About what kind of contamination do you talk? Mycoplasma, bacteria, fungi? Are you doing primary cell culture or use "standard" cell lines?

Most of the causes of contamination in cell culture come from handling. You have to be really careful when you work under the hood, and prepare all your reagents...moreover if you work regularly without antibiotics in the medium ( which I highly recommend).

What kind of contamination are you talking about? Depending on that you should proceed in different ways. Bacteria contamination can be removed ( depending of course of the kind of bacteria and if they are resistant or not to antibiotics) treating the cells, with antibiotics ( the most common for cell culture is a mixture of penicillin/streptomycin) for aprox 5-7 days, changing medium every day with two or three washes with PBS. Also making a huge dilution of the cells after doing the treatment helps sometimes (sometimes there are few bacteria living after treatment and if you dilute the cells (for example to a 96 well plate) you have the chance of having wells bacteria-free.

For funghi there are also anti-funghi compounds and for mycoplasma there are kits for decontamination.

Anyway if you have enough stock of non contaminated cells I would throw away the contaminate cells and defreeze a new crio-stock.

The source of contamination might be the improper handling of cells in during splitting, seeding, changing mediums and also from the frozen stock, mistakes during all the processes of cell culture. You have to be careful at each and every step. Always wear gloves and use 70% aclohol spray over the your hand each time you take out from the hood during cell handling. Use at least 20 mins of UV exposure in the hood, cleaning of al hood surface with Virkon and then wipe all the surface with 70% aclohol before starting your work. Wipe all the flasks and medium bottles with alcohol before putting into the hood. The source of contamination might be incubator itself. Clean thorooughly all the incubator with antifungal and antibactericidal solution or follow the cleaning protocol of incubator. Always pay attention during the changing medium not touch anyhting with pipet superficially... The mouth must be above the glass pan of the hood to avoid any direct breathing before hood if the glass pan is not sufficient low, use the mask... Put the medium in a flask alone just to chek if there is any problem with medium. Sometimes medium is contaminated... Never leave any contaminated flask inside the incubator it will spoil all other flasks... Just be carefull. You need to chack whther you might do mistake and accordingly, take care of that... All the best... :-)

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

I agree with Kadasia that the most common cause of contamination is mycoplasmas though bacterial infections may also be a major cause. Bacterial causes are generally observed in cases where the cells have not been handled properly and I personally have observed that bacterial contamination generally develops in later stages of culture, usually accompanied by smelly feeling in culture and change of colour in upper surface of medium. If there is development of mucos/slime it is probably an amoeba. The control of all types of contaminations start from hygiene of hands and travel through all steps of culture. You may find many references on how to sanitize the culture table, lab, your hands and so on. Of course, dont forget to control your breathing while culturing and proper sterilization of the culture medium before culture.

More important is, can you save your culture once an infection has developed.....YES.... you may do it. In most of the cases simply isolation of the cultured cells by lifting it along with a small portion of culture medium using a sterilized spatula and then treating it with an antibiotic/fungicide, followed by culturing in fresh sterilized medium recovers the culture successfully.

What if bacterial contamination comes right after transfection and not in control group? Also we checked all reagents and plasmids, tubes and tips. But they are there after each transfection.

You should clean the working area with 70% ethanol and turn on UV in the hood for 20 min , sterilize equipments , avoid leaving bottles opened for long time (media, solutions .......etc) close it well Immediately after use , avoid using water bath for thawing reagents because it is a source of contamination , use filtered tips not the usual tips and micrometer filters in the pipetting gun

Use( fungizone 50 micron +Penicillin-Streptomycin 100 micron) for each 10 ml of complete media , this will give you a good help with fungi and bacteria caused contamination

Salma