Cleaning, Disinfection and Sterilization Technology in Tissue Culture

1. All kinds of glassware used for cleaning plant tissue culture, especially culture bottles and culture medium containers, must be strictly cleaned to prevent oil stains, heavy metal ions, acids, alkalis and other harmful substances from remaining in the bottle and affecting the growth of the culture. Used glassware should be cleaned in time, the first dirt such as culture medium, culture, and then immersed in water, and then washed. The washing of glassware can be carried out in different ways according to the degree and nature of the contamination of the ware, usually with alkali washing and acid washing. Where there is microbial contamination of the utensils, must first high-pressure disinfection and boiling, in order to kill the bacteria, otherwise it will produce spores flying, pollution of the environment, to bring serious difficulties to tissue culture. After washing, the utensils should be dried or air-dried and placed in the specified place for easy access.

Commonly used sterilization methods can be divided into physical and chemical categories, namely: physical methods such as dry heat (baking and burning), humid heat (normal pressure or high pressure cooking), radiation treatment (ultraviolet, ultrasonic, microwave), filtration, cleaning and rinsing with a large amount of sterile water, etc. Chemical methods are treated with mercuric chloride, formaldehyde, hydrogen peroxide, potassium permanganate, bleach, sodium hypochlorite, antibiotics and alcohol chemicals. These methods and agents should be based on the work of different materials and different purposes of appropriate selection of hot and humid sterilization (culture medium) culture medium in the preparation of 24h after the completion of the sterilization process. The principle of autoclaving is: in a closed steamer, the steam can not overflow, the pressure continues to rise, so that the boiling point of water continues to increase, so that the temperature in the pot also increases. Under the pressure of O.1MPa, the temperature in the pot reached 121 ℃. At this vapor 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 vapor in the pot can be completely sterilized. There are several different ways of high pressure sterilization, but the purpose is to exhaust the air, so that the pot evenly heated, to ensure complete sterilization. The common method is: close the air release valve, after power, when the pressure rises to O.05MPa, open the air release valve and release the air, and then close the air release valve after the pointer of the pressure gauge returns to zero. After three times of bleeding, the valve shall be closed and then energized. When the pressure gauge rises to 0.1-0.15Mpa, it shall be maintained for 15-20min. • For items that do not deteriorate after autoclaving, such as sterile water, cultivation media, and inoculation tools, the sterilization time can be extended or the pressure can be increased. The culture medium should strictly abide by the pressure holding time, not only to maintain the pressure thoroughly, but also to prevent the deterioration of the components in the culture medium or reduce the effectiveness, and can not extend the time at will. For some cloth products, such as laboratory clothes, masks, etc. can also be autoclaved. Wash and dry with high pressure plastic bags, high pressure sterilization 20-30min. The pH of the medium before and after autoclaving decreased by 0.2-0.3 units. The direction and magnitude of changes in the pH of the medium after high pressure depend on a variety of factors. The opposite is true for raising the pH to a predetermined value with a base prior to autoclaving. When the medium contains a single component and the medium contains high or high concentration substances, the pH value after autoclaving changes greatly, even more than 2 pH units. Changes in environmental pH greater than 0.5 units are likely to have significant physiological effects. Autoclaving usually hydrolyzes the sucrose in the medium to monosaccharides, thereby changing the osmolality of the medium. In the range of 8%-20% sucrose, the medium after autoclaving was about 0.43 times higher. Iron in the medium catalyzes the hydrolysis of sucrose when autoclaved, allowing 15%-25% of sucrose to be hydrolyzed to glucose and fructose. The value of the medium is less than 5.5, and the amount of hydrolysis is more. When 0.1% activated carbon is added to the medium, the hydrolysis of sucrose under high pressure is greatly enhanced, and when 1% activated carbon is added, the hydrolysis rate of sucrose can reach 5%.

Caring sterilization (instruments used for aseptic operation) • During aseptic operation, immerse forceps, scissors, scalpel, etc. in 95% alcohol, and remove the burning bacteria in the alcohol lamp flame before use. After cooling, use immediately. 250 or 500ml jars can be used in the operation, with 95% alcohol in order to insert the tool.

Dry heat sterilization (glassware and heat-resistant appliances) Dry heat sterilization is the use of oven heating to 160-180 ~ C temperature to kill microorganisms. Because under dry heat conditions, the heat resistance of the vegetative cells of bacteria is greatly improved, which is close to the heat resistance level of the bud, it is usually sterilized at 170 ℃ for 90min. Dry heat sterilized items should be washed and dried in advance, and tools should be properly wrapped to avoid re-contamination when used after sterilization. Wrapping can be made of high temperature resistant plastic. The temperature shall be increased gradually during sterilization, and the time shall be recorded after reaching the top 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 can be opened until it is fully cold and cool, so as not to break the utensils due to sudden cooling. Dry heat sterilization energy consumption is too large, a waste of time.

Filter sterilization (heat-labile substances) Some growth regulators, such as gibberellin, zeatin, abscisic acid and certain vitamins, are heat-labile and cannot be autoclaved. Filter sterilization is usually used. Sterile nitrocellulose membrane with pore size of 0.20-0.45um can be used to filter bacteria. When the solution passes through the filter membrane, bacterial cells and fungal spores are blocked due to being larger than the diameter of the filter membrane. When the amount of liquid to be filtered and sterilized is large, a suction filtration device is often used. When the liquid volume is small, a syringe can be used. Sterilize it under high pressure before use, install the filter membrane at the needle tube of the syringe, load the liquid to be filtered into the syringe, push the piston rod of the syringe, press the solution out of the filter membrane, and the solution pressed out from the needle tube is sterile solution.

Ultraviolet and Fumigation Sterilization (Space) •(1) Ultraviolet Sterilization Use an ultraviolet lamp to sterilize in the inoculation room, ultra-clean table or inoculation box. Ultraviolet sterilization is the use of radiation factor sterilization, bacteria absorb ultraviolet light, protein and nucleic acid structure changes, causing bacterial chromosome variation, resulting in death. The wavelength of ultraviolet light is 200-300nm, of which 260nm has the strongest sterilization ability, but because the ability of ultraviolet light to penetrate substances is very weak, it is only suitable for sterilization of air and object surfaces, and it is required that the distance from the irradiated object should not exceed 1.2m.

(2) fumigation sterilization with heating incineration, oxidation and other methods, so that the chemical agent into a gas state diffusion into the air, in order to kill the air and the surface of the microorganism. This method is simple, only need to close the disinfection space tightly. The commonly used fumigant is formaldehyde. When fumigating, the room is closed tightly. According to the dosage of 5-8ml/m3, formaldehyde is placed in a wide-mouth container and 5g/m3 potassium permanganate is added for oxidation and volatilization. When fumigating, the room can be pre-sprayed wet to enhance the effect. Glacial acetic acid can also be heated fumigation, but the effect is not as good as formaldehyde. There are many types of chemical disinfectants. They denature the proteins of microorganisms, or compete with their enzyme systems, or reduce their surface tension, increase the permeability of the plasma membrane of bacterial cells, and cause the cells to rupture or dissolve. Generally speaking, the higher the temperature, the longer the action time, the better the sterilization effect. In addition, because the disinfectant must be dissolved in water to play a role, so to make a water-soluble state, such as mercury and potassium permanganate. And the concentration of disinfectant is generally the greater the concentration, the stronger the sterilization ability, but carbolic acid and alcohol exception.

Spray sterilization (surface of object) The surface of an object can be wiped and spray sterilized with some agents. For example, desktop, wall surface, hands, plant material surface, etc. can be repeatedly wiped and sterilized with 70% alcohol, and l%-2% lysol solution and 0.25%-1% negerin can also be used.

2. Aseptic operation

Plant tissue culture is a kind of aseptic technology. Therefore, it not only requires the whole operation process, all appliances, materials, culture medium, culture room and staff's clothes to be sterile, but also requires the staff to abide by the aseptic operation procedures in the operation process. If there is little negligence, it will cause irreparable consequences and make all previous efforts come to an end. Staff in the aseptic operation is to strictly observe the following points:

a. Before entering the sterile room, wash your hands and remove the dirt from your nails.

B. When entering the room, wear sterilized work clothes, hats, masks and shoes.

C. before the operation to use 70 of alcohol scrub the hands of the staff, the operation should often use alcohol scrub hands. Do not talk or breathe into the operating area to avoid microbial contamination of materials, media and appliances. The tool must be sterilized on the flame every time it is re-operated.

D. must be operated in the alcohol lamp flame, such as open the bottle mouth, transfer material. Before capping the bottle, burn the bottle mouth on the flame, then burn the lid on the flame, and then cover it.