Bacteria contamination will release LPS in media. LPS, which is a bacterial toxin will strongly change the cellular metabolism that will lead to a false cell response in you experimental model. However, for further culture, you can use 0.2% Normocin™ from Invivogen. The only scientifically correct option is to reinitialize the culture!

Hi Bivek, treating is the only option you have? What about thawing some new stock?

I faced this problem before with another cell line, and I solved it by freeze it for a week and thawed it again, and I did this 2 times, and it worked well and contamination was removed.

u can try it, and good luck,

Thankx Aminake and Bashir..

Bashir, How the freezing of cell removes contamination. By freezing, does bacteria get killed? I think they are also freezed and when thawed they can retain properties and grow. Isn't it?

Aminake, I have very limited vials left for thawing the cell. That made me think more. Otherwise I used to thaw the cell. I think the incubator might be contaminated or media might be contaminated. So I must check them..

Regards,

Bivek, the best option is to clean everything in your culture room and to check the media are free of bacteria. When you´re sure all is ok, take a vial of cells from the nitrogen container and put them to grow. When plate is ready, split it to get more plates, so you can prepare more vials to freeze. In case you don´t have more vials, I guess you can contact any lab in your area, those cells are frequently used.

I had the same problems with some very precious HEK293 cell samples... I washed it intensively two reduce the amounts of bacterial cells as much as possible and added ampicillin 100 ug/ml for 2 weeks.

When you have no back-up stocks, this is a real problem. No one likes to have to subject their cell cultures to antibiotics as these always have some effect on growth. I've used P/S (as you are doing) in combination with: Amp (100 micrograms/ ml), Kanamycin (75 micrograms/ml), erythromycin (100 micrograms/ml), and chloramphenicol (10 micrograms/ml). For moderate contamination levels or when addition of either antibiotic alone has no effect, i.e, you have encountered resistance, than this mixture should be used for at least one week but for the best results treat for two weeks even if the contamination appears to be gone. You may find that using either Amp or Kan or these together (along with the P/S) is enough as these two usually give the best results in my hands even after only 3 - 5 days of treatment. Remember that the bacteria is causing problems with your cells. Bacteria wall components can set into play a number of pathways that generate products that fend off infection. The bottom line is that these signals are changing the normal growth characteristics of your cells. The antibiotics suggested here at these concentrations have been tolerated but a variety of cell lines that I've used including primary cancer cell lines (directly out of tissues), myoblasts, as well as HEK293 cells. Good luck. (Oh yes, do what has been suggested by someone else, be sure to clean your incubator and all cell culture equipment.)

Discard them and thaw a new vial after you clean everything.

Hi Bevik,

I had a similar situation before (with primary cell culture from mouse tumor tissue), what I did was

1. washing the cells with HBSS or DPBS or DMEM w/o FBS 1-2 times, to dilute the bacteria.

2. Then cells were washed with regular medium with 10x higher concentrated P/S twice. Then add mew culture medium with regular concentration of P/S plus another antibiotic, in my case gentamicin.

3. After incubation O/N, I changed back into regular culture medium and the bacteria were gone.

Hope this would help.

Good luck!

Bacteria contamination will release LPS in media. LPS, which is a bacterial toxin will strongly change the cellular metabolism that will lead to a false cell response in you experimental model. However, for further culture, you can use 0.2% Normocin™ from Invivogen. The only scientifically correct option is to reinitialize the culture!

I guess first thing to do is to autoclave the incubator, make fresh medium (use freshly made antibiotic). Clean the laminar hood, including below, have the filter checked. Autoclaving the pipet you use for cell culture if they are autoclavable is also a good idea. A clean environment is essential, otherwise even if you cure the cells the contamination will return (particularly important is to have the hood efficiency checked)

And then start a fresh culture, it is very hard to get rid of bacteria completely and the danger is that low level contamination affects the results of experiment (particularly ROS studies will not give reproducible results).

I agree with Andrei. My main concern is the cells behavior after the contamination. It's not worth risking getting unreliable data so if I were you…I would clean everything and start a new line! did you have penn-strep from the beginning? If you didn't, I would on your fresh batch to avoid this situation again. From my experience penn-strep (1% of 10,000 unit) alone usually keeps those buggers away. good luck!

Thank you all for nice suggestions. I am going to check incubator and hood by taking sample using LB agar plates and will autoclave incubator and suction head and will make the fresh media. I have ordered new HEK flask from other lab too.

The cells are treated with Cipro and It's about a weak. So, I splitted the Cipro treated HEK Cell in two; one was with cipro and the other was with without cipro, I have seen some few bacterial contamination after removal of cipro in one flask and the other flask with cipro are good without contamination. I hope, I will get the knowledge of source of contamination.

Regards,

Bivek

For freezing, I used DMSO which was opened earlier and kept in shelves. Can it be the source of contamination of freezed vials?

Why bother testing the incubator and hood? That's just a waste of time. Just clean them up. Wipe the hood throughly, including the walls and sash with alcohol, then switch ON the UV light. Do the same for your incubator. Dismantle all the shelves and and walls and wipe everything out with alcohol.

Your DMSO could be the probable source of your contamination. It happened to me as well. When I was starting out cell culture I froze a batch of cells using DMSO I found on the shelf (non-sterile). Every time I recover cells from the stocks in liq N2, two days later they're contaminated. Since then, I used sterile DMSO cell culture grade from ATCC. This solved my problem.

Like most people suggested here that you literally have to start fresh by decontaminating your hood and incubator and purchasing fresh cells, new media, reagents and DMSO ...etc. If possible, try and shut down your facility for a bit before you introduce fresh cells in.

The key point is to try and purchase cells from a trusted source!! Avoid getting the cells from another lab because you may introduce another type of contamination to your lab that may not be visible under the microscope. For example, mycoplasma contamination. It's a nasty contamination that slows down the growth of your cultures and changes the biology of your cells. That's why it's recommended that you purchase fresh cells from a trusted source that tests for such contaminations.

Good luck

I agree with the above statements. However, It would be better to check the PBS, which is the predominant cause of contamination. Another way to recover the contamination from your plates or flasks, is by washing with autoclaved PBS twice and add L-Glutamine–Penicillin–Streptomycin solution and gentamycin containing media into the cells. I do hope it will remove the bacterial contamination.

I agree with Shaima, and would not take cells with an unknown hygiene status. In the worst case you end up with an supressed contamination or mycoplasma contamination.

To be one save side I would order new cells from a company (e.g. ATCC) it is worth to pay the price.

To have enough cells for your entire work, and if you enough space in the nitrogen you can think about to frezze them as cell bank (50-100 vials).

For nitrogen storage I would always do this in gas phase never store the vials in liquid phase. This is one of the biggest source for contamination.

Check and filter the Fetal Bovine Serum which is used in your media ,most of the time the serum is the important and common part of contamination

We had the same problem once that was controlled when we applied Gentamicin (500uL/5mL) directly on the T25 flasks, changing it each days up to 3 days. Likewise, you should verify your stock solutions every week, performing sterile tests.

Good luck

About 12% Mycoplasma strains are resistant to Ciprofloxacin. We use also Plasmocin, works well. What is the method of Mycoplasma detection in your lab? The simplest way seems to be DAPI staining, but you should make PCR too. Make all equipment clean with the use of alkohol and use fresh and sterile liquids. You should remember also that, the ciprofloxacin is a potent topoisomerase inhibitor and it may be cause of genotoxicity.

Hi Lídia Andrade, Thank you for worthy explanation.

As you mentioned that 3 days of treatment of 500uL/5ml Gentamycin.. Does this time of treatment is sufficient?

And how we can do sterile test from storage vials each week. Is there any easy methods.

Regards,

Bivek

Thank you Michal. I appreciate your suggestion.

I can do DAPI staining. I did gram staining of the contaminated sample and found Gram negative small rods. Yes you right that why using cipro, The cells are also growing slowly.

I am using pipet controller which was also uses sometimes outside culture hood.. I thnk that may be the source of contamination, isn't it?

I agree with Tarlan Jafarpour, Zahir Shah, Ryan Pekson .. Thank you..

Hi Bivek

thank you very much. This concentration is enough to kill bacterias. However, if the contamination stays lower. When you find high number of bacterias you must to throw it away immediately. HEK 293 is sensitive cell line for that reason you need pay more attention. Sterile test is easy and cheap. Firstly you take a small volume of your stock solution (5mL is enough), and transfer it to a sterile conic tube (Falcon). Keep it for one week at C02 incubator, taking a look each day. If the liquid begins cloudy it means that your stock solution is contaminated and you can discharge it. At the ends of week you take a look at the microscope if there is any microorganisms.That`s it. Good luck.

Cheers

Lidia

Thank you Lidia for nice suggestion. I knew so many reason for contamination and their check and removal.. I am going to implement the aseptic techniques and routine monitoring..

Cheers :)

Greats :)

"As you mentioned that 3 days of treatment of 500uL/5ml Gentamycin.. Does this time of treatment is sufficient?" - gentamycin is not efficient against most Mycoplasma strains. During cipro treatment, you should use it at least 1 week.

" am using pipet controller which was also uses sometimes outside culture hood.. I thnk that may be the source of contamination, isn't it? " - you have to clean it, if the pipet controller is not autoclavable, try to take it to pieces, then clean it with the use of alkohol, change filter (smaller than 0,2um if you can get it). All actions under the hood.

Through them away¡, start a new culture, you will save time and money.

Of course Gentamycin is not to treat Mycoplasma infections. We had another kind of bacterial infections sensitive to Gentamycin. However, to detect and treat Mycoplasma infection, please, find in attachment the protocol.

Thanks for your comments.

Cheers

Michal Kielbus, good question.

What drug will we use, if there is fungal contamination like Candida?

Thankx Lidia Andrade for the document. But still detection is robust. So It will be better to revive new vial.

Regards

Hi Bivek

in case of fungal contamination you need to throw your flask away, to clean your CO2 incubator and start up with a new vial.

Good luck.

Cheers :)

Cheers :)

Hello Bivek,

I am afraid that I have bad news. The only way to remove the bacterial contamination is to kill the affected cells, discard all media and solutions that you used and start from the start.

I have been down that road trying to recover a cell line from contamination and I used a variety of antibiotics. However, this is time consuming, you spend a lot of money and resources with no results.

So, I think that you should start from the beginning and try to eliminate possible sources of contamination.

I hope that helps

Discard everything, decontaminate your lab, and start with a new batch of cells. All other maneuvers are waste of time.

If contaminations occur repeatedly, you have to search for the source of contaminations (check the filter in the laminar flow hood, decontaminate the incubator, make fresh media, salt solutions, trypsin solutions, etc.)

Thank you Iraklis and Gerhard.

We have fumigated the room with formaldehyde, decontaminated incubator by heating cycle and wiped with 70% ethanol and checked the filter.

Could you please suggest the proper method of decontamination of Room?

Can we fumigate incubator too?

Regards

As I said, if the contamination was a single event, forget it.

More importantly: have enough frozen stocks of cells in liquid nitrogen.

I agree strongly with Francisco, throw them away and carry out a thorough clean of your facility (hoods, pippettes, incubators). take care with your technique and ensure that media/PBS etc is also not contaminated (if multiple users of media etc this can lead to cross contamination: if possible have your own stocks). Bring out a fresh vial and re-grow. If cell lines are important (and you have the space) we freeze down cells immediately as a token freeze (~5). Take 1 vial and grow this up and freeze vials as a master cell bank (10+ or more if possible). Check for contamination i.e bacteria, mycoplasma all the way through the process. Take a master cell vial and grow up to freeze stocks for a Working cell bank (10+ or more if possible). Use this stock to grow from and carry out experiment. Simply grow more vials from the Master cell stock to replenish working stock. This way you can ensure the integrity of your cell line and keep the passage # relatively the same therefore lowering culture variations and also be assured that your cells are contamination free.

good luck

I have had this problem a lot in summers. You have to be very cautious with every step. Check your media, PBS and other stuff you use regularly after you use them for cell culture. You can check the sterility of media

by keeping the media overnight in incubator. Sometimes media look clean when we keep it in refrigerated. But yes for now, as everyone else says, start a new culture and keep everything sterile. Good luck.

It is always good practice to aliquot all media and reagents into "single-serving" sizes in order to prevent both bacterial and cross contamination. Tragically, I had to learn the hard way a few years back. Sorry to hear about your contamination, but it might be less time consuming (and less of a headache) to start over. Good Luck!

I didn't read through all the answers, but most are focusing on decontaminating the earth. :-) Usually the main problem is technique, unless the problem is fungi and spores have spread everywhere. Then rigorous decontamination is necessary.

Sorry to say, I agree that there is no cure. Make everything fresh, filter sterilise if necessary (e.g., expensive reagents), and start over. We actually culture cells without any antibiotics (unless they are primary cells from animals), and always passage cells so backup cultures can go a few more days. By then (since no antibiotics) you will know whether your passage is contaminated, and can go to the backup culture before you go to the frozen backups. Another thing you can do to trace the source of contamination is to have a 'duel' with someone who isn't having problems. Exchange various reagents, etc. If they use your reagents and don't get contamination, and you use theirs but do have problems, then it's technique. Otherwise you should be able to identify the reagent which is the source.

Follow the fololwing steps

first fumigate laminar and co2 incubator

check the water in incubator

go for mycoplasma screening throughly

adding more antibiotics will be stressful to cells in long time

How do you get the contamination is bacteria?

Hi Nasrin, I did Gram staining and found Gram negative..

Dear Meeta, I used follow the earlier steps but not mycoplasma screening. Please suggest me easy and cost effective method for mycoplasma screening. thankx

Regards,

Bivek

Bivek, if the contamination is Gram-negative bacteria, there is no need to decontaminate dry surfaces. Only Gram-positive bacteria and fungi form spores. Gram-negative bacteria live on wet surfaces and in water baths. Mycoplasma live inside cells and usually come from animal products (serum, saliva). Knowing what contamination is present indicates how it is transmitted. Throw out infected cultures, decontaminate humidified incubators, clean and disinfect water baths, and practice better technique (have a 'duel').

Have you heard the story of King Midas? Everything he touched turned to gold. Tissue culture is like King Midas in reverse. Everything you touch turns to shit.

Bivek Timalsina, Do you see any changing in color of supernatant (from purple to orange) during your cell culture?

Confirm mycoplasma by pcr primers are available

Gerald, this seems like you said.. main culprit will be water bath in my case.. because I got fishy smell after some days inside waterbath.. and the last line is absolutely right.. but we have to touch for shit also that make us aware of shit too.

Hi Nasrin, as you said, the change of media colour from pin-red to yellow is so fast as compared to earlier. this became more yellowish when I left for overnight.

Thankx Meeta, but in my lab, no one is working on that.. I short out the problem now.

regards

First find the source of contamination, Media, cells, incubator, hood

Is other cells grown in same media doing fine? or other cells in the incubator is fine?

First suggestion, sterilize everything, incubator hood water bath etc...

Make fresh good media with fresh Penicillin/ Streptamycin.

Revive the cells which dont had the contamination earlier.

If you dont have other cells then go for cleaning steps for your contaminated cells.

1. Test for mycoplasma

2. If you found mycoplasma in your cells treat cells with MRA (Mycoplasma Removal Agent)/ Plasmocin etc

3. For removal of mycoplasma use less cells to treat mycoplasma removal agents

4. If you dont want to remove the above steps just make a serial dilution to get a single colony of your cells and expand them (This is not solution to completely remove mycoplasma).

Goodluck!!

Best thing is revive a fresh vial from liq N2 and make sure all the reagents are contamination free or filter sterilize before you start!!

good luck!!

Dear in case if your contamination is not too much ; you can increase the amount of antibiotic up to 3%. But if contamination is too high, better to throw the flask and thaw new cryovial. TQ

Thank you all for such great sugestion..

Happy.. Problem solved.. but encountered with other problems.. like very low transfection efficiency with pIndie-C1 DNA.

Any idea?

Regards,

The best solution is thawing a new batch, or getting new cells by purchasing or asking other lab etc. I would not suggest elimination of contaminants to use the cells.

If you think you have to eliminate bacteria, many antibiotics could be tried--Kanamycin, streptomycin, tetracycline, gentamicin, etc. If bacteria are contaminated, the media would get opaque. You do not see opaque media culturing several days; the contamination is gone. However, the character of cells may change due to exposure of bacteria and antibiotics. Mycoplasma contamination is difficult to find by just observation of culture media.

Toss out the contaminated culture and start again. You have nothing to gain mucking with antibiotics and .....

Hi Bivek,

low transfection efficiency can have many reasons. I can give a little list what I check when I have to deal with this problem:

- to many cells plated

- cells are not healthy (bacteria or mycoplasma contamination)

- depending on the transfection method you choose could be that on of the solutions is not ok e.g. pH

- bad quality a of the DNA

- amount of the DNA to less or tp high

- backbone of your plasmid

I hope this helps you and best wishes

Sibylle

Adding antibiotics and irradiation may be OK, but speaking as an editor and reviewer of your work, it will NEVER be accepted. The golden rule is start a new culture and recheck your growth media.

When cells do not grow as rapid as they usually they do and the transfection is poor I would think on mycoplasma contamination. You could check it by DAPI staining and you could treat your cells with plasmocin (Invivogen), although the best solution it would be to discard your cells and de-freeze a new vial.

Adjust the concentration of the antibiotic and choose the best one

Easier and more predictable/dependable/higher quality to start over

If you are sure that HEKs are contamined with bacteria, I would strongly suggest to throw them away, clan everything in your cell room and the workflow, throw away serum and media you used to colture them and start a new colture. In this way you'll avoid contamination of other cell lines in colture in the same incubator.

However low growing rate often asociated with low transfection efficiency are more frequently associated with mycoplasma contamination which has to be treated with the right antibiotics. Usually, when coltures are contaminated with bacteria, growth medium turn to yellow and you can see little black dots floating in the medium if you look at the cells at the microscope. It's a very evident contamination.

Good luck!

MG

Dear,

It is not advisable to wash your cells with antibiotics (above the required concentrations) because the bacteria will attain resistance to the family of antibiotics used. Then it will be a head ache to your future cell cultures also. Try to find the source of contamination and rule it out accordingly. And restart your cell culture.

The short answer is you can't. Toss out your culture and any media you were using to grow them and start fresh. Filter everything if you must. Clean out your culture hood thoroughly with 10% bleach followed by sterile water and then 70% ethanol. Mist everything that goes into your hood with 70% ethanol including your gloves. Decontaminate/disinfect the water jacket in your incubator and thoroughly disinfect the inside before starting up new cultures. Change your gown often. Go back to the basics. It is best not to use antibiotics or anti-fungals at all if you can because they can mask low level, underlying, contamination that might be altering your cell responses. It is better to know if you have contamination and work accordingly. LIke another poster suggested, testing your cultures for mycoplasma is advisable as well. Kits can be purchased reasonably and a quick test of your culture supernatant will let you know if you have contamination.

I would prefer to reautoclave all the things, fumigate the cell culture room and start all over again with a fresh batch of cells, media, serum and Penstrep as well.

Some times more antibiotic could slow down the growth too.

Hi there

I have little to add that hasn't been previously stated however as a general rule:

(i) don't try to "cure" a cell culture of microbial infestation - like it or lump it, no experiment is so important that you can pretend bacteria / yeast didn't affect it.

(ii) throw everything away that is associated with the culture - the culture itself, medium, FCS aliquot, flasks if open, pipette tips, pipettes, previously-harvested supernatants - everything.

(iii) Clean everything like a maniac.

(iv) Get fresh cells from your liquid nitrogen and bring them up without antibiotics to ensure that are not carrying a low level contamination.

(v) If the ARE contaminated, throw all the ampules of that batch away and get in a fresh stock of cells. Get them from ATCC or ECACC, not (repeat, "not") from a friend/colleague/"good lab" - start fresh with what is as close to the original as possible.

(vi) Grow your routine cell cultures without antibiotics of any description (except obviously if you're selecting for a expression plasmid). If you can't routinely do this, learn how to do it. It isn't hard.

(vii) Consider it a lesson learned and move on.

Good Luck

G

I have got the same issue, and I did try to solve the problem by adding Gentamycine, but it did not work unfortunately .

Truly people - routine use of antibiotics is a hangover from the old days when equipment was not optimal, particularly air-flow. In addition - the old "Pen&Strep" that I grew up with did not last very long, unlike things like Gentamycin, which is much more stable.

Cell culture is a skill like any other - it has to be learned and practiced, you can't just expect to be able to do it. The routine use of antibiotics will mask imperfections in your technique, and while this may protect you from *some* bacterial contamination in short-term culture, only good technique will protect you from fungal/yeast contamination, or keep your long-term cultures clean and healthy.

I like my students (of all levels) to hone their skills and so we never use antibiotics.

One of the original masters of mammalian cell culture is R. Ian Freshney. He was developing cell culture methods when people were still using Chick Embryo Extract. While there are many methodology books related to cell culture techniques, Ian'a is still the one you should read - it's currently in its sixth edition:

"Culture of Animal Cells" by R. Ian Freshney, published by Wiley-Blackwell

Work hard and build your skills!

Good luck

G

I agree with Grant Gallagher. With meticulous technique, even the most valuable cell cultures do not require antibiotics and contamination issues certainly cannot be solved by treating with them.

As i know in cell culture, When a dish of cell become contaminated you should out it immediatly and clean all things and environment that were in related with it.

If your cells are too precious to be destroyed, you may want to try treatement with Normocure from InvivoGen. This is the only product that will kill multi-resistant bacteria. As previous researcher mentioned, treatement is only a temporary option. Cleaning is the only way forward !

I have also used in the past high concentration of G418 for 2-3 weeks; G418 is usually used to get your stable cell line.  I experienced that this treatment it's quite useful; however I want to suggest you to use new cells because, as above said, contaminated cells change their methabolism. 

The only option is to start the culture again. You will waste a lot of time trying to remove contamination and never be sure. Time should be spent on assessing how this happened. Make sure all workers cell culture technique is good and keep culture lab clean/tidy. Simple things/rules can be set up to ensure a clean lab.

Also you run the risk of contaminating other workers cells lines by keeping these cells growing.

bw

Ian

better you should start again with fresh cell line instead of wasting time on removing bacteria from contaminated cell culture. antibiotic tretment will surely decrese the bacterial number but it will not eliminate completely. even a single bacteria left it will grow again very fast.

Hi Xi Feng! May I know what concentration of Gentamicin did you use for de-contamination? Thanks. :)

what about  the water in  CO2  INCUBATOR .? Did you cheek its sterility. if not you should change it with sterile  supply

Rinse the cells with PBS thoroughly and change fresh media with antibiotics. If problem persists better start again with fresh cells.

Although it is an old question, I would like to add my empirical method here:

Contaminated cell line: Capan-1;

The contaminating worms looks like Achromobacter described in the paper: Article Got black swimming dots in your cell culture? Identification...

Unfortunately, there was no backup stock in my lab and Freezing-and-thawing did not work well in my case. So I proceeded with rescue as following:

1. Wash the cultured cells with PBS 3 times;

2. Add the new media with 10x PS (routinely the final concentration is 100 I.U./mL Penicillin and 100 ug/mL Streptomycin. Here the final concentration is 1000 I.U./mL and 1 mg/mL respectively);

3. Repeat step 1&2 once daily.

After 3 days, the worm-like contamination got disappeared. Switch back to the routine culture media.

Good luck to everybody coming here intentionally.