rellenburg wrote:As for the autoclaving media section I would...
1. Pour prepared media into a pot (or other container that is safe for a stove top or hotplate)
2. Heat on stove or hotplate almost to a boil while stirring media until everything dissolves.
3. Dispense hot liquid (you can let it cool a little) into vessels and secure lids.
4. Autoclave at 15psi for 20-25 min (depends on how much media you're making)
5. When cooled and gelled it is ready for use.
I second this procedure as it is the most contamination free method normally used, even in big laboratories. Pouring media is used only with disposable petri dishes.
Also, a minor change in order:
1- Dissolve the MS+vit powder in 900mL of destilled water and stir
2- Add the mix for plant tc
3- Add 30g of suggar and stir
4- Add food coloring and stir
5- Complete with destilled water up to 1L mark
6- Get the ph of the solution and adjust until ph5.6
7- Add 2,5g of gellan gum powder(see picture) and stir
8- get to boil in microwave 8min/L
That because the gel normally damages the electrode in the meter by clogging the tiny pores it has, causing errors in future uses. And the gel need to get dissolved before autoclaving to have best results, that is done even when using the pouring medium procedure after autoclaving.
1- wash the explant in current water about 2 minutes (optional With soap in water, antibacterial
2- wash the explant in destilled water about 2 minutes
Dip into 10% v/v NaClO for 5-7 minutes
wash the explant in destilled water about 2 minutes
3- Put the explant in a esterilized plate and cut off top(trap)/botton(white tissue) parts
4- wash the explant in destilled water+PPM about 4 minutes
4- wash the explant in destilled water about 2 minutes
5- wash the explant in destilled water about 2 minutes
6- Put the explant into the media and cap the tube using a plastic film as a seal.
PPM instructions say that you do not wash the explant after using PPM, you use it and transfer to media, so do not do the Red section.
Also, PPM is just a bacteriostatic agent, it does not kill bacteria, as you are using a plant, that tissue has too much bacteria to handle it with just PPM, it is ok with seed but not tissue. That is why I added the green section.
Matt has written a very good protocol for introducing tissue, you may follow it and have good results.