First, the cleaning of glassware <br> In the process of preparing the medium, first use some glassware, such as test tubes, flasks, petri dishes, beakers and straws. These utensils must be cleaned according to different conditions and processed according to different conditions before use. Some have to be packaged and ready for use after sterilization.
1. Newly purchased glassware <br> After removing the contaminated dirt from the package, first wash it with hot soapy water, rinse it with water, and then soak it in 1-2% industrial hydrochloric acid for several hours to remove the free alkaline substance. Rinse with running water. For larger vessels, such as large flasks, measuring cylinders, etc., after washing, inject a little concentrated hydrochloric acid, rotate the container to make the inner surface of the mixture with hydrochloric acid, pour hydrochloric acid after a few minutes, then rinse with running water, and place it on the washing rack. Dry the water and use it.
2, used glassware <br> Where there are no pathogens or vessels not contaminated with fungi, can be washed at any time after use, pipettes with chemical reagents, can be soaked in clean water, and then concentrated after a certain amount Wash it. Vessels that may be contaminated with pathogens must be properly sanitized, the dirt removed, washed with soap, and rinsed with running water. If the dish that has not been washed with soap is used, it can be soaked in the washing liquid for a suitable period of time and then washed with water. The main components of the lotion are potassium dichromate and concentrated acid, which act to oxidize the organic matter to a soluble substance for rinsing. The lotion has a strong corrosive effect and should be used with care to avoid splashing on clothing, body and other items.
Second, the type of medium <br> Any nutrient substrate formulated in the laboratory suitable for the growth or accumulation of microorganisms or metabolites, called the medium (Media). Due to the different nutritional requirements of various microorganisms, the purpose of culture and the needs of detection are different, and thus there are many kinds of culture media. We can classify a wide variety of media into several types according to certain criteria.
1. According to whether the chemical composition of the constituent materials of the medium is completely understood, the medium can be divided into a natural medium, a synthetic medium and a semi-synthetic medium.
1) Natural medium Natural medium refers to a raw material using various animals, plants or microorganisms, and its composition is difficult to know exactly. The main raw materials used for this medium are: beef extract, wort, peptone, yeast extract, corn flour, bran, various cake powder, potato, milk, serum and the like. Although the medium prepared by these substances cannot know exactly its chemical composition, in general, the nutrition is relatively abundant, the microorganisms grow vigorously, and the source is wide, and the preparation is convenient, so it is more commonly used, especially suitable for the preparation of laboratories. Medium. The stability of this medium is often affected by factors such as the manufacturer or batch number.
2) Synthetic medium Synthetic medium is a type of chemical composition and a well-known medium, which is prepared from chemicals of known chemical composition. The chemical composition of this type of medium is precise and reproducible, but it is expensive, and the microorganisms grow slowly. Therefore, it is only suitable for some scientific research, such as nutrition and metabolism research.
3) Semi-synthetic medium In a synthetic medium, one or several natural ingredients are added; or in a natural medium, one or several known ingredients are added as a semi-synthetic medium. For example, potato sucrose medium. This medium is used most in production practices and laboratories.
2. According to the physical state of the medium, it can be divided into solid medium, liquid medium and semi-solid medium.
1) The medium prepared by the liquid medium is liquid, and the components thereof are substantially soluble in water, and there is no obvious solid matter. The nutrient composition of the liquid medium is evenly distributed, and it is easy to control the growth and metabolism state of the microorganism.
2) Solid medium A suitable amount of coagulant is added to the liquid medium to form a solid medium. Commonly used as a coagulant are agar, gelatin, silica gel, etc., and agar is most commonly used. Solid media is used in a wide variety of applications. In the laboratory, it is used for the separation, identification, inspection of bacteria, counting, preservation, bioassay, etc. of microorganisms.
3) Semi-solid medium A semi-solid medium is prepared if a small amount of coagulant is added to the liquid medium. Taking agar as an example, its amount is between 0.2 and 1%. This medium can sometimes be used to observe the dynamics of microorganisms and sometimes to preserve the species.
3. According to the use of the medium, it can be divided into selection medium, proliferation medium, identification medium and the like.
1) Selection medium A medium in which a substance is added to the medium to kill or inhibit the growth of an undesired strain, which is called a selection medium. Such as streptomycin, chloramphenicol and the like inhibit the growth of prokaryotic microorganisms; while nystatin, griseofulvin and the like can inhibit the growth of eukaryotic microorganisms; crystal violet can inhibit the growth of Gram-positive bacteria.
2) Proliferation medium In nature, different kinds of microorganisms often live together. In order to separate the microorganisms we need, some nutrients that some microorganisms particularly like are added to the common medium to increase the reproduction speed of the microorganisms. Other microorganisms are gradually phased out. This medium is called a proliferation medium, and this medium is often used for strain screening and selection of bacteria. To some extent, proliferation media is also a selective medium.
3) Identification medium The addition of certain reagents or chemicals to the culture medium makes the indistinguishable microorganisms show significant differences after being cultured, thus facilitating the rapid identification of certain microorganisms. Such a medium is referred to as an identification medium. For example, eosin blue medium for checking the presence of intestinal pathogenic bacteria in drinking water and dairy products is a commonly used discriminating medium.
Some media have a dual role of selection and identification. For example, the MacConkey medium commonly used in food testing is an example. It contains bile salts, lactose and neutral red. Bile salts have the effect of inhibiting bacteria other than intestinal bacteria (selectivity), lactose and neutral red (indicator) can help distinguish lactose-fermenting intestinal bacteria (such as E. coli) and intestinal pathogenic bacteria that cannot ferment lactose ( Such as Salmonella and Shigella).
In addition, depending on whether the nutrient content of the medium is "complete", it can be divided into a basic medium, a complete medium, and a supplementary medium, and such terms are mainly used in microbial genetics. According to the purpose of the medium for production, it can be divided into a seed medium and a fermentation medium. There are also living tissue culture media dedicated to the cultivation of parasitic microorganisms such as viruses, such as chicken embryos; and inorganic salt culture media for the cultivation of autotrophic microorganisms.
Third, the basic methods and precautions for the preparation of the medium
1. Selection of medium formula <br> The formula of the same medium often has some differences in different works. Therefore, in addition to the standard methods used, it should be prepared in strict accordance with its provisions. Generally, the relevant materials should be collected as much as possible, and compared and checked, and then selected according to their own purposes, and the source should be recorded.
2. Preparation of the culture medium <br> Each preparation of the culture medium should have a record, including the name of the medium, the formula and its source, and the grades of the various ingredients, the final pH value, the temperature of the disinfection and the date of preparation of the time and The preparer, etc., the record should be copied, the original record is kept for reference, and the copy record is stored together with the prepared medium to prevent confusion.
3, the weighing of the composition of the medium <br> The various components of the medium must be accurately weighed and should be taken care of to prevent confusion, preferably completed in one go, do not interrupt. The formula can be placed on the side of the sputum. Each time a component is weighed, it is marked in the matching army, and the drugs to be weighed are taken at one time, placed on the left side, and after each weighing is completed, it is moved to Right. After the complete weighing is completed, an inspection should also be carried out.
4. Mixing and melting of various components of the culture medium<br> The chemicals used in the medium should be chemically pure. The cooking pot used must not be a copper pot or an iron pot to prevent a trace amount of copper or iron from being mixed into the medium to make the bacteria difficult to grow. It is best to use a stainless steel pot to heat and dissolve. It can be placed in a large beaker or a large flask and placed in a high pressure steam sterilizer or a mobile steam sterilizer to cook and dissolve. When it is dissolved in the pot, it can be heated with warm water and disturbed at any time to prevent coking. If coking is found, the medium can not be used, and should be re-prepared. After most of the solid components have dissolved, all the ingredients are completely dissolved with less fire, and then boiled. If the agar is dissolved, dissolve the other ingredients with another portion of water and then mix the two solutions thoroughly. During the heating and melting process, the water lost due to evaporation must be supplemented.
5, the initial adjustment of the pH of the medium <br> Because the medium in the heating and disinfection process, the pH will change, after the components of the medium are completely dissolved, the initial adjustment of the pH should be carried out. For example, beef infusion can reduce pH by about 0.2, while intestinal infusion pH will increase significantly. Therefore, for this step, the operator should always pay attention to exploring the experience, in order to grasp the final pH of the medium and ensure the quality of the medium. After the pH adjustment, the medium should also be boiled for a few minutes to facilitate the precipitation of the culture medium precipitate.
6. Filtration and clarification of the culture medium<br> The liquid medium must be absolutely clarified, and the agar medium should also be transparent and not significantly precipitated. Therefore, filtration or other clarification methods are required to meet this requirement. Generally, the liquid medium can be filtered by a filter paper, and the filter paper should be folded into a folding fan or a funnel to avoid cracking of the filter paper due to uneven hydraulic pressure.
The agar medium can be filtered hot with a clean white tissue cloth. It can also be filtered with a double layer of gauze sandwiched between thin layers of absorbent cotton. When immersing fresh meat, liver, blood and potatoes, the slag must be filtered with flannel and filtered repeatedly with filter paper. If the filtration method does not meet the clarification requirements, the egg white clarification method must be used. The medium to be cooled to 55~60 °C should be placed in a large conical flask. The amount of the medium should not exceed 1/2 of the volume of the flask. Add 1 to 2 eggs of protein per 1000 ml of medium, and shake vigorously 3~5. Minutes, set in a high-pressure steam sterilizer, heated at 121 ° C for 20 minutes, remove the heat and filter with flannel.
7. Packing of medium <br> The packing of the medium should be divided into appropriate containers such as test tubes and flasks according to the purpose and requirements of use. The dispensing capacity shall not exceed 2/3 of the container loading capacity. The mouth of the container can be sealed with a cotton plug padded with moisture-proof paper, and it must be wrapped with waterproof paper (the test tube is generally more useful with a screw cover). It is best to use a semi-automatic or electric quantitative dispenser when dispensing. When dispensing agar slant medium, the amount of the package should be equal to the amount of 2/3 of the bottom layer and 1/3 of the slope. The dispensing container should be pre-cleaned and dry-sterilized to facilitate thorough sterilization of the medium. Each batch of medium should be separately dispensed with 20 ml of medium in a small glass bottle, and the batch medium is simultaneously sterilized to determine the final pH of the batch.
8. Sterilization of the medium <br> The general medium can be autoclaved at 121 ° C for 15 minutes. In the various medium preparation methods, if there is no special regulation, it can be sterilized by this method.
Some components that are afraid of heat, such as sugars, should be formulated as a 20% or higher concentrate, sterilized by filtration or intermittent sterilization, and then added to the medium by aseptic technique. Gelatin media is also sterilized at lower temperatures. Blood, body fluids, and antibiotics should be extracted by aseptic technique and added to a medium that is cooled to about 50 °C.
The agar slant culture medium should be taken out immediately after sterilization, and when it is cooled to 55 ° C - 60 ° C, it is placed into a suitable slope until it is naturally solidified.
9. Quality test of culture medium<br> After each batch of medium is prepared, it should be checked carefully. If it is found that cracking, moisture intrusion, abnormal color, cotton plug is contaminated by the medium, etc., it should be picked and discarded. And determine its final pH.
All the culture medium was placed in an incubator at 36 ± 1 ° C overnight, and if bacteria were found to grow, they were discarded.
Inoculate 1~2 tubes or flasks of medium with the relevant standard strains, and culture for 24 to 48 hours, such as sterile growth or poor growth. The cause should be traced and the inoculation should be repeated once. If the result is still the same, the medium should be discarded and cannot be used.
10. Preservation of the culture medium<br> The culture medium should be stored in a cool dark place, preferably in a normal refrigerator. The placement time should not exceed one week, and the poured medium should not exceed 3 days. Each batch of media must be accompanied by a copy of the batch preparation record or a clear label.
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