Usually, bacterial contamination of in vitro cultures is much easier to cure. Since contaminants are rarely plant pathogens (they are usually human pathogens such as Staphylococcus and Streptococcus), one can use antibiotics for human use to cure plant cultures. In our laboratory cephotaxim (300 to 500 mg per liter of medium, depending on plant species, sensitivity of bacteria to antibiotic and intensity of contamination) was used. Water solution of the antibiotic must be added to medium after sterilization in autoclave, after it was cooled down to about 40 degrees of Celsius, by filter-sterilization or directly, in aseptic environment. It is very important to use antibiotic through more then one subculture, and to lower antibiotic concentration gradually (it takes at least 3-5 subcultures), in order to achieve complete bacteria eradication (although the possibility of latent presence of bacteria inside explants always exists). Contamination by fungi (usually by Penicillium and yeasts) is hard to cure, since anti-fungal agents are highly toxic for plants. Therefore, we usually do not apply fungicides in vitro, but only fungistatics, such as Nystatin for human use (500 mg per liter), only if it is necessary, i.e. if cultures are valuable. Nystatin can be used as sterile solution to rinse contaminated explants, and rarely is added to media. The best way to rescue plant cultures in vitro from fungal contamination is to isolate upper parts of explants (to cut them if shoots are developed), in aseptic conditions, and to transfer them to fresh medium. It is successful only if fungi are in the early stage of development, i.e. if sporulation did not start yet.

Moreover, both bacterial and fungal contamination could be prevented if very diluted water solution of sodium hypochlorite (bleaching agent) is added on medium surface when plant cultures are already developed (unfortunately, I cannot find the reference from Plant Cell Reports, but I know for sure that this method is used in commercial production of sugar cane).

Of course, all I mentioned is efficient for shoot and callus cultures. I have no experience with cell suspensions and protoplast cultures.

Hi Ammar,

I have been doing plant tissue culture involved in genetic transformation for many years. I worked on carrots, Arabidopsis, tobacco and cotton. For these 4 species, in the tissue culture medium, I only add antibiotics for the purposes of selection and killing Agrobacteria after genetic transformation (Note: the explants are derived from plantlets germinated from sterilized seeds). I never add extra agent such as antifungal agents in the media. They are doing just fine, barely see contamination (however, I am not sure if the antifungal agents are needed for other plant species). Sometimes, we do see the 'overgrowth' of Agrobacteria or the carry-over of Agrobacteria to the next generation seeds. In that case, we continued to add small amount of Carbenicillin and Cefotaxime in the 'rooting' medium and 'germinating' medium. And generally speaking, situations were under controlled.

We add antibiotics later after the medium was autoclaved. The antibiotics were filter-sterilized (see attached pictures) before adding to the cooled-down medium in a sterilized hood. The filter is disposable and commercially available.

I think the easiest way to establishe an axenic culture is to use apex or apical tips  as explant rather than the use of bacterial antibiotics.

Contamination is a common word in tissue culture. However, contamination can be minimized by practicing proper procedure  eg:  selection of healthy mother plant, maintaining of these mother plants in clean environment, selection of proper sterilization agent and proper sterilization, minimizing the error during culture establishment, maintaining  a good culture room for culture establishment and proper maintenance of culture room.

Regarding the use of antibiotic, I have used antibiotics for black pepper  tissue culture and found that most of the antibiotics are toxic to plant tissues. therefore it is always advisable to  adhere proper tissue culture procedure iin order to minimize the contamination

Hi Ammar,

I have used anti-fungal and anti-bacterial agents before autoclaving in my tissue culture media of orchids and it worked well. In some specific cases you may need to add the anti-microbial agents after autoclaving (depending upon the chemical you are going to use, i.e, it is heat sensitive or not), in that case you can go for the filter sterilization.

I agree with the suggestions given by others. It is very important to follow all the procedural steps to minimize the occurrence of contamination like selection of explants from healthy source(s), thorough washing of explants in presence of a suitable detergent, sterilization of explants etc in case you collect the material from natural source(s). If contamination occurs while using explants from in vitro sources then please maintain aseptic conditions as much as possible like flame sterilize inoculating forceps/needles etc several times before transfer, wipe hands thoroughly with alcohol, please ensure that the autoclave you are using is clean and working properly. Use of antibiotics and anti-fungal agents in the medium should be the last option as they are often toxic to the growing tissues and if used please standardize the inhibitory concentration of the antibiotics. Filter sterilize while adding in the medium. 

Use of antibiotics or antifungal agents in the medium should not be a common practice as they  may bring out changes in the cultures.  You may use them for treating the explants during the surface sterilization procedure where the explants are exposed to them only for a short period. Adding them in the medium is to expose them for a very long time. 

Hi,

till now I have used successfully Cefotaxime. But you should establish appropriate concentration - to inhibit the bacteria growth, but not to influence negative the plant tissue growth.

I agree with my colleagues. Plant tissue culture involves a lot of procedures which should be optimized to reduce contamination. Highly determinant is the selection of the donor plant and the stablishment of a donor bank; the selected plants should be grown preferably far away from the field where they were taken, in nursery conditions.

I remember in 2000 when I was working on propagation of Mentha in Chihuahua, Mexico. The original plants were taken from a field which had been affected by black waters from husbandry. I didn´t know that, but I realized when the contaminant bacteria were identified as Streptobacillus and Enterobacter,  typical from the internal tract of cattle. Then I had to develop a procedure with chloramphenicol to disinfect the explants before putting them in culture medium, and also to add the antibiotic to the medium.

But, as a rule, avoid use of antibiotic and antifungal agents as constituents of culture medium (you may use them before implantation, by washing the explants). They are selective agents leading to survival of resistant tissues but potential killers to the susceptible ones. They will restrain growth and proliferation, thus impeding massive multiplication in micropropagation systems. There is also danger of adaptation of tissues to the antibiotics, with subsequent death of explants when the antibiotic is absent from the medium.....

In any case, try to identify which microorganisms are you commonly having as contaminants, and the source (as in example given above). Then try to improve the procedure in the critical points representing the source of contamination. Good luck !!!!

It is frequent to observe contamination of plant tissue cultures with fungal (70%) and bacterial (30%) growths. Proper surface sterilization of explants with broad spectrum fungicide, in low concentrations (to be standardized), followed by a mild detergent wash and mercuric chloride (0.1 - 0.2%) dip for 5-7 min. will minimize the contamination. The microbe-free portion of the explants need to be isolated and sub-cultured within 2 weeks to prevent the emergence of microbes from the inner tissues of the original explants.  

Hi!

I have been doing plant tissue culture for quite a few years now and i too have come across with the problem of having contamination. I personally wouldn't recommend you to use any antibiotic/antifungal substances in the growth media. Its not a selection if you are not transforming the plant tissues. In the worst case scenario if you are using them then do not add to the media before autoclaving. Autoclave the media, cool it to 55 degrees centigrade and then add filter sterilized antibiotic/antifungals.But still as I said I wouldn't recommend it to use if theres no transformation involved.

The best way to get rid of contamination is to sterilize the explants. Use 0.1%-2% bleach (magnesium chloride is toxic to plants too) depending on the tissue types. wash it for 5-20 minutes and then wash of the bleach by using autoclaved water. Pat dry your sample on autoclaved blotting paper as wet explants might become a source of contaminations, better practice of using biosafety cabinet or laminar air flow is required. always flame the mouth of the testube/conical flask where you are going to insert your explant for culture.  culture in batches so that even if you get contamination in one batch others can save your time.

wish you good luck with your plant tissue culture

You can add antibiotics (after autoclave) to prevent bacterial contamination but no chemical to control fungal contamination as it a eukaryote just like your plant. Put the UV on in the hood for 30 min before you work in the laminar hood, check if the filters of your hood are Ok and working, wipe the hood bench with alcohol and bleach and wear gloves and wipe them with alcohol. Anything inside the hood should be wiped with ethanol, still use a flame inside the hood, as flame is the best sterile condition, autoclave long enough, at least for 30 min at 121C. Keep the head out of the hood and try to maintain the hood room as clean as possible with minimum entries.

Usually, bacterial contamination of in vitro cultures is much easier to cure. Since contaminants are rarely plant pathogens (they are usually human pathogens such as Staphylococcus and Streptococcus), one can use antibiotics for human use to cure plant cultures. In our laboratory cephotaxim (300 to 500 mg per liter of medium, depending on plant species, sensitivity of bacteria to antibiotic and intensity of contamination) was used. Water solution of the antibiotic must be added to medium after sterilization in autoclave, after it was cooled down to about 40 degrees of Celsius, by filter-sterilization or directly, in aseptic environment. It is very important to use antibiotic through more then one subculture, and to lower antibiotic concentration gradually (it takes at least 3-5 subcultures), in order to achieve complete bacteria eradication (although the possibility of latent presence of bacteria inside explants always exists). Contamination by fungi (usually by Penicillium and yeasts) is hard to cure, since anti-fungal agents are highly toxic for plants. Therefore, we usually do not apply fungicides in vitro, but only fungistatics, such as Nystatin for human use (500 mg per liter), only if it is necessary, i.e. if cultures are valuable. Nystatin can be used as sterile solution to rinse contaminated explants, and rarely is added to media. The best way to rescue plant cultures in vitro from fungal contamination is to isolate upper parts of explants (to cut them if shoots are developed), in aseptic conditions, and to transfer them to fresh medium. It is successful only if fungi are in the early stage of development, i.e. if sporulation did not start yet.

Moreover, both bacterial and fungal contamination could be prevented if very diluted water solution of sodium hypochlorite (bleaching agent) is added on medium surface when plant cultures are already developed (unfortunately, I cannot find the reference from Plant Cell Reports, but I know for sure that this method is used in commercial production of sugar cane).

Of course, all I mentioned is efficient for shoot and callus cultures. I have no experience with cell suspensions and protoplast cultures.

And don't forget to change the filters of the hood, where the air flow goes in, after certain length of time. To make sure the air flow inside the hood is sterile, the hood need to be certified every period of time from a specialty company. They have the adequate equipment for detecting and measuring the air quality in the hood. 

By proper surface sterilization protocol and adding antimicrobial agents to the media

if the contamination occur after the plant has grown then what would i do, and the contamination on the plant surface not in media how to remove this contamination from plant surface please tell me...

I am afraid that, in the situation you are describing, the only one option is to do (in the sterile environment) surface sterilization of contaminated plants (removed from culture vessels) by solution of some bleaching agent and subsequent rinsing with sterile water, separately, i.e.  plant by plant, likewise it is done with plant material that one intend to introduce to in vitro culture. After that, plants should be returned to vessels with fresh sterile medium – one plant per vessel – and situation should be monitored during further development of plants. If contamination is still there, you have two options: to repeat surface sterilization or to give up from those plants that are contaminated. This procedure gives uncertain results, so it should be used only in the case the plants from in vitro culture are valuable and cannot be substituted by new ones. You should use very diluted solution of bleaching agent (less than 5 % water solution) and apply it only for short time period (up to 1 minute), since plant tissues are more sensitive to chlorine from bleaching agents then bacteria and their spores, so they can be killed during procedure.  

In the case that infection is not spread all over the plant, you can try to isolate plant tips in situ, without removal of plants from culture vessels, in sterile environment, and to place them on fresh medium. This method is the only one possible way to rescue plants from fungal contamination, before sporulation has been started.

I have no experience with other ways to rescue in vitro plants from infection that is not at the level of the medium, but assume that maybe some kind of spraying by antibiotics or other chemicals could exist.   

Addition of antibiotics specially Cefotaxime@250-400 mg/L or 4% Sodium hypochlorite @1-2 ml/L in culture media could be an effective strategy to encounter microbial contamination in tissue cultured plantlets. I have tried it and got satisfactory results.

I got contamination free cultures of Tagetes by adding PPM or anti microbial supplements @ 1ml/Lt in the culture media before autoclaving. Few researchers reported the negative effects of PPM on shoot regeneration and culture growth but in my case i didn't observed any negative effects.

Dear Ammar, I will tell you what I came up with many years ago in all the laboratories that I ran, as a preventive method that was used routinely with great success in all of them, particularly when we had more complicated tissues.

Sugar-free culture media are prepared and the tissues are left in them for a couple of days. Then it is transferred to medium with the chosen sugar concentration. If the case is that you already have the crops going and you want to apply antibiotics, the scenario is completely different. The first action is to identify what type of bacteria are generating the contamination. To do this, use the typical means for identifying Gram + and gram -. With that information you go to the SIGMA catalogs. In them you will find the antibiotics applicable in each case. In a second stage identify them and with it you will specify with certainty what to apply. Antibiotics should be applied in the chamber and sterilized by filtration and stored at 4 ° C for short periods. Never autoclave them. I hope I've helped

You have also received a lot of good opinions and experience from many colleagues, that you will surely be successful in overcoming this frequent problem in vitro. Good luck!

Please also have a look at these useful links.

https://www.researchgate.net/post/How_to_control_bacterial_infection_in_a_plant_tissue_culture

https://www.researchgate.net/post/How_to_control_fungal_infection_in_plant_tissue_culture

https://www.researchgate.net/post/Getting_rid_of_bacterial_contamination_in_tissue_culture

https://www.researchgate.net/post/Does_anyone_know_a_good_protocol_to_avoid_fungal_contamination_for_an_in_vitro_propagation_on_media_culture

https://www.researchgate.net/post/Contamination_in_Plant_Tissue_culture_Media_MS_Media_after_autoclave

https://www.researchgate.net/post/What_is_a_good_way_to_control_bacterial_contamination

https://www.ars.usda.gov/ARSUserFiles/4630/Contaminants/4.%20Plant%20Tissue%20Culture%20and%20Biotechnology%203%20137-142%20(1995)%20Bacteriology%20review.pdf

Hi dear

There are a lot of things to keep in mind while doing Tissue culturing.

Some important Steps must be followed.

Always Use apical meristem or buds in tissue culture.

Use autoclaving according to the standard methods. e.g If we sterilize 1L of water (in a single flask) in outoclave for 20 minuts it will never remove contamination, you must instead do the process for 40 min at least and so on.

Clean the Laminar Air flow hood with ethanol/Spirit, every time before work.

Always use UV light for 30 minuts after placing all the media and glassware in side the hood.

Never move hands over the media or explants.

Handling is Very Very Important for preventing Contamination.

Thanks

We always use 'shan nong 1 hao' , a kind of antibiotics in china, to prevent the microbial contamination.

I got a lot of good information from the responses. Thanks to all.