Perhps you could try Copper or acibenzolar-S-methyl (it is a resistance inducer)

Hi Mohammad

How are you managing withoug a laminar flow? Alot of contaminants are already in the air and without a laminar you will be exposed to all of them. Surface sterlization procedures defer with different plant species but 70% ethanol for about 2 minutes followed by 12.6% jik (hypochlorite) for 10 minutes with shaking works wrll for most explants. Thourough rinsing with sterile distilled water in between washes is necessary and making sure all yor tools are autoclaved. However due to endogenous contamination you may need to do serial subculturing of your explants

Assalam-u-alaikum brother

I can understand your problem. Although laminar-hood is imp. for tissue-culture purposes, anyway now you don't have access, so no need to worry.

Do one thing, after tween-20 wash for 20-30 minutes under normal running tap water, rinse 2-3 times with DD water and treat your explants with 0.1 % mercuric chloride or 0.1 % sodium hypochloride for 2 minutes, then with 0.5% streptomycin sulphate for 1.5 to 2 minutes & then carefully dip them in 70% ethanol for not more than 30 seconds, & lastly rinse with double-distilled autoclaved water 3-4 times. You will get the results, Insha-Allah.

To start with, make sure that your starting material is free of bacteria.

Special attention should be given to slow growing endogenous bacteria that often do not interfere with initial in vitro multiplication but only appear after prolonged culture and/or stress.

How to screen? This includes careful observation of material during subculturing and streaking freshly cut ends onto bacteria growth medium. At the laboratory for Tropical Crop Improvement, we use a growth medium (BACT medium) containing 23 g/L Difco® Bacto nutrient broth, 10 g/L glucose and 5 g/L yeast extract (Van den houwe and Swennen, 2000). These plates are incubated for 3 to 8 weeks at 28°C. Cultures responding positively are always discarded.

You could try adding other antibiotics, which you can filter-sterilise as a concentrated stock solution, then add after autoclaving when the media has cooled to below 60C. In some cases you can even make a very concentrated stock solution in 80% ethanol and add that to the media for final concentration around 100-200ppm. To control bacteria when isolating fungal cultures I use penicillin at 100ppm and streptomycin sulphate at 200ppm - both need to be added to media after autoclaving.

Streptomycin suphate is a good idea, we use this since 40 years, sometimes also in doubled haploid work. The basic idea for us came from Dr. George papavizas who published a paper in this matter in 1967. However, this will not replace to work as steril as possible. details see here and in the previous comment by Jon West.

We use this product with excellent results:

http://www.ppm4plant-tc.com/distributors.htm

Aminoglycosides antibiotics (gentamycin, streptomycin....) in some cases could affect plant material with few effects on bacteria control. As an additional inconvenient these antibiotics lose quickly their activity in aqueous solutions. I recommend you to apply a two-step disinfection. First you have “to remove” the external contamination from visible contaminated material. Afterward you should make a chronic disinfection using, for example, cefotaxime (up to 300 mg/L are ok) and rifampicin or combining both. These antibiotics are thermolabile hence you have to sterilize them by filtration. As you don´t have laminar hood you have to add antibiotic close to a flame and when the temperature of culture medium are between 50 to 55ºC. In this case a chronic treatment should be more effective than acute one so you should probably repeat this protocol several times. For a better comprehension of contaminants in tissue culture read the papers of Pious Thomas and Barbara Reed, for example. Good luck!!!!!!!!!!!

For plant tissue culture work, laminar flow cabinet is needed since all procedures starting from surface sterilization and explanting must be done under sterile condition. I’m surprise that you managed to produce sterile cultures of Begonia, Echinacea Purpurea, Lilium Ledebourii without contamination. In our laboratory we deals with all kind of plant samples from horticultural plants (mostly ornamentals and fruit trees) to herbal and medicinal plants and forest species. Some of the ornamental plants, particularly roses, harbour internal or endogenous bacteria, whereby cultures from cuttings may look clean after the first week and later we can see bacterial contamination. Initially I used all kind of antibiotics but they were not effective and I was advised not to use them because the bacteria my develop immunity or resistance to the antibiotics. PPM is quite effective to reduce bacterial contamination. However, with roses and pineapple suckers taken direct from the field, the use of PPM alone is less effective. Therefore we used combination of mercuric chloride at a dose of 0.3mg/l which totally get rid of bacterial contamination. However, since mercuric chloride can be toxic, precaution must be taken by wearing gloves and face mask and we normally do this when nobody is around in the lab or we do it during the weekend. Also try to use the smallest possible explant for starting materials since the bigger the explant the more chances of contamination.

Hi Mohammad

I am enclosing one paper and one reference for your guidance. I was successful in establishing these two plants (Jatropha and Piper betle) after a lot of difficulties. The protocol developed for the removal of latent bact contamination in Jatropha is a foolproof tested protocol and remained successful for many other infected cultures of different plant species. Addition of antibiotics in liquid media with regular subculture after 48 h and continuously decreasing the concentration of the antibiotic is another option. This was followed for Piper which is having a problem of phenolics also. You can use the antibiotics which you are having and follow the jatropha protocol, otherwise, try to get Augmentin. This antibiotic doesnt affect regeneration efficiency. Wish you all the best.

Sorry, other reference could not be attached.

Also, you can try to make a disinfection protocol in your in vivo plants for 2-3 weeks before in vitro introduction, for example:

monday: kasugamycin (atb+antimycotic) 2.5 ml/l, wednesday: agrimicyn (atb sistemic) 0.2 g/l, friday: benomyl (fungi.) 1g/l.

Good luck.

Thank you my Scientist friends , your method are all very nice and interesting , my infected explants have show very nice results specialy Echinacea Purpurea and i want to save them .PPM is expensis and that company don't ship to Iran , i am seeking for similar chemical called Kathon CG that is used in plant tissue culture again companies don't acept to ship to Iran or they sell it very expensive .I need more experiences and new techniques to give me a way to add thermolabile antibiotics to my medium or get the currect biocide .everythings are hard to find if you live in Iran when trade embargo is increasing everyday .

You can use wide spectra antibiotics or combinations of more than one antibiotic in the solution in low concentrations and keep your material for few hours before inoculation. Add another antibiotics combination in the media. instead one antibiotic compound, the use in combination of more than one will give better results.

You can use thermotherapy on your tissue before the treated with chlorine and posterior cleaning in sterile water. After only cut the dome or meristem on clean area without contamination. You can treat to do the cut on stereoscope.

I dont know if you are working with plant transformation?anyway the main problem in general with tissue culture is contamination so what you have to do all the work of tissue culture under a very high degree of aseptic technique in laminar hood then you need to treat the tissue culture media with PPM(1mg/l) this is the begining of experiment said the starting of the tissue culture experiment.If you are treat the tissue culture with bacteria so you need to add to the second media either cefotaxime or carbenicillin at concentration 250mg/l it depend on your experiment to kill the bacteria then reduce the rate of contamination in plant.

I just find new idea and will use a this method for sterilizing antibiotics instead of filter sterlization, this is cold method so thermolabileI antibiotics may be stable with this method , this method must be working if sterilizing chemicals don't destroy antibiotics , hope it work and resolve my problem .Will report the results if it work .

Thank you very much for your information

There is very important point that you can not autoclave the antibiotics because in this case you inhibit the effect of any antibiotics so you need to add the antibiotics to the media after autoclave and cool the ,before pour the media in any type of container in your prtocol , you can use sterilise filter if you like.Another point that you cannt use Gentamycin to inhibit the bacterial growth.

Dear Dr.

In our times though we had laminar flow etc. but for getting rid off bacterial contamination sometimes we used to apply lactic acid near the contamination and it worked well. You may also try this.

Dear Arun

is it possible to mix Lactc Acid with medium as a ingredient ?Is it possible to autoclave Lactic Acide ?I am seeking for a biocide ingredient that i can add it to medium always to prevent microbial contamination .Sadly Kathon proders don't answer my request for 250ml of Kathon CG .

Just want to inform everybody that after resterilizing and recuture to a medium with antibiotic and NADCC now bacterial contamination is controled , now i have to reculture plants to a medium without antibiotic and NADCC to help them to grow normal .I don't know which (NADCC or Antibiotic) were helpful but it is important that contamination is controled .

How many subculture you did with NADCC and antibiotic? I recommend to you to make at least 3 subculture under this conditions. A final test, inoculating segments in an indexing medium is the best way to assuere that your culture are clean. Afterward you have to remove NADCC and antibiotic from culture media. Good luck.