Making sterile agar plates

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elaruu
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Making sterile agar plates

Post by elaruu »

Hi everyone. When you make up nutrient agar for pouring (sterile) plates, is heating it up to boiling to dissolve the agar also enough to sterilise the agar? Or is it necessary to pressure cook or autoclave the agar before pouring the plates?

Thanks!
RosalieL
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Re: Making sterile agar plates

Post by RosalieL »

I wash the glassware in metho then place cotton wool loosely in the top so nothing can fall in while the agar is dissolving and boil it for a few minutes by moving it on and off the hotplate when it starts to bubble up too much. It's time consuming and you have to be vigilant but it works for me.

(Edited to add: I do this in a 1L or 2L conical flask depending on how much I need - 2L flask if I'm making 1L of agar)

I have also made up a mixture of 50:50 nutrient agar and plain agar and use that for my plates since the plain is cheaper. There is still enough nutrient in the mixture to work for school experiments. Use the instructions on your original bottles to calculate how much you'll need.

Eg. if your original recipes call for 26g/L for nutrient agar and 16g/L for plain agar, your new amount will be:

13g + 8g = 21g/L for the agar to set properly.
Last edited by RosalieL on 04 Feb 2025, 11:33, edited 1 time in total.
LabbieSeth
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Re: Making sterile agar plates

Post by LabbieSeth »

elaruu wrote: 04 Feb 2025, 10:43 Hi everyone. When you make up nutrient agar for pouring (sterile) plates, is heating it up to boiling to dissolve the agar also enough to sterilise the agar? Or is it necessary to pressure cook or autoclave the agar before pouring the plates?

Thanks!
I personally autoclave the agar after as I heat and mix my agar in a beaker, meaning it is susceptible to contamination from airborne particles. This way I ensure that no contaminants will grow on/in the agar after pouring and cooling (assuming I haven't contaminated it during another step). That being said, I'm sure there is a way to make agar without leaving it open to airborne particles thus eliminating the need to autoclave it.

Take with a grain of salt as I'm sure someone on here has a pretty bullet proof method on how to make agar that is far superior to mine haha.
Regards, Labbie Seth :thumbup:

Lab Tech NSW Sydney
Marama T
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Re: Making sterile agar plates

Post by Marama T »

I never sterilise the agar after the initial step of boiling it. It does mean it's best to prepare the plates as close to the day of use as possible. I always make a few extra plates so that if there are a few that are contaminated, I still have enough for the class(es). Works for me. Just be sure to store the plates in the fridge, of course.
I think most contamination occurs in the pouring and cooling of the plates, so it doesn't really matter whether you autoclave it or not.
Last edited by Marama T on 05 Feb 2025, 09:04, edited 1 time in total.
PeterK
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Re: Making sterile agar plates

Post by PeterK »

I always make it in a schott bottle in the autoclave.

Steps/warnings:
Quick initial mix
Make sure you a bottle at least double your volume (eg. 1L bottle for 500ml of agar)
Tighten the schott bottle to just tight, then turn the lid back a quarter turn.
Indicator tape
Autoclave
Cool and pour.

Nice and easy and you don't have to worry about watching it to make sure you don't burn your agar.
Nads343
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Re: Making sterile agar plates

Post by Nads343 »

I boil the agar in a Schott bottle first using a hot plate, then place in the autoclave for 15 minutes at 121 degrees with autoclave indicator tape (as PeterK said, double the volume and ensure the lid is loose by tightening first, and then loosening by 2 turns where the lid is still on but loose). Once slightly cooled, pour into plates.
I pour agar in my fume cupboard after sterilising the surface and flaming the neck of the Schott bottle (unsure if this is correct, but I get sterile plates every time) and let them cool and gel completely before placing with the agar facing up in the fridge until it is needed.

I did ask a company where I purchase my nutrient agar from if boiling before autoclaving was entirely necessary which I believe they said it is recommended but not entirely necessary. I haven't tried making plates without boiling first so I can't really comment, but I don't think it will really affect the sterilisation process if you have stirred the nutrient powder into the distilled water properly enough and that there are no clumps.
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