I had another child free day to continue my orchid seed sowing today. I wanted to sow some more seed and prepare more plates and flasks, while also transplanting the protocorms which had already developed.My planned work order was:
Step 1 Oats Medium
- Mix up 1l oats mix with distilled water in large mixing vessel
- Microwave as per instructions
- Pour into polypropylene growth vessels
- Pour into 250ml flasks with foil and autoclave tape for transferring to Petri dishes.
- Sterilise for 20 mins at max. with tools and
- Allow to cool
- Prepare plating up area with
- Latex gloves
- Bowl of 5% bleach
- Sheet of absorbent material
- Cut parafilm into 1” x 5” strips
- Meanwhile prepare glove box and continue with step 2.
Step 2 Sow seed in prepared plates and flasks
- In glove box, get all the prepared plates and flasks, ready and when available the tools and sterile water.
- In the meantime start on the seed sowing, adding seed to two sowing tubes plus 2ml 5% bleach, agitated for about 10 - 15 mins, then rinsed twice with 2ml clean sterile water. Add 5ml water and then sow into the medium. Then wash thoroughly and rinse and move onto the next species.
Step 3 Plate up new Petri dishes and flasks
- Lay out Petri dishes in rows from the back of the cabinet
- Work from back to front and left to right pour medium into each petri dish 2/3 full
- Replace lid so half covered allow to cool.
- Innoculate petri dishes and flasks with B1 fungus.
Step 4 Malmgren’s Medium – not done.
I also inspected the O. apifera seeds previously sowed. They looked like they may have been infected with possibly a yeast infection, but it wasn't very clear.
I managed to plate up 9 new petri dishes, plus 6 flasks with oats medium. I sowed the remaining Ophrys apifera plus Dactylorhiza fuchsii and D praetimissa seed in, this time all in petri dishes.
- The new Glove box was a bit of a marked improvement on the previous model. The absorbant surface of the plywood prevented the slippery surface and the using the alcohol also helped, while access and space to move was much better. If anything it is too big - I think storage will be a problem. Finally, at the moment the visibility was also good. The removable base was useful and makes cleaning easier, but hopefully it will not compromise the sterile environment. Longer angle brackets at the base would improve this (with foam gasket?).
- I now have a pipette and some dilute sulphuric acid to modify the pH. Not required this time but next time I make some more asymbiotic flasks it should help.
- This was a much more manageable process – the most time consuming part of the overall process is making the asymbiotic medium. Since this does not require using a glove box it makes sense to do this seperately.
- The 250ml flasks worked really well, and two are enough to do a pack of 10 petri dishes. They are easy to pour and handle and fit well in both the pressure cooker and the glove box. I checked with John Haggar and the petri dishes cannot be autoclaved. I'm not sure how they can be reused – maybe they can't but I'm sure the answer is out there on the internet...
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