Hello,
I always get great information from researchgate.
It seems that i sometimes forget grateful responses to those who replied my questions and I am sorry for that.
Today i want to ask optimal ratio of antibiotics in cell cultrue media.
From previous answers, I learned that antibiotics is only for
prevention of contamination. But i know that there is optimal ratio of
antibiotics added. If I mix medium with antibiotics and same kind of medium
without antibiotics, the ratio of antibiotics would reduce and i wonder that
if the ratio of antibiotics is not at the optimal ratio, i wonder it would affect
results of my research. In my opinion, because antibiotics is just for
prevention of contamination, such mix and change of ratio of antibiotics
does not matter, but considering there is 'optimal ratio' of antibiotics,
I wonder whether there would affect my research results
Thank you
I would use "concentration" instead of "ratio". Try different concentrations of the antibiotic on your bacteria and see what happens. Maybe you will find some resistant mutants. Maybe you will not see the most efficient killing of the bacteria using the highest concentration of the antibiotic . . .
Hi,
It depends on type of antybiotic as well as on type of cells you are willing to treat.
You have to check both of them, usually commercial AB have very good description you can start with, it might be useful to check your cell line as well (e.g. ATCC or some research articles for conditions).
To heal contaminated lines (always last resort), keep them sparse and treat for 2 weeks, there are commercial mixes for that purpose or set your own as described above.
Regards,
Hi,
To make sure that antibiotics does not affect your results, you have to select a readout. Viability and microscopic observation should be fine. You can use a dye to discriminate between live and dead cells (for example, Trypan Blue). This assay will give you information about the toxicity of the antibiotics. Check datasheets and bibliography to have a reference concentration and make a curve with this point right in the middle.
Of course , you have to be sure that the selected concentration also prevents contamination.
Good luck!
You do often see effects on assay results from antibiotics in the cell culture media - I would say that you don't need antibiotics at all if you are just growing cell lines - your sterile technique should be good enough to not get contamination. Adding antibiotics just masks any contamination, encourages antibiotic resistance and allows the contaminant to spread further before being detected. Adding antibiotics to select for cells expressing your gene of interest should be the only time that they are required for cell line growth. Of course if you are growing primary cultures especially from post natal or adult tissue then antibiotics are very helpful to cut down on contamination but I would still try it without to make sure you really need them. You should always use the antibiotics at the concentration recommended for that particular one.
Dear Vee Mitchell, thank you for your answer.
I am quite impressed with your knowledge. I want to ask additional question to you, if you don't mind.
So you mentioned that the recommended concentration of antibiotics is required
at all times. Then, what effects(Effects on research results)
happen if i add medium without antibiotics
so the recommended concentration is diluted to lower concentration?
Thank you again.
Dear Martin Klavana, thank you for your help and advice.
I reminded about the choice of scientific word from your reply.
I appreciate again.
Dear Martin Somoza,
Thank you for your helpful and kind response.
I learned a lot from your description.
I appreciate again.
Dear Wojciech Witarski,
I am grateful for your great support by answering my question.
So you mentioned that "It depends on type of antybiotic as well as on type of cells you are willing to treat."
I work with HUVECs.
what effects(Effects on research results)
happen if i add medium without antibiotics
so the recommended concentration is diluted to lower concentration?
( I know such mix is definitely not the optimal)
I want to ask additional question if it does not matter with you.
Thank you again.
Dear sir:
In your question you mentioned "affect results of my research", do you mean it will affect the growth of your cells or interact with other added materials ?
I usually use 1% antibiotic and antimycotic solution to prevent bacterial and fungal contamination. This solution contains 10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Fungizone (amphotericin B)...You need to check by using different antibiiotic concentration on the same media and cell no.. the check the absorbance using an ELISA reader.
FOR CELL CULTURE
1. Gentamycin (50 mg/ml) - use 1 ml/1000 ml.
Flow's Penicillin-Streptomycin (5,000 I.U./ml
and 5,000 mcg/ml).
USE:
Add 10 ml per 500 ml of medium.
Final concentration: 100 I.U. Penicillin/ml.
100 mcg Streptomycin/ml.
2. Fungizone (Gibco, 250 mcg/ml).
USE:
Add 5 ml per 500 ml of medium.
Final concentration: 2.5 mcg/ml.
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