The idea of ​​shuttle running was developed by the Dane Jengs Bengsbo and consists of intense interval physical activity (interval endurance test, keyword "intense") and the ability to recover after its completion (interval recovery test, again after intense physical activity), and when taking heart rate readings, also determine the individual characteristics of the athlete’s body in aerobic (to a greater extent) and anaerobic mode.

Yo-Yo test:

Chips represent three lines (see photo). 5 meters for recovery and 20 meters for running. The test involves running between chips located at a distance of 20 meters from each other, back and forth. Start on command. The time between signals, i.e. the time to travel a 40-meter segment, is gradually reduced depending on the level. The time between rest and main run is 5 or 10 seconds depending on the type of Yo-Yo test. The athlete starts the test from a low level, where he alternates with rest and passes segments for a certain time. Then comes the next level, where the number of segments per level increases, and the time to complete a 40-meter segment decreases and the running speed increases. That is, athletes repeatedly, without stopping, perform jerks with recovery, and with an increase in level. The time allowed to complete the segment decreases. The player quits the Yo-Yo test when he can no longer complete the segments in the allotted time. The end of the Yo-Yo test is the total distance the athlete ran before retiring.

Excellent: Speed ​​19 - 20 km/h Distance: > 2320 m. Number of forty-meter segments: >58
Very good: Speed ​​18-19 km/h Distance: >2000 m. Number of forty-meter segments: >50
Good: Speed ​​17-18 km/h Distance: >1680 m. Number of forty-meter segments: >42
Bad: Speed ​​16-17 km/h Distance: >1360 m. Number of forty-meter segments: >34
Very bad: Speed ​​15-16 km/h Distance: >1040 m. Number of forty-meter segments: >26

The idea of ​​testing is neutralized or, one might say, discredited by the reluctance of football players to undergo it or the formal approach to participation in it. The majority of players go through 40-meter segments without reaching the maximum heart rate, and when a threshold comes, crossing which requires effort, they simply leave the race. They believe that there is no point in straining yourself once again: “What are the complaints against me, I ran away.”

Experienced football players are especially susceptible to such conclusions. Therefore, 75% of the results of the Yo-Yo test after being carried out in Russian teams can be thrown into the trash bin, since they do not provide any information, and on the contrary, they confuse their results and lead the coaching staff and team doctors in the wrong direction regarding the functional readiness of the team players .

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Disc diffusion method

No more than 6 disks impregnated with antibiotics are placed on the surface of a dense nutrient medium seeded over a continuous lawn with the crop under study, at a distance of at least 2 cm from each other. The results are recorded after 18-24 hours of incubation in a thermostat according to the diameter of the zone of no growth around the disks with antibiotics. The presence of growth around the disc indicates the insensitivity of this microbe to the antibiotic. Special tables are used to interpret the results.

Figure 1. Determination of sensitivity

microorganisms using the disk diffusion method:

1 – microorganism sensitive to an antibiotic;

2 – microorganism moderately resistant to an antibiotic;

3 – microorganism stable to an antibiotic.

E-test method

Principle of the method. Determination of the sensitivity of a microorganism is carried out similarly to testing by the disk diffusion method. The difference is that instead of an antibiotic disk, an E-test strip containing a gradient of antibiotic concentrations from maximum to minimum is used. At the intersection of the ellipsoidal zone of growth inhibition with the E-test strip, the minimum inhibitory concentration (MIC) value is obtained.

Figure 2. Determination of the sensitivity of microorganisms using E-tests

Serial dilution method in broth medium

In test tubes containing 1 ml of Mueller-Hinton broth, prepare serial two-fold dilutions of the antibacterial drug, for example 100 µg/ml - 1st, 50 µg/ml - 2nd, 25 µg/ml - 3rd, 12.5 µg /ml – 4th, etc. Then 0.1 ml of the tested bacterial suspension is added to each tube. At the same time, growth control is administered (1 ml of Mueller-Hinton broth and 0.1 ml of bacterial suspension). The crops are incubated at 37°C for 18-24 hours, after which the results are noted. The absence of turbidity in the medium indicates a retardation of bacterial growth in the presence of a given concentration of the drug.

Figure 3. Determination of the MIC value by dilution in a liquid nutrient medium

Minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic (in μg/ml or mg/l) that completely inhibits visible bacterial growth in vitro.

2 Determination of the sensitivity of different strains of staphylococci to antibiotics using the standard disk method

Antibiotic	Growth inhibition zone, mm	Strain characteristics

The culture under study is sensitive to ___________________________________________________

moderately resistant to ________________________________________________________________________,

resistant to _________________________________________________________________________________.

3 Determination of the minimum inhibitory concentration (MIC) of penicillin by the method of serial dilutions.

Conclusion: The MIC of penicillin for the strain under study is _____________________________________

Advantages of the method:______________________________________________________________________________

Disadvantages of the method:______________________________________________________________________________

4 Identification and registration of the antagonistic effects of different types of bacteria.

An antagonist microbe and test strains perpendicular to it are inoculated with a streak along the diameter onto a plate with MPA. The results are recorded one day after sowing. The presence and degree of antagonistic action is determined by the size of the growth inhibition zones of the test cultures.

Line sowing_________________________________

Conclusion: the greatest antagonistic effect was detected for test strains (specify species) ______________________________________________________________________________________________

LESSON No. 8

SUBJECT: FINAL LESSON ON THE TOPIC: “HISTORICAL STAGES IN THE DEVELOPMENT OF MICROBIOLOGY, MORPHOLOGY, PHYSIOLOGY AND GENETICS OF MICROORGANISMS.”

Shapes and sizes of true bacteria. Characteristics of spherical, rod-shaped and convoluted forms of true bacteria.

Structure of bacteria. The main differences between a prokaryotic cell and a eukaryotic cell.

Cell wall of gram-positive and gram-negative bacteria.

Types of microscopic preparations. Technique for preparing fixed preparations.

Technique of microscopy in a light microscope. Study of the morphology of microorganisms in an electron microscope.

Tintorial properties of microbes. Dyes. Simple methods of staining fixed preparations.

Principles of classification of pathogenic prokaryotes (Burgee, 2001).

Protective devices in microorganisms. Spores, stages and conditions of spore formation, biological significance.

Bacterial capsules, their meaning.

Flagella, their structure. Cilia. Sex-drinking.

Complex painting methods. Gram, Ziehl-Neelsen, Burri-Gins, Neisser staining techniques.

Methods for studying microorganisms in a living state. CON test. Principle of the method.

Spirochetes. Systematic position and morphology of spirochetes. Features of ultrastructure and chemical composition. Research methods.

Actinomycetes, morphology, ultrastructure, chemical composition. Pathogenic species. The role of actinomycetes in nature and medicine. Detection methods.

Taxonomy of chlamydia. Morphology, structure, detection methods. Chlamydia development cycle.

Rickettsia, morphology, ultrastructure, chemical composition. Pathogenic species.

Mycoplasmas. Classification. Phylogenesis. Methods of detection.

Defective forms of microbes: protoplasts, spheroplasts, L-forms.

Nutrition of bacteria. Nutrients are sources of carbon and nitrogen. Classification of bacteria by type of nutrition Autotrophs and chemoorganotrophs

Growth factors and their sources. Sources of mineral elements.

Methods and mechanisms of nutrient transfer through the membrane.

Energy requirements of bacteria. Ways of obtaining energy from autotrophs (photosynthesis, chemosynthesis). Sources and ways of obtaining energy in chemoorganotrophs.

Aerobic and anaerobic types of biological oxidation in bacteria. Aerobic, anaerobic, facultative anaerobic and microaerophilic bacteria. Methods for creating anaerobic conditions.

Objectives, stages, advantages and disadvantages of the bacteriological (cultural) research method.

Growth and reproduction of microorganisms. Reproduction methods. Binary (simple) fission mechanism. Reproduction of bacterial populations.

Principles and methods of bacterial cultivation. Nutritional needs of microbes.

Nutrient media for the cultivation of bacteria. Requirements for nutrient media. Classification of nutrient media.

Conditions and techniques for cultivating bacteria. Technique of sowing on nutrient media. Patterns and nature of bacterial growth on solid and liquid nutrient media.

Methods for isolating pure cultures of aerobic and anaerobic bacteria.

Properties of microorganisms used to identify isolated cultures.

Bacterial enzymes, classification. Methods for studying the biochemical properties of microorganisms. Practical use of biochemical activity in bacterial identification

Determination of saccharolytic properties, composition of Hiss media; determination of proteolytic properties, determination of catalase and oxidase activity.

The operating principle and features of the use of devices for automatic identification of bacterial cultures (hemocultivator, automatic analyzer).

Features of the cultivation of rickettsia and chlamydia.

Bacteriophages (phages). History of discovery. Morphology, structural features, chemical composition and properties of phages.

Virulent phages. Phases of interaction with a bacterial cell. Results of interaction between phage and cell. Temperate phages. Prophage. The phenomenon of lysogeny. Phage conversion.

Methods for isolating and titrating bacteriophages on solid and liquid nutrient media. Application of phages in microbiology and medicine. Phage diagnostics and phage typing.

Heredity. Organization of the genetic apparatus in bacteria (nucleoid, plasmids, Is-sequences, transposons).

Principles of functioning of the bacterial genome. Organization of the operon. Genotype and phenotype.

Plasmids, classification, structure and properties of plasmids. R-plasmid, features of structure and function. Bacteriocinogeny plasmids.

Microbial variability. Modifications in bacteria, significance, main manifestations and properties (non-hereditary nature, adaptability, high frequency of direct and reverse changes, many inducing factors).

Genotypic variability. Mutations and their classification. Mutagens. Phenotypic manifestations of mutations. The fate of mutants. Dissociation in bacteria. The influence of selection. Genome damage repair system.

Recombination variability. Mechanisms of formation of combined genomes. Frequency of changes in individual characteristics. Transformation, transduction, conjugation.

Practical significance of knowledge about the genetics of microbes. Principles of genetic mapping.

Genetic analysis methods (molecular hybridization, polymerase chain reaction, blotting, sequencing).

The concept of genetic engineering and the use of its methods in microbiology and biotechnology. Production and use of genetically engineered vaccines and cytokines.

Antimicrobial measures. The influence of environmental factors on microbes. The action of physical factors (temperature, drying, radiation, ultrasound, osmotic pressure). Action of chemical factors.

Goals, methods, means and objects of sterilization and disinfection in medical and microbiological practice. Disinfection quality control. Control of sterilization and sterility. Methods of carrying out.

Antiseptic. Definition. Antiseptic agents, requirements, origin, properties, groups, mechanisms of action on microbes. Types of antiseptics. Therapeutic antiseptics. Preventive antiseptics.

Chemotherapy drugs. Properties. Main groups of chemotherapy drugs. Mechanisms of action on bacteria. The concept of selectivity and “targets” of action.

Antibiotics. Definition. Producers of antibiotics. Synthetic and semi-synthetic antibiotics.

Main groups of antibiotics by chemical structure. Beta-lactam antibiotics Tetracyclines. Aminoglycosides. Macrolides and azolides. Ansamycins (rifampicins). Levomycetin. Fluoroquinolone antibiotics. Lincomycin. Polymyxins. Glycopeptides

Classification of antibiotics based on the mechanism of action on the bacterial cell.

Mechanisms of resistance of microorganisms to antibacterial drugs.

Methods for determining the sensitivity of bacteria to antibiotics and other chemotherapy drugs. Technique for setting, recording and assessing sensitivity using the disk method, E-test, serial dilutions.

LESSON No.9

SUBJECT: ECOLOGY OF BACTERIA. INFECTION. PATHOGENIC MICROORGANISMS. MICROBIAL TOXINS. BIOLOGICAL (EXPERIMENTAL) METHOD.

CHECKLIST

Microflora of the human body . Normal (resident) human microflora. Autochthonous and allochthonous, parietal and luminal microflora. Formation and development of normal microflora. Functions of normal microflora: anti-infective, metabolic, immunobiological, antitoxic.

Dysmicrobiocenosis (dysbacteriosis), causes, types, principles of correction.

Concept of infection. Definition, general characteristics. Differences between infectious diseases and non-infectious diseases.

The role of the microorganism in the infectious process. Infectious dose. Methods of infection. Entrance gate. Pathogenicity and virulence. Genetic control of pathogenicity and virulence. Factors that increase and decrease the virulence of microbes.

Pathogenicity factors. Methods for determining virulence, units. Obligate pathogenic and conditionally pathogenic microorganisms.

Toxicity and toxigenicity of microorganisms. Endotoxins, properties, production, application. Exotoxins, properties, production, units of measurement. Types of exotoxins, mechanism of action.

The role of the macroorganism in the development and course of infectious diseases. Hereditary factors. Anatomical and physiological state of the body. The role of living conditions in the development and course of infectious diseases. Natural factors. Social factors.

Classification of infectious processes by severity, nature of the pathogen, source of infection, mode of transmission of the pathogen and mechanism of infection, and prevalence. Classification of infectious processes according to the localization of the microbial focus, duration of course and frequency of infection.

Dynamics of the infectious process, its features.

Biological (experimental) research method, stages, evaluation. Laboratory animals. Methods of infection.

LABORATORY WORK

1 Study of normal microflora.

A) Sowing to study the normal microflora of the skin of the hands on Endo medium and blood agar replica method.

Principle of the method: moisten sterile pieces of filter paper 1x1 cm in a Petri dish with sterile saline. solution. Using sterile tweezers, place a piece of paper on the surface of the skin to be examined for 0.5 minutes. Place the paper on the surface of the dense nutrient medium (print) for 1 minute. Remove the paper. Incubate the plates with prints at 37 0 C for 24-48 hours.

C) Conduct a record of microflora inoculation, prepare preparations from different types of colonies, Gram stain, microscope (in demonstration inoculations).

Accounting for microflora inoculation:

Microscopy of preparations:

A drug______________ _______________________ Coloring _______________ _______________________

A drug______________ _______________________ Coloring _______________ _______________________

2 Adhesiveness assessmentE. coliby their ability to adsorb on the surface of red blood cells

Principle of the method: The test culture of microorganisms is added to the erythrocyte suspension. After incubation, smears are prepared, stained, and the average number of bacteria adsorbed on one red blood cell is determined under a microscope.

In this case, red blood cells are used as a model cell of a susceptible microorganism.

3 Determination of invasiveness enzymes in staphylococci

1. Plasmocoagulase

Principle of the method: The test culture is added to a test tube containing citrated rabbit blood plasma. After incubation in a thermostat, the result is taken into account. If the result is positive, the plasma clots (coagulates).

2.Fibrinolysin

Principle of the method: The test culture is added to a test tube with fibrin (a blood clot washed from red blood cells). After incubation in a thermostat, the result is taken into account. If the result is positive, the clot dissolves.

3.Hyaluronidase

Principle of the method: The test culture is added to a test tube with hyaluronic acid (HA). After incubation in a thermostat, a reagent that causes coagulation of the HAA is added and the result is taken into account. If the result is positive (due to the breakdown of HAA), no clot is formed.

4.Lecitovitellase (lecithinase)

The principle of the method: the isolated staphylococcus cultures are inoculated on yolk-salt agar, which contains 7.5% sodium chloride and a yolk suspension. If the result is positive, a rainbow halo forms around the colonies of virulent staphylococci due to the breakdown of lecithin contained in the yolk of a chicken egg.

Conclusion: (list the virulence enzymes of each of the two strains studied) ________________________________________________________________________________________________

_____________________________________________________________________________________________

Bacterial toxins

Toxicity ___________________________________________________________________________________

Toxigenicity _________________________________________________________________________________

Endotoxin ___________________________________________________________________________________

Endotoxic shock ___________________________________________________________________________

Practical application of endotoxins:

Exotoxin ___________________________________________________________________________________

Anatoxin _____________________________________________________________________________________

Scheme for obtaining exotoxin and toxoid.

1.____________________________________________________________________________________________

2.____________________________________________________________________________________________

4.____________________________________________________________________________________________

Practical use of toxoids:

1.____________________________________________________________________________________________

2.____________________________________________________________________________________________

3.____________________________________________________________________________________________

The lecture discusses the main methods for determining sensitivity in vitro microorganisms to antimicrobial drugs (disk diffusion, E-tests, dilution methods). Approaches to empirical and etiotropic prescription of antibiotics in clinical practice are reflected. The issues of interpreting the results of sensitivity determination from a clinical and microbiological point of view are discussed.

Currently, in clinical practice, there are two principles for prescribing antibacterial drugs: empirical and etiotropic. Empirical antibiotic prescription based on knowledge of the natural sensitivity of bacteria, epidemiological data on the resistance of microorganisms in the region or hospital, as well as the results of controlled clinical studies. An undoubted advantage of empirical prescription of chemotherapy is the possibility of rapid initiation of therapy. In addition, this approach eliminates the costs of additional research.

However, if the ongoing antibacterial therapy is ineffective, in case of nosocomial infections, when it is difficult to guess the pathogen and its sensitivity to antibiotics, they tend to carry out etiotropic therapy. Etiotropic prescription of antibiotics involves not only isolating the infectious agent from clinical material, but also determining its sensitivity to antibiotics. Obtaining correct data is possible only with the competent implementation of all stages of bacteriological research: from taking clinical material, transporting it to a bacteriological laboratory, identifying the pathogen to determining its sensitivity to antibiotics and interpreting the results obtained.

The second reason for the need to determine the sensitivity of microorganisms to antibacterial drugs is to obtain epidemiological data on the structure of resistance of pathogens of community-acquired and nosocomial infections. In practice, these data are used in the empirical prescription of antibiotics, as well as for the formation of hospital formularies.

Methods for determining sensitivity to antibiotics

Methods for determining the sensitivity of bacteria to antibiotics are divided into 2 groups: diffusion and dilution methods.

When determining sensitivity by the disk diffusion method, a bacterial suspension of a certain density (usually equivalent to the McFarland turbidity standard of 0.5) is applied to the surface of an agar in a Petri dish and then disks containing a certain amount of antibiotic are placed. Diffusion of the antibiotic into the agar leads to the formation of a zone of suppression of the growth of microorganisms around the disks. After incubating the dishes in a thermostat at a temperature of 35 o -37 o C overnight, the result is taken into account by measuring the diameter of the zone around the disk in millimeters ().

Picture 1. Determination of the sensitivity of microorganisms using the disk diffusion method.

Determining the sensitivity of a microorganism using the E-test is carried out similarly to testing by the disk diffusion method. The difference is that instead of a disk with an antibiotic, an E-test strip is used containing a gradient of antibiotic concentrations from maximum to minimum (). At the intersection of the ellipsoidal zone of growth inhibition with the E-test strip, the minimum inhibitory concentration (MIC) value is obtained.

Figure 2. Determination of the sensitivity of microorganisms using E-tests.

The undoubted advantage of diffusion methods is the ease of testing and accessibility in any bacteriological laboratory. However, given the high cost of E-tests, the disk diffusion method is usually used for routine work.

Breeding methods are based on the use of double serial dilutions of antibiotic concentrations from maximum to minimum (for example, from 128 μg/ml, 64 μg/ml, etc. to 0.5 μg/ml, 0.25 μg/ml and 0.125 μg/ml) . In this case, the antibiotic in various concentrations is added to a liquid nutrient medium (broth) or agar. A bacterial suspension of a certain density, corresponding to the McFarland turbidity standard of 0.5, is then placed in an antibiotic broth or on the surface of an agar plate. After incubation overnight at a temperature of 35 o -37 o C, the results obtained are recorded. The presence of microorganism growth in the broth (broth turbidity) or on the surface of the agar indicates that the given concentration of antibiotic is insufficient to suppress its viability. As the antibiotic concentration increases, the growth of the microorganism deteriorates. The first lowest concentration of antibiotic (from a series of serial dilutions), where bacterial growth is not visually determined, is considered to be minimum inhibitory concentration (MIC). MIC is measured in mg/l or μg/ml ().

Figure 3. Determination of the MIC value by dilution in a liquid nutrient medium.

Interpretation of sensitivity results

Based on the obtained quantitative data (diameter of the antibiotic growth inhibition zone or MIC value), microorganisms are divided into sensitive, moderately resistant and resistant (). To distinguish between these three categories of sensitivity (or resistance) the so-called borderline concentrations(breakpoint) antibiotic (or boundary values ​​​​of the diameter of the zone of inhibition of microorganism growth).

Figure 4. Interpretation of the results of determining the sensitivity of bacteria in accordance with MIC values.

Boundary concentrations are not immutable values. They may be revised depending on changes in the sensitivity of the microbial population. The development and revision of interpretation criteria are carried out by leading specialists (chemotherapists and microbiologists) who are members of special committees. One of them is the US National Committee for Clinical Laboratory Standards (NCCLS). Currently, NCCLS standards are recognized throughout the world and are used as international standards for assessing the results of determining the susceptibility of bacteria in multicenter microbiological and clinical studies.

There are two approaches to interpreting susceptibility results: microbiological and clinical. Microbiological interpretation is based on the analysis of the distribution of antibiotic concentrations that suppress the viability of bacteria. Clinical interpretation is based on assessing the effectiveness of antibiotic therapy.

Sensitive microorganisms (susceptible)

Clinically, bacteria are classified as sensitive (taking into account the parameters obtained in vitro), if when treating infections caused by these microorganisms with standard doses of an antibiotic, a good therapeutic effect is observed.

In the absence of reliable clinical information, division into sensitivity categories is based on a joint account of the data obtained in vitro, and pharmacokinetics, i.e. on the antibiotic concentrations achievable at the site of infection (or in the blood serum).

Resistant microorganisms

Bacteria are classified as resistant (resistant) when, when treating an infection caused by these microorganisms, there is no effect of therapy even when using maximum doses of antibiotics. Such microorganisms have resistance mechanisms.

Microorganisms with intermediate resistance (intermediate)

Clinically, intermediate resistance in bacteria is implied when infections caused by such strains may have different therapeutic outcomes. However, treatment may be successful if the antibiotic is used in a higher than standard dosage or the infection is localized to an area where the antibacterial drug accumulates in high concentrations.

From a microbiological point of view, bacteria with intermediate resistance include a subpopulation that, in accordance with the MIC values ​​or zone diameters, is between sensitive and resistant microorganisms. Sometimes intermediate-resistant strains and resistant bacteria are combined into one category of resistant microorganisms.

It should be noted that the clinical interpretation of bacterial sensitivity to antibiotics is conditional, since the outcome of therapy does not always depend only on the activity of the antibacterial drug against the pathogen. Clinicians are aware of cases where, when microorganisms are resistant, according to research in vitro, received a good clinical effect. Conversely, if the pathogen is sensitive, therapy may be ineffective.

In certain clinical situations, when the results of sensitivity testing by conventional methods are insufficient, the minimum bactericidal concentration is determined.

Minimum bactericidal concentration (MBC)- the lowest concentration of antibiotic (mg/l or μg/ml), which during the study in vitro causes the death of 99.9% of microorganisms from the initial level over a certain period of time.

The value of MBC is used in therapy with antibiotics that have a bacteriostatic effect, or in the absence of effect from antibacterial therapy in a special category of patients. Special cases for determining MBC may be, for example, bacterial endocarditis, osteomyelitis, or generalized infections in patients with immunodeficiency conditions.

In conclusion, I would like to note that today there are no methods that would allow us to predict with absolute certainty the clinical effect of antibiotics in the treatment of infectious diseases. However, these sensitivity results can serve as a good guide for clinicians to select and adjust antibacterial therapy.

Table 1. Criteria for interpreting bacterial susceptibility

Classification, general approaches to implementation. Diffusion methods: paper disk method, E-test.

Methods for determining the sensitivity of bacteria to antibiotics are divided into 2 groups:
1. Diffusion methods:
. using antibiotic discs
. using E-tests
2. Serial dilution methods:
. dilution in liquid nutrient medium (broth)
. dilution in agar medium
Methods for determining sensitivity were developed in the second half of the 60s - early 70s of the 20th century and since then have not undergone fundamental changes from a methodological point of view.
The following steps are common to all methods:
- preparation and quality control of nutrient media
- preparation of a suspension of test microorganisms (inoculum)
- inoculation
- for diffusion methods - the stage of applying E-test disks or strips to a solid nutrient medium.
- incubation
- recording and interpretation of results
- formulation of treatment recommendations
Diffusion methods are based on the diffusion of an antibacterial drug (ABP) from a carrier into a solid nutrient medium inoculated with a microorganism, and recording the diameter of the zone of inhibition (delay) of growth of the microorganism under study.
. The method is less sensitive and less accurate than the serial dilution method, but is used more often in practice due to its simplicity. Posted on ref.rf.
. The rate of diffusion of any drug into agar depends on its structure, molecular weight, presence of impurities, composition and pH of the medium.
Method of paper disks with antibiotic (disc diffusion method).
. To carry out this method, standard discs containing a certain amount of antibiotics and a standard nutrient medium necessary for the growth of this type of microorganism are used. Within certain limits, the diameter of the growth inhibition zone is inversely proportional to the MIC. . A bacterial suspension of a certain density is applied to the surface of the agar in a Petri dish. . Discs containing a certain amount of antibiotic are placed. . Incubate under conditions favorable for each specific microorganism. . The diameters of growth inhibition zones around the disc are measured in millimeters (taking into account the diameter of the disc). . The result is assessed using a special table by comparing the diameter of the growth inhibition zones of the tested crop with the boundary values ​​of the zone diameter in the table. . The culture under study is classified into one of three categories: sensitive, moderately sensitive and resistant.

E-test (E-test or epsilometric method)
The method is close in technology to the paper disk method.
. A narrow strip of polymer (0.5x6.0 cm) is used as a carrier, onto which a gradient of ABP concentrations is applied (from minimum to maximum). The ABP concentration values ​​in each section of the strip are marked on the outer (facing the researcher) surface.
. Inhibition of microorganism growth around the carrier strip occurs in the zone where the concentration of antibiotic diffusing from the carrier is higher than the MIC.
. At the intersection of the ellipsoidal zone of growth inhibition with the E-test strip, the MIC value is obtained.
The E-test combines the simplicity of the paper disk method with the accuracy of the serial dilution method.

Methods used for comparative in vitro evaluation of antimicrobial therapy drugs: method of serial dilutions in liquid and solid nutrient media.

Serial dilution methods:
. They allow one to quantify the sensitivity of the isolated microorganism to antibacterial agents and determine the MIC of the drug.
. Used for comparative assessment of the in vitro antimicrobial activity of the generic drug under development and the original drug.
. To determine the MIC value, specified concentrations of antibiotics are added to the nutrient medium, which is then inoculated with a culture of the microorganism under study. After incubation, the presence or absence of visible growth is assessed.
. Based on the use of two-fold serial dilutions of ABP concentrations from maximum to minimum (for example, from 128 μg/ml, 64 μg/ml, etc. to 0.5 μg/ml, 0.25 μg/ml and 0.125 μg/ml ).
. They are carried out in liquid and agar nutrient media. Method of serial dilutions in liquid nutrient medium (broth)
There are 2 options for this method:
macromethod (test tube) and micromethod (plate).
Macro method.
. Testing is carried out in test tubes in a final volume of 1 ml for each dilution.
. The nutrient broth is poured into 0.5 ml into each test tube. The number of tubes is determined by the required dilution range of the ABP.
. Preparation of a suspension of the studied microorganisms:
- A working suspension (~ 10 6 CFU/ml) is prepared from a standard suspension of each microorganism under study (~ 10 8 CFU/ml). Preparation of two-fold serial dilutions of ABP: - prepare a stock solution of ABP of the test generic drug and the reference drug (original) at a concentration of 1000 μg/ml and higher (taking into account the content of the active substance). - from the basic solutions of the ABP of the generic drug under study and the reference drug (original), working solutions of the ABP are prepared using a liquid nutrient medium. (The concentration of working solutions is calculated based on the required maximum concentration in a series of serial dilutions, taking into account the dilution factor during subsequent inoculation with a suspension of the microorganism) - serial dilutions are prepared: 0.5 ml of the working solution of ABP is added to the first test tube containing 0.5 ml of broth. Stir. Using a new pipette (tip), transfer 0.5 ml of ABP solution in broth into a second test tube containing 0.5 ml of broth, etc., until the entire required series of dilutions has been prepared. 0.5 ml is removed from the last tube. Thus, a series of test tubes with ABP solutions are obtained, the concentrations of which differ in neighboring test tubes by 2 times. Inoculation: 0.5 ml of a microbial suspension with a microorganism concentration of ~ 10 6 is added to each test tube with 0.5 ml of an appropriate dilution of ABP. The final concentration of the microorganism in each tube is ~ 5x10 5 CFU/ml. . Control - a test tube with broth and a microorganism culture (growth control). Negative control - a tube with broth (sterility control). . Incubation: All tubes, sealed with stoppers or caps, are incubated under conditions that ensure growth of the test microorganisms. . Recording and interpretation of results: test tubes with cultures are viewed in transmitted light. The growth of the culture in the test tube with ABP is compared with the control tube. - the presence of microorganism growth in the broth (clouding of the broth) indicates that this concentration of the antibiotic is insufficient to suppress its viability. - as the antibiotic concentration increases, the growth of the microorganism worsens. The first lowest concentration of the antibiotic (from a series of serial dilutions), where bacterial growth is not visually determined, is considered to be the minimum inhibitory concentration (MIC). drugs for antibacterial therapy - compare the results obtained for the original drug and the generic drug under study. A conclusion is made about their equivalence in terms of the spectrum (list of microorganisms used) and the degree of antimicrobial activity (MIC values).

. Determination of MBC: from the last few tubes with growth retardation, inoculate with a loop onto the sectors of a Petri dish. The MBC, which, as a rule, is several dilutions less than the MIC, is taken to be the concentration of the drug in the last test tube, the culture from which did not produce growth. . Disadvantage of the method: low productivity - application is limited to studies of a small number of microorganisms.
Micromethod
.The test procedure is similar to that when using the macromethod
.The final volume is up to 0.2 ml. Availability of appropriate laboratory equipment: a 96-well plate with sterile lids, multi-channel pipettes. Working solutions of ABP can be added to the wells of the plate in advance, and then stored sealed in polyethylene at a temperature below 60°C until moment of use. . Advantages of the method: - high productivity - possibility of long-term storage of pre-prepared tablets - saving on consumables. Posted on ref.rf
Method of serial dilutions in agar medium. The test principle is similar to the broth dilution method. Preparation of a suspension of test microorganisms: - a standard suspension of each test microorganism should contain ~ 10 8 CFU/ml. - the standard microbial suspension for the experiment is diluted ~ 10 times to obtain a microorganism concentration of ~ 10 7 CFU/ml. The preparation of two-fold serial dilutions of ABP for the original drug and the generic drug under study is carried out similarly to the method of dilutions in broth. The agar medium is melted and cooled to a temperature of 45-50°C. . Preparation of dishes with agar medium and ABP dilutions: mix the agar medium and ABP solutions directly in a Petri dish (for plastic dishes with a diameter of 90 mm, add 18 ml of melted and cooled agar to 2 ml of ABP solution). . Inoculation and incubation: a bacteriological loop is used to transfer 1-2 μl of a suspension of the microorganisms under study to the surface of the agar medium. Thus, the final inoculum dose is ~10 4 CFU (a standard bacteriological loop with a diameter of 3 mm carries 1-2 µl of liquid). . A spot with a diameter of 5-8 mm forms on the surface of the agar. After drying, the dishes are turned over and incubated under conditions favorable for the growth of the microorganisms under study. . Recording and interpretation of results: similar to the broth dilution method. Petri dishes are placed on a dark, non-reflective surface. The concentration of ABP that caused complete inhibition of visible growth is taken as the MIC. . Control: agar plates inoculated with a suspension of microorganism cultures without ABP (growth control). Negative control: agar plates (sterility control). Advantages of the method: the sensitivity of several microorganisms can be determined on one plate.

Scope of research on the comparative assessment of in vitro antimicrobial activity for generic and original antimicrobial agents.

Scope of research on the comparative assessment of in vitro antimicrobial activity of generic antimicrobial drugs:
.Objective of the study: confirmation of the compliance of the generic drug with the reference (original) in terms of spectrum (microorganisms) and degree (MIC, MBC value) of antimicrobial activity.
.Set of tested microorganisms: 1-2 strains of each microorganism included in the spectrum of action
- reference collection strains
- clinical strains isolated in hospitals
.The values ​​of MIC and MBC are determined
.Control: reference drug - original drug
.Expected result: the MIC and MBC of the developed generic antimicrobial drugs are within the acceptable ranges of values ​​and completely coincide with the MIC and MBC of reference drugs (original drugs) in relation to collection and clinical strains.
The procedure for studies to determine in vitro antimicrobial activity of new antimicrobial compounds:
.Primary assessment of sensitivity to new compounds of reference strains of various types of gram-negative and gram-positive microorganisms (4-5 strains for each species);
.Detailed study of the degree of antibacterial activity of compounds against strains of gram-negative and gram-positive microorganisms from international collections with known resistance mechanisms (serial dilution method);
.Study of activity against clinical strains of opportunistic and pathogenic microorganisms in comparison with known drugs of a similar chemical group or similar in antimicrobial effect:
- in case of predominant activity against gram-positive microorganisms, control - natural penicillins, cephalosporins of the first and second generations, macrolides, lincosamides; - for activity against gram-negative microorganisms, control - polymyxin B, aztreonam; - for broad-spectrum drugs, control - semisynthetic penicillins, aminoglycosides, tetracyclines, cephalosporins of III - IV generations
. Assessment of antimicrobial activity against problematic pathogens: methicillin-resistant staphylococci, benzylpenicillin-resistant Streptococcus pneumonia, multidrug-resistant enterobacteriaceae, aminoglycoside-resistant bacteria of the genus Pseudomonas, etc.
.Initial therapeutic concentrations of new drugs are established taking into account the toxicity determined in experiments studying acute toxicity;
. The comparative degree of antibacterial activity of drugs is assessed by the MIC or MBC value, determined at no less than 2 values ​​of the seed dose: minimum - 10 4 - 10 5 CFU/ml and maximum - 10 6 - 10 9 CFU/ml, depending on the type of pathogen ; To date, there are no methods that would allow us to predict with absolute certainty the clinical effect of antibiotics in the treatment of infectious diseases. However, these sensitivity results can serve as a good guide for clinicians to select and adjust antibacterial therapy.

Lecture, abstract. Methods for determining the sensitivity of microorganisms to antibiotics - concept and types. Classification, essence and features.

Book table of contents open close

1. Pharmaceutical microbiology. Subject and tasks of pharmaceutical microbiology.
2. Pharmacy and pharmaceuticals: history of origin and development.
3. Medicine: definition, classification.
4. Composition of medicines | pharmaceutical substance, excipient.
5. Original and generic medicines. Name of medicines.




10. Effect of damaging factors on microorganisms. The influence of the temperature factor and its use in pharmaceuticals.
11. Effect of radiation on microorganisms, types of radiation.
12. Influence of chemical damaging factors on microorganisms
13. Sterilization. Sterility Assurance Level (SAL). Criteria for choosing a sterilization method.
14. Thermal and chemical sterilization
15. Monitoring the effectiveness of sterilizing devices.
16. Industrial disinfection
17. Disinfectants and antiseptics. Requirements for chemical disinfectants and antiseptics.
18. Preservatives and their use in pharmaceutical production

Table of contents of the topic "Methods for determining sensitivity to antimicrobial agents. Side effects of antibiotic therapy.":

New methods for determining the sensitivity of bacteria to chemotherapy. Automatic systems for recording the results of the serial dilution method. E-test.

Currently computerized systems developed, automatically conducting isolation of bacteria from test samples and determining their sensitivity to various drugs. Their main advantage is the release of bacteriological laboratory personnel from a large volume of routine research and clear standardization of the results obtained.

The widespread use of these devices in domestic practice limits their cost. Among the more accessible methods, the most widespread are Alamar system And E-test, combining the advantages of the serial dilution method and the disk method.

New methods for determining the sensitivity of bacteria to chemotherapy. Automatic systems for recording the results of the serial dilution method">

Automatic systems for recording the results of the serial dilution method(for example, Baxter MicroScan AutoSCAN-4) - automated incubation systems with built-in photometers that nephelometrically record bacterial growth or its absence 24 hours after introducing microorganisms into the wells of micropanels. The principle of operation is based on taking into account the difference in the optical density of the medium in wells where there is bacterial growth and in wells where there is no growth. Currently, devices have been developed (for example, VITEK) that provide results within 4-10 hours.

Alamar system is a panel with wells, each containing filter paper discs containing different concentrations of antimicrobial agents and impregnated with the Alamar Blue indicator. After introducing bacteria into the well, the disk turns blue, and with their further growth, its color changes to pink. The order of placing the disks in the wells corresponds to double serial dilutions of the drug. The last well with a blue disk, preceding the wells with pinkish disks, corresponds to the MIC of the drug.

E-test[from English ellipse, ellipse, since in the presence of sensitivity, an elliptical-shaped zone of growth inhibition is formed] - a modification of the disk method, but instead of the latter, strips of filter paper impregnated with various concentrations of drugs are used, each of these zones has appropriate markings. The strips are placed on the surface of the agar. If bacteria are sensitive to the action of the drug, an ellipsoidal zone is formed around the areas of the strip containing its inhibitory concentrations. Its form is due to the action of several concentrations of the drug at once. The MIC corresponds to the section of the strip where it is crossed by the boundary of the growth inhibition zone.