How to Resolve Possible Contaminant in RAW 264.7 Cell Culture?

Alvianisa Edriana Kumala @Alvianisa-Kumala

21 January 2025 2 136 Report

Hello, I am an undergraduate student currently conducting a thesis that involves RAW 264.7 macrophage cells. The composition of the complete medium I use includes DMEM, FBS, penicillin-streptomycin, and amphotericin B. However, I am facing difficulties as the cells cannot survive for more than one week due to contamination (black spots in Figure or gb 1).

I have tried filtering the medium using a 0.22 µm CA filter. I also have decontaminated the incubator, fumigated the laboratory room, replaced the incubator filter, and evaluated aseptic techniques. Despite these efforts, contamination persists when I test the medium only-no cells (Figures or gb 2 and gb 3).

Does anyone know about the possible contaminant and how to resolve this issue? Thank you.

Raghad Mouhamad

To address contamination in RAW 264.7 cell culture:

Test for Contaminants:Perform mycoplasma testing (PCR or fluorescence). If positive, use removal agents like Plasmocin™.

Replace Reagents:Discard current batches of DMEM, FBS, penicillin-streptomycin, and Amphotericin B, and replace them with fresh ones from reliable suppliers.

Sterilize Water:Use ultrapure water (e.g., Milli-Q) for media preparation.

Enhance Sterilization:Use a 0.22 µm PES filter, autoclave all equipment, disinfect the incubator with 70% ethanol, and run a UV sterilization cycle if available.

Adjust Antibiotics:Add gentamicin (50 µg/mL) temporarily and increase Amphotericin B to 1.25 µg/mL if fungal contamination is suspected.

Aseptic Techniques:Ensure strict adherence to sterile techniques during media preparation and cell handling.

If contamination persists, isolate the environment and reassess all procedures.

Lucian Miles

Hi Alvianisa！

Was the filtration performed on the complete medium, or was the basal medium filtered first, followed by the addition of serum?

In this case, the issue may be with the serum. Before use, did you notice any abnormalities in the serum, such as flocculent precipitates?

Generally, serum precipitates can be categorized into several types, which typically do not affect cell culture. However, given the current situation, it is suspected that the serum might be problematic. It is recommended to centrifuge the serum at 400g, collect the supernatant, and then prepare the culture medium accordingly.

After filtering the complete medium, perform an empty incubation to check if the contamination persists. If contamination is still present, mycoplasma testing should be conducted. If the test confirms contamination, a new batch of serum may need to be purchased.

Fibrin: The most common large precipitate, up to 1-2 mm, visible to the naked eye. Some sera contain fibrinogen, a soluble precursor that can form flocculent fibrin. It remains dissolved during collection and rapid processing but may precipitate after filtration and aliquoting.

Calcium phosphate: This type of precipitate causes turbidity in the serum and increases when incubated at 37°C. Under the microscope, these appear as small black dots that move due to Brownian motion, which can sometimes be mistaken for microbial contamination.

Cholesterol, fatty acid esters, and proteins: These are also common causes of precipitates in serum.

Hope this helpful, feel free to reach out if u need more details!

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