Microbial inoculation, culture, separation
vaccination
The microbe is fed into an artificial medium suitable for its growth and reproductionmediumThe process in living organisms is called inoculation.
Vaccination and separation tools
1. Inoculation needle 2. Inoculation ring 3. Inoculation hook 4.5. Glass coating rod 6. Inoculation ring 7. Inoculation hoe 8. Small scalpel
Common vaccination methods include the following:
1. Scribe vaccination
This is the most commonly used inoculation method. That is, the surface of the solid medium can be moved back and forth in a straight line to achieve the role of inoculation. Commonly used inoculation tools include inoculation rings, inoculation needles, etc. This method is commonly used in bevel inoculation and plate scribing.
2. Three-point vaccination
This method is often used when studying the morphology of molds. This method involves inoculating a small amount of microorganisms on the surface of the plate into three points of an equilateral triangle, allowing them to form bacteria independently to observe and study their morphology. In addition to three points, there are also one or more points for inoculation.
3. Puncture vaccination
This method is often used in the preservation of anaerobic bacteria or the study of the power of microorganisms. When doing puncture inoculation, the inoculation tool used is the inoculation needle. The medium used is usually a semi-solid medium. Its method is to dip a small amount of bacteria in the inoculation needle and make a straight puncture along the center of the semi-solid medium to the bottom of the tube. If a bacterium has flagella and can move, it can grow around the puncture line.
4. Mixed vaccination
The method is to put the microorganisms to be picked up into a petri dish first, then pour in a solid medium cooled to about 45 ° C, shake quickly and gently, so that the bacterial liquid can achieve the purpose of dilution. After the plate solidifies and incubates at a suitable temperature, a single microbial colony can grow.
5. Coating and inoculation
It is slightly different from pouring mixed inoculation, that is, first pour the plate, let it solidify, and then pour the bacterial liquid into the plate, quickly use the coating rod to coat the surface back and forth, so that the bacterial liquid is evenly distributed, and a single microbial colony can grow.
6. Liquid inoculation
Washing the bacteria from the solid medium, pouring them into the liquid medium, or pipetting the bacterial liquid from the liquid culture into the liquid medium, or moving the bacterial liquid from the liquid culture into the solid medium, can be called liquid inoculation.
7. Injection
The method is to transfer the microorganisms to be transferred to a living organism by injection, such as in humans or other animals. The common vaccination method is to use injections to access the human body to prevent certain diseases.
8. In vivo vaccination
In vivo inoculation is a method specifically used to grow viruses or other pathogenic microorganisms because viruses must be inoculated into a living organism in order to grow and reproduce. The living organism used can be a whole animal; it can also be an isolated living tissue, such as a monkey kidney, etc.; it can also be a developing chicken embryo. Inoculation is done by injection, or it can be fed with a mix of ingredients.
Note: Vaccination must be aseptic
After the medium is autoclaved, the bacteria-containing materials (such as samples, fungi or bacterial suspensions, etc.) are inoculated on the medium under sterile conditions with sterilized tools (such as inoculation needles and straws, etc.). This process is called aseptic inoculation operation. Various inoculations in laboratory tests must be aseptic operations.
(1) Inoculation and sterilization (2) Opening the tampon (3) Sterilization at the nozzle (4) Pick up the moss (5) Inoculation (6) Plug the tampon.
separation and purification
A culture containing more than one type of microorganism is called a mixed culture. If all cells in a colony are from one parent cell, then the colony is called a pureculture. In the identification of strains, the microorganisms used are generally required to be pure cultures. The process of obtaining a pure culture is called isolation and purification, and there are many methods.
1. Pouring plate method
First, the microbial suspension is passed through a series of dilutions, and a certain amount of dilution is taken and fully mixed with the melted nutrient agar medium maintained at about 40-50 °. Then, the mixed liquid is poured into a sterile petri dish. After solidification, the plate is inverted and cultured in a constant chamber. A single cell is multiplied many times to form a colony, and a single colony is taken to make a suspension. Repeat the above steps several times to obtain a pure culture.
2. Coating plate method
First, the microbial suspension is diluted properly, and a certain amount of the dilution is placed on a sterile solidified nutrient agar plate. Then, the dilution is evenly coated on the surface of the medium with a sterile glass scraper, and a single colony can be obtained by constant temperature culture.
3. Flat scribing method
The simplest way to isolate microorganisms is the plate scribing method. Use a sterile inoculation ring to take a little culture and scribe it on the plate. There are many scribing methods, and the common scribing methods that are more likely to appear a single colony include the slash method, the curve method, the square method, the radiological method, and the four-grid method. When the inoculation ring moves back on the surface of the medium, the bacterial liquid on the inoculation ring is gradually diluted, and finally individual cells are dispersed on the scribed line. After culture, each cell grows into a colony.
4. Enrichment culture method
The method and principle of the enrichment culture method are very simple. We can create conditions that allow only the desired microorganisms to grow. Under these conditions, the required microorganisms can effectively compete with other microorganisms and far exceed other microorganisms in terms of growth capacity. If you want to isolate some obligatory parasitic bacteria, you must inoculate the sample into the corresponding sensitive host cell population to make it grow in large quantities. Pure parasitic bacteria can be obtained by repeated transplanting.
5. Anaerobic method
In the laboratory, in order to isolate certain anaerobic bacteria, a test tube containing the original culture medium can be used as a culture container, and the test tube can be heated in a boiling water 0 bath for several minutes in order to expel the dissolved oxygen in the medium. Then quickly cool and inoculate. After inoculation, sterile paraffin wax is added to the surface of the medium to isolate the medium from the air. Another method is to use n2 or co2 to replace the gas in the medium after inoculation, and then seal the tube mouth on the flame. Sometimes in order to isolate certain anaerobic bacteria more effectively, the isolated sample can be inoculated on the medium, and then the petri dish is placed in a completely sealed anaerobic culture device.
6. Single cell (or single spore) separation method
Microscopic separation is used to directly separate a single cell or individual from a mixed population for culture to obtain pure culture. Larger microorganisms can be extracted from a single individual by capillary. Microorganisms with relatively small individuals need to use a micromanipulator to pick a single microbial cell or spore under a microscope with capillary tubes or micro needles, hooks, loops, etc. to obtain pure culture. Single cell separation method has relatively high requirements for operation technology.
cultivate
1. According to whether oxygen is required during culture, it can be divided into two categories: aerobic culture and anaerobic culture.
Aerobic culture: also known as "aerobic culture". That is to say, this microorganism needs oxygen to be added when it is cultivated, otherwise it will not grow well. In the laboratory, inclined culture is to obtain sterile air from the outside through cotton plugs. Most of the liquid cultures in triangular flasks are shaken by a shaker, so that the outside air can continuously enter the bottle.
Anaerobic culture: also known as "anaerobic culture". This type of microorganism does not require oxygen to participate in the culture. During the culture of anaerobic microorganisms, the most important point is to remove the oxygen in the medium.
2. According to the physical state of the medium, it can be divided into solid culture and liquid culture.
Solid culture: It is a method of connecting strains to a loose and nutritious solid medium for microbial culture under suitable conditions.
Liquid culture: In experiments, liquid culture allows microorganisms to multiply rapidly and obtain a large number of cultures. Under certain conditions, it is also an effective method for microbial selection to increase bacteria.
Conventional identification technology
Observation of morphological structure and culture characteristics
1. The morphological structure of microorganisms is mainly observed through staining, and its shape, size, arrangement, cell structure (including cell wall, cell membrane, nucleus, flagella, spores, etc.) and staining characteristics are observed under the microscope to intuitively understand the morphological structure characteristics of bacteria, and to achieve the purpose of distinguishing and identifying microorganisms according to the differences in morphological structure of different microorganisms.
2. The cell population formed by bacterial cells on the surface of solid medium is called a colony (cOlony). Different microorganisms grow and multiply in a certain medium, and the colony characteristics formed are very different, while the same bacteria have certain stability in culture characteristics under certain conditions. This can distinguish and identify different microorganisms. Therefore, the observation of microbial culture characteristics is also an important content in microbial inspection and identification.
Physiological and biochemical tests
Microbial biochemical reaction: refers to the use of chemical reactions to determine the metabolites of microorganisms. Biochemical reactions are often used to identify microorganisms that are not easily distinguished in morphology and other aspects. Therefore, microbial biochemical reactions are one of the important bases for microbial classification and identification.
Biochemical reactions commonly used in microbiological testing include:
1. Glycolysis test
The ability of different microorganisms to decompose and utilize carbohydrates varies greatly, whether it can be used or not, and whether it can be used or not. Indicators and fermentation tubes can be used to test.
2. Starch hydrolysis test
Some bacteria can produce enzymes that break down starch, which is hydrolyzed into maltose or glucose. Starch does not turn blue when hydrolyzed with iodine.
3. V-P test
Some bacteria can decompose glucose in glucose peptone water medium to produce pyruvate, pyruvate condensation, decarboxylation to acetyl methanol, which is oxidized into diacetyl in the air under strong alkali environment, and diacetyl forms a red compound with guanidine in peptone, called v-p (+) reaction.
4. Methyl red test
Enterobacteriaceae can ferment glucose and produce pyruvate in the process of decomposing glucose. During further decomposition, due to the different pathways of sugar metabolism, a large number of acidic products such as lactic acid, succinic acid, acetic acid and formic acid can be produced, which can reduce the pH of the medium to below ph4.5 and make the methyl red indicator turn red.
5. Indigo matrix (imdole) test
Some bacteria can break down tryptophan in peptone to produce indoles. The presence of indoles can be expressed by a color reaction. Indole binds to p-dimethylaminobenzaldehyde to form roseindole, a red compound.
6. Nitrate reduction test
Some bacteria have the ability to reduce nitrate, which can be reduced to nitrite, ammonia or nitrogen, etc. The presence of nitrite can be tested with nitric acid reagents.
7. Gelatin liquefaction test
Some bacteria have gelatinase (also known as proteolytic enzymes), which can first hydrolyze gelatin into peptides, and then further hydrolyze it into amino acids, losing its gel properties and liquefying it.
8. Urease test
Some bacteria can produce urease, which breaks down urea and produces two molecules of ammonia, making the medium alkaline and phenol red pink. Urease is not an inducible enzyme because bacteria can synthesize this enzyme regardless of the presence or absence of substrate urea. Its activity is optimum for pH 7.0.
9. Oxidase test
Cytochrome oxidase, also known as cytochrome oxidase, is the terminal respiratory enzyme of the cytochrome respiratory enzyme system. The oxidase first oxidizes cytochrome c, and then the oxidized cytochrome c oxidizes p-phenylenediamine to produce a color reaction.
10. Hydrogen sulfide (H2S) test
Some bacteria can decompose sulfur-containing amino acids or sulfur-containing compounds in the culture medium to produce hydrogen sulfide gas, which can form black deposits when exposed to lead salts or low iron salts.
11. Iron trisaccharide (TSI) agar test
12. Hydrogen sulfide-indigo matrix-dynamic (sim) agar test
III. Chemical composition analysis
microbial preservation
The basic principle is to artificially create an environment conducive to dormancy on the basis of selecting excellent pure cultures and making them dormant, so that they can still maintain the original excellent characteristics of the strains after long-term storage. The basic measures are low temperature, vacuum and drying.
Preservation method:
1. Periodic transplantation
Also known as the subculture preservation method, it refers to a short-term preservation method in which strains are inoculated on a suitable inclined medium, cultured under the most suitable conditions, and cultured at 4 to 6 ° C and transplanted at a certain interval (3-6 months).
2. Liquid paraffin method
It refers to a kind of bacteria preservation method in which the bacteria are inoculated on a suitable inclined surface medium, cultured under the most suitable conditions, and then injected with sterilized liquid paraffin wax to cover the entire inclined surface, and then placed upright in a dry place at low temperature (4~ 6 ° C).
3. Vacuum freeze-drying method
It refers to a long-term strain preservation method in which the preserved strain cells or spores are suspended in a protective agent, the water is sublimated under vacuum conditions after pre-freezing, and then stored after vacuum storage.
4, -80 ℃ low temperature freezing method
A long-term preservation method of bacterial species suspended in a protective agent and frozen in a refrigerator at -80 ° C.
5. Liquid nitrogen cryogenic freezing method
It refers to a long-term preservation method in which strains suspended in protective agents are stored in liquid nitrogen at -196 ° C or liquid nitrogen gas at about -150 ° C after programmed cooling.
