Commonly used sterilization methods can be divided into physical and chemical types, namely: physical methods such as dry heat (baking and burning), moist heat (normal pressure or high pressure cooking), radiation treatment (ultraviolet, ultrasonic, microwave), Filtration, cleaning and a large number of sterile water rinses; chemical methods are treated with mercury, formaldehyde, hydrogen peroxide, potassium permanganate, lycopene, bleaching powder, sodium hypochlorite, antibiotics, alcohol chemicals. These methods and medicaments should be appropriately selected depending on the different materials used in the work. Table 1. Minimum time required for autoclaving of medium 2. The instrument used for aseptic operation is sterilized by burning In aseptic operation, scorpion, scissors, scalpel, etc. are immersed in 95% alcohol, and then sterilized by burning on an alcohol lamp flame before use. Use immediately after cooling. A 250 or 500 ml jar can be used in the operation, and 95% alcohol is placed in order to insert the tool. 3, glassware and heat-resistant appliances using dry heat sterilization dry heat sterilization is the use of oven heating to 160-180 ° C temperature to kill microorganisms. Since the heat resistance of the vegetative cells of the bacteria is greatly improved under dry heat conditions, close to the heat resistance level of the spores, it is usually sterilized by using 170 ° C for 90 minutes. Dry heat-sterilized items should be pre-washed and dried, and tools should be properly wrapped to avoid re-contamination when taken after sterilization. Wrapped with high temperature resistant plastic. The temperature should be gradually increased during sterilization, and the time should be recorded after reaching the predetermined temperature. The number of items placed in the oven should not be too much, so as not to hinder the heat convection and penetration. After the power is cut off at the specified time, the oven should be fully cooled to avoid the rupture of the vessel due to the quenching. Dry heat sterilization consumes too much energy and wastes time. 4. The heat-resistant substance is filtered and sterilized. Some growth regulators, such as gibberellin, zeatin, abscisic acid, and certain microorganisms are not heat resistant and cannot be autoclaved, usually by filtration sterilization. Some chemical components will degrade under high temperature and high pressure and lose their effectiveness or reduce their efficiency. After autoclaving, the activity of gibberellin GA3 was only 10% of that of the fresh solution not sterilized by high temperature. Sucrose is partially degraded to d-glucose and d-fructose after high temperature, and fructose can be partially hydrolyzed to produce a substance that inhibits the growth of cultured plant tissues. High temperatures also allow carbohydrates and amino acids to react. Vitamins have varying degrees of thermal stability, but if the pH of the medium is above 5.5, vitamin b1 is rapidly degraded. Calcium pantothenate, plant tissue extracts, etc. should be filtered and sterilized, and can not be sterilized by high temperature, otherwise it will lose its effect. The diameter of the mesh of the antibacterial filter is less than 0.45 μm. When the solution passes through the filtrate, the spores of bacteria and fungi are blocked by the diameter of the filter, and are often used when the amount of liquid to be sterilized by filtration is large. Filter device; when the volume is small, a syringe can be used. Before use, autoclave the filter, place the filter on the needle tube of the syringe, put the liquid to be filtered into the syringe, push the plunger of the syringe, press the solution out of the filter, and the solution from the needle is a sterile solution. . Filtration and sterilization operation steps: First, wrap the filter and the liquid bottle in paper, and the filter can be wrapped in paper in the culture dish. Before use, autoclave at 121 °C for 30 minutes. On the ultra-clean workbench, install the filter device, place the filter on the separator with sterile toothless tweezers, and the filter is rough facing up; The sterilized liquid is injected into the filter, and the vacuum pump can be started to filter and sterilize. The filtrate is cultured to prove that it can be stored for future use after aseptic growth. 5, the space is UV and fumigation (1) UV sterilization is sterilized by UV lamp in the inoculation room, on the clean bench or in the inoculation box. UV sterilization is the use of radiation factor sterilization. After the bacteria absorb ultraviolet light, the protein and nucleic acid undergo structural changes, causing chromosomal variation of the bacteria and causing death. The wavelength of ultraviolet light is 200-300nm, of which 260nm has the strongest bactericidal ability, but because of the weak penetration ability of ultraviolet light, it is only suitable for the sterilization of air and object surface, and it is better to not exceed 1.2 meters from the irradiated object. . (2) Fumigation sterilization by means of heating incineration, oxidation, etc., so that the chemical agent becomes a gaseous state and diffuses into the air to kill the air and the microorganisms on the surface of the object. This method is simple, and it is only necessary to close the disinfection space. There are many types of chemical disinfectants, which denature proteins of microorganisms, or compete with their enzyme systems, or reduce their surface tension, increase the permeability of bacterial membranes of cells, and cause cells to rupture or dissolve. In general, the higher the temperature, the longer the action time and the better the sterilization effect. In addition, since the disinfectant must be dissolved in water to function, it must be made into a water-soluble state, such as mercury and potassium permanganate. Also, the concentration of the disinfectant is generally the higher the concentration, the stronger the bactericidal ability, except for carbolic acid and alcohol. The commonly used fumigant is formaldehyde. When fumigation, the room is closed tightly. According to the dosage of 5-8ml/m3, the formaldehyde is placed in a wide-mouth container, and 5g/m3 potassium permanganate is added to oxidize and volatilize. When fumigation, the room can be pre-sprayed to enhance the effect. Glacial acetic acid can also be heated and fumigated, but the effect is not as good as formaldehyde. 6, some of the surface of the object with spray sterilization The surface of the object can be coated with some chemicals and spray sterilized. Such as table tops, walls, hands, surface of plant materials, etc., can be repeatedly smeared with 75% alcohol, 1% - 2% of the solution to the sulphate and 0.25% - 1% of the new germination. 7. Sterilization of plant material surface with disinfectant Plant materials selected from the outside or indoors are provided with various microorganisms to varying degrees. Once these sources are brought into the medium, they can cause contamination of the medium. Therefore, the plant material must be subjected to strict surface sterilization treatment and then inoculated onto the medium through aseptic processing, a process called inoculation. The inoculated plant material is called an explant. Wash with detergent powder, then rinse with tap water. The washing powder can remove the dirt slightly attached to the surface of the plant, remove the lipid substance, and facilitate the direct contact of the sterilizing liquid. Of course, the most desirable cleaning material is the surfactant Tween. The second step is to infiltrate the surface of the material. To be finished in a clean bench or inoculation box, prepare a beaker, glass rod, 70% alcohol, disinfectant, sterile water, watch, etc. for disinfection. Soak for 10-30 seconds with 70% alcohol. Since alcohol has the effect of soaking the surface of plant material, and 70% alcohol penetration is strong, it is also easy to kill plant cells, so the infiltration time should not be too long. There are some special materials, such as real, flower buds, bootings containing bracts, loquat leaves, dormant buds of multi-layered scales, etc., and mainly using internal materials, can be treated with only 70% alcohol for a slightly longer time. After the treated material is under aseptic conditions, the outer layer is peeled off after the alcohol is evaporated, and the internal material is taken. The third step is to use a sterilizing agent. There are many types of surface sterilizing agents, and one or two types can be used depending on the situation (Table 2). Table 2. Comparison of concentrations and effects of commonly used sterilizing agents Plant growth regulators are organic compounds that are artificially synthesized (or natural extracted from microorganisms) and have growth and development regulation similar to natural plant hormones. Insen Biotech is specilized in Plant Growth Stimulants with large capacity and competitive price, including 1-naphthylacetic acid, Indole acetic acid(IAA), Indole butyric acid(IBA), Triacontanol, Diethyl aminoethyl hexanoate(Da-6), Brassinolide, 6-benzyladenine(6-BA), Forchlorfenuron(CPPU), Gibberellin acid(GA3). Plant Growth Regulator,Indol Butyric Acid,Plant Growth Stimulant,Indole Butyric Acid Hunan Insen Biotech Co., Ltd , https://www.insenhealth.com
1. The medium is sterilized by using a moist heat sterilization medium within 24 hours after preparation. The principle of autoclaving is: in a closed steamer, the steam can not overflow, the pressure rises continuously, the boiling point of the water is continuously increased, and the temperature inside the pot is also increased. The temperature in the pot reached 121 ° C under a pressure of 0.1 mPa. At this steam temperature, various bacteria and their highly heat-resistant spores can be quickly killed.
Pay attention to completely eliminate the air in the pot, so that all the water in the pot is water vapor, and the sterilization can be thorough. There are several different practices for autoclaving and deflation, but the purpose is to remove the air and evenly heat up the pot to ensure complete sterilization. The common method is to close the bleed valve. After the power is turned on, when the pressure rises to 0.05mpa, open the vent valve and release the air. After the pressure gauge pointer returns to zero, close the bleed valve.
After the valve is turned off again, when the pressure gauge rises to 0.1 mpa, the timing is started, and the pressure is maintained at 0.1-0.15 mpa for 20 minutes.
The holding time varies depending on the size of the container. See Table 1. The numbers listed in this table are safe and sterilized numbers. If the container is large, but the number is small, it can also reduce the time.
For articles that are not degraded after autoclaving, such as sterile water, cultivation media, and inoculation tools, the sterilization time or pressure can be prolonged. The medium should strictly adhere to the pressure holding time, and it is necessary to maintain the pressure thoroughly, and to prevent the deterioration or effectiveness of the components in the medium, and not to prolong the time.
For some cloth products, such as laboratory clothes, masks, etc. can also be autoclaved. Wash and dry, put it in a high-pressure plastic bag, and press it for 20-30 minutes.
The pH of the medium before and after autoclaving decreased by 0.2-0.3 units. The direction and magnitude of the change in pH of the medium after high pressure depends on a variety of factors. When the components in the medium are single and the medium contains high or high concentration substances, the pH value after autoclaving varies greatly, and may even be greater than 2 ph units. A change in the environmental pH value greater than 0.5 unit may have a significant physiological impact.
Autoclaving typically hydrolyzes sucrose in the medium to monosaccharides, thereby changing the osmotic pressure of the medium. In the range of 8%-20% sucrose, the medium after autoclaving was increased by about 0.43 times.
Iron in the medium catalyzes the hydrolysis of sucrose during autoclaving, which can hydrolyze 15%-25% sucrose to glucose and fructose. When the medium value is less than 5.5, the amount of hydrolysis is more. When 0.1% activated carbon is added to the medium, the sucrose hydrolysis is greatly enhanced under high pressure. When 1% activated carbon is added, the hydrolysis rate of sucrose can reach 5%.
The following methods can be used to prevent some of the above changes caused by autoclaving:
(1) Always pay attention to the collection of information on the composition of the medium affected by autoclaving so that effective measures can be taken in a timely manner.
(2) When designing the medium formula, try to use a stable reagent with similar effect and accurately grasp the dose. If mannitol is avoided by using fructose and sorbitol, IAA is replaced by IBA, and the amount of activated carbon (below 0.1%) is controlled to pay attention to the influence of pH on the components in the medium under autoclaving.
(3) Appropriate grouping and order of addition of ingredients should be noted when preparing the medium. For example, phosphorus, calcium and iron are added at the end.
(4) Pay attention to the change and recovery dynamics of the pH value of the medium after autoclaving. For example, the ph value after autoclaving is often raised from 5.8 to 6.48. And after 96 hours, it will drop to around 5.8. This can be mastered in this experiment according to this law.
Kaiwei Yicheng's plant tissue culture antibacterial agent (PPM) is a broad-spectrum antimicrobial agent that kills bacterial and fungal cells, prevents fungal spores from germination, and eliminates endogenous contamination at higher concentrations. Explants.
First, remove the unused parts of the plant material and carefully clean the required parts, such as brushing with a suitable brush. Cut the material to the appropriate size, ie a sterile container can be placed. Rinse the tap water for a few minutes to several hours, and the rinse time should be based on the cleanliness of the material. Easy to float or small material, can be washed in a gauze bag. Flowing water rinses are especially useful when there is severe pollution.
When sterilizing, transfer the unwashed plant material into a beaker or other utensils, remember the time, pour in the disinfection solution, and gently stir it with a glass rod from time to time to promote the full contact of the various parts of the material with the disinfecting solution to remove air bubbles. Make disinfection thorough. 1-2 minutes before the time is approaching, start pouring the disinfectant into a prepared large beaker. Be careful not to pour out the material. Pour it into sterile water immediately after pouring it and gently rinse it. The sterilization time is from the time the liquid is poured into the disinfectant until the time it is poured into sterile water. Recording time also makes it easier to compare the disinfection effects in order to correct them. The sterilizing solution should be fully immersed in the material. It is better to use more sterilizing liquids. Do not use too much material to sterilize in a small container.
It is better to add Tween-80 or triton X to the sterilizing solution. The main function of these Surfactants is to make the agent easier to spread and easier to immerse into the surface of the sterilized material. However, the damage to the material after the addition of Tween is also increased. The dosage of the Tween and the sterilization time should be noted. Generally, 0.5% of the sterilizing solution is added, that is, 15 drops are added in 100 ml.
The last step is to rinse with sterile water. The rinse requires about 3 minutes, depending on the type of disinfectant used, and rinses about 3-10 times. Sterile water rinsing is a side effect of eliminating disinfectant killing of plant cells.
Container volume / ml
Minimum time required to sterilize at 121 ° C / min
20-50
15
75-150
20
250-500
25
1000
30
After reaching the dwell time, the power can be cut off. When the pressure reaches 0.5mpa, the steam can be slowly released. Care should be taken not to make the pressure drop too fast, causing intense decompression boiling and overflowing the liquid in the container. When the pressure drops to zero, the lid can be opened, the medium removed, and placed on the platform for condensation. Do not deflate for a long time, causing changes in the composition of the medium, so that the medium can not be inclined. Once placed for too long, the cover cannot be opened due to the negative pressure inside the boiler. As long as the bleed valve is opened, the atmospheric pressure is entered, the internal and external pressures are balanced, and the cover is easy to open.
Sterilizer name
Use concentration
Sterilization time / min
Sterilization effect
alcohol
70-75%
0.1-3
it is good
Mercury chloride
0.1-0.2%
2-10
well
bleaching powder
saturated solution%
5-30
well
Calcium hypochlorite
9-10%
5-30
well
Sodium hypochlorite
2%
5-30
well
hydrogen peroxide
10-12%
5-15
it is good
Antibiotic
4-50mg/L
30-60
better
The above sterilizing agent should be temporarily prepared before use, and mercury chloride can be stored for a short period of time. Sodium hypochlorite and calcium hypochlorite are sterilized by decomposition of chlorine gas, so it is better to use a jar to sterilize when sterilized; mercury is sterilized by heavy metal mercury ions; hydrogen peroxide is released by decomposition. Oxygen to sterilize, the effect of this agent residue is small, after sterilizing, it can be rinsed 3-4 times with sterile water; due to the material sterilized by mercury, it is difficult to remove the mercury residue, so it should be used The bacteria water is rinsed 8-10 times, each time not less than 3 minutes, in order to remove residual poison as much as possible.