I would strongly advise against using "decontaminated" cell lines. The best cell culture technique uses no antibiotics (or antimycotics) because if your technique fails, the infection is a warning; analogous to pain in a sprained ankle or knee. You want to know about the infection (injury). Even if you were to succeed in completely eliminating the infection (low probability), you might still have a mycoplasma infection as well. This would change the behavior of your cells and make future experiments unrepeatable by other labs, and probably even your own. Get your aseptic lab and technique in order, then keep your stock cells in antibiotic-free culture conditions. Of course, you should have a stock frozen and maintained in liquid nitrogen, so that you can replace your growing stock every 3 to six months (depending on cell doubling times). This protects against genetic drift of your stock cultures and will enable you to repeat experiments decades later and still get the same results.

Are you positive it is bacterial and not fungus/mould? have you tried adding an anti-fungal?

You can see this link. This has good methods

https://www.goldbio.com/documents/1086/Antibiotic+Treatment+of+Contaminated+Cell+Culture.pdf

Dear Mona Dehhaghi,

Dear Nathan Rout-Pitt

Thank you so much for your advices

As I experienced, it must be bacteria but not fungus or other contamination.

In our complete medium, we always add antibiotics-antimycotics that can eliminate both bacterial and fungal contamination.

After contamination, together with this antibiotics-antimycotics we treated cell with Kanamycin and Ampicilin as well but the trouble can not be solved yet.

So, along with antibiotics treatment, do you have any other solution for this.

Thank you so much for your help!

Article Cell Culture Contamination

Article Cell culture contamination: Sources, consequences, preventio...

# Dear Aamal Ghazi Mahdi Al-Saadi

Thank you so much!

# viji Viji

Thank you so much for your advice!

I would advise against trying to remove bacterial (or fungal/mycolasma) from your cultures.

1. It take can a long time and a lot of testing

2. You can never be sure and you may well still have a low level contamination which is not always visible if you passage cells often and use Pen/Strep.

3. Effect of low level contamination on your cells can not be guaranteed

4. Most (not) all labs use antibiotics Pen/Strep and other high strength antibiotics: this can mask poor technique.

Problem (usually lies) in the operators technique or others in the lab: poor skills, dirty lab, or just an off day in cell culture!! etc. I would work on solving these problems first. Set up training for all users before being aloud to use the facilities, set up cleaning rotor.

# Dear Ian R Wilkinson,

Thank you so much!

I appreciate your advice, and now I would try the other way to solve this problem other than removing bacteria.

Thank you for your help!

FACS methods will purify your cell line, but it requires particular instrumentation and some expensive materials. You may opt for this depending on how important that cell line is. Our university has such an instrument, and I can put you in touch with an expert, if you want.

Here's a place to start for information:

Article Purification of Specific Cell Population by Fluorescence Act...

I would suggest that you isolate the contaminating microbe by streaking out on nutrient agar plates and test it for antibiotic susceptibility.

Preferably, you should use bactericidal antibiotics (ones that kill the bacteria, rather than just stopping their growth). Ciprofloxacin, third-generation cephalosporins and Vancomycin might be useful.

If you don't have the facilities, ally yourself with the microbiology department, or get the microbiology department of the local hospital to help you.

Good luck

Andrew

If you are going to rely on antibiotics to get rid of the bacteria, then remember that many of the contaminating bacteria will be intracellular and penicillin/ampicillin and gentamicin/ kanamycin/streptomycin/vancomycin will not enter your cells to kill the bacteria. First, find out the contaminating bacteria by culture, then choose antibiotics to which they are susceptible, and that will give enough intracellular concentration. Use two at once. You can use rifampicin, ciprofloxacin, trimethoprim, minocycline, linezolid. However, best to discard all and start again.

I had pretty good luck with removing bacteria contaminated media, washing once with PBS, and then incubating the cells with 2X pen-strep in PBS for 25 min. Longer incubation in 2X is detrimental to the cells however so longer incubation time is not better. I put media with 1X pen-strep in thereafter and it appears to be "clean". We always add 1X pen-strep in the media for that particular cell line now. I don't believe the contamination is gone by any means but we can do some preliminary studies with them and can buy fresh if this cell line proves interesting..

# Dear Felise Wolven,

# Dear Andrew Jenkins,

# Dear Roger Bayston,

# Dear Gail Sudlow,

Thank you all so much for your kind helps!

I intended to abandon my effort to remove bacteria from cell culture, but after reading your suggestion, I think I would try once more!

And # Dear Felise Wolven, if eventually I could not get rid of bacterial contamination, I would like to contact you for further purification of my cell line.

Once again, thank you all in advance for your kind helps!

I would strongly advise against using "decontaminated" cell lines. The best cell culture technique uses no antibiotics (or antimycotics) because if your technique fails, the infection is a warning; analogous to pain in a sprained ankle or knee. You want to know about the infection (injury). Even if you were to succeed in completely eliminating the infection (low probability), you might still have a mycoplasma infection as well. This would change the behavior of your cells and make future experiments unrepeatable by other labs, and probably even your own. Get your aseptic lab and technique in order, then keep your stock cells in antibiotic-free culture conditions. Of course, you should have a stock frozen and maintained in liquid nitrogen, so that you can replace your growing stock every 3 to six months (depending on cell doubling times). This protects against genetic drift of your stock cultures and will enable you to repeat experiments decades later and still get the same results.

# Mr Dear Christopher Lange,

Thank you so much for your helpful advice.

I recognize that I have been using a wrong procedure for cell culturing.

I think the only solution now I would do is to re-setup my culture technique with the new cell source.

I am grateful for you suggestion.

Best regards!

For human lymphocyte cultures for chromosome studies we add pen_ strep to filter sterilized medium in bulk and also to individual culture tubes.Usually it gives ample sterilisation

Dear Asha, your addition of pen + strep to the culture medium is to prevent contamination, but we have been asked here about an established contamination in a cell line, which is different. Ideally, antibiotics should not be a necessary component of cell culture media, and I have always manipulated my cell cultures on an open bench, with stringent aseptic precautions, without contamination (or antibiotics), but I am a microbiologist. Much of the contamination is from direct inoculation by instruments, pipettes, fingers etc rather than air (unless it's filthy).

I am considering that usual addition of "pen + strep" and/or "strep + kanamycin" may not be suitable. I recently have added Amphotericin B into the medium of Caco-2 cell in order to prevent the contamination by fungus or yeast (please see file; JCB Fucoidan transport).

I also have recently answered to Dr. Parin Mehta (Drexel University, Department of Biochemistry & Molecular Biology, Philadelphia, PA, USA) in this ResearchGate as follows; i.e.,

" I recently have found that cultured fetal hepatocyte Hc cells already have infected by Caulobacter vibrioides, Lesionella pneumophila, Treponeme pallidum, Yersinia enterocolitica, Pseudomonas aeruginosa PA7, Helicobacter pylori J99 etc. (please see file; HepG2 Fucoidan). These bacteria seem to be able to swim. Then, New Quinolone, Erythromycin, and Clarithromycin may be effective. Healed hepatoma HepG2 (cultured with fucoidan) contains large virus of Mimivirus, which may not swim. Fucoidan seems to be strongest medicine to reduce virus and bacteria of the cultured cells (please see again; HepG2 Fucoidan and Fucoidan bacteria). Amphotericin B is very effective to prevent contamination by fungi and yeast (please see file; JCB Fucoidan transport)".

Hi,

Add kanamycin and streptomycin or vancomycin

Dear Xoan Hoang, I think you are on the wrong track and you need to take account of Ian Wilkinson's post at the top of this list, especially about clean technique, training etc.

Thank you all for your comments and suggestion.

I decided to start all over again with the fresh cell stock with strict procedure of sterilization.

I hope this would work.

By the way, I am still confused that so far, we have been culturing all cell lines (even without any contamination) in the presence of antibiotics. But, according to many suggestions, this would affect a lot of cell conditions. So should I exclude antibiotics from the culture medium?

Once again, thank you all so much for your kind help and advice

Best regards!

1. You should exclude antibiotics from the culture medium.

2.Strict sterilized technique is the best way to avoid contamination.

3.Do not put the plates beside many plates.

4.The temperature should be controlled in the incubator.

5.Limit opening the incubator many time by many colleagues having similar plates inside the incubator.

If contamination still occurs despite all preventative procedures (rigorous aseptic techniques) being undertaken effectively. One cost effective and simple preventative method could be regular filtering of culture media for the removal of unwanted particles that may have accumulated over time. Use of 0.2 µm filters is recommended for protection against bacteria and fungi; 0.1 µm filters will additionally remove mycoplasma.

It is recommended that filtration options are explored, enabling the best membrane selection for the specific needs of the laboratory or research application.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4298231/

I am concerned by the paper Huda provided for this discussion. Two problems: First, the scale of antibiotic effectiveness covers ON A LINEAR scale, 30 to 0. For elimination of microbiological contaminants, one should use a logarithmic scale and see at least 5 orders of magnitude (powers of 10) depletion to have any confidence that a minor contaminant had been removed. Based on the data shown, I would be surprised if the contaminant did not reoccur with further culturing.

Second, even if the antibiotic reduced the growth potential of the contaminating microbes, one is in danger of establishing a co-culture of the test organism and contaminant, such that if their growth rates are comparable, neither outgrowing the other, and all experimental results with this population will not be representative of those of the pure culture.

@Xoan Hoang,

You should remember, that when handling sterile materials in your laminar flow (preferably vertical flow in potential pathogens are involved), look at the direction of air flow and ensure that nothing that is not sterile is allowed to pass upstream of an open container of sterile material. This is important even if you wash your hands with alcohol (70% ethanol), don gloves (nitrile preferred), washed your hands again in alcohol with the gloves on, all in a hood that had been turned on, swabbed down with alcohol, and then had the UV-lamp irradiate it for 20 minutes before anyone uses it. Any container being introduced into the hood (biosafety cabinet) should also be swabbed down with alcohol to sterilize its outside.

Hope this helps.