What are the specific methods of sterilization?

A method of killing microorganisms (exceeding the maximum growth temperature) by high temperature. Principle of heating sterilization: When high temperature acts on microorganisms, the physiological and biochemical reaction rate in cells will be accelerated at first, and temperature-sensitive substances such as protein and nucleic acid in the body will be irreversibly damaged with the increase of temperature, which will eventually lead to the change of protoplast and the destruction of enzyme structure in cells, thus making cells lose the coordination of life functions and stop growth and development. Under the continuous action of high temperature, the intracellular protoplasm coagulates, the enzyme structure is completely destroyed, the activity disappears, the biochemical reaction stops, the metabolic activities such as osmotic exchange disappear, and the cell dies. Heat sterilization can be divided into dry heat sterilization and wet heat sterilization.

1) dry heat sterilization The sterilization method using burning or dry heat air without saturated steam is called dry heat sterilization. Dry heat sterilization is widely used in production because of its convenient use and simple method. For example, flame sterilization: microorganisms are directly burned to death by flame, so it is also called incineration sterilization. This method is thorough and rapid, but it is only suitable for the treatment of metal inoculation tools, test tube mouths and contaminated items. Hot air sterilization method: that is, dry hot air sterilization is used in an electric constant temperature drying box.

2) Wet heat sterilization is a method of steam sterilization. Wet heat sterilization can be divided into four types: high pressure, normal pressure, intermittent sterilization and pasteurization.

① High-pressure steam sterilization Because of the strong penetration of high-pressure steam, the temperature is higher than normal pressure, which can greatly shorten the sterilization time and improve the work efficiency. In addition, protein is easily denatured under hot and humid conditions. Under the condition of hot steam, all bacterial spores can be killed at 120℃ after 20 ~ 30 minutes. If the sterilized material is too large to penetrate, the pressure can be increased to 0. 152 MPa and extended to 1 ~ 2 hours. In high pressure steam sterilization, the sterilization temperature increases with the increase of steam pressure (Figure 2-6).

Figure 2-6 High pressure steam sterilizer

When using the autoclave, the air in the autoclave should be completely exhausted and replaced by saturated steam. If the air is not clean, the temperature in the pot will be lower than the temperature generated by pure saturated steam under the same pressure, which will affect the sterilization effect.

This method is suitable for sterilization of various heat-resistant articles, such as general culture medium, physiological saline solution, glassware, work clothes, etc. The steam pressure and time used should be determined according to the nature, volume and container type of the articles to be sterilized.

(2) Atmospheric steam sterilization This is a method of sterilization at 100℃ by using natural pressure and steam. It has simple equipment and low cost, and is the most widely used at present. As long as you build a stove and buy 1 ~ 2 cauldrons, which are made of bricks and cement, you can also use large iron drums and wooden barrels. The size can be decided by yourself, but it can't be too big. It is best to pack 800 ~ 1500 bottles. Problems that should be paid attention to when designing atmospheric pressure pot: the size depends on the production scale, and it is best to make the top of the pot into an arch shape so that condensed water can flow down the inner wall of the pot without wetting the cotton plug; Kitchen cabinets should have shelf structure, so that they can be loaded and sterilized in layers; A thermometer should be installed on the furnace to observe the temperature change in the furnace at any time; Because of the long sterilization time, the water in the pot is not enough to evaporate, so a water adding device should be installed in the large-capacity furnace; The sealing degree of the stove bin should be as high as possible, which not only improves the sterilization effect, but also saves fuel. Mushroom growers often use small steam generators in their production. After the steam is pumped out, it directly flows into the bacterial bag pile with wooden strips at the bottom and sealed with multi-layer plastic sheets for atmospheric sterilization. This method is simple and labor-saving without building a furnace (Figure 2-7). Normal pressure sterilization usually lasts for 8 ~ 10 hour after water is boiled, and it can be stuffy overnight.

Figure 2-7 Simple atmospheric sterilization method

(3) Atmospheric pressure intermittent steam sterilization method This is a method of repeated sterilization with atmospheric pressure steam. The specific method is to put the articles to be sterilized into a pot, and treat them at 100℃ for about 1 hour to kill the vegetative cells of microorganisms, and let them cool to about 30℃, at which time spores will germinate, and then heat them three times in the same way to achieve the purpose of sterilization. The method can be used for sterilization of drugs, nutrients and special culture media that are not resistant to high temperature.

(4) Low temperature pasteurization, that is, the method of killing harmful microorganisms at 60 ~ 70℃ for a certain period of time. Suitable for disinfection of articles that are not resistant to high temperature. Some media will be destroyed at high temperature. This method can not only kill the vegetative body of pathogenic microorganisms, but also prevent the components of the culture medium from being seriously damaged. In the production of edible fungi, the accumulation and fermentation process of culture materials is to use this principle to kill diseases, insects and miscellaneous bacteria.

(2) filtration sterilization method

It can be divided into two types: liquid filtration and air filtration, that is, a sieve with filter holes smaller than bacteria is designed by mechanical method to make various filters. Through mechanical filtration, only liquid culture medium or air flows out of the sieve holes, and all kinds of microbial cells remain on the sieve, thus achieving the purpose of sterilization. This method is suitable for the sterilization of liquid culture media (such as animal serum, protein, enzymes, vitamins, etc.). ) and thermally unstable gases. The working principle of Clean bench is to filter and sterilize the air with bacteria to form sterile air, which is blown out of the wind tunnel to create a sterile state within the workbench. The biggest advantage of filtration sterilization is that it does not destroy the chemical composition of various substances in the culture medium. Commonly used filters are diatomite, asbestos, ceramic soil, collodion and nitrocellulose filter membrane.

(3) radiation sterilization method

The method of sterilization by using the energy generated by radiation is called radiation sterilization. Radiation can be divided into ionizing radiation and non-ionizing radiation. Alpha rays, beta rays, gamma rays, X rays, neutrons, protons and microwaves are ionizing radiation, while ultraviolet rays, ozone and sunlight are non-ionizing radiation.

1) ultraviolet sterilization The principle of ultraviolet sterilization is to use ultraviolet radiation. Direct irradiation of bacteria with lamp tube causes photochemical reaction and induces the formation of thymine dimer in the cytoplasm of bacteria, thus inhibiting DNA replication and causing degeneration and death. On the other hand, ozone (O3) produced by air under ultraviolet irradiation also has a certain bactericidal effect. The effective range of ultraviolet rays is 1.2 ~ 2.0 meters. Generally, ultraviolet lamps are hung above the inoculation room or culture room, and the number depends on the size of the room. 1 ~ 2-person inoculation room can be equipped with 30 watt ultraviolet lamp. Before each inoculation, all necessary instruments should be put into the inoculation room (box) together, and then the ultraviolet lamp should be turned on for irradiation. If the inoculation room is large, turn on the light for 2 hours to achieve sterilization effect; If it is very small, it only takes about half an hour to turn on the light to achieve the sterilization effect. Because of the weak penetration of ultraviolet rays, even ordinary glass can not be filtered, so it is only suitable for sterilization of air or object surface. Ultraviolet rays can kill human skin, especially eyes, so avoid looking directly and turn off the ultraviolet light at work. During ultraviolet disinfection, if the workplace is slightly dark, the sterilization effect can be improved. Bacteria will be exposed to visible light immediately after receiving deadly ultraviolet radiation, and some bacteria may revive. Stem cells are more resistant to ultraviolet rays than wet cells, and spores are more resistant to ultraviolet rays than their vegetative cells.

2) Microwave sterilization Because the cells of microorganisms contain 70% ~ 90% water, water molecules are polarized in the microwave electric field and rotate with the change of electric field direction. In the process of rotation, high-speed friction between molecules produces heat energy, which is different from external heating, and can make cells explode in a short time, while the temperature of the object itself only rises slightly, thus achieving the sterilization effect. Using YM760 1 microwave oven can kill 1.92 million Escherichia coli in food within 60 seconds.

3) Ozone generator disinfection ozone (O3) has a strong oxidation effect, which can destroy the cell membrane and nucleic acid of microorganisms. O3 is also a transient substance, which can be naturally decomposed at room temperature and reduced to oxygen. Its sterilization principle is actually very similar to ultraviolet disinfection.