Is the small black spot in fetal bovine serum under the microscope in cell culture contaminated or precipitated?

Long lead times and expensive, don't return the goods yet. I've encountered similar situations. South American serum shows significant batch-to-batch variation in precipitates, especially after overnight thawing at 4°C, flocculent material appears at the bottom of the bottle. If not mixed well during aliquoting, black spots are likely to appear. You can place the currently aliquoted tubes in a 37°C water bath and gently swirl them. After warming, many calcium phosphate precipitates will dissolve or decrease; then centrifuge at low speed and collect the supernatant. For unopened bottles from the same batch, thaw at 4°C in advance, then rewarm at 37°C, filter through 0.45μm, and aliquot to reduce repeated freeze-thaw cycles. When communicating with the supplier, directly request the COA and the shipping records for that batch, explain the amount, size of black spots and the impact on culture, and ask whether you can exchange for the same batch or an equivalent amount from a new batch. If the cells are in good condition, this batch can continue to be used, but it is recommended to keep photos and records to prevent subsequent fluctuations in cell condition.

Don't rush to switch lots just yet. You've just thawed and aliquoted the serum. The most common cause of serum precipitate is calcium phosphate or lipoprotein complexes, characterized by small black dots of varying sizes that are basically immobile and do not increase in number after overnight incubation at 37°C. Bacterial contamination typically causes rapid turbidity, and the black dots will increase in number as the bacteria proliferate. You can take 2 mL of serum containing black dots and add it to sterile liquid culture medium, then incubate at 37°C for 48 h. If it remains clear, bacterial contamination can essentially be ruled out. For 293T cells, if it is only precipitate, after aliquoting, centrifuge at low speed (500g, 5 min) and take the supernatant, or let it stand and take the supernatant; this usually does not affect Western blot or viral packaging. However, viral packaging is sensitive to cell status, so it is recommended to first culture one passage with the treated serum and assess cell refractility, doubling, and transfection efficiency before deciding. If you did not mix well during aliquoting, the precipitate at the bottom of the bottle will concentrate in the last few tubes, and these tubes are best handled separately.

The black dots "basically not moving" look more like precipitation, but mycoplasma cannot be seen on microscopy, so this cannot be used to rule out mycoplasma. Among the batch you have on hand, there are still unopened ones. It is recommended to do two things first: first, take a small amount of serum and send it for mycoplasma PCR or perform Hoechst staining yourself to look for punctate fluorescence outside the nuclei; second, perform Gram staining or ordinary plate culture on the black dot sample. If contamination is not a concern, you can choose 0.45 μm filtration if you continue to use it; direct 0.22 μm filtration is not recommended—0.22 μm will retain more proteins and lipids, which may not have much effect on viral packaging but is unnecessary. A better approach is to aliquot and centrifuge at 400g for 10 min, then take the supernatant for use; for 293T, serum added to DMEM after centrifugation generally will not cause a drop in attachment or yield. For Western blot, as long as the cell number is sufficient, it is fine; for viral packaging, it is recommended to run a small-scale comparison of titers.