Medium preparation and sterilization
The following information is provided by Kameshu (Shanghai) Biotechnology
1. Purpose
1.1 Understand and master the preparation and distribution methods of culture medium
1.2 Master various laboratory sterilization methods and techniques.
2 Principle
The medium is a mixed nutrient for microbial growth, reproduction and metabolism. Because microorganisms have different nutritional types, the requirements for nutrients are also different, and the purpose of experiments and research is different, so there are many types of culture media, and the raw materials used are also different, but from a nutritional perspective, the culture medium Generally contains the carbon source, nitrogen source, inorganic salts, auxin and water necessary for microorganisms. In addition, the culture medium should also have a suitable pH value, a certain buffering capacity, a certain redox potential and a suitable osmotic pressure.
Agar is a colloidal substance extracted from seaweed and other seaweeds, and is the most widely used coagulant. The medium made with agar is melted at 98-100 ° C and solidified below 45 ° C. However, it melts repeatedly, and its coagulability decreases.
Once any kind of culture medium is prepared, it should be sterilized thoroughly in time to prepare for pure culture. General medium sterilization uses high-pressure steam sterilization.
3 Materials
3.1 Utensils and materials
Balance, weighing paper, croissant, precision pH test paper, measuring cylinder, graduated enamel cup, test tube, Erlenmeyer flask, funnel, dispensing rack, pipette and pipette barrel, petri dish and petri dish box, glass rod, beaker , Test tube rack, wire basket, scissors, alcohol lamp, cotton, string, kraft paper or newspaper, gauze, latex tube, electric stove, sterilization pot, drying box.
3.2 Pharmaceutical reagents
Peptone, beef extract, NaCl, K2HPO4, agar, NaNO3, KCl, MgSO4, FeSO4, sucrose, maltose, xylose, glucose, galactose, lactose, potato juice, sprout meter, ammonium phosphate, 5% NaOH solution, 5% HCl Solution.
4 Process
Weigh the drug → dissolve → adjust the pH value → melt agar → filter and pack → bandage the mark → sterilize → slope or pour the plate.
5 steps
5.1 Preparation of medium
5.1.1 Weighing medicines
Accurately weigh various drugs according to the medium formula, put them into a beaker of appropriate size, and do not add agar. Peptone is extremely hygroscopic, so it should be weighed quickly.
5.1.2 Dissolution
Use a graduated cylinder to take a certain amount (about 1/2 of the total amount) of distilled water into a beaker, heat it on a small fire in an electric furnace with asbestos mesh, and stir with a glass rod to prevent liquid overflow. After the various drugs are completely dissolved, stop heating and make up the water. If there is starch in the formula, first adjust the starch into a paste with a small amount of cold water, and heat and stir on the fire, then add enough water and other raw materials, and make up the water after it is completely dissolved.
5.1.3 Adjust pH
According to the pH requirements of the medium, adjust to the desired pH with 5% NaOH or 5% HC1 solution. The pH can be measured with pH test paper or acidity meter.
5.1.4 Dissolved agar
A certain amount of agar must be added to the solid or semi-solid medium. After the agar is added, stir on the electric stove while heating, until the agar is completely melted, then stop stirring and make up the water (water needs to be preheated). Pay attention to control the fire power not to overflow or scorch the medium.
5.1.5 Filtering and packaging
Install the filter device first. If it is liquid culture medium, put a layer of filter paper in the glass funnel, if it is solid or semi-solid medium, you need to put multiple layers of gauze in the funnel, or two layers of gauze with a thin layer of absorbent cotton to filter while hot. Dispense immediately after filtering. When dispensing, be careful not to contaminate the culture medium with the mouth of the tube or bottle, so as not to wet the cotton plug,
Cause pollution. The liquid filling height should be about 1/4 of the test tube height. The solid volume is 1/5 of the tube height. The semi-solid volume test tube is generally 1/3 of the height of the test tube; the volume of the triangle flask is not more than half of the volume of the triangle flask.
5.1.6 Bandage mark
After filling the medium, add a cotton plug or a test tube cap, then wrap a layer of moisture-proof paper, and fasten it with a cotton rope. Mark the medium name, preparation group and name, date, etc. on the packaging paper.
5.1.7 Sterilization
The above medium should be sterilized in time according to the conditions specified in the medium formula. The common medium is 121 ℃ for 20 minutes to ensure the sterilization effect and not damage the effective components of the medium. After the medium is sterilized, if it is necessary to make a bevel solid medium, it should be placed into a bevel immediately after sterilization (Figure 3-2). After sterilization, vertically condense into semi-solid deep agar.
5.1.8 Invert the tablet
Cool the culture medium to be poured into the plate to 45 ～ 50 ℃ in a water bath, and immediately pour the plate (Figure 3-3).
5.2 Sterilization method
Sterilization refers to killing or destroying all microorganisms in a certain environment. There are two types of sterilization methods: physical and chemical sterilization. This experiment mainly introduces one of the physical methods, namely heat sterilization.
Heat sterilization includes wet heat and dry heat sterilization. By heating, the proteins in the bacteria are solidified and denatured, thereby achieving the purpose of sterilization. The solidification of protein is related to its own water content. The higher the water content, the lower the temperature required for solidification. At the same temperature, the bactericidal efficacy of damp heat is greater than that of dry heat, because in the case of damp heat, the bacteria absorb moisture and make the protein easy to solidify; at the same time, the penetration of wet heat is strong, which can increase the sterilization effect.
5.2.1 Damp heat sterilization
5.2.1.1 Boiling disinfection method
This method can be used for syringes and dissecting instruments. Wrap the syringe etc. with gauze first, then put it in the boiling sterilizer and add water to boil. Boil the vegetative body of bacteria for about 15 to 30 minutes, for spores it takes about 1 to 2 hours.
5.2.1.2 High-pressure steam sterilization method
High-pressure steam sterilization has a wide range of uses and high efficiency. It is the most commonly used sterilization method in microbiology experiments. This sterilization method is designed based on the principle that the boiling point of water increases with the increase of steam pressure. When the steam pressure reaches 1.05kg / cm2, the temperature of the steam rises to 121 ° C, and after 15 to 30 minutes, it can completely kill all kinds of microorganisms and their spores or spores on the items in the pot. General culture media, glassware, infectious specimens and work clothes can be sterilized by this method (Figure 3-4).
5.2.1.3 Operation methods and precautions are as follows
5.2.1.3.1 Add water
Open the lid of the sterilization pot and add water to the pot to the water level line. It is best to use boiled water for the vertical sterilization pot in order to reduce the accumulation of scale in the pot. Pay attention to add enough water to prevent the dry pot during the sterilization process.
5.2.1.3.2 Loading and capping
After putting the sterilization material in place, close the sterilizer cover and use the diagonal type to evenly tighten the screw on the pot cover to make the steam pot tightly closed without air leakage.
5.2.1.3.3 Exhaust
Open the exhaust port (also called the purge valve). Heat in an electric furnace. After the water is boiled, steam and air are discharged from the exhaust hole together. When a large amount of steam is discharged, maintain it for 5 minutes to completely drain the cold air in the pot.
5.2.1.3.4 Boost, hold and buck
When the cold air in the pot is exhausted, the exhaust valve can be closed and the pressure starts to rise. When the pressure rises to the required pressure, control the voltage to maintain the constant temperature, and start to calculate the sterilization time. After the time reaches the requirements (general medium and utensil sterilization control at 121 ℃, 20min), stop heating and wait for the pressure to drop When approaching "0", open the purge valve. Be careful not to vent the air too soon or too soon, otherwise the liquid in the bottle will flush out of the container because the pressure in the bottle drops more slowly than in the pot.
5.2.1.3.5 Blank culture of sterilized medium
The sterilized medium is put in a 37 ° C incubator for cultivation. After 24 hours of aseptic growth, it can be kept for later use. After the oblique surface medium is taken out, it is placed in an oblique surface immediately after blank culture; the semi-solid medium is vertically placed and condensed into half After solid deep agar, blank culture.
5.2.2 Dry heat sterilization
The method of killing microorganisms by using dry hot air is called dry heat sterilization. Generally, after the items to be sterilized are ready for packaging, they are placed in an electric oven for baking, that is, heated to 160 to 170 ° C for 1 to 2 hours.
Dry heat sterilization is often used to sterilize empty glassware and metal utensils. All items with rubber skin, liquid and solid medium can not be sterilized by this method.
5.2.2.1 Preparation before sterilization
Glassware, etc. must be properly wrapped and stoppered before sterilization, to ensure that glassware is not contaminated by external bacteria after sterilization. Commonly used glassware bandage and stoppering methods are as follows: the plate is wrapped with paper or packed in a metal plate cylinder; the triangular bottle is wrapped with thick paper outside the cotton plug and the mouth of the bottle, and tied tightly with a cotton string to prevent the bottle after sterilization The mouth is contaminated by external bacteria; the straw is placed at the center of the cotton with a straightened paper clip end, gently poke into the mouth of the tube, the tightness must be moderate, the cotton fibers exposed at the mouth of the tube are burned through the flame, and the straw is loaded during sterilization For sterilization in the metal tube, you can also use a paper strip to obliquely wrap up from the tip of the straw, and gradually roll up. The paper roll at the end is flattened and twisted a few times, and then the wrapped straw is sterilized centrally.
5.2.2.2 Dry box sterilization
Put the wrapped items into the drying oven, taking care not to place them too tightly, so as not to obstruct the air circulation; do not allow the containers to directly contact the inner bottom plate of the oven. Raise the temperature of the oven to 160 ～ 170 ℃ and keep it at a constant temperature for 1 ～ 2h. Be careful not to make the temperature too high. If it exceeds 170 ℃, the paper and cotton wrapped outside the utensils will be burnt by burning. If it is for baking glassware, the temperature is 120 ℃ for 30 minutes. When the temperature drops to 60 ～ 70 ℃, you can open the door and take out the items, otherwise the glassware will burst due to the sudden cooling.
When using this method for sterilization, never use oil or wax paper to wrap items.
5.2.2.3 Flame sterilization
Direct flame sterilization, rapid and thorough. For the inoculation ring, inoculation needle or other metal utensils, it can be sterilized by burning directly to the red heat on the flame of the alcohol lamp. In addition, during the inoculation process, the mouth of the test tube or flask is also sterilized by flame.
6.1 Record the sterilization methods and sterilization conditions (temperature, pressure, etc.) used for various items.
6.2 Try to describe the process and precautions of high-pressure steam sterilization.
7 Thinking
7.1 What is the general procedure for preparing media?
7.2 After doing this experiment, what problems do you think you should pay attention to when preparing the medium?
7.3 What is the significance of sterilization in the operation of microbiology experiments?
4 Describe the operation method and principle of high-pressure steam sterilization.
5 What should be paid attention to when autoclaving?
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