, 2010 3:53 pm, edited 2 times in total.

Wolfy

Re: Using Agar Slants - In Pictures

Post by Wolfy » Fri Aug 27, 2010 3:50 pm

While you can use any metal wire (such as a paper clip) an inoculation loop makes yeast transfer much easier.
These can be purchased as metal or plastic from usual lab supply places, however you can also make your own easily and cheaply (I got this idea from mitch from a post he made in another thread).

I purchased some 2m of 24SWG NiChrome wire from Ebay for a couple of $.
Image
Then with some pliers and a small drill bit to wrap the wire around I formed some new loops:
Image
the metal loops can be sterilized much easier with a flame, than the old plastic loops that I had before.

I use the handle from an x-acto craft knife for my loops, but any similar handle or attachment can be used:
Image
The small refillable hand held butane torch, is from a crème brulée kit that I found at a cheapo-shop and makes an ideal flame source.

The flame source can be used to sterilize inoculation loops and to provide a 'safe' working space when working with slants.
If you work within the 'cone' of heat generated by the flame there is very little risk of falling dust/bugs/nasties that might infect your sample, however just be careful you don't stick your fingers or clothing into the flame (especially if your favorite sweat-shirt is made from some sort of flammable nylon material).
Last edited by Wolfy on Fri Aug 27, 2010 3:59 pm, edited 2 times in total.


sladeywadey
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Re: Using Agar Slants - In Pictures

Post by sladeywadey » Fri Aug 27, 2010 3:57 pm

thank you very much for taking the time to post this procedure - I've often wondered what was really involved in doing this as it looked a bit black art-ish when I initially had a look into doing it and I may try it when I get more time.

Wolfy

Re: Using Agar Slants - In Pictures

Post by Wolfy » Fri Aug 27, 2010 5:17 pm

sladeywadey wrote:I've often wondered what was really involved in doing this as it looked a bit black art-ish
I think I made this one a bit more complicated than it needs to be.
But, make slant, heat paperclip with cigarette lighter, stick paperclip in yeast, roll yeast-coated paperclip on slant surface, grow yeast and refrigerate, does not have the same impact. ;)

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Hogarth
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Re: Using Agar Slants - In Pictures

Post by Hogarth » Fri Aug 27, 2010 6:17 pm

Another excellent photo essay, Wolfy. Great stuff. I'd be good if these could be stickied or permanently flagged somehow.

malc

Re: Using Agar Slants - In Pictures

Post by malc » Fri Aug 27, 2010 8:01 pm

cheers Wolfy will give this a go in the very near future, great post mate!

that's some malt stash you have there!

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Re: Using Agar Slants - In Pictures

Post by Horden Hillbilly » Fri Aug 27, 2010 8:28 pm

Hogarth wrote:Another excellent photo essay, Wolfy. Great stuff. I'd be good if these could be stickied or permanently flagged somehow.
I agree, excellent posts Wolfy, I have made them both stickies.

196osh

Re: Using Agar Slants - In Pictures

Post by 196osh » Tue Feb 01, 2011 12:28 am

What a great description. Nice work mate.

Wolfy

Re: Using Agar Slants - In Pictures

Post by Wolfy » Wed Mar 02, 2011 8:48 pm

If I had followed my own instructions as outlined above, I'd not have had this problem, but yeast is not the only thing that likes living on the wort/agar media, so one must be pedantic and careful about sanitation and storage at all points of the process:
Image

With the 4 slants on the left, the green mould seems to have grown (inside a sealed zip-lock bag, inside the fridge) on both sides of the slightly damp masking tape, which allowed the condensation inside the tube to contaminated the wort/agar media. The three slants on the left were most likely contaminated when they were made and the growths are result a tiny infection having a year to grow. Screw-top tubes sealed with Parafilm, and more pedantic sanitary procedures when making the slants, should have prevented these problems, so I edited the first post to more clearly explain that.

Also to note - in the picture - is that on the 5 month old slants (on the left) the remaining yeast still looks fairly healthy and viable, however the yeast on the year old slants (on the right) does not appear to be as healthy and is quite dried out.

Luckily the contaminated slants represent less than 10% of the total number I have stored, and there was also a second uncontaminated slant (and frozen sample) of each yeast strain stored. :)

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Bobba
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Re: Using Agar Slants - In Pictures

Post by Bobba » Wed Mar 02, 2011 11:59 pm

=D> =D> =D>
FANTASTIC POST Wolfy! Thanks very much for taking the time and effort to make such an informative post. Perfect timing also, I literally inoculated some slants with Wyeast 1469 3 days ago, and was wondering how they should look before putting them in the fridge. Was also wondering if the bottom shelf might be cold (risk of freezing) for the little blighters, but from what you say, it must be ok.
Thanks also for answering my questions in the other yeast post mate ;)

FV: -
Conditioning: AG34 Randy's Three Nipple Tripel 9.2%, AG39 APA for a mate's wedding
On bottle: AG32 Homegrown Northdown ESB, AG33 Homegrown Cascade Best
On tap: -
Garden: 2x cascade, 2x Farnham whitebine (mathon), 2x northdown, 1x first gold

Wolfy

Re: Using Agar Slants - In Pictures

Post by Wolfy » Thu Mar 03, 2011 10:45 am

Bobba wrote:=D> =D> =D>
I literally inoculated some slants with Wyeast 1469 3 days ago, and was wondering how they should look before putting them in the fridge. Was also wondering if the bottom shelf might be cold (risk of freezing) for the little blighters, but from what you say, it must be ok.
Limited release yeasts (such as Wyeast's Private Collection) such as Wy1469 are ideal candidates for putting on slants to store at home, since you can't buy them all year round.
They should look like the ones at the top of the page, if they look green and fuzzy like the recent ones, something is wrong. ;)

If there is any risk of freezing, do NOT put the slants there, even one freeze cycle will kill 50% (or more) of the yeast. Things on the top shelf of my fridge (where the cooling vents are) they are likely to freeze, so for me the bottom shelf is much better. Cold is good, since it slows the yeast's metabolic rate and minimises changes or mutations (which is why cryogenic storage is best) but without adequate protection (such as a glycerin mix) freezing will kill the yeast by rupturing their cell walls.

TheMumbler

Re: Using Agar Slants - In Pictures

Post by TheMumbler » Fri Apr 08, 2011 11:01 am

Great post Wolfy =D> Thanks for taking the time.

barney

Re: Using Agar Slants - In Pictures

Post by barney » Tue Mar 06, 2012 5:06 pm

What is the limiting factor affecting growth of the yeast on an Agar slope? Does the yeast just run out of oxygen or is there another reason for the growth arresting?

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