From reading the answers above, I think a lot of what I call "lab voodoo" is going on in tissue culture rooms all over the world. I agree with Antonio that technique is key to preventing contamination.

Reagents: Aliquot for novices to prevent domino effect contamination Give each researcher their own bottle of media.

Flame: If using, do not overflame, flame mouths of flasks and bottles upon opening and before closing. Flame caps before closing unless using filter caps

No flame: if not using flame, do not pour reagents and use an aspirator to remove spent media

Using the laminar flow cabinet: Work well into the cabinet. That grating at the front of the hood is part of the air flow - it is not a clean area. Remind coworkers to avoid walking too fast near the hood when it is in use and avoid making fast movements in and out of the hood. Ensure that everything you need is in the hood before you begin.

DO NOT pass anything over an open container that is meant to be kept sterile. I personally think that this is the biggest source of contamination.

There is nothing wrong with making your own media. I have done so for over 20 years. If you make your own media, you must make the media at least five days before you need it and make a media sterility test flask or dish (5 ml media, 0.5 ml FBS) from your filtered media. Make sure there is no contamination before putting the media into use. I have NEVER had a media test come up contaminated.

I only ever use the UV in my hood after I have been working with infectious material (to protect my coworkers, not the cells). It is completely unnecessary for the purpose of preventing contamination. Spraying alcohol all over the place is not necessary either, but as I tell my techs, if it makes you happy...

Are you getting one sort of contamination, or is it something new every time? Bacteria, yeast, mold? Yeast and mold spores are small and thus easily can cause contamination if there is a lot of movement in and near the hood. Bacteria likely dropped into your open container as you passed your hand over a bottle or some scenario like that. Test all your solutions for sterility and in the meantime, use fresh I like the idea of having someone watch you. I usually watch newbie for a month before cutting them loose.

The lab and the hood should be cleaned and becareful when autoclaving the sample .... For liquid media we usually leave it after autoclaving for one day to make sure that it is contamination free

I don't know as to what kind of culture I am looking at, but given the eukaryotic cell culture I can suggest some possible causes. Do have them into consideration specifically in all types of cultures:

All these are in addition to Noha's answer-

1. Wash your hands with alcohol (90%) upto you elbows, twice.

2. Wear full sleeved coats, such that the coats are kept inside the culture room and worn only after having a good UV bath for atleast 30 mins.

3. Use room UV for 45mins prior culture, specially if you hail from a humid part of the planet.

4. Do not talk inside the culture room. If so, use masks. Your mouth has fungi that can be possible conta source.

5. test whether your media is contaminated by keeping a parallel empty media in the incubator. If it turns negative, then the cells you use are contaminated.

6. Most importantly, use double sterilized tips and parts that come in direct contact with the media.

Hope these will help.

Problems:

1) You froze down contaminated cells (probably easiest to start again though you might be able to fix depending on bug). Test different lots of cells and identify problematic ones.

2) You have contaminated media - incubate some for a couple of days to see if it grows, or the easier fix - sterile filter it last step before use.

3) You have a contaminated incubator - unlikely if copper inside, clean and autoclave the removable metal bits and decon the inside with bleach (10%) then clean with Ethanol (70%).

4) You have a faulty hood - get your hood tested and certified (you can check if there is stuff there by leaving TC media/agarose plates out in the hood for a while then seeing what grows). If the hood is a problem you are likely to end up getting the company engineer in to service it.

4.5) some item of equipment (lab coats are a favorite) is contaminated. Clean everything up, launder lab coats (which should never be used for other experiments) and sterilize things before putting them in the hood (esp. important clean waterbath and spray down bottles prior to putting them in hood).

5) Your sterile technique is flawed - easiest test for this (once you do 1-4) is to have all the lab workers culture some cells without antibiotics. People can then see if they are making mistakes and get pretty immediate feedback.

Hey Abhijit,

You already have received some very valuable suggestions regarding contamination. I believe you might have even put them into practice. However, I still would like to add some, just from my own lab experience in both humid (India) and dry/cleaner (Canada) environments.

1. The most common source of contamination that I found was water (both in the water bath and incubator). Therefore, I suggest you to clean your water bath as often as possible (may be twice in a week). Also, replace water inside your incubator once every week. Use only autoclaved Milli-Q water for the incubator. Add water-treatment liquid (or even copper sulphate) in the water to get rid of contaminants.

2. Make sure that the media bottles are dry and wiped with 70% alcohol before they are transferred from the water bath to the biosafety cabinet.

3. Regarding media, it is always better to buy a prepackaged bottled. However, if you are preparing your own media, filter it using a 0.2micron filter. Keep an aliquot of it (without adding any antibiotics) inside the incubator for at least 72h before you use it.

Best of Luck

I culture primary human cells that are adherent. Make sure that if you are working in culture flasks, that the neck and lids have no liquid in them. If the lids are vented caps, make sure the filter isn't wet, or this could also serve as a breeding ground for contaminants. I feed my cells from two different bottles of media to ensure that if one is contaminated, that I most likely won't lose all my cells due to contamination. I still use two different bottles even after sterile filtering the media AND performing sterility tests. I use glass pipets that have been cleaned and autoclaved, and I only use one pipet per flask. This ensures that the pipet is not transferring any possible contaminants.

In addition to all the good advices, everything that goes in the laminar is sprayed with 70% EtOH. Also for primary cells - we are isolating cells form skin that not the most sterile tissue - usually tissue samples are kept in solution containing penicillium/streptamycin and fugizone.

I know some people clean their hands (up to elbow) with EtOH before opening CO2 incubator.

I wish you best of luck in solving this problem!

I am sorry Shinde, but i have not had this problem in a few years, i believe that is the way one uses the technique that contaminates or not the cell culture. I think i am quite lucky in not having had a cell contamination in 15 years. the only thing i can say is be careful and always aware of what you touch, spray and so on. careful with media preparation and if you need to use antibiotics (which i don't) make sure you get the dilution right and sterile. so i believe that is down to the person that is doing cell culture. one more thing i noticed through the years that the more people are paranoid about sterility the easier becomes to contaminate everything. so take the plunge and get on with cell culture at the best you can and ask people to look at you when you're doing cell culture so they can point out any mistakes you are doing

Thank you everybody!

We have been trying most of things out of those suggested by all of you, but i noticed couple of things that I should look in to. I will see what could further be done with these suggestions.

Thanks again for all those valuable suggestions!

Abhijit

From reading the answers above, I think a lot of what I call "lab voodoo" is going on in tissue culture rooms all over the world. I agree with Antonio that technique is key to preventing contamination.

Reagents: Aliquot for novices to prevent domino effect contamination Give each researcher their own bottle of media.

Flame: If using, do not overflame, flame mouths of flasks and bottles upon opening and before closing. Flame caps before closing unless using filter caps

No flame: if not using flame, do not pour reagents and use an aspirator to remove spent media

Using the laminar flow cabinet: Work well into the cabinet. That grating at the front of the hood is part of the air flow - it is not a clean area. Remind coworkers to avoid walking too fast near the hood when it is in use and avoid making fast movements in and out of the hood. Ensure that everything you need is in the hood before you begin.

DO NOT pass anything over an open container that is meant to be kept sterile. I personally think that this is the biggest source of contamination.

There is nothing wrong with making your own media. I have done so for over 20 years. If you make your own media, you must make the media at least five days before you need it and make a media sterility test flask or dish (5 ml media, 0.5 ml FBS) from your filtered media. Make sure there is no contamination before putting the media into use. I have NEVER had a media test come up contaminated.

I only ever use the UV in my hood after I have been working with infectious material (to protect my coworkers, not the cells). It is completely unnecessary for the purpose of preventing contamination. Spraying alcohol all over the place is not necessary either, but as I tell my techs, if it makes you happy...

Are you getting one sort of contamination, or is it something new every time? Bacteria, yeast, mold? Yeast and mold spores are small and thus easily can cause contamination if there is a lot of movement in and near the hood. Bacteria likely dropped into your open container as you passed your hand over a bottle or some scenario like that. Test all your solutions for sterility and in the meantime, use fresh I like the idea of having someone watch you. I usually watch newbie for a month before cutting them loose.

Thank you so much Elizabeth for your valuable inputs!

Abhijit

You are most welcome!

Good luck!

For contaminants you are talking mostly about bacteria, fungi and mycoplasma. What contamination you have will dictate what antibiotics you need. While proper technique is important, look for other sources. A major one many people overlook is the presence of cardboard boxes (for pipettes, flasks etc) these are a major feeding ground for bug. So make sure to remove all cardboard from your culture room. Use appropriate sterile technique. There are lots of youtube videos on this.

Thank you so much Neil for your inputs.

Abhijit

In my experience (15 years), multiple users of a facility is the biggest challenge. Establish firm protocols for all handling steps, train all users for weeks to months, if youre new in the field, get trained by a competent person. And when infection hits you, do not try to "save" your cultures, if one culture tray is infected the chance of its neighbour being infected is quite high. Get rid of cells and everything which potentially can be contaminated. Log all infection events, and ask all users to do the same. As suggested above, regular change of water in waterbaths, regular cleaning of the facility, regular cleaning of the LAF-bench, regular decontamination of incubators and general monitoring of the facility with agar plating tests certainly helps..

It is depending on the handling and manipulation first of all the lab. Location and availability of sterility and access on the lab. The most difficult and most important the contamination with mycoplasma be very careful

May be time to validate laminar air flow unit , or validate autoclave to circumvent ur problem .

what about the U.V lamp and the air curtain in the entrance of the lab and the disposable vessels.

The most important inital step is to identify the key organism(s) causing the contamination (as different microbes require different methods of decontamination!). The comments from colleagues re: multi-use facility are valid - ensure all personnel from Professors to students are familiar with and compliant with the standard operating procedures and H&S policies in the laboratory. Also, check laminar flow hoods, autoclaves are working fully. Training and re-training are also recommended. Then, if these all fail, zap the lab with a decontamination protocol, usually involved formaldehyde and shutting the lab down for 3-5 days (but works!).

For the most part, uv lamps are pretty useless. They have power only enough to decontaminate a few cm from the lamp, so never rely on the lamp for sterility. Also make sure than any alcohol you use is at 70% as higher than this leads to dehydrated bacteria which can recover. 10% bleach is probably the best, left for 10 min before wiping up with alcohol. Unfortunately the biggest source of contaminants are people. Make sure everyone is using appropriate techniques. They are your cells, so you need to approach people who are not using proper technique.

Just to add that no "cardboard box" into your cell culture room, this brings a lot of contaminations. Change cleaning solutions (spray on benches and hoods) every 6 months to kiill all germs.

Avoid culturing animal and human cells within the same incubator.

Do not cultivate viruses and cells within the same incubator besinde infection experiments or virus production. Te rest can be provided by training given by institutions or private labs.

I already answered someone else on the same topic. Doing primary cell culture on a daily basis, I would suggest to make sure you keep everything as sterile as possible, let someone look you doing cell culture so can point out which mistakes you are making, make sure that once you have the gloves you don't touch anything else apart from sterile things once in the hood. remember that some primary cell need anitmicotic (cartilage extraction) because they are more prone to infections. then need to keep in mind all the basics, no cardboard boxes, the tissue culture as free of junk as possible, clean hood/incubators, correct aliquots of sterile antibiotics etc. do not linger too much on those things because the more paranoid you are the easier is to have contamination, so get on and do it then correct a bit at the time any possible mistake

Doing tissue culture the last 18 years I had to deal with different scenarios in TC.

some suggestions.

1) Clean, dust free tissue culture room (dust= fillamentous fungi). TRy to have as little clutter as possible. Wear designated TC lab coats and only for that room. Contamination does not just appear out of nowewhere, it is people that usually bring it in on their skin, clothes etc.

2) Sterility in the technique everyone is using. Maybe will help to identify a competent person to train everyone else.

3) antibiotics in the medium (most commonly used penicilin/streptomycin and fungizon, should give you a good cocktail against bacteria and fungi), however, make sure they do not interfere with your assays.

4) set up a weekly rota for all hoods and incubators to be cleaned.

5) use copper sulfate (works as anti-fungal) in the water (if using) of your humidified incubator.

6) if there is contamination: throw away the contaminated flask or plate. Check the rest stuff in the same incubator. if more contaminated bin directly.

wipe everything else in there (flasks and surfaces with bleach and ethanol. Put fresh clean water + copper sulfate in the tray (if there is one)

7) check your medium (just put some in a flask and incubate for 1-2 days, if anything grows in it bin the stock bottle.

I hope that helps

Hi Abhijit,

I used (Falcon, 35-3002) for infant lung fibroblastscell cell culture and (Falcon, 35-1001)for infant lung type II cells. because fibroblasts need more time than type II cells, it most likely to be contaminated. anyway, I used Penicillin G and streptomycin sulphate to final concentrations of 0.0715 g.L-1 and 0.1525 g.L-1, respectively.

I afoided chloramphinicol because its effect on cell shrinking. It works and NO contamination occured.

wish all the best.

George

All of the above are great suggestions. Have been doing this for over 16 years and have found that one of the items typically overlooked is to check and change out the filter in your Pipet Aid on a periodic basis. The filters typically range in size from 0.2-0.45 um pore sizes and are either sterile or non-sterile, depending on your needs.

This might sound foolish, but if you warm your media in a water bath before feeding cells be sure to wipe media bottles down thoroughly with 70% ethanol before bringing the bottles into the laminar flow hood. Make sure that you wipe the bottles down from top to bottom. Once the wipe has touched any part of the media bottle that came in contact with the water in the water bath, consider it contaminated and get a new wiper. Water baths are notoriously good breeding grounds for all kinds of microbes including bacteria, yeast, fungi and molds.

When starting the air to circulate (when the hood is turned on in the morning), allow the air in the hood to circulate for 5 minutes before use. This will allow any contaminants to hopefully enter the laminar flow and not end up in your media or dishes.

Hi Abhijit

I think,first of all. you should check strility of your materials such as media,FBS,PBS,ETC,and your equipments such as pipets,and Tips ,After that you should try to work in very,very clean conditions specially with 30 cm distance of laminar hood edge.another recommend is good cleaning and use UV light of your hood before starting.I hope you can find and remove infection source

I think you have to check the hepa filter it may be expired or damaged some how

Penelope does not mention her recommended concentration of copper sulfate, but I would not recommend its use. Careful technique will avoid necessitating the use of toxic compounds in the water tray, that one cannot legally put down the drain, at least where I live. That means you would need to package the water from the incubator as hazardous waste and pay to dispose of it.  Not an environmentally sound practice, and absolutely not necessary if careful technique is employed. If you feel the need for disinfectant in the water tray, use something a bit more friendly, at the recommended concentration to avoid making hazardous waste unnecessarily and avoid personal exposure.

What precautions must one take to avoid contamination

in plant tissue culture