In the experiment: prevention of cell contamination and post-contamination countermeasures - Database & Sql Blog Articles
In scientific research experiments: pollution has always been a big problem in cell culture. Preventing or controlling pollution is a key factor in the success of cell culture. When we are in cell culture, we must pay great attention to the pollution problem at the beginning stage, otherwise we will give up our efforts beforehand, which will not only waste time, but also waste manpower and material resources. In this chapter, Shanghai Jinma Bio will analyze the countermeasures against cell contamination and pollution in the experiment. (a) What kinds of pollution are there? Contamination during cell culture refers not only to microorganisms, but also to all substances, including organisms and chemicals, that are mixed into the culture environment and are harmful to cell survival or cause cell impureness. 1 Bacterial contamination Bacterial contamination is a common contamination in laboratory cell culture. Even if antibiotics are added to the cell culture fluid, it may cause contamination due to careless handling. Gram-positive bacteria such as Bacillus subtilis and Gram-negative bacteria such as Escherichia coli and Pseudomonas are the most common, and Staphylococcus aureus is more common. When the cultured cells are contaminated with bacteria, the culture solution becomes cloudy. pH change. After the contamination, the cells undergo pathological changes, the intracellular particles increase, thicken, and finally become round and die. 2 Fungal contamination Fungal contamination is the most common form of cell culture. The most common fungi are Aspergillus fumigatus, Aspergillus niger, Confucius, Mucor, Candida albicans and yeast. After the cultured cells are contaminated by fungi, white or light yellow dots floating in the culture solution are observed, and some are scattered, and the culture solution generally does not turbid; under the inverted microscope, filiform, tubular or dendritic hyphae can be seen interlaced at Some cells are arranged in a chain between cells or in the medium. After fungal contamination, cell growth slows down, but eventually the cells shed and die due to nutrient depletion and toxicity. 3 Mycoplasma contamination Mycoplasma is the smallest microorganism that can live independently between bacteria and viruses, the smallest diameter 0.2μm Generally, it is impossible to remove it by filtering and sterilizing, and it is difficult to see its morphological structure under the light microscope. It is not easy to find at first, can survive under alkaline conditions, and is resistant to penicillin. More adsorbed on the surface of the cell or scattered between cells. After the cultured cells are contaminated by mycoplasma, some sensitive cells can be seen to grow and proliferate slowly, and some of the cells become rounded and fall off the bottle wall. However, most cells do not change significantly after contamination, or change slightly. If not treated in time, cross-contamination will also occur. 4 Virus contamination In the process of tissue cell culture, if the potential virus is not removed, virus contamination will occur. At present, it has been found in the cultivation of primary monkey kidney cells. 20 a serum virus. Tube-polluted cells do not affect primary culture, but vaccine production is not safe. Therefore, the potential virus is a problem in the mass production of cells and the production of biological products such as vaccines and interferons. 5 Non-same cell contamination Since the equipment and solutions required for each cell strain are not strictly separated during the cell culture operation, one cell is often contaminated by another. At present, there are dozens of cells in the world. HeLa The contamination of the cells has caused many experiments to be invalidated. Contamination of chemical components caused by cell cultures also occurs occasionally, mostly due to the incomplete cleaning and disinfection of the items required for cell culture, and the introduction of some toxic chemicals. ( b) the difference in pollution types 1 , bacterial, fungal contamination detection ( 1 ) Visual observation ---- Bacterial and fungal contamination often occurs after open operations such as passage, liquid exchange, and sample loading, and the proliferation is rapid. If there is pollution, 48 Obviously observed within hours, for example, the culture solution became cloudy, or a little swayed with a lot of floats floating. ( 2 ) Inoculation observation ---- Inoculation with ordinary broth or inoculation with a medium without an anti-antibiotic is also found to be contaminated. ( 3 Under the microscope Under the high power microscope of the inverted microscope, a large number of spherical particles floating in the culture solution can be seen, which is bacterial contamination. Filamentous, tubular, dendritic or ovoid substances between cells are often contaminated with fungi. 2 Detection of mycoplasma contamination ( 1 Phase contrast microscopy---- A small amount of culture droplets were taken on the slide, and then covered with a cover sheet. The mycoplasma was dark microscopic particles under the microscope, mostly located between the cells and the cells, and sometimes showed similar behavior to Brownian motion. Note the difference between the contents of the cell disruption and overflow, such as mitochondria. ( 2 ) Fluorescent staining observation ----- Fluorescent dyes Hoechst33258 , this dye can be DNA Specific binding, which can be used in the protoplast DNA Coloring, under the fluorescence microscope, mycoplasma is a small green dot scattered around the cell or attached to the cell surface. ( 3 Electron microscopy ---- Conditions permit, can be observed by scanning electron microscope or transmission electron microscope. Generally in cell culture 48 ~ 72 After the cells are close to confluence, the cells are digested with trypsin to prepare a cell suspension, which is fixed, embedded, and sliced ​​before observation. ( 4 Culture test ---- Cell suspension 5mL Join 45mL Mycoplasma broth culture, culture 14 After the day, observe the broth culture with or without a misty precipitate, and then take 0.5ml Join has cooled down to 50 ° C In the medium, separate culture is carried out using agar medium. 37 ° C to cultivate 3 Day observation " Poached egg †Colonies appeared. 3 Virus detection ( 1 Rapid diagnosis of animal virus disease by electron microscopy ( 2 Reverse transcription _ Polymerase chain reaction RT_PCR Detecting virus (3) Pollution removal methods 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; after searching for the cause, thoroughly disinfect the operation room, resuscitate or repurchase the cells, and then culture. The value of contaminated cells is large and difficult to recover. The following methods can be used to remove them. 1. Bacterial and fungal clearance Antibiotics are more effective at killing bacteria. Combination therapy is better than medication alone. Preventive medication is better than medication after contamination. Preventive medications generally use double antibiotics, and the use of drugs after contamination should be greater than the usual amount. 5 ~ 10 Double washing method, after the addition of drugs twenty four ~ 48 Hours, then change the regular medium. This method is effective in the early stages of pollution. 2. Mycoplasma clearance ( 1 ) use MRA deal with use MRA ( Mycoplasma Removal Agent ) process cells, each 4 Change the liquid once a day , Continuous processing 15 Days to ensure that the cells are pure and healthy, the effect is good. ( 2 Method for removing mycoplasma contamination by washing and purifying Cell nutrition domestication → Screening of high quality cell populations → Cell cleaning → Repeated centrifugation. The principle is to use the centrifugal force, the cell, the microbial quality and the buoyancy difference of the suspension to achieve the purpose of removing mycoplasma. Due to the small size of the mycoplasma and the extracellular parasitic , Therefore, the number of potential mycoplasma is reduced to the limit by repeated washing of cells and low-speed centrifugation. . If combined with the anti-killing effect of sensitive antibiotics, better results can be achieved. (3) Drug-assisted heating treatment ----- After the drug is treated, the contaminated tissue culture is placed 41 ° C to cultivate 18 In hours, it kills mycoplasma, but it has an adverse effect on cells. 4 Using mycoplasma-specific serum use 5% Rabbit mycoplasma immune serum can remove mycoplasma contamination, because specific antibodies can inhibit the growth of mycoplasma, so after treatment with antiserum 11 Day turns negative, and 5 It remains negative after a month. However, this method is more troublesome, and it is better to use antibiotics for convenience and economy. (4) 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 Add antibiotics 2 , 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. 3 Starting from the operator ( 1 Wash your hands with soap before entering the sterile room and wear a gown as required. Work must be used first 75% Alcohol cotton balls rub the hands, wipe the mouth and burn the mouth of the bottle. ( 2 The operator must be light, and the bottle opening must be opened in the sterile area around the flame and the bottle mouth rotated to burn. Try not to talk during operation. If you sneeze or cough, turn to the back. ( 3) Always change the straw when handling, and discard it if it is found to be in contact with the hand and other contaminated items. After the experiment, wipe the table with gauze soaked in disinfectant water. 4 To prevent cell cross-contamination (1) When performing various cell culture operations, the instruments used should be strictly distinguished. ( 2) When changing or substituting, do not touch the syringe and the dropper to the bottle of the cell culture bottle, so as not to bring the cells into the culture solution to contaminate other cells. (3) Once the cells are purchased or introduced from elsewhere, they should be stored early and frozen, and once regenerated, they can be resuscitated. 5 Thorough disinfection of sterile rooms ( 1 ) 0.1% New cleansing thoroughly cleans the sterile room thoroughly; ( 2 Formaldehyde fumigation method: Formaldehyde is a broad-spectrum sterilizing agent. Its aqueous solution and gas-killing have a killing effect on various bacteria, spores and fungi. (3) Cell culture, first of all to avoid contamination, but in the process of cell culture, the pollution is removed by appropriate methods, which can also ensure the success of cell culture.
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