Section 1 Preparation of Medium
First, the preparation of principles and types
(A) The principle of medium preparation
1. Medium: The medium is a substrate that provides nutrients and specific environmental conditions for the growth and development of edible fungi and other organisms, such as mushroom fungi are grass-rotating bacteria in organic nutrition. The preparation of the culture medium should not only consider the carbon-containing compounds, nitrogen-containing compounds, mineral salts, and growth factors it needs, but also the water and pH environment, and whether they should be cultured in solid or liquid culture. Therefore, the sterilized nutrient matrix prepared according to the ratio required for the growth and development of fungi can be called a culture medium. In other words, the medium must have three conditions: it contains the nutrients needed for the growth and development of the strain; it has environmental conditions for growth of the fungi; it is sterilized and remains sterile.
2, the choice of medium: In the whole process of preparation of the bacteria, from the mother to the original species, cultivars, the purpose of the various stages of the different requirements, the choice of the ratio of the medium should also be different. In general, the mother primordial mycelium is more tender and weak, and its ability to decompose nutrients is poor, requiring nutritious and complete nutrition. The ratio of nitrogen source and vitamins should be high. Must use substances that are easily absorbed by mycelium, such as glucose, sucrose, potato, peptone, inorganic salts, and auxin, etc., while the original species, cultivars need more, and the hyphae decomposition ability is strong. A large amount of crop straw, manure, cottonseed husk, bran, rice bran and other raw materials are used as the culture medium.
(b) Types of medium
1, according to the source of nutrients, the medium can be divided into inflamed natural medium, semi-synthetic medium and synthetic medium.
(1) Natural medium The natural medium refers to a medium prepared by using natural organic substances whose chemical composition is not yet known or whose chemical composition is not constant, such as various agricultural and sideline products and leftovers—wheat, soybeans, corn flour, and bran Skin, rice bran, crop stalks, sawdust, cottonseed hulls, sugar cane bagasse, etc., as well as extracts from animal and plant tissues - beef extract, broth, potato juice, wort, bean sprouts, etc. can all be used to prepare natural medium. The medium has a wide source, low cost, and rich nutrition, and is suitable for the large-scale cultivation of fungi in production. However, due to the differences in the origin, species, and growing period, the chemical composition of these natural organic substances cannot be declared. Each batch of ingredients is also unstable, and should not be used to make detailed scientific experiments. Separation and domestication of edible mushroom culture medium
(2) Semi-synthetic medium In order to promote the growth and development of edible mycelium, it is often added a suitable amount of inorganic salts in the natural medium, or a certain amount of certain natural organic substances are added to the synthetic medium to become a semi-synthetic medium. . This type of medium has a wide range of applications and is widely used. It is also the most commonly used medium for growth strains and practical cultivation.
(3) A synthetic medium refers to a medium prepared by using organic substances (carbohydrates, nitrogen-containing compounds, organic acids) or inorganic substances with known chemical compositions. The composition and content of this type of culture medium are clear and the price is relatively expensive. Generally, it is used in the laboratory to do nutrient, metabolism, strain identification, and so on. There is a physiological and biochemical research on edible fungi.
2. According to the physical state after the culture medium is made, the culture medium can be divided into liquid culture medium, semi-solid culture medium and solid culture medium.
(1) Liquid medium According to the nutrients required by the edible fungi, a certain proportion of the nutrient solution is prepared and called a liquid medium. The liquid medium can be used for screening experiments of nutrient sources, as well as physiological and biochemical aspects such as enzyme activity, etc. Liquid culture medium-producing strains can also be used in production.
(2) Curing Medium After a variety of nutritional ingredients are formulated into a nutrient solution in proportion, an appropriate amount of a curing agent, such as agar, can be formulated into a solidifying medium. The use of this slant tube can preserve and expand the strains, and can also be made into various strains of plate-solidifying medium.
(3) Solid medium A variety of agricultural and forestry wastes rich in cellulose and lignin such as sawdust, cottonseed husks, straw, and corn kernels are used as the main raw materials, and appropriate amounts of rice bran or bran and inorganic salts are added to make solids. Culture medium. The medium can be used as a culture medium for the production of second, third, and class bacteria, and it can also be used as a culture medium for fermented grass powder deposits.
Second, a kind of medium
The primary species is also known as the mother species. Its culture medium generally uses test tubes as containers, so it is also called test tube slant culture medium. This type of culture medium is often used for the separation and purification of strains, expansion, transfer of tubes and preservation of bacteria, etc. Strain production commonly used formula.
1. Potato Dextrose Agar (PDA)
Potatoes (peeled) 200 g Glucose 20 g Agar 18-20 g Water 1000 ml
2. Potato Comprehensive Culture Medium (CPDA)
Potato 200g Glucose 20g Potassium Dihydrogen Phosphate 3g Magnesium Sulfate 1.5g
Vitamin B1 0.05 mg agar 18-20 g water 1000 ml
3. Potato yeast extract medium (PDYA)
Potato 200 g glucose 20 g yeast extract 1.5 g agar 18-20 g water 1000 ml
4. Bran glucose media
Bran 50-70g Glucose 20g Agar 18-20g Water 1000ml
5. Composting dip glucose medium
Compost (dry) 100 g glucose 20 g agar 18-20 g water 1000 ml
6. Corn flour synthesis medium
Corn flour 20-30 g Glucose 20 g Potassium hydrogen phosphate 1 g Magnesium sulfate 0.5 g
Peptone 1 g agar 18-20 g water 1000 ml
9.EBLamberr medium
Glucose 10 g magnesium sulfate 0.5 g potassium dihydrogen phosphate 1.9 g agar 20 g water 1000 ml
Suitable for the spore germination of Agaricus bisporus
III. Second and third seed media
The second and third kinds of medium usually use natural nutrients such as straw, cottonseed husks, bagasse and other crop wastes, and add appropriate amount of inorganic salts, etc. to make solid medium. Now the commonly used medium formula is introduced as follows.
Fecal culture medium
Fermented wheat straw 72% Fermented cow dung 20% ​​Bran 5% Sugar 1% Calcium phosphate 1% Gypsum 1% Moisture content 62-65% Suitable for secondary species
2. Cow dung powder culture medium
Fermented cow dung powder 98% Calcium carbonate 2% Moisture content 62-65% Suitable for secondary species
3. Cottonseed husk medium?
Fermented Cotton Seed Hull 97% Superphosphate 1% Calcium Carbonate 1% Lime 1% For Second and Third Grades
4. Sheep manure medium
Dry sheep feces 98% Calcium carbonate 2% Moisture content 62-65% Suitable for secondary species
5. Mineral culture medium
Expanded Perlite 1450g Bran 1650g Gypsum Powder 200g Calcium Carbonate 50g Water 1650g pH6.2-6.4 Suitable for Secondary Production
6. Grain medium
Wheat grains (wheat, barley, oats, sorghum, etc.) 97% calcium carbonate 2% gypsum 1% water content 50-55% pH6.5- 7.0 suitable for second and third grades
7. Wheat rice medium
88% wheat 10% calcium carbonate 2% suitable for three-grade species
8. Wheat cow dung powder medium
88% wheat dung powder 10% calcium carbonate 2% suitable for three-grade species
Section 2 Disinfection and Sterilization
First, the concept of disinfection and sterilization
In the extended world, microbes are everywhere and permeable, such as raw materials, water, tools, equipment, hands, clothing, and space used for seed production. Therefore, to separate and cultivate edible mushroom purebreds, first the medium must be sterilized to a sterilized state, and all links and environments of the production process must also be disinfected, so as to reduce the survival of other microorganisms and cause production pollution. Sterilization and sterilization are one of the most important key technologies that must be mastered during the production of strains.
(A) Disinfection has the effect or method of killing pathogenic microorganisms, called disinfection. For the production of edible mushroom strains, disinfection refers to the use of physical or chemical methods to kill or eliminate microorganisms that are harmful to the growth and development of edible fungi. Usually, only a part of microorganisms on the surface or in the environment can be eliminated, and all microorganisms can be eliminated.
(b) Sterilization refers to thorough sterilization, ie, the use of physical or chemical methods to kill all microorganisms on an object, as well as their spores and spores, to make the object sterile.
Second, commonly used disinfection and sterilization methods
(I) Physical Sterilization Methods Sterilization or sterilization methods using high tide, radiation, microwaves, and instruments are physical sterilization methods.
1. Heat sterilization The method of using climax to kill microorganisms is called heat sterilization. The most important components of microbes are proteins, nucleic acids, etc. When climax, it will cause irreversible denaturation or coagulation of proteins and nucleic acids, which will cause cells to lose their physiological functions, stop their growth and development until the biochemical melon stops and ends their lives. In addition, the high temperature can still destroy other components of the cell, or cause the fat film of the cell to be dissolved by heat to form a very large pore, resulting in leakage of cell contents and causing death, thereby achieving the effect of climax sterilization.
Heat sterilization often uses two bactericidal indices: heat dead and heat dead. The former refers to the temperature at which all cells in the bacterial suspension can be killed within 10 minutes, and the latter refers to the time required to kill all cells at a certain temperature. Under the same temperature conditions, microorganisms with high thermal death temperatures have a slow thermal death rate, and microorganisms with a low thermal death temperature have a rapid thermal death rate. Increasing the sterilization temperature can speed up the thermal death rate. Therefore, the sterilization time can be shortened by increasing the sterilization temperature, or the sterilization time can be extended to reduce the sterilization temperature.
Heat sterilization is a cost-effective, simple and easy method. It is the most commonly used method. It can be divided into two kinds of heat sterilization and dry heat sterilization.
(1) Wet heat sterilization refers to the use of hot steam to kill microorganisms. Because steam penetrates stronger than hot air, protein and protoplasmic colloids tend to denature and coagulate under hot and humid conditions, and the enzyme system is easily destroyed. Steam enters the cells and condenses into water, which releases potential heat to increase the temperature, and enhances sterilization. . Commonly used hot and humid sterilization methods are as follows.
A. High pressure steam sterilization method using high temperature and high pressure steam sterilization. Autoclave sterilization kills all microorganisms, including bacterial spores, fungal spores or dormant individuals, and other heat-resistant individuals. The temperature of the sterilized steam increases as the steam pressure increases, increasing the steam pressure, and the sterilization time can be greatly reduced. Therefore it is one of the most effective and widely used sterilization methods. The steam pressure and sterilization time used for autoclaving should be based on the specific sterilization material. When the liquid medium is sterilized, generally 1.0 Kg/cm2 is used, the temperature is 121.30° C., and sterilization is performed for 30 minutes; when a solid medium such as a parent species or a cultivar is sterilized, 1.5 Kg/cm 2 is usually used, and the temperature is 129° C. Sterilization 1 -2.5 hours. However, some solid mediums have poor thermal conductivity and there are many heat-resistant microorganisms in the medium, such as river mud drying medium for mushroom cultivation, sterilization time or sterilization pressure should be increased, usually the pressure is 2Kg/cm2. The sterilization time is 4 hours.
When using autoclave sterilization, the following points should be noted:
a. The air conditioner in the sterilizer must be drained. The coefficient of thermal expansion of the cold air is large. If there is cold air in the sterile pan, when the sterilizer is sealed and heated, the cold air is rapidly heated and the pressure rises, resulting in inconsistent pressure and temperature in the sterilizing pan, resulting in false steam Pressure, the pot temperature is lower than the corresponding temperature indicated by the steam pressure, so that sterilization can be thorough. Especially when using an sterilizer with only a pressure gauge and no thermometer, care should be taken.
There are two ways to eliminate cold air: slow exhaust and centralized exhaust. The slow exhaust method, which starts the heat sterilization, opens the exhaust valve. As the temperature in the retort gradually rises, the cold air in the pan gradually drains. When the temperature in the pan rises to 100°C, a large amount of steam flows from the exhaust valve. When it is discharged, the exhaust valve can be closed and pressure sterilized. Centralized exhaust method, that is, when the heat sterilization is started, the exhaust valve is closed first. When the pressure rises to around 0.5Kg/cm2, the exhaust valve is opened, the air is exhausted, the pressure is reduced to zero, and a large amount of steam is discharged. At that time, close the vent valve again for boost sterilization. When the solid medium is sterilized, a centralized exhaust method can be used. When the liquid medium is sterilized, it is generally not suitable to avoid the liquid rushing out of the bottle mouth
b. The medium in the sterilizer must be loosely arranged so that the steam can flow freely. Whether the steam in the sterilizing pot is unimpeded is related to whether the sterilization temperature is uniform or not. If the sterilization material is put too much and too dense, it will hinder the circulation of steam, affect the uniformity of the temperature distribution, cause the temperature in the local area to be low, and even form a temperature "dead angle", can not achieve thorough sterilization, often leads to hand miscellaneous Bacterial contamination.
c. Sterilization should be done slowly. The high pressure steam sterilization after the end of the exhaust pressure can not be too fast, if the exhaust is too fast, the pressure difference between the inside and outside the bottle will increase, causing the bottle to rush out of the bottle; liquid medium sterilization, the liquid will suddenly boil Dirty stoppers; plastic bags should be allowed to cool naturally when they are sterilized. When the pressure drops to about 0.5Kg/cm2, the exhaust valve is opened again to prevent air bags from being generated inside or outside the decompression process. The pressure difference breaks down the plastic bag, causing contamination of the bacteria in the future.
d, pay attention to tampon moisture. When a sterilized bottle or test tube is sterilized in a high-pressure steam sterilizer, condensed water is easily generated around the wall of the pot and inside the pot lid due to the hot steam in the pot. Condensed water flows down the pot wall and lid, and Wet tampon. In the course of cultivation, wet tampons are prone to grow molds such as Neurospora spp. and contaminate cultures. In order to prevent the tampon from being wet, the tampon of the bottle cannot contact the wall of the sterilizer when sterilizing, and the aluminum cap or waterproof oil paper is covered on the bottle to prevent the water under the lid from flowing to the tampon. At the end of the sterilization, hot pot water should be drained first, and then the steam should be slowly discharged. If the tampon is wet, it can be baked in the pan for a while and then removed.
B. Atmospheric pressure steam sterilization method using natural pressure steam sterilization. Arnold circulation steam sterilizers are commonly used equipment in microbiological technology. In the production process of edible fungus strains in China, some use steamer sterilization, some use reinforced steel cement to build large-scale circulating steam sterilization stoves, and can sterilize thousands of bottles of parent species or cultivating culture materials at a time.
When sterilizing with normal pressure steam, the sterilized articles can not be arranged too tightly in the sterilizing pot, and it is necessary to ensure that the steam can circulate uniformly in the pot. The temperature of circulating steam is generally about 100°C. To kill heat-resistant spores, it is necessary to extend the sterilization time, which is generally 6-8 hours. The biggest advantage of this kind of dedicated circulation steam sterilizer is: large capacity, simple structure, low cost, and self-construction. Disadvantages are long sterilization times, large energy consumption, and little attention is paid to incomplete sterilization.
C. Intermittent sterilization refers to a method of intermittent sterilization for a certain period of time. This method is used to sterilize high-temperature (100°C or so) culture media. To kill the spores of the culture medium, it can be sterilized in circulating steam for 20-30 minutes. The vegetative cells in the medium are killed first, and then the culture medium is incubated at room temperature or in an incubator to grow the spores into a vegetative body. Then use circulation steam to sterilize for 20-30 minutes to kill the newly grown vegetative cells. In this way, 3 consecutive times, all the spores in the medium can be killed and the sterility can be achieved.
(2) Dry heat sterilization Sterilization by burning or dry hot air is called dry heat sterilization. Although the penetrating power of the dry main hot air is not as strong as the hot and humid steam, it is easy to use and is suitable for sterilizing glassware, porcelain, etc. Therefore, it is widely used in laboratories and production practices. There are two main types of dry heat sterilization.
A. Flame Sterilization is a method of sterilizing by high temperature flame burning. Heat-resistant inoculation loops, inoculum shovels, inoculation spoons, inoculation needles, etc., can be completely sterilized by flame burning, test tube mouth and glass bottle mouth, through several flames, temperature up to 200 °C above, all microorganisms and spores, All can be killed to achieve sterility.
B. Dry heat sterilization is a method of sterilization using heated high-temperature air. Dry hot oven is a commonly used instrument for dry heat sterilization. It is heated and regulated by electric heating wire. It can not only be used for sterilization, but also used for drying materials such as utensils. Dry heat sterilization is suitable for solid material sterilization and is not suitable for liquid material sterilization. Due to the poor penetration of dry hot air, combined with microbial protein under dry conditions, not easy to coagulation and deterioration, so dry heat sterilization temperature, generally required to master at 160 °C, maintain 2 hours. However, it should be noted that if the dry heat sterilization temperature exceeds 170°C, the paper or cotton cloth used to sterilize the product will be scorched by the hot air and may even burn.
2, radiation sterilization The use of radiation generated energy to sterilize the method, said radiation sterilization. The application of radiation sterilization methods in production mainly refers to external sterilization.
Ultraviolet sterilized UV is non-ionizing radiation, with a wavelength of 136-400 nm, and the sterilization is the strongest at a wavelength of 265-266 nm. The nucleic acid bases in the microbial cells are particularly strong in ultraviolet absorption ability, causing nucleic acid mutations after irradiation, thereby inhibiting the replication of DNA and causing death; at the same time, some of the oxygen in the air generates ozone under ultraviolet irradiation, which is also very strong. Sterilization. In addition, ultraviolet irradiation of microbial cells produces a photochemical oxidation reaction in the presence of oxygen to generate hydrogen peroxide (H2O2), which can strongly oxidize and cause cell death to achieve sterilization. Due to the poor ability of UV to penetrate substances, a layer of ordinary glass or water can filter out a large amount of ultraviolet rays, so it is only suitable for the sterilization of air and the surface of the object, generally requires that the UV lamp is not more than 1.0 meters from the irradiated object. The inoculation room and the inoculation box are commonly used as ultraviolet and air disinfection on the desktop.
3, filter sterilization is the use of mechanical retention method to remove the microorganisms in the medium. The equipments used in the production of strains mainly include ultra-clean benches and air cleaners, all of which are sterilized by air filtration. The ultra-clean bench is mainly used to achieve partial sterilization of the operating table, and the air self-cleaner is used to make the inoculation room. The air is filtered to achieve sterility throughout the space.
(b) Chemical sterilization methods
Using chemicals to sterilize or create a sterile environment is also a very important sterilization method. Many performance medicines can denature bacterial proteins, and some can hinder a certain metabolic link of microorganisms. Therefore, chemicals can be bactericidal or bacteriostatic. Direct addition of the medium can act as a sterilizing agent without affecting the chemical composition of the medium components. According to reports, β-propiolactone (BPL) and diethyl pyrocarbonate (PKE) have such effects, but application less. Some of them have bactericidal and bacteriostatic effects, but they hinder the growth and development of Lentinus edodes after they are added to the medium and cannot be used. Chemicals commonly used in mushroom production and specific methods of use are as follows.
1, formaldehyde sterilization mechanism is a strong reduction. Formaldehyde binds to the amino group of microbial proteins (including proteases) to denature proteins. The reaction formula is: protein-amino+formaldehyde (gas)→protein-amino and formaldehyde. The formaldehyde solution has stronger bactericidal power than stearic acid. The formaldehyde solution of 1:200 can kill all bacterial spores in 6-12 hours; it has extremely strong lethality to fungal spores and vegetative bodies, and is not affected by organic substances. . Because it is corrosive and irritating, it should not be used by the human body. Commonly used formalin sterilized empty cans, sterile rooms or sterile boxes.
The use of heating fumigation method, the use of formalin boiling point 96! 0 characteristics, the formalin placed in glass, ceramic or metal containers, heated with alcohol lamp or electric furnace, the liquid evaporated, immediately remove the heat source .
The addition of potassium permanganate oxidant fumigation method, that is, formaldehyde is a reducing agent, so the reaction with the oxidant can generate a lot of heat. This heat in turn volatilizes formaldehyde. The specific method is to measure the ratio of 10 ml of formalin and 1.5 g of potassium permanganate per cubic meter of volume in the crucible (or using a petri dish), allow the formaldehyde to evaporate, and close the doors and windows for a night or longer. Time, to achieve a good purpose of fumigation sterilization. After fumigation, the interior smells of irritating nose. When it is necessary to drive off indoor formaldehyde odor, ammonia can be sprayed into the room. The concentration of ammonia is 25-28%. The smaller the spray mist, the better. The amount of ammonia sprayed is half that of fumigation, about 30 minutes, ammonia and air. The residual formaldehyde combined to form a white odorless powder. If there is no ammonia, ammonium chloride or ammonium sulfate plus lime water can be used to produce ammonia to neutralize formaldehyde. The amount of ammonium salt is 5-6 grams per cubic meter of space.
2. Sulphur and its method of fumigation sterilization Commercial sulphur is a shiny pale yellow powder (or lumps), insoluble in water, and has a melting point of 363°C and generates sulphur dioxide after combustion. Sulfur dioxide is a colorless gas, has a stimulating odor, strong penetration, soluble in water, generating sulfurous acid, helps to increase the sterilization rate, while killing insects, killing.
The method of use is to measure 15-20 grams per cubic meter of space, placing sulfur in a porcelain or metal container higher than the ground, ignite sulphur to produce sulphur dioxide, the density of sulphur dioxide is greater than air, it is easy to sink, so we must pay attention Place sulphur containers in high-spatial locations. In order to improve the sterilization effect, a small amount of water mist should be sprayed on the space and walls before fumigation, which will help increase sulfur dioxide and generate sulfite, thereby improving the effectiveness of fumigation sterilization.
After the ignition of sulfur, it is irritating to human respiratory tract mucosa and conjunctiva. Attention should be paid to protection. Sulfur dioxide has a corrosive effect on fibers and metal objects. Therefore, erodible articles should be withdrawn when fumigating the house.
In addition to fumigation and internal suction, other chemical sterilization methods can be summarized as surface sterilization. Their toxic effects on microorganisms are mainly the rapid destruction of cell surface proteins, nucleic acids and related enzymes.
3, ethanol ethanol is also known as alcohol. 70-80% ethanol has a strong bactericidal capacity. The bactericidal capacity of high-concentration ethanol is reduced, because 95-100% of ethanol has a strong dehydration function. When the microbial cells are encountered, the cell surface dehydrates quickly to form a protective film, and the ethanol molecules do not penetrate the protective film to sterilize. effect. For example, adding dilute acid or dilute alkali to ethanol can increase the germicidal efficacy. Adding 1% sulfuric acid or sodium hydroxide in 70% ethanol can kill Bacillus subtilis spores within 1-2 days. Ethanol will only kill bacterial vegetative bodies. In the production of shiitake mushrooms, it is mostly used to sterilize the skin and instruments of the hands when inoculated. When used, wipe the surface-dried article with 70-75% ethanol; use 85% ethanol for the slightly wetted surface. Ethanol can reduce bactericidal capacity such as phenol and formaldehyde, but it can enhance the bactericidal efficacy of sulfur and mercury.
4, phenol and its derivatives are commonly used stone carbonic acid, also known as phenol, pure colorless or white needle, block or triangular prism crystal, soluble in water, with a special smell. Carbonic acid can make the skin white, fingers paralyzed and can damage the skin and mucous membranes, toxic to humans and animals. A 0.5-1% aqueous solution of stony carbonic acid is used as a spray to treat sterile chambers (tanks), which can act as a dust-removing, sterilization, and air-disinfecting agent; it can also be sterilized by soaking in a 3-5% aqueous solution of stony carbonic acid. The addition of 0.85-0.9% of salt in 5% aqueous solution of mineral carbonic acid can increase the effect of stearic acid. If ethanol is present, the efficacy is greatly reduced.
The principle of stone carbonic acid sterilization is to denature bacterial proteins, and also has a surfactant effect, which destroys the permeability of cell membranes and can cause spillage of cellular contents. When the concentration of carbonated carbonic acid solution is large, it is a lethal factor; otherwise, it acts as a bacteriostatic effect.
Cresol is a derivative of phenol, also known as sol (saponin), which is a cresol that is emulsified with soap. Its bactericidal effect is 4 times greater than that of phenol. A 3-5% solution is commonly used to disinfect skin, tables, and utensils.
5. Lime and Alkali Lime is divided into quicklime (CaO) and hydrated lime (Ca(OH)2). The bactericidal effect is the hydroxide ions in slaked lime, which can hydrolyze proteins and nucleic acids, impair the enzyme system and structure of microorganisms, and decompose sugars in the bacteria.
The use of quick lime should be added with water, or the quick lime is exposed to moist objects. Both will produce mature lime. Its chemical reaction formula is: CaO+H2O→Ca(OH)2+ heat. If slaked lime is exposed to air for a long time, slaked lime may absorb carbon dioxide in the air to generate bactericidal calcium carbonate. The reaction formula is: Ca(OH)2+CO2→CaCO3+H2O
From the reaction formula, it can be clearly seen that if we want to sterilize, we can only use quicklime to react with water and produce mature lime to sterilize. Generally 1-3% lime water can play a very good bactericidal effect. For example, soaking rice straw with 0.5% lime water for 2 hours or more can also play a role in sterilizing and softening fibers. When mold contamination is found on the fungus tube or the bacteria brick, the quick lime can also be directly sprinkled on the surface of the contaminating bacteria, and the sterilization effect is good.
6, oxidants and reducing agents Potassium permanganate, hydrogen peroxide, halogen (chlorine, bromine, iodine) and certain compounds (or complexes), such as bleach powder are all oxidants, can be sterilized. Sulfurous acid and sulfites are reducing agents that can act as bactericidal agents through redox reactions.
(1) Potassium permanganate strong oxidants, with concentrations of 1:1000 and 1:250, have bactericidal action. A 0.5-1% aqueous solution of potassium permanganate kills most bacteria within 5 minutes; a 5% aqueous solution kills bacterial spores within 1 hour. Potassium permanganate has an increased bactericidal effect in acidic solutions. For example, 1% potassium permanganate and 1% hydrochloric acid solution can destroy anthrax spores within 30 minutes. Potassium permanganate is reduced to manganese dioxide without bactericidal action in the event of organic matter. Therefore, it can be used for direct seasoning sterilization. Potassium permanganate has a poor killing effect on mold.
Potassium permanganate is limited to the disinfection of skin, mucous membranes, and glassware, and must not be used for the direct seasoning and sterilization of raw materials for mushroom production (wood chips and other fiber materials).
(2) Iodine and its compounds (complexes) 3-7% iodine is dissolved in 70-83% ethanol and is an effective disinfectant for skin and small wounds. Aqueous solutions of 5% and 10% potassium iodide are also effective skin disinfectants. Organic iodides such as iodine fumarate have a good bactericidal effect, they can only kill common bacteria and fungi, and can inactivate viruses.
(3) Bleaching powder and its use method The bleaching powder (chlorine lime) is a white granular powder that dissolves in water and easily precipitates. The effective chlorine content of the efficient bleaching powder is 40-80%. Bleach is used at a concentration of 5% and is usually measured in 1 gram of high-performance bleach per cubic metre of space. When using, 1 gram of high-efficient bleaching powder was dissolved in 1 liter of water, allowed to stand still for 1 hour, and the supernatant was spray-sterilized.
The bactericidal principle of bleaching powder is that bleaching powder dissolves in water to generate hypochlorous acid and hypochlorite, and can release oxygen to oxidize bacterial proteins or enzymes. At the same time, the released chlorine can also replace the hydrogen ions in the protein amino group. Denature proteins to kill microorganisms.
7. Heavy metals Some heavy metals and their compounds can destroy the protein synthesis of microorganisms, inactivate enzymes and replace the main elements of cell structure, causing the growth of microorganisms to suffer from sputum or death.
The commonly used surface disinfectant is dichlorination, also known as mercury. 1:500-1:2000 mercury-injection solution has a homogenous effect on most bacteria. As mercury has a corrosive effect on metalware, it is highly toxic to humans and animals. At present, thimerosal and metaphen which are low toxicity to human are synthesized.
Copper sulfate, which is commonly used in the production of shiitake mushrooms, is also a good disinfectant for preventing and controlling fungi. The use concentration is lower than 2%, and the surface of the object is sprayed to prevent and control mainly penicillium fungi. Such as the copper sulfate, lime, water, according to the ratio of 1:1:100 made of Bordeaux late at night, is a mushroom house, bed surface and other good surface disinfectant.
8. Surfactants Substances with reduced surface tension are called surfactants. Common soap and sodium dodecyl sulphonate (such as washing powder) bactericidal capacity is equivalent to 2-3 times the carbonic acid.
Commercially available benzalkonium chloride is a quaternary ammonium salt surfactant. The chemical name is dodecylamine bromide, a pale yellow, scented colloidal body. It is generally possible to dilute the stock solution (5%) and disinfect the device or hand and the appliance that does not get hot. It has better killing ability against bacteria and viruses, but it has no good effect on killing fungi. Frequent use of benzalkonium chloride can cause dry skin, irritation to mucous membranes, corrosiveness to aluminum products, and no disinfectant to rubber. Therefore, it is not possible to carry out the basidiomycete strain preservation with a benzalkonium disinfectant plug.
The bactericidal mechanism of surfactants is to change the cell permeability, so that the bacteria are completely broken; when they accumulate on the surface of bacteria, they affect the metabolism of cells and destroy the activity of cell proteins and enzymes.
9, aerosol disinfection box
Third, the disinfection and sterilization effect inspection
The effectiveness of disinfection and sterilization is directly related to the level of the finished product. Therefore, in addition to strictly sterilizing and sterilizing, it is also necessary to strictly implement the inspection system. Both are indispensable.
(A) Inspection of the sterilization effect of the medium
1. After the test tube mother culture medium is sterilized, randomly select five from different parts of the autoclave and place it in a thermostatic chamber at 282°C for 5-7 days. If there are no molds, bacteria, yeasts and other bacteria, then Sterilization is considered thorough.
2. After the culture medium of the original species and cultivars is sterilized, samples are taken from the upper, middle, and lower layers of the sterilizing pan, and 5 bottles (bags) are randomly sampled from each diagonal line, and the constant temperature incubator at 28-30°C is placed. Incubate for 5-7 days and check for mold colonies. If you check for bacteria that do not kill, you must use bacterial culture medium for testing. Bacterial medium: 10 g of beef extract (or beef juice), 10 g of peptone, 15 g of NaCl, 14 g of agar powder, 1000 ml of distilled water, dissolved by heating, and placed in a test tube, sterilized at 121°C After 15 minutes, the beveled test tubes were then arranged. The above-mentioned samples were aseptically taken out of a small amount of culture medium to enter the bacterial culture medium, and then cultured at 37° C. for 24 hours. No bacterial colonies were produced as a result of the inspection, indicating that the sterilization was thorough. For example, the presence of bacteria in a sample may be a “dead corner†in the pot, or the structure of the sterilizer may be unreasonable, or it may not accumulate as required by the operation, or the material may be sprayed, or the material may be thrown too thick, or the bacteria Bottles and bags are too crowded and the circulation of the steam is uneven. If there are no parts, most or almost all bottles and bags contain germs. This indicates that the sterilization temperature or sterilization time is not enough. If it is an autoclave The phenomenon indicates that the cold kiln has not been cleaned out.
(B) Inoculation room (sterile room), box sterilization effect test method
1. Petri dish method First, a permanent mold culture medium such as CPDA, PDA, BSA, etc. is prepared. After sterilization, a sterile plate culture medium (abbreviated as plate) is conventionally produced. Check whether the inoculation chamber (box) is sterilized or not. Randomly take several sets of sterile petri dishes (3-5 plates per group) to open the placed dishes at different positions on the inoculation chamber (box). One of the groups is not The petri dish lid was opened as a control, the lid was allowed to stand for 5 minutes, and then the lid was covered, and the control group was incubated at 30°C for 72 hours to check whether there was mold colony on the plate culture medium. The experimental group was less than 1 colony and considered to be sterilized.
The method of checking bacteria is the same as above, and MPSA medium is generally used. The difference was that the plates were incubated at 37°C for 24 hours up to 48 hours.
2. Take the 3-5 pieces of the conventional fungal and bacterial culture media slopes in a test tube set as a group, place them in the inoculation room (case), and open the tampon of the experimental group by aseptic operation, and then after 30 minutes, Sterile tampon was stoppered and incubated at 30°C and 37°C (bacteria and mold) simultaneously with the control group. Bacteria were observed after 24 to 28 hours of cultivation, and the molds were observed after 72 hours of culture. No colonies were observed in the experimental groups, and the inoculation chambers and boxes were sterilized well.
Agaricus bisporus cultivation heavy four water
First, bacteriostatic water should be timely emergence of hyphae on the bed surface, indicating that the mycelium has been sent to the bottom of the culture and has been fully developed in the soil, already have the ability to mushroom, at this time should be timely spray bacteria water. Method: Increase the ventilation, so that the wind blows into the mushroom shed, so that the surface of the mycelium lodging; according to the situation of the soil layer to control the amount of water spray, usually about 1 kg per square meter bed of water spray, divided into morning and evening 1 Conduct 1/2 of the thickness of the soil. After spraying water, the hyphae no longer drilled out of the bed, combined with strong ventilation, so that the top of the bed about 0.5 cm in thickness of the surface of the water loss, slightly white, mycelium in the soil below the thickness It can grow laterally, connect, and reach a physiological maturity.
Second, the mushroom water to re-spray mushroom water spray in the spray bacteria 3 days after spraying. According to the water content of the culture materials and soil layers, the mushroom water is sprayed in 4 doses of about 3 kg per square meter, until the cover soil is wet and permeable, but no seepage water prevails. When spraying mushroom water, ventilation should be strengthened, and compulsory ventilation measures should be taken. Two days after the water spray, there were small buds appearing.
Third, mushroom water should be sufficient water and spraying method of mushroom water similar to mushroom water. At this point there have been mushroom buds, pay attention to the water temperature to be similar to the greenhouse temperature, not to make the temperature difference is too large. In addition, there should be a small amount of water spray in the place where there are mushroom buds, and the total amount should be sufficient. However, it is important to note that there should be no water penetration into the culture in the soil.
4. Maintaining water should always be combined with maintaining the humidity of the mushroom shed. Every day, the number of water sprays and water consumption should be controlled according to conditions such as temperature and ventilation. The humidity in the shed should be maintained at about 90%, and the overburdened surface should be basically moist. It is normal if there is whitening at the vents and near the doorway. With the continuous growth of fruiting bodies, the number of water sprays and the amount of water used should also continue to increase.
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