PPM significantly simplifies the tissue culture working procedures as follows:
1. Media containing PPM may be dispensed outside the laminar flow hood (LFH) exposed to the ambient air.
The plates should be covered soon after agar solidification. In the event that media dispensing is done by a pump, we recommend passing autoclaved hot water through the hoses prior to and after media dispensing.
2. Heat sensitive or heat stable liquid media containing PPM does not need to be filter sterilized or autoclaved provided that it will be stored in sterile containers and that the stock solutions are not contaminated. In rich media containing 200 mg/liter or more of amino acids or proteins, it is recommended to filter the media with the PPM.
3. If working in the LFH the utensils (forceps or scalpels) do not need to be flamed. They should be periodically dipped in 70% alcohol. The LFH does not need to be certified and the work can be done as well outside the LFH on a clean surface for a period not exceeding 1 hour.
4. PPM is less effective when exposed to high density of bacteria or fungi spores found regularly on seed’s coat. For in vitro germination, seeds should be conventionally surface sterilized with EPA registered bleach.
Therefore, in the presence of PPM (in the germination medium), the seeds can be rinsed under tap water in a non-sterile strainer and left to dry preferably in the LFH. If the utensil ends have touched active bacteria, fungi culture or otherwise suspected of being contaminated, they should be sterilized by autoclave or by use of an electric heating element.
5. General Dosage levels: With the exception of endogenous contamination, the recommended dose range is 0.05%-0.2%. (For callus proliferation, organogenesis and embryogenesis, the recommended range is 0.05-0.075%.) To eliminate higher endogenous contamination densities, higher doses of PPM are needed (see paragraph 6 below).
6. Endogenous Contamination: (a) For explants: gently and routinely shake / stir 1 cm. long explants (or shorter) for 4-12 hours in 4-5% v/v PPM solution supplemented as above with full MS strength basal salts without pH ing and without Tween 20. Without rinsing, insert into a medium supplemented with 0.05 – 0.1% PPM for herbaceous plants and 0.2% PPM for woody plants.
Paragraphs 6(b) through 10 below are intended for ornamental plants only.
(b) For tubers, bulbs and scales: shake / stir the entire tuber / bulb / scale in bleach. Rinse with water (can be
done under non-sterile conditions). Slice the tuber / bulb / scale to thin slices. Shake / stir for 12-24 hours in 4 -5% PPM solution supplemented with full strength basal salts without pH ing and Tween 20. Without rinsing,
insert into a medium supplemented with 0.1 – 0.2% PPM.
7. In cases where the above protocols do no yield satisfying results (especially thick explants, highly infested
explants, seeds), we recommend the following:
(a) Shake / stir the explants in water (1hr for soft tissues and 2 hr for hard tissues).
(b) Shake / stir the explants in (50%) PPM supplemented with full strength MS basal salts (without pH ing and without Tween 20) for 5 -10 minutes.
(c) Without rinsing, insert the explants into the medium. In fungal contamination, the addition of PPM to the medium is optional. However, with bacterial or mixed contamination, the addition of 0.05 – 0.2% PPM to the
medium in the first month is essential. Do not discard highly oxidized explants as approximately 50% of the explants will recover within 4 – 6 weeks.

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