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Method for separating and purifying microbial strains commonly used in laboratories
The process of obtaining a microorganism containing only one or a certain strain from a mixed microbial population is called microbial isolation and purification. In the research and application of molecular biology, it is not only necessary to separate specific microorganisms from the mixed natural microbial population through separation and purification techniques, but also must pay attention to maintaining the "pure" of the pure culture of microorganisms and preventing the incorporation of other microorganisms.
1. Separation and purification with solid medium
A single microorganism grows on the surface or inside of a suitable solid medium to a certain extent to form a population of sub-cells that are visible to the naked eye and have a certain morphological structure, called a colony. When many colonies on the surface of the solid medium are connected into one piece, it becomes a lawn. Colonies or lawns formed by different microorganisms growing on a specific medium generally have stable characteristics and can be an important basis for classification and identification of the microorganisms. Most bacteria, yeasts, and many fungi and unicellular algae can form isolated colonies on solid media, and pure cultures are readily obtained by suitable plate separation methods. The so-called flat plate, that is, the abbreviation of the culture plate, refers to a plate in which a solid medium is poured into a sterile plate and cooled and solidified to hold a solid medium. This method involves separating and immobilizing a single microorganism on or in the solid medium. Solid medium A medium solidified with agar or other gel material. Each isolated living microorganism grows and multiplies to form colonies, and the formed colonies are easy to transplant. The most commonly used solid medium for isolating and cultivating microorganisms is an agar solid medium plate. The technique developed by Kock using plate-separating microbial culture is simple and easy, and has been the most common means of separation of various strains for more than 100 years.
1.1 Dilution plate method
First, the microbial suspension is diluted in series (such as 1:10, 1:100, 1:1000, 1:10000), then take a little different dilutions, and agar medium that has been melted and cooled to about 50 °C. Mix, shake, and pour into the sterile culture dish. After the agar is solidified, make agar plates that may contain bacteria. After incubation for a certain period of time, colonies will appear. If properly diluted, a single colony can appear on the surface of the plate or in the agar medium. This colony may be formed by the propagation of a bacterial cell. The single colony is then picked, or the above operation is repeated several times to obtain a pure culture.
1.2 Coating plate method
Because the microbial suspension is first added to the hot medium and then the flat plate is liable to cause the death of some heat-sensitive bacteria, and the dilution of the plate method will also cause some strict aerobic bacteria to be fixed in the middle of the agar without oxygen. It affects its growth, so the pure seed separation method commonly used in microbiology research is the coating plate method. The method comprises the steps of: pouring the melted culture medium into a sterile plate to form a sterile plate, and after cooling and solidifying, adding a certain amount of microscopic suspension to the surface of the plate, and then uniformly dispersing the bacterial solution with a sterile glass coating rod. Disperse to the entire surface of the plate and pick up a single colony after incubation (Figure 1).
Figure 1 coating plate method
1.3 flat line method
The simplest method for isolating microorganisms is to perform a plate scribing method in which a small amount of material to be separated is aseptically treated with an inoculating loop, and a continuous scribing is performed on the surface of the sterile plate (Fig. 2), and the number of microbial cells will follow The number of lines is reduced and gradually dispersed. If the scribing is appropriate, the microorganisms can be dispersed one by one, and after cultivation, a single colony can be obtained on the surface of the plate. Sometimes such single colonies are not all propagated from a single cell, so it is necessary to repeatedly separate multiple times to obtain a pure species. The principle is to dilute the microbial sample on the surface of the solid medium multiple times to "separate from line to line" for separation purposes. There are many methods for scribing, and the common scribing methods for single colonies are slash method, curve method, square method, radio method, and four-frame method.
Figure 2 flat line method
1.4 Dilution shake method
Separation of strict anaerobic bacteria by solid medium is special. If the microorganism does not die immediately after exposure to the air, the plate can be prepared by a usual method and then placed in a closed container, and the oxygen in the container can be chemically. Physical or biological methods are removed. For those anaerobic microorganisms that are more sensitive to oxygen, the pure culture separation can be carried out by dilute shaker culture, which is a variant of the dilution plate method. First, a series of tubes filled with sterile agar medium are heated to melt the agar, and then cooled and kept at about 50 ° C. The materials to be separated are diluted with these tubes, the tubes are shaken rapidly, and after condensation, they are poured on the surface of the agar column. A mixture of sterilized liquid paraffin and solid paraffin separates the medium from the air. After the cultivation, the colonies were formed in the middle of the agar column. To pick and transplant a single colony, first remove the liquid paraffin-paraffin cover with a sterile needle, then insert a capillary into the agar and the wall, blow in sterile oxygen-free gas, and agar column. Aspirate, place in a Petri dish, and cut the agar column into thin slices with a sterile knife for observation and colony transplantation.
2. Separation and purification with liquid medium
Most bacteria and fungi, which are separated by plate method, are generally satisfactory because most of their species grow well on solid media. However, not all microorganisms have so far been able to grow on solid media, such as some large cell bacteria, many protozoa and algae, etc. These microorganisms still need to be separated by liquid medium to obtain pure culture.
The dilution method is a commonly used method for separation and purification of liquid medium. The inoculum was serially diluted in liquid medium to achieve a highly diluted effect, so that one microorganism could not be dispensed in one tube. If there is no microbial growth in most of the diluted tubes, the culture obtained from the tubes with microbial growth may be pure cultures. If the proportion of microbial growth in the diluted tube is increased, the chances of obtaining a pure culture will drop dramatically. Therefore, liquid separation using the dilution method must be performed in many parallel tubes of the same dilution, and most (generally should exceed 95%) behave as no growth.
3. Single cell (spore) separation
It is an important disadvantage of the dilution method to isolate only the dominant species in the mixed microbial population. In nature, many microorganisms are a minority in mixed populations. At this time, single cells or individual individuals can be directly isolated from the mixed population by microscopic separation to obtain a pure culture, which is called a single cell (or single spore) separation method. The difficulty of single cell separation is inversely proportional to the size of cells or individuals. Larger microorganisms such as algae and protozoa are easier, and individuals with smaller bacteria are more difficult.
For larger microorganisms, a single individual can be extracted by capillary extraction and washed several times in a large amount of sterile medium to remove contamination by smaller microorganisms. This can be done under a low power microscope, such as a dissecting microscope. For microorganisms with relatively small individuals, a micromanipulator is used, and a single microbial cell or spore is picked up under a microscope with a capillary or a microneedle, a hook, a loop, or the like to obtain a pure culture. In the absence of a micromanipulator, single-cell separation can also be performed under the microscope by some alternative methods. For example, the appropriately diluted sample is prepared into small droplets and observed under a microscope, and a liquid containing only one cell is selected. Separation of pure cultures. The single cell separation method has relatively high requirements for the operation technology, and is mostly limited to highly specialized scientific research.
4, choose culture separation
No medium or a culture condition can meet the needs of all microbial growth, and to a certain extent all mediums are selective. If the growth requirements of a microorganism are known, it is also possible to design a specific environment suitable for the growth of such microorganisms, and thus it is possible to select such microorganisms from a mixed population of microorganisms, although in a mixed microbial population Microorganisms may only be a minority. This technique of performing microbial pure culture separation by selective culture is called selective culture separation, and is particularly suitable for separating and searching for useful microorganisms from nature. In nature, in most cases, the microbial community is composed of a variety of microorganisms, and it is very difficult to separate the specific microorganisms required, especially when a certain microorganism exists in a very small amount compared with other microorganisms. The general plate dilution method is almost impossible. In order to isolate such microorganisms, it is necessary to adopt a method of selective culture separation according to the characteristics of the microorganism, including nutrition, physiology, growth conditions and the like. Or inhibiting the growth of most microorganisms, or causing the growth of the bacteria, after a certain period of time, the number of the bacteria in the community is increased, and then it is purely cultured and separated by means of plate dilution.
4.1 Direct separation using a selection plate
Different culture conditions can be selected depending on the characteristics of the microorganism to be separated, and various methods can be employed. For example, to isolate high-temperature bacteria, culture can be carried out under high temperature conditions; to isolate an antibiotic-resistant strain, it can be isolated on a plate with antibiotics; some microorganisms such as spirochetes, myxobacteria, cyanobacteria, etc. can be surfaced on agar plates. Or sliding inside, they can be separated and purified by their sliding characteristics, because the sliding can separate themselves from other microorganisms that cannot move. The microbial community can be planted on the plate, the microorganisms can be slid, and the inoculum can be inoculated from the gliding front and repeated to obtain a pure culture.
4.2 Enrichment culture
The principles and methods of enrichment culture method are very simple. Different characteristics of life activities between different microorganisms are used to formulate specific environmental conditions, so that microorganisms that are only suitable for this condition grow vigorously, so that the number in the community is greatly increased, and it is easy to Separate to the specific microorganisms required. The enrichment conditions can be selected from physical, chemical, biological, and comprehensive aspects depending on the characteristics of the microorganism to be separated, such as temperature, pH, ultraviolet light, high pressure, light, oxygen, nutrition, and the like. Under the same medium and culture conditions, after repeated seeding, the finally enriched strain easily grows single colonies on the solid medium. If you want to isolate some obligate parasites, you must inoculate the sample into the corresponding sensitive host cell population to grow it in large quantities. Pure parasites can be obtained by repeated seedings.
5, binary culture
The purpose of the separation is usually to obtain a pure culture. However, in some cases this is very difficult to do. However, binary cultures can be used as a substitute for purification culture. Cultures containing more than two microorganisms are referred to as mixed cultures, and if the culture contains only two microorganisms, and a culture that consciously maintains a specific relationship between the two is called a binary culture. For example, binary cultures are the most effective way to preserve viruses because they are the strict intracellular parasites of cellular organisms. Some microbes with cells are also strict intracellular parasites of other organisms, or special symbiotic relationships. For these organisms, the binary culture is the closest culture method that can be achieved under laboratory controlled conditions. In addition, protozoa that hunted small microbes are also easily cultured in a laboratory using a binary culture method consisting of both protozoa and the microbes it preys. For example, ciliates, amoeba, and slime molds.
Among the several methods described above, plate separation is commonly used for the separation and purification of laboratory microorganisms. A single colony formed by the growth of microorganisms on a solid medium, usually an aggregate produced by one cell. Therefore, a pure culture can be obtained by picking up a single colony. The method of obtaining a single colony can be accomplished by techniques such as dilution coating of a plate or scribing of a plate. It is worth noting that a single colony that is isolated from a microbial population and grown on a plate does not necessarily guarantee a pure culture. Therefore, in addition to observing the characteristics of colonies, the determination of pure culture can be determined by combining microscopic examination of individual morphological characteristics. Some pure cultures of microorganisms can be obtained through a series of separation and purification processes and various characterizations.