Since tween 20 is no doubt an excellent surfactant to remove surface tension and provide better exposure of plant surface for sterilization, by removing the bubbles which are more problematic in case of plants having plenty of hairs and trichomes. I wonder if in case of unavailability of tween 20, can a common detergent or dish soap provides the same benefit or there is any other cheaper compound as a replacement?
Hello, Amina!
We use 70% ethanol for 1-2 minutes, then treatment by 70% bleach for 15-20 minutes on a shaker and further rinsing in sterile water 1-1,5 l (running through sterile sieve). This procedure always was good for seeds sterilisation even cotton seeds. Good luck.
Amina Ilyas
Yes, Triton-x100 can be used, as already suggested by Mª Angeles Zorrilla Lopez-Perea . Sigma sell them for $13.9 (5 mL) or $42.3 for (100 mL) and other sizes. But, I don't think you need to buy a big quality of them, since only *drops* of it needed for sterilization purpose.
We use PROCLIN in Jose M. Colmenero laboratory.
ProClin 200 is a highly effective biocidal preservative offered for the control of microorganisms in reagents and products intended for in vitro use. It has a broad spectrum of activity, excellent compatibility and stability, and low toxicity at use levels. At low concentrations it eradicates bacteria, fungi, and yeasts for prolonged periods of time, increasing the shelf life of a product. In addition, it does not affect the functionality of most enzymatic or antibody-linked reactions, and will not interfere with the assay of indicators.
Within minutes of contact with a microorganism, the active components penetrate the cell wall and inhibit specific enzymes (at the points marked in red in the Krebs cycle).
Growth, macromolecule synthesis and respiration are inhibited, ultimately leading to cell death.
Application:
1. 5-15 minutes with 5.77% Proclin (0.865% ITZ) = 50% PPM.
a. 5.77% = 577 µl / L + 9423 µl / L sterile water.
2. Transfer to in vitro culture medium with 0.023% Proclin (0.00346% ITZ) = 0.2% PPM
a. 0.023% = 230 µl / L of medium
Differences PPM vs ProClin
1. PPM and Proclin are not identical. Proclin is a biocide intended to be used exclusively for in vitro diagnostic applications, whereas PPM is manufactured and tested especially for Plant Tissue Culture.
2. PPM was extensively tested on plant tissue culture by independent laboratories. HPLC testing demonstrated the absence of detectable residues of PPM present in plant tissue culture. To my knowledge, these tests were not carried out with Proclin and therefore, supplementing the medium with this substance is a risk.
3. Only PPM has EPA approval for plant tissue culture. This approval ensures that the product is safe for this application. Proclin does not have approval for this application which means that the use of Proclin is illegal in most jurisdictions.
4. Finally, the use of isothiazolone molecules in plant tissue culture and the method of use is internationally patented and protected. The worldwide patent rights are owned by Plant Cell Technology. Patent infringement in many jurisdictions is subject to severe penalties, including treble damages.
Proclin MUST NOT be used as a PPM Substitute.
Hi, You can use either triton-x 100 or teepol. Teepol is also a very effective cleaning agent and not expensive.
Thank you all, especially J.D. Franco-Navarro for your valuable suggestions.
If you have a vacuum chamber you could try doing a quick vacuum removal of bubbles. Might be a bit tricky to set up the first time but could be well worth it.
Ideally the sample chamber would need to go in the clean hood, then your plant samples would be immersed in the wash solution, a brief (not too much pressure change) vacuum might be enough to remove some pesky bubbles
We know, surfactants such as polysorbate 80, Pluronic F-68, Tetronic, ProClin 200, Nonoxynol, Tweens (20, 40, 60, 80), Triton-X-100 and so on is useful for sterilization, but according to my experience, using a few drops of dishwashing liquid (soap) as a detergent has the same result and on the other hand is cheaper, more accessible and more cost-effective.
Dishwashing liquid- unscented for preference. Fine for sterilising explants and way cheaper than Tween etc