I noticed from some research articles that by addition of Ag nanoparticles in the MS medium could replace entire autoclaving process. Experts please comment on this, I want to know and try this.
@ Mahipal Sir, adding silver nanoparticles in the MS medium may reduce the contamination chances. Definitely, in my view, it will not replace the entire autoclaving process. One more thing according to the literature by adding silver nanoparticles in media may reduce the regeneration and growth rate.
Dear Mahipal Shekhawat,
I have recently used Silver nanoparticles (4000 ppm) in the MS culture medium (5, 10, and 15 ml/L, added after autoclaving) to control endophytic contaminations of fig single node explants. Silver nanoparticles drastically caused the growth stoping of explants. However, the SN treatments had a significant effect on the elimination of fungi and bacterial contaminations. Nonetheless, I don't recommend the use of SN instead of autoclaving. It is better you use a systemic fungicide and a suitable antibiotic for the elimination of contaminations. Also, some commercial products as ProClin 300 and PPM are very effective in this subject.
Regards
Dear Dr. Phanikanth and Dr. Ruhollah Abdolinejad,
Thank you for your comments.
Please go through the following article:
Article Silver nanoparticles as the sterilant in large-scale micropr...
It depends on concentration of AgNo3. over 10 ppm may suppress explant's response. You can try it starting 1.0, 5.0, 10, and 100 ppm of AgNo3. It never substitutes the Autoclave.
I expect that the sufficient concentrations of Ag against micro-organisms will strongly exceed the threshold values of phytotoxicity for plant explants. In any case, growth inhibition will always manifest itself from certain concentrations, even with commercial products such as the well known biocides of Plant Protection Mixture. They can prevent large-scale spreading when the inoculum is small, but it will never be possible to drop autoclaving for propagation purposes.
Dear Dr. Buhara Yucesan and Dr. Stijn Everaert,
Thank you for your valuable comments and ideas.
Please go through this recent article:
- DOI:10.1007/s11627-021-10163-7
- After reading the above article, I wanted to know better about this topic and started the discussion.
Autoclaving must never be replaced by Ag NPs because it is the standard procedure for sterilization of nutrient media. Although Ag NPs could prevent the growth of bacteria or fungi owing to their bactericidal and fungicidal behavior, still not appropriate method to be replaced by the standard method of autoclaving.
Rabia Javed
Thank you for your valuable comments and ideas.
Please go through this recent article:
- DOI:10.1007/s11627-021-10163-7
Ag nanoparticles can not be used instead of autoclaving because still there are some strains of fungi and bacteria resistant to them. on the other hand it may be not safe for the plant genome!! you should examine it first.
Fadwa Waleed Abdulqahar Thank you for your comments, please go through the following article
DOI:10.1007/s11627-021-10163-7
In theory, AgNPs can be used to bypass autoclaving process however, its concentration needs to be optimized. Still, AgNPs might not guarantee that there would be no contamination. Secondly, high concentration of AgNPs would also negatively affect the results of pertinent study. Plant growth may be retarded, or even inhibited in the presence of high concentrations of AgNPs in MS Media.
@Syed Salman Hashmi, thank you for your comments.
Please go through the following article and comment again.thank you🙏
DOI:10.1007/s11627-021-10163-7
Most of colleagues here have refused, or ammended, the results shared by Mahipal Shekhawat . Beyond these results must be consdiered under the focus of the particular conditions of everyone (doses, scopes, species, in vitro protocol...), I would like to ask if someone has any other reference regarding some preservative substance (different PPM and antibiotics) that could help to control or to prevent the in vitro flourishing of microorganisms. I must to point out that my question don't would consider the substitution of autoclaving for sterilization of culture media.Thanks beforehand.
Dear friends, as I have gone through the recently published article "Silver nanoparticles as the sterilant in large-scale micropropagation of chrysanthemum" by Tung Thanh Hoang I wanted to know the other's experience on the use of Ag nanoparticles in PTC media formulation. It may be revolutionary that Ag nanoparticles may replace the entire process of autoclaving of the nutrient media,
as claimed by the authors in the last line of the abstract "The successful use of AgNP as a sterilizer and as a component of culture media would reduce the cost of micropropagation and improve plants' quality". These are just the opposite results of my experiments, that's why I wanted to know the other's experience in this area of research. I found that all the researchers got the same type of results and not as claimed by the authors of the article.
Friends, it is for a healthy discussion and must be continued for the growth of science...
Thanks and regards
Mahipal Shekhawat
I don't read the paper yet. However, in my opinion we must to assume that the statments of paper are true, as those posted here. Sometimes results could be completely different from a lab to another, from a protocol to other. Small details can also make the differences. It would be worth if results could be compared on the base of protocol described in the paper and those procedures followed for those colleagues that have used this procedure unsuccesfully. Otherwise, in my opinion, the proposed discussion will not render scientific benefits.
Methods, Non-Sterilized Medium Supplemented with AgNP
The MS medium supplemented with 30gL−1 sucrose, 8 gL−1 agar, pH 5.8,sterilized in anautoclave at 121°C, and 15 psi for 20 min, was used as control (AuM1 system).
The same MS media supplemented with different AgNP concentrations (1, 2, 3, 4, or 5ppm) and boiled for 4min to dissolve the agar (not sterilized in autoclave) were used as treatments to investigate the disinfectant effects of AgNP (NoM0 system).
@Tung Thanh Hoang thank you🙏, it's wonderful observation.. It may change the scenario of entire plant tissue culture research and industry. We appreciate your work and trying to replicate it. We will contact you in case of any clarification.
@Mahipal Shekhawat, though I am not an expert when it comes to Plant tissue culture, I did design a study to investigate the effect of AgNPs on phenology and sec. metabolite content of some plants. But since we did not bypass the autoclaving step, we cannot be sure to claim that AgNPs had greater role in reducing the contamination. The study you referred to is quite remarkable but still, much work would be needed to standardize the use of AgNPs to bypass Autoclaving step.
Since you mentioned getting the opposite results to what was claimed in the research you referred to, that might be due to various factors like difference in plant species, difference in AgNPs size, difference in NPs synthesis protocols and above all, difference in the conditions, environment and handling.
Syed Salman Hashmi, you are right sir,
Thank you for your valuable comments.
As far as I know we can only use Ag nanoparticles for surface sterilization of new plant materials that we want to establish in culture and we can not replace it with autoclave.
Nano-silver as an antifungal or/and antibacterial activity that used for reducing mother explant contamination at 50-200 ppm for 10min, but as we know, in concentrations above 100 ppm, it reduces viability. I completely agree with Professor, Dr. Kourosh Vahdati, because assuming that it can be sterilized, adding it to the culture medium will have a negative effect on the explant, and can not justify this method.
Never. I used 5 ppm AgNo3 + gentamicin 40 ppm in MS medium but fungal contamination covered the plates. But the same formula was effective for gr(-) bacteria, no more microbial growth appeared until we transfer the philodendrons into normal MS medium. Endophytic bacterial pathogens were suppressed by AgNo3 at 5 ppm for philodendron samples.
Well, , science is to talk nearing exceptions and serendipity... One would need to ascertain whther the properties are antiphyto too?? It would be interesting to see how would it influence morphogenesis in vitro and comparatively to ex vitro treatments. Can nanops cause mutations as well, can they lead to epigenetic remodellings.. etc.. somebody do it please.
I don't think the use of AgNP will replace autoclaving. Fungal contaminants are not easy to eradicate,. The Ag can also affect plant growth and development especially somatic embryogenesis.
As you know microbes are diverse with diverse characteristics, and AgNPs can be used for surface disinfection of explants as well as it has a very good antibacterial and antifungal effect. When we came to PTC, it is a very sensitive technique (the entire tissue culture) and recommended to use autoclaving. Since research is always in progress, i really appreciate the attempt but the real function can not be conclusive; it depends on the nature and quality of the laboratory (standard). In areas that is most exposed to fungus (Actually every tissue culture lab is exposed, saying relative) only AgNPs will not completely replace autoclaving, but can minimize the load of infection.
Dear all, it has been passed a long time since Mahipal Shekhawat asked about this question. This question came just in the moment in which I was looking for an alternative to reduce the losses caused by microbial contaminations. In an early beginning I was suspicious about the possibility the replacement of autoclaving as a sterilization’s method; moreover, I was seeking a way to reduce losses by contaminations, not to remove autoclaving. Therefore, I read several papers, among them that from Tung Thanh Hoang; however, I don't have any doubt that the results presented there aren't right. Thus, some weeks ago I began a trial using AgNps. Some of treatments deal with the use of non-autoclaved culture media. It's early to have a conclusion; however, I've to say that it's possible.
Ricardo Julian Licea-Moreno, I am eagerly waiting for your results and final remark in this regard. Thank you.
Mahipal Shekhawat and colleagues, because of a confidentiality agreement, I could share the results, but not the methodology. I'm still far to obtain a conclusion; however, after the first approaching I can say that it's possible. There're a lot of variables that must be controlled, as some of us have mentioned. Check the NPs source and concentration, plant species, as well as the preparing methodology.
Also check https://iopscience.iop.org/article/10.1088/1755-1315/875/1/012053/pdf
Please check this.
Article Silver nanoparticles as the sterilant in large-scale micropr...
Please see this.
Article Effects of antimicrobial activity of silver nanoparticles on...
No, definitely not. Silver nano-particles cannot remove all the contaminants.
I can't absolutely say that AgNPs could replace autoclaving. However, I disagreed with the statements that it's not possible at all. Many factors (the kind of NPs, the used dose, the state of cultures, the water quality, the plant species...) might influence a result, or another. But I can say that under my conditions, I've maintained free of microbial contaminants non-autoclaved culture media using AgNPs by 2 subculture cycles. A key note is that it seems that I could increase the dose of NPs, which would increase its sterilant effect. The only procedure followed has been heating culture media to gelling. I don't have sterilized the culture vessels at all. Clean cultures have been inoculated. Unfortunately, I can't give more details, but none can say that I don't have obtained these results. I don't say yes, I only could say that it's possible.
Ricardo Julian Licea-Moreno
I disagree - it is absurd to offer that autoclave sterilization can effectively be replace by Ag nanoparticles. Not only will resistant dormant forms of microorganisms passively resist such treatment, actively growing bacteria have already developed resistance to such nanoparticles - exampkes follow.
The efficacy of autoclave sterilization need is well known. Article Mechanisms of Resistance to Silver Nanoparticles in Endodont...
andhttps://www.nature.com/articles/s41565-021-00929-w and
Article Extracellular Synthesis of Crystalline Silver Nanoparticles ...
Phil Geis it could be absurd for you, but not for me. I don't offering nothing, I just say that it's possible because my results support it by now. I could add several papers stating the contrary, but it's not the point. Have you heard before about Vitrofural or G1? If not I recommend to serch for it. It's also recommended for substitution of autoclaving , and it works. It's a fact, absurd or not for someone, but culture media can be sterilized using G1, without autoclaving.
Phil Geis
I can't understand why you think that you can recommend me this. I don't try to demostrate anything. If you have followed this discussion from the beginning, it arose for a question from Mahipal Shekhawat , and mostly based on a paper from Tung Thanh Hoang . When I read this paper, I'd some doubts. However, before to say it's imposible, I decided to assess the role of AgNPs under my conditions. My first goal was to reduce the effects of microbial contaminations, incorporating NPs in autoclaved culture media, but at the same time I decide to follow the recommendations of Tung, and you know, every morning that I enter to my culture room to see my experiments, I don't observe (after the subculture an indexing in bacteria growing medium will be performed) the growing of contaminants in treatments with AgNPs in which culture media were not autoclaved: and it's enough for me. I only try to say to other colleagues that the absolute true doesn't exist. If someone say that it doesn’t has sense, it's ok. But when I read that it's impossible, I just think that who say that has not right.
I agree with Ricardo Julian Licea-Moreno and the discussion is based on the paper from Tung Thanh Hoang
Mahipal Shekhawat Ricardo Julian Licea-Moreno
The original question asked offered no paper. or context. Tung Thanh Hoang's post did not include a paper and offered "disinfection" - not sterility. Marzena Parzymies 's paper spoke of "reduction" not sterility and also noted effect on implant - effectively a confounding variable
Suggest you organize your thoughts and offer a specific context and application. application. Any microbiologist addressing this question as Mahipal offered it will say "no".
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