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24 Vinegar and Hydrogen Peroxide Sterilant in Plant Tissue Culture

Hydrogen peroxide and vinegar, when mixed, makes a gentle but effective decontaminate. It works as an oxidizer attacking cell walls and destroying the cell walls of fungus and bacteria.  Since it will oxidize cells, remember that you and I are made up of cells.  Wear gloves and glasses as a safety precaution.  While this is quite mild extended exposure isn’t good.

Hydrogen peroxide from the pharmacy is usually a 3% solution that will in itself sterilize many delicate seeds. When plain white vinegar is added it becomes stronger. White vinegar has about 5% acetic acid. Both are stable at room temperature, readily available, and mostly safe to store and use without prolonged exposure.

When creating this mixture use a 4 to 1 ratio. Easily find the amounts of each by the volume of solution you want to make. If you want to make 500 ml divide by 5 and you get 100ml of white vinegar (5% acetic acid) and 400 ml of hydrogen peroxide (3% hydrogen peroxide).

Heat the vinegar in the microwave for around 1 minute to the point of boiling. This does a couple of things. It helps sterilize the vinegar, accelerate the reaction between the vinegar and hydrogen peroxide, and to destroy any catalytic enzymes or proteins in the vinegar. Pour the room temperature hydrogen peroxide into the vinegar.

Now, this is a stronger oxidizer than chlorine so you must use the same caution as you would when using bleach or other strong chemicals. Store the mixture in the refrigerator in a dark bottle. It will lose its effectiveness in a weeks time so only make as much as you can use in a few days.

This mixture can be used by spraying a work surface with a fine mist and draining away excess liquid. Explants can be dipped for extended periods of time and it also is a good solution to use in sterilizing stubborn explants.

Explant soft tissue can be disinfected with a 1 to 4 min soaking time. Woody and hard plants may benefit from 5 to 10 minutes. Leave hard and woody tissue in long enough to penetrate surface tissue as it is likely more contaminated than soft cuttings.

Sensitive explants may require rinsing before initiation with a sterile water bath but most plants can be initiated without rinsing. This both saves time and reduces residual contamination in the vessel. With hard to get or sensitive plants it is best to use caution and time your first attempts to keep a log on the outcome. In the event that you kill your explant, you can reduce the soak time for the next attempt.

Another use of this mixture is that it can be added on top of a contaminated vessel in an attempt to save the culture. Enough can be added, under sterile conditions, to cover newly forming fungus or other contamination. This can lower the pH and can affect your culture and the effectiveness of the mixture, however. Use as little as you have to in covering contaminants under your clean hood. It may save an otherwise contaminated and useless culture.

Some have had success adding several drops to the top of new cultures to create a thin covering for an effect much like Plant Preservative Mixture (PPM), to keep contamination down. Some plants benefit from it and some don’t. Experimentation and note-keeping is important when experimenting with these methods. In the long term, they will save time and effort.

Orchid seedlings that have become contaminated can sometimes be saved using this solution. Rinse the contaminated seedlings in distilled water. Wash the seedlings in the 4:1 mixture for 30 seconds to 1 minute. Using sterile distilled water rinse the seedlings three times and replate.

Some plants it works well with but others the pH or oxidation can’t handle so some experimentation may be needed to find what is best for the particular plant you are working with. Overall it is a very useful tool in your kit for sterilizing explants, tools, and surfaces. It is easy to make and inexpensive. Most importantly it works.

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