Preparation of microbial bacterial culture medium
In the process of preparing the culture medium, first use some glassware, such as test tubes, triangular bottles, petri dishes, beakers and straws. These vessels must be treated and washed according to different conditions before use. Some also need to be packaged and sterilized before they can be used.
1. Newly purchased glassware
After removing the dirt contaminated with the packaging, first wash it with hot soapy water, rinse it with running water, and then soak it in 1 to 2% industrial hydrochloric acid for several hours to remove the free alkaline substances, and then rinse it with running water. For large-capacity utensils, such as large flasks, measuring cylinders, etc., after washing, inject a little concentrated hydrochloric acid, turn the container so that its internal surface is stained with hydrochloric acid, pour out the hydrochloric acid after a few minutes, rinse it with running water, turn it upside down on the washing rack and dry the water before use.
2. Used glassware peiyangji preparation technology glassware diagram
All utensils that are indeed free of pathogenic bacteria or not contaminated by bacteria can be rinsed at any time after use. Straws that have absorbed chemical reagents can be soaked in water first, and then cleaned in a concentrated manner after a certain amount. Utensils that may be contaminated by pathogenic bacteria must be properly disinfected, the dirt must be removed, washed with soap, and then rinsed with running water. If utensils that cannot be washed with soap can be soaked in washing liquid for an appropriate period of time and then washed with water. The main ingredients of the washing liquid are potassium dichromate and concentrated stream acid, which are used to oxidize organic matter into soluble substances for rinsing. The washing liquid has a strong corrosive effect, and special care should be taken when using it to avoid splashing on clothes, body and other items.
II. Types
Any nutrient matrix formulated in the laboratory that is suitable for the growth and reproduction of microorganisms or the accumulation of metabolites is called a medium. Due to the different nutritional requirements of various microorganisms, the purpose of culture and testing needs are different, so there are many types of media. We can be divided into several types according to certain criteria.
1. According to themediumWhether the chemical composition of the constituent substances is fully understood can be divided into natural, synthetic, and semi-synthetic media.
1) Natural medium refers to the use of various animal, plant or microbial raw materials, the composition of which is difficult to know exactly. The main raw materials are: beef paste, malt juice, peptone, yeast paste, corn flour, bran, various cake powders, potatoes, milk, serum, etc. Although the chemical composition of these substances cannot be precisely known, in general, nutrients are relatively rich, microorganisms grow vigorously, and they come from a wide range of sources. It is convenient to prepare, so it is more commonly used, especially suitable for the preparation of media commonly used in laboratories. Stability is often affected by factors such as production plant or batch number.
2) Synthetic medium is a type of medium with a well-known chemical composition and quantity, which is prepared with chemicals with known chemical composition. This type of medium has precise and reproducible chemical composition, but it is expensive, and microorganisms grow slowly, so it is only suitable for doing some scientific research, such as nutrition and metabolism research.
3) In the synthetic medium, add some or several natural ingredients; or in the natural medium, add one or several chemicals with known ingredients to form a semi-synthetic medium. For example, potato sucrose medium, etc. This medium is most used in production practice and laboratories.
2. According to the physical state, it can be divided into solid medium, liquid medium and semi-solid medium.
(1) Liquid medium, prepared in a liquid state, with the ingredients basically soluble in water and no obvious solids. The nutrients in the liquid medium are evenly distributed, making it easy to control the growth and metabolism of microorganisms.
2) Add an appropriate amount of coagulant to the liquid medium to form a solid medium. Substances commonly used as coagulants include agar, gelatin, silica gel, etc. Agar is the most commonly used and is widely used in practice. In the laboratory, it is used for the isolation, identification, detection of bacteria, counting, preservation, bioassay, etc.
3) If a small amount of coagulant is added to the liquid medium, a semi-solid is formed. Take agar as an example, its dosage is between 0.2 and 1%. This medium can sometimes be used to observe the power of microorganisms, and sometimes it is used to preserve strains.
3. According to the application, it can be divided into selection, proliferation, and identification media.
1) Adding a substance to the medium to kill or inhibit the growth of unwanted strains is called selection medium. Such as streptomycin, chloramphenicol, etc. inhibit the growth of prokaryotic microorganisms; while nystatin, griseofulvin, etc. can inhibit the growth of eukaryotic microorganisms; crystal violet can inhibit the growth of gram-positive bacteria, etc.
2) In nature, different kinds of microorganisms often live together. In order to isolate the microorganisms we need, some nutrients that microorganisms particularly like are added to the ordinary medium to increase the reproduction rate of this microorganism and gradually eliminate other microorganisms. It is called a proliferation medium, which is often used for species screening and selective growth. To some extent, a proliferation medium is also a selection medium.
3) Adding certain reagents or chemicals to the culture medium makes indistinguishable microorganisms show significant differences after cultivation, thus helping to develop a rapid identification of a certain microorganism, which is called an identification medium. For example, eosin blue, which is used to check whether drinking water and dairy products contain intestinal pathogens, is a commonly used identification medium.
Some media have the dual function of selection and identification. For example, McConkay medium, which is commonly used in food inspection, is an example. It contains bile salts, lactose, and neutral red. Bile salts have the effect of inhibiting bacteria other than intestinal bacteria (selectively), and lactose and neutral red (indicators) can help distinguish between lactose-fermenting intestinal bacteria (such as Escherichia coli) and intestinal pathogens that cannot ferment lactose (such as Salmonella and Shigella).
In addition, according to whether the nutritional content is "complete", it can be divided into basic, complete and supplementary media, such terms are mainly used in microbial genetics. According to the purpose of production, it can be divided into seed media and fermentation media. There are also living tissue media specially used to cultivate parasitic microorganisms such as viruses, such as chicken embryos, etc.; inorganic salt media specially used to cultivate autotrophic microorganisms, etc.
III. Basic methods and precautions for preparation
1. Selection of the formula
The formula of the same medium often has some differences in different works. Therefore, in addition to the standard method used, which should be strictly formulated according to its regulations, it is generally necessary to collect relevant information as much as possible, compare and check it, and then choose it according to your own purpose of use, and record its source.
2. Preparation records
Each preparation should be recorded, including the name, formula and its source, as well as the grades of various ingredients, final pH value, temperature and time of disinfection, date of preparation, and preparation. A copy of the record should be made, and the original record should be kept for future reference. The copy record should be stored with the prepared medium to prevent confusion.
3. Weigh the ingredients
The various components of the medium must be accurately weighed and attention should be paid to prevent confusion. It is best to complete the formula at one time without interruption. The formula can be placed on the side. After each component is weighed, it is marked on the side of the preparation, and the medicines to be weighed are taken at one time and placed on the left side. After each weighing is completed, it is moved to the right side. After the complete weighing is completed, an inspection should be carried out.
4. Mixing and melting of various ingredients
The chemicals used in the medium should be chemically pure. The cooking pot used should not be a copper pot or an iron pot to prevent trace amounts of copper or iron from being mixed into the medium, making it difficult for bacteria to grow. It is best to use a stainless steel pot for heating and melting, and it can be boiled and melted in a large beaker or a large flask with a high-pressure steam sterilizer or a flowing steam sterilizer. When melting in the pot, it can be heated with warm water first and disturbed at any time to prevent coking. If coking is found, the medium cannot be used and should be re-prepared. After most of the solid ingredients are melted, use a small amount of fire to completely dissolve all the ingredients until boiling. If agar is melted, use another part of water to dissolve the other ingredients, and then mix the two solutions thoroughly. During the process of heating and melting, the water lost due to evaporation must eventually be replenished.
5. Initial adjustment of pH
Because the pH of the medium will change during the heating and disinfection process, the pH should be adjusted initially after the ingredients are completely dissolved. For example, the pH of the beef infusion can be reduced by about 0.2, while the pH of the intestinal infusion will increase significantly. Therefore, for this step, the operator should always pay attention to the exploration experience, so as to master the final pH and ensure the quality of the medium. After the pH adjustment, the medium should also be boiled for several minutes to facilitate the precipitation of the medium sediment.
6. Filtering clarification
The liquid medium must be absolutely clear, and the agar medium should also be transparent without significant precipitation. Therefore, filtration or other clarification methods need to be used to achieve this requirement. Generally, the liquid medium can be filtered by filter paper, and the filter paper should be folded into a folding fan or funnel shape to avoid filter paper cracking due to uneven hydraulic pressure.
The agar medium can be filtered hot with a clean white flannel. It can also be filtered with a double-layer gauze with a thin layer of absorbent cotton sandwiched in the middle. When soaking new meat, liver, blood and potatoes, the debris must be filtered off with a flannel first, and then filtered repeatedly with filter paper. If the filtration method cannot meet the clarification requirements, the egg white clarification method must be used. The medium that is about to be cooled to 55-60 ° C is placed in a large triangular flask, and the amount should not exceed 1/2 of the flask capacity. Add 1-2 egg whites to every 1000ml of medium, shake it vigorously for 3-5 minutes, put it in a high-pressure steam sterilizer, heat it at 121 ° C for 20 minutes, take it out and filter it with a flannel while it is hot.
7. Separate packaging
It should be divided into suitable containers such as test tubes and flasks according to the purpose and requirements of use. The amount of dispensing should not exceed 2/3 of the container's capacity. The mouth of the container can be sealed with a tampon with moisture-proof paper, and the outside must be wrapped with waterproof paper (the test tubes are generally used with screw caps). It is best to use a semi-automatic or electric dosing dispenser when dispensing. When dispensing agar bevel medium, the amount of dispensing should be appropriate to form 2/3 of the bottom layer and 1/3 of the bevel. The dispensing container should be cleaned in advance and dry-baked and sterilized to facilitate thorough sterilization. Each batch should be divided into an additional 20ml of medium in a small glass bottle, and sterilized at the same time with the batch, for the purpose of determining the final pH of the batch of medium.
8. Sterilization
Generally, the medium can be sterilized by autoclaving at 121 ° C for 15 minutes. In various medium preparation methods, if there are no special regulations, this method can be used for sterilization.
Certain heat-resistant ingredients, such as sugars, should be prepared separately in a concentrate of 20% or more, disinfected by filtration or batch sterilization, and then added to the medium by aseptic operation techniques and quantification. Gelatin media should also be sterilized at a lower temperature. Blood, body fluids, and antibiotics should be drawn and added to the medium cooled to about 50 ° C by aseptic operation techniques.
The agar ramp should be taken out immediately after sterilization, and when cooled to 55 ° C to 60 ° C, it should be placed in an appropriate ramp until it solidifies naturally.
9. Quality testing
After the preparation, it should be carefully checked, such as the discovery of rupture, water immersion, abnormal color, tampon contaminated with culture medium, etc., should be picked out and discarded. And determine its final pH.
All media were incubated overnight in a 36 ± 1 ° C incubator, and discarded if bacterial growth was found.
Inoculate 1-2 tubes or bottles of medium with relevant standard strains for 24-48 hours, if aseptic growth or poor growth. The cause should be traced and the inoculation should be repeated. If the results are still the same, the batch of medium should be discarded and cannot be used.
10. Save
It should be stored in a cool and dark place, preferably in a normal refrigerator. The storage time should not exceed one week, and the poured plate should not exceed 3 days. Each batch of product must be accompanied by the preparation record of the batch of medium subsheet or obvious label.
