Hello everyone
I have been working with the Caco-2 strain for a few months, but unfortunately, the cells were already contaminated. I already tried to decontaminate with penicillin/streptomycin 10 times concentrated, for three passes, but it didn't work, I changed the antibiotic, so I tried Normocin (Invivo gen), for two passes, but I still observe bacteria.
During cultivation and medium changes, I wash thoroughly with PBS culture, and the plate background is free of bacteria. But when I trypsinize, bacteria appear that “apparently” before trypsinization were not present or not in the same microbial load.
The cells have a compatible morphology, there is no turbidity in the typical medium, due to bacterial contamination, and the growth rate of the cells is reasonable.
When they reach 60% of confluence, vacuoles appear.
I don't know what else to try or what antibiotic to use. Thank you in advance for any help.
Hello Luciana C Teixeira
You need to discard contaminated cultures immediately. If the contamination is detected early, it is possible to eradicate it with antibiotics. However, bacterial contamination is not easily treatable once the bacteria begin log phase of growth. Bacteria often produce toxins that disrupt cell function and ultimately destroy cell cultures. The cells become unfit to be used in any type of cell-based assays and may give you misleading results.
If you feel that the contamination is not severe and it may be possible to get rid of it, then the most commonly used antibiotics in cell culture that may help are penicillin/streptomycin (pen/strep), gentamicin, kanamycin, and ampicillin. All four are effective against both gram-positive and gram-negative bacteria.
Normocin is a good choice. It is a broad-spectrum antibacterial. Normocin contains three compounds. Two of these compounds act on Gram+ve bacteria, Gram-ve bacteria, and mycoplasmas The third compound eradicates yeasts and fungi by disrupting ionic exchange through the cell membrane.
Have you tried Normocure? Simply add to contaminated cell cultures at the recommended concentration (100 μg/ml) for 2 weeks. After 3 passages, the bacterial contamination is totally eliminated. You may add 10 μl of Normocure (100 μg/ml) in a 25 cm^2 flask containing 5 ml of culture medium. Normocure is also active against multidrug-resistant bacteria.
You may refer to the link below for more details on Normocure.
https://www.tamar.co.il/broad-spectrum-preventing-treatment/#:~:text=Normocin%E2%84%A2%20%E2%80%93%20Antimicrobial%20Reagent&text=Normocin%E2%84%A2%20is%20an%20innovative,mycoplasma%2C%20bacterial%20and%20fungal%20contaminations.you
Do not overtreat the cells with antibiotics as it may have a toxic effect on the cells.
Are you sure the culture is contaminated with bacteria? You may check by streaking a few microlitre of the so-called contaminated culture media on agar plate and incubate the plate overnight at 37 degree C. The next day you will notice bacterial colonies if there is bacterial contamination. Since you have not observed any turbidity in the culture media, the reason for the cells to be stressed may not be contamination. It may be due to some other reason. You need to be for sure that it is contamination.
Best.
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