10 replies to this topic

[seanspotatobusiness]

I tried to plate some transformants this evening using some plates that were prepared a few hours earlier by a technician and a new student. A couple of the plates were fine and another couple seemed fine, until I stroked them the wrong way and the horrible truth became clear - they were practically liquid! I carried on as best I could, and tried spreading by rotating the plate around. I will investigate what happened tomorrow but I suspect that the agar wasn't mixed properly? Then again, I don't see how that could be an issue after autoclaving and cooling, would diffusion not have been sufficient during all this time? Does anyone else have experience of some plates not solidifying due to improper mixing before pouring? It's never happened to me.

In any case, if they weren't mized properly, then I guess the ampicilin wasn't distributed properly either...

[eberthella]

All, good points and you were prob right - inadequate mixing before autoclaving. As you said, don't count on the amipicillin to be even distributred. Merely combining ingredients and autoclaving will give the result you saw.
Pity - and so many of our colleagues give techncians such credit.

[phage434]

Diffusion is definitely NOT sufficient to thoroughly mix agar. Even if you mix the agar prior to autoclaving, it still must be mixed after autoclaving. Of course, it must be also to mix in the antibiotic. Perhaps you should spend some time teaching the student and technician.

[seanspotatobusiness]

phage434, on Sep 2 2009, 03:40 AM, said:

Diffusion is definitely NOT sufficient to thoroughly mix agar. Even if you mix the agar prior to autoclaving, it still must be mixed after autoclaving. Of course, it must be also to mix in the antibiotic. Perhaps you should spend some time teaching the student and technician.

It was generally agreed that the agar was not mixed properly before pouring. The student was left alone to pour the plates but didn't realise or forgot to mix.

[pito]

phage434, on Sep 2 2009, 03:40 AM, said:

Diffusion is definitely NOT sufficient to thoroughly mix agar. Even if you mix the agar prior to autoclaving, it still must be mixed after autoclaving. Of course, it must be also to mix in the antibiotic. Perhaps you should spend some time teaching the student and technician.

Mixed after autoclaving?

I never mix it after autoclaving.

I only mix it before.... and after autoclaving I simply start pouring. (or when adding antibiotic, I gently mix it by turning the bottle around a bit, very slowly)

Normally autoclaving is enough to dissolve and mix the agar perfectly. At least thats how I have been told and also I never had that kind of problem or problems in general.


Besides that: when working with antibiotics , you cant mix it anyway. You add the antibiotics and then you gently turn the bottle around a little bit, because when you mix it, you will get to many airbubbles in your agar..... and thus you will need to wait even longer before you can pore the plates.

seanspotatobusiness are you sure you added enough agar ??

Edited by pito, 02 September 2009 - 06:11 AM.

[gebirgsziege]

pito, on Sep 2 2009, 04:07 PM, said:

phage434, on Sep 2 2009, 03:40 AM, said:

Diffusion is definitely NOT sufficient to thoroughly mix agar. Even if you mix the agar prior to autoclaving, it still must be mixed after autoclaving. Of course, it must be also to mix in the antibiotic. Perhaps you should spend some time teaching the student and technician.

Mixed after autoclaving?

I never mix it after autoclaving.

I only mix it before.... and after autoclaving I simply start pouring. (or when adding antibiotic, I gently mix it by turning the bottle around a bit, very slowly)

Normally autoclaving is enough to dissolve and mix the agar perfectly. At least thats how I have been told and also I never had that kind of problem or problems in general.


Besides that: when working with antibiotics , you cant mix it anyway. You add the antibiotics and then you gently turn the bottle around a little bit, because when you mix it, you will get to many airbubbles in your agar..... and thus you will need to wait even longer before you can pore the plates.

seanspotatobusiness are you sure you added enough agar ??

(
I usually add a magnetic stirrer to the agar bottle and let it stirr SLOWLY (to avoid bubbels) until it is cool enough to add antibiotics or simply pour it. This works perfectly as long as the pH of the medium is high enough (sometimes I need media with a low pH...this means slurry plates to work with...so just an idea was your pH too low??)

[DrRob]

Why not just flame your plates with a Bunsen after pouring them to break the bubbles? Then you can mix your antibiotic in to your hearts content!!

[DrRob]

gebirgsziege, on Sep 3 2009, 06:24 AM, said:

I usually add a magnetic stirrer to the agar bottle and let it stirr SLOWLY (to avoid bubbels) until it is cool enough to add antibiotics or simply pour it. This works perfectly as long as the pH of the medium is high enough (sometimes I need media with a low pH...this means slurry plates to work with...so just an idea was your pH too low??)

To pour low pH plates, I used to use a separate mixture of 1% agarose (which has the gelling strength of 2% agar) and mixed this in equal parts with my (low pH) autoclaved nutrients before pouring

[Quasimondo]

I do that regularly and NEVER MIX THEM, just pour the agar into the medium-containing flask, and then autoclave. I also don't mix after autoclave, except gently mixing after adding antibiotics. I think your problem is because of the insufficient agar concentration.

Edited by Quasimondo, 05 November 2009 - 11:02 PM.

[backwoodsman]

Maybe when the person pour liquid medium into the plate, the liquid was just became into colloid. So the agar deposit down. At first the couple of the plates can not be solid, but the following couple of plates is firmly solid because have overfull of agar.

[arera]

In my opinion, u should double check your menu on agarose powder... how much did u add to the mixture before autoclaving....