Methods for isolating a pure culture of the pathogen. Determination of the number of cells by seeding on solid nutrient media (Koch plate method)

Pasteur method Koch method Biological Physical

(has historical (lamellar)

meaning) wiring) Chemical Method of Shchukevich

Modern

Sowing with a loop Sowing with a spatula

(Drigalski method)

Methods for isolating pure cultures (Scheme 11):

1. Mechanical release methods are based on the separation of microbes by sequential rubbing of the test material over the surface of an agar.

A) Pasteur's method– has historical significance, provides for the sequential dilution of the test material in a liquid nutrient medium using the rolling method

b) Koch method– plate method – based on sequential dilution of the test material with meat-peptone agar, followed by pouring test tubes with the diluted material into Petri dishes

V) Drigalski method– when sowing material richly contaminated with microflora, use 2-3 cups for sequential sowing with a spatula.

G) Sowing with a loop in parallel strokes.

2. Biological methods based on the biological properties of pathogens.

A) Biological– infection of highly sensitive animals, where microbes quickly multiply and accumulate. In some cases, this method is the only one that allows isolating a culture of the pathogen from a sick person (for example, with tularemia), in other cases it is more sensitive (for example, isolating 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 acid solution. Only tuberculosis bacilli will grow, since acid-resistant microbes died under the influence of acid.

V) Physical method based on the resistance of spores to heat. To isolate a culture of spore-forming bacteria from
mixtures, the material is heated at 80°C and inoculated on a nutrient medium. Only spore bacteria will grow, since their spores remained alive and gave rise to growth.

G) Shchukevich method– is based on the high mobility of Proteus vulgaris, capable of producing creeping growth.

Method of reseeding from colonies onto slanted agar and MPB:

A) Transferring from colonies to agar slant

Open the lid of the dish slightly, remove part of a separate colony with a calcined, cooled loop, open a test tube with sterile slanted agar, holding it in your left hand in an inclined position, so that you can observe the surface of the medium. Transfer the loop with the culture into the test tube without touching the walls, rub it over the nutrient medium, sliding along the surface from one edge of the test tube to the other, raising the strokes to the top of the medium - streak seeding. The test tube is closed and, without letting go, the name of the inoculated microbe and the date of inoculation are signed.

b) Transferring from the colony to meat-peptone broth

The technique for reseeding on MPB is basically the same as when sowing on solid media. When sowing on the MPB, the loop with the material on it is immersed in the medium. If the material is viscous and cannot be removed from the loop, it is rubbed on the wall of the vessel and then washed off with a liquid medium. The liquid material, collected with a sterile Pasteur or graduated pipette, is poured into the 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 in complying with the rules of the anti-epidemic regime and safety precautions

2. Disinfect the material, disinfect hands

3. Prepare preparations from bacterial colonies

4. Microscopy colonies

5. Gram stain microorganisms

LESSON 8

SUBJECT. Methods for isolating pure cultures (continued). Enzymatic activity of bacteria and methods for studying it.

If, based on certain symptoms on 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 accompanying organisms, i.e., there is a mixed population. To obtain the pathogen in the form of a separate growing colony, tissue macerate should be streaked onto the medium.

Sowing with a touch. Using a calcined inoculation loop, take a small amount of plant tissue macerate containing bacteria and, with light movements, without damaging the agar surface, apply 4-6 strokes to the prepared nutrient medium. Having re-calcined the loop, the cup with the medium is turned 90° to the right and then another 4-6 strokes are applied from the second stroke, the needle is calcined again and the third sowing is carried out. This achieves a dilution of the starting material such that the bacteria, after incubation in a thermostat for 48-72 hours at 28 °C, form individual colonies of various shapes and colors. The colonies are then transferred to agar slant tubes for further examination. Using a calcined loop, take a colony and apply it onto nutrient agar with a careful movement in the form of a snake or zigzag.

Koch pouring method. The Koch plate method ensures that each colony is formed from a single bacterial cell. It is best to prepare a suspension from the starting material in sterile water and use 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 a second test tube, carefully open it over the burner flame, and use large loops to transfer three portions of the substrate into it from the first test tube. After firing the neck and stopper, the contents of the test tube are poured into the first Petri dish, opening the lid of the dish just enough to insert the neck of the test tube under it. Immediately after pouring, close the cup and carefully distribute the nutrient medium evenly.

Having thoroughly mixed the contents of the second test tube, take a third test tube and transfer six portions of the substrate from the second into it with a loop. The contents of the test tube are poured into a cup, and the contents of the test tube, after mixing, are poured into the cup. The dishes 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 soil, then serial dilution is used. Sterile nutrient medium (15 ml per cup) is poured into cups, 0.1 ml of the last three dilutions of the suspension is 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 settle for a few seconds, and serial dilutions are prepared from the suspension. Using this method, the number of microorganisms in each sample can be determined.

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Cultural properties of bacteria

A colony is an isolated collection of cells of the same type that grew from one cell (clone of cells). Depending on where the microorganism grows (on the surface of a dense nutrient medium or in its thickness), surface, deep and bottom colonies are distinguished.

Colonies grown on the surface of the medium are diverse: they are species-specific and their study is used to determine the species of the crop under study.

When describing colonies, the following characteristics are taken into account:
1) the shape of the colony - round, amoeboid, rhizoid, irregular, etc.;

2) size (diameter) of the colony - very small (pointed) (0.1-0.5 mm), small (0.5-3 mm), medium-sized (3-5 mm) and large (more than 5 mm in diameter );

3) the surface of the colony is smooth, rough, folded, wrinkled, with concentric circles or radially striated;

4) colony profile - flat, convex, cone-shaped, crater-shaped, etc.;

5) transparency - dull, matte, shiny, transparent, powdery;

6) color of the colony (pigment) - colorless or pigmented (white, yellow, golden, red, black), especially note the release of pigment into the medium with its coloring;

7) edge of the colony - smooth, wavy, jagged, fringed, etc.;

8) colony structure - homogeneous, fine- or coarse-grained, streaky; the edge and structure of the colony are determined using a magnifying glass or at low magnification of a microscope by placing a Petri dish with the inoculation on the microscope table with the lid down;

9) consistency of the colony; determined by touching the surface with a loop: the colony can be dense, soft, growing into agar, mucous (stretches behind the loop), fragile (easily breaks when in contact with the loop).

Deep colonies most often look like more or less flattened lentils (oval shape with pointed ends), sometimes lumps of cotton wool with thread-like outgrowths into the nutrient medium. The formation of deep colonies is often accompanied by rupture of the dense medium if microorganisms release gas.

Bottom colonies usually look like thin transparent films spreading along the bottom.

The characteristics of the colony may change with age; they depend on the composition of the medium and the cultivation temperature.

The growth of microorganisms on liquid nutrient media is taken into account using four- to seven-day cultures grown under stationary conditions.

In liquid nutrient media, with the growth of microorganisms, turbidity of the medium and the formation of a film or sediment are observed.

When growing on semi-liquid (0.5-0.7% agar) nutrient media, mobile microbes cause pronounced turbidity, immobile forms grow only during sowing by injection into the medium.

Often the growth of microbes is accompanied by the appearance of odor, pigmentation of the environment, and the release of gas. The characteristic odor of cultures of some types of bacteria is associated with the formation of various esters (ethyl acetate, amyl acetate, etc.), indole, mercaptan, hydrogen sulfide, skatole, ammonia, butyric acid.

The ability to form pigments is inherent in many types of microorganisms. The chemical nature of pigments is diverse: carotenoids, anthocyanins, melanins. If the pigment is insoluble in water, only the cultural plaque is stained; if it is soluble, the nutrient medium also becomes colored. It is believed that pigments protect bacteria from the harmful effects of sunlight, which is why there are so many pigmented bacteria in the air; in addition, pigments are involved in the metabolism of these microorganisms.

In nature, there are so-called phosphorescent bacteria, cultures of which glow in the dark with a greenish-bluish or yellowish light. Such bacteria are found mainly in river or sea water. Luminous bacteria - photobacteria - include aerobic bacteria (vibrios, cocci, rods).

Isolation of pure cultures of microorganisms

The material under study (water, soil, air, food or other objects) usually contains associations of microbes.

Isolation of a pure culture makes it possible to study the morphological, cultural, biochemical, antigenic and other characteristics, the totality of which determines the species and type of the pathogen, i.e., its identification is made.

To isolate pure cultures of microorganisms, methods are used that can be divided into several groups:
1. Pasteur's method - sequential dilution of the test material in a liquid nutrient medium to the concentration of one cell in the volume (has historical significance).

2. Koch method (“plate wiring”) - sequential dilution of the test material in molten agar (temperature 48-50 C), followed by pouring into Petri dishes, where the agar solidifies. Inoculations are made, as a rule, from the last three or four dilutions, where bacteria become few and later, as they grow on Petri dishes, isolated colonies appear, formed from one initial mother cell. From isolated colonies deep in the agar, a pure culture of bacteria is obtained by subculture onto fresh media.

3. Shukevich method - used to obtain a pure culture of Proteus and other microorganisms with “creeping” growth. The test material is inoculated into condensation water at the base of the agar slant. Mobile microbes (Proteus) are able to rise up the slanted agar, immobile forms remain to grow below, at the site of sowing. By reseeding the upper edges of the culture, you can obtain a pure culture.

4. Drigalsky method - widely used in bacteriological practice, in which the material being studied is diluted in a test tube with sterile saline solution or broth. One drop of the material is added to the first cup and spread over the surface of the medium with a sterile glass spatula. Then, with the same spatula (without burning it in the burner flame), the same sowing is done in the second and third cups.

With each inoculation of bacteria, less and less remains on the spatula and, when sowing on the third cup, the bacteria will be distributed over the surface of the nutrient medium separately from each other. After 1-7 days of keeping the dishes in a thermostat (depending on the growth rate of microorganisms), on the third dish, each bacterium produces a clone of cells, forming an isolated colony, which is subcultured onto slanted agar in order to accumulate a pure culture.

5. Weinberg method. Particular difficulties arise when isolating pure cultures of obligate anaerobes. If contact with molecular oxygen does not immediately cause cell death, then seeding is carried out according to the Drigalsky method, but after this the dishes are immediately placed in an anaerostat. However, the breeding method is more often used. Its essence lies in the fact that the dilution of the material under study is carried out in a molten and cooled to 45-50 oC agar nutrient medium.

6-10 successive dilutions are made, then the medium in the test tubes is quickly cooled and the surface is covered with a layer of a mixture of paraffin and petroleum jelly to prevent air from penetrating into the thickness of the nutrient medium. Sometimes the nutrient medium, after sowing and mixing, is transferred into sterile Burri tubes or capillary Pasteur pipettes, the ends of which are sealed. With successful dilution, isolated colonies of anaerobes grow in test tubes, Burri tubes, and Pasteur pipettes. To ensure that isolated colonies are clearly visible, clarified nutrient media are used.

To extract isolated colonies of anaerobes, the tube is slightly heated by rotating it over a flame, while the agar adjacent to the walls melts and the contents of the tube in the form of an agar column slide out into a sterile Petri dish. The agar column is cut with sterile tweezers and the colonies are removed with a loop. The extracted colonies are placed in a liquid medium favorable for the development of isolated microorganisms. The agar medium is blown out of the Burri tube by passing the gas through a cotton plug.

6. Hungate method. When they want to obtain isolated colonies of bacteria with particularly high sensitivity to oxygen (strict aerobes), the Hungate rotating tube method is used. To do this, the molten agar medium is inoculated with bacteria at a constant current through a test tube of inert gas freed from oxygen impurities. The tube is then sealed with a rubber stopper and placed horizontally in a clamp that rotates the tube; the medium is evenly distributed over the walls of the test tube and solidifies into a thin layer. The use of a thin layer in a test tube filled with a gas mixture allows one to obtain isolated colonies that are clearly visible to the naked eye.

7. Isolation of individual cells using a micromanipulator. A micromanipulator is a device that allows you to remove one cell from a suspension using a special micropipette or microloop. This operation is controlled under a microscope. A moist chamber is installed on the microscope stage, into which the hanging drop preparation is placed. Micropipettes (micropoops) are fixed in the holders of operating stands, the movement of which in the field of view of the microscope is carried out with micron precision thanks to a system of screws and levers. The researcher, looking through a microscope, removes individual cells with micropipettes and transfers them into tubes containing a sterile liquid medium to obtain a cell clone.

L.V. Timoschenko, M.V. Chubik

Special environments.

In bacteriology, industrially produced dry nutrient media 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 them closer to resolving the issue of media standardization. The medical industry produces dry media 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, chamber, double walls, between which air or water circulates. The temperature is regulated by a thermostat. The optimal temperature for the reproduction of most microorganisms is 37°C.

LESSON 7

TOPIC: METHODS FOR ISOLATING PURE CULTURE OF AEROBES. STEPS OF ISOLATION OF PURE CULTURE OF AEROBIC BACTERIA BY MECHANICAL DISSOCIATION METHOD

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 of isolating pure cultures, to teach how to sow with a loop, strokes, and an injection

Guidelines for the demonstration

In their natural habitat, bacteria are found in associations. In order to determine the properties of microbes and 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. Mechanical separation methods are based on the separation of microbes by sequential rubbing of the test material over the surface of the agar.

a) Pasteur’s method - has historical significance, provides for the sequential dilution of the test material in a liquid nutrient medium by the rolling method

b) Koch's method - the plate method - is based on the sequential dilution of the test material with meat peptone agar, followed by pouring test tubes with the diluted material into Petri dishes.

c) Drigalsky method - when sowing material richly seeded with microflora, use 2-3 cups for sequential 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 quickly multiply and accumulate. In some cases, this method is the only one that allows one to isolate the pathogen culture 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 pathogen of tuberculosis in guinea pigs).

b) Chemical – based on the acid resistance of mycobacteria. To free the material from accompanying flora, it
treated with acid solution. Only tuberculosis bacilli will grow, since acid-resistant microbes died under the influence of acid.

c) The physical method is based on the resistance of spores to heat. To isolate a culture of spore-forming bacteria from
mixtures, the material is heated at 80°C and inoculated on a nutrient medium. Only spore bacteria will grow, since their spores remained alive and gave rise to growth.

d) Shchukevich's method - based on the high mobility of Proteus vulgaris, capable of producing creeping growth.

Method for preparing plate agar

MPA is melted in a water bath, then cooled to 50-55°C. The neck of the bottle is burned in the flame of an alcohol lamp, the Petri dishes are opened so that the neck of the bottle fits in without touching the edges of the dish, 10-15 ml of MPA is poured in, the lid is closed, the dish is shaken so that the medium is evenly distributed, and left on a horizontal surface until it hardens. After drying, plate agar plates are stored in the cold.

Loop sowing

Using a sterile cooled loop, take a drop of material, open one edge of the cup with your left hand, bring the loop inside and make a few strokes in one place with a loop at the opposite edge, then tear off the loop and inoculate the material in parallel strokes from one edge of the cup to the other with an interval of 5-6 mm. At the beginning of sowing, when there are a lot of microbes on the loop, they will give confluent growth, but with each stroke there are fewer and fewer microbes on the loop, and they will remain solitary and produce isolated colonies.

Sowing according to the Drigalsky method

This method is used when inoculating material heavily contaminated with microflora (pus, feces, sputum). To sow using the Drigalsky method, take a spatula and several cups (3-4). A spatula is a tool made of metal wire or glass dart, bent into a triangle or L-shape. 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 such sowing, the first cup will have confluent growth, and isolated colonies will grow in subsequent cups.

Main stages in the development of microbiology, virology and immunology

These include the following:

1.Empirical knowledge(before the invention of microscopes and their use for studying the microworld).

J. Fracastoro (1546) suggested the living nature of agents of infectious diseases - contagium vivum.

2.Morphological period took about two hundred years.

Antonie van Leeuwenhoek in 1675 first described protozoa, in 1683 - the main forms of bacteria. The imperfection of instruments (the maximum magnification of X300 microscopes) and methods for studying the microworld did not contribute to the rapid accumulation of scientific knowledge about microorganisms.

3.Physiological period(since 1875) - the era of L. Pasteur and R. Koch.

L. Pasteur - study of the microbiological foundations of fermentation and decay processes, development of industrial microbiology, elucidation of the role of microorganisms in the circulation of substances in nature, discovery anaerobic microorganisms, development of principles asepsis, methods sterilization, weakening ( attenuation)virulence and receiving vaccines (vaccine strains).

R. Koch - isolation method pure cultures on solid nutrient media, methods of staining bacteria with aniline dyes, discovery of the causative agents of anthrax, cholera ( Koch comma), tuberculosis (Koch sticks), improvement of microscopy techniques. Experimental substantiation of the Henle criteria, known as the Henle-Koch postulates (triad).

4.Immunological period.

I.I. Mechnikov is the “poet of microbiology” according to the figurative definition of Emil Roux. He created a new era in microbiology - the doctrine of immunity (immunity), developing the theory of phagocytosis and substantiating the cellular theory of immunity.

At the same time, data was accumulated on the production in the body antibodies against bacteria and their toxins, which allowed P. Ehrlich to develop the humoral theory of immunity. In the subsequent long-term and fruitful discussion between supporters of the phagocytic and humoral theories, many mechanisms of immunity were revealed and the science was born immunology.

It was later found that hereditary and acquired immunity depends on the coordinated activity of five main systems: macrophages, complement, T- and B-lymphocytes, interferons, the main histocompatibility system, which provide various forms of immune response. I.I. Mechnikov and P. Erlich in 1908. the Nobel Prize was awarded.

February 12, 1892 At a meeting of the Russian Academy of Sciences, D.I. Ivanovsky reported that the causative agent of tobacco mosaic disease is a filterable virus. This date can be considered a birthday virology, and D.I. Ivanovsky is its founder. Subsequently, it turned out that viruses cause diseases not only in plants, but also in humans, animals and even bacteria. However, only after the nature of the gene and genetic code were established, viruses were classified as living nature.

5. The next important stage in the development of microbiology was discovery of antibiotics. In 1929 A. Fleming discovered penicillin and the era of antibiotic therapy began, leading to revolutionary progress in medicine. Later it turned out that microbes adapt to antibiotics, and the study of the mechanisms of drug resistance led to the discovery of a second extrachromosomal (plasmid) genome bacteria.

Studying plasmids showed that they are even more simply structured organisms than viruses, and unlike bacteriophages do not harm bacteria, but provide them with additional biological properties. The discovery of plasmids has significantly expanded the understanding of the forms of existence of life and possible paths of its evolution.

6. Modern molecular genetic stage The development of microbiology, virology and immunology began in the second half of the 20th century in connection with the achievements of genetics and molecular biology, and the creation of the electron microscope.

Experiments on bacteria have proven the role of DNA in the transmission of hereditary traits. The use of bacteria, viruses, and later plasmids as objects of molecular biology and genetic research has led to a deeper understanding of the fundamental processes underlying life. Clarification of the principles of encoding genetic information in bacterial DNA and establishing the universality of the genetic code made it possible to better understand the molecular genetic patterns characteristic of more highly organized organisms.

Decoding the genome of Escherichia coli has made it possible to design and transplant genes. By now Genetic Engineering created new directions biotechnology.

The molecular genetic organization of many viruses and the mechanisms of their interaction with cells have been deciphered, the ability of viral DNA to integrate into the genome of a sensitive cell and the basic mechanisms of viral carcinogenesis have been established.

Immunology has undergone a genuine revolution, going far beyond the scope of infectious immunology and becoming one of the most important fundamental medical and biological disciplines. To date, immunology is a science that studies not only protection against infections. In the modern sense Immunology is a science that studies the mechanisms of self-defense of the body from everything genetically foreign, maintaining the structural and functional integrity of the body.

Immunology currently includes a number of specialized areas, among which, along with infectious immunology, the most significant include immunogenetics, immunomorphology, transplantation immunology, immunopathology, immunohematology, oncoimmunology, ontogenesis immunology, vaccinology and applied immunodiagnostics.

Microbiology and virology as basic biological sciences also include a number of independent scientific disciplines with their own goals and objectives: general, technical (industrial), agricultural, veterinary and those of greatest importance for humanity medical microbiology and virology.

Medical microbiology and virology studies the causative agents of human infectious diseases (their morphology, physiology, ecology, biological and genetic characteristics), develops methods for their cultivation and identification, specific methods for their diagnosis, treatment and prevention.