Hello! My colleague insisting, that first two passages after unfreezing of new cryovial must be without any antibiotics (gentamicin, pen/strep, etc...).
For all of my work I never got bothered by this question - with of without any ab's my cultures grew same.
Is there any reason to unfreeze cells without antibiotics?
I was always taught not to have antibiotics with my cells, whether they are being defrosted or just passaging. The reason being if you have contamination without antibiotic, it is easier to get rid of the bacteria by adding antibiotic, if you have contamination with antibiotic already added, the bacteria are already resistent and more difficult to get rid of. So I would agree with your colleague, try not to use antibiotics.
I was always taught not to have antibiotics with my cells, whether they are being defrosted or just passaging. The reason being if you have contamination without antibiotic, it is easier to get rid of the bacteria by adding antibiotic, if you have contamination with antibiotic already added, the bacteria are already resistent and more difficult to get rid of. So I would agree with your colleague, try not to use antibiotics.
In some labs, researchers assess if culture is mycoplasma/contamination free after every thawing of cells. Before performing such test, you have to grow cells in antibiotic-free medium.
Actually I prefer to culture cells without antibiotics for whole culturing time. Use of antibiotics is not always a guarantee for contamination-free culture. Antibiotics can only suppresses bacterial growth which can lead to hidden contamination. If you use appropriate aseptic technique, you shouldn't have problems with contaminations.
I use antibiotics in all my cell line cultures and primary cultures. we check for mycoplasma every month. I agree that the gold standard might be to culutre without antibiotics, and you will create antibiotic resistant bacteria but its not always possible experiments without them. Contamination can cause large set backs in experiments. When you come to publish, if you are concerned about the effect of antibiotics on your data you could always repeat key experiments. All the largest journals accept use on antibiotics.
Hi, I would say that the mist important reason is that most of the antibiotics are only bacteriostatic. I.e. they dont kill the contamination, but only slow it down. So, you can get "hidden" contamination, you will be using contaminated cells over a long time, you can even again store contaminated cell line. And after time, you can end up with all stocks contaminated. Moreover, you can select atb-resistant bacteria in your culture by passaging them. So if you need to use atb, it is better practice to do treatment with atb for few passages, then transfer to atb-free media, and after few passages you can start using the cells.
The effects of antibiotics on cell metabolism is also a big question.
I used atb-free cultivations all the time, with proper technique it is not necessarry, and if the contamination happens, you will most likely see it clearly following day and just dispose the contaminated flask.
Btw the contamination is just a matter of time when it happens, so it is better to see it when it came
Thank you all! So, it seems that this is just another step to check if cells were contaminated during freezing or pre-freezing passages.
Yes, i do agree with the all suggestion given to you. But if you use antibiotic during unfreezing the cells to grow in culture they will be no harm in it.
IF the your culture is already contaminated by mycoplasm infection. It will still come in that culture also.
I support Steve Hawkins' words, one by one. Antibiotics are expensive and toxic to cells. Plus, if you have antibiotics you're more likely to mask minor contaminations that you definitely don't want to have during your experiments. I always maintained cell lines without antibiotics and never got any problem. You just need to extreme basic aseptic habits during handling. Additional upside: you'll feel more confident and proud of your hands if, after long time w/o AB, you got nothing contaminated. No merit at all if AB are present :-)
Yes, I do agree with the word of Steve Hawkin's and Angel L. Alvarez's.
Handling the cell line under the aseptic conditions is the important factor.
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