Making Agar Slants (Slopes) - In Pictures

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gregorach
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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Mon Oct 17, 2011 10:25 am

If they were unopened since autoclaving, then you didn't cook them for long enough. Those vials should be completely airtight if they're tightened properly.
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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Mon Oct 17, 2011 2:30 pm

I've noticed these ones don't tolerate much internal pressure when you fill with wort and shake up your slant. Tend to fizz out. So probably not absolutely airtight

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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Mon Oct 17, 2011 2:53 pm

Well, you ideally should be using containers that are absolutely airtight. Your agar will dry out otherwise.

I still don't think there's much chance of mould spores getting in though - if they could, Pasteur's swan-necked flask experiment wouldn't have worked.
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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Mon Oct 17, 2011 3:07 pm

These were cooked at max for 25 mins from solid steam hiss. However I also sterilise my rinse jars in the same cook which are 2/3rds full of water. So perhaps should aim for 40 mins...

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Re: Making Agar Slants (Slopes) - In Pictures

Post by nastyphoenix » Sat Oct 22, 2011 10:16 pm

What is this all about? What is slanting? Why do it?

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Befuddler » Sun Oct 23, 2011 3:50 am

nastyphoenix wrote:What is this all about? What is slanting? Why do it?
It's a method of isolating and storing healthy yeast.
"There are no strong beers, only weak men"

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Goulders » Wed Nov 02, 2011 10:59 pm

This is a really interesting topic. Must be my school science background coming back to me! Found this on YouTube Maybe not exactly the same as Wolfy's method, as the yeast is captured from a bottle. However, he also shows how to make a starter from a slant in another video.

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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Thu Nov 10, 2011 12:02 am

gregorach wrote:Well, you ideally should be using containers that are absolutely airtight. Your agar will dry out otherwise.

I still don't think there's much chance of mould spores getting in though - if they could, Pasteur's swan-necked flask experiment wouldn't have worked.
The rest of that batch were all not sterile. It took months at room temp but eventually they all failed. The worrying thing is some of that batch have been inoculated so I should really discard the lot, even though no infection is evident in the refrigerated slants. Must try harder....I best identify the relevant dates. If there is no visual clue to a small rogue colony on the chilled slants would you still discard?

After all if you slant from recovered bottle yeast it is not completely free of foreign microbes anyway, nearly but not 100%.

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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Thu Nov 10, 2011 10:44 am

I'd probably streak some plates to isolate clean yeast from the suspect slants.
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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Thu Nov 10, 2011 12:24 pm

Good idea...but its a lot of work aint it?

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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Thu Nov 10, 2011 12:46 pm

Yeah, it's a right pain in the arse sometimes... ;)
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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Mon Dec 19, 2011 5:03 pm

gregorach wrote:Right, first off, if nobody's said this yet: you clearly need the yeast book.

This is (more or less) what a streaked plate is supposed to look like:

Image

It's not perfect, because the colonies from the last set of streaks aren't as well isolated as I would like, but it's not bad. i'm not getting any of the really good ones out long enough to photograph... ;)

I've never had very good results with the streaking patterns usually recommended - rather than using a zig-zag pattern, I streak a number of not-quite-parallel lines at each step. Hopefully you can see the pattern in the photo. One thing to remember is that you need to flame the loop between each "set" of streaks.

1: Take a tiny sample from your yeast source, pick up the plate, and run the loop back and forward several times in a short line near the edge of the plate - you need to be able to track where this is, so I do it next to my left thumb when holding the plate. Put the plate back down and rinse your loop. (I like to rinse at this stage so that you don't end up with crud burnt onto the loop.) Rotate the plate clockwise through about 45 degrees.
2. Flame the loop. Pick up the plate (keeping it inverted for now), cool the loop by touching it to an area of the plate you're not going to be working near (if you've picked the plate up in your left hand, this is likely to be somewhere around the edge of your palm on the far side from your thumb. You can see the distorted area where I cooled my loop on this plate near the bottom of the photo.) Turn the plate over and quickly draw a set of streaks across the line you did in step 1. Put the plate back down and rotate it clockwise again.
3. Using the same technique, do another two sets of streaks, flaming the loop each time.

Incubate for two or three days, and you should have a lovely plate like mine.
Am having a go again at getting some isolated yeast colonies from a slant. So 2- 3 days at 20C is best for the colonies from single cells to be evident ? Noticed at 1.5% agar the plates were pretty liable to be torn with the loop unless you were very light handed...I'll pop a photo up .... if its worth posting!

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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Mon Dec 19, 2011 5:20 pm

Yeah, I think 2 or 3 days usually does the job... You can always leave it longer if need be.

You need to have a pretty light touch with all of this sort of stuff, but you could up the proportion of agar to produce a stiffer medium if need be.
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Re: Making Agar Slants (Slopes) - In Pictures

Post by greenxpaddy » Mon Dec 19, 2011 5:31 pm

Regarding storage they say leave them upside down. However that means that mould spores can fall down the crack between the lid and bottom (now top). I have left them upside down but in a plastic bag and with florists parafilm round. Being paranoid again!

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Re: Making Agar Slants (Slopes) - In Pictures

Post by gregorach » Mon Dec 19, 2011 5:47 pm

Yeah, I always keep them wrapped. I just use cling film.
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