Disinfection
(or Disinfestation) of Orchid Seeds
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1. Using a homemade filter unit:
This setup includes the filter unit, 70% isopropyl alcohol, 10% bleach solution and sterile water plus sterile plate and forceps |
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| Pour dry seeds
into filter unit (go to http://www.kitchenculturekit.com/filter.htm
for instructions on making this). Dip unit up and down into 70% alcohol until all seeds are wet. Spray down inside of filter with 70% alcohol such that interior is disinfected completely. |
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| Disinfect forceps
in 70% alcohol. Use forceps to lift filter unit out of alcohol and
transfer to solution of 10% bleach + detergent.
Dip unit up and down again to wet all the seeds with the bleach solution. Continue this for 10 minutes. |
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| Using a sterile
forceps, lift the filter unit out of the bleach solution and allow
to drain slightly
Place on a sterile plate. Using a sterile spatula, small spoon, or rounded butter knife, transfer some seeds to a sterile jar of medium. Spread the seed over the surface of the medium. Add some sterile water with the sterile spoon to the medium surface to help spread the seeds (if necessary). Cap and seal with tape. |
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2. Using a paper
coffee filter: |
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| Seed are placed
in a test tube or small jar. A solution of 10% bleach + few
drops detergent are added and the seeds and solution are mixed for
10 minutes.
The mixture is poured into a funnel lined with a paper coffee filter which has been first wetted down with more bleach solution. Sterile water is poured over the seeds several times to rid them of bleach. |
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Using a sterile spatula, small spoon, or rounded butter knife, transfer some seeds to a sterile jar of medium. Liquid medium can also be used if you have a shaker unit. The vessel shown here is a Combiness unit which works great for the in vitro life of the plants.
Spread the seed over the surface of the medium. Add some sterile water with the sterile spoon to the medium surface to help spread the seeds (if necessary). Cap and seal with tape.
3. Using sucrose and peroxide: This setup requires a 5% solution of sugar, small test tube or small container, hydrogen peroxide (3%), and a transfer pipette. |
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| Seeds are placed in a small test tube or container and 5% sucrose solution (with a few drops detergent) is added. Five percent sucrose is 5 grams of sugar ( a gently rounded teaspoon) in 100 ml water. This is allowed to "incubate" for 12 hours. The sucrose aids in germination of fungal spores. |
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| The sucrose is then removed with a transfer pipet. Hydrogen peroxide (3 %) is added and the seeds are allowed to disinfect in the hydrogen peroxide for 30 minutes. This should kill the bacteria and germinating fungal spores. | |
| 1. Mix the
solution with the pipet and add a few drops to each media bottle.
2. Tilt the bottle
back and forth to distribute the seeds over the top of the agar
medium. Cap and seal.
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| 4.
Using a green seed pod: This setup requires 70% isopropyl alcohol, 10% bleach solution and sterile water plus sterile plate and forceps |
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| Green pods can be cleaned with a tooth brush and then subjected to 70% isoproyl alcohol for a few seconds followed by 10% bleach solution for about 20 minutes. | |
| The pod is removed and placed on a sterile surface. |
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| It is then cut in half and the seeds are gently removed with a forceps or knife blade and put on media as described above. |
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