ACTIVITY #4
TOPIC: PHYSIOLOGY OF MICROORGANISMS. BACTERIOLOGICAL (CULTURAL) RESEARCH METHOD. BIOCHEMICAL PROPERTIES OF MICROORGANISMS.
CHECKLIST
Nutrition of bacteria. Nutrients are sources of carbon and nitrogen. Classification of bacteria by types of nutrition Autotrophs and chemoorganotrophs
Growth factors and their sources. Sources of mineral elements.
Ways and mechanisms of transfer of nutrients through the membrane.
Energy requirements of bacteria. Ways of obtaining energy in 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. Ways to create anaerobic conditions.
Tasks, 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 cultivation of bacteria. The nutritional needs of microbes.
Nutrient media for the cultivation of bacteria. nutrient requirements. Classification of nutrient media.
Conditions and techniques for cultivating bacteria. Technique of sowing on nutrient media. Regularities and character of bacterial growth on dense and liquid nutrient media.
Methods for isolating pure cultures of aerobic and anaerobic bacteria.
Properties used to identify isolated cultures.
INDEPENDENT AND LABORATORY WORK
Bacteriological method(stages):
1 1st stage isolation of a pure culture of aerobic bacteria: A) Microscopy of pathological material.
Gram staining of smears from pathological material. Drug sketch.
B) Mastering, under the guidance of a teacher, the technique of sowing pathological material with a bacteriological loop and a spatula on plate nutrient media.Inoculation of pathological material with a bacteriological loop on lamellar meat-peptone agar (MPA) to obtain isolated colonies.
Classification of culture media(specify areas of application)
1. By consistency:liquid (meat-peptone broth, bile, sugar broth), dense (2-3% agar) and semi-liquid (0.15-0.7% agar) media.
2. By origin:natural - from milk, meat. eggs, potatoes, human blood serum, animal and other products; artificial - 1) natural balanced mixtures of nutrients in concentrations and combinations necessary for the growth and reproduction of microorganisms, a universal source of nitrogen and carbon - peptones - products of incomplete breakdown of proteins using pepsin or various hydrolysates (fish, casein, yeast, etc.) .2) synthetic caccurate chemical composition Soton for mycobacteria, 199 for cells.
3. In composition: simple culture media (meat-peptone broth-MPB, meat-peptone agar-MPA) and with false (KA = MPA + 5-10% of animal blood)
4. By appointment:
BUT) general purpose - universal, intended for cultivation of any microorganisms (MPA,KA)
B ) Specialfor growing microorganisms that do not grow on universal media, differentiation of species and selective isolation of certain types of microorganisms:
elective (selective) to isolate certain types of microorganisms and suppress the growth of related ones - (salt agar for staphylococci).
differential diagnostic (DDS)-environments that make it possible to distinguish between types of bacteria by enzymatic activity; They With possess: 1) universal nutrient medium (MPA, KA); 2) differentiating factor - a chemical substrate (for example, carbohydrate), a different relationship to which is a diagnostic feature for a given microbe. 3) An indicator whose color change indicates a biochemical reaction. (environments of Endo, Ploskirev, Giss and others).
differential selective (DS) - environments that allow allocate bacteria of a certain species according to their physiological characteristics and differentiate from other species according to enzymatic activity They contain: 1) MPA 2) elective a chemical substrate that inhibits the growth of other types of bacteria . 3) differentiating factor - substrate to which is a diagnostic feature for this microbe;) 4.) An indicator whose color change indicates a biochemical reaction. (Wednesdays for staphylococci, ICA for salmonella, Ploskirev for shigella and salmonella).
B) Enrichment media for the reproduction and accumulation of bacteria of a certain type in clinical material (blood in 20% bile broth = salmonella, throat discharge in 10% serum + 2% tellurite = corynebacteria.)
D) Transport medium for the collection and delivery (preservation) of clinical material = 48 hours (Amies medium - semi-liquid agar + activated charcoal).)
Nutrient media(examples):
Wednesday Endo Medium type differential diagnostic for enterobacteria Nutrient Base MPA differentiating factor lactose 1% Indicator basic fuchsin decolorized with sodium sulfite. E.s oli decompose lactose to acid - colonies are red with a metallic sheen, pathogenic colorless;
Salt agar Medium type selective for isolation of staphylococci Nutrient Base MPA elective factor sodium chloride 10%
Wednesday Ploskirev Medium type differential selectivefor enterobacteria
Nutrient Base MPA elective factor bile salts differentiating factor lactose
Indicator neutral red
Yolk-salt agar Medium type differential selective for S . aureus _
Nutrient Base MPA elective factor sodium chloride 10%
differentiating factor egg yolk
Indicator No
2 Stage 2 bacteriological research method (isolation of pure culture):
A) The study of isolated colonies (escherichia, staphylococcus) on lamellar MPA.
|
Studied cultural properties |
1 type of colonies |
2 type of colony |
|
colony shape |
Regular shape, round |
Correct form |
|
Consistency |
homogeneous |
homogeneous |
|
Colony size |
medium (size 2-4 mm) | |
|
The nature of the edge |
with smooth edges |
with smooth edges |
|
Surface nature |
convex |
B) Preparation of smears from selected colonies (Gram stain).
C) Transfer of isolated colonies to slanted MPA for the accumulation of pure culture.
3 Isolation of a pure culture of anaerobic bacteria: inoculation of a soil suspension on the Kitta-Tarozzi medium to isolate pathogenic clostridia
Kitt-Tarozzi medium consists of nutrient broth, 0.5% glucose, and pieces of liver or minced meat to absorb oxygen from the medium. Before sowing, the medium is heated in a boiling water bath for 20-30 minutes to remove air from the medium. After sowing, the nutrient medium is immediately filled with a layer paraffin
Methods for creating anaerobiosis:
1.Physical- pumping out air, introducing a special oxygen-free gas mixture (usually N 2 - 85% CO 2 - 10%, H 2 - 5%), preliminary boiling of nutrient media, inoculation in a deep column of agar, filling the media with vaseline oil to reduce oxygen access, the use of hermetically sealed vials and test tubes, syringes and laboratory glassware with an inert gas, the use of tightly closed desiccators with a burning candle
2. Chemical- chemical oxygen scavengers are used.
3. Biological - joint cultivation of strict aerobes and anaerobes (aerobes absorb oxygen and create conditions for the reproduction of anaerobes - the Fortner method).
Wednesday Kitt - Tarozzi consists of a nutrient broth, 0.5% glucose and pieces of liver or minced meat to absorb oxygen from the environment. Before sowing, the medium is heated in a boiling water bath for 20-30 minutes to remove air from the medium. After sowing, the nutrient medium is immediately filled with a layerparaffin or vaseline oil to isolate from oxygen access.
4. Mixed - use several different approaches.
Special devices are used to create anaerobic conditions - anaerostats. Currently the simplest and most efficient equipment for creating anaerobic and microaerophilic conditions is a chemical method with special bags acting on the principle of absorbing atmospheric oxygen in hermetically sealed containers .
Wilson-Blair medium (tubes, cups):
Nutrient Base MPA Respiratory substrate glucose
Reducing factor sodium sulfite and ferric chloride sodium sulfiteNa 2 SO 3 → Na 2 S
For the Wilson-Blair environment, the base is agar with addition glucose , Clostridia form on this medium colonies black color due to restoration sulfite before sulfide - anion , which is connected with cations gland (II) gives black salt. Typically black on this education medium colonies , appear in the depth of the agar column .
Thioglycol medium (medium for sterility control): (tubes):
Nutrient Base BCH Respiratory substrate glucose Reducing factor sodium thioglycolate
Indicator resazurin
Zeissler blood glucose agar: (cups): Nutrient Base MPA, blood
Respiratory substrate glucose Reducing factor hemoglobin
The term "anaerobes" was introducedLouis Pasteurwho discovered in 1861bacteriabutyric fermentation.
BUTLecture 3 Physiology of microorganisms. Metabolism of bacteria .
The physiology of microorganisms includes :
types of food;
types of breathing;
cultivation (conditions, environments, character and growth rate);
biochemical activity;
variability;
release of biologically active substances, toxins and other pathogenicity factors;
sensitivity to antibiotics, bacteriophages, bacteriocins;
other biological properties.
Metabolism of bacteria - a set of physical and chemical processes (chemical transformations and reactions) aimed at reproducing structures and ensuring the vital functions of a microbial cell, such as:
growth and reproduction;
deposition of reserve food material;
transport of nutrients into the microbial cell;
release of metabolic products (toxins, enzymes, antibiotics and other biologically active substances);
traffic;
spore formation;
adhesion on sensitive receptors of host cells and penetration into them;
various adaptive responses to changes in the external environment.
Anabolism- a set of biochemical reactions that carry out the synthesis of cell components.
catabolism- a set of reactions that provide the cell with energy.
Metabolism study scheme - stages:
1. Initial (peripheral) metabolism - the penetration of substances into the cell from the outside and decay to intermediate products.
2. Amphibolism (intermediate metabolism) - the formation of intermediate metabolic products common to catabolic and anabolic pathways.
3. The final, strictly specialized stages of constructive metabolism (lead to the construction of cell structures) and energy metabolism (ATP formation).
Mechanisms for the penetration of nutrients into the cell:
Simple diffusion (for true solutions). energy independent process.
Facilitated diffusion ("steam downstream") - in the direction of the concentration gradient with the participation of carrier proteins. energy dependent process.
Active transport is against the concentration and electrochemical gradient with the participation of permeases (amino-, hydroxy-acid, ionic, etc.). The process goes with the expenditure of ATP energy, depends on the charge of substances and their transformation in the process of transfer.
Microorganisms are divided into two groups according to their ability to absorb carbon sources: autotrophs (lat. autos - myself, trophy - nutrition) synthesize all the carbon-containing components of the cell from CO 2 as the only source of carbon and heterotrophs (lat. heteros - the other, “feeding at the expense of others”) use a variety of organic carbon-containing compounds.
Depending on energy sources, microorganisms are also divided into phototrophs (photosynthetic), capable of using solar energy, and chemotrophs (chemosynthetic), receiving energy through redox reactions.
Depending on the electron donors used, bacteria are divided into lithotrophs (using inorganic electron donors) and organotrophs (using organic compounds).
Prototrophs- microorganisms capable of synthesizing all the organic compounds they need from glucose and ammonium salts.
Auxotrophs- microorganisms incapable of synthesizing any organic compounds. They get these compounds ready-made from environment or the human body.
Enzymes(from the Greek. fermentum-sourdough) - highly specific protein catalysts present in all living cells, without which life and reproduction are not possible. Enzymes recognize their respective metabolites (substrates), interact with them, and accelerate chemical reactions. Enzymes are proteins.
The enzyme composition of a microorganism is determined by the genome and is a fairly stable trait. The determination of enzymes is widely used for the biochemical identification of bacteria.
Endoenzymes catalyze metabolism within the cell.
Exoenzymes are secreted by the cell into the environment.
Constitutive enzymes are constantly synthesized at certain concentrations.
inducible enzymes are enzymes whose concentration increases with the intake of the corresponding substrate.
Enzymes of aggression: hyaluronidase, fibrinolysin, neuraminidase, collagenase, lecithinase (licitovitellase), coagulase, urease, amino acid decarboxylases, deoxyribonuclease.
cultivation- obtaining cultures of microorganisms in an artificial nutrient medium.
Cultivation goals:
obtaining pure cultures of pathogenic microorganisms and their identification;
accumulation of biomass of BAS producers (vitamins, hormones, amino acids, antibiotics, etc.);
obtaining diagnostic and prophylactic preparations (vaccines, diagnosticums);
storage of reference museum cultures;
in sanitary microbiology to determine sanitary-indicative microorganisms - indicators of environmental pollution.
culture- a population of microorganisms grown on a nutrient medium.
pure culture- a population of one type of microorganisms grown from an isolated colony on a nutrient medium.
Most pathogenic microbes are grown on nutrient media at 37°C for 1-2 days.
Classification of culture media
By consistency: liquid, semi-liquid, dense.
Origin: natural (milk, potatoes), artificial, semi-synthetic, synthetic
In composition: simple (MPA, MPB, vegetables, milk), complex (1% glucose, 10-20% serum, 20-30% ascitic fluid, 5-10% defibrinated blood).
By appointment:
universal - media on which many types of bacteria grow well. These include meat-peptone broth (MPB) and meat-peptone agar (MPA);
special - media specially prepared to obtain the growth of bacteria that do not grow on universal media;
differential diagnostic - environments that make it possible to distinguish one type of bacteria from others by enzymatic activity;
selective - media containing substances used by microorganisms of certain species and preventing the growth of other microorganisms. Selective media allow you to select certain types of bacteria from the material under study;
differential-selective - environments that combine the properties of differential diagnostic and selective environments;
preservative;
concentrating.
Reproduction of bacteria on liquid and dense nutrient media.
Growth coordinated reproduction of all components of a bacterial cell and an increase in its biomass. reproduction- reproduction and increase in the number of cells, leading to the formation of a bacterial population.
Bacteria are characterized by a high rate of reproduction. The rate of reproduction depends on the species, composition growth medium, pH, temperature, aeration.
On dense nutrient media, bacteria form clusters of cells called colonies. Colonies of different species differ in size, shape, consistency, color, nature of the edges, nature of the surface, transparency.
The nature of growth on liquid nutrient media: filmy (formation of a film on the surface of the nutrient medium), diffuse turbidity, near-bottom (precipitation).
Phases of development of a bacterial population
Initial stationary phase (~ 1-2 hours). The number of bacteria does not increase, the cells do not grow.
Lag phase or breeding delay phase (~ 2 hours).
Log-phase - logarithmic or exponential phase (~ 3-5h). The population is dividing at maximum speed and there is an increase in individuals exponentially.
Phase of negative acceleration (~ 2 hours). Associated with the depletion of the limiting metabolite or the accumulation of toxic metabolic products.
Stationary phase of the maximum. The number of formed and dying cells is the same.
Phase of accelerated death (~ 3 hours).
Logarithmic death phase (~5).
The phase of decreasing the rate of death - the remaining living individuals go into a dormant state.
Energy metabolism of bacteria
Aerobes- microorganisms that use the aerobic (oxidative) type of biological oxidation of substrates. Metabolism of aerobes is carried out only in the presence of a high concentration of free oxygen in the habitat, which acts as the final acceptor of electrons taken from the substrate. The cultivation of aerobes is carried out on media with full access to atmospheric oxygen.
obligate anaerobes- microorganisms using anaerobic type biological oxidation(fermentation). Metabolism occurs only in environments with a low redox potential in the absence of oxygen.
An increase in the oxygen concentration in the environment leads to the death of vegetative forms.
The amount of energy extracted during fermentation is small, so obligate anaerobes are forced to ferment a large amount of substrate.
Facultative anaerobes- microorganisms capable of extracting energy from substrates by aerobic (oxidative) and anaerobic (fermentative) pathways of biological oxidation. Metabolism can be carried out both under conditions of full access of oxygen to the environment, and under conditions of anaerobiosis.
Methods for creating anaerobiosis
Physical
sowing in a column of sugar MPA;
boiling (regeneration) of liquid nutrient media followed by oil coating;
mechanical removal of oxygen in anaerostats;
replacement of oxygen by an indifferent gas;
Veillon-Vignal tubes.
Chemical
apparatus of Aristovsky;
candle Omelyansky ( alkaline solution pyrogallol);
the use of chemical oxygen acceptors: glucose, pyruvic acid, sodium formic acid, etc.
Biological
Kitta-Tarozzi Wednesday
Fortner method
Anaerobes - organisms that obtain energy in the absence of access oxygen by substrate phosphorylation , the end products of incomplete oxidation of the substrate can be oxidized with more energy in the formATP in the presence of a terminal proton acceptor by organisms that .
Anaerobic respiration- aggregate biochemical reactions, occurring in the cells of living organisms when used as the final proton acceptor, does not oxygen, and other substances (for example, nitrates) and refers to the processes energy metabolism(catabolism,dissimilation), which are characterized oxidationcarbohydrates,lipids and amino acids to low molecular weight compounds.
BUT 
aerobic and anaerobic bacteria are preliminarily identified in a liquid nutrient medium by the O2 concentration gradient:
1. obligate aerobic(oxygen-demanding) bacteria mainly gather at the top of the tube to absorb the maximum amount of oxygen. (Exception: mycobacteria - film growth on the surface due to the wax-lipid membrane.)
2. obligate anaerobic bacteria gather at the bottom to avoid oxygen (or not grow). 3. Facultative bacteria are collected mainly in the upper ( oxidative phosphorylation is more beneficial than glycolysis), however, they can be found throughout the medium, since they do not depend on O 2. four . microaerophiles are collected in the upper part of the tube, but their optimum is a low concentration of oxygen. 5. Aerotolerant anaerobes do not react to oxygen concentrations and are evenly distributed throughout the test tube.
D 
for measurement capacity environments M. Clark proposed to use the pH20 value - negative logarithmpartial pressure gaseous hydrogen. The range characterizes all degrees of saturation of an aqueous solution with hydrogen and oxygen. Aerobes grow at a higher potential, facultative anaerobes, and obligate ones at the lowest.)
Classification of anaerobes, distinguish:
Facultative anaerobes
Capneistic anaerobes and microaerophiles
Aerotolerant anaerobes
Moderately strict anaerobes
obligate anaerobes
If an organism is able to switch from one metabolic pathway to another (for example, from anaerobic respiration to aerobic and vice versa), then it is conditionally referred to as facultative anaerobes .
Until 1991, a class of capneistic anaerobes was distinguished in microbiology, requiring a reduced concentration oxygen and increased concentration of carbon dioxide (Brucella bovine type - B. abortus)
Cultural research method is the isolation from the nutrient medium of bacteria of a certain type by cultivation, with their subsequent species identification. The type of bacteria is determined taking into account their structure, cultural and environmental data, as well as genetic, biochemical and biological indicators. For bacteriological diagnostics, schemes are used that are approved by the Ministry of Health.
New species of bacteria derived from a nutrient medium, the properties of which have not yet been determined, are called pure culture. After the final identification of their characteristics, bacteria derived from a certain place and at a certain time are given a name. strain. In this case, a slight difference in the properties, place or time of isolation of a strain of one species is allowed.
Purpose of the method:
1. Etiological diagnosis, that is, the isolation and identification of a pure culture of bacteria.
2. Determination of the number of microorganisms and their special characteristics. For example, a specific reaction to antibiotics.
3. Identification of intrageneric differences of microorganisms, based on their epidemiological and genetic component. This is necessary to determine the commonality of microorganisms isolated in different places and different conditions, which is important for epidemiological purposes.
This research method has a certain number of stages, which are different for aerobic, facultative and obligate aerobic bacteria.
Breeding pure culture for aerobic and facultative aerobic bacteria.
Stage 1
BUT) Preparatory activities. This stage includes the collection, storage and transportation of the material. Also, if necessary, it can be processed, depending on the properties of the studied bacteria. For example, when examining material for tuberculosis, alkali or acid solutions are used to identify acid-resistant microbacteria.
B) Enrichment. This stage is optional and is carried out if the number of bacteria in the test material is not enough to conduct a full-fledged study. For example, when isolating a blood culture, the test blood is placed in a medium in a ratio of 1 to 10 and stored for a day at a temperature of 37°C.
AT) Microscopy. A smear of the test material is stained and examined under a microscope - the microflora, its properties and quantity are examined. In the future, from the primary smear, it is necessary to separately isolate all the microorganisms in it.
G) Creation of separate colonies. Material is applied to the cup, with a special, selective medium, for this, a loop or a spatula is used. Next, set the cup upside down to protect the colonies from condensation, and store in a thermostat for about 20 hours, maintaining a temperature of 37 o.
Important! It should be remembered that in the process of research, it is necessary to adhere to the rules of isolation. On the one hand, to protect the test material and the bacteria to be removed, and on the other hand, to prevent contamination of surrounding persons and the external environment.
As for conditionally pathogenic microorganisms, when they are removed, their quantitative characteristics matter. In this case, quantitative seeding is carried out, in which several hundredfold dilutions of the material are carried out in isotonic sodium chloride solution. After that, sowing is carried out in Petri dishes of 50 μl.
Stage 2
BUT ) The study morphological properties colonies in media and their microscopy. The dishes are examined and the properties of microorganisms, their numbers, growth rates, and the most suitable nutrient medium are noted. For study, it is best to choose colonies located closer to the center, and if several types of pure cultures are formed, then study each separately. To study the morphotype purity of the culture, a colony smear is used, it is stained (usually using the Gram method or any other) and carefully microscopic.
B) Accumulation of pure culture. To do this, colonies of all morphotypes are placed in separate test tubes with a nutrient medium and kept in a thermostat at a certain temperature (for most microorganisms, a temperature of 37 o is suitable, but in some cases it may be different).
Kligler's medium is often used as a nutrient medium for accumulation. It has a "slanted" appearance in test tubes, where 2/3 of its parts are in the form of a column, and 1/3 is a beveled surface, colored light red. Compound:
· MPA
· 0.1% glucose
· 1% lactose
· Special reagent for hydrogen sulfide
· Phenolic red indicator.
Stage 3
BUT) Level of growth and purity of culture. In the general order, the derived pure culture has a uniform growth and, under microscopic examination, the cells have the same morphological and tinctorial structure. But there are some types of bacteria with pronounced pleophorism, while there are cells that have a different morphological structure.
If Kligler's medium was used as a nutrient medium, then biochemical characteristics are determined by changing the color of the column and the beveled part. For example, if lactose decomposes, the beveled part turns yellow, if glucose - yellowing of the column; with the production of hydrogen sulfide, blackening occurs due to the transition of sulfate to iron sulfide.
As you can see in the figure, the Kligler medium tends to change its color. This is due to the fact that the breakdown of nitrogenous substances by bacteria and the formation of alkali products occur inhomogeneously both in the column (anaerobic conditions) and on the sloping surface (aerobic conditions).
In an aerobic environment (slanted surface) more active alkali formation is observed than in an anaerobic environment (column). Therefore, when glucose is decomposed, the acid on the sloping surface is easily neutralized. But, with the decomposition of lactose, the concentration of which is much higher, the acid cannot be neutralized.
As for the anaerobic environment, very little alkaline products are generated, so here you can observe how glucose is fermented.
Rice. Kligler's nutrient medium:
1 - initial environment,
2 - growth E. coli
3 - growth S. paratyphi B,
4 - growth S. Typhi.
E.coli- promotes the decomposition of glucose and lactose with the formation of gases, does not produce hydrogen . Causes yellowing of the entire medium with discontinuities.
S. paratyphi - promotes the decomposition of glucose with the formation of gases, lactose-negative. The beveled part does not change color, the column turns yellow.
S. paratyphi A- does not produce hydrogen sulfide.
S. paratyphi B - hydrogen sulfide is produced (a black color appears during the injection).
S. typhi - glucose decomposes without gas formation, hydrogen sulfide is produced, lactose-repellent. The beveled part does not change color, the column turns yellow and the medium turns black during the injection.
Shigella spp.- lactose-negative, glucose-positive, hydrogen sulfide is not produced. The column acquires a yellow tint, and the beveled part remains the same.
B) Final identification of pure culture and its response to antibiotics. On the this stage biochemical, biological, serological and genetic properties of culture are studied.
In research practice, there is no need to study the full range of properties of microorganisms. It is enough to use the simplest tests to determine whether microorganisms belong to a particular species.
GOAL:
1. Study methods for isolating pure cultures of bacteria
2. Master the bacteriological method of diagnosing infectious diseases.
THEORETICAL REFERENCE
Bacteriological method is the main method for diagnosing infectious diseases. Its essence is to determine the type of infectious agent, therefore, based on the results of the bacteriological method, an etiological (final) diagnosis can be made. The main disadvantage of the method is the duration of the study - from 3 to 5 days, and in some cases even more.
The success of the bacteriological method largely depends on the preliminary stage, including the sampling of the test material and its transportation, and the design of a referral to a bacteriological laboratory. In this case, a number of rules must be observed.
1. Fence of the test material must be carried out before the start of antibiotic therapy or 8-10 hours after the last dose of the antibiotic. To avoid contamination of the sample with microflora of the environment, it is necessary to observe the strictest asepsis. To do this, use sterile material: a) cotton swabs for taking material from the wound, from the mucous membranes (eyes, pharynx, nose); b) a wire loop for taking material from the vagina, anus; c) a syringe for taking blood, pus; d) sterile dishes for direct collection of urine, sputum, feces into it.
2. Transportation The received material should be produced as soon as possible (2-3 hours) in special biks or pencil cases.
3. Direction attached to the clinical sample as an accompanying document. It contains the basic information necessary for conducting a microbiological study:
Surname, name, patronymic, age of the patient;
Proposed diagnosis of the disease;
Prior antimicrobial therapy;
The nature of the material;
Date and time of taking the material;
Purpose of the study;
The name of the medical institution, the number of the department, ward;
Physician's signature.
The bacteriological method is carried out in two stages (Fig. 2.1.):
1. Isolation of a pure culture of the pathogen (1-2 days);
2. Identification of pure culture (1-3 days).
At the first stage, the test material is sown on a solid or liquid nutrient medium, cultural properties are assessed, suspicious colonies are selected and screened on a slant agar. The identification stage includes a mandatory study of the morphology, biochemical properties and antigenic structure of the isolated pure culture, as well as additional studies to determine antibiotic sensitivity, phage sensitivity, phage typing, pathogenicity and persistent properties.
PREPARATION QUESTIONS:
1. Rules for the collection and transportation of the test material for bacteriological examination.
2. Rules for issuing a referral for bacteriological examination.
3. Methods for isolating pure cultures of microorganisms.
4. Bacteriological diagnostic method. Target. Stages. diagnostic value.
INDEPENDENT WORK PLAN:
1. Study the tables "Methods for isolation of pure cultures of bacteria" and "Isolation and identification of pure cultures".
2. Isolate a pure culture from a mixture of bacteria and carry out its identification - master the bacteriological diagnostic method (Work 1)
TOPIC: Sterilization, asepsis, antisepsis, disinfection.
Principles, methods of cultivation of microorganisms and isolation of pure cultures.
Bacteriological research method. Stage 1.
1. Familiarize yourself with the main methods of disinfection and sterilization used in microbiology and medicine.
2. Know the characteristics of the metabolism of microorganisms, the principles of their cultivation in the laboratory.
3. Master stage 1 of the bacteriological method for diagnosing infectious diseases.
1. Methods, devices and modes of sterilization of nutrient media, laboratory glassware, medical instruments.
2. The main groups of disinfectants, their mechanism of action, area and method of application.
3. Appointment of nutrient media in microbiological practice.
4. The principle of obtaining pure cultures of microorganisms and the essence of the bacteriological method as the "gold standard" in the diagnosis of infectious diseases.
5. The purpose and sequence of performing the 1st stage of the bacteriological method for isolating pure cultures of microorganisms.
1. Select the means, mode of sterilization and disinfection in accordance with specific tasks.
2. Describe the proposed nutrient media used in microbiological practice.
3. Carry out the 1st stage of the bacteriological method for isolating pure cultures of aerobic microorganisms.
1. Bacteriostatic and bactericidal effect of low and high temperatures on microorganisms.
2. Influence of chemicals various classes on microorganisms. Antiseptics and disinfectants.
3. Concepts: sterilization, disinfection, asepsis, antisepsis.
4. Methods, equipment and modes of sterilization, their choice depending on the properties of the object to be sterilized.
5. The main groups of disinfectants and the tactics of their use in healthcare facilities.
6. Principles and methods of cultivation of microorganisms.
7. Nutrient media: concept; requirements for them; classification.
8. The concept of a species, strain, colony, pure culture of microorganisms.
9. The essence of the bacteriological method and the scope of its application.
10. The purpose and sequence of the 1st stage of the bacteriological method for the isolation of aerobes.
Tasks performed during the lesson (UIRS):
1. Familiarize yourself with the devices used for sterilization in medical and microbiological practice: steam sterilizer (autoclave), Pasteur oven.
2. Select devices and modes of sterilization of nutrient media, laboratory glassware, medical instruments.
3. Familiarize yourself with the disinfectants used in medical and microbiological practice. Select disinfectants and disinfection mode for the proposed objects.
4. Familiarize yourself with various nutrient media used in microbiological practice, characterize them in terms of composition, consistency, and purpose.
5. Carry out stage 1 of the bacteriological method for isolating pure cultures of aerobes:
5.1. Prepare a fixed preparation from the test material, stain by Gram, microscopically and identify the identified microorganisms by morphological and tinctorial properties.
5.2. Sow the test material using the "stroke with a platform" method.
5.3. Sow the test material using the Drygalski method (demonstration).
6. Familiarize yourself with the set of tools for collecting and transporting pathological materials.
Guidelines to the research task:
1. Acquaintance with the devices used for sterilization: steam sterilizer (autoclave), Pasteur oven.
1.1. Steam sterilizer (autoclave) - steam sterilization under pressure.
The most reliable and universal method of sterilization in medical and microbiological practice is pressure steam sterilization. It is produced in an autoclave, in which the objects to be sterilized are heated with saturated steam at a pressure above atmospheric. Between the readings of the manometer and the temperature of saturated steam there is the following relationship:
Zero pressure is considered normal atmospheric pressure (760 mm Hg).
Sterilization can only be achieved if the autoclave is fully operational and properly operated by specially trained personnel. Therefore, it is necessary to constantly monitor the sterilization regime, which is carried out by physical (maximum thermometer, etc.), biological (biotest with spores of test cultures of microorganisms) and chemical (chemical tests, indicators such as IS) methods.
Control of the mode of sterilization of autoclaves is carried out by chemical means each time the autoclave is loaded. Chemical test - glass tube with chemical having a certain melting point: antipyrine, resorcinol - 110 ± 1 °, benzoic acid - 120 ± 2 °, benzamide - 126 ± 1 °, urea, nicotinamide, D (+)-mannose - 132 ± 2 °. Part chemical tests an aniline dye (magenta, gentian violet, etc.) is introduced, which uniformly colors the substance when it is melted. Currently, indicators of the IS type (Vinar, Russia) are more often used, representing a strip of paper with a layer of indicator mixture applied to it and designed for operational visual control not only of temperature, but also of sterilization time (IS-120, IS-132) . The sterilization regimen is monitored quarterly using a bioassay with spores of the test culture Bacillus stearotermophilus BKM B-718.
1.2. Pasteur oven - dry heat sterilization.
In the Pasteur oven, products made of glass, metals and rubbers based on silicone rubber are sterilized. Sterilization mode: 160°C - 150 min; 180°С - 60 min. Sterilization mode control at each cycle is carried out with the help of sterilization indicators IS-160, IS-180; quarterly - using a biotest with test culture spores Bacillus licheniformis PCS. G BKM B-1711 D.
2. Fill out at home table number 1.
Table 1.
Sterilization
3. Familiarize yourself with disinfectants and fill in class table No. 2, using applications No. 1, 2.
Table 2.
Disinfection
4. Familiarize yourself with the nutrient media and fill in class table No. 3 "Nutrient media".
Table 3
5. Clinical microbiology as a branch of medical microbiology solves two main tasks: the etiological diagnosis of an infectious disease and the rational choice of etiotropic therapy.
The main method of microbiological diagnostics to solve these problems is bacteriological method. The essence of the bacteriological method is to isolate a pure culture of the pathogen, determine its type and sensitivity to antimicrobial drugs.
Selection of the material under study depends on the type of disease and the predominant localization of the pathogen at a certain stage of its development (pathogenesis). The material can be blood, cerebrospinal fluid, wound discharge, sputum, feces, urine, etc. The material sampling technique has great importance in obtaining reliable results.
The success of isolating a pure culture is determined by the correctness choice of nutrient medium and cultivation conditions. There is no universal nutrient medium, the use of which will make it possible to isolate any microorganisms from any test material. Therefore, taking into account the physiological characteristics of possible pathogens, the material is sown on a specific nutrient medium or a complex of nutrient media (special, elective, differential diagnostic). Some microorganisms also require special cultivation conditions (anaerobic, microaerophilic, with a high content of carbon dioxide).
Pathological material from a patient is often a mixture of microorganisms. In this regard, the task is to separate them and getting isolated colonies. An isolated colony, as a result of the reproduction of one microbial cell and consisting of one type of cell, is the basis for obtaining a pure culture. In microbiological practice, various methods are used to obtain isolated colonies. The following are most commonly used:
1. Sowing the test material "stroke with pad" method- the test material is applied to the surface of a dense nutrient medium in a limited area, and then distributed by sowing with frequent parallel strokes.
2. Drygalski method- the material applied to the first cup with a nutrient medium and sown with a spatula is sequentially inoculated with the same spatula, without sterilizing it, for another 1-2 cups.
3. Sector crop method– the studied material is sown sequentially with one loop on several sectors. At the same time, a certain sowing technique (method gould) allows not only to obtain isolated colonies, but also to determine the number of microorganisms in 1 ml (g) of the test material, which is important in assessing the etiological role of opportunistic microorganisms (OPM).
5.1. Carrying out the 1st stage of the bacteriological method for the isolation of aerobes:
Prepare a fixed preparation from the test material, stain according to the Gram method, microscopically, identify the detected microorganisms by morpho-tinctorial properties; pay attention to the number of microorganisms. Record the results in the protocol and draw a conclusion;
Sow the test material on half a cup with a dense nutrient medium using the “stroke with a platform” method;
· sow the test material on three plates with nutrient media using the Drygalski method (demonstration);
Label the dishes with the date of inoculation and place them upside down in a thermostat at 37°C for 18-24 hours.
6. Means for the collection and delivery of pathological material
Note: * - used if the delivery time of the material to the laboratory after its receipt exceeds 1.5-2 hours.
Questions for self-control:
1. Name and justify the principles of cultivation of microorganisms.
2. Why are elective, differential diagnostic and accumulation media used for sowing pathological material?
3. Justify the principle of obtaining pure cultures of microorganisms.
4. Why is the bacteriological method the "gold standard" in microbiological diagnostics of infectious diseases?
5. What are the chemical and biological methods for obtaining pure cultures of microorganisms based on?
6. What is the difference between sterilization and disinfection?
7. Justify the purpose of sterilization and disinfection in microbiological and medical practice.
8. Justify the advantage of using thermal indicator systems for monitoring the sterilization regime (on the example of an IS indicator from Vinar, Russia).
Literature:
Tutorials:
1. Borisov L. B. Medical microbiology, virology, immunology. - M.: MIA LLC, 2002. - S. 26-29, 63-66, 150-159.
2. Pozdeev O. K. Medical microbiology / Ed. acad. RAMS V. I. Pokrovsky. - M.: GEOTAR Medicine, 2001. - S. 76-77, 126-130, 253-265.
Additional literature:
1. Safety of work with microorganisms of III - IV groups of pathogenicity and helminths: Sanitary rules. – M.: federal center State Sanitary and Epidemiological Supervision Ministry of Health of Russia, 1999. - 107p.
Lectures on microbiology.
Tests.
Bacteriological research method. Isolation of a pure culture of aerobic bacteria (1-2 stages)
1. The essence of the bacteriological method
3. Methods for obtaining isolated colonies
4. Methods for obtaining pure culture
1. Carry out primary seeding by the Koch and Drygalski method
2. Isolate pure culture
Work plan:
1. Bacteriological research method
2. Stages of the bacteriological method
3. Concept: pure culture, strain, colony
4. Methods for obtaining isolated colonies
5. Methods for obtaining a pure culture of aerobic bacteria
The main method of laboratory diagnosis of infectious diseases is the bacteriological method. The essence of the bacteriological method is the isolation of pathogens from the material under study and its identification (definition of the genus, species, varieties).
The bacteriological method allows you to determine:
1. Type of pathogen and make a diagnosis of the disease
2. Antibiotics that kill the pathogen and prescribe the appropriate treatment
3. Phagovars and serovars of the pathogen to identify the source of infection
The first stage is the primary inoculation of the test material to obtain isolated colonies. Primary sowing is carried out on storage media and DDS (cup media).
The second stage is the characterization of isolated colonies and obtaining a pure culture from them by reseeding onto a slanted column of the main medium.
The third stage is the identification of a pure culture - the determination of the genus, species, varieties of the pathogen, the determination of antibiotic sensitivity, the determination of fagovars.
The culture of microorganisms is bacteria grown on nutrient media. A pure culture is one species of bacteria grown on nutrient media. Within a species, there are varieties that differ in one trait:
1. Morphovars - differ in morphology
2. Chemovars - differ in biochemical properties
3. Serovars - differ in antigenic properties
4. Fagovars - differ in sensitivity to phages
A pure culture is necessary for identification, determination of serovars, fagovars, sensitivity to antibiotics. A strain is one species of bacteria isolated from a specific source (Volga River, sick Ivanov, etc.). Colony - a visible accumulation of bacteria of the same species, formed during the reproduction of one bacterial cell on a dense nutrient medium.
The first stage of the study is the primary inoculation of the test material to obtain isolated colonies. Before the initial inoculation, microscopy of the test material is carried out. The test material usually contains a mixture of various bacteria, including pathogens. To isolate the pathogen, it is necessary to obtain isolated colonies (bacteria of the same species) by selecting a nutrient medium and using special seeding methods.
There are two methods for obtaining isolated colonies:
1. Drygalski method
