Special environments.
In bacteriology, dry nutrient media of industrial production are widely used, which are hygroscopic powders containing all components of the medium, except water. For their preparation, tryptic digests of cheap non-food products (fish waste, meat and bone meal, technical casein) are used. They are convenient for transportation, can be stored for a long time, relieve laboratories from the enormous process of preparing media, and bring the issue of standardization of media closer to resolution. The medical industry produces dry media of Endo, Levin, Ploskirev, bismuth sulfite agar, nutrient agar, carbohydrates with BP indicator and others.
thermostats
Thermostats are used for cultivating microorganisms.
A thermostat is a device that maintains a constant temperature. The device consists of a heater, a chamber, double walls between which air or water circulates. The temperature is controlled by a thermostat. The optimum temperature for the reproduction of most microorganisms is 37°C.
ACTIVITY 7
TOPIC: METHODS FOR ISOLATION OF A PURE AEROBIC CULTURE. STAGES OF ISOLATION OF A PURE CULTURE OF AEROBIC BACTERIA BY THE METHOD OF MECHANICAL UNCOUPLING
Lesson Plan
1. The concept of "pure culture" of bacteria
2. Methods for isolating pure cultures by mechanical separation
3. Biological methods for isolating pure cultures
4. Methods for identifying bacteria
Purpose of the lesson: To acquaint students with various methods for isolating pure cultures, to teach how to do crops with a loop, strokes, prick
Guidelines for the demonstration
In their natural habitat, bacteria are found in associations. In order to determine the properties of microbes, their role in the development of the pathological process, it is necessary to have bacteria in the form of homogeneous populations (pure cultures). A pure culture is a collection of bacterial individuals of the same species grown on a nutrient medium.
Methods for isolating pure cultures of aerobic bacteria
Pasteur Method Koch Method Biological Physical
(has historical (plate wiring)
Meaning)
Chemical Method
Shchukevich
Modern
Sowing with a loop Sowing with a spatula
(Drigalski method)
Methods for isolating pure cultures:
1. Methods of mechanical separation are based on the separation of microbes by successive rubbing of the test material over the surface of the agar.
a) Pasteur's method - is of historical importance, provides for the sequential dilution of the test material in a liquid nutrient medium by the rolling method
b) The Koch method - the method of plate layouts - is based on the sequential dilution of the test material with meat-peptone agar, followed by pouring test tubes with diluted material into Petri dishes.
c) Drygalski's method - when sowing material abundantly seeded with microflora, use 2-3 cups for successive sowing with a spatula.
d) Sowing with a loop in parallel strokes.
2. Biological methods are based on the biological properties of pathogens.
a) Biological - infection of highly sensitive animals, where microbes rapidly multiply and accumulate. In some cases, this method is the only one that allows you to isolate the culture of the pathogen from a sick person (for example, with tularemia), in other cases it is more sensitive (for example, the isolation of pneumococcus in white mice or the causative agent of tuberculosis in guinea pigs).
b) Chemical - based on the acid resistance of mycobacteria. To free the material from accompanying flora, it
treated with an acid solution. Only tubercle bacilli will grow, as the acid-tolerant microbes are killed by the acid.
c) The physical method is based on the resistance of spores to heat. To isolate cultures of spore-forming bacteria from
mixtures, the material is heated at 80°C and inoculated on a nutrient medium. Only spore bacteria will grow, because their spores remained alive and gave growth.
d) Shukevich's method - based on the high mobility of the vulgar proteus, capable of creeping growth.
Agar Plate Preparation Method
MPA is melted in a water bath, then cooled to 50-55°C. The neck of the vial is burned in the flame of an alcohol lamp, the Petri dishes are opened so that the neck of the vials enters, without touching the edges of the dish, pour out 10-15 ml of MPA, close the lid, shake the dish so that the medium is evenly distributed, leave it on a horizontal surface until solidified. After drying, plates with plate agar are stored in the cold.
Loop sowing
A drop of material is taken with a sterile cooled loop, one edge of the cup is slightly opened with the left hand, a loop is inserted inside and a few strokes are made at the opposite edge in one place, then the loop is torn off and the material is inoculated with parallel strokes from one edge of the cup to another with an interval of 5-6 mm. At the beginning of sowing, when there will be a lot of microbes on the loop, they will give confluent growth, but with each stroke of the microbes on the loop, there will be less and less, and they will remain solitary and give isolated colonies.
Sowing according to the Drygalski method
This method is used when sowing material abundantly seeded with microflora (pus, feces, sputum). For sowing according to the Drygalsky method, a spatula and several cups (3-4) are taken. A spatula is a tool made of metal wire or glass dart, bent in the form of a triangle or L-shaped. The material is introduced into the first cup with a loop or pipette and evenly distributed with a spatula over the surface of the medium, with the same spatula, without burning it, the material is rubbed into the nutrient medium in the second cup, and then in the third. With this inoculation, the first plate will have confluent growth, and isolated colonies will grow in subsequent plates.
If, on the basis of certain symptoms on the plants and the results of microscopic examination, it is suspected that the causative agent of the disease is a bacterium, the next step should be to isolate it.
In this case, it is assumed that the pathogen is contaminated with associated organisms, i.e., there is a mixed population. To obtain the pathogen as a separate growing colony, the tissue macerate should be streaked onto the medium.
Stroke sowing. A small amount of plant tissue macerate containing bacteria is taken with a calcined inoculation loop and with light movements, without damaging the surface of the agar, is applied to the prepared nutrient medium with 4-6 strokes. Having re-ignited the loop, the cup with the medium is turned 90 ° to the right and then 4-6 more strokes are applied from the second stroke, the needle is re-ignited and the third inoculation is carried out. This achieves such a dilution of the starting material, in which the bacteria, after incubation in a thermostat for 48-72 hours at 28 ° C, form separate colonies of various shapes and colors. The colonies are then transferred for further study into slant agar tubes. A colony is taken with a calcined loop and, with a careful movement in the form of a snake or zigzag, is applied to nutrient agar.
Koch pouring method. The Koch pouring method ensures that each colony is formed from a single bacterial cell. It is best to suspend the starting material in sterile water and apply the Koch method only with this dilution. A small amount of the suspension is transferred into the first test tube with a nutrient medium cooled to 60 °C. Then the contents of the tube are mixed with the inoculum by rotating it between the palms. Next, take the second test tube, carefully open it over the flame of the burner and transfer three portions of the substrate into it from the first test tube with large loops. The contents of the test tube after firing the neck and stopper are poured into the first Petri dish, slightly opening the lid of the cup just enough to insert the neck of the test tube under it. Immediately after pouring, the cup is closed and the nutrient medium is evenly distributed with careful movements.
After thoroughly mixing the contents of the second test tube, take the third test tube and transfer six portions of the substrate into it with a loop from the second. The contents of the test tube are poured into a cup, and the contents of the test tube, after mixing, are poured into a cup. Cups with the medium are incubated in a thermostat at 28°C, after a few days the bacteria contained in the starting material form colonies.
Serial breeding. If, for example, it is necessary to isolate bacteria from the soil, then serial dilution is used. Sterile nutrient medium (15 ml per dish) is poured into dishes, 0.1 ml of the last three dilutions of the suspension are applied to the hardened agar and spread over the surface with a glass spatula.
To isolate bacteria, 1 g of soil is suspended in 9 ml of water, shaken well, allowed to stand for a few seconds, and serial dilutions are prepared from the suspension. With this method, the number of microorganisms in each sample can be determined.
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Pasteur Method Koch Method Biological Physical
(has historical (lamellar
value) wiring) Shchukevich Chemical Method
Modern
Sowing with a loop Sowing with a spatula
(Drigalski method)
Methods for isolating pure cultures (Scheme 11):
1. Mechanical release methods based on the separation of microbes by successive grinding of the test material over the surface of the agar.
but) Pasteur Method– is of historical importance, provides for the sequential dilution of the test material in a liquid nutrient medium by the rolling method
b) Koch method- method of plate layouts - based on the sequential dilution of the test material with meat-peptone agar, followed by pouring test tubes with diluted material into Petri dishes
in) Drygalski method- when sowing material abundantly seeded with microflora, use 2-3 cups for successive sowing with a spatula.
G) Loop sowing with parallel strokes.
2. biological methods based on the biological properties of pathogens.
but) Biological- infection of highly sensitive animals, where microbes multiply rapidly and accumulate. In some cases, this method is the only one that allows you to isolate the culture of the pathogen from a sick person (for example, with tularemia), in other cases it is more sensitive (for example, the isolation of pneumococcus in white mice or the causative agent of tuberculosis in guinea pigs).
b) Chemical– based on the acid resistance of mycobacteria. To free the material from accompanying flora, it
treated with an acid solution. Only tubercle bacilli will grow, as the acid-tolerant microbes are killed by the acid.
in) physical method based on the resistance of spores to heat. To isolate cultures of spore-forming bacteria from
mixtures, the material is heated at 80°C and inoculated on a nutrient medium. Only spore bacteria will grow, because their spores remained alive and gave growth.
G) Shchukevich's method- based on the high mobility of the vulgar proteus, capable of creeping growth.
The method of transferring from colonies to slant agar and BCH:
but) Transferring from colonies to agar slant
The lid of the cup is slightly opened, a part of an individual colony is removed with a calcined cooled loop, a test tube with sterile slant agar is opened, holding it in the left hand in an inclined position, so that the surface of the medium can be observed. The loop with the culture is transferred into the test tube without touching the walls, rubbed over the nutrient medium, sliding over the surface from one end of the test tube to the other, raising the strokes to the top of the medium - sowing with a stroke. The tube is closed and, without letting go, the name of the seeded microbe and the date of sowing are signed.
b) Transferring from the colony to meat-peptone broth
The technique of reseeding on MPB is basically the same as when sowing on a dense medium. When sowing on the BCH, the loop with the material on it is immersed in the medium. If the material is viscous and is not removed from the loop, it is rubbed on the vessel wall, and then washed off with a liquid medium. Liquid material collected with a sterile Pasteur or graduated pipette is poured into a nutrient medium.
As a result of independent work, the student should know:
1. Methods for isolating a pure culture of microorganisms
2. Methods for cultivating microorganisms
Be able to:
1. Skills to comply with the rules of the anti-epidemic regime and safety precautions
2. Disinfect the material, treat hands
3. Prepare preparations from bacterial colonies
4. Microscope the colonies
5. Gram stain microorganisms
ACTIVITY 8
TOPIC. Methods for isolating pure cultures (continued). Enzymatic activity of bacteria and methods for its study.
Relatively few methods are known for isolating bacteria as pure cultures. This is most commonly done by isolating single cells on solid growth media using the streak-inoculation method or by pouring a small amount of liquid culture into plates ( dilution method). However, obtaining a separate colony does not always guarantee the purity of the culture, since colonies can grow not only from individual cells, but also from their clusters. If microorganisms form mucus, then foreign forms often attach to it. For cleaning, it is preferable to use a non-selective medium (MPA) because contaminants grow better on it and are easier to detect.
Obtaining isolated colonies on a solid nutrient medium is achieved either by sieving a suspension of microorganisms with a spatula ( Koch method), or with the help of a bacteriological loop ( debilitating stroke method). As a result of mechanical separation of microbial cells, each of them can give rise to an isolated colony of one microbial species.
Sieving with a spatula (Koch method) produced in the following sequence:
1) a drop of the enrichment culture is applied to the surface of the nutrient medium in cup No. 1 with a sterile pipette and distributed with a sterile spatula;
2) the spatula is taken out, the cup is quickly closed and the spatula is transferred to cup No. 2 without sterilizing it. Simulate the distribution of the culture over the entire surface of the medium by touching its surface with the same side of the spatula that was previously used to distribute the sample;
3) exactly the same actions are carried out in cup No. 3, after which the spatula is sterilized;
4) seeded cups are placed in a thermostat and incubated at the optimum temperature.
After a certain time, the cups are removed from the thermostat and the growth of microorganisms is studied. Usually, a continuous growth of bacteria is observed in dish No. 1, colonies are noted in subsequent dishes.
Loop sieving (depleting stroke method) involves inoculation of a bacteriological loop from an enrichment culture onto the surface of an agar medium in Petri dishes. At the first stage, a loop with the culture is applied with a series of parallel strokes on the agar medium (Figure 4.2, BUT). The loop is sterilized, cooled on the uninoculated part of the agar medium and a series of strokes is drawn in the direction perpendicular to the first (Figure 4.2, B). Then the loop is sterilized again, cooled and strokes are applied in the direction IN(Figure 4.2), and after the next sterilization - in the direction G(Figure 4.2). The cups are placed in a thermostat and after a certain time the results are taken into account. Usually on strokes BUT And B a large number of colonies grow (sometimes continuous growth), while on the strokes IN And G isolated colonies are formed.
Figure 4.2 - Scheme of sieving bacteria with strokes to obtain isolated colonies
Serial dilutions in solid media- the simplest method of plate inoculation, which consists in the fact that after inoculating the sample into a test tube with sterile melted and cooled agar, the medium is mixed, poured into a Petri dish and allowed to solidify. To obtain well-isolated colonies, a series of successive ten-fold dilutions is prepared and 1 ml of samples is added immediately to the dish, 15–20 ml of molten agar medium is added and mixed by shaking the dish. Sometimes individual colonies are immersed in agar and can only be removed mechanically. It is also bad that the bacteria are in the medium at the temperature of molten agar for some time.
A pure culture is a collection of microorganisms belonging to the same species. Obtaining this material is a necessary moment of research in the implementation of laboratory diagnostic techniques. To isolate pure cultures of bacteria, smears from blood, sputum, feces are used, purulent, as well as other biological material is examined. In natural conditions (in air, water, soil), food products, in corpses (human, animal) associations of various types of microorganisms are contained.
The separation of pure culture is important for a detailed study of the properties of microbes: morphological, cultural, biochemical, and also antigenic. Based on the results of these research methods, it is possible to establish that the pathogen belongs to a certain type and type, in other words, to perform its identification.
The principles of biological separation of bacteria imply taking into account the characteristics of the vital activity of microbes in order to choose the most optimal research methods. The selected methods must match the pathogen in certain parameters.
- Breath type. Among bacteria, groups of aerobic and anaerobic representatives are distinguished. To obtain anaerobic microbes, the research material is heated. The next stage is the cultivation of the microbe under anaerobic conditions. Methods for obtaining aerobic and anaerobic bacteria are different.
- Sporulation. The ability of some bacteria to sporulate ensures the protection and safety of microorganisms.
- The resistance of bacteria to the aggressive effects of alkalis and acids. The resistance of some types of bacteria to these substances ensures the maximum purification of the test material from the admixture of other bacteria using alkali or acid solutions.
- Mobility of bacterial organisms. To separate a pure culture of motile microorganisms, the methods of separating cultures of microbes in a drop of condensate are used.
- Susceptibility of microorganisms to antibiotics, certain chemicals and other antimicrobial agents. For some pathogens, culture isolation methods using selective (specific) nutrient media are most preferred.
- Antibiotics. Purify the material from additional microorganisms.
- The ability of bacteria to penetrate intact skin. This property is inherent in some varieties that have aggression factors.
- Susceptibility of animals to certain diseases of infectious origin.
Pathogen Assessment Methods
A number of methods are used to obtain pure cultures of bacterial cells. Each of them is used in a particular situation and has successive clear steps to obtain colonies of the pathogen. Consider the most popular.
Pasteur technique
This is the dilution of pure cultures in bacterial growth medium (liquid consistency) to a concentration of 1 cell per volume of liquid. This method of selection is of great historical importance.
Koch's technique
Also known as the "plate spread" technique. It is a dilution of the material for research in agar (in a molten state with a temperature of 48-50 ° C). Next, the resulting composition is poured into Petri dishes, where it should solidify.
Crops are usually carried out from the last 3-4 dilutions, since there are already few bacteria there. Thus, during the growth of microbes on a nutrient medium, isolated colonies of microorganisms appear, which are born from a single mother cell. Then, an isolated colony can be selected from the depth of the agar and transferred to a fresh nutrient medium. The next step is the identification and isolation of the pathogen.
Shukevich's technique
It is used when it is necessary to isolate a pure culture of Proteus aerobes or any other microbes characterized by "creeping" growth. Plant the cultures in water condensed at the base of the agar slant.
With this method of separating a pure culture, mobile cellular organisms (Proteus) rise to the top of the agar slant, and immobile forms do not change their location on the seed medium. By subculturing the upper edges of the germinated culture, a pure bacterial culture can be obtained.
Drygalsky's technique
The Drygalsky method is quite widely used in the study of biological material by diluting cultures in a test tube with broth or saline.
The next step: using a sterile glass spatula, one drop of the resulting solution is distributed evenly over the surface of the nutrient medium in the 1st cup. Then, without burning the spatula on the fire, they do the same sowing on the 2nd and 3rd bowl. The essence of the Drygalsky method is that with each subsequent sowing of cultures, the concentration of bacteria (aerobes) is less and less. On the 3rd plate, they are distributed quite apart, each bacterial cell gives rise to clonal cells (in the form of an isolated colony). They are subcultured on slant agar for the accumulation of pathogens.
Weinberg technique
Certain difficulties are caused by the isolation of pure cultures of anaerobic pathogens if the microorganisms do not die upon contact with oxygen molecules. The essence of the technique is to remove anaerobic microbes (in the composition of the material) in a slightly cooled (45-50 ° C) molten nutrient medium (agarosed). Spend 6-10 dilutions. Then comes the next stage: it is necessary to quickly cool the composition in the test tube and fill its surface with a mixture of petroleum jelly and paraffin oil, which prevents air from penetrating and promotes the development of anaerobic conditions.
With favorable sowing, isolated colonies germinate on the second day, which can be removed from the tube by heating it with a burner. Cultures are looped from the cut agar column for further examination. The resulting colonies are placed in a liquid nutrient medium, at which the steps for isolating the colony of anaerobic pathogens can be completed.
Hungate technique
It is often used to isolate aerobic microbes with high oxygen sensitivity. In carrying out such isolation of the culture of aerobes, the technique of rotating test tubes is used.
Methods for inoculation of aerobic bacteria involve breeding them on a molten agar medium. The presence of an inert gas in a test tube makes it possible to grow isolated columns of aerobic bacteria in such a way that the isolated cultures of aerobes can be clearly seen even with the naked eye for 2-3 days.
Micromanipulator
This is a technique for isolating individual bacterial cells using a micromanipulator. The micromanipulator is a special device that can catch one cell from a suspension, as well as provide the possibility of seeding a culture in a test tube.
Further identification of the pathogen is carried out taking into account all the listed properties of the pathogen (aerobes and anaerobes), as well as the features of their interaction with various substances.
