First, the removal of pollution
Once cultured cells are contaminated, most are more difficult to handle. If the value of the contaminated cells is not large, it should be discarded; if the cell line is retained or can be purchased, the operation room can be thoroughly disinfected after the cause is found, and the cells can be resuscitated or re-purchased and cultured. If the value of contaminated cells is large and difficult to regain, the following methods can be used to remove them.
(a) the use of antibiotics
Antibiotics are more effective at killing bacteria. Combination therapy is better than medication alone. Preventive medication is better than medication after contamination. Prophylaxis is generally treated with double antibiotics (penicillin 100u/mL plus streptomycin 100μg/mL). After the contamination, the drug should be washed 5~10 times larger than the usual amount. After the drug is added, the effect is 24 to 48 hours. Culture medium. This method may be effective in the early stages of pollution. In addition to penicillin and streptomycin, gentamicin, kanamycin, polymyxin, tetracycline, and nystatin can be used. It is usually treated with 400-800 μg/mL kanamycin or 200 μg/mL tetracycline, and the solution is changed once every 2 to 3 days, and the treatment is carried out for 1 to 2 generations. In recent years, there have been reports of three antibiotics such as 4-fluoro, 2-hydroxyquinoline (Cip), and pleu-romutilinderivative (BM-Cyclin2: BM-1 and tetracycline derivatives (BM-2). It can be used alone or in combination to kill mycoplasma. All three antibiotics are formulated into 250X concentrate with PBS, and stored at -20 °C for use. The concentration of Cip is 10 μg/mL, BM-1 is 10 μg/mL, and BM-2 is 5 μg. /mL. When using, firstly remove the contaminated culture solution, add RPMI1640 culture solution containing BM-1, and then aspirate the culture solution after 3 days, add RPMI1640 culture solution containing BM-2, and culture for 4 days, thus 3 consecutive rounds. Once, until the mycoplasma was cleared by 33258 fluorescent staining microscopy, the culture medium was cultured for 3-4 times.
(2) Heating treatment
Incubation of contaminated tissue cultures at 41 ° C for 18 hours killed mycoplasma but had an adverse effect on the cells. Therefore, pre-test should be carried out before treatment to find the heating time that can kill the mycoplasma to the utmost and minimize the influence on the cells. This method is sometimes unreliable. If it is treated with a drug first, it is better to heat it at 41 °C.
(3) Use of mycoplasma-specific serum
Mycoplasma contamination can be removed by using 5% rabbit mycoplasma immune serum (hemagglutination titer 1:320 or more). Because specific antibodies can inhibit the growth of mycoplasma, it is negative after 11 days of antiserum treatment, and remains after 5 months. Negative. However, this method is more troublesome, and it is better to use antibiotics for convenience and economy.
(4) Other methods
In addition to the above methods for removing pollution, there are animal inoculation sterilization method, macrophage phagocytosis method, bromine uracil added to the contaminated culture bottle, and light irradiation method, and filtration method, etc., but both are troublesome and the effect is not determine. Therefore, once the mycoplasma is contaminated, unless it has a particularly important value, it is generally discarded and re-cultivated.
Second, the prevention of pollution
Prevention is the best way to prevent contamination during cell culture. Only when prevention work is done before can the possibility of pollution be minimized. General prevention can be started from the following aspects:
(1) Starting from the sterilization of articles and supplies
The items used for cell culture should be thoroughly cleaned and disinfected. The sterilization of various solutions should be carefully performed and used only after the sterility test is negative. The operating room and the remaining sterile equipment should be cleaned and disinfected regularly.
(2) Starting from the operator
1. The operator has a strong sense of responsibility, be careful and steady, and be proficient in operating techniques. Wash hands with soap or 5% chlorhexidine for 5 minutes before entering the sterile room. Wear a gown as required. After entering, close the door and sit down and walk as little as possible. Work begins with a 75% alcohol cotton ball, hand rubbing the mouth and cauterizing the mouth. Strictly check equipment, solutions and cultures in advance, and do not bring contaminated or unsterilized items into the sterile room, and do not use them casually to avoid a large amount of pollution.
2, the operator should be light, you must open the bottle mouth in the sterile area around the flame, and rotate the bottle mouth to burn. Try not to talk during operation. If you sneeze or cough, turn to the back.
3. Always change the straw when operating. Do not use a straw to do it. Once the mouth of the pipette is found to be in contact with hands and other contaminated items, it should be discarded. After the experiment is completed, keep the laboratory clean and tidy, and finally wipe the countertop with gauze soaked in disinfectant water.
(three) to prevent cell cross-contamination
When performing a variety of cell culture operations, the instruments used should be strictly differentiated, and it is best to mark them for easy identification. And operate in order to avoid confusion when working together.
When changing or substituting, the syringe and the dropper should not touch the mouth of the cell culture bottle, so as not to bring the cells into the culture solution to contaminate other cells.
Once all cells have been purchased, introduced from elsewhere, or established by themselves, they should be kept frozen as soon as possible. Once the contamination occurs, the resuscitation can be re-cultured.
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