I had prepared my media RPMI1640 with FBS and Pen-Strep in the most appropriate way and made sure I don't do anything that could cause contamination. But when I had kept one plate with the media for sterility check there were cells in them and a bit of contamination. Is there any way to solve this?
I suggest doing the sterility check separately on the medium, FBS and even the pen-strep to make sure your stock solutions are OK. If the contamination occurs while making the complete medium, use sterile filtration immediately after adding the FBS and P-S.. Also make sure all measuring instruments(pipettes, etc) are sterile.
Dear Researcher
May be you have fungal contamination. You can add a little antifungal drug like Amphotericin B
It should be added aseptically to the cell culture medium at concentrations up to 10ml per liter.
Hello Hanni Grace F
I would not know how well you carried out the cell culture media preparation while taking the necessary precautions. Here are some measures which you could follow while preparing cell culture media or working with mammalian cells in the cell culture laboratory.
1. Culture media prepared from powders must be sterilized by filtration, although liquid media are often supplied sterile and at ready-to-use concentration, sterile filtration is recommended, particularly if sera or other supplements are to be added before use. Following sterilization, aseptic techniques should be used when handling culture media to prevent cross-contamination or the growth of unwanted microorganisms.
2. Turn on the hood for at least 15 minutes before you start any tissue culture work and make sure that clean air is flowing in. The vent for the in-flowing air is normally visible and you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half an hour can help keep your hood contamination free, but you should keep in mind that UV light only kills microorganisms that it strikes directly. Wipe the hood with a disinfectant (use 70% ethanol) before and after your work.
3. While it’s likely that you could be the main source of contamination for your experiments. So, added precautions are necessary to keep away contamination. Wipe down everything that’ll be brought under the hood with 70% ethanol, including your glove-covered hands! You should additionally remove all jewellery as well as the watch you’re wearing before you start. Also, avoid unnecessary talking because talking generates microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the hood is another great way to prevent contamination. With proper planning, you’ll know exactly what materials you’ll need to bring into the hood and can wipe them all down before you start. This will also help you avoid moving things in and out of the hood while work is in progress.
4. Laminar hoods should not be used as storage cabinets. Clutter not only makes it difficult to clean your workspace properly but can also disrupt the laminar air flow around your work area. Make sure to only bring required materials into the hood. Also, keep all liquid containers closed when they aren’t in immediate use. Avoid sticking pipettes into solution bottles directly, rather decant the solutions into disposable sterile tubes first. Not only does this make things more manageable, but it also keeps stock solutions from getting contaminated.
If you follow these measures, hopefully you should not have any contamination problem while working in cell culture laboratory in the future.
Best.
Separate contamination check needs to be done post sterilizing the culture room with separate media, stocks, FBS and others culture biologics. Addition of Amphotericin B in a bit higher ratios with proper sterilized procedures under hood may prevent the contamination further.
Hi,
I suggest that it is better to prepare a new media bottle (RPMI1640+FBS+Antibiotics) and also check the contamination in your stock solutions. I also think it is best to check for the absence of mycoplasma using a commercially available kit (e.g. MycoAlert PLUS Mycoplasma Detection kit).
Best Regards,
Hi Hanni,
I am not sure about your sterile filtration condition. Please do the filtration in a hood, add antifungal drug and use either 0.25 micron or less filter. You need a vacuum pump to do the filtration and check its connection.
Best,
P. Ramaraj
Hi,
For avoiding cells contamination there are lots of parameters you need to keep in mind, while for cell culture medium, after performing all basic necessary preparation steps I would suggest filtering your medium through a 0.42nm filter before using it for cell culturing.
Best
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