Sterile nutrient agar plates

[cathy]

Hi, How do you sterilise a batch of nutrient agar using a pressure cooker? Is it similar to an autoclave where you just put the nutrient agar powder and water in a sealed schott bottle and 'cook' it for the appropriate time? If so, how long do you give it for say 400mL of NA? Then, do you pour the plates within a 10cm radius of a bunsen flame or just go somewhere relatively clean to pour them and hope? Thanks for your help in advance, Cathy

[estelle]

Hi Cathy,
do you have a copy of the chemical safety in schools folder?There should be one somewhere in the science dept. It was put out several years ago. when CSIS came into schools.
In it in section 3.2 science there is a section .6 on page 57 that has some good information about biological materials, agar plate etc. how to use and dispose etc. If you don't let me know and maybe I could fax that section to you.
Estelle.

[chemical]

Hi Cathy

I made the agar plates today. After I read the NSW school manual, thought and asked a person that had made them when she was on the job for a while. I simply boiled the water (demuire water) to a warm then added Ajdar, Peptone(I think it was) and vegimite.

You simply do in on a hot plate with a magnetic stir. Bring the water to the near boil then add the other slowly and cook for about 5 minutes until all has dissolved.
Before making the agar mixture Prepare the bench with bleach, lay new petri dished out carefully.

Only open them enough to pour the agar mixture. Let them stand for a while, there will be condensation in the petri dishes. When cool stack on a tray in the fridge I will be using the plates in 24 hours. I made the plates early so they were set for the pract for tomorrow. I have also got to go and get the 4 food mould bits the 4 water examples. So yes you do your best and it will work but ask lots of question.

I wouldnot be without this site.
Lindy

[Labbie]

Do you Sterilise your Agar plates?? After mixing all the items together?? Before pouring your plates??

This can be done two ways, Put foil on top of flask, ans simmer the liquid for 30 Minutes on a lower heat on your Hot plate. Stirring all the time, (need to keep an eye on it), Remove from heat and allow the medium to cool to about 50c then pour into plates.

Or Put in a glass screw top bottle, in the microwave, and cook again on high for (I think it is 10 mins) but please do a search of this site, as I may be wrong.

Regards

[Ocean Breeze]

I dont sterilise under pressure. For school purposes, simply boiling on hotplate or in microwave is plenty hot enough(near 100 deg C).
The trick is to work aseptically while pouring. So wash down benches with bleach/dettol or similar first.
Keep the base of the plate covered over with the lid of the plate, so that no organisms "fall" down throught the air and into your sterile plate while you are pouring.
Do work within the updraught of a bunsen flame.. as close as possible.
Pour when agar has cooled to 50 deg C, or just a bit above. That way you will have less problems with condensation on the plates.

If you pop a thermometer(100 deg) into your flask of hot agar, as soon as you finish boiling it, and then replace the cotton wool plug in the flask again to support the thermo, your agar will stay sterile. Leave the cotton wool plug in the neck of the flask until you are ready to pour. That way you wont get contamination while its cooling.