|My sterile transfer chamber|
I had been looking forward to getting stuck into the tissue culture process the entire week! Friday finally came and I eagerly set about getting everything laid out that evening for Saturday and ensured that everything that I needed I had. That evening I mixed up a litre of Phytotechnology Labs’ orchid multiplication medium which is an agar-based and ready to use medium for encouraging vegetative growth of stem nodes. Once heated for just enough time to allow the agar to melt, I poured the mix into various jars and some McCartney bottles to be sterilised. All containers of medium and a bottle or two of tap water and my dissection kit were placed into my Fagor pressure cooker for sterilisation for 30 minutes. After the time was up I let everything cool down for a bit inside the cooker and then opened it up and moved everything into the sterile transfer chamber. The chamber had been pre-sprayed with a 10% bleach solution and had been irradiated with UV for 30 minutes. After everything had been placed inside I switched on the UV lamp again for a further 30 minutes considering that my hands were probably a source of potential contamination.
The next morning the media had cooled and set. I prepared two jars of bleach solution, one with 10% and the other with 5% to which I added 3 to 4 drops of Tween 20 from Phytotech. These jars of bleach solution were for the sterilisation of nodal sections of the flowering stems of my parent plants. As these jars would initially be used outside of the sterile transfer chamber, I immersed them in a larger container of 10% bleach solution to ensure that the outside of the jars would not pose a contamination risk. In addition, I prepared a third jar of Virkon S using a 2% solution. This jar too was immersed in 10% bleach to avoid a possible contamination source. Once this was done I set off to the greenhouse and selected my plants for culture. The flowering stems were cut and placed into a jar of water and brought into the kitchen for washing. Each flowering spike was thoroughly rinsed and washed under normal tap water and then cut into nodal sections. Additional sections of internode were also cut. All these cuttings were placed into the Virkon S solution and agitated while I prepared to sterilise my hands with alcohol-based hand sanitiser (the new one by Dettol). Once I was happy with my hands I put a pair of surgical gloves on and repeated the procedure with the hand sanitiser and lit a candle next to the chamber for flaming my tools.
|Phalaenopsis nodal sections|
Here’s holding thumbs!