Figure 8-1 Pipette wrapping pipette wrapping: in the pipette at the top end of the plug into a small piece of cotton (do not use cotton wool), its role is to avoid the outside world and the mouth of the bacteria into the tube, and to prevent the bacteria from sucking into the mouth. Stuffed into this small section of cotton should be about 0.5cm from the mouth of the tube, the length of the cotton itself is about 1 ~ 1.5cm. stuffed with cotton. Can be used to straighten the periphery of a curve pin, a small amount of cotton into the mouth of the tube. Cotton should be stuffed with the appropriate tightness, blowing in order to be able to ventilate and not make the cotton slide down shall prevail. Newspaper cut into long strips of paper about 5cm wide, and then the tip of the pipette has been stuffed with cotton on the end of a long strip of newspaper, about 45 ° C angle, folded paper wrapped around the tip, with the left hand to hold the body of the pipette, with the hands of the pipette pressed tightly. Roll forward on the desktop, wrapped up in a spiral. The remaining paper strips on the upper end, folded and knotted, ready for sterilization. (B) liquid and solid medium preparation process 1. liquid medium preparation 1) weighing: generally available 0.01g balance weighing the preparation of the medium required for a variety of drugs, first calculated according to the formula of the medium of each component of the dosage. 2) weighing, and then accurately. Then accurately weighed. 2) Dissolve: put the weighed drugs in a beaker, first add a small amount of water (according to the experimental needs of tap water or distilled water), stirring with a glass rod, heat to dissolve. 3) Volumizing: to be all the drugs are dissolved, poured into a cylinder, and add water to the required volume. If the dosage of a certain drug is too small, it can be pre-prepared into a concentrated solution, and then suck a certain volume of solution according to the proportion and add it to the culture medium. 4) pH adjustment: Generally, pH test paper is used to determine the pH of the culture medium. cut out a small section of the pH paper with scissors, and then use tweezers to take the section of the paper, dip it into the culture medium to see its pH range, such as acidic or alkaline culture medium, use 1 mol/L NaoH or 1 mol/L HCl solution for adjustment. When adjusting pH, NaOH or HCI solution should be added drop by drop to prevent localized over-acidic or over-basic, destroying the components in the medium. Stir while adding, and test with pH paper from time to time until the desired pH is reached.5) Filtration: Filter the medium with filter paper or multi-layer gauze. Generally no special requirements, this step can be omitted.2. Preparation of solid culture medium When preparing solid culture medium, the liquid medium should be heated and boiled, and then add the weighed agar (1.5~2%) and stir constantly with a glass rod, so as not to paste the bottom of the burnt. Continue to heat until all the agar melted, and finally make up for the loss of water due to evaporation. (C) the partition of the culture medium according to different needs, can have been prepared culture medium into test tubes or triangular bottles, partitioning, pay attention to not make the culture medium stain the mouth of the tube or bottle, resulting in contamination. If the operation is not careful, the culture medium stained the mouth of the tube or bottle, can be used tweezers clip a small piece of cotton wool, wipe off the mouth of the tube or bottle mouth of the culture medium, and the cotton wool will be discarded. 1. test tube filling take a glass funnel, mounted on an iron frame, under the funnel is connected to a rubber tube, rubber tube, rubber tube, the lower end of the tube and then connected with another glass tube, rubber tube, the middle of the tube plus a spring clamp. When loading, the left hand to hold the empty test tube in the middle, and the funnel under the glass tube mouth into the test tube, with the right thumb and forefinger to open the spring clamp, the middle finger and ring finger clip Zuo glass tube mouth, so that the medium flows directly into the test tube. The amount of culture medium loaded into the test tube depends on the size of the test tube and the need to be determined, if the size of the test tube used for 15 × 150 mm, the liquid culture medium can be divided into test tubes up to the height of the left 1/4 appropriate; such as the distribution of solid or semi-solid culture medium, in the agar completely melted, it should be divided into test tubes while hot. The amount of solid medium used for making slant is 1/5 of the height of the tube (about 3-4 mI), and the amount of semi-solid medium is 1/3 of the height of the tube.2. Dispensing of triangular flasksFigure 8-2 Dispensing of medium When used for oscillating culture of microorganisms, 50 m1 of liquid medium can be added to 250 m1 triangular flasks; when used for making plate culture medium, 150 m1 of liquid medium can be added to 250 m1 triangular flasks. If used to make plate culture medium, add 150 ml culture medium into 250 m1 triangular flask, then add 3g agar powder (calculated as 2%), and the agar powder in the flask will be melted at the same time when it is sterilized. (D) the production of cotton plugs and test tubes, triangular flasks, bandaging In order to cultivate aerobic microorganisms, it is necessary to provide excellent aeration conditions, while in order to prevent contamination of stray bacteria, it is necessary to pass into the test tube or triangular flasks of air pre-filtering sterilization. The usual method is in the test tube and triangular vial mouth with cotton plugs, etc. 1. test tube cotton plug production, should be used to make cotton plugs, should be selected size, thickness of a piece of ordinary cotton, spread in the left hand thumb and forefinger buckle into a group of holes, the right hand forefinger will be pressed from the center of the cotton into the group of holes made of cotton plugs. Then pressed directly into the test tube or triangular bottle mouth. You can also borrow a glass rod into the plug, can also be folded and rolled plug method to produce cotton plugs. The production of cotton plugs should be close to the wall of the tube, leaving no gaps, in order to prevent the outside world microbial invasion along the gaps, cotton plugs should not be too tight or too loose, stuffed with hand-held cotton plugs, test tubes do not fall shall prevail. Cotton plug 2 / 3 in the test tube, 1 / 3 outside the test tube. At present, there is also the use of silicone plug instead of cotton plug directly over the mouth of the test tube. Fill the culture medium and plug the cotton plug or silicone plug test tubes into a bundle, wrapped in a layer of kraft paper. Use a marker to indicate the name of the medium and the date of preparation, and sterilize it for use.
Figure 8-3 Production of cotton plugs
Figure 8-4 Correct and incorrect cotton plugs 1. metal plugs 2 correct 3, 4 incorrect 2. triangular bottle cotton plugs production is usually wrapped in a layer of gauze outside of the cotton plugs, and then plugged in the mouth of the bottle. Sometimes in order to carry out liquid oscillation culture to increase aeration, it can be used 8 layers of gauze instead of cotton plug wrapped in the mouth of the bottle, there are also using silicone plug directly on the bottle. In the loaded culture medium and plugged cotton or wrapped eight layers of gauze or covered with silicone plug on the mouth of the triangular flask, and then wrapped with a layer of kraft paper and bundled with string, sterilized for use. (E) Sterilization of culture medium culture medium after packing and wrapping, should be immediately prepared in accordance with the provisions of the sterilization method of sterilization conditions for autoclaving. (F) the production of slant and plate 1. the production of slant will have been sterilized test tube with agar medium, while hot, placed on a wooden stick or glass rod, so that the appropriate slope, solidification is made into a slant. The length of the beveled surface does not exceed the length of the test tube l / 2 is appropriate. Such as the production of half-group or solid deep medium, after sterilization should be placed vertically until solidified. 2. the production of the plate will be mounted in a triangular bottle or test tube has been sterilized agar medium melting, to be cooled to about 50 ℃ poured into the sterile petri dish. When the temperature is too high, too much condensation on the lid of the dish; temperature below 50 ℃, the medium is easy to solidify and can not make the plate. The production of the plate should be carried out next to the fire, the left hand to take the petri dish, the right hand to take the bottom of the triangular bottle or test tube, the left hand at the same time with the little finger and palm of the cotton plug to open, cauterize the mouth of the bottle, with the left thumb will be opened to the petri dish lid a slit to the mouth of the bottle just to reach in, pour into the 10 ~ 15mL of medium, quickly cover the lid of the petri dish, placed on the table, and gently rotate the petri dish to make the medium uniformly distributed throughout the petri dish, the condensation will be made into After condensation, the plate will be formed. (VII) Sterilization of the culture medium check after sterilization of the culture medium, generally need to carry out aseptic examination. It is best to take out 1~2 tubes (bottles), placed in a 37 ℃ incubator culture 1~2 days to determine the sterility before use. (H) the preparation of sterile water in each 250mL triangular bottle filled with 100mL of distilled water and plugged with cotton or silicone stopper. Fill each test tube with 4.5mL of distilled water and plug with cotton or silicone stopper. Then wrap a piece of kraft paper on the cotton plug and autoclave sterilize it. 0.1MPa sterilize it for 20~30min. v. Experiment 1. Clean various glassware according to the requirements and wrap it. 2. Prepare various culture media according to the requirements. 3. Dry heat sterilize the glassware with electric blast drying oven. 4. Use autoclave sterilizing pot to sterilize the prepared culture media. 5. The laboratory report 1. briefly describe the pipettes and petri dishes packing precautions. 2. briefly describe the basic steps and precautions for the preparation of culture media. 3. why the microbiology laboratory pipette mouth or dropper mouth of the upper end of the pipette need to be stuffed with a small piece of cotton, and then wrapped in newspaper, after autoclave sterilization can be used? 4. why microbiology laboratory triangular bottles used in the mouth of the vials or the mouth of the test tubes have to be plugged with a cotton plug ( silicone plug) to be used? 5. why the microbiology laboratory used in the mouth of the vials or test tubes have to be used? Why are cotton plugs (silicone plugs) used in microbiology laboratories? 5. Why is it necessary to adjust the pH when preparing culture media? 6. Why does autoclaving require lower temperatures and shorter times than dry heat sterilization? Appendix Sterilization techniques I. Purpose: To understand the principles of disinfection and sterilization, and to master the operation of various sterilization methods. II. Experimental materials 1. Instruments or other utensils: Petri dishes, test tubes, pipettes, etc., wrapped as above, electric blast drying oven, autoclave, microporous membrane filter, 0.22 μm filter membrane, syringes, tweezers, glass coated rods. 2. Culture medium: culture medium prepared as above and saline. 3. Fundamentals In microbiological experiments, it is necessary to carry out a pure culture without any contamination of stray bacteria, and it is important to know how to sterilize the culture medium. Therefore, it is necessary to sterilize and disinfect the equipment used, the culture medium and the workplace. Sterilization refers to the killing of all microorganisms in a certain environment, including microbial nutrients, germs and spores. Commonly used sterilization methods in the laboratory include direct burning, constant temperature drying oven sterilization, autoclaving, intermittent sterilization, boiling sterilization and other methods. The basic principle of these methods is to heat the microbial body protein coagulation denaturation, so as to achieve the purpose of sterilization. Fourth, the operation step (a) dry heat sterilization method dry heat sterilization is in the electric drying oven using high temperature dry air (160 ℃ ~ 170 ℃) for sterilization, it is the use of high temperature to make the microbial cell protein coagulation and denaturation to achieve the purpose of sterilization. This method is applicable to the sterilization of glassware such as pipettes, test tubes and petri dishes. Culture medium, rubber products, plastic products can not be sterilized by dry heat. Protein coagulation within the cell is related to its own water content, in the bacterium heat, when the environment and the cell water content is greater, the protein coagulation is faster, and vice versa, the smaller the water content, the slower the coagulation. Therefore, compared with moist heat sterilization, dry heat sterilization requires high temperature (160 ℃ ~ 170 ℃), long time (1 ~ 2h). But the dry heat sterilization temperature can not exceed 180 ℃, otherwise, the paper or cotton plugs will be burned, or even cause combustion. Dry heat sterilization using an electric drying oven (dry box). Specific operational steps are as follows: 1. Load the items to be sterilized: put the packaged items to be sterilized into the electric drying box, close the door. Stacked to leave a gap, do not place the items too crowded, so as not to impede air circulation, sterilization items do not touch the inner wall of the electric drying box iron plate, temperature probe, in order to prevent the wrapping paper scorched fire. 2. warming up: turn on the power, open the switch, open the top of the electric drying box properly vent, rotate the thermostat regulator, so that the temperature rises gradually. When the temperature rises to 100 ℃, close the air vent. In the process of temperature rise, if the red light is off and the green light is on, it means that the electric heating drying box stops heating, at this time, if it has not yet reached the required 160℃~170℃, it is necessary to rotate the thermostat regulator to make the red light on, and so on repeatedly to adjust until it reaches the required temperature.3. Constant Temperature: When the temperature rises to 160℃~170℃, it will maintain this temperature with the help of automatic control by the thermostat regulator for 2 h. Dry Heat Sterilizing In the process, strictly prevent the automatic control of thermostatic regulation failure and cause safety accidents. 4. cooling: cut off the power supply, natural cooling. 5. open the box to take things: to be the temperature inside the electric drying box to 60 ℃ or less before you open the door and take out the sterilized items. At the same time, the temperature adjustment knob should be set to zero, and open the vent. The temperature in the electric drying oven has not been reduced to 60 ℃ before, do not open the door, so as not to sudden cooling lead to glassware cracking. (B) autoclave sterilization autoclave sterilization is placed in a closed high-pressure steam sterilization pot, under a certain pressure to maintain 15 ~ 30 minutes for sterilization. This method is suitable for culture medium, sterile water, work clothes and other items of sterilization, can also be used for glassware sterilization. The items to be sterilized are placed in a closed pressurized sterilizer, and steam is generated by heating the water between the jacket of the sterilizer to boiling. To be water vapor dramatically the cold air inside the pot from the exhaust valve, and then close the exhaust valve, continue to heat, at this time because the steam can not overflow, and increase the pressure inside the sterilization pot, thereby increasing the boiling point, obtaining temperatures higher than 100 ℃, resulting in the bacterial protein coagulation and denaturation to achieve the purpose of sterilization. At the same temperature, the effect of moist heat sterilization is better than dry heat sterilization. There are three reasons: in the humid heat sterilization in the bacterial absorption of water, protein coagulation and denaturation, protein with the increase in water content, the required coagulation temperature is reduced; humid heat sterilization in the steam penetration than the dry air; steam in the surface of the object to be sterilized condensation, the release of a large amount of latent heat of vaporization, can quickly increase the temperature of the surface of the sterilized object, thereby increasing the effectiveness of sterilization. Laboratory commonly used sterilizing pots are non-autonomous portable autoclave and autoclave, its structure and working principle is the same. This experiment introduces the non-autonomous portable autoclave, autoclave can refer to the manufacturer's instructions for the use of autoclave. Specific operating steps are as follows: 1. Add water: first remove the inner pot, and then add an appropriate amount of water to the outer pot, so that the water surface does not exceed the heating snake tube, and the triangular shelf level is appropriate. Do not forget to check the water level, add too little water, the sterilization pot will be burned dry caused by blowing up the accident. 2. loading: put back the inner pot, and load the items to be sterilized. Be careful not to load too crowded, so as not to hinder the steam flow and affect the sterilization effect. Containers with culture medium should be placed to prevent liquid spillage, triangular bottles and test tubes should not be in contact with the barrel wall, so as to avoid condensation wetting the wrapping paper and penetrate into the cotton plugs. 3. lid: the lid and the exhaust hole connected to the exhaust hose inserted into the exhaust groove of the inner pot, the lid, align the screw, and then symmetrically and simultaneously tighten the two bolts opposite each other to make the bolts the same tightness and do not make leakage, and open the exhaust valve. 4. air venting: put back to the inner pot, and fill in items to be sterilized. Exhaust: Turn on the power to heat the sterilizer, boil the water, so that the cold air and water vapor inside the pot together from the exhaust holes. It is generally believed that when the discharged air flow is very strong and there is a hush sound, indicating that the air inside the pot has been exhausted, boiling takes about 5 minutes. 5. pressure: after the cold air is completely exhausted, close the exhaust valve, continue to heat the pot, the pressure began to rise. 6. pressure: when the pressure gauge pointer reaches the desired pressure, control the power supply, start the clock and maintain the pressure to the desired time. Such as this experiment using 0.1Mpa, 121.5 ℃, 20 minutes sterilization. Sterilization of the main factor is the temperature rather than pressure, so the cold air inside the pot must be completely exhausted before closing the exhaust valve to maintain the required pressure. 7. pressure reduction: to reach the required time for sterilization, cut off the power supply, so that the temperature of the sterilization pot naturally fall, when the pressure gauge pressure drops to "0", before opening the exhaust valve, exhaust the remaining When the pressure gauge pressure drops to "0", then you can open the exhaust valve, exhaust the remaining steam, loosen the bolt, open the lid, take out the sterilized items, pour out the leftover water in the pot. Pressure must be reduced to "0" before opening the exhaust valve, open the lid to take things. Otherwise, it will be due to the sudden drop in pressure in the pot, so that the culture medium or reagents in the container due to internal and external pressure imbalance and rushed out of the mouth of the container, resulting in contamination of the mouth of the bottle, or even burn the operator.8. Aseptic check: the sterilized medium into the 37 ℃ constant temperature incubator incubator for 24h, after checking that there is no growth of stray bacteria, it is ready for use. (C) filter sterilization of some substances, such as antibiotics, serum, vitamins, sugar solution, etc., using heat sterilization method, easy to be thermally decomposed and destroyed, and thus to use filter sterilization method. Filtration sterilization is a method of filtering out bacteria in liquid or gas by mechanical action, the method's greatest advantage is that it does not destroy the chemical composition of various substances in solution. Filtration sterilization method in addition to the laboratory for solutions, reagents, sterilization, microbiological work in the use of purification bench is also based on the principle of filtration and sterilization of the design, according to different needs to choose different filters and filter plate materials. The most widely used types of filters are: 1. microporous membrane filter: a new type of filter, which consists of upper and lower two respectively with the outlet and inlet connecting device of the plastic box, the outlet can be connected to the needle, the inlet can be connected to the syringe, the use of the filter membrane loaded into the two plastic boxes between the box, tighten the lid, when the solution is injected into the filter from the syringe, a variety of micro-organisms are blocked above the microporous filter membrane, while the liquid and small molecules pass through the membrane, thus achieving the goal of filtering the bacteria, and the microorganisms. When the solution is injected into the filter from the syringe, all kinds of microorganisms are blocked on the microporous filter membrane, while liquid and small molecules pass through the membrane, thus achieving the purpose of sterilization. The filter membrane is made of nitrocellulose, cellulose acetate and other films, there are a variety of specifications with different pore sizes (such as 0.1 μm, 0.22 μm, 0.3 μm, 0.45 μm, etc.), the pore size of the pore size of the microporous membrane used for the sterilization of the laboratory is generally 0.22 μm, according to the amount of solution to be decontamination of the amount of different sizes of filters can be selected. The advantages of this filter are small adsorption, i.e., less loss of substances in the solution, fast filtration, and each membrane is used only once without cleaning.2. Cai's (Seitz) Filter: It is a filter funnel made of metal, and its filtration part is a kind of sheet structure pressed with asbestos fibers and other fillers. Bacteria in the solution are removed by adsorption and filtration of asbestos fibers, but the adsorption of other substances in the solution is also large, and each fiber sheet can only be used 1 time. 3. Glass filter: is a filter funnel made of glass, and its filtration portion is a plate-like structure made of fine glass powder sintered. There are many specifications of glass filters, No. 5 (pore size 2~5μm) and No. 6 (pore size <2μm) are suitable for filtration of bacteria, the advantage of which is that the amount of adsorption is small, but each time after the use of washing and reuse. This experiment uses microporous membrane filter for filtration and sterilization, the specific operation steps are as follows: 1. assembly, sterilization: the 0.22?0?8m pore size of the membrane into a clean plastic filter, screwed and flattened, packaged and sterilized to be used (0.1Mpa, 121.5 ℃, sterilized for 20 minutes). 2. connection: the inlet of the sterilized filters under aseptic conditions, aseptically operated connected to the Connection: Connect the inlet of the sterilized filter to the syringe with the solution to be filtered under sterile conditions, connect the needle to the outlet and insert it into the sterile test tube with rubber stopper. 3. Pressure filtration: Squeeze the solution to be filtered in the syringe under pressure into the filtered sterile test tube, and then pull out the needle after the filtration. The pressure should be appropriate, not too strong too fast, so as not to avoid bacteria being squeezed through the membrane. 4. aseptic check: aseptic operation to suck up the sterilization filtrate 0.1mL in broth peptone plate, evenly coated, placed in a 37 ℃ incubator incubation for 24 hours, check whether there is the growth of stray bacteria. 5. cleaning: discard the microporous filter membrane on the plastic filter, the plastic filter clean, and replaced with a new microporous membrane, assembled and wrapped, and then sterilized by the microporous membrane. The plastic filter should be cleaned and replaced with a new microporous filter membrane, assembled and wrapped, and then sterilized for use. The whole process should be in strict accordance with the aseptic operation, in order to prevent contamination, filtration should be avoided at the connection of the phenomenon of permeability. From Baidu, I organize, can not be on the map!