Have you maintained sterility and aseptic technique?

Are alfalfa extracts sterile?

Phil Geis yes of course, i think i have applied sterilization and aseptic technique according to the procedure. for alfalfa extract before use i filtered it using membrane filter

what do you think i should do? i am still confused about that

Mold did not appear spontaneously. I'd culture every component individually including the cells as well as putting settle plates out during culture procedures.

Phil Geis If it is due to lack of sterilization and aseptic techniques, can the dislocation technique of the experimental animal and the isolation process of the tissue also affect it?

Anything that goes into the culture could be the immediate source, including the fibroblast cells.

Are there any suggestions or input that I should do to minimize the risk of continued contamination? Phil Geis

Think you need to find the source. check everything that goes into the dish and the air (by settle plates) while you do execute the test.

Phil Geis thank you very much for your positive feedback and suggestions and than that's so really helpful for me, i hope you stay healthy~

Your culture might be contaminated due to surface contaminant ie. In your lab or it may be mainly due to adding of plant extracts without sterilization

1. Replace the alfalfa extract with a filter-sterilized batch

Prepare the alfalfa extract and sterilize it by passing it through a 0.22 μm membrane filter. This method effectively removes microbial contaminants without exposing the extract to heat, which could degrade sensitive compounds.

2. Add amphotericin B to the culture medium

Incorporate amphotericin B into your culture medium at a concentration of 2.5 μg/mL to inhibit fungal growth. This antifungal agent is commonly used in cell culture to prevent contamination.

3. Re-isolate fibroblasts using stricter aseptic protocols

Ensure all instruments and surfaces are sterile, and perform all procedures within a laminar flow hood to maintain aseptic conditions. This minimizes the risk of introducing contaminants during fibroblast isolation.

4. Validate the sterility of all components (FBS, DMSO, extract)

Conduct sterility testing on all reagents, including fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), and plant extracts, by incubating samples in culture media and monitoring for microbial growth. This ensures that all components are free from contamination before use.

Zahra Mirzaeipour Mareeswari Petchimuthu thank you so much your feedback for me