Orchids — Fundamentals Of Flasking
Fundamentals of Flasking
Flasking is not for the faint hearted.
First, you need seed.
Once pollinated, seed can take anywhere from 3 - 18 months for the seed pod to develop and ripen/mature, depending on genera.
Plants will typically grow in laboratory conditions for at least 12 - 18 months before being ready to deflask (in this time, they can also be re-plated 2-3 times).
ALL INFORMATION IS GIVEN IN GOOD FAITH AND IT IS YOUR RESPONSIBILITY TO READ CHEMICAL AND EQUIPMENT LABELS, ETC.
IT IS IMPORTANT TO FOLLOW ALL SAFETY PRECAUTIONS GIVEN ON LABELS.
ALWAYS FOLLOW THE MSDS GIVEN ON CHEMICALS AND EQUIPMENT.
IF YOU DO NOT FEEL CONFIDENT OR DO NOT UNDERSTAND ANY INFORMATION ON LABLES OR MSDS, DO NOT PROCEED.
Orchid seed do not have a nutrition pack to survive and grow like other plants. Most orchids need a fungus to grow and germinate. This fungus is commonly found upon the roots of orchids.
IMPORTANT: With flasking EVERYTHING MUST BE 100% STERILE.
Picture of part of our Lab holding around 2000-3000 flasks
You will need a few things to get started:
- Laminar flow cabinet (preferred)
- Dedicated room (preferred)
- Dedicated sink
- Racks (to hold flasks)
- A washable chair
- An air conditioner (running 24/7)
- Grow lights
- Vinyl or tiled floor (to help clean and wipe floor from any split chemicals, etc)
- Scales and balance
- PH tester
- Microscope / stereo microscope
- Dedicated blender (Must only be used for blending media and must not be used for food).
- Alcohol @ 70% (not metho because long term use may cause eye damage)
- Tools for replating and cutting seeds (scalpels, long tweezers, micro spoons, loops, cutting plate, etc)
- Lab coat or dedicated clean new clothes
- Spray bottle for alcohol
- Paper towel
- Small bucket for dunking flasks into bleach
- Sterile wash
- 2 x ice cream containers
Laminar Flow Cabinets
Our Flasking Method:
1. Make up a batch of mix (we use boiling water as this helps to activate the agar).
2. Autoclave your flasks (we allow a minimum of 20-25min @121 degrees Celsius or just above for approx 12-24 flasks per batch. This is something that can change/vary greatly for everyone depending on their materials such as the size of flasks, amount of mix, types of filters, etc. Too much may burn the mix and not enough may not sterilize and may contaminate the mix).
3. When removing flasks we use two clean cloths or paper towel (one to hold the bottom and one to tighten loose lids as you pull them out of the autoclave. When doing this you must be gentle, careful and do not shake. You do not want any of the mix to touch the filter otherwise you will get contamination).
4. Allow to cool and write date and type of mix on lids.
5. Move the flasks to your lab and leave under lights.
6. Autoclave your clean tools (we do this for 25min). The time you autoclave your tools depends on what mass/size/volume is to be autoclaved.
7. Turn on flow cabinet.
8. Spray and wipe out inside and around front of flow cabinet with alcohol.
9. Turn on UV lights (beware that you can get very easily sunburnt from UV lights). It is best practise to run the UV lights for approximately 20mins before use as this allows time for UV to take affect.
10. Get your seed sterilizing under way:
A) Get a new tooth brush.
B) Wash the green seed with a detergent in an ice cream container with the new tooth brush for about two minutes.
C) Now wash the seed in an ice cream container with a 50:50 mix of "Aldi" bleach and water, with another new tooth brush for approximately two minutes.
D) Make up a 50:50 mix of bleach and water in a bottle and fill to the top.
E) Put washed seed in bottle for 10 minutes.
Orchid seed under a Microscope
11. Make up bleach solution in bucket 1:10 bleach to water. This is for dunking your flasks into before putting into flow cabinet. PUT A TEMPORY LID ON BUCKET. (Bleach has a shelf life if not used, throw out after 6 months and always keep the lid on).
12. All of your movement around your work area should be as slow and as gentle as possible to reduce air movement.
13. Turn off UV lights when time expires.
14. Once your tools are ready, take them out of the autoclave and place them in the flow cabinet.
15. Scrub your hands and arms with soap.
16. Now sterile wash your hands and then spray with alcohol.
17. Put gloves on and spray again with alcohol. (This makes a sterile barrier for you to work.)
18. Remember to not touch anything other than if it is in the flow cabinet or if sterile as it will be contaminated. If you drop your tools in the cabinet or if they touch something other than the direct work area discard and use another as they may be contaminated.
19. Dunk the flasks, seed bottle and alcohol sprayer into the bleach bucket one at a time making sure that all of the outside surface are in contact with the solution.
20. Place these into the flow cabinet.
21. Spray the work area/surface of the cabinet.
22. Remove the seed lid and the mix lid.
23. Cut seed capsule and place some of the seed onto the mix in the flask. Remember that tools, inside, rim and lid of flask and seed must be kept from being contaminated at all times. (We make up two flasks of seed with different tools to reduce the chance of contamination to one flask).
How to know whether seed is ready:
This seed above is not ready.
This seed is no good. As you can see with the seed, they were picked too early.
This seed is ready.
24. Place lids on flasks and tighten.
25. Once finished, remove all flasks and label with date, type of seed, batch number, etc.
26. Clean all tools and work areas.
27. Turn off cabinet, drain water and clean autoclave.
28. Put flasks under grow lights, cross your fingers and hope that all goes well!
Pictures above are the different stages of flask development
Sometimes you will find that not all or any seed are viable and will not germinate. This has a lot to do with plant parentage and time that the seed capsule was picked (too early and seed is not viable).
Once you have growth, to re-plate, do the same as above but always remember to change or keep tools sterile as you go. It is also important to not over-crowd your work area as this affects air flow from the HEPA filter.
Note: For mould that is visible within 2 days and is growing on top of the mix, it is a good chance that the breathers/filters are not sealing/working properly. If mould is taking longer than 1-2 weeks to develop and if the mould looks like it is leeching out of the mix, it is a good chance the flasks have not been autoclaved properly.