Orchid seed sowing at home

I took a day off work yesterday to ensure I had a child free environment to start my orchid seed sowing setup. I had to, since I had no idea how long it would take and I knew there were going to be bowls of bleach etc plus pressure cookers and as sterile an environment as possible. THey do not mix well with small children...

My planned work order was:

 Step 1 Prep

• Sterilise distilled water in pressure cooker
• Sterilise other equipment (spatula, forceps) wrapped in foil
• Sterilise for 20 mins at max.
• Meanwhile clean inside of glove box with sterilant with 5% bleach (alcohol might be better)

Step 2 Oats Medium

• Mix up 1l oats mix with distilled water in large mixing vessel
• Microwave as per instructions
• Pour into larger growth vessels (washed honey jars, kilner jars etc)
• Pour into bottles for transferring to Petri dishes.
• Sterilise for 20 mins at max.
• 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

Step 3 Plate up Petri dishes

• 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

Step 4 Malmgren’s Medium

• Ophrys: Swede 4cc per 100ml and 1% Pineapple. For 1 liter = 40cc (2cm x 2cm x 10cm) plus 10ml
• Dactylorhiza: Potato 2cc / 100ml and 2% pineapple juice. For 1liter = 20cc (2cm x 2cm x 5cm) plus 20ml
• Measure potato / swede and cut up into 5mm x 5mm cubes, add pineapple juice plus 100ml of sterile water.
• Microwave for 1 min until soft, then blend.
• Add 1liter deionised water and mix
• Test pH – adjust to 5.8
• Heat in microwave for about 10 mins on high
• Pour Hot medium into jar, unscrew top by half and cap with aluminium foil. If using kilner jars use a strip of aluminium foil to stop the top from sticking.
  

I managed to plate up 10 petri dishes, plus 3 jars with oats medium and 2 with asymbiotic medium(one each for Ophrys and Dactylorhiza). I sowed some Ophrys apifera seed in one of the symbiotic jars.  

 

 
Lessons learnt:

 

• Glove box was a bit of a disaster it was very difficult to get material inside due to the limited size of the entrance holes, the spraying down procedure meant that the bottom became a slick of bleach solution which the Petri dishes slid around on making the process maddeningly difficult. The box was too small to accommodate 10 dishes with the lids ajar plus implements and other necessary items. And the sheet of plastic was so opaque that I had to cut it open to see what I was doing, probably compromising the process. Solution: build a new glove box using the spec in the back of Seaton. Note that alcohol might be better .
• I was very clumsy with the pH adjustment –the  Ophrys batch (which is unfortunately the one I kept) was changed from pH 5.3 to 7.9 by carelessly adding too much Household Ammonia! I used pineapple juice since it was the only source of acidity available, and again was clumsy and it plummeted to 4.5! A few more drops of ammonia were sufficient to rebalance at 5.8 but god knows what effect this will have on the chemistry overall. Solution: carry no more than a couple of drops in the pipette; find a more suitable source of acidification.
• Lengthy process – in the long run it would be better to have more vessels and possibly another pressure cooker. A lot of the prep work could be done before hand – if no petri dishes are used then all the flasks can be made in advance. Note try using the pipette  seed sowing method – this must give greater control over dispersing the seed. In this case all the flasks can be prepared, then sterilised and only later on do they need to be worked on in the glove box.
• Worth having some more vessels to handle the medium to be poured into the Petri dishes. The bottles I used worked but did not fit into the pressure cooker upright and needed to be tilted. This may have compromised the sterilisation process, I don’t know. Perhaps a standard flask with cotton wool, autoclave tape and foil would work better. I cooled the bottles in a cold water bath and the bottom third of each bottle set! It worked out that there was enough in the two bottles for all the Petri dishes, but obviously some was wasted. I guess about 500ml would have been sufficient for all the plates. Ideas: have ordered some flasks and autoclave tape. Given that the Petri dishes can be autoclaved perhaps smaller batch sizes would be better?
• Difficult to take notes – I had the camera there with a big memory card. Why not record the whole process? Difficult to find the pertinent bits though…look into other methods (e.g. voice activated Dictaphone app?)