Making Agar Slants (Slopes) - In Pictures

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

Post by Cobnut » Wed Dec 16, 2020 4:07 pm

Resurrecting this thread in the hope of getting some further guidance re: infections in "blank" slants.

I made a batch of about 20 slants using (essentially) the method described at the start of this thread. I "sterilised" the slants with slightly loosened lids for about 20 mins after the pressure cooked came to the boil (don't know what pressure as it only came with a single weight).

I then tightened the lids and laid the slants to set.

They have been stored in a plastic sandwich box since then in my (home) office, albeit I noticed quite a lot of condensation had collected, so I removed each lid and poured out the excess liquid next to a gas burner (kitchen hob).

I've since innoculated a few with yeast from a whitelabs liquid yeast pack using a "loop".

However, as can be seen in the attached pictures, I clearly have a contamination issue with some of them, but not all.
Slant1.jpg
Slant2.jpg
Re-reading the initial post from wolfy, he says to "autoclave" for 30 mins. Is that my error? If so, why have some of them remained "clean" and others gone furry?

Or does the Hive Mind think I've "infected" them by opening them to remove the condensation? How else to get rid of the condensation?
Fermenting: nowt
Conditioning: English IPA/Bretted English IPA
Drinking: Sunshine Marmalade, Festbier, Helles Bock, Smokey lagery beer, Irish Export StoutCascade APA (homegrown hops), Orval clone, Impy stout, Duvel clone, Conestoga (American Barley wine)
Planning: Dark Mild, Kozel dark (ish), Simmonds Bitter, Bitter, Citra PA and more!

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Hanglow » Wed Dec 16, 2020 5:08 pm

Most pressure cookers in the UK won't get to high enough pressure to sterilise unfortunately, so I think that would be your problem

McMullan

Re: Making Agar Slants (Slopes) - In Pictures

Post by McMullan » Wed Dec 16, 2020 5:24 pm

When a pressure cooker seals and the pin pops up all that means is it’s sealed. It's nowhere near at the temperature (121*C) required to sterilise (over 20 minutes) at this point. That actually takes a while, even in a proper autoclave. I’d recommend 45-60 minutes from when it seals. Hanglow is partly right, if you were to follow a standard autoclave protocol, but if you extend the time it is possible to sterilise in a bog standard pressure cooker.

I don’t like the look of those tubes. Have you been reusing them? I prefer glass if I’m reusing.

In a lab, condensation is dealt with by allowing the moisture to escape by loosely fitting the lids and leaving them in a warm area overnight. At home I’d recommend a big lunch box or similar that can be sterilised with iodine solution. Try not to pour to molten agar when it’s really hot. That creates more condensation.

The key is to keep everything sterile. Working around a flame, even a small camping stove, can maintain a sterile envelope to work in. Let it burn for about 15 minutes or so in a clean, draft free area. Plan how you work, don’t work over open vials and if you have to put a cap down put it faced down. At least wash your hands and forearms very thoroughly. You’ll be surprised how many microbes we shed, especially from our hands. Ideally, wear a clean apron or even a cheap lab coat. We’re walking microbial ecosystems.

I think your contamination problem (looks like a mold) is most likely airborne so the last paragraph is likely the most relevant, but it’s all good practice and promotes success here.

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Cobnut » Wed Dec 16, 2020 6:23 pm

McMullan wrote:
Wed Dec 16, 2020 5:24 pm
When a pressure cooker seals and the pin pops up all that means is it’s sealed. It's nowhere near at the temperature (121*C) required to sterilise (over 20 minutes) at this point. That actually takes a while, even in a proper autoclave. I’d recommend 45-60 minutes from when it seals. Hanglow is partly right, if you were to follow a standard autoclave protocol, but if you extend the time it is possible to sterilise in a bog standard pressure cooker.
Better make sure SWMBO is out when I'm doing this next time!
McMullan wrote:
Wed Dec 16, 2020 5:24 pm
I don’t like the look of those tubes. Have you been reusing them? I prefer glass if I’m reusing.
Brand new tubes - first use.
McMullan wrote:
Wed Dec 16, 2020 5:24 pm
In a lab, condensation is dealt with by allowing the moisture to escape by loosely fitting the lids and leaving them in a warm area overnight. At home I’d recommend a big lunch box or similar that can be sterilised with iodine solution. Try not to pour to molten agar when it’s really hot. That creates more condensation.

The key is to keep everything sterile. Working around a flame, even a small camping stove, can maintain a sterile envelope to work in. Let it burn for about 15 minutes or so in a clean, draft free area. Plan how you work, don’t work over open vials and if you have to put a cap down put it faced down. At least wash your hands and forearms very thoroughly. You’ll be surprised how many microbes we shed, especially from our hands. Ideally, wear a clean apron or even a cheap lab coat. We’re walking microbial ecosystems.
These are good tips. Thanks.
McMullan wrote:
Wed Dec 16, 2020 5:24 pm
I think your contamination problem (looks like a mold) is most likely airborne so the last paragraph is likely the most relevant, but it’s all good practice and promotes success here.
I agree that it looks like mould. I'm just a little surprised that some of the slants have grown a substantial covering of mould, but other seems still to be pristine.

Certainly makes me think I need to improve my technique to reduce contamination risk!

Grist to the mill :)
Fermenting: nowt
Conditioning: English IPA/Bretted English IPA
Drinking: Sunshine Marmalade, Festbier, Helles Bock, Smokey lagery beer, Irish Export StoutCascade APA (homegrown hops), Orval clone, Impy stout, Duvel clone, Conestoga (American Barley wine)
Planning: Dark Mild, Kozel dark (ish), Simmonds Bitter, Bitter, Citra PA and more!

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Hanglow » Fri Dec 18, 2020 11:37 am

McMullan wrote:
Wed Dec 16, 2020 5:24 pm
Hanglow is partly right, if you were to follow a standard autoclave protocol, but if you extend the time it is possible to sterilise in a bog standard pressure cooker.
Thanks for the correction, I've put off doing this sort of thing before because every time I'd searched for a high pressure cooker the only ones I thought suitable were expensive US made ones. If a standard one works fine then I'll probably just get one of them

McMullan

Re: Making Agar Slants (Slopes) - In Pictures

Post by McMullan » Fri Dec 18, 2020 12:05 pm

I use a certified Prestige media autoclave and a Prestige standard pressure cooker when I need to. To be honest, if there was any responsibility attached, such as selling sterile consumables, pure yeast slants, etc., I'd use an autoclave, but for our own personal uses in DIY microbiology a standard pressure cooker is fine. I get very similar looking results when extending the time in a pressure cooker and I have no problems to report.

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Re: Making Agar Slants (Slopes) - In Pictures

Post by Carnot » Fri Dec 25, 2020 5:55 pm

More! More! More! Don't feel like you're 'labouring it' because you're totally taking the piss

Your exact words lest you forgot.You might be an accomplished biochemist but I doubt your water chemistry. Quid pro quo. B:O

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