Hi all,
is there anyone who experienced a persistent and slow-growing bacterial contamination? It has been 4 months now that I have been struggling with what I think is a bacterial infection (picture attached - 20x bright-field microscope).
I am using DMEM low glucose to culture my cells added with 2% FBS and 1% P/S, with or without 33 mM glucose. I continue to find bacterial contamination after 5 or 7 days from the beginning of my experiments on 24wp. However, I have never seen contamination in cells cultured on flasks.
I have already changed my media bottles three times, filtered all solutions I used, changed falcon tubes, clean the hood, change the incubator, and used different cell batches. Additionally, I also tested my media many times leaving samples in a plate, finding only a couple of random contaminations (even with media I didn't use for experiments). Overall, the media appeared to be clean all the time. However, I still have the issue.
The contamination appeared as a cloudy thing in the well, without colour changes of the media.
Does anyone have any clue about what this contamination could be and what I can do to get rid of it?
Thank you so much!
Gaia
You could add antibiotics. Bacteria tend to grow faster at different pH, which may be the reason why they show slow growth. You could monitor pH change by looking at the color of DMEM. You may want to look for recipes of antibiotics for your culture system.
Hello Gaia Lugano
Preventing contamination is always better. Special care must be taken when culturing cells in 24-well plate because unlike T-25/T-75 flask, plate or petri dish is considered as an open system. So before commencing work with 24-well plate make sure the biosafety cabinet is well cleaned and UV irradiated for 15-30 minutes. Ensure that you are working well inside the laminar hood. Generally, the culture medium in 24-well plate should not fill the wells more than halfway
(1ml per well should suffice). Flame the lid of the plate before you place it on the plate. The purpose of flaming the lid is to warm the air above the plate. The warm rising air prevents micro-organisms on suspended dust particles from entering the plate.
Ensure that the design of the lid is oriented correctly when placed on top of the plate. The design of the plates should be such that it includes spaces between wells to avoid cross-contamination. Needless to mention that all good quality
24-well plates are gamma ray sterilized to prevent contamination and are certified pyrogen-free. Try to minimize the exposure of the plate to outside air, especially when you have to observe cells under the microscope.
So, in addition to the precautionary measures that you have mentioned, you need to take extra care when it comes to culturing cells in plates or dish to avoid contamination.
I hope this information is helpful.
Best.
In the picture, it seems like mycoplasma contamination, and for mycoplasma common P/S doesn't work. So try adding 1:1000 anti-mycoplasma to your media to cop with that issue.
Good luck.
Thank you everyone for your help! I am trying with fungizone now because I have been told it can possibly be a fungal infection. Thank you again!
Kindly read this advert
https://timesofindia.indiatimes.com/india/prevention-of-fungal-infections-key-area-of-focus-in-treating-covid-patients-aiims-director/articleshow/82660926.cms