One should distinguish endogenous contaminants living inside the tissue (usually in the vascular tissues and in the intercellular spaces), and exogenous ones inhabiting the surface of plants. The latter can be killed with the proper mixture of antibiotics. Antibiotics will not penetrate the tissues in sufficient concentrations to kill the endogenous pathogens so in this case they will not help. Adding antibiotics though prevents bacteria from overgrowing the plant nutrient medium. PPM is in these respects similar to antibiotics. At the same time you should remember that many bacteria species die when inoculated on plant medium.
Geert-Jan
Hi Dear Jimmy,
The microbes that contaminate animals and human are differ from those cause contamination for plants. antibiotics may be harmful for plants. if we want to test (if applicable) as the plant tissue is much more sensitive than animals. caution must be taken to account and MIC and MIC and MBC must be considered.
I would like to introduce the below pdf file which is about antibiotic therapy in herbs.
Good Luck
hi,
You can try streptomycin and related antibiotics in very low concentration while the sterilization ,It will give you good result(from my experience) but there are many things you have to take care of
(1)High concentration or long sterilization time will damage your plant.
(2)antibiotics will cause genetic variation.
Most of the time I didn't get any response in my culture,so be careful with the use ,I don't think that using antibiotics in media it self is a good idea.
good luck
Hi,
Yes you sould do try timentin 50 um/l in the first culture, during calli induction. Or just afther sterilization procedire.
We use PlantPreservativeMixture at 0,2 % in micropropagation culture without harmful effects to the plant
Antibiotics does not prevent contamination in most cases in my experience. Also they are very expensive. There wont be much harmful effect in micropropagation but they interfere in organogenesis and embryogenesis.
It depends on what type of contamination you are having problems with. For fungal contaminants we also use Plant Preservative Mixture at 0.2% when initiating cultures and have found it more effective than the use of a systemic fungicide. You might want to check the plant tc list serve archives for more information on in vitro contamination problems.
Our results allow to infer that over the 30 days of culture, the antibiotic gentamicin, resulted in the microstumps development decrease and was not effective in controlling bacterial manifestations. The antibiotics rifampicin and ciprofloxacin although were not effective in controlling the bacterial manifestation, affected the development of microstumps. It was concluded that the biocide chloromethyl-methyl-Isothiazolinone + Isothiazolinone effectively controlled the bacterial manifestations without modifying the morphophysiology of the microstumps, well as the timentin, which besides controlling the manifestations, provided greater vigor of microstumps due to better assimilation of nutrients.
One should distinguish endogenous contaminants living inside the tissue (usually in the vascular tissues and in the intercellular spaces), and exogenous ones inhabiting the surface of plants. The latter can be killed with the proper mixture of antibiotics. Antibiotics will not penetrate the tissues in sufficient concentrations to kill the endogenous pathogens so in this case they will not help. Adding antibiotics though prevents bacteria from overgrowing the plant nutrient medium. PPM is in these respects similar to antibiotics. At the same time you should remember that many bacteria species die when inoculated on plant medium.
Geert-Jan
If you have interest, I suggest give a look: LEONE, Gabriela Ferraz. Estabelecimento de protocolo para controlar a manifestação de bactérias endofíticas no processo de multiplicação in vitro de eucalipto. 2013. Dissertação (Mestrado em Fisiologia e Bioquímica de Plantas) - Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, 2013. Disponível em: . Acesso em: 2013-10-01.
http://www.teses.usp.br/teses/disponiveis/11/11144/tde-10092013-160116/>.
Yes you can use. Before using the antibiotics, you should identify that bacteria/fungi are exogenous or endogenous. If it is exogenous more than 90% will be eliminated by sterilization method. If it is endogenous you have to identify the organism by culturing the explant on fungal/bacterial medium, it will certainly come out, and then you identify the organism, find out antibiotics to stop it. The same can be used in tissue culture medium to stop the the growth of the contaminants. We have experienced in Chrysanatheum cinerarifolium (produce pyrethrins) which has endosymbiont for the production of secondary metabolites. We could be able to standardize the micropropagation protocol successfully without contamination.
Yes you can use but from my experience, it depends so much on the kind of contaminants you want to control. Exogenous bacterial contaminants are very easy to control and usually do not present much problem. So most likely, your contaminants are endogenous. Before you try antibiotics, i will advise to try a different sterilization protocol which is less harmful like low concentration of commercial bleach because the plant tissue usually get overwhelmed by harsh sterilant and therefore unable to defend themselves against endogenous bacteria. Antibiotics should be the last option, they don't work well.
Hello
I used antibiotics (penicillin and ampicillin of powder)for banana tissue culture,
it is very useful for removing of tissue contamination, but you should use a very little (0.2%) because proliferation and grown will decrease.
thanks
Farah Farahani
Antibiotic do not kill all of microorganism. please prepare plant by using fungicide (systemic and contract) to plants that we will use for explants. tHis will get clean plant or less microorganism before culturing.
To every culture species one must try to see if any antagonistic effects do occur and if it is worthwhile using. Of course, good practices are essential.
I have typically used Claforan (Cefotaxime) 500 mg/L in cell culture media to get rid of Agrobacterium after transformation of the cell line. It does a fairly good job of clearing bacterial contaminants, but very little against fungi.
Some years ago I studied the genetic tranformation in olive mediated by Agrobacterium and we carried out the selection by using Kanamicin.
Of course you can even you have to but it depends on what type of culture and which type of contamination that you have the most general and common used is the kanamicine , ampiciline
Well, for initiation of cultures in case of difficult to eradicate endophytes, you may use antibiotics or plant preservative mixture if you are sure it will kill the contaminant. Many do not, only inhibit or arrest microbial growth! Once the culture is clean, remove the antibiotics as soon as possible to avoid induction of resistance. Many antibiotics have detrimental effects on propagation rate and rooting percentage.
Use of antibiotics is necessary to get rid of contamination. If we do not know the the source (exo or endo origin) but cultures are contaminated. It is clear that such cultures are useless for cell suspension culture. Therefore use antibiotic (200- 500 mg/l) for three successive transfers (passages of same medium including antibiotic) and then transfer in liquid medium and see the development of healthy cultures or you get contamination. In case of contamination, make plating of the cultures already grown for three passages on antibiotics, on antibiotic containing medium. We have used this method for getting Commiphora wightii cultures contamination free.
depends on the types of contaminants under query. use Carbenicillin - anti-bacterial
As Letícia mentioned before, you MUST do all the precautions. Than íoine those collegues, who suggest the addition of Claphoran (Cefotaxime) - we apply it for long time. But before you use it a preliminary test for the sensitivity of your plant tissues should be carried out.
Really as said the objective (s) is important. Is one doing selection of transformants or something else. However, depending on the sensitivity they can or can not be used.
Though, antibiotics difinitly sterlized the media but it could vary in resposive to host. Ohter factors must be given importance like the expensiveness, effectiveness and or asociated residual effect on the cultural media/ exx-plant.
Antibiotic like cefotaxime can be used to controlled contamination during sterilization of explants and in media preparation, but its concentration in the culture media highly depends upon the type of contamination and explants. Otherwise antibiotic though control contamination but it can also restrict the explant growth.
Yes, you can add using Gentamycine sulfate which is slightly affected by autoclaving, the level should not be high so as notto harm the plant ( 10 mg/L)
Streptomycin at 1000 g 1 for a period of 10 days was effective against endophytic bacteria and less phytotoxic than gentamicin, neomycin and rifampicin. However, antibiotics have been shown to restrict rooting, general growth and multiplication in plant cultures (Leifert et al., 1994). Antifungals, such as binomyl has been shown to reduce fungal infection when used with mercuric chloride (Mederos and Lopez, 1991).
But before you use it a preliminary test for the sensitivity of your plant tissues should be carried out.
Filter sterilized H2O2 at 1ml/l was very effective to control endophytic bacteria
As per my experience inclusion of streptomycin (10-100mg/l ) in medium was effective in cleaning the cultures in a number of species. However after 3-5 subculture, we streptomycin was excluded from the medium.
Please see our publication on this topic. We studied the phytotoxicity of about 8 broad spectrum antibiotics and evaluated their efficacy in reducing bacterial growth. Conclusion: rigorous sterile techniques are more effective than antibiotics since microbial communities shift rapidly.
Smart, D., A. Ferro, K. Ritchie, and B. Bugbee. 1995. On the Use of Antibiotics to Reduce Rhizoplane Microbial Populations in Root Physiology and Ecology Investigations. Physiol. Plantarum 95:533-540.
Additionally to this, once you use antibiotics, there is a risk that you have to use it always for that specific culture. Generally, antibiotics do not kill spores. This means that if after a few subculture cycles you use a medium devoid of antibiotics there is is risk that your spored will germinate. Moreover when antibiotics are used in the media you will not detect new infections. So my suggestion try to avoid antibiotics.
A last reason to avoid has to do with human health issues; the more antibiotics are used (for whatever reason) the higher the chance that resistant strains will appear.
I agree totally with Bugbee and Panis to limit the use of antibiotics.
Bruce and Bart are completely right. One has to bear in mind that many of the contaminations seen during (mass) propagation of plants in tissue culture are human saprophytes, and sometimes even human pathogens. Using antibiotics in this phase will result in generating resistant microbial strains. Try to imagine the effect that has on the operators behind the LAF-cabinets when tissue-culture containers with sporulating resistant microbes are opened. Therefore, use of antibiotics should only be permitted during culture initiation when no other method is available to obtain clean cultures. A rigid discipline to autoclave and to throw away contaminated cultures should be maintained at all times.
In addition I suggest to check strictly the hand manipulations in aseptic conditions. It's very easy to make mistakes during the cultures manipulation.
I do totally agree with Micheli, but I will add to that you have to use healthy, young and clean explant source, most of contamination source is the explant it self specially when taken from old plant.
You can incorporate antibiotics in medium, which are safe for the plant tissue (there is a good number of antibiotics that are safe for plant tissues). You may like to test the sensitivity of bacteria twowards the antibiotics and then carefully select the effective ones. This wwill only be helpful for the exogenous bacteria. It has limited effect on endogenous contaminations
To our experience antibiotics do not really help as they -some comments mentioned that- are not really penetrating the tissue. On the other hand antibiotics only may control for a while bacteria but not fungi. In addition, as most antibiotics target prokaryotic replication or translation, they ma also harm the chloroplasts and mitochondria. Sterility is more important, so it pays of if the clean bench filters are cleaned routinely etc.
I think it is not necessary to add antibiotics in plant tissue culture media if strict hygienic steps were taken during media preparation. The addition of antibiotics to the media may have effect on the tissues of the explants.
In addition to that Oseni says I concern about possible addiction phenomena on the plant cultures
The plant species you are working with and also the bacteria involved in the problem are important and you may not get the same response with the same treatment.
Indeed the best is to prevent contaminations from occurring by proper sterile procedures. Dont use it preventatively. If you do have a contamination issue and you HAVE to rescue the material, you can consider surface sterilization (depends on the material, if you use bleach, ethanol, antibiotics and in which conc), and THEN add antibiotics to the media. Identify your contamination first (is it bacterial or fungal), then either add carbenicillin (for example, = antibacterial, ~100mg/l), or nystatin and/or miconazole (=antifungals, ~20mg/l). You can look them up in literature.
Yes, we can add antibiotics to avoid contamination. Though it negetively affects regenration efficiency but effective against some bacterial contamination.
Normally we do not put them in media, but we use to clean explants again with Clorox and a few drops of surfactant and some time put some antibiotic in solution, and take longer time for soaking. We used low concentration of Clorox to prevent Clorox damaged explants. Rinsed 2-3 times with steriled water to remove Clorox solution.
Yes, you can and it helps. However, according to my experience you should keep the antibiotic all the time. Most of this contaminations are endogenous bacteria and when you tranfer an explat from an apparently clean media to another media without antibiotics, the contamination appears again. I did not see harmful effects.
Núria. This shows that the antibiotics do not kill the contaminants. You probably are not working in a biohazard cabinet when transferring the plants but in a crossflow cabinet, so you will inhale the spores of the bacteria that are present in the plant cultures on antibiotics. When these bacteria have developed resistance to the antibiotics, what then? It has already been shown that human pathogens are also transferred in this way. Ever looked at the disease index of the people working in the transfer room?
You can. But it's not very effective. Better way is to take out the contaminated media and prepare the new one after checking and keeping sterility.
Depends on the type of antibiotic, contaminant and plant species. Cell-wall antibiotics (eg. carbencillin) may be used, but this is not very effective as problems with contamination seldom are due to one single (sensitive) bacterial species, and breakdown products are negatively influencing plant growth. Other types of antibiotics (protein synthesis inhibitors, eg. kanamycin) will hit both the bacteria and the plant (chloroplast), although sensitivity may differ. The tobacco cv. SR1 is a streptomycin-resistant mutant that allows routine use of strep in media.
must used scientific way to prevent contamination. Yes ..in some time antibiotic is useful for isolation/pure culture.
but it shows little effect also
antibiotics can be added to plant tissue culture media, but they should be used as the last source to get rid of contamination. They do have harmful effects on plant tissues in majority of cases, but for systemic contamination, it may be useful to use them. But for others, it is better to go for alternative surface sterilization methods and make sure that there is no contamination. For surface sterilization, multiple treatments could be more useful
You should used sterilizer and then after use cefataximin, ppm (plant preservative mi ) and so many other chemical .
In potato cultures we used carbencellin to take rid of endogenous bacteria. it works quite well. In in-vitro germplasm conservation where sub-culture period is 1-2 years, carbencellin helped in keeping the cultres free of bacterial contamination. No adverse effect was observed.
Antibiotics can certainly be useful in establishing and maintaining cultures. Knowing whether bacteria are present in cultures or not is difficult to determine, since slow growing bacteria can remain covert in cultures for years without detection e.g. ppfms. Only a fraction of existing bacteria are known to us and unless they appear as an infection, they are not considered as relevant. Bacteria are however capable of synthesizing large amounts of plant growth substances and other substances that affect cell growth. Present in small quantities, these bacteria may be undetectable, but none the less influential. There have been suggestions that covert bacteria may be necessary for normal cell growth and development. In that case your putatively axenic culture may need to remain as an ecosystem to flourish.
No
provided that molecular targets of antibiotic should not be present or similar to that of plant.
I also added antibiotic to initiate yellow bamboo shoots. It was successful and I haven't seen any harmful effect to explants or plantlets.
What i came to know from your question is that you should be perfect in autoclaving your medium and distilled water used for surface sterilization of explant. In fact wew go for autoclave of medium according to the volume. This is applicable to distilled water also. You just try to find loop holes in your autoclaving procedure. Addition of antibiotics in medium may not be helpful. As some of our friends suggested it may be harm your tender explants or shootlets.
Hi
yes sure.I added.I used Sefitaxim (antibacterial) to the media to control contaminaion.Also I used it in washing stage.So I think u can use antibiotecs too.but u should find the exact concentration of it from articles.because it differs for any material.
I hope I could help u.
I used for roses.no.It didnt have any harmful problem for my plant
The short answer is that it depends.
I've had very mixed results with antibiotics and it really depends on the nature of the plant and the contaminant(s) you are dealing with. The trick is to find the right type and concentration of antibiotic that will kill the contaminant while not hurting or killing the plant, it is a bit of a balancing act. I have had some good success with rifampicin, but again you will need to test it on your specific organism(s).
PPM can also be quite useful, but some plant species hate it and will not grow. I've also found that PPM often prevents bacterial growth, but as soon as you transfer the plants onto PPM free medium the bacteria resumes growth. I suspect that it mostly just prevents growth rather than killing many microbes.
Dear jimmy
you can add but so as to avoid harmful effect one has to calculate or standardize optimum concentration of antibiotics with respect genotype and species of plant
Of course you can add antibiotics to the medium however you should first determine the concentration (ppm) which would not interfere the growth of the tissues. Higher levels mimic or retard the growth of the tissues. Sometimes you can use a mixture such as RAN (Rifamycin, ampicillin and and neomycine)
Antibiotics can be used to control the contamination but not always. It is to be controlled by following proper tissue culture techniques such as selection of the explant from a disease free plant, cleaning of the explant, standardization of surface sterilization method, inoculation and incubation method, etc. In spite of following these methods, if one gets contamination then use of antibiotics can be done. There are certain disadvantages of antibiotics as they create stress to the tissue, if exposure to antibiotics is in high concentration and long duration. It is necessary to standardize the concentration and duration so as to make the explant comfortable.
We have tested a number of different agents and each has to be optimised for the species of plant you use and even then some tissues can be more sensitive than others. We have used PPM (Plant Preservative Mixture) extensively with good success in liquid cultures as it combats a wide range of micro-organisms including yeasts, fungi and bacteria (there are a couple of papers on this on my RG page)
There are few antibiotics which have been tested for plant and have been found no side effect on plant / explants. the examples are carbenicillin, cifotaxime. if you are going to add any antibiotic to culture media, first be sure if they are harmful or not.
Generally antibiotics affect the bacterial system and also plant system since the plant cells have mitochondria which is of bacterial origin i.e antibiotic affects plant.
Our experience on PPM effects demostred the strictlly connection with the plant genotypes. It is effective in several species, but also toxic in others. The positive/negative effect is also concentration-depending.
Cefotaxime works great. Our plants tolerate as high as 2500 ppm. Bacterial contamination goes away with one cycle and then plants can be moved back to antibiotic free medium
Yes we can use minimum quantity. but endophytic organisms may survive you have consider that. Antibiotic kill only endophytic bacteria. Grow explant clone in lab disease free condition and use those plant for your tissue culture.
Antibiotic can be added to the seed before planting for treatment of the seed for better productivity
In plant tissue culture there has in general two types of contaminations: bacterial contamination and fungal contamination;
antibiotics work against bacterial contamination. In the case of fungi antibiotics is not effective.
Yes we are using streptomycin it is effective against bacterial contamination
No need to add antibiotics in the tissue culture media... by doing proper sterilization of media and surface sterilization we can avoid the contamination... also by increasing the concentration of the Mgcl2 we can reduce...
I just heard of the use of Milton solution for surface sterilisation of seeds and explants and for incorporation into tissue culture media. this is sterilant used to sterilize baby's bottles and is being advocated by SAPS (Science and Plants for Schools) in Cambridge. I have not experimented with this but it is worth considering.
use of antibiotic in TC media has its own merits and demerits. You should therefore assess the type of contamination you are experiencing i.e systemic (emanating from inner tissues) or surface contamination. This way, you will be able to decide whether you need to use antibiotics. In the event its necessary to use antibiotics make an informed choice since some antibiotics have been proved to interfere with callus induction, organogenesis and rooting either positively or negatively.
yes.u can use.I agree with Mehrdad khatami.but u should know which antibiotic and also the concentration to not have toxic effects on the plants.
I recommend the use of antibiotic only if the source of contamination is systemic (endogenous in nature).Otherwise I recommend that you vary the concentration regimes of the sterilant and time of exposure to the sterilant. This is likely to reduce the contamination. If this fails you might consider changing the sterilant type before using antibiotic
Dear All
shall we add any one antibiotic or mixed antibiotic to get rid of this problem?. Which antibiotic is broad spectrum and whats is the concentration is best to get rid the same.
Dear my freiends
I used for contamination remove in banana tissue culture, it was very useful in banana micropropagation, I suggest penicilin antibiotic (1g/l) is better than other antibiotics, this antibiotic is powder and you can buy from pharmacy.
good luck
its possible to add antibiotics in culture media but in most cases after sub-culturing the plant the bacteria will resume growth and make the situation worth.
In my oppinion, it's kinda gmbeling to use antibiotics!
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