Making the medium!
SO, today was the day that I managed to make the growing mediums up for my orchids. Its been a great learning curve and I am now proud to say that I will be able to do it again, hopefully, the only help I will need is the calculations.
Now, the "Murashige and Skoog" needed to be made up. I needed to add the gelling agent, otherwise known as agar and the Sucrose which is the recommendation from the book "Growing Hardy Orchids" by the micro-propagation lab at Kew Gardens. The packets we had for this one was 4.4g of medium for 100ml. I had to add 25g/L of Sucrose to this and 12g of agar. Mandy, the lady who helped me, suggested that I use 500ML jars to make it up as this would be easier to pour it into the petri dishes. So I halved everything, so we had 2.2g of medium for 500ml, 12.5g of Sucrose, 6g of Agar. Water needs to be added to help mix it and it also helps it set. Once this is done, it is well mixed and then is left for a bit because I have to make the other one. The other medium, given to me by Phytotechnology laboratories, already has all the necessary ingredients mixed within it, as well as the gelling agent, so all I needed to do was add the water; again, it was in 500ml jars, so i needed to divide the medium between the two. With this one it is 28.44g per litre so each jar needed 14.22g of medium.
Once it was all mixed up and waiting to go into the autoclave (will explain all), I started getting all the bits and bobs ready that I need for sowing the seed for when the medium is ready!!!.
Now, the "Murashige and Skoog" needed to be made up. I needed to add the gelling agent, otherwise known as agar and the Sucrose which is the recommendation from the book "Growing Hardy Orchids" by the micro-propagation lab at Kew Gardens. The packets we had for this one was 4.4g of medium for 100ml. I had to add 25g/L of Sucrose to this and 12g of agar. Mandy, the lady who helped me, suggested that I use 500ML jars to make it up as this would be easier to pour it into the petri dishes. So I halved everything, so we had 2.2g of medium for 500ml, 12.5g of Sucrose, 6g of Agar. Water needs to be added to help mix it and it also helps it set. Once this is done, it is well mixed and then is left for a bit because I have to make the other one. The other medium, given to me by Phytotechnology laboratories, already has all the necessary ingredients mixed within it, as well as the gelling agent, so all I needed to do was add the water; again, it was in 500ml jars, so i needed to divide the medium between the two. With this one it is 28.44g per litre so each jar needed 14.22g of medium.
Once it was all mixed up and waiting to go into the autoclave (will explain all), I started getting all the bits and bobs ready that I need for sowing the seed for when the medium is ready!!!.
Once the medium was made up, I needed to make sure the PH was just right for the orchid seeds, in the books that I read, it was recommended that for Dactylorhiza seeds the Ph needs to be between 5.5 and 5.9. In order to do this, we needed to test it to see if it was acidic. Both mediums were slightly too acidic so myself and Mandy added some Sodium Hydrochloride to make it more alkaline. a few drops sufficed and it was perfect.
The medium was put on a a heating plate and had a magnet put in it so that it was mixed adequately when the Sodium Hydrochloride was added. The heat wasn't used as it was merely used for the PH.
Ready to sterilize it!!
Once all the bits and bobs were done and the medium ready, I got all the stuff i needed for the sowing. I will explain all of that when the seeds have been sown. These all needed to be sterilized in an autoclave, this is a type of machinery a little bit like a kiln that heats up to about 275 degrees, this will guarantee that everything is sterile to reduce the amount f bacteria in the medium. This also needs to be done for any other materials being used. Anything other than the medium needs to be covered in tin foil, the main reason for that is so when it all comes out, nothing gets into the newly sterilized supplies.
The lids for the medium jars are un screwed to stop them exploding because the autoclave is also like a pressure chamber, it is heat and steam that helps to sterilize. It takes about two hours to run.
The lids for the medium jars are un screwed to stop them exploding because the autoclave is also like a pressure chamber, it is heat and steam that helps to sterilize. It takes about two hours to run.
The medium is made up!!
Once the medium was made, it was time to put it into the petri dishes to set. This is done in a flow cabinet, a flow cabinet is a unit that allows to sow seeds in sterile conditions. It has an air flow that blows air out and allows any fungus or germs to be evacuated from the cabinet, all though, it doesn't stop them all from being in the area so it needs to be cleaned with alcohol to help it stay cleaner than it would be. This bit is the tricky bit, you can't lean into it, otherwise bacteria from hair, clothes or other things can penetrate the flow cabinet and infect anything that has already been cleaned!!! The same process happens when sowing seeds.
Now the medium comes out liquid, but the gelling agent helps it set when its cooled down, which creates perfect growing conditions for seeds, especially orchids, as you read before.
The medium needs to be poured but before that you need to sterilize the top of the jar by burning it with a bunson burner, which helps with the obvious, burn away an extra bacteria.
Once the liquid has been poured in, the lids need to be closed to stop any other possible germs or bacteria to land on the media, I'll know in a few weeks if any infection has happened. But to let it set properly, the lids need to be opened slightly and left in the flow cabinet to allow the medium to dry and also to let the condensation dry up, otherwise it is a perfect environment for fungus or mould. The medium is then closed again and left over night, and should be set and dry, perfect for when I sow the seeds!
All the medium is now ready and stored in the fridge waiting for the seeds to be sown!
Now the medium comes out liquid, but the gelling agent helps it set when its cooled down, which creates perfect growing conditions for seeds, especially orchids, as you read before.
The medium needs to be poured but before that you need to sterilize the top of the jar by burning it with a bunson burner, which helps with the obvious, burn away an extra bacteria.
Once the liquid has been poured in, the lids need to be closed to stop any other possible germs or bacteria to land on the media, I'll know in a few weeks if any infection has happened. But to let it set properly, the lids need to be opened slightly and left in the flow cabinet to allow the medium to dry and also to let the condensation dry up, otherwise it is a perfect environment for fungus or mould. The medium is then closed again and left over night, and should be set and dry, perfect for when I sow the seeds!
All the medium is now ready and stored in the fridge waiting for the seeds to be sown!
After a longs day work this is what I look like!