In cell culture, it is often seen that there are some black particles of different sizes and shapes in the cells and culture fluids. If the microscopic examination (100x) counts more than 10 per field, cell growth may be affected, and if the number is increased, the cells may stop proliferating and then die.
It is often referred to as such particles as black worms or poisoned granules.
Description of black worm
Classification description
For the classification of black rubber worms, the academic community did not explicitly give a statement. Regarding the classification of black worms, most people currently believe that black worm infection is a microbial infection. In some articles, the black rubber worm is classified as a biological pollution type, but it is not classified as a certain taxonomic type, but the black rubber worm is independent of an unknown organism. According to the Chinese Institute of Virology and the Academy of Military Sciences, it is a kind of protozoa that is parasitic in bovine serum. It seems to have similarities with paramecium and amoeba, but it cannot continue because of insufficient funding, and ultimately there is no strong proof. Black worms belong to the protozoa. Some scholars do not think that the so-called black worm pollution is a kind of biological pollution, but it is considered to be a cell debris, which is caused by cell rupture during cell culture. It is also reported in the literature as a similar silica in glass bottles. The substance, which is rarely found in the literature; it is also reported that the scorpion worm is a nano-scale bacterium.
Morphological description
Morphologically similar to rod-shaped bacteria, but the length is longer than bacteria, the diameter is about 0.5 ~ 1 micron, the microscope is black, the rubber worm is linear after maturity, and the shape is elliptical.
Movement form
In the 400x inverted microscope, there is a typical Brownian motion (irregular in-situ small-distance jitter), which is seen by many cell culturers, like black bugs swimming around. It can penetrate the filter membrane or spread through the air. It is black dot at low magnification, and the black bug can swim around at high magnification. But physically, the particles are small enough to do Brownian motion in the liquid, which is not enough to show that the Brownian motion of the black rubber bug proves that it is a creature.
Physiological description
Resistance
Antibacterial and antifungal drugs are ineffective against black worms, and it can be conservatively determined that black worms are not bacteria.
Environmental resistance
Bake the culture equipment at 150 degrees for 8 hours, which can be eliminated, while the black glue insects are not dead. It can be seen that the black worm can withstand high temperature and high pressure, and there is a bit of acidophilia. If it is confirmed to be a creature, it is likely to be an extreme microorganism, an archaea.
Nutritional condition
"Blackgel insects" can be parasitic on animal cells, can also survive in the culture medium, rely on the nutrition of cells and culture medium, and pass through with the passage of cells. It can be seen that "black rubber bug" may be a heterotrophic organism.
Various guesses about black rubber bugs
Non-biological
Cell debris
During cell culture, due to cell metabolism, material exchange, and changes in the surrounding environment, especially during the observation from the 37-degree incubator, the liquid medium temperature is higher than the outside temperature due to the larger specific heat of the liquid medium. , causing cold and heat exchange, the formation of liquid flow in a small range is intensified, of course, these small years will be stirred up to form a feeling of "walking"; as cell culture continues, some cells begin to decline, cell membrane structure rupture, rupture Subsequent cell contents are leaked into the culture solution. In particular, the destruction of lysosomes causes damage to other cells and organelles in a continuous manner. If the liquid exchange is not very diligent, further damage and residue appear. Further, the problem of "black rubber insects" is many cell cultures. The problems have been discovered, but the reagents and means for monitoring and elimination have not been found so far. This is because the so-called "gelworm" pathogens are difficult to collect and capture at first, which proves "black glue insects" from another aspect. The problem is difficult to determine; any foreign microbe in the medium will have the reaction and performance of life activities, rather than the appearance of simple movement. To give an example: if the movement status of "black rubber bug" has reached the extent that it can be observed with a microscope, its nutrient metabolism and energy demand can be imagined. Thus, the influence consumption, the degree of acid-base content, and the like in the culture solution are all significantly changed. For example: rapid, large-scale precipitation, rapid decline in pH, cell death and death, and other types of microbial infection. These conditions are difficult to observe in the so-called "gelworm" infection. At the same time, the appearance of "black glue insects" affecting the state of the cells does not seem to have strong evidence. However, when the state of the cells decreased, the "black rubber bugs" increased significantly. It is very likely that the cell fragments produced by cell decay when the cell state declines. Therefore, it is more likely and reasonable for black worms to belong to cell debris.
Some silicon oxide-like things in glass culture flasks
There is an article on the Internet that black worms are not a kind of organism, they are inorganic substances, and their essence is silicon particles in glass culture bottles. Can affect cell growth, when the cell state is good, not obvious. The state of the cells is poor, and it is easy to see when the number is small. The best way is to use a disposable culture bottle to eliminate the effects of black worms.
Serum polymer product
Gibco's serum indicates that this is a serum polymerization product, not a microorganism.
Biological class
Mycoplasma
Some people think that black worms may be mycoplasma pollution, because relatively authoritative website articles indicate that black worms can pass through the filter and can spread in the air. It happens that the oral mycoplasma is the normal flora in the human mouth, so the pollution of the laboratory operators may also be the source of mycoplasma pollution. However, some people have done experiments to prove that the black rubber insects will not be mycoplasma for the following reasons:
1, visible under the light microscope, because the volume is wrong, mycoplasma can not be seen under ordinary microscope.
2, a variety of staining can be seen, even hoechst can dye it, in general, mycoplasma is not easy to color with ordinary dyeing, with Giemsa staining very shallow, Gram staining is negative. Therefore, it is less likely that the black worm is a mycoplasma.
Fungus
Many people have found similar black worms, but it is finally determined to be a fungus, and amphotericin B is effective. That means that the absence of black worms is only a fungal infection in cell culture, or that black worm infection is a fungal pollutant. If the black worm is a fungus, the resulting pollution will not cause so much attention, even if it is special. But this does not rule out those who think that "black rubber insects" are fungi, and only see general fungal infections. Since the cells are infected, it is not as simple as it is theoretically. It is easy to cause the culture to fail if it is not noticed, so it is impossible for the black worm to be a fungus.
Parasitic protozoa
Black gum is a parasitic protozoa, not a bacterium, a mycoplasma, nor a complement, cell debris or protein precipitate. Some people can clearly see that the rubber worm has two different forms under the high magnification of 400 times, one is larger, the movement is slower, and the red light is red; the other is smaller, the movement is faster, the red is green, A small distribution around a large, probably a male around the female. This kind of organism can't survive without leaving the cell. Some people have done such a test. The serum of the contaminated colloidal worm is cultured for 4 weeks until the black slag-like rubber worm is full. The feeling is that the rubber worm has the same clustering as the cell. If the density of the worm is low, the growth is slow. If the hand is lazy, it will not change for a few days. After the worm grows to a certain concentration, it will fly rapidly. The degree of infection is also large. At this time, besides being visible in the medium, the cell surface is usually covered with acne, so some people think that certain factors secreted by the cells can inhibit the growth of the rubber worm. It is said that the Chinese virus institute and the Academy of Military Sciences are a kind of protozoa that is parasitic in bovine serum. It seems to have similarities with paramecium and amoeba. However, due to insufficient funds, it cannot be continued. Therefore, it is not clear. The evidence proves the correctness of this guess.
Analysis
From the previous description of the scorpion worm, it can be seen that the scorpion worms that people encounter are not all the same, like bacteria, like mycoplasma, like protozoa, like fungi, and like cells. Fragmented. The equipment conditions in the domestic laboratories are uneven, and the quality of the experimental personnel is not the same. The same phenomenon may be slightly different. The difference in the phenomenon described may become larger or smaller. Therefore, many people see that cells are contaminated by bacteria, which are not easy to handle, but the phenomenon described is similar to that of the spread of black worms. It will attribute the failure of culture to untreated scorpion worm contamination, and may even say it. Found is a real black rubber bug. There are so many things like this, and the black rubber bugs will be artificial variants, like everything. Whether or not black rubber worms exist remains to be scientifically developed. But one thing is certain. Most people find that the "gelworm" is not the real unknown gelatin, but the common bacteria, fungi, protozoa and other microbial contamination.
Report on the authority of black rubber insects
In the Journal of Cell Biology, there is a paper on "Study on the Properties of a Black Moving Particle in Cell Culture". After various experiments, it is pointed out that "there are often different sizes and shapes observed in tissue culture. The black moving particles, which have an adverse effect on the growth of cultured cells, sometimes even lead to cell death, and can increase the amount of the cells under certain conditions as the culture time increases. People often call this particles black worm or poisoned granules. We took black moving particles from serum, culture medium and other materials used for cell culture, and observed the morphology by optical microscopy, transmission electron microscopy and scanning electron microscopy; and observed the activity by microphotography; Electrophoresis analysis by electrophoresis dialysis showed that the particle was a membrane-free structure, no protein component, positively charged under neutral environment, and the spectrum was analyzed by scanning electron microscopy to be a silicon composite. We call it silicon body (B). It is proved that the activity is Brownian motion. This paper further analyzes the culture solution and double distilled water by inductively coupled plasma emission spectroscopy.
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