(1) heating sterilization method
The use of high temperature to kill microorganisms (over the maximum growth temperature) method. The principle of heating sterilization: when the high temperature effect on microorganisms, the first cause of physiological and biochemical reactions within the cell rate accelerated, the body of temperature-sensitive substances such as proteins, nucleic acids, etc., with the increase in temperature and suffered irreversible damage, which led to changes in intracellular protoplasmic changes in the structure of the enzyme destruction, so that the cells lose the coordination of the function of life, stop the growth and development. With the continued action of high temperature, intracellular protoplasm will be solidified, the enzyme structure is completely destroyed, the activity disappears, the biochemical reaction stops, the osmotic exchange and other metabolic activities disappear, the cell death. Heating sterilization can be divided into two categories: dry heat sterilization and moist heat sterilization.
1) Dry heat sterilization The use of burning or dry hot air sterilization without the participation of saturated water vapor sterilization method is called dry heat sterilization method. Because dry heat sterilization is easy to use, simple methods, so widely used in production. Such as flame sterilization method: the direct use of flame to burn microorganisms to death, it is also known as incineration sterilization method. Sterilization using this method is both thorough and rapid, but only applies to metal inoculation tools, test tube mouths and contaminated items. Hot air sterilization method: that is, in the electric constant temperature drying oven using dry hot air to sterilize.
2) moist heat sterilization that is the use of steam sterilization method. Moist heat sterilization is divided into high-pressure, atmospheric pressure, intermittent sterilization and pasteurization of four kinds.
①High-pressure steam sterilization Because high-pressure steam has a strong penetrating power and higher temperature than atmospheric pressure, can greatly shorten the sterilization time, improve efficiency, coupled with proteins are easily denatured in hot and humid conditions, in the hot steam conditions, bacterial spores at 120 ℃, after 20 ~ 30 minutes can be all killed. If the sterilized material is large, not easy to be penetrated, the pressure can be increased to 0.152 MPa, extended to 1 to 2 hours. In autoclaving, the sterilization temperature rises with increasing steam pressure (Figure 2-6).
Figure 2-6 Autoclave
When using an autoclave, it is important to completely remove the air from the pot and replace it with saturated steam. If the air is not removed, the temperature inside the autoclave will be lower than that produced by pure saturated steam at the same pressure, which will affect the sterilization effect.
This method is applicable to the sterilization of various heat-resistant items, such as general culture medium, saline and other solutions, glassware, work clothes. The steam pressure and time used should be decided according to the nature of the items to be sterilized, volume and type of container.
② atmospheric steam sterilization This is the natural pressure, 100 ℃ steam sterilization method. It is simple equipment, low cost, the current use of the most widely used. Just build a stove, buy 1 to 2 pots, above the brick and cement, can also be used in large iron drums, wooden barrels, etc., the volume size can be decided, but should not be too large, in order to load 800 to 1500 bottles as well. Design of atmospheric stove should pay attention to the problem: size according to the production scale to determine the top of the stove is best made of arched round, so that condensate can be downstream along the inner wall of the stove and will not wet cotton plugs; stove warehouse should be a layer of shelf structure, in order to be layered into the sterilized material; stove should be installed on the thermometer, you can always observe the stove temperature changes; due to the sterilization time is long, not enough evaporation of the water in the pot, the stove should be installed to add water to a stove with a large capacity; the warehouse warehouse should be closed as high as possible, so as to fill 800 ~ 1500 bottles good. The degree of closure should be as high as possible, so as to improve the sterilization effect and save fuel. Mushroom farmers in the production of a small steam generating device is also commonly used, after the introduction of steam directly to the lower side of the moat with wooden bars, surrounded by a multi-layer plastic sealing of the pile of bags for atmospheric sterilization. This method does not need to build a stove, simple and labor-saving (Figure 2-7). Atmospheric sterilization generally boil water to keep 8 to 10 hours, smothered overnight can be.
Figure 2-7 simple atmospheric sterilization method
3 atmospheric intermittent steam sterilization method This is the use of atmospheric steam sterilization method repeated several times. Specific approach is to put the items to be sterilized in the pot, 100 ℃ treatment for about 1 hour to kill the nutrient cells of microorganisms, let it cool to about 30 ℃, at this time the buds will sprout, and then the same method of heat treatment, repeated three times, can achieve the purpose of sterilization. This method can be used for sterilizing drugs, nutrients and special culture media that are not resistant to high temperature.
4 low-temperature pasteurization method that is 60 ~ 70 ℃, after a certain period of time, the method of killing harmful microorganisms. Adapted to the sterilization of items that do not tolerate high temperatures. Some of the culture medium, destroyed at high temperatures, with this method can kill the nutrients of disease-causing microorganisms, but also to make the composition of the medium will not be seriously damaged. Edible fungi production in the culture material pile fermentation process, is to use this principle to kill the pests and stray bacteria in it.
(2) filtration sterilization method
And divided into liquid filtration and air filtration of two kinds, that is, the use of mechanical methods, the design of a pore smaller than the bacterial sieve, made into a variety of filters, through the mechanical filtration, only to let the liquid medium or air from the sieve out of the holes, a variety of microbial organisms stay on the sieve, so as to achieve the purpose of sterilization. This method is suitable for the heat instability of the small volume of liquid culture medium (such as animal serum, protein, enzymes, vitamins, etc.) and the sterilization of gas. The working principle of the ultra-clean bench is to form sterile air by filtering and sterilizing the germ-carrying air, and blowing it out from the wind tunnel to cause the sterile state of the bench range. The biggest advantage of filter sterilization is that it does not destroy the chemical composition of various substances in the culture medium. Commonly used filters are made of diatomaceous earth, asbestos, ceramic clay, but also made of fire cotton glue, nitrocellulose filter membrane.
(3) radiation sterilization method
The use of radiation generated by the energy sterilization method called radiation sterilization. Radiation can be divided into ionizing radiation and non-ionizing radiation, alpha rays, beta rays, gamma rays, X-rays, neutrons and protons, microwaves and other ionizing radiation, ultraviolet light, ozone, sunlight for non-ionizing radiation.
1) ultraviolet sterilization The principle of ultraviolet sterilization is the use of ultraviolet radiation. Direct irradiation of bacteria with lamps to cause photochemical reactions, the bacterial cytoplasm induced the formation of thymine dimer, thereby inhibiting the replication of DNA and the occurrence of denaturation, lethal. On the other hand, ozone (O3) produced by air under UV irradiation also has a certain bactericidal effect. The effective action distance of UV light is 1.2 to 2.0 meters. Ultraviolet light is generally suspended in the inoculation room or culture room above, the number of room size depends on the inoculation room for 1 to 2 people to operate, the installation of a 30-watt ultraviolet light can be. Before each inoculation, the required equipment should be put into the inoculation room (box), and then turn on the ultraviolet light irradiation. If the inoculation room is large, turn on the lamp irradiation for 2 hours to achieve sterilization effect; if smaller, only need to turn on the lamp for about half an hour can achieve sterilization effect. Due to the weak penetration of ultraviolet light, even ordinary glass can not filter, therefore, only suitable for air or object surface sterilization. Ultraviolet light on human skin, especially the eyes have a killing effect, should avoid looking directly at the work should be turned off the ultraviolet lamp. If the workplace is in a slightly dark and lightless situation during UV disinfection, it can improve the bactericidal effect. Some bacteria may be revived when given visible light immediately after receiving a lethal dose of UV irradiation. Stem cells are more resistant to UV than wet cells, and spores are more resistant to UV than their trophoblasts.
2) microwave sterilization As the microbial cells contain 70% to 90% of the water, water molecules in the microwave electric field is polarized, and with the change in the direction of the electric field and the rotation of the molecules in the process of rotation of the high-speed friction between the heat generated by the thermal energy, which is different from the external heating, can be a short period of time to make the cell bursting and the object's temperature itself is only a very slight increase in the sterilizing effect. The effect of sterilization can be achieved. It takes only 60 seconds to kill 1.92 million E. coli bacteria in food with YM7601 microwave oven.
3) Ozone generator disinfection Ozone (O3) has a strong oxidizing effect, can destroy microbial cell membranes and nucleic acids. O3 is also a transient substance, room temperature can be naturally decomposed, reduced to oxygen. The principle of sterilization is actually very similar to that of ultraviolet disinfection.