The Orchid Seedbank Project
PO Box 7042
Chandler, AZ 85246

Making and Storing Hypochlorite Solutions

There have been a lot of misunderstandings when it comes to making  and storing hypochlorite solutions for disinfection of orchid seeds.  This discussion concerns only calcium hypochlorite (versus sodium  hypochlorite, or household bleach) solutions.

Calcium hypochlorite is favored by many growers, for different  reasons. It has been thought by many growers that calcium  hypochlorite (CaH) solutions must be used immediately after making  them, or at least no longer than 24 hours after creating the  solution. Thanks to Fred Bergman and his comprehensive series of  papers on the subject in the Orchid Digest, many myths have been dispelled.

First off, CaH solutions may be created from any form of calcium  hypochlorite; for most growers, this will be from pool chlorine, such  as HTH (High Test Hypochlorite). Be very careful that the contents  read calcium hypochlorite, and are not some other form of chlorine or  other disinfectant. This will be a granular powder that is caustic  and otherwise bad to deal with in the home. Be very careful when  using it, and take caution in storing it and using it only as the  manufacturer suggests.

There is some disagreement in how soluble calcium hypochlorite is;  for our purposes, we will be creating a strong, not saturated,  solution, so it is not terribly important to weigh it out. I use a 1  liter flask, and place about 15 grams of the compound in the bottom,  and fill to 1 liter. I then let this stir using a magnetic stir bar  for 15 minutes, making sure there is still plenty of undissolved  compound at the bottom of the flask. After stirring, let it sit for  15 minutes or more.

While letting the solution settle, prepare a funnel with filter paper  suited to the filtration of the solution. A coffee filter is fine.  Carefully pick up the flask so as not to disturb the fines at the  bottom, and pour it through the filter. Leave the last 10% of the  solution in the flask, so that the fines don't clog the filter. The  filtrate should then be placed in a clean glass jar (preferably  PVC-coated to prevent spillage if the glass is broken), which is then  tightly capped and placed in the refrigerator.

The solution should remain cold and dark, but above freezing to  prevent shattering of the vessel. Keep it tightly capped, and do not  introduce anything into the bottle. Kept this way, the solution will  remain stable for at least a year.

For disinfection of seed, a small amount of liquid is withdrawn from  the bottle, and placed in with the seed. I use small test tubes  (about 20 ml capacity), and place a tiny amount of seed in the  bottom. It is difficult to express how much seed to use, as it  depends upon many parameters: the size of the seed, size of the  flask, number of embryos found in the seeds, and so forth. Some  suggest a quantity about the size of a grain of rice, which will work  well for now. To this I add 1-3 ml of CaH solution, and then swipe  the bottom of the stopper across the top of a dishwashing detergent  bottle to add a very tiny amount of detergent.

Seed is disinfected for 6-15 minutes, agitating regularly. The  solution (disinfectant and all) is dumped into the sterile flask. If  too much seed stays behind in the test tube, I add 1-2 ml more CaH  solution, and dump this into the flask as well. If there is too much  liquid in the flask, I decant whatever I can without spilling too  many seeds. The solution should be deposited directly on the media,  to allow for as many seeds as possible to be placed on the agar, and  not on the walls of the flask.

It should be noted that this technique can kill seeds if there is too much solution put on the media. If you are using small flasks (baby food jars, Magenta GA-7’s, etc.) rather than large Mason jars, the seed should be washed first with sterile distilled water. In fact, germination will almost certainly be better if you do so. Decant the hypochlorite solution, either by spilling it or using a sterile bulb to draw up excess solution. Replace this with autoclaved distilled water. Shake gently. This wash can be repeated, or the seeds can be deposited on the media, carried along with the distilled water.

DCCA (dichloroisocyanuric acid, Sigma chemical company D-2536) may  also be used for disinfection at 5.0 grams dissolved in 1000 mL of  distilled water. Disinfection times are the same as with other  hypochlorites. This solution has the benefit of being slightly acidic, increasing the activity of the chlorine.

Disclaimer: if anything I suggest is illegal, or falls outside the  labeling on the products, don't do it. I'm not responsible if you put  your eye out. Be careful with storing calcium hypochlorite, as it  reacts violently with organics, and must be stored according to the label.