Worm like contamination (see photo) - serious problems (Jun/28/2007 )
Stardust
Edit: I used gentamycin which is tested for cell culture cells so there shouldn't be any problems about the purity
Dear Starust,
Titration of the dose of any antibiotic is a good idea in theory. However they all will affect the cells in question, along with the bacterial toxins released. Gentamycin, a aminoglycoside antibiotic, is well known to be cytotoxic and can cause :
< in cellular glutathione levels
> concentration of reactive oxygen species
< in cellular proliferation
Induction of morphological changes
Induction of apoptosis
CELL DEATH.
You must be very careful with antibiotic usage. It is always best to throw the cells away and start again.
Stardust
Edit: I used gentamycin which is tested for cell culture cells so there shouldn't be any problems about the purity
Dear Starust,
Titration of the dose of any antibiotic is a good idea in theory. However they all will affect the cells in question, along with the bacterial toxins released. Gentamycin, a aminoglycoside antibiotic, is well known to be cytotoxic and can cause :
< in cellular glutathione levels
> concentration of reactive oxygen species
< in cellular proliferation
Induction of morphological changes
Induction of apoptosis
CELL DEATH.
You must be very careful with antibiotic usage. It is always best to throw the cells away and start again.
Thanks Stardust for sharing your experiences with us. I really appreciate your valuable comment.
Sincere thanks to Rhombus for his worthy responses.
Warmest regards
Thapa
Could be eukaryotic (i.e. yeast or fungal) contamination, which is why it's resistant to pen-strep. You can add fungicidal reagents to your culture, but use them cautiously, as they can also be very unhealthy for your cell line...
Ginger
did you use antibiotics? it is the only way to get rid off bacteria...
yes we use 1% penicillin/streptomycin.
Hi,
Had that problem in my lab before. Pin-pointed the source of contamination.... waterbath.
We've taken quite a few precautions in the lab, namely...
1. Used filtered tips for pipetting
2. Used disposable sterile serological pipettes for maintaining cells
3. Used a large beaker and wash with detergent and change the water in the beaker daily before using it to thaw and temperature equilibrate complete medium, solution, FBS/FCS, pen/strep etc.
4. Seal all Falcon tubes containing medium/solution with parafilm before you immerse them into preheated water inside aforementioned beaker in a waterbath.
5. Used 70% ethanol non-sparingly.
6. Never place bottle of DMEM (500 ml) into waterbath to equilibrate temperature to 37C when preparing complete medium, especially when you can't finish using it on the day itself. Preserve the low temperature for the said medium. I'd prefer to prepare a complete medium first (e.g. DMEM + FBS + Pen-Strep) in aliquots of 50 ml Falcon tubes, then place tube(s) containing full medium into the waterbath to equilibrate the temp before use.
Bests
Had that problem in my lab before. Pin-pointed the source of contamination.... waterbath.
We've taken quite a few precautions in the lab, namely...
1. Used filtered tips for pipetting
2. Used disposable sterile serological pipettes for maintaining cells
3. Used a large beaker and wash with detergent and change the water in the beaker daily before using it to thaw and temperature equilibrate complete medium, solution, FBS/FCS, pen/strep etc.
4. Seal all Falcon tubes containing medium/solution with parafilm before you immerse them into preheated water inside aforementioned beaker in a waterbath.
5. Used 70% ethanol non-sparingly.
6. Never place bottle of DMEM (500 ml) into waterbath to equilibrate temperature to 37C when preparing complete medium, especially when you can't finish using it on the day itself. Preserve the low temperature for the said medium. I'd prefer to prepare a complete medium first (e.g. DMEM + FBS + Pen-Strep) in aliquots of 50 ml Falcon tubes, then place tube(s) containing full medium into the waterbath to equilibrate the temp before use.
Bests
Thanx a ton----I love MSGs,
I really love your details regarding aseptic measures of possible contaminations. It gonna really helpful for me for the days to come.
And Ginger spice, your suspect is not bad. But here, we concluded as a bacterial contamination possibly E. coli (yet we didnt culture and didnt perform other biochemical and serological tests).
Interesting thing in our case is that the outbreak of contamination's source was TomR cells, which was actually brought from another university. So everything and almost all cells was contaminated. We discarded almost every media and solutions plus cells, that were used for culture. Next we decontaminated out incubator, and we are going to change hepafilter very soon. Then only we will start from new lyophilized stock. I will be happy to share with you what will happen next.
LOVE
Thapa
Dear Rhombus,
I'm well aware of the fact that antibiotics in general are harmful for the cells. This is the reason why i normally culture everything completely without Pen/Strep (primary cells after the first week) or any other antibiotics/antimycotics. My point is, that if you have an irreplacable cell line (like I said, I'm working a lot with primary cultures) sometimes you can clean it with gentamycin instead of throwing it away. Routinely I would put them in the bin if they are easy to replace but not if this is not possible (like patient samples). Concerning Fungizone or similar (amphotericin): I have never managed to destroy yeast/fungus after it strated growing in a culture. I always had to throw them away. But it seems to prevent the growth if added to medium right from the point of primary cell isolation. If my cultures are clean after the first week I omit everything (Pen/Strep/Gentamycin/Amphotericin) from the medium not to harm the cells.
Stradust
I'm well aware of the fact that antibiotics in general are harmful for the cells. This is the reason why i normally culture everything completely without Pen/Strep (primary cells after the first week) or any other antibiotics/antimycotics. My point is, that if you have an irreplacable cell line (like I said, I'm working a lot with primary cultures) sometimes you can clean it with gentamycin instead of throwing it away. Routinely I would put them in the bin if they are easy to replace but not if this is not possible (like patient samples). Concerning Fungizone or similar (amphotericin): I have never managed to destroy yeast/fungus after it strated growing in a culture. I always had to throw them away. But it seems to prevent the growth if added to medium right from the point of primary cell isolation. If my cultures are clean after the first week I omit everything (Pen/Strep/Gentamycin/Amphotericin) from the medium not to harm the cells.
Stradust
Dear Stardust,
We all do things slightly differently. When I was growing primary Aortic endothelial cells (porcine) which were very precious at the time, I quarantined the cells rather than adding any antibiotics/antifungals i.e. cells from one aorta went into 1 flask, 8 aorta's/day.....5 days a week. The cells were grown for 4/5 days as a screen for infection, then pooled and used as one
In the very early days I tried using Fungizone as a profilactive agent. Fungus contamination from the abattoir was a major problem. It turned out that the fungizone completely inhibited the process I was interested in (eNOS release of Nitric oxide)
Reference S. Moncada Nature Vol 327 pages 524-526 June 1987.