# Using Agar Slants - In Pictures



## Wolfy (28/8/10)

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 or another (older) slant 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.





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:





Store the slants at room temperature (20-30DegC is good):




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:





Another day or two and the yeast will be visible:





Depending on temperature and conditions after 3-6 days the yeast should have covered most of the surface:




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 can be sealed with sticky tape, electrical tape, masking tape or cling wrap, however I found that most tape does not stick well and that parafilm works best.
Parafilm is a stretchy elastic 'tape' that will stick to itself often used in labs, however rather than paying (~$30 a roll), florists parafilm (~$2 a roll) works just as well:




(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:




Slants stored in the fridge will 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.


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## Wolfy (28/8/10)

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:





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:




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 individal yeast cells/colonies can be easily distinguished:


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## Wolfy (28/8/10)

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 purchased some 2m of 24SWG NiChrome wire from Ebay for a couple of $.





Then with some pliers and a small drill bit to wrap the wire around I formed some new loops:




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:




The small refillable hand held butane torch, is from a crme brule 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).


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## Wolfy (28/8/10)

References and further reading:
http://www.maltosefalcons.com/tech/yeast-p...s-and-practices
http://brewery.org/brewery/library/yeast-faq.html
http://www.alsand.com/beer/yeast/index_E.html
http://hbd.org/brewery/library/SterileDW1096.html
http://braukaiser.com/wiki/index.php?title...g_Plates_Slants
http://www.realbeer.com/spencer/yeast-culturing.html
http://www.tigereye.net.au/bluedog/slants.html
http://www.unm.edu/~draper/beer/slantuse.html


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## Synthetase (28/8/10)

Mmmmm microbiology at home...... :icon_drool2: 

Great post!


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## ekul (28/8/10)

great post wolfy. After reading your other two topics this was the piece of info that i needed to get started. Had a search on here today but couldn't find it.
thanks


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## MaltyHops (28/8/10)

Wolfy said:


> Gently rub the loop over the surface of the agar slope, being careful not to touch the sides of the tube.


Congrats - A+ work.

Why though the need to avoid touching the tube sides? I had bought
these tubes from a lab supply place that are only 15mm diameter (outside)
and avoiding touching the in-sides is difficult. Also my slants seem to
release enough liquid to give a path for the yeast to grow to the sides
anyway.

Tom.


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## Wolfy (28/8/10)

MaltyHops said:


> Why though the need to avoid touching the tube sides? I had bought
> these tubes from a lab supply place that are only 15mm diameter (outside)
> and avoiding touching the in-sides is difficult.


You're right the inside of the tubes should be sterile (especially if you autoclave them after filling them), the 15ml plastic tubes are also narrow and more difficult to work with (which is why I prefer the larger 30ml tubes for slants).
However, it's still best practice, better for sanitation and better if you can place the yeast directly onto the agar slope surface where you want it.
I tend to find if there is water inside the tube it will make the yeast 'run' all over the surface and it makes it difficult to spot any infections or problems since it does not look like 'normal' growing yeast.
But another method to inoculate slants is to simply drop a drop of yeast-liquid onto the surface and swirl it around, maybe I'm just a control freak and want everything to do what I tell it to.


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## Synthetase (28/8/10)

Depending on the length of your loop/handle and the tube, if you touch the sides, you may touch it with the handle of the loop which is not sterile. Also definitely avoid touching the opening/inside of the tube with your fingers.

If there's water on your agar, you haven't dried it properly. When I dry plates, I usually open them face down in a warm room with as little air flow as possible. Obviously, this is more difficult with a slant, but I would suggest that after your slants have been poured and set, that you loosen the lid a bit to allow water vapour to pass and leave them in a warm room for a couple of days to dry prior to use.

Having water on the surface of the agar isn't a major problem. However, it makes it difficult to isolate single colonies as the water allows the cells to flow across the surface of the agar, resulting in a lawn of growth rather than a patchwork of individual colonies.


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## MaltyHops (28/8/10)

Synthetase said:


> ...
> If there's water on your agar, you haven't dried it properly. When I dry plates, I usually open them face down in a warm room with as little air flow as possible. Obviously, this is more difficult with a slant, but I would suggest that after your slants have been poured and set, that you loosen the lid a bit to allow water vapour to pass and leave them in a warm room for a couple of days to dry prior to use.
> ...


Interesting - I haven't come across a mention of needing to dry (moderately, I guess)
agar before. The unm.edu link also links to another hbd.org FAQ that suggests storing
slants/petri's upside down so any water collects away from the agar. That FAQ also
suggests what contaminants to look for:

_"Things to look for: Colors: creamy off white. (red, yellow, etc likely to be contaminants)
Textures/Shapes: Mostly roundish, like a demi-sphere. (Fuzzy=bad mold, flat=maybe bad).
Light Transmittance: Hold the plate up to the light. Look for colonies which are transluscent
- let light pass. If there are opaque ones (darker) consider them contaminants. You can
still pick a pure colony off of a plate with a contaminant elsewhere on the plate (unless you
have fuzzy fungal hyphae and spores all over)"_


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## Synthetase (28/8/10)

Moderate drying is standard operating procedure in a microbiology laboratory. We incubate plates upside down to stop them drying out too much whilst they're in use. Water vapour comes off the agar and then rises and condenses straight back onto it again.


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## rendo (28/8/10)

Hey Wolfy,

Am quite interested in your frozen yeast sample. Is that just yeast and wort in there, taken from a brew or a starter maybe?

I always thought that the freezer would rupture and kill the yeast. Let us know what you do to store the yeast in the freezer. 
How much success/failure have you had with yeast in the freezer

Any ideas on how long it will last in the freezer? I imagine AGES?

rendo



Wolfy said:


> (The plastic tubes are frozen yeast samples not slants.)


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## wakkatoo (28/8/10)

as with the other 2 threads, subscribed! B)


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## Wolfy (28/8/10)

rendo said:


> Am quite interested in your frozen yeast sample. Is that just yeast and wort in there, taken from a brew or a starter maybe?
> 
> I always thought that the freezer would rupture and kill the yeast. Let us know what you do to store the yeast in the freezer.
> How much success/failure have you had with yeast in the freezer
> ...


I've started freezing my yeast with this procedure (it's similar to JZ/JP's suggestions on a recent TBN podcast - but without the centrifuge):
Slant - 10ml starter (ferment 2-3 days, fridge 2-3 days) - decant all but the yeast and 1.5-2ml wort/beer - add 30-50% glycerin - mix/freeze.
At that ratio of glycerin the samples do not actually freeze solid in the -20degC (standard) freezer (and it's not frost free), in theory the samples should remain viable and (hopefully) with little mutation for some years, but since I've only just started doing it can't be sure - will test them in a few years and let you know.


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## ekul (28/8/10)

Wolfy said:


> I've started freezing my yeast with this procedure (it's similar to JZ/JP's suggestions on a recent TBN podcast - but without the centrifuge):



I saw a ghetto centrifuge made from a bicycle wheel once. You can get cheap/free broken bikes at the tip a dime a dozen.


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## Gopha (29/8/10)

Wolfy said:


> Slants stored in the fridge will 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.



Hi, You refer "noticeable autolysis" can you describe what I should be looking for, is it the colour, shape or does just it just stand out that a colony may have autolyised.
Fantastic resource of info, well done - Cheers


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## Wolfy (29/8/10)

Gopha said:


> Hi, You refer "noticeable autolysis" can you describe what I should be looking for, is it the colour, shape or does just it just stand out that a colony may have autolyised.


I find the yeast-colour goes darker and the slants also tend to dry out a little, fresh yeast on the slants is very easy to select or scrape off with a loop, but the older yeast/slants tend to be 'stiffer' ... not really a good description I know, so you might have to store a few for yourself and see how they change over a year or so.
However the dead yeast is most noticeable when making a starter - I scrape all the yeast on the slant into the starter - if it's more than a few months old the first step(s) of the starter often have a burnt-rubber type aroma. But after stepping up the starter a few times, the viable yeast have reproduced sufficiently and there is no issue.


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## Wolfy (3/3/11)

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:





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 (that answers *Gopha*'s query) is that in the picture 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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