Determination of bacterial motility. Microbial cell derivatives
10. Motility of bacteria, methods for detecting mobility.
Bacteria that form flagella wives. Therefore, the mobility of bacteria can be judged by the presence
flagella.
Methods for detecting mobility:
1. Staining of flagella according to Leffler.
2. Study of intact culture:
a) “crushed drop” method - a drop of a daily culture of bacteria is applied to the middle of a glass slide and carefully covered with a glass slide so that the liquid does not spread beyond its edges and air bubbles do not get into it.
b) “hanging drop” method: a drop of bacteria is applied to the middle of the cover glass, a special glass slide is placed on it with a recess smeared around with Vaseline so that the drop is in the center of the well, then the preparation is carefully turned over.
11. Principles of microbial taxonomy. Systematic position of microbes. Taxonomic categories. Concept and criteria of the type.
Taxonomy establishes the position of living beings in the organic world, and also develops principles, methods, rules for classification, nomenclature and identification of microorganisms.
1) Monophylithic principle - all living things come from one ancestor.
2) Genetic principle - establishing connections between organisms at the genetic level and their hierarchy, division into groups, connections among themselves.
Taxonomy approaches: gene systematics, chemosystematics, phenosystematics, etc.
Nomenclature establishing subordination and communication between MBs.
The organic world is divided into: superkingdoms, kingdoms, types, classes, orders, families, genera, species,
All taxa up to species are defined by one word, species is a binary name (the first word is generic
name, the second - specific), subspecies three spruces (genus, species, name of subspecies).
In reality, only a species exists - a set of freely interbreeding populations descending from
one ancestor, having a common gene pool, ecological unity and reproductive isolation.
Type criteria:
a) morphological; b) territorial properties (ability to color); c) biochemical, d)
serological (antigenic structure); e) biological; f) environmental, g) geographical
Classification of microorganisms:
I. Overkingdom Prokaryotes
1 kingdom of bacteria
1.1. Scotobacter type
1.1.1. class Bacteria
1.1.1.1. order True bacteria
1.1 1.2 order Spirochetes
1.1.1.3 order Acginomycetes
1.1.2. Ricetti class
1.1.2.1. Ricchetti's order
1.1.2.2. Chlamydia order
1.1.3. class Molicutes
1.1.3.1. Mycoplasma order
II. suprakingdom of eukaryotes
III. Kingdom of Viruses
1. Kingdom of Mushrooms
2. kingdom Protozoa.
According to Bergey's classification, the kingdom Prokaryotes is divided into four divisions:
1. Gracilicutes - thin-walled, gram-negative
2. Firmicutes - thick-walled, gram-positive
3. Tenericutes lack a cell wall (this includes mycoplasmas)
4. Mendosicutes - archaebacteria, defective walls, lacking peptidoglycan, structural features of ribosomes, membranes and RNA.
12-16. Systematic position and morphology of spirochetes, actinomycetes, mycoplasmas, rickettsia, chlamydia. Study methods.
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Spirochetes |
Actinomycetes |
Mycoplasmas |
Rickettsia |
Chlamndia |
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Gram-ownership |
They do not have a cell wall. Gram- | ||||
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Diagnostic methods |
staining according to Romanovsky-Gimta, silvering method according to Morozov, dark-field or phase-contrast microscopy |
Simple methods, Gram stain, Ziehl-Neelson stain |
Phase contrast microscopy Curtural and serotological methods |
According to the Zdrodovsky method, according to Gram, electron microscopy |
According to Romanovsky-Giemsa. |
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Morphology |
Thin spirally crimped threads, bent around the central axis, up to 50 µm |
Thread-like, twisted, rod-shaped cells |
Small or large spherical, ovoid or filamentous cells |
Small polymorphic bacteria of coccoid, rod-shaped or filamentous shape |
Elementary spherical bodies (outside humans) and reticular bodies (intracellular) |
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Features of structural organization |
Her typical cop, no dispute |
They do not have flagella or endospore capsules. |
There is no typical CS, does not form spores and does not have flagella |
The CS is built according to the type of Gram bacteria |
Capsuleless |
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Representatives |
Pathogenic and saprophyte; Treponema (8-12 curls), Borrelia (3-8 curls), Leptospira (20-30 curls) |
Most are saprophytes, the pathogenic genera are Actinomycetes and Nocardia |
Pathogenic and non-pathogenic forms, widely distributed in nature | ||
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Caused diseases |
Syphilis, relapsing fever, leptospirosis |
Nocardiosis, cutaneous mycetomas |
Acute respiratory infections, atypical pneumonia and |
Rickettsial diseases, typhus |
Trachoma, ornithosis. inguinal lymphogranulomatosis |
6. Flagella, villi, pili. Methods of movement of prokaryotes. Methods for determining motility in bacteria
Flagella are thin, long, thread-like, protein formations. From the protein labellin. It has contractility. The flagella are arranged according to their character and their number is different: monotrichs (one polarly located flagellum), lophotrichs (a bunch of flagella at one end), anphytrichs (1 or a bunch at opposite ends of the cell), peritrichs (over the entire surface), atriches (stationary). They are typical for young crops; with age or with a lack of nutrition, the flagella are lost. The mobility of the bacteria was determined by micro and macroscopic methods. With microscopy, smears of crushed or hanging drops are prepared. macroscopic - by injection, inoculation on semi-liquid agar. Flagella consist of 3 components: basal body, hook, spiral flagellar filament. The base body consists of a system of special rings. In gr-bacteria there are 2 pairs: external L and P and internal S and M. In gr+ S and M, as a result of their rotation relative to each other, the flagellum rotates. The hook is a curved protein cylinder that functions as a flexible connecting link between the basal body and the rigid filament of the flagellum. The basal body is a complex structure consisting of a central rod and rings. The movement of prokaryotes is carried out by rotational, translational, and flexion movements. They drank. Bacteria that carry plasmids have thread-like structures of a protein nature. Constructed from the protein pillin. The synthesis of these villi is under the control of plasmid genes. Pili are a conjugation apparatus; with their help, contact is established between donor cells and recipient cells. There are 2 classes of pili - sexual and general type (fimbriae). Sex drinking – 1.2 and more than 5 per 1 class. Villi (fimbriae). Short threads, the number of which can reach many thousands. With their help, bacteria attach to certain surfaces. For many pathogenic bacteria, fibria are a pathogenicity factor, because With their help, bacteria attach to sensitive tissue and form adhesion. Causes agglutination of red blood cells.
7.Simple and complex methods for staining microorgans
Practical significance. Preparations are stained using simple and complex methods. Simple method. For coloring, use any one coloring solution. A solution of one dye is applied to a fixed smear: methylene blue for 4 minutes, gentian violet for 2 minutes, magenta for 1 minute. The paint is washed off with water, and the smear is dried with filter paper. A drop of immersion oil is applied to the finished smear and examined under a microscope. Simple staining allows you to quickly familiarize yourself with the morphology of bacteria. Complex methods. Several solutions of dye and reagents are used. They make it possible to determine the morphology of bacteria, their tinctorial features and the presence of structural elements of cells, which has important differential diagnostic significance. One of the methods is Gram staining: filter paper soaked in gentian violet is applied to a fixed preparation for 2 minutes. The paper is removed and Lugol's solution is applied for 2 minutes. Drain, treat the smear with alcohol for 30 seconds, wash with water and stain with fuchsin for 1 minute. Based on the staining result, the type of cell wall was determined. Spore staining methods: Meller's method: a fixed smear is etched with chrome acid for 2 minutes, washed with water, dried with filter paper, filter paper is placed on the smear and magenta is applied, the preparation is heated, stained for 7 minutes, the paper and paint are drained and treated with sulfuric acid 5 sec, washed with water, stained with methylene blue for 4 min, washed with water, dried with filter paper. Microscopy: spores are pink-red, vegetative cells are blue. Zlatogorov’s method is the same, only without treating it with chrome. Peshkov's method: the smear is fixed, stained with methylene blue with heating, washed off with water, stained with neutral red solution for 10 seconds, washed off with water, dried with filter paper. Spores are blue, CL is red.
8. Cultivation of aerobic microorganisms in the laboratory. Methods for isolating pure cultures of aerobic microorganisms
Microorganisms grown on artificial feeds are microbial cultures, and obtaining their growth on feeds is by cultivation. Conditions required for cultivation are: optimal temperature, taking into account which group the species of bacteria under study belongs to, appropriate nutritional conditions, aerobiosis (or anaerobiosis). Thermostats are used to ensure a constant optimal temperature. A laboratory thermostat is a cabinet with double walls, the outside lined with a non-heat-conducting material (plastic), the inner wall is metal. Between two metal walls there is water (or air) heated by electricity. From the heated water, heat enters the thermostat through the inner metal wall. Inside there are mesh shelves on which racks with test tubes, Petri dishes, etc. are placed. A constant temperature is maintained using thermostats - when the temperature reaches a given level, the device automatically turns off; When the temperature drops, the thermostat turns on again automatically. In addition to ensuring the temperature regime, the type of respiration of microorganisms should be taken into account: with the aerobic type of respiration, no additional conditions need to be created. Isolating one type of microbe from a mixture is isolating a pure culture. One of the first methods proposed by Pasteur was the dilution method. The test material is sequentially diluted in a liquid pit medium: a number of test tubes with MPB are taken, the test material is added to the first test tube, mixed, transferred from it to the second, etc. Pasteur assumed that in the last test tube the growth of one type of microbe was possible. But that's not true. Koch's method - a dense medium is used - using Pasteur's principle, the test material is diluted in 4-5 test tubes with molten and cooled MPA, the contents of the test tube are carefully poured into a Petri dish and the medium is distributed in a thin layer, the cup is closed, and when A cools down, it is turned upside down. Place it in the thermostat. Where the concentration of microbes is lower, colonies isolated from each other grow. On the reverse side, the desired colony is marked, inoculated on MPB and MPA, and a pure culture grows. The Drigalski method is a plate sowing method. take 4-5 Petri dishes. The agar medium is melted in a flask, poured into cups and placed upside down in a thermostat. Use a Drigalski spatula or Pasteur pipette to evenly rub a drop on the surface of the medium. Rub the same spatula on the surface of the second cup, etc. Place it in the thermostat upside down. The desired crop is sown in the MPA and MPB. Biological method - the test material is injected into a susceptible living person. If a pathogenic microbe is present, they die alive, they are opened and cultured. Shukevich's method - a mobile microbe moves to surface A from the condensation liquid, inoculations are made from the upper edge of the grown culture and a pure culture is obtained. Chemical method - chemical ingredients are added to pita, the cat has a lethal effect on some, growth is delayed in others, and still others are not susceptible.
9. Chemical composition of bacterial cells
75-85% water, 25-15% dry residue. The leading role belongs to the 4th basic element, the cat is called organogens: oxygen - 30% of the dry residue, H - 6-8%, C-45-55%, N-8-15%. Water in a cell can be in 2 states: free water, which will dissolve crystalline substances and the movement of ions occurs in it; bound water, the cat is part of proteins, fats and carbohydrates. In bacteriological cells, the share of proteins is 60-70%, carbohydrates - 20%, lipids -1-2%. The increased lipid content gives the cell acid-alcohol-alkali resistance. Proteins are high-molecular polymer compounds formed during the hydrolysis of amino acids - complex - proteins, simple - proteins. Protein functions are the main structural material for all cell membranes, they provide motor functions, transport and nourish substances through the membrane. Carbohydrates - polyhydric alcohols (mannitol, dulcit) and polysaccharides (glycogen). They play an energetic role in the class. Lipids – fatty and neutral fats, phospholipids of the cytoplasmic membrane. It is a reserve cell, used as a starting component for protein synthesis. Min. content – 3-10% dry residue. Micro and macroelements. Microbial functions. Head of the saint: specificity and thermolability. In a microorganism, the set of functions is genetically fixed and is inherited. Difference between functions: 1. exophths - release of cells into the external environment and the catalyst is decomposed of complex substances of the substrate to simpler ones. 2. endophytes – are localized in the cell itself and participate in intracellular metabolic processes. 3. constitutive - they are a constant component of CL and can be detected even in the absence of the substrate they catalyze in the medium. 4. adaptive - cells are produced only when an appropriate substrate appears in the environment. There are: oxireductase, transferase, hydrolase, lyase, isomerase, ligase and kinase. The presence of functions can be detected using special media.
The other two compartments, separated by a membrane, are the pyrrellulose or riboplasma, which contains ribosomes and associated proteins, and the ribosome-free paraphoplasma (Glockner, 2003). 3. Characteristics of the general properties of microorganisms Microorganisms are organisms invisible to the naked eye due to their small size. This criterion is the only one that unites them. The rest of the world...
By the number of matching features. This approach to the taxonomy of microorganisms is quite objective, but its implementation requires extensive mathematical calculations using electronic computers. After detailed study, the microorganism is given a scientific name, which must be expressed in two Latin words, as required by the binomial nomenclature proposed...
Under autotrophic conditions. According to the oxidizable substrates, such groups of chemolithoautotrophs as hydrogen, nitrifying, sulfur bacteria, and iron bacteria are distinguished. Chemolithoautotrophic microorganisms that oxidize H2 also include many methane-forming bacteria, individual representatives of acetate-forming, sulfate- and sulfur-reducing bacteria. Various possibilities are revealed...
It is also suggested that mesosomes do not actively participate in the processes of cellular metabolism, but perform a structural function, providing compartmentalization of the prokaryotic cell, i.e., spatial delimitation of intracellular contents into relatively separate compartments, which creates more favorable conditions for the occurrence of certain sequences...
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To determine the mobility of bacteria, the “hanging drop” and “crushed drop” methods are used.
Hanging drop method. A drop of an 18...20-hour broth culture or a drop of agar culture condensate is applied to a coverslip. A special glass slide with a recess (well), the edges of which are lightly greased with Vaseline, is used to cover a drop of culture so that the cover glass sticks to the slide. The preparation is turned over with the cover glass up, and the drop “hangs” above the well (Fig. 14).
Microscopy the preparation in a dry lens system with a slightly darkened field of view (use the diaphragm and the condenser lowered). Under low magnification, find the edge of the drop, then, lifting the tube, put the medium magnification lens (40...60) into working condition, carefully
under the control of the eye (looked from the side), the tube is lowered until the frontal lens of the objective comes into contact with the coverslip
glass. Then, looking through the eyepiece, carefully lift
macrometric screw the tube and find it in the field of view
a drop. Next, use a micrometer screw to adjust the microscope until the microbes are optimally visible. Rice. 14. Hanging drop preparation.
“Crushed drop” method. A drop of a daily bacterial culture is applied to a regular glass slide and carefully covered with a cover slip so that no air bubbles form between the slides and the drop of culture does not spread beyond the edges of the cover slip. Carefully lower the medium magnification lens and examine the microscope.
In both cases, the movement of microbial cells is clearly visible against the grayish background of the field of view.
Preparation of dyes and coloring of smear preparations. Methods for subculture of microorganisms.
Microscopy microbes in living and non-living states. To study the morphological and tinctorial properties of microorganisms, a specially colored preparation is prepared using various aniline dyes.
Paints And coloring solutions. The following aniline dyes are most often used in microbiological practice: magenta (basic), methyl red, neutral red - they are red in solution; carbolic crystal violet, methyl violet, gentian violet, ready-made liquid paint Giemsa (azur-eosin) violet; methylene blue, diamond and malachite green.
Aqueous or alcoholic solutions of paints are prepared from dry crystalline or powdery dyes. The latter are usually prepared for future use, as they are well preserved in the dark (dark glassware, dark room). To enhance the effect of dye solutions on the microbial cell, various disinfectants are used, which are added to the dye solution (phenol, caustic potassium) or they are used to treat the preparation before staining (weak solutions of hydrochloric, sulfuric or chromic acids). Also, for the purpose of etching, the preparation with paint poured onto it is heated or filled with a preheated paint solution. Paints that are unstable in solution and do not last for a long time are prepared only immediately before use in the form of a 1 ... 2% solution.
Alcohol-water solutions. Carbolic fuchsin (Tsil fuchsin). Basic fuchsin crystals are pre-dissolved in 96% ethyl alcohol. First, prepare a saturated alcohol solution (100 ml of alcohol per 5...10 g of paint). For better and faster dissolution, the paint crystals are first ground in a porcelain mortar in a small amount of alcohol with the addition of a few drops of glycerin. A pure alcohol solution is unsuitable for painting, so an alcohol-water solution is prepared: add 100 ml of distilled water with 5% phenol (mordant) to 10...20 ml of a saturated alcohol solution of fuchsin. The resulting fuchsin solution is filtered through filter paper. In some cases, before use, Ziehl fuchsin is diluted again with distilled water (1:10) and its working solution is obtained (Pfeiffer fuchsin).
Carbolic crystal violet, metipviolet, gentian violet. The first two dyes in solution precipitate very quickly and, when stained, can distort the microscopic picture. More often they use gentian violet, which is obtained by mixing methyl and crystal violet with the addition of dextrin; it gives a more even color. To prepare an alcohol-water solution, 1 g of dry gentian violet is dissolved in 10 ml of alcohol, ground in a mortar with glycerin and phenol crystals (2%), then distilled water is added. To avoid the formation of sediment when storing the solution, sheets of filter paper are soaked in a saturated alcohol solution of paint, dried in air, cut into small strips or squares and stored in a dark jar with a ground stopper.
When coloring the preparation, place a dried strip of gentian violet on it, pour a few drops of water on top, leave for 2...3 minutes.
Methylene blue solution(Leffler's alkaline blue). To prepare a solution, 3 g of paint is infused for a long time (3...4 months) in 100 ml of 96% alcohol, then 30 ml of a saturated solution is diluted in 100 ml of distilled water containing 1 ml!% solution of potassium hydroxide (mordant ). Filtered.
Aqueous solutions. 2% safranin: 2 g of dry dye is poured into 100 ml of hot distilled water, filtered through a paper filter and immediately use a fresh solution for dyeing.
1% solution of malachite green: 1 g of crystalline paint is dissolved in 100 ml of hot distilled water, filtered, cooled and used for coloring.
Ready-made liquid paint azur-eosin (Giemsa paint) used for special methods of staining bacterial preparations. Before use, it must be diluted with distilled water (1:10), but a sediment will immediately form. To prevent the latter from affecting the preparation, staining is carried out, according to Romanovsky’s recommendation, as follows: glass rods or matches with broken heads are placed on the bottom of a Petri dish, the preparation is placed on them with a smear down, the paint solution is poured under the preparation (Romanovsky-Giemsa method).
To effectively influence the bacterial composition of the environment, it is necessary to have reliable information about its qualitative and quantitative content. There are many methods for identifying bacteria, and the choice of how to examine a sample depends on what results you want to obtain. The method for determining lactic acid bacteria differs from the method for identifying Listeria monocytogenes, and the determination of enzymatic activity is carried out differently than the determination of biochemical properties.
Determination methods based on the results obtained can be divided into two large groups:
- determination of the number of microbes;
- qualitative research.
The results of the analysis of the bacterial composition of the samples are expressed as the total microbial number expressed in CFU (colony-forming units).
Microbial count analysis, depending on its capabilities, can determine:
- the number of all microorganisms contained in the sample;
- only viable microbes.
Depending on the method of obtaining the result, methods for determining the number of microorganisms are divided into:
- straight (microscopic);
- indirect.
In turn, indirect methods are divided depending on the criterion used as:
- optical research methods (spectrophotometry, nephelometry) – the measured parameter depends on the number of microorganisms;
- plating is a method of measuring the colonies that have formed.
Methods for determining the total number of bacteria are based on the titer value of the sample.
titer technique
The method of limiting sample dilution (titer method) allows you to determine the quantitative value of a group of microorganisms with high accuracy.
The essence of the technique is that the test sample is diluted in a certain way and inoculated into microorganism-specific media. This creates favorable conditions for growth. Over time, samples are examined to determine the maximum dilution at which bacteria of a certain group are detected. Conclusions are drawn based on specific changes in the nutrient substrate.
A similar technique, taking into account the individual properties of microbes, has proven itself well in the detection of E. coli microorganisms and related species.
Direct counting
The method is convenient for studying soil and water samples. Direct counting is carried out in counting chambers designed for this purpose, on membrane filters or fixed smears. The method does not require complex equipment, is short in time and minimal in cost.
A limitation of the method is the required high concentration of microbes in the samples.
A method for optical determination of the number of bacteria based on the determination of light scattering by a sample suspension. This method allows you to determine the number of cells in a sample, which makes the method popular in microbiological studies.
Nephelometry method
Counting viable microbes
The technique is based on inoculating a certain number of bacteria in the form of a suspension onto an agar medium. After this, the formed colonies are counted, keeping in mind that each of them is the offspring of a viable bacterium.
There are two types of sample inoculation method:
- the test sample is added to the agar medium and mixed;
- the sample is sown on the surface layer of agar.
Motility as an important factor in bacterial identification
A significant factor in the identification of bacteria is motility, which is provided by flagella. Since the number and location of flagella that provide motility can be different, all microbes with flagella are divided for ease of identification into:
- monotrichous - one flagellum at the pole;
- lophotrichs - a bundle of flagella located at one of the poles;
- amphitrichous - flagella or bundles are located at both poles;
- peritichi - flagella are located along the perimeter of the cell.
Determination of bacterial mobility is carried out in cultures no older than one day. In older cultures, the ability to move is lost.
Determination of qualitative bacterial composition
Determining the qualitative composition of bacteria is based on several factors.
Biological and chemical characteristics of microorganisms
The study of the biochemical properties of microorganisms helps determine the qualitative composition of bacteria.
Identification of microorganisms is facilitated by knowledge of biochemical processes. Methods for determining the quantity and quality of bacteria are based on the proteolytic and saccharolytic properties of microorganisms, as well as toxin and pigment formation.
Microbial enzymes
One of the factors in the life of bacteria is enzymatic activity: the enzyme composition and properties are regulated by the genome of the microorganism and is a stable criterion for identifying microbes. Therefore, the detection of proteolytic, saccharolytic and other enzymes is of great importance in the identification of microorganisms.
For example, a criterion for the proteolytic activity of microorganisms is the ability of bacteria to break down protein into deep decomposition products (hydrogen sulfide and indole). A method for determining the number of microorganisms, which is of great practical importance, is based on this result of enzymatic activity.
Pigment formation property
Another persistent genetic trait of bacteria is pigment formation. This property is intended to protect the bacterial cell from exposure to ultraviolet rays.
Most pathogenic microorganisms do not have such protective properties - pigment formation is not typical for them.
Study of milk microflora
The definition of bacteria is important in practical human activities, for example, in the food industry. Thus, bacterial contamination of milk is the main indicator of the sanitary conditions of its production. If the threshold number of microorganisms in milk is exceeded, the quality of the product is reduced.
Since 1987, the EEC countries have adopted uniform standards for the degree of bacterial contamination of milk, dividing the product into three categories:
- A – 20 thousand/ml;
- B – 100 thousand/ml;
- C – over 100 thousand/ml.
In this case, the numbers indicate the maximum possible number of microorganisms in 1 ml of milk (contamination).
Bacterial contamination of milk directly depends on the sanitary conditions of receipt and initial processing of the product. Thus, the use of reusable filters for milk purification can lead to additional bacterial contamination.
The presence of somatic cells in milk is an important quality criterion. These cells are particles of animal biomass. They are formed in the udder and reflect the natural processes of aging and renewal of the body.
The number of somatic cells in milk increases when animals have injuries, gastrointestinal diseases or other pathologies, which leads to an increase in the rate of bacterial contamination of milk.
Determination of lactic acid bacteria
Lactic acid microorganisms are of great importance in the food industry due to their antagonistic and proteolytic activity.
For example, the plastic consistency and pronounced taste of various types of cheese are associated with the proteolytic activity of lactic acid microbes in the starter culture. Research into the activity of lactic acid bacteria in this area is given great importance, but so far it has not been possible to create a criterion for determining strains of lactic acid bacteria in sourdough based on proteolytic activity.
The antagonistic activity of lactic acid microbes is used not only in the food industry, but also in medicine, veterinary medicine, agriculture, etc.
Examples of the use of lactic acid bacteria as antagonists to certain microorganisms:
- cheese production - antagonists to oil microbes and E. coli;
- baking production - antagonists to the spore bacillus, the causative agent of the “potato disease” of bread;
- lactic acid products are antagonists to bacteria that provoke the development of gastrointestinal infections.
If it is necessary to count the number of lactic acid bacteria in the starter, use the method of sowing microbes on agar and milk with the addition of chalk. The resulting lactic acid dissolves the chalk, and light zones appear around the colonies of lactic acid bacteria.
To count lactic acid streptococci, the limiting dilution method is used by inoculating them in milk. Depending on the thermophilicity of lactic acid bacteria, the optimal thermal sowing regime is selected. Samples with curdled milk are used to prepare microscopic slides. After this, the minimum dilution containing lactic acid rods is detected.
Air microbiological control
The air environment is not favorable for the life of bacteria, but most microorganisms, when released into the air, are able to temporarily retain activity and their properties. Among them are pathogenic bacteria such as the causative agents of measles, scarlet fever, whooping cough, smallpox, tuberculosis, pneumonic plague and other respiratory tract infections transmitted by airborne droplets.
Microbiological control of the air environment evaluates the general bacterial contamination of the air and develops preventive methods to reduce the number of pathogens of infectious diseases.
The objects of study of the degree of bacterial contamination in enclosed spaces are hospitals and clinics, children's institutions and places where people constantly gather (cinemas, gyms, etc.). Determination of the degree of bacterial contamination of indoor air is carried out using proven methods, including the following actions:
- sample collection;
- transportation and sample preparation;
- bacterial culture;
- determination of microorganisms through identification.
If a high degree of bacterial contamination is detected, various methods are used to reduce the number of bacteria:
- chemical - treating the room with nitrogen dioxide, ozone or a suspension of lactic acid;
- mechanical – forced air filtration;
- physical - ultraviolet irradiation.
Study of the bacterial composition of water
Water analysis is carried out for the presence of the following groups of microorganisms:
- coliform microbes – microorganisms of the Escherichia coli group, used as markers of fecal contamination (dissemination);
- clostridia are microbes that are highly resistant to disinfection; reference indicator (landmark) - if there are no clostridia in the sample, then there are no other pathogenic microbes;
- viruses;
- Giardia.
Coliform bacteria are gram-negative coliform bacteria found in the intestines of mammals and birds. They enter the water with feces and are able to exist in it for weeks, but lose their ability to reproduce.
The presence of coliform microorganisms in a water sample indicates a high probability of the presence of wastewater. The presence of virulent (pathogenic) strains of coliform bacteria is an indicator of the risk of disease.
Coliform microbes include a group of thermotolerant coliform microorganisms that have the ability to survive at high (45°C) temperatures. Thermotolerant coliform bacteria are an indicator of recent fecal contamination and are easily detected analytically.
According to SanPiN, coliform bacteria cannot be present in water supply systems, and the presence of coliform microorganisms indicates either poor quality treatment or secondary fecal contamination. The presence of coliform microbes in an amount of no more than 5% of the total is considered the norm. The criterion for the effectiveness of treatment of fecal wastewater is thermotolerant coliform bacteria, which are easily identified.
Identification of the causative agent of listeriosis
The bacterium Listeria monocytogenes is the causative agent of listeriosis, an infectious disease of humans and animals.
Listeria monocytogenes
The causative agent of listeriosis is the highly motile, non-spore-forming, gram-positive Escherichia coli Listeria monocytogenes. In humans, infection with Listeria monocytogenes occurs as acute sepsis, affecting the central nervous system, lymphatic system, tonsils, spleen and liver. Listeria monocytogenes infection in humans can occur in both acute and chronic forms.
Statistical risk group for Listeria monocytogenes:
- elderly people;
- pregnant women;
- persons with weakened immune systems, HIV-infected and cancer patients;
- alcoholics and drug addicts.
Listeria monocytogenes infection is determined by PCR (polymerase chain reaction) - Listeria monocytogenes DNA is detected in the blood plasma. Confirmation of the presence of listeriosis is the identification of a specific DNA region of Listeria monocytogenes. The specificity of Listeria monocytogenes DNA determination is 100%; the method is highly sensitive.
Interaction of bacteria with antibiotics
Determining the sensitivity of bacteria to antibiotics is of great practical importance for calculating the dosage of drugs in the prevention and treatment of infectious diseases.
To determine the sensitivity of microbes to various antibiotics, two main methods are used:
- determination of sensitivity to antibiotics using discs;
- studying the sensitivity of microorganisms to antibiotics through serial dilution.
Method using disks
To determine the degree of sensitivity to antibiotics, the bacteria being studied are inoculated into a nutrient culture. Discs containing various antibiotics in known dosages are placed on the surface.
The sample is kept at the optimal temperature (37°C) for a day, after which, by comparing the diameter of the annular germ-free zones around various disks, a conclusion is drawn about the sensitivity of bacteria to various antibiotics and their concentrations.
To obtain reproducible results, standard disks and culture media should be used, and reference strains should be used as controls. The disc technique does not allow obtaining reliable results, and is also very critical to weakly diffusing antibiotics (ristomycin or polymyxin).
Serial breeding
The sensitivity of microbes to antibiotics by serial dilution allows us to identify the minimum concentration of a drug that has a therapeutic effect. The sensitivity of microorganisms is determined as:
- sensitive strains - the vital activity of bacteria is suppressed by the usual dosage of antibiotics in the blood;
- moderately resistant strains – to inhibit bacteria it is necessary to use maximum doses of antibiotics;
- resistant bacteria - the vital activity of bacteria is not suppressed even at the maximum concentration of the antibiotic, that is, lack of sensitivity.
Determination of bacteriophage sensitivity
Bacteriophages are natural enemies of bacteria. The nature of the interaction of the bacteriophage with bacteria is described as:
- virulent, causing lysis (death) of the microbial cell;
- moderate – transition of the bacterium to a non-infectious form of phage (prophage).
Bacteriophages are specific to certain groups of microorganisms, which is reflected in their name - streptococcal phages, staphylococcal phages, etc. A method for determining the number of bacteriophages per unit volume is the agar layer method. It is simple to perform and has sufficient accuracy.
Thus, there are a large number of methods for determining microorganisms. The choice of the optimal one depends on the given selection criterion.
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Purpose of the lesson. Learn methods for staining spore-forming, capsule-forming bacteria, as well as determining the motility of bacteria.
Materials and equipment. Suspensions of bacteria with anthrax vaccine strain, clostridia, ready-made preparations with capsule-forming bacteria, mobile broth cultures of Escherichia 18 hours of growth, slides and cover glasses, posters, 2% safranin solution, aqueous solution of malachite green, Ziehl carbolic fuchsin.
Guidelines. Each student prepares smears from suspensions of microorganisms and stains them according to the Trujillo, Olt method, microscopes and sketches; prepares a preparation for studying the mobility of microorganisms using the “crushed” and “hanging” drop method.
Spore coloring. Under unfavorable conditions for microbes (lack of a nutrient medium, drying, unfavorable temperature, etc.), spores form in the cytoplasm of some microorganisms. They are formed inside the vegetative cell, being endospores. Rod-shaped gram-positive microorganisms that form round spores, the diameter of which does not exceed the width of the microbial cell, belong to the genus Bacillus and are called bacilli. Microorganisms of the genus Clostridium have spores whose diameter exceeds the width of the microbial cell and are called clostridia. They are oval and round in shape (Fig. 5).
The spores are resistant to high temperatures, chemicals, and drying out; they persist in the soil for a long time, which is explained by their special structure and chemical composition, especially its shell. Therefore, the spores are resistant to dyes.
All methods of staining spores are based on ensuring the penetration of the dye through the difficult-to-stain spore shell. Therefore, a mordant is used. After cooling, the shell becomes dense again and does not allow additional dye to pass through.
Technique for staining spores using the Trujillo method. A small piece of filter paper is placed on the fixed smear and an aqueous solution of malachite green is applied to it.
Rice. 5. Spores of various types of microorganisms
Heat the preparation on a burner flame until vapor appears and leave for 3 minutes, wash with water and finish painting with a 0.25% aqueous solution of basic fuchsin for 1 minute. Wash with water and dry. Micro picture: spores are green and vegetative cells are red.
Capsule coloring. The body of the microbial cell is covered with a loose mucous layer. In some types of microorganisms this layer develops very strongly and is then called a capsule. The capsule is a mucin-like substance, a high-molecular polysaccharide, and is a derivative of the outer layer of the shell. The presence of a capsule is an important diagnostic sign in the identification and differentiation of pathogens of certain infections (anthrax, pneumococcal pneumonia, etc.) (Fig. 6). Pathogenic microorganisms form a capsule in the infected body. It is a virulence factor and protects the bacterial cell from phagocytosis and the bactericidal effect of blood serum. The capsular substance does not stain well. Therefore, when preparing a drug to detect a capsule, the following rules are followed:
a) the smear is prepared from fresh material, since the capsule is quickly lysed;
b) fix the smear chemically; for coloring, metochromotic paints are used, that is, when used, the cytoplasm is painted in one color, and the capsule in another;
c) the smear should be washed lightly and briefly with water.
Technique for coloring capsules using the Olt method. A fresh hot 2% solution of safranin is applied to a fixed smear and stained for 5-7 minutes. Rinse quickly with water and dry. The cell body is painted red brick, the capsule is yellow-orange. Determination of bacterial motility.
Bacterial mobility is an important species characteristic and is used in diagnostic studies: the result is taken into account when identifying microorganisms. In mobile species, the ability of independent translational (and rotational) movement is due to the presence flagella- special thin thread-like formations.
Fig. 6. Capsule in bacteria
a - anthrax bacillus; b - diplococcus
Flagella come in different lengths.
Their diameter is so small that they are invisible in a light microscope (less than 0.2 microns). Different groups of bacteria have different numbers and locations of flagella. Flagella do not accept dyes well. Complex staining methods distort the true appearance of flagella, so flagella are not stained in laboratories, but bacteria are examined in a living state. Depending on the location and number of flagella, microbes are divided (Fig. 7):
A) monotrichs- microorganisms that have one flagellum at one of the poles, active, translational movements (pseudomonas);
Rice. 7. Types of arrangement of flagella in bacteria
b) lophotrichs- microbes that have a bundle of flagella at one of the poles (Listeria);
V) amphitrichs- microbes that have flagella at both poles of the microbial cell;
G) peritrichous- microbes in which flagella are located over the entire surface of the cell (E. coli).
There are types of microorganisms that have mobility, but do not have flagella (spirochetes, leptospira). Their movement is caused by impulsive contractions of the fibrillar motor apparatus of the microbial cell.
To determine the mobility of bacteria, it is necessary to use a culture no older than one day old, since old cultures lose the ability to move.
Determination of bacterial motility using the hanging drop method. A drop of a young (18-20 hour) broth culture of bacteria is applied to a cover glass using a bacteriological loop. A drop of culture is covered with a special glass slide with a recess (well) so that the cover glass with the drop is in the center of the well and sticks to the slide (the edges of the well are first lightly greased with Vaseline). The drug is turned upside down, and the drop “hangs” over the hole (Fig. 8). The specimen is examined microscopically under a darkened field of view, first at low, then at medium or high magnification. On a light background, microbes are dark gray. Shukevich's method. To do this, a drop of microbial suspension is applied to the condensate of a slanted dense nutrient medium in a test tube. Mobile microorganisms, moving from the condensate, grow on the surface of the medium; non-motile species reproduce only in the condensate of the medium (“without going” to the surface of the agar).
“Crushed drop” method. A drop of bacterial suspension is applied to a regular glass slide, carefully covered with a coverslip and pressed lightly with a finger. Microscopy is carried out in the same way as in the hanging drop method.
Method of sowing by injection into semi-liquid agar. To do this, a bacteriological loop is used to inoculate the culture under study by injecting it to the bottom of a test tube with a semi-liquid nutrient medium. The mobile culture grows throughout the nutrient medium, forming a uniform turbidity, and the stationary culture grows only along the prick in the form of a rod, maintaining the transparency of the uninoculated area of the medium.
LESSON 5. Laboratory glassware and its preparation. Nutrient media. Methods for preparing and sterilizing culture media. Methods for sterilizing laboratory glassware.
Purpose of the lesson. Prepare the dishes. Prepare nutrient media. Determine the pH of the medium. Familiarize yourself with methods of sterilizing culture media and laboratory glassware.
Equipment and materials. Racks, test tubes, microbiological loops, pipettes, Petri dishes , paper. Autoclave, drying cabinet. Set of media and chemical reagents. pH meter.