Hope you had a good week. This time I am going to share a method I use for flasking, which is simple and doesn’t necessarily need a Laminar hood. I use 6% sodium hypochlorite solution (Chlorox) as stock solution for seed sterilization and surface sterilization of the sowing agar surface. Let me try my best to break it down.
A little history first. I got into flasking about 2 years ago and after several attempts at building my own laminar flow hood with axial fans, centrifugal fans with HEPA filters and having initial hits and misses and lots of frustration, I decided to go the chemical way. Since a lot of information was available for sodium hypochlorite, I decided to try and optimize the dose of NaClO (sodium hypochlorite) that is seed safe and provides the necessary surface sterilization.
I produce my own seeds and so far had success with Phalaenopsis, Dendrobiums, Phaphiopedilums, and Miltonia. I collect dry seeds from ripe pods so I can store them longer. After I scrape of the seeds and separate the chaffe, I put them in micro centrifuge tubes and refrigerate them.
Note: Please, wear the proper personal protective equipment as needed, safety glasses, gloves, etc. I am not responsible for any injuries or incidents caused by improper use of chemicals or equipment.
Media and sterile water preparation
I begin by preparing my media in flasks at least a day before I intend to sow. I developed my own media, which is a simpler variation of P668. Autoclaving is performed using a regular pressure cooker. I autoclave three elements namely; 1) Flasks with media , 2) Water for seed sterilization and, 3) water for seed sowing. For 2 and 3, I add a dab of dish detergent to help lower surface tension of water and help improve seed wetting.
For this step, it is very important to determine the wettability properties of your seeds. Seeds of Stanhopea, Gongora, Phaps and some others that are “floaters”, need to be soaked overnight to completely wet them and ensure they sink. The seed sterilizing and washing is performed in microcentrifuge tubes.
Water for seed sterilization (Tube A)
Make a 4% solution of Chlorox using the autoclaved Water for seed sterilizing (number 2 in the autoclaving elements). This can be done by adding 2ml of Chlorox (6% solution of NaClO) to 50ml of sterilized water and mixing well. This gives us an active chlorine concentration of about 0.25%. We will use this solution for seed sterilization. Perform the addition in the sterile enclosure, if you can.
Water for seed sowing (Tube B)
Make a 1% solution of Chlorox using the autoclaved Water for seed sowing (number 3 in the autoclaving elements). This can be done by adding 0.5ml of Chlorox (6% solution of NaClO) to 50ml of sterilized water and mixing well. This gives us an active chlorine concentration of about 0.063%. We will use this solution for seed sowing.
Fill the centrifuge tubes to 3/4 level with the contents of Tube A we just prepared. Now use a toothpick or a similar tool and dip it into the seed storage microcentrifuge tube and pick a few seeds(as shown). Make sure the toothpick is completely dry when you pick seeds.
Now place the seeds in the sterilizing tube, close the tube and shake well. We want the seeds to eventually sink to the bottom say in about 15 minutes (as shown).
So observe what the seeds are doing. Keep the tube upright and place it inside an enclosure (as shown). This enclosure is where we will be doing all the transfers, henceforth. Also, at this point, we will place inside the enclosure;
- the autoclaved media flask,
- small container for waste water,
- Tube B,
- microtube with seed being sterilized,
- a pipette that is full of the 4% bleach solution (we are sterilizing the pipette this way)
Now spray the enclosure with 70% isopropyl alcohol to sterilize the surfaces and wait for 15 mins while the seeds are being sterilized.
After 15 mins, put on your gloves and sterilize them with 70% isopropyl alcohol. Now slowly enter the enclosure and empty the contents of the pipette into the waste water container. Then, slowly open the microtube with the seeds and pipette out the 4% bleach supernatant, making sure you do not disturb the seeds that are by now sunk to the bottom of the tube. Dispose the supernatant into the waste water container.
Now pipette about 1.5ml of the contents of Tube B and place it in the microtube with seeds we just sterilized. Pump the pipette a couple of times to resuspend the seeds. We now are ready to sow. Pipette out the contents of the tube, slowly open the flask lid to where you only have space for the pipette, inject the contents and close the lid. All actions should be slow as possible. Notice how I keep the tip of the pipette right at the rim of the flask and not further than I have to. Gently swirl the seeds inside making sure the whole top surface of the agar is wet with the solution. This is to evenly distribute the seeds and also to bring in contact with NaClO any spores that might have landed on the dry area, which will eventually proliferate, if not treated
That’s all my friends. Any little contamination you may have introduced should be kept in check with the NaClO. I still have (very little) contamination from time to time using this method but way way better than before. Also, I do not replate because with this method, I can control the amount of seeds so I do not get overcrowding.
Also, if your seeds are sensitive to NaClO, you can try lowering the dosage of Bleach in Tube B to 0.75% or lower.
Please, let me know if you have any questions. Thanks for reading
-Paco de Lucia