What are the main ways of microbial contamination in plant tissue culture? The main measures to prevent plant tissue culture pollution are:
(1) Improve environmental conditions. The inoculation room and culture room should be disinfected and purified regularly, and the workbench or inoculation box should be opened for more than 30min before inoculation. The relative humidity of the culture room should be controlled at about 70%. When the relative humidity is too high, dehumidifier can be used to dehumidify.
(2) The training of vaccinators is very important, and aseptic operation should be strictly carried out. Tools needed for inoculation must be strictly disinfected before use. Don't put too much material in the operation area of the clean workbench to avoid airflow obstruction. Also regularly check the working quality of clean workbench [8].
(3) Strictly check the inoculation materials. Eliminate inoculation materials contaminated by fungi and bacteria.
(4) Check the sterilization quality of the sterilizer frequently, and repair it immediately when problems are found. The pressure gauge of the sterilizer can't be taken out immediately after it drops to zero. Due to the negative pressure effect caused by the action of cold and hot air, the cold air in the external environment is sucked back into the sterilized culture bottle, causing fungal pollution. In order to avoid this phenomenon, the sterilized culture bottle should be taken out of the pot after the pot is slightly cooled [9].
(5) Check whether there is any problem with the culture container. Culture containers are usually sealed with plastic covers, rubber plugs, cotton plugs, films, etc. Plastic covers are prone to aging and poor sealing when used for a long time, which will also cause pollution. Lin Sheng et al. formulated a "No.4 disinfectant" to disinfect the bottle mouth, which can control the contamination rate of the culture medium below 0.3% [10].
Prevention and control of pollution in subculture
The sterile materials obtained in the initial stage are sterile in theory, but they will also be contaminated in the later stage or subculture. Besides careless operation, they may also be contaminated by fungi spread by mites in the culture room. This kind of pollution can be prevented from two aspects: (1) There should be a reasonable plan when expanding reproduction. After obtaining the original sterile culture, some of them should be preserved as "original species", propagated in batches, and then provided for mass production after re-inspection. (2) Antibacterial agents can be added to the culture medium to prevent [3]. Xu Wanfang and others used carbendazim to inhibit bacteria and promote growth in tissue culture of Anoectochilus roxburghii, and the effect was obviously better than metalaxyl, thiophanate-methyl and their mixtures. Anoectochilus roxburghii grown in the medium containing carbendazim was not contaminated by microorganisms, and the sterilization rate was 100%, with no albino seedlings and strong seedlings [1 1].
Endophytic bacteria in plants have deep potential and cannot be eliminated by surface disinfection. Sometimes, in the initial culture of explants, including the subculture of previous generations, it is not easy to be detected by naked eyes on the culture medium. With the increase of subculture times, the amount of bacteria gradually accumulated and developed, and then appeared on the culture medium [12]. Huang Xiaorong and others think that the cause of bacterial contamination in plant tissue culture is the result of dormant bacterial spore germination [13]. The pollution of endophytic bacteria such as bacteria can be prevented and reduced by adding antibiotics to the culture medium, cultivating stem tips and lowering PH value.
Faye et al. added 200mg/L penicillin GK during the rapid propagation of colored taro to the 4th-5th generation, which can effectively inhibit the growth of endophytes without affecting the propagation coefficient [14]. In the tissue culture of ivy, Zhai Jianzhong and others used streptomycin to inhibit the endophytic bacterium Xanthomonas. Better than gentamicin and cefazolin sodium. The simultaneous use of these two antibiotics in culture medium is beneficial to prevent bacteria from producing drug resistance and control the development of bacterial pollution for a long time. However, the increase of streptomycin dose will cause toxicity to plants [15].
It is very important to add antibacterial agent to the culture medium and choose the appropriate concentration. Low concentration will have poor effect, while high concentration will easily cause toxicity to plants, affect proliferation or blacken the culture, resulting in dead seedlings and albino seedlings. The combination of two or more antibiotics can prevent and control the drug resistance of bacteria. Antibiotics and carbendazim are generally not resistant to high temperature and need to be filtered and sterilized, so it is not convenient to add them in production. Sodium benzoate, potassium sorbate and sodium propionate are commonly used preservatives in food, which are resistant to high temperature and high pressure and convenient to use in tissue culture production.
Endophytes in plant materials can also be removed by repeated shoot tip culture. Because the distribution of pathogenic bacteria in plants is uneven, the meristem of shoot tip is sterile through vascular bundle transmission. Through repeated shoot tip culture, both endophytic bacteria and viruses can be removed.
Except Erwinia, most bacteria can't grow when the PH of the medium is less than 4.5. In the tissue culture of Zi Yuan, Iris and Rosa, adjusting the PH value of the culture medium from 5.8 to 3.9 ~ 4.3 can prevent a lot of bacterial pollution [7]. Hu Qing and others were cultured in water with low PH value (3. 10), and the ventilation condition of the container was improved, which could make the Hulk block with serious bacterial pollution proliferate normally and was harmless to the growth of a single plant. The proportion of rooting seedlings is higher than that of conventional solid culture, which basically solves the problem of bacterial production after serious bacterial pollution [16].
For contaminated cultures, sometimes it takes a lot of time because of the scarcity or repeated appearance of plant materials, and larger plants can also be cut and disinfected and inoculated again. Li et al. soaked the bacterial contaminated seedlings in tissue culture of Populus alba with 4 million or 6 million units /L penicillin sterile aqueous solution for 60min or 40min respectively, which could effectively prevent bacterial contamination in tissue culture. When the treated seedlings are subcultured, there is no repeated bacterial pollution and the seedlings have strong differentiation ability. When rooting, the root system is developed and the survival rate of transplanting is high [17]. Mars et al. proved that if there is only fungal pollution in subculture, it can be eliminated by soaking in 3% carbendazim sterile solution for more than 0.5h, then inoculating with sterile water or directly inoculating without washing with sterile water. If bacteria, bacteria and fungi are contaminated at the same time, the sterile seedling system can be re-established by HgCl2 disinfection method. The contaminated seedlings are cut into long stems as explants, washed with tap water for 0.5h, and then treated with ethanol and HgCl2 for a short time on the ultra-clean bench [18].
4. Reduce the organic components in the culture medium
The existence of organic matter in culture medium is an important cause of pollution, and removing organic matter is also a way to reduce pollution. In the tissue culture of pistachio, Song Fenghui and others removed organic components (such as VB 1, VB6, nicotinic acid, etc. ) In the culture medium, after 2 ~ 3 generations of culture, it can inhibit the growth of bacteria and has no effect on the growth and proliferation of clustered buds. The reason is that these are all necessary substances for the growth of some bacteria. After removing these organic substances, bacteria can't grow and develop, and the death gradually decreases [19]. Sugar-free tissue culture and rapid propagation of plants initiated by Professor Toyuki Kozai in Japan completely removes the organic components in the culture medium, inputs a controllable amount of CO2 gas as a carbon source, and promotes the photosynthesis rate of plants by controlling environmental factors, so that plants can be transformed from heterotrophic to autotrophic, and the plants can grow well and the pollution rate is obviously reduced [20]. Because of the removal of sugar, the culture of tissue culture seedlings has changed from glass bottles to box containers, and the whole culture room can also be used as a culture container, even without strict aseptic operation, with little pollution. This technique is very successful at least in the rooting promotion stage of many plant tissue culture seedlings.
The United States imported Puweixin Tmall to effectively prevent smog and go out for protection.
The dust blocking efficiency of the mask is based on its blocking efficiency for fine dust, especially for inhalable dust below 2.5 microns. Because dust with this particle size can directly enter alveoli, it has the greatest impact on human health. Gauze mask, its dustproof principle is mechanical filtration, that is, when dust collides with gauze, some large particles of dust are blocked in emery cloth through layers of barriers. For some fine dust, especially dust smaller than 2.5 microns, it will enter the respiratory system through the mesh of gauze. Dust mask, its filter material is composed of activated carbon fiber felt pad or non-woven fabric, and those inhalable dust smaller than 2.5 microns pass through this filter material.
What are the main methods of sterilization in tissue culture? 1. The moist heat sterilization method increases the steam temperature by pressurizing, and the high-pressure steam sterilization has strong penetration, high temperature and the best sterilization effect. Note: 1. Completely eliminate the cold air in the autoclave. When there is cold air, the temperature reached at the same gauge pressure will be lower, and the less cold air discharged, the lower the temperature. In high-pressure steam, in order to ensure the specified temperature, cold air must be completely excluded. Otherwise, although the pressure has reached and the temperature has not reached the specified requirements, sterilization will not be complete. 2. The ultraviolet sterilization spectrum is broad, but the penetration is weak, and there are many influencing factors. The sterilization effect is limited to some extent. The germicidal efficacy of ultraviolet rays is related to factors such as ultraviolet intensity, irradiation time, temperature and humidity. China stipulates that the irradiation intensity distance of ultraviolet lamp at 1m is not less than 70μw/cm2. Generally, the ultraviolet lamp will be replaced if it is used for more than 100h. Dust on the surface reduces the sterilization effect. The sterilization temperature of ultraviolet lamp is 20~40℃. The relative humidity is 40%~60%. 3. Dry heat sterilization. Burning and flame sterilization: Burning is mainly used for tool sterilization, and burning on flame can completely sterilize. Flame sterilization is usually used for aseptic operation. Test tube mouth, glass bottle mouth, silicone plug, etc. The nozzle is disinfected by flame repeatedly to prevent the nozzle from being polluted. As an auxiliary sterilization method in aseptic operation. 4. Dry-drying sterilization: using thermal radiation and dry hot air for sterilization. Generally, articles to be sterilized, such as metal, glass and ceramic products, can be sterilized in an oven after packaging. Usually, they can be completely sterilized by heating to 160℃ and keeping this temperature for 2 hours. But it cannot exceed 170℃, and the glass measuring tool is easy to deform. If the temperature drops too fast, it is easy to suddenly cool down.
What are the factors that cause the pollution of tissue culture materials in plant tissue culture?
Incomplete sterilization of culture medium
Incomplete sterilization of containers
Irregular operation
It was not sealed properly after inoculation.
What factors can lead to pollution in plant tissue culture? Cause of pollution 1. 1.
explant
(1) Age, type and size. Adult plants carry more bacteria; Older materials carry more bacteria; Exophytes and plants with diseases and insect pests are easy to be polluted; There are many bacteria in the materials grown outdoors; Materials with soil on the surface and underground organs carry more bacteria; Materials with large surface area carry more bacteria than materials with small surface area [1-2].
② Time and place of sampling. Fungi thrive in the rainy season, when there are many bacteria in the material; When the sun is strongest, the ultraviolet rays are stronger and the sterilization effect is good. At this time, the material carries less bacteria. Choose materials with more bacteria for unclean and poorly growing parts [1].
③ Sterilization effect. Sterilization of explants is the key to tissue culture, and the sterilization effect directly determines the success or failure of tissue culture. Improper sterilization method and sterilization agent treatment lead to poor sterilization effect and bacteria on explants. 1.2
In the process of tissue culture, the operation of each technical link is not standardized ① culture medium. Preparing a culture medium by using a mother liquor of long bacteria; cultivate
High pH and new organic components will lead to bacterial pollution. The culture medium is not sealed in time after sub-packaging or sterilized immediately after sealing; The mouth of the culture container is not sealed, which causes sterilized bacteria and fungal spores to enter the culture container again and pollute the culture medium; The plastic cover and sealing film for sealing are easy to age and crack under long-term irradiation of fluorescent lamp, resulting in poor sealing performance, and careless use may lead to culture medium pollution; Rubber bands are easy to loosen and break under the irradiation of fluorescent lamps for a long time, and miscellaneous bacteria may take the opportunity to enter the culture container; Improper sterilization method leads to medium pollution; Too short sterilization time leads to incomplete sterilization of culture medium; When the sterilization time is up, open the sterilization pot immediately, resulting in excessive pressure difference in the container, which may lead to external miscellaneous bacteria being sucked back into the container [1]; After filtering more solution, the membrane filtration effect is poor, which makes the filtered solution contain bacteria and causes pollution after adding culture medium.
② culture container and inoculation apparatus. The culture container is not clean; The culture container is not completely disinfected after being polluted, which leads to pollution when it is used again; The culture container and inoculation apparatus in the autoclave or oven are too full, which affects the sterilization effect; The sterilization time of culture containers and inoculation instruments is too short, which leads to incomplete sterilization; Turn on the autoclave or oven immediately after sterilization. Strong cold and hot convection will cause cold air to be sucked into the envelope, causing pollution and bacteria again [1].
③ Inoculation personnel. Nails are too long; Unclean hands; There is a lot of dust on clothes and hair, so don't change slippers, vaccination clothes, hats and masks when vaccinating; Inadvertently bring the contaminated culture into the clean workbench and pollute it again after transfer; There are too many inoculated cultures in the culture container; Too many and too high objects on the ultra-clean workbench block the airflow, resulting in poor sterilization effect; Talking or coughing during vaccination will dial out a lot of bacteria; Hands are outside the workbench when operating; Touch the edge of the culture container, put your hands above the utensils and inoculation utensils, so that microorganisms fall into the culture container; When the plug, cover or sealing film of the culture container is opened, air enters and brings dust; During the inoculation process, the equipment was contaminated by hands or bacteria-bearing materials without disinfection, and the materials were contaminated after repeated use; Fungi fall into the container with the wind because the wind direction under the clean workbench is not operated [3]; After I left clean bench, I continued to vaccinate immediately. I took the culture medium, instruments and other items to a clean workbench for inoculation, with dust, which drifted into the vessel with the wind. Clothes, hats, masks and slippers are dirty after long-term wear, and there is a lot of dust on the surface, which will cause pollution after repeated wear without cleaning.
④ Inoculation room and culture room. There are a lot of bacteria and fungi in the air of inoculation room and culture room due to the entry and exit of personnel, especially when non-staff members enter and leave during inoculation; Unreasonable design of inoculation room leads to indoor dust and bacteria exceeding the standard; The clean workbench is placed in a place with too much dust, and the filter device fails due to too long use time and improper maintenance; The doors and windows of tissue culture room and inoculation room are not tightly closed, and flies fly in, which is conducive to the spread of miscellaneous bacteria; The culture room is easy to be polluted in a large area, and the indoor temperature is high, the air humidity is high, and the fungi multiply vigorously, which will also aggravate the pollution.
What factors can lead to pollution in plant tissue culture? How to prevent 98 pollution (Chinese words) editing
Pollution, a Chinese word, refers to getting dirty or harmful substances or being influenced by bad thoughts.
Chinese name pollution mbth pollution spelling w Sr m:n phonetic ㄨㄖㄢˇ synonym turbid pollution antonym.
1. [Pollution; Be * * * ear; Defile; Dirt: To stain with dirt or harmful substances.
Radiation polluted the whole area.
2. [Opinion poll] refers to being influenced by bad ideas.
Thought is polluted.
3. [* * * ears; Involve]: refers to framing or implicating [1]
What factors can lead to pollution in plant tissue culture? First, improper treatment of explants caused pollution.
Second, improper handling of contaminated Petri dishes caused secondary pollution.
Third, tools such as tweezers and scalpels are contaminated, the culture medium is not sterilized, and the workbench is not sterilized.
Fourth, there is no bacteria falling near the flame bottle mouth during inoculation, and the operation is not skilled to increase the pollution rate.
Fifth, the experimenters are noisy and work hard.
Large-scale pollution in intransitive verbs laboratory
What are the methods to control air pollution and their characteristics? Air pollution control can be divided into atmospheric dust removal, impurity removal, deodorization and disinfection.
Dust removal includes bag dust removal, electrostatic dust removal and spraying.
Impurity removal is waste gas treatment, such as desulfurization, including wet method, semi-dry method and dry method.
Deodorization includes biological deodorization, biological filter tower deodorization, activated carbon deodorization and so on.
What are the control methods of automobile noise pollution? There seems to be no way to see if you are at that angle. If you are in the car, you can only change the exhaust pipe list, and the effect may not be good.