3. Start up your laminar flow cabinet. Turn on the clean air filter. The filter should run for not less than half an hour before you start working. If the cabinet is equipped with a UV light, this should also be switched on at the same time. Beware of the UV light. Never look into the light. Permanent damage to your eyes can be the result.
4. Prepare the sterilizing solutions from bleach. Two strengths of bleach solution are needed. One to sterilize your work area and items being moved in and out of the cabinet and one to sterilize the seeds.
Use the 1000 ml graduated container and pour in 200 ml bleach (e.g. Snowite, Chlorox) and add 500 ml distilled water. Normal tap water is also okay. If normal tap water is used let it run for a minute or so in case there is any debris in the pipe and use it then. Fill the spray bottle with this mixture and thoroughly spray the inside of the laminar flow cabinet - but not the clean air filter - until it is soaking wet. Place the cotton dish washing cloth in the center of the cabinet and spray it with the bleach solution.
Decant a small amount of this solution in a deep dish, large enough to stand one of the flasks inside. Decant another quantity in an empty flask or glass and place syringes inside. Place the dish and the glass inside the cabinet.
For the seed sterilizing solution decant 100 ml of the solution in the 200 ml container and fill it up to 200 ml. Get a toothpick and obtain a minute amount of dish washing detergent. Add this to the mixture. It will help to break the surface tension of the water and allow the sterilizing solution to get into the entire surface of the seeds to be sterilized. Too much detergent will result in excessive foaming.
5a) When the medium has turned solid you are ready to start work. Cut small pieces of self-adhesive tape and stick them on the flasks. Write the identification number of the seed on the tape. Place bottle with distilled sterilized water in deep dish inside cabinet.
Switch off UV light. Put on the dish washing gloves and sit in front of the cabinet. Spray the inside of the cabinet, the cloth and both gloved hands thoroughly with the bleach solution
Get your assistant to pass the flasks to you by first placing them into the deep dish. You take them out form the dish and spray them thoroughly with the bleach solution. You are now working in a sterile environment. Keep it sterile, use the bleach solution liberally and avoid sudden movements. Never cross your arms, learn to work from left to right or vice versa, whatever is convenient.
Your assistant will now fill one of the tablet tubes containing seed with a small amount of sterilizing solution from the 200 ml container. Turn the timer to 8 minutes and start shaking the tube - outside the cabinet. When the time is nearly up have the tube placed in the deep dish, take it out, spray it thoroughly with the bleach solution and observe whether the seed is sinking, floating or stays suspended in the liquid.
Prepare the syringe by attaching the needle. Keep everything sterile. Extract the sterilizing solution with the seed from the tube and eject the sterilizing solution leaving the seeds behind. This is sometimes quite tricky and will take some practice not to decant the seed with the liquid. With the seed inside the syringe suck up a little of the distilled, sterilized water and wash the seeds. Depending on how many flasks are to be seeded leave 1 ml of distilled water per flask inside the syringe.
The next step needs to be done quickly. Unscrew the lid of the flask to be seeded but leave the lid on the flask. Hold syringe in right hand, with your left hand lift lid of flask, shale syringe and quickly eject about 1 ml of liquid with seed into the flask and close lid immediately. Repeat with the remaining flasks. Now with both hands tightly close lid of flask and spray flask with bleach solution, especially under the rim of the lid.
6. Store flasks in a dry place with normal light. Important is that the storage place is not subject to severe changes in temperature. Around 22-25 degrees C. is most adequate for orchids. Do not place flasks in direct sun. Observe flasks daily for any sign of contamination. Write down the date of germination in the notebook and also if and when contamination occurred and its colour.