Hi Daniel Pannwitz

The video is not very clear, but it does look like bacterial contamination. Since it appears consistently after a couple of weeks, it may be coming from the original source rather than being introduced each time during handling.

One way to troubleshoot is to systematically check where the contamination might be introduced. Possible sources include the cell stock you thawed them from, media or supplements, the biosafety cabinet, the incubator, or even the water/bead bath.

To troubleshoot, I’d recommend:

• Plating your media and supplements on LB agar and incubating overnight to see if anything grows.
• Thawing a fresh vial of your cells and monitoring closely.
• Deep-cleaning and disinfecting the biosafety cabinet, incubator, and water/bead bath.
• If the contamination is not present in the media or supplements, the issue may be with the batch of cells themselves.

If it’s truly inside the cell stock, then it will be very hard to rescue them. In that case, you may want to obtain a fresh vial from a reliable frozen stock or request cells from another lab. I know some ambitious lab mates that have tried to use stronger antibiotics with several passaging rounds and gotten successful but it might just be easier to do the former.

All the best!

The video shows non-cellular particles and granular debris dispersed across the field, with some blue-purple granules and irregular structures that do not resemble healthy SCP-1 cells.

The bright circular formations with internal granules and a yellow nucleus-like structure suggest foreign cell-like entities, possibly protozoa, yeast, or mycoplasma-infected cells.

Based on the growth pattern and resistance to standard penicillin/streptomycin (P/S), the most probable culprit for your SCP-1 cell contamination is mycoplasma.

• Growing in cells: Mycoplasmas are extremely small bacteria (0.15–0.3 μm) that lack a cell wall, allowing them to pass through standard 0.22 μm filters and adhere to or invade host cells.
• Takes time to notice: Mycoplasma contamination often doesn't cause immediate visual cues like turbidity (cloudiness) or a rapid pH shift. This means it can go undetected for weeks, manifesting as subtle changes in cell morphology or growth rate.
• P/S resistance: Since mycoplasmas lack a cell wall, they are naturally resistant to antibiotics like penicillin that target cell wall synthesis.

Other, less common possibilities include specific slow-growing intracellular bacteria or viruses, but mycoplasma is the most frequent and stealthy suspect for this type of issue.

How to combat mycoplasma contamination

To avoid another outbreak, establish a routine prevention protocol.

• Routine testing: Implement a regular testing schedule for mycoplasma (e.g., every 1–3 months), especially for newly received cell lines or if your cells' growth behavior changes.
• Use certified reagents: Always use mycoplasma-free serum and reagents from reputable suppliers.
• Change technique: Handle only one cell line at a time in the biosafety cabinet and avoid sharing media or reagents between different cell lines.
• Quarantine new cells: Test any new cell line you receive from an external lab before introducing it to your general cell culture area.