# Making Agar Slants - In Pictures



## Wolfy (16/8/10)

I've had the camera out again since the slant-to-starter thread, this time making agar slants. 

Agar slants are an easy way to store yeast strains. 
Once inoculated with a yeast sample and refrigerated, agar slants should keep the yeast viable for months or longer.
While there is noticeable autolysis after a few months, I usually reculture slants after 1 year, others have suggested they recultured viable yeast from slants up to 4 years old.

Here is what we'll need to make our agar slants.
Clean test tubes/vials, (1.030-1.040)wort/nutrient mix, agar-agar (from an Asian supermarket), Pyrex jug and a syringe.






Most slants I make are in glass 50ml test tubes, using 10ml of wort/agar in each, but for the the smaller 15ml plastic test tubes I use 5ml wort/agar.
250ml wort will make 20 larger slants (allowing for some losses).

After measuring the required amount of wort into the Pyrex jug (add some yeast nutrient if you have some), add the agar agar powder.
For the powdered agar-agar shown in the above picture, I use 1.5% agar, which works out to be about 3.75g of Agar in 250ml wort.





The agar will not dissolve in the cold liquid, so you need to bring it to the boil, I do this in the microwave.
I microwave the wort/agar for about 30 secs on high, then stir, and repeat the process until it just comes to the boil.
When it gets to the boil the agar will dissolve into the wort and the mixture will be very runny:





While it is still hot, measure the wort/agar mix into the test tubes, try not to drip the wort/agar mix down the sides of the tubes if you can help it.
You could tip it in from the jug but using a syringe makes it much easier:




The reason I prefer agar over gelatin (which also works) is that agar exhibits hysteresis, meaning that it melts at ~85C but it does not solidify until it cools to ~40 C which means its easy to work with in liquid form, but once it has cooled and solidified it will not melt again (until it's heated over ~85C).

Now we need to sterilize our slants.
An autoclave is the best way to do this, but not too many people have one at home, so most people sterilize our slants by moist heat in a pressure cooker or similar.
I have a (99c Ebay) pressure cooker, so I load the slants in that and 'boil' them for about 30mins at 15PSI.
Before I had the pressure cooker, I just put a couple of phone books ontop of my mini-mash-pot and did the best I could and never seemed to have any problems, but to be more pedantic I should have used the Tyndallization process:





After the slants have been 'autoclaved', let them cool enough so you can handle them safely, but not enough so they start to solidify.
Take them out of the 'autoclave' and lay them on a slanted surface:





Any slanted surface will do, cardboard box, tray, etc, but try to maximize the slope surface by adjusting the angle they are on:





Cover with a clean tea towel (or similar) and let them cool (only takes about 1 hour now its winter), and they should solidify like so:




When you fill, autoclave and lay out the slants try not to shake, tilt, or mix the slants because the wort/agar will coat the side of the tubes and make things messy, if you treat them gently you should get nice clean slopes to work with. If possible leave the caps on losely while 'autoclaving' and cooling the slants, that way there should not be too much moisture buildup inside the slants.

Since the slants should be sterile there is no need to store them in the fridge, I usually put mine into a plastic container - just to keep any contaminants off - until I need to use them. It's also not a bad idea to use tape or parafilm to seal the caps - again just to ensure they do not get contaminated.

I use the 50ml glass push-top test tubes because I have lots of them, and I got them cheap, however if I was going to buy more tubes to make slants with, I'd get screw top polypropylene vials like these ones:


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## Fourstar (16/8/10)

Wolfy said:


> I use the 50ml glass push-top test tubes because I have lots of them, and I got them cheap, however if I was going to buy more tubes to make slants with, I'd get screw top polypropylene vials like these ones:



Nice photo of my slants there wolfy! 

Also i make my wort/nutrient alot lower in OG so the growth isnt OTT. the last lot i did worked out to being 1.005~ takes around 1 wwek to get to full growth but its a perfect fine film on the slant. :icon_cheers:


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## potof4x (17/8/10)

> I use the 50ml glass push-top test tubes because I have lots of them, and I got them cheap, however if I was going to buy more tubes to make slants with, I'd get screw top polypropylene vials like these ones:



What is the actual size of these screw top vials?


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## Florian (17/8/10)

Very nice post Wolfy, and just in time, as I'm trying to get my head around the whole thing. There are two and a half things that still aren't a 100% clear to me:

1. Could you use a slant several times if you only take a little yeast out with an inoculation loop?

2. How do you actually inoculate the slant? Do you just pour yeast into it?

2.5 The only advantage of slants vs. test tubes filled with yeast (as described in Tony's thread) is that slants can be kept longer, is that correct? 

Thanks,
Florian


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## Fourstar (17/8/10)

potof4x said:


> What is the actual size of these screw top vials?



50ml IIRC

30ml IIR Incorrectly


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## Wolfy (17/8/10)

Fourstar said:


> Nice photo of my slants there wolfy!
> 
> Also i make my wort/nutrient alot lower in OG so the growth isnt OTT. the last lot i did worked out to being 1.005~ takes around 1 wwek to get to full growth but its a perfect fine film on the slant. :icon_cheers:


I didn't have any of those vials of my own, but IMHO if buying tubes for slanting those ones are much better than the glass ones I have.
Since I'm (attempting to) freeze yeast for longer storage, I'm using the agar slants as a growth medium, so I'm happy with fairly good growth on the slants.


Florian said:


> 1. Could you use a slant several times if you only take a little yeast out with an inoculation loop?
> 
> 2. How do you actually inoculate the slant? Do you just pour yeast into it?
> 
> 2.5 The only advantage of slants vs. test tubes filled with yeast (as described in Tony's thread) is that slants can be kept longer, is that correct?


1) Yes, some people do that, someone on the UK forums has used the same slant - just taking a little at a time - for 4 years now. However - IMHO - if you take just a little yeast, it adds an extra step/complexity to your starter procedure and (the few times I've tried it) the starter is more fickle since you are reculturing from only a tiny bit of yeast. I prefer to make enough slants to last me for a few months and use the entire thing (and have had no problems doing it that way even with slants up to a year old) then make fresh slants as/when required.

2) I collect yeast on an inoculation loop and rub/smear it over the surface of the slant (culturing yeast and inoculating slants is the next set of photos I was looking to make, the time-frame is just a bit longer so it's not finished yet). If I have a fresh smackpack/vial/slant I just run the loop through the yeast in/on that and end up with an abundance of cells/growth on the slant. If I have a non-virgin source (bottle/reculture etc) I like to streak out the yeast, then select only individual (healthy/normal looking) colonies and transfer only 1 or 2 colonies to the 'storage' slant (given I don't have a microscope that step might be a bit trivial but I'm happy doing it).

2.5) If you 'just' want to save money by reusing liquid yeast, I'd suggest that splitting the pack as soon as it's open into sterile tubes (as per Tony's method) is the best and most efficient way to do that; its quick, easy, simple and allows you to use the same pack up to 4-6 times. However, if you have a non-virgin source of yeast (bottle/reculture etc) then slants are a good storage media, they are also useful if you wish to keep a 'yeast bank' and store yeast for long periods of time (by reslanting as required you can keep the strains for years).


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## WarmBeer (17/8/10)

Fourstar said:


> Nice photo of my slants there wolfy!
> 
> Also i make my wort/nutrient alot lower in OG so the growth isnt OTT. the last lot i did worked out to being 1.005~ takes around 1 wwek to get to full growth but its a perfect fine film on the slant. :icon_cheers:


Nice work, Wolfy. Enjoying the whole "Introduction to advanced yeast management" series. 

Fourstar, where did you purchase your vials? I assume they're "autoclaveable" using the pressure-cooker method, as well as the lids?


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## wakkatoo (17/8/10)

subscribed.

I'm in the same boat as Florian so this thread could not have come at a more perfect time! 

Thanks Wolfy!


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## Florian (17/8/10)

Thanks for the detailed explanations Wolfy! 
Think I will order a heap of 10ml sterile screw top vials to divide smack packs, and a few bigger vials for slants so I can store yeast strains that aren't always available (PC). I guess 10ml vials are to small for slants?

If I understood correct then re-slanting (after a year or so) has the same effect as building up a vial with slurry and dividing it into new vials. So in both cases you would have 2nd generation yeast?

Florian


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## Wolfy (17/8/10)

Florian said:


> Think I will order a heap of 10ml sterile screw top vials to divide smack packs, and a few bigger vials for slants so I can store yeast strains that aren't always available (PC). I guess 10ml vials are to small for slants?
> 
> If I understood correct then re-slanting (after a year or so) has the same effect as building up a vial with slurry and dividing it into new vials. So in both cases you would have 2nd generation yeast?


I made some slants with the 15ml plastic tubes (the ones with green lids in the 1st picture) that came from the recent Bulk Buy. While I was able to make them, they are smaller and so a little more difficult to work with, but they are good for splitting yeast packs, so they may work for both purposes. But what you've suggested sounds like the easiest and most practical scenario for anyone who does not want to get too involved with 'yeast ranching'.

I think yeast 'generation' is less important than yeast mutating over time, which also depends on how it is stored, but unfortunately without testing it against the original sample (with microscopes, DNA, brewing tests, or whatever methods are used) it's not going to be easy to know how much the yeast has changed. I guess the argument with 'generations' is that the yeast is more likely to mutate and change while it's fermenting a large batch of beer, than it will when stored in small samples (agar/wort). If I save yeast on agar, there are more 'generations' involved than if it's taken directly from the fermenter, however the agar saved sample has also been isolated and in theory is more 'pure'.

I've just started to freeze the first of my yeast samples (in glycerin) since - in theory - it should mean less long term maintenance combined with less yeast mutation over time, when compared to other storage methods.


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## Fourstar (17/8/10)

WarmBeer said:


> Nice work, Wolfy. Enjoying the whole "Introduction to advanced yeast management" series.
> 
> Fourstar, where did you purchase your vials? I assume they're "autoclaveable" using the pressure-cooker method, as well as the lids?



proscitech!


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## mb83 (18/8/10)

For months now I have been plagued with blank slants being infected by microbes (I must have scrapped 50 slants, sometimes whole batches).
I think my lab technique is pretty good thanks to 1st year uni biology classes, but couldn't work out how my slants were being infected.
Then I read this on one of your links:

_Tindalization[4] /Tyndallization[5] named after John Tyndall is a lengthy process designed to reduce the level of activity of sporulating bacteria that are left by a simple boiling water method. The process involves boiling for a period (typically 20 minutes) at atmospheric pressure, cooling, incubating for a day, boiling, cooling, incubating for a day, boiling, cooling, incubating for a day, and finally boiling again. The three incubation periods are to allow heat-resistant spores surviving the previous boiling period to germinate to form the heat-sensitive vegetative (growing) stage, which can be killed by the next boiling step. This is effective because many spores are stimulated to grow by the heat shock. The procedure only works for media that can support bacterial growth - it will not sterilize plain water._

Now I realise that the spores weren't necessarily airborne and got in while pouring the agar into the vials, but are most likely heat resistant spores.

Does anyone know if you can reheat, reliquify and reboil solid agar medium?

Cheers,

Michael


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## Wolfy (18/8/10)

mb83 said:


> Now I realise that the spores weren't necessarily airborne and got in while pouring the agar into the vials, but are most likely heat resistant spores.
> 
> Does anyone know if you can reheat, reliquify and reboil solid agar medium?


I try to use sanitised equipment/procedures even when pouring the slants.
But you can reheat, reliquify solid wort/agar with no problems. 
The last time I made some I did a double batch, filled all the tubes with wort/agar and 'autoclaved' only half, by the time that was finished the second batch were totally solid, but reheating them and sterilizsng them liquified them with no problems so they could laid out to set as slants.


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## praxis178 (18/8/10)

A link to Proscitech and the relevant page at that....

linky 

Not a bad buy for $15 per 50 tubes plus postage ($6 for my last order of tubes and flasks <back one page for the curious>).

Enjoy! B)


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## manticle (18/8/10)

Picture threads make processes much easier to follow and are invaluable.

Suggestion for this and Tony's thread to be made into a wiki - maybe combined as 'splitting and storing yeast' or something?


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## Nick JD (19/8/10)

Awesome tutorial thread. 

It'd be nice if a homebrew shop sold sterile slants ready to go...


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## Fourstar (19/8/10)

Nick JD said:


> It'd be nice if a homebrew shop sold sterile slants ready to go...



Heck, next thing you know they will be selling fresh wort kits and brewing them for you!


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## Nick JD (19/8/10)

Fourstar said:


> Heck, next thing you know they will be selling fresh wort kits and brewing them for you!



Not to mention it's probably not in their best interests to encourage yeast farming.


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## Fourstar (19/8/10)

Nick JD said:


> Not to mention it's probably not in their best interests to encourage yeast farming.




thats one way to decrease your profits! kinda like producing the everlasting lightbulb! :lol:


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## jakub76 (30/9/10)

Wolfy you're a legend for posting this thread!

Yesterday I finally got my science on and made up some agar slants and petri dishes.




Is the condensation inside the tube a problem? Does it end up being taken up by the growth medium or does it just sit there?

I ended up making four batches of agar solution. 1st couple didn't dissolve properly and left the slants really chunky so I washed the tubes all out and started again. By the third time I was satisfied with the consistency of the medium plus I had enough practice to be pretty confortable with the whole process too. Fun little project. Now I'm making a loop out of a thin guitar string and I'll start innoculating this weekend :icon_cheers:

PS: How do you keep your petri dishes sanitary? Is it enough to just tape across them keeping pressure between the halves? I've wraped in glad wrap and taped but it makes it hard to see plus it's not exactly elegant.


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## Wolfy (30/9/10)

jakub76 said:


> Is the condensation inside the tube a problem? Does it end up being taken up by the growth medium or does it just sit there?
> 
> I ended up making four batches of agar solution. 1st couple didn't dissolve properly and left the slants really chunky so I washed the tubes all out and started again. By the third time I was satisfied with the consistency of the medium plus I had enough practice to be pretty confortable with the whole process too. Fun little project. Now I'm making a loop out of a thin guitar string and I'll start innoculating this weekend :icon_cheers:
> 
> PS: How do you keep your petri dishes sanitary? Is it enough to just tape across them keeping pressure between the halves? I've wraped in glad wrap and taped but it makes it hard to see plus it's not exactly elegant.


Looks good *jakub76 *- I have no idea how you got the same tubes as I have, I thought mine were some sample batch they just wanted to get rid of. 

The condensation is both a problem, and it does seem to get absorbed by the agar over time.
If you inoculate your slants while there is condensation it can cause the newly inoculated yeast to 'run' all over the slant and rather than individual colonies it will just be a carpet of yeast - not really an issue except that it 'looks' different to the 'usual' yeast growth and so it's harder to tell if you have an infection or the like.

I find that 1.5% agar agar works well for me, and it will usually dissolve when the wort gets to boiling point - just need a bit of practice to get it all right. 

I use floritsts parafilm (it's much cheaper than the lab stuff) on mine.





But wrapping freshly made plates in cling-film is a common thing to do.


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## Bribie G (27/5/11)

Bumperty bump

Great guide Wolfy

Due to bad back I can't do any brewing as such but a bit of slanting might be the go, to take the mind off things.

So now I have a couple of packets of Agar threads, some 30ml test tubes , just got a pressure cooker. 

I have 4 smack packs of 1469 which are starting to show their age so I'd better get them slanted while the going is good as the 1469 long long drought is upon us once again. 
For one smacko would you suggest:


Smack it and use the resulting liquid to inoculate a heap of slants
or
Smack it, do a micro brew with LDME for a few days and use the slurry to inoculate a heap of slants?

The reason I ask is that with an older smacko, even when proved and swollen it can still take an appreciably longer time to build up to pitchable as opposed to an ultra fresh one that can be crawling out of the jar in the same day.
So I'm worried that just using the liquid out of the pack would result in slants that could take forever to culture up.

Or does the actual growing on the agar surface result in a similar yeast count independent of the age of the pack?

I don't have a bunsen burner but I expect a "creme brulee" little blowtorch and some thin gal wire would be good for inoculating?

cheers


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## Wolfy (27/5/11)

*BribieG* to inoculate multiple slants you'd not need more than a single drop of the yeasty-liquid from a single smack pack.
Unless the pack was years old and most of the yeast was dead, I can't imagine it would need to be smacked or propagated into a starter.
Since individual cells can be propagated onto malt-agar, there should be many many viable cells on just the one drop of yeasty-liquid (probably too many actually).


I use a similar blowtorch and a loop made from nichrome wire, I guess gal would work fine but, stainless-steel would be better (even a paperclip that bent into an appropriate shape).

*Edit*: If it was me, I'd wait until you are ready to use one of the packs to make a batch of beer, rather than opening it to take a few drops to make some slants from.


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