Technical instructions for preparation of culture medium for microbiology laboratory
1. Cleaning of glassware
In the process of preparing the medium, we should first use some glassware, such as test tube, triangle bottle, Petri dish, beaker and straw. These containers should be cleaned and cleaned according to different situations before they are used. Some have to be packed and sterilized before they can be used.
1. Newly bought glassware
After removing the contaminated dirt from packaging, brushing with hot soapy water, rinsing water, and soaking in 1 - 2% industrial hydrochloric acid for several hours, remove the free alkaline substance and rinse with water. For the containers with large capacity, such as the large flask, the measuring cylinder, and so on, a little hydrochloric acid is injected after washing, and the inner surface of the vessel is turned into hydrochloric acid. After a few minutes, the hydrochloric acid is tilting to the hydrochloric acid, then the water is washed with water, and the water is dry on the washing rack and can be used.
2. Used glassware
The utensils, which have no pathogenic bacteria or not contaminated by bacteria, can be rinsed at any time after use and absorb the straw of chemical reagents. They can be soaked in clean water first and then be cleaned after a certain amount. Containers that are likely to be contaminated by pathogens must be properly sterilized to remove dirt, wash them with soap solution, and rinse them with water. If you can't wash the soap with liquid soap, wash it with suitable lotion and wash it with clean water. The main components of lotion are potassium dichromate and concentrated acid, which are used to oxidize organic matter to soluble substances for washing. The lotion has a strong corrosive effect and should be especially careful when used. Avoid splashing onto clothes, body and other articles.
Two. Type of culture medium
In the laboratory, any nutrient matrix suitable for microbial growth, reproduction or accumulation of metabolites is called Media. Because of the different nutritional requirements of all kinds of microorganism, there are many different kinds of culture media for the purpose of cultivation and detection. According to certain criteria, we can divide a large number of culture media into several types.
1. The culture medium can be divided into natural medium, synthetic medium and semi synthetic medium based on the complete understanding of the chemical composition of the medium.
1) natural medium is a natural medium that uses all kinds of raw materials of animals, plants or microorganisms. The main ingredients used for this medium are beef paste, malt juice, peptone, yeast extract, corn flour, bran, various cake powder, potato, milk, and serum. Although the medium made with these substances can not exactly know its chemical composition, in general, the nutrition is rich, the growth of microbes is strong, and the source is wide, the preparation is convenient, so it is more commonly used, especially suitable for the preparation of laboratory medium. The stability of this medium is often influenced by factors such as production plants or batch numbers.
2) synthetic medium is a kind of medium with a complete knowledge of chemical composition and quantity, which is made up of chemical drugs known as chemical components. This kind of medium is accurate and reproducible, but it is expensive, and the microorganism is slow to grow. So it is only suitable for some scientific research, such as nutrition and metabolism.
3) semisynthetic medium is added to a synthetic medium by adding some or several natural ingredients; or in a natural medium, a chemical known as a known component is added to a semisynthetic medium. For example, potato sucrose medium. This medium is the most widely used in production practice and laboratory.
2. According to the physical state of the medium, it can be divided into solid medium, liquid medium and semisolid medium.
1) the medium prepared by the liquid medium is liquid, and the components of the medium are basically dissolved in water, there is no clear solid, the nutrient composition of the liquid medium is evenly distributed, and it is easy to control the growth and metabolism of microorganism.
2) solid medium is added to the liquid medium to form a solid medium with a suitable amount of coagulant. Agar, gelatin and silica gel are commonly used as coagulants. Agar is the most commonly used material. Solid medium is widely used in practice. In the laboratory, it is used for isolation, identification, examination of bacteria, counting, preservation and biometrics.
3) if semisolid medium is added to a liquid medium with a small amount of coagulant, a semisolid medium will be made. Take agar as an example, and its dosage is between 0.2~1%. This medium can sometimes be used to observe the power of microorganisms and sometimes to preserve bacteria.
3. According to the use of culture medium, it can be divided into selective medium, proliferating medium and differential culture medium.
1) choose a medium to add or kill certain substances in the medium to kill or inhibit the growth of an unnecessary strain. Such as streptomycin, chloramphenicol and other inhibition of the growth of prokaryotic microorganisms; and nystatin and chyx can inhibit the growth of eukaryotic microbes; crystal violet can inhibit the growth of Gram-positive bacteria.
2) the proliferating medium often lives together in the natural world. In order to separate the microbes we need, we add some specific microbes to the common culture medium in order to increase the rate of reproduction and gradually eliminate the other microbes. This medium is called increasing. The culture medium is often used in screening and selecting bacteria. To some extent, the proliferation medium is also a selective culture.
3) a variety of reagents or chemicals are added to the medium in the culture medium to make the indistinguishable microorganisms show obvious differences after culture, thus helping to quickly identify a certain microorganism. Such medium is called the differential medium. For example, the eosin methylene blue medium used to check whether water and dairy products contain enteric pathogens is a commonly used differential culture medium.
Some media have dual functions of selection and identification. For example, Makanke culture medium, 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 (selectivity), lactose and neutral red (indicator) can help distinguish between lactose ferment enterobacteriella (e. E. coli) and intestinal bacteria that can not ferment lactose (such as Salmonella and Shigella).
In addition, according to whether the nutrient components of the medium are "complete", they can be divided into basic medium, complete medium and supplemental medium. These terms are mainly used in microbial genetics. According to the purpose of culture medium, it can be divided into seed medium and fermentation medium. There are also living tissue culture media, such as chicken embryos, specially used to cultivate parasitic microorganisms, such as viruses.
Three. The basic methods and matters needing attention in the preparation of medium.
1. Selection of culture medium formula
There is always some difference in the formula of the same medium in different works. Therefore, in addition to the standard method, it should be made up strictly according to its provisions. Generally, we should collect relevant information as far as possible, check it, and then select and record its source according to the purpose of his own use.
2. Record of culture medium
Each preparation culture medium should have a record, including the name of the medium, the formula and its source, the number of various ingredients, the final pH value, the date and the preparation of the temperature and time of disinfection, the records should be copied, the original records are preserved, and the copy records are stored together with the prepared medium in order to prevent the confusion.
3. The name of the medium composition of the culture medium
The ingredients of the medium must be accurately weighed and attention should be paid to preventing confusion. The formula can be placed beside the side, each of which is called a mark with the square face, and the required medicine is collected at the left, each of which is removed to the right. After the completion of the test, a check should also be made.
4. Mixing and melting of the components of the medium
The chemicals used in the culture medium should be chemically pure. The cooking pot used shall not be copper pots or iron pots, so that no trace copper or iron can be mixed into the medium so that the bacteria are not easy to grow. It is best to use stainless steel pots to be heated and melted, and can be dissolved in a large beaker or large flask in a high pressure steam sterilizer or a mobile steam sterilizer. When melted in a pot, it can be heated with warm water and disturbed at any time to prevent coking. If coke is found, the medium can not be used, and it should be re prepared. After most of the solid components are melted, use less firepower to dissolve all the components until boiling. If it is dissolved for agar, dissolve some other components with another part of water, and then mix the two solution together. In the process of heating and melting, the water lost by evaporation must be supplemented finally.
5. Preliminary correction of culture medium pH
Because the medium will change during the process of heating and disinfection, pH will be changed after the ingredients are completely dissolved. PH should be adjusted preliminarily. For example, beef extract can reduce pH0.2 and intestinal extract pH will increase significantly. Therefore, for this step, the operator should pay attention to exploring experience at any time so as to master the final PH of the medium and ensure the quality of the medium. After PH is adjusted, the medium should be boiled for several minutes to facilitate the precipitation of the media.
6. Filtration and clarification of culture medium
The liquid medium must be absolutely clarified, and the agar medium should also be transparent and no significant precipitation. Therefore, filtering or other clarification methods should be used to achieve this requirement. The general liquid medium can be filtered by filter paper. The filter paper should be folded into a folding fan or funnel shape to avoid the rupture of filter paper caused by uneven hydraulic pressure.
Agar medium can be filtered with clean white flannelette. It can also be filtered by double-layer gauze sandwiched with thin absorbent cotton sandwiched in the middle. When making fresh meat, liver, blood and potatoes, it is necessary to filter the residue with flannelette and filter it repeatedly. If the filtration method fails to achieve the clarification requirement, the egg white clarification method must be used. The culture medium that is about to be cooled to 55~60 C is put into a large triangular flask, the loading amount should not exceed the 1/2 of the capacity of the flask, the protein of 1~2 eggs is added to each 1000ml medium, the strength is shaking for 3~5 minutes, and the high pressure steam sterilizer, 121 degree C is heated for 20 minutes, and the heat is removed with the flannelette.
7. The separation of culture medium
The media should be packed in test tubes, flasks and other appropriate containers according to the purpose and requirements of the use. The amount of separation shall not exceed 2/3 of the container's full capacity. The mouth of the container can be blocked by a cotton stopper with wet proof paper, and it must be wrapped with waterproof paper. It is better to use semi-automatic or electric quantified distributor when loading. When the agar slant medium is packed separately, the amount of packing should be consistent with the amount of 2/3 bottom and 1/3 slope. The separated containers should be cleaned in advance and dry baked and sterilized, so as to facilitate the complete sterilization of the medium. Each batch of medium should be separately packed with 20ml and cultured in a small glass bottle, and then sterilized with the batch culture medium to determine the final pH of the medium.
8, sterilization of culture medium
The general medium can be sterilized by 121 degree C high pressure steam for 15 minutes. In all kinds of medium preparation methods, without special regulations, this method can be sterilized.
Some of the hot ingredients, such as carbohydrates, should be separated into 20% or more high concentration liquid, which are sterilized by filtration or intermittent sterilization, and then used in aseptic technique and added to the culture medium. The gelatin medium was also used for low temperature sterilization. Blood, body fluids and antibiotics should be extracted and added to the medium with a cooling of about 50 degrees C by aseptic technique.
The agar slant media should be removed immediately after sterilization. When the temperature reaches 55 -60 C, the agar should be placed in a suitable inclined plane until it solidifies naturally.
9, quality test of culture medium
When each batch of medium is prepared, it should be examined carefully, such as breakage, water immersion, color and lustre abnormality, and cotton plugs being stained with culture foundation. And the final pH was determined.
All the medium was incubated in a 36 + 1 degree C incubator overnight.
Inoculate 1~2 tube or bottle medium with standard strains, and cultivate 24~48 hours, such as aseptic growth or poor growth. The cause should be traced and vaccinated repeatedly. If the result is still the same, the batch should be abandoned and not used.
10. Preservation of culture medium
Medium should be stored in cold and dark places, preferably in ordinary refrigerators. The placement time should not exceed one week, and the plate medium should not exceed 3 days. Each batch of culture medium must be attached to the batch medium to prepare a record page or a marked label.