Knowledge

In cell culture experiments, various problems are often encountered, among which the "black spot" problem is particularly troublesome. The reason why black spots have attracted much attention is that their number will continue to increase with the extension of culture time. When the number accumulates to a certain extent, it will not only affect the normal growth of cells, but also may cause deviations in experimental results. In some cases, it may even hinder progress of scientific research. Therefore, timely discovery and resolution of the "black spot" problem is crucial to ensure the quality of cell culture and the progress of experiments.

So, what are black spots? Are they bacteria? Mycoplasma? Or other problems? Should cells with black spots be disposed? This article will analyze the causes of the "black spot" problem in detail and provide effective solutions to help experimenters easily deal with this problem and ensure the quality of cell culture and the smooth progress of experiments.

What are "black spots"?

Cell fragments

Performance:

▼Some cells are broken and blurred under the microscope;

▼A large number of black particles of different sizes and irregular shapes appear in the intercellular space;

▼Most of the particles can be removed by changing the medium and washing;

▼The culture medium is not turbid and the color does not change significantly.

Causes:

▼Poor cell state and decreased growth vitality;

▼Improper operation during freezing or resuscitation leads to cell damage;

▼Improper handling during digestion causes mechanical or chemical stimulation of cells;

▼Changes in the culture environment caused by external factors, such as abnormal temperature and pH value.

Serum precipitation

Common components of serum precipitation include fibrin (flocculated precipitation), lipoprotein, fetuin and calcium phosphate (small black spots).

Causes:

▼The appearance of small black spots in serum is usually related to improper serum dissolution.

Solution:

▼Effectively remove the precipitate by centrifuging the supernatant or filtering the culture medium.

Impact:

▼The small black spots formed by calcium phosphate deposition in serum will not affect cell proliferation, and experimenters can use them with confidence.

▼However, since these small black spots are in an "active" state under the action of Brownian motion, they are easily mistaken for "black gum worm" contamination.

Bacterial contamination

The contamination speed is extremely fast, and the number of bacteria increases rapidly in a short period of time.

Performances:

▼A large number of movable black particles or rods can be seen under the microscope;

▼When the culture dish is slightly shaken, the contaminants move like quicksand;

▼In the early stage of bacterial contamination, the culture medium is not turbid but the color gradually turns yellow. As the contamination worsens, the culture medium will eventually become turbid;

▼The cell state may appear normal or abnormal.

Mycoplasma contamination

Performances:

▼After mycoplasma contamination, the culture medium turns yellow quickly, but it is generally not turbid;

▼Under the microscope, the intercellular space or surface may be filled with black particles, and the cells proliferate slowly;

▼Some cells may have vacuoles or drawing phenomena.

Conventional concentrations of antibiotics cannot inhibit the growth of mycoplasma, so once mycoplasma contamination is suspected in cells, it should be confirmed by PCR testing.

If mycoplasma contamination is confirmed, the treatment method should be determined according to the state and preciousness of the cells. For conventional cell lines, it is recommended to discard them directly to avoid the spread of contamination and affect the experimental results; for precious cells, you can try to save them according to the situation, but you need to be careful.

Solution

Suspension cells

Centrifuge slowly (500-600 rpm/min, 5-6 minutes) to collect the cell supernatant, and then replace with new culture medium.

Adherent cells

▼Wash the cells 2-3 times with buffer, tap the culture flask to help remove loose debris and particles.

▼After discarding the buffer, use low-concentration trypsin (such as 0.05% trypsin) to digest for about 1 minute to remove particles and debris in the cell gap.

▼After removing the low-concentration trypsin, digest the cells normally.

▼Centrifuge the collected cell suspension slowly (500-600 rpm/min, 56 minutes), and replace with new culture medium and culture flask.

Tips for routine culture experiments

▼ Catch the best time for cell passaging: pass the cells when they reach about 80% confluence to avoid excessive cell growth and aging.

▼ Control digestion time: ensure that digestion is not excessive to avoid the generation of cell debris.

▼ Reduce the number of freeze-thaw cycles of reagents: minimize the number of freeze-thaw cycles of reagents such as serum to maintain their activity.

▼ Adjust the pH value of the culture medium: ensure that the pH of the culture medium is in the optimal state to facilitate cell growth.

▼ Strictly control water quality and vessel cleanliness: use pure water and keep the vessels clean to avoid contamination and affect experimental results.

Based on the characteristics of the black spots (growth, movement and distribution), the cause can be preliminarily determined. If you are not sure about the specific cause, you can take a sample of the culture for microscopic observation or microbial testing to eliminate contamination factors. Ensuring the sterility of the experimental environment and using high-quality consumables and reagents are the key to preventing problems in cell culture.

Although cell culture seems simple, every detail may affect the growth of cells and the effectiveness of the experiment. I hope this article can help everyone improve work efficiency and smoothly promote the progress of scientific research experiments.