Staining of smears according to the Gram method The most common method of staining bacteria, which makes it possible to classify cells according to the type of wall staining as gracilicutes (gram-negative) or firmicutes (gram-positive).

A piece of filter paper is placed on a fixed smear, a carbolic solution of gentian violet is poured onto it for 30-50 seconds. The dye is drained, the paper is removed, Lugol's solution is applied for 60 seconds. Discard Lugol's solution. Rinse the drug in 95 ° ethanol for 30-50 seconds until the dye stops leaving, which depends on the thickness of the smear. The preparation is washed with water, stained for 30-60 seconds with Pfeiffer fuchsin, washed with water, dried and microscoped. Microscopic picture: gram-positive bacteria - purple, gram-negative - red.

Practice shows that cleaner preparations without prejudice to differentiation are obtained if, after staining with gentian violet, the smear is slightly rinsed with water, the remnants of which are shaken off and only then Lugol's solution is applied.

Modification according to A.P. Sineva. A strip of paper with a dye is placed on a fixed smear, 2-3 drops of water are poured and stained for 2 minutes. Then proceed as described above.

Kerry modification. The smear is stained for 1-2 minutes with an alcohol solution of gentian violet, treated with iodine solution for 1-2 minutes, then with ethanol for 30-40 seconds. Washed. Repeatedly treated for 1 minute with iodine solution, washed, stained for 1-2 minutes with fuchsin or a mixture of safranin with brilliant green.

Burke's modification. Solution A: 1% crystal violet solution in distilled water.

Mordant: crystalline iodine - 1 g, potassium iodide - 2 g, distilled water - 100 ml.

Discoloration solvent: ethyl ether - 1 volume, acetone - 3 volumes.

Additional dye: 2% solution of safranin in distilled water.

Methodology. The smear fixed by heating is immersed in solution A. The smear is rinsed in iodine mordant and covered with fresh mordant for 2 minutes. Rinse with water and then with bleaching solvent until the effluent is free of paint. After drying, stain with additional dye for 5-10 seconds, rinse with water, and microscope.

Hooker modification

Solution A: crystal violet - 2 g, 95% ethanol - 20 ml.

Solution B: ammonium oxalate - 0.8 g, distilled water - 80 ml.

Solution A and B in prepared volumes are mixed in a dark vial and left at room temperature for 24 hours.

Solution B: 2.5% safranin solution (in 96% ethanol) - 10 ml, distilled water - 100 ml.

Methodology. The smear fixed by heating is immersed in a mixture of solutions A and B for 1 min, after which the preparation is washed with water and treated with Lugol's solution for 1 min, then washed again with water and dried; 96% ethanol is applied for 30 s, the preparation is thoroughly washed with water, dried, placed in solution B for 10 seconds, slightly washed with water, dried, microscoped.

Potassium hydroxide test to monitor bacterial Gram stain results. If the Gram stain is not clear, this method allows you to refine the result.

Setting up the test. 1-2 drops of a 3% KOH solution are applied to a glass slide. The agar culture is introduced into KOH with a bacteriological loop, mixed, and then the loop is lifted up. If the culture in the form of a "thread" reaches for the loop - gram-negative, in the absence of this phenomenon - gram-positive bacterium.

Methods for staining acid-fast bacteria

Ziehl-Nielsen staining. Carbol fuchsin dye is prepared: basic fuchsin - 0.3 g, 95% ethanol - 10 ml, phenol (crystals melted when heated) - 5 ml, distilled water - 95 ml.

The basic fuchsin is dissolved in ethanol, phenol dissolved in water is added, mixed, kept for 5-7 days at room temperature; filtered through filter paper before use.

Discoloration solvent (hydrochloric alcohol): ethanol 95% - 97 ml, concentrated hydrochloric acid - 3 ml. Additional dye: methylene blue - 0.3 g, distilled water - 100 ml.

Methodology. Carbol-fuchsine dye is poured onto a smear fixed by heating, heated until the vapors escape (but do not boil!). Washed with water after 5 minutes. Then the smear is treated with a bleaching solvent for 30-50 seconds, washed with water, after which the film of bacteria on the smear becomes pale pink.

The smear is stained for 3-5 minutes with an additional dye (methylene blue, etc.), washed with water, and dried.

Acid-fast microorganisms in the smear are red, others are blue.

Auramine stain. Solution A: auramine - 1.5 g, rhodamine B - 0.75 g, glycerin - 75 ml, melted phenol - 10 ml, distilled water - 50 ml.

Dyes: phenol and glycerin are dissolved in 25-30 ml of water, the remaining volume of water is added and filtered through glass wool.

Solution B: 70% ethanol - 99.5 ml, concentrated hydrochloric acid - 0.5 ml.

Solution B: potassium permanganate - 0.5 g, distilled water - 99.5 ml.

Methodology. A smear fixed over a burner flame is stained for 15 minutes with solution A at room temperature or at 37-38 ° C. Washed under running water until discoloration. Solution B is poured onto the smear for 3-5 minutes, washed and stained for another 2-4 minutes with solution C. Washed, dried, microscopically in a fluorescent microscope under immersion (exciting light filter BG-12, blocking OG-1).

Acid-fast bacteria yellow-orange on a dark background.

Fly coloring. The drug withstands 24 hours in a solution of methyl violet (10 ml of a saturated alcohol solution of methyl violet, 100 ml of a 2% aqueous solution of carbolic acid). Lugol's solution is applied for 1-2 minutes, then a 5% solution is applied for 1 minute nitric acid and 10 seconds 3% solution of hydrochloric acid. Then, a mixture of ethanol and acetone (1:1) is applied to the preparation until the paint stops coming off, washed with water, dried and microscoped.

Spore staining methods

Aujeszky's method. An air-dried smear without fixation is treated for 2-3 minutes (when heated) with 0.5% sulfuric acid, washed and fixed over a flame. It is stained for 7-8 minutes with Ziel's carbol fuchsin when heated, the paint is drained. The smear is treated for 5-7 seconds with a 5% sulfuric acid solution, washed and stained with methylene blue for 4-5 minutes. Under microscopy, the vegetative part of the cell is blue, the spore is red.

Meller's method. A smear fixed over a flame is etched for 2-3 minutes with 5% chromic acid, washed, and stained with Ziel's carbolic fuchsin. Bleach and stain in the same way as the Aujeszky method.

Peshkov method. The fixed smear is stained with methylene blue heated to boiling. The paint is washed off and stained with a 1% aqueous solution of neutrality for 10 seconds. Washed, dried. Spores are blue, vegetative cells are red.

Schaeffer-Fulton method. A 0.5% aqueous solution of malachite green is poured onto a smear fixed by heating, covered with filter paper. The smear is placed for 5 minutes over boiling water. Washed and counterstained for 30 sec with 2% safranin aqueous solution. Washed, dried. Spores are bright green, vegetative cells are red-brown.

Trujillo method. The smear is fixed over a burner flame, covered with a saturated aqueous solution of malachite green, heated until steam appears and stained for 3 minutes. The paint is washed off with water and the smear is stained with a 0.25% aqueous solution of basic fuchsin for 1 min. Washed, dried. Spores are green, vegetative cells are red.

Dorner method. A smear fixed on a burner flame is stained for 5-10 minutes with carbol fuchsin when heated, discolored for 1 minute with hydrochloric acid alcohol (3 ml of concentrated hydrochloric acid and 97 ml of 96% ethanol). The drug is washed, dried with filter paper and poured onto the smear with a thin layer of a 10% solution of nigrosine in a 0.5% formalin solution. Without washing, the smear is dried in air and microscoped. Spores are red, vegetative cells are colorless on a black background.

Waldman's method. Lefleur blue (alkaline) is applied to the fixed preparation and heated to a boil, then cooled and washed with water. Stain with 1% neutral solution for 30 seconds. Microscopic picture: spores - blue, vegetative cells - red.

Ozheshka method. A 0.5% solution of HC1 is poured onto an unfixed smear and heated for 1-2 minutes. The acid is drained, the preparation is washed with water, dried, fixed on a flame and stained according to the Ziehl-Neelsen method. Microscopic picture: spores - red, vegetative cells - blue.

Capsule Coloring Methods

Romanovsky-Giemsa method. A smear fixed in ethanol or Nikiforov's liquid is placed in Romanovsky-Giemsa paint (15-20 drops of paint per 10 ml of distilled water) for 15-20 minutes. Washed, dried. The body of the bacteria is stained dark blue, the capsules are pink.

Olt's method. Fixed smears over the flame of the burner are stained for 1-3 minutes with a 2% aqueous solution of safranin (safranin is dissolved in hot water, filter, apply a freshly prepared solution). The body of the bacteria is stained brown, the capsule is pale yellow.

Rebiger's method. An unfixed smear is stained for 15-20 seconds with a solution of gentian violet in formalin (15-20 g of gentian violet is dissolved in 100 ml of 40% formalin, settled, filtered), washed with water. The body of the bacteria is dark purple, the capsule is reddish purple.

Anton's way. The smear is stained for 2 minutes with a 1% aqueous solution of crystal violet. The paint is washed off with a 20% aqueous solution of copper sulfate (CuSO 4 ·5H 2 O), the excess solution is shaken off and the smear is dried with filter paper without rinsing with water. Microbes - dark purple, capsule - light blue.

Gins method. A drop of black ink diluted 1:3 with distilled water is applied to a glass slide and centrifuged at 3000 rpm for 15 minutes. In a drop of carcass, the culture is suspended with a loop and a smear is made (similar to a blood smear). Dry, fix over the flame of a burner, stain with Ziel's carbol fuchsin, diluted 1:3. Bacterial bodies are red. The capsule on a dark background is visible as a light halo around the bacteria.

Mikhin's method. The smear is stained for 3-5 minutes with Leffleur's blue (only paint that has been stored for at least 20-30 days is suitable, since azure is formed in it during storage, giving metachromaticity). The bacterial bodies are blue, the capsule is pink.

Giss method. A drop of normal blood serum of cattle or horses (or a drop of skimmed milk) is applied to a glass slide, the culture is introduced into it with a loop, suspended and a smear is prepared. The film of the bacterial suspension is dried in air, fixed over a burner flame and stained with 0.1% aqueous crystal violet for 1 min. The smear is washed with a 20% aqueous solution of copper sulfate, dried with filter paper. Capsules in a smear are pale blue, bacteria bodies are dark purple.

Dugid's method. A drop of black ink is placed on a glass slide, and the culture is suspended in it. The drop is covered with a cover glass, excess ink is removed with filter paper. On a brown-black background, light-refracting bacteria are visible, surrounded by a capsule in the form of a transparent zone.

Jon's way. A drop of ink diluted 1:2-1:4 with distilled water is applied to one end of the glass slide surface. Bacteria are suspended in a drop and a smear is prepared (like a blood smear). The preparation is dried in air, fixed over a burner flame and stained for 2 minutes with a 2% aqueous solution of gentian violet or a 1% solution of fuchsin. Washed with water, treated with 2% acetic acid solution for 8-10 seconds and washed again. In a smear, an unstained capsule is visible around intensely stained bacteria.

flagellum staining method

The presence of flagella in bacteria is judged by indirect signs, for example, by the mobility of cells in the preparations "crushed drop", "hanging drop", as well as by the nature of the growth of the test culture in semi-liquid agar (0.15-0.3%). In the latter case, the test culture is inoculated with a prick in the pancreas and incubated for 18-24 hours. In the process of growth, motile bacteria migrate from the injection line, causing cloudiness of the medium, while immobile bacteria grow only along the injection.

In some cases, staining methods will be used that allow direct observation of flagella

For all staining methods, slides should be prepared from young agar cultures (14-20 hours of growth). A 0.5% aqueous solution of peptone is added to tubes with culture on slant agar for 30-40 minutes. The liquid is settled, for the sedimentation of microbes that have passed into it, it is centrifuged. The sediment is poured with saline and centrifuged again. In order to prevent the loss of flagella, the washed precipitate is resuspended in 10% formalin solution to obtain a slightly cloudy suspension.

Flagella stained with osmic acid. On a clean defatted object or watch glass, a drop of a suspension of bacteria is mixed with a drop of a 2% solution of osmic acid. The resulting drop of the mixture is applied to a clean, fat-free glass slide with a thin capillary of a Pasteur pipette. Air dry and fix on flame (avoid overheating!).

A mordant prepared in a few days is poured onto a fixed preparation (1 ml of a saturated alcoholic solution of fuchsin, 10 ml of a 20% aqueous solution of tannin and 5.5 ml of a saturated aqueous solution of iron sulphate of ammonia). After 15 minutes, the mordant is washed off with water, stained with fuchsin for 4 minutes, washed and microscopically.

Coloring flagella with silver. A thin smear is prepared from the suspension, dried and fixed for 2-3 minutes with a 2% solution of osmic acid. The drug is washed by immersion for 2-3 s in a 0.5% aqueous solution of silver nitrate and, without washing, dipped for 2-3 s in a pickle (pyrogallol or gallic acid - 2.0 g, tannin - 1.2 g , sodium acetate - 4.0 g, distilled water - 150 ml). It is taken out of the mordant and again immersed in a solution of silver nitrate, keeping until the preparation turns black. After washing, microscopy is carried out.

Coloration of flagella according to Plimmer. Paint is prepared: tannin - 5.0 g, zinc chloride - 5.0 g, aluminum chloride - 10.0 g, fuchsin powder - 0.75 g of a sample of the listed components are ground in a mortar, gradually adding 40 ml of 60% ethanol until completely dissolved. Before use, the paint is diluted with water (1:5).

A strip of filter paper is placed on a dried thin smear and filled with prepared paint. After 2 minutes, the paint is washed off with water and additionally stained with Ziehl carbol fuchsin. Bacteria and flagella are red.

Coloration of flagella according to Benichetti. 3 solutions are prepared in advance. Solution 1: zinc sulfate - 0.5 g, tannin - 8.0 g, distilled water - 50 ml. Solution 2: saturated solution of potassium alum. Solution 3: Saturated solution of gentian violet or crystal violet. Before use, the solutions are mixed (5:5:3). A mixture of solutions is poured onto the dried smear and heated until steam appears. Wash after 3 minutes and microscope. Violet-black flagella.

Coloring according to Valenti. The smear is treated for 3 minutes with a 20% tannin solution when heated, washed, stained for 10 minutes with carbolic fuchsin when heated. Washed, microscopic. Bacteria and flagella are red.

Gray coloring. Solution A: tannic 20% aqueous acid - 2 ml, saturated aqueous solution of potassium alum - 5 ml, mercury chloride, saturated aqueous solution - 2 ml, basic fuchsin 3% in 96% ethanol - 0.4 ml . The dye (magenta) is added to the rest of the ingredients immediately before use, and after its dissolution, the resulting solution is filtered through filter paper. Solution B: Ziel's carbolic fuchsin (basic fuchsin 3% in 95% ethanol - 10 ml, phenol - 5 ml, distilled water - 95 ml).

Methodology. A drop of bacteria (suspension) in formalin is applied to the glass and allowed to drain. Air dry. A strip of filter paper is placed and solution A is poured for 4-6 minutes. Washed with water and stained with solution B for 3 minutes through filter paper. Washed, dried, microscopically. Flagella and bacteria are red.

Coloring according to Lifeson. Prepare 3 solutions. Solution 1: 1.5% sodium chloride solution in distilled water. Solution 2: 3% tannic acid solution in distilled water. Solution 3: acetic acid rosaniline - 0.9 g, hydrochloric acid prozanilin - 0.3 g, 96% ethanol - 100 ml. Solutions 1 and 2 are mixed equally and then 2 volumes of this mixture are poured into 1 volume of solution 3. The mixture is stored in the refrigerator for up to 3 months.

Methodology. A drop of the slurry is applied to a well degreased glass and allowed to drain. After drying, the borders of the bacteria film are outlined with a pencil on the glass, the area is filled with dye for 7-15 minutes. Withstand until the surface of the dye acquires a golden color, and the film of bacteria is not covered with sediment. Washed, dried. Bacteria and flagella are red.

Cell wall staining methods

Cell wall staining is of diagnostic value in differentiating mycoplasmas from bacteria.

Gutstein method. The drug is fixed for 1-2 hours in Bouin's liquid, kept for 2-3 minutes in a 5-10% aqueous solution of tannin, rinsed with water and microscoped in a drop of a 0.01% aqueous solution of crystal violet.

Peshkov method. The drug is fixed for 15 minutes in a liquid of the following composition: ethanol 90% - 60 ml, chloroform - 30 ml, acetic essence - 10 ml. Next, the preparation is kept for 2-5 minutes in a 10% aqueous solution of tannin, washed with water, dyed for 30-60 seconds with Pfeiffer magenta and, without stirring, dried and microscoped.

Method) Robinow. The studied microorganism is grown on an agar medium in a Petri dish, an agar block is cut out and placed on a coverslip with its surface, placed overnight in Bouin's liquid. The next day, the agar is removed, the glass is placed face down on the surface of a 5-10% aqueous solution of tannic acid for 20-30 minutes. Then the preparation is washed with water, a cover glass with bacteria cells down is placed on the surface of a 0.02% aqueous solution of crystal violet for 5-10 seconds. As a result, the cell membranes and partitions are stained dark blue or black, and the cytoplasm remains unstained.

Methods for staining cytoplasmic inclusions

When identifying microorganisms, data on the presence of inclusions in cells reflecting the type and direction of constructive metabolism are of particular importance. Cytoplasmic inclusions include poly-β-hydroxybutyric acid, grains of metachromatin (volutin) and polysaccharides.

Poly-β-hydroxybutyric acid is a polyester of beta-hydroxybutyric acid, which accumulates in the cytoplasm of cells in the form of round or oval granules 200-800 nm in size. Metachromatin is polyphosphates, and polysaccharides are glucans, consisting of D-glucose, up to 200 nm in size. The listed inclusions act as a kind of reserves of cell substances and can be detected by special staining methods.

Detection of poly-beta-hydroxybutyric acid

A smear fixed over a burner flame is stained for 5-15 minutes with a 0.3% solution of Sudan black B in ethylene glycol. The paint is drained and, after drying in air without washing with water, rinsed in xylene. After allowing the smear to dry, it is lowered for 5-10 seconds into a 0.5% aqueous solution of safranin. Washed with water, dried and microscoped. Inclusions of poly-β-hydroxybutyric acid are visible against the pink background of the cytoplasm as black-blue formations.

Detection of metachromatin (volutin)

Neisser method. Prepare 4 solutions. Solution A: methylene blue - 0.1 g, 95% ethanol - 2 ml, glacial acetic acid - 6 ml, distilled water - 100 ml. Solution B: crystal violet - 1 g, ethanol 95% - 100 ml, distilled water - 300 ml. Solution B crystalline iodine - 1 g, potassium iodide - 2.0 g, distilled water - 300 ml. Solution D: chrysoidin - 1 g, distilled water - 300 ml (chrysoidin is dissolved in hot water). A mixture of solution A and B in a ratio of 2:1 is poured onto a smear fixed above the flame of a burner for 1 min (the mixture is prepared before painting). The paint is drained, treated for 1 min with solution B (Lugol's solution) and washed with water, dried, microscopically. Bacterial cells are light yellow, volutin grains are dark blue.

Ruskin's method. Prepare the paint of the following composition: Tsilya carbolic fuchsin - 4 ml, saturated alcohol solution methylene blue- 4 ml, glacial acetic acid - 5 ml, ethanol 96% - 95 ml, distilled water - 92 ml. A dye is poured onto a smear fixed above the burner flame, heated over the burner until alcohol is burned, washed with water, dried and microscoped. Bacterial cells are stained light red, volutin grains are stained black and blue.

Polysaccharide detection

Stained with Schiff's reagent. Prepare 4 solutions. Solution A (periodate solution): 4% iodic acid solution 20 ml, 0.2 M aqueous sodium acetate solution 10 ml, 96% ethanol 70 ml. The solution is sensitive to light, so it should be stored in a dark bottle in the dark.

Distinguish between simple and complex methods of coloring.

Simple staining methods called pre-staining

paraty any one dye. Some microorganisms (spirochetes) that are difficult to detect with positive stains are easily detected when stained with negative stains.

Cooking technique drug is as follows. The fixed preparation is placed on parallel glass slats (bridge) that lie above the cuvette. A 1% aqueous solution of fuchsin or methylene blue is applied to the smear using a dropper for 1-2 minutes. Make sure that the dye solution does not dry out during staining. After staining is completed, the dye is drained. The drug is washed with water until the flowing water becomes colorless. Then the drug is dried. To do this, the lower side of the preparation is blotted with filter paper, and the upper side is carefully dried from the sides, without touching the smear. The drug is finally dried in air or high above the flame of an alcohol lamp. To obtain more pure preparations, the dye is applied to a smear covered with filter paper. Staining method in the Sineva modification allows you to use filter paper pre-impregnated with it instead of a dye solution. In a correctly stained and well-washed preparation, the visual field is bright and clean, only cells are stained. Microscopic preparations with an immersion system.

At complex (differentiating) methods staining on the same preparation is affected by several coloring substances, one of which is called the main, others - additional. In addition to dyes, various bleaching agents are used: alcohols, acids, acetone, etc. Using complex staining methods, cytological features of microorganism cells (cellular structures, inclusions, etc.) are revealed.

Gram stain is the most versatile of the complex staining methods. Coloring is the basis for bacterial differentiation and reflects the ability of cells to perceive and retain the coloring complex of gentian violet and iodine inside the cell, or lose it after treatment with alcohol. Accordingly, gram-positive ( bacillus, Clostridium, Staphylococcus, Streptococcus, Lactobacillus, Sarcina, etc.) and gram negative (Escherichia, Pseudomonas, Erwinia, Neisseria, Rickettsia, etc.) forms.

Gram-staining smears must be prepared from young, actively growing (usually one-day-old) cultures to obtain reliable results, as cells from old cultures sometimes give an unstable Gram reaction. Gram-negative bacteria may appear as Gram-positive if the bacterial film (smear) is too thick and alcohol discoloration is not complete. Gram-positive bacteria may appear as Gram-negative if the smear is discolored with alcohol.

Method Essence . On the surface of the cell cytoplasm in gram-positive microorganisms there is a complex of protein and magnesium ribonucleate, which is absent in gram-negative bacteria. Gram-staining on the surface of gram-positive cells forms a strong complex of protein, magnesium ribonucleate, gentian violet and iodine, which is not destroyed by alcohol treatment. Such bacteria remain blue-stained. purple. Gram-negative bacteria do not have the ability to retain paint and become discolored when treated with alcohol. To identify them, the preparation is stained with fuchsin. Gram-negative bacteria stain red.

Stages of differentiated Gram staining:

A fixed smear is covered with a piece of filter paper (1.5 x 1.5 cm) and a carbolic solution of gentian violet or crystal violet is applied to it until completely wetted (the Sinev modification is more often used). Staining is carried out for 1-2 minutes.

The paper is removed, the dye is drained and, without washing the preparation with water, Lugol's solution is applied to the preparation for 1-2 minutes until the smear turns black.

The Lugol solution is drained, the stained smear is decolorized with 96º ethyl alcohol. To do this, the drug is placed 2–3 times in a glass with alcohol until the purple streams stop coming off (discoloration time is no more than 30 s), or 2–4 drops of alcohol are poured onto the smear for 30–45 s.

The drug is quickly washed with water (as described above).

The smear is additionally stained for 1-2 minutes with an aqueous solution of fuchsin (Pfeiffer's fuchsin).

The dye is drained, the preparation is washed with water, dried with filter paper.

microscoped with an immersion system.

Gram-positive bacteria stain purple, Gram-negative bacteria stain pink.

Nucleoid. It is difficult to detect a nucleoid in a bacterial cell using a light microscope. For selective nucleoid staining, fixed cells are pre-treated with ribonuclease or dilute hydrochloric acid to degrade ribosomal RNA. Subsequent staining with the main dye makes it possible to reveal the nucleoid in the form of dense bodies with irregular outlines located in the center or at the poles of the cell.

Ziehl-Neelsen staining of acid-fast bacteria reflects the characteristics of mycobacteria and nocardia.

The principle of the method. The acid resistance of these bacteria is associated with high content lipids in the cell wall. Acid-resistant bacteria are stained with Ziehl carbolic fuchsin when the preparation is heated red and retain this color after bleaching with sulfuric acid. Non-acid-fast bacteria are decolorized with acid and subsequently stained blue with methylene blue.

For detection of capsules using a variety of methods, including Burri method and Burri Guinsa . The method is based on negative contrast. Since the capsule or body of the microbial cell does not perceive dyes, only the background of the micropreparation is stained with ink. The capsule is visible as an unstained area against the dark background of the ink smear. According to the Burri-Gins method, the bodies of microbial cells are additionally stained red with fuchsin.

For flagellum staining Several methods have been proposed, the common step for which is the etching of the preparation (usually with solutions of tannin, KAl(SO 4) 2 , HgCl 2) and subsequent coloring (often with a carbolic solution of fuchsin). As a result of this, the dye is deposited on the flagella, due to which both an increase in their thickness and a decrease in transparency are simultaneously achieved. One of the proposed methods for staining flagella is Leifson's method. The preparation is microscoped with an immersion system. Bacterial cells turn red, flagella take the form of thick filaments extending from the cell.

Questions for self-control

1 List the main morphological cell types of microorganisms.

2 Describe the schematic structure of a bacterial cell.

3 List the stages of preparation of fixed preparations of microorganisms, describe them.

5 Classify the dyes used in microbiology, characterize them.

Practice 4

Target: familiarization with the methods of preparation of fixed preparations, simple and complex staining methods; familiarization with the morphology and cytology of various microorganisms.

Materials and equipment: biological microscopes, microscope slides and coverslips, spirit lamps, bacterial loops, immersion oil, sterile pipettes, tweezers, filter paper, matches, hygienic cotton swabs, markers, sterile Petri dishes, per group - in a 50 ml flask sterile tap water, distilled water for washes, washers, cuvettes, bridges, a set of ready-made paint solutions in a stand, alcohol 96 0, pipettes, infusions from natural materials: meat, peas, etc.

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Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-2.jpg" alt="(!LANG:> Safety in the microbiology room (laboratory) Rules"> Техника безопасности в кабинете (лаборатории) микробиологии Правила работы со спиртовкой ØЗаправленную спиртовку хранят плотно закрытой притертым колпачком, чтобы предотвратить испарение спирта с фитиля. ØПри зажигании спиртовки нужно снять колпачок и поджечь фитиль спичкой. При этом нельзя сдвигать держатель фитиля, т. к. пламя проскочит внутрь спиртовки, что вызовет сильную вспышку и разбрызгивание горящего спирта. ØНельзя зажигать спиртовку от другой уже горящей спиртовки, т. к. при этом может сдвинуться держатель фитиля и вылиться спирт. ØНельзя дуть на пламя, чтобы погасить спиртовку. Для этого следует аккуратно закрыть спиртовку колпачком. ØНеобходимо следить за тем, чтобы спиртовка не перегревалась. Это ведет к испарению спирта и опасности взрыва. ØПри длительном нагревании лучше пользоваться двумя спиртовками. ØНе следует при работе наклонять голову над спиртовкой, т. к. при накоплении паров спирта под держателем фитиля может происходить самопроизвольное вспыхивание.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-3.jpg" alt="(!LANG:> Rules for working with live culture. 1. The main thing when sowing live culture - this is"> Правила работы с живой культурой. 1. Главное при посеве живой культуры – это оградить посев от посторонних микробов и распространение культуры микроорганизма с питательной среды или материала во внешнюю среду. 2. Работать следует быстро, но без резких движений, усиливающих колебания воздуха. 3. Во время посевов нельзя разговаривать, перемещаться по лаборатории. 4. Вся посуда и оборудование, подвергшиеся обсеменению во время работы с культурой, обрабатываются на месте путем фломбирования в пламени спиртовки или сброса в емкость с дез. средством. 5. По окончанию посевов рабочие поверхности столов подвергаются дезинфекции, обрабатываются руки. 6. Лабораторная посуда с посевами помещается в термостат: пробирки в штативе, а чашки Петри переворачивают дном вверх. Использование материалов и средств личной гигиены, раздражающих кожу рук, запрещается.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-4.jpg" alt="(!LANG:> Rules for the preparation and storage of glasses (subject, covers). Subject and coverslips"> Правила приготовления и хранения стекол (предметных, покровных). Предметные и покровные стекла должны иметь абсолютно чистую поверхность и быть хорошо обезжиренными. Капля воды, нанесенная на обезжиренное стекло, растекается равномерно, а на плохо обезжиренном стекле распадается на мелкие капельки. Предметные и покровные стекла рекомендуется мыть и ополаскивать в резиновых перчатках, чтобы не загрязнять их жиром, находящимся на поверхности кожи. Стекла предметные и покровные, не бывшие в употреблении, моют в теплой мыльной воде и после споласкивания заливают смесью Никифорова.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-5.jpg" alt="(!LANG:> save"> Если стекла, вынутые через 2- 3 дня из смеси Никифорова, сохраняют следы жира, их складывают в фарфоровую чашечку, заливают 2- 5 % раствором гидрокарбоната натрия или едкого натра, ставят на слабый огонь и кипятят, считая от момента закипания воды, в течение 20- 30 мин. После кипячения в !} alkaline solution glasses are rinsed with running tap water, dipped for 10-15 minutes in a 5-10% solution of hydrochloric acid and then washed with running water. Used slides and coverslips contaminated with paint and immersion oil are treated in the following way: they are immersed for 2 hours in concentrated sulfuric acid or chromium mixture, then thoroughly washed with running tap water; pour 5% solution of sodium bicarbonate or alkali (caustic potash or caustic soda), put on low heat and boil for 30-40

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-6.jpg" alt="(!LANG:> STEPS OF PREPARATION. 1. Preparation of the smear 2. Drying 3."> ЭТАПЫ ПРИГОТОВЛЕНИЯ ПРЕПАРАТА. 1. Приготовление мазка 2. Высушивание 3. Фиксация 4. Окрашивание!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-7.jpg" alt="(!LANG:> Recipes for making dyes Carbolic"> Рецепты приготовления красителей Карболовый фуксин Циля: - насыщенного спиртового раствора основного фуксина – 10 мл - раствора карболовой кислоты 5% - 90 мл Внимание! Карболовую кислоту вливают в краситель, а не наоборот. Смесь встряхивают, фильтруют и сливают во флакон! Или - основной фуксин в порошке - 1 гр - карболовая кислота кристаллическая -5 гр - глицерин -0, 5 мл - спирт 96% - 10 мл - вода дистиллированная - 100 мл 2. Насыщенные спиртовые растворы (исходные): - красителя - 1 гр - спирта 96% - 10 мл Смесь помещают в термостат на несколько дней до полного растворения. Взбалтывают ежедневно, хранят с притертыми пробками. Фуксин Пфейффера: - фуксин Циля – 1 мл - воды дистиллированной – 9 мл Готовят непосредственно перед употреблением, т. к. раствор нестойкий. Бумага по Синеву: - 1%спиртовой раствор кристаллического фиолетового: на 100 мл 96% спирта добавляют 1 гр сухого красителя и 3 мл глицерина Полоски фильтровальной бумаги пропитывают раствором и высушивают.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-8.jpg" alt="(!LANG:>"> АЛГОРИТМ ПРИГОТОВЛЕНИЯ МАЗКА ИЗ КУЛЬТУРЫ, ВЫРАЩЕННОЙ НА СКОШЕННОМ АГАРЕ(«КОСЯЧКЕ») Предметное стекло аккуратно берут за ребро и с обратной стороны нанесения мазка наносят его границы и номер культуры. Зажигают спиртовку, горелку, предварительно выпустив пары спирта. Фламбируют поверхность стекла, где будет располагаться мазок. Стекло помещают вблизи спиртовки на чашку Петри В !} left hand take a test tube with a sterile physical solution (distilled water) and place it between fingers 1 and 2 in such a way that the palm is under the test tube and the neck of the test tube is directed to the alcohol lamp zone. Take a bacterial loop on the right, like a pencil. The loop is flambéed to redness, first horizontally, then vertically. releasing loops, little finger right hand press the cork physical. solution to the palm of your hand and carefully remove it from the test tube. The movements should be smooth. The throat of the test tube is burned in a flame and a loop is inserted. They are taken with a physical outlet loop. solution and put a couple of drops on the glass within the boundaries of the smear Close the cork, having previously passed the cork and throat of the test tube through the flame of the spirit lamp at the same time Place the test tube in a tripod

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-9.jpg" alt="(!LANG:> Take the culture tube in the left hand and place it like the tube"> В левую руку берут пробирку с культурой и помещают, как и пробирку с физ. р-ром петля в правой руке Петлю фламбируют Над пламенем спиртовки спокойно вынимают пробку с культуры одновременно обжигая пробку и горло пробирки Вводят в пробирку петлю, охлаждая ее о стенки пробирки Осторожно закрывают и захватывают петлей культуру, не повреждая агара, и вынимают петлю, не касаясь ею стенок пробирки Петлю с культурой вносят в каплю физ. р-ра и осторожно эмульгируют, незаходя за границы мазка Бактериальную петлю с остатками культуры прожигают до покраснения в пламени спиртовки Прожигают горло пробирки пробку в пламени, одновременно. Закрывают пробирку. Пробирку с культурой и петлю ставят в штатив Приготовленный мазок высушивают либо на воздухе либо высоко над пламенем спиртовки!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-10.jpg" alt="(!LANG:>"> Примечание: 1. Приготовление мазка из культуры, выращенной на жидкой питательной среде, не требует использования физ. р-ра. 2. При приготовлении мазка из культуры, выросшей на чашке Петри необходимо: отметить изолированную колонию со стороны дна чашки чашку берут в левую руку и в сторону спиртовки приоткрывают крышку, придерживая ее 1 и 2 пальцами левой руки прокаленную петлю вводят под крышку чашки, остужая ее о крышку осторожно откалывают часть выделенной колонии и, не задевая края чашки, выносят на стекло с физ. р-ром. При подсушивании мазка следует помнить: высокая температура может нарушить структуру клетки!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-11.jpg" alt="(!LANG:>Smear Preparation Scheme">!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-12.jpg" alt="(!LANG:> Types of fixation q Physical - in the flame of an alcohol lamp. q Chemical"> Виды фиксации q Физический – в пламени спиртовки. q Химический – в растворах спирта, ацетона, смеси Никифорова (1: 1 спирт и эфир) Цели фиксации Обеззараживание патогенных микробов Закрепление клеток на стекле Убитые микроорганизмы лучше воспринимают красители!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-13.jpg" alt="(!LANG:> Staining methods: Simple Complex (Gram, Ziehl-Nielsen, Orzeshko , Buri-Gins).">!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-14.jpg" alt="(!LANG:> SIMPLE METHODS FOR COLORING SMEARS Aniline dyes (basic, sour"> ПРОСТЫЕ МЕТОДЫ ОКРАСКИ МАЗКОВ Для окрашивания микробов используют анилиновые красители (основные, кислые и нейтральные). Микроорганизмы лучше окрашиваются основными красками. Для окраски препаратов готовят спиртовые, водно-спиртовые и водные растворы. В некоторых случаях добавляют в качестве протравы карболовую кислоту, щелочь и др. Наиболее часто употребляемыми красителями являются следующие: Синие - метиловый синий, водный синий, опаловый синий; красные - фуксин основной, конго красный, сафранин, нейтральный красный, фуксин кислый; фиолетовые - метиловый фиолетовый, кристаллический фиолетовый, генцианвиолет; зеленые - малахитовый зеленый, бриллиантовый зеленый, светло-зеленый; желто-коричневые - хризоилин, везувин.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-15.jpg" alt="(!LANG:> With a simple staining method, a few drops of any alcohol or water"> При простом методе окраски на фиксированный мазок наливают несколько капель какого-либо спиртоводного или водного раствора красок на 1- 2 минуты; чаще всего для этой цели применяется фуксин 1: 10 или леффлеровская метиленовая синька. Затем краску смывают дистиллированной водой и мазок обсушивают между двумя полосками фильтровальной бумаги. Обычно Фуксином I: 10 красят 10- 30 секунд, а метиленовой синькой – 2 -10 мин. Фуксин окрашивает мазки более интенсивно. А при окраске метиленовой синькой получаются нежные, более изящные препараты. Мазок после обсушивания фильтровальной бумагой должен быть совершенно сухим, в противном случае при соприкосновении оставшейся влаги с кедровым маслом образуется эмульсия и при микроскопии получится неясное изображение. Вообще же продолжительность окраски зависит от вида и качества красящего раствора, степени восприимчивости микроба к окраске и толщины мазка.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-16.jpg" alt="(!LANG:>With simple coloring, microbial bodies perceive the color of the applied paint as intensely as like"> При простой окраске микробные тела воспринимают цвет применяемой краски так же интенсивно, как и ядра клеток; в то же время цитоплазма и весь фон мазка (если это не мазок из !} pure culture) are painted in the same color, but somewhat paler. Magenta and gentian violet are more intensely coloring paints; methylene blue stains much paler. The perception of color depends not only on the properties of the colors, but also on the properties of the microbes being dyed. Most microbes are easily and quickly stained with water or alcohol-water solutions of paints. To increase the coloring ability, the paint is exposed to high temperature (heating) until vapors appear (up to boiling). By varying the degree of heating, it is possible to obtain different degrees of color strength. Extending the exposure time of the coloring solution to the object can also enhance the degree of coloring to a certain extent.

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-17.jpg" alt="(!LANG:> COMPLEX Staining Methods Based on the features of the physicochemical structure of the microbial cell. The essence of these methods"> СЛОЖНЫЕ МЕТОДЫ ОКРАШИВАНИЯ Основаны на особенностях физико-химического строения микробной клетки. Сущность этих методов заключается в воздействии на мазок двух красящих веществ, из которых одно является основной, главной краской, а другое дополнительной - контраст. После воздействия первой краской мазок обесцвечивают и только после этого подвергают дополнительной окраске. В качестве обесцвечивающих веществ может быть применен целый ряд химических реагентов (кислоты, щелочи, спирт, ацетон и др.). Обмывание мазка простой водой также является в какой-то степени чисто механическим процессом обесцвечивания, но для этого необходимо продолжительное время; кроме того, обесцвечивание получается неполное. По отношению к обесцвечивающим веществам можно разбить микробов на группы легко и трудно обесцвечиваемых. Не все виды микробов одинаково относятся к обесцвечивающим реагентам; некоторые являются кислото - и спиртоустойчивыми, другие только кислотоустойчивы. Наконец, некоторые, как, например, споры, энергично противостоящие воздействию всех обесцвечивающих веществ, относятся к группе краскоустойчивых.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-18.jpg" alt="(!LANG:> GRAM METHOD v Apply gentian violet on a fixed swab"> СПОСОБ ГРАМА v На фиксированный мазок нанести генцианвиолет (бумага по Синеву) на 1 -2 мин. v Краску слить и нанести раствор Люголя на 1 мин. v Раствор Люголя слить и на мазок нанести 96% спирт на 15 -20 мин в зависимости от толщины мазка. v Спирт смыть дистиллированной водой. v Мазок дополнительно окрасить разведенным фуксином Пфейффера на 2 -3 мин. v Краситель смыть водой, препарат высушить, промикроскопировать с иммерсией Микроскопия с иммерсией. Генцианвиолет связывается с пептидогликаном клеточной стенки Толстый слой пептидогликана грамположительных бактерий связывает много красителя, тонкий слой – грамотрицательных – мало. Раствор Люголя фиксирует краситель за счет образования комплекса – краситель-пептидогликан-йод. При обработке мазка спиртом грамотрицательные микроорганизмы быстро теряют краситель и обесцвечиваются, а грамположительные остаются окрашенными в синий цвет. Дополнительный краситель окрашивает грамотрицательные микроорганизмы в красный цвет Грамположительные бактерии окрашиваются в сине-фиолетовый цвет. Грамотрицательные бактерии окрашиваются в красный цвет.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-19.jpg" alt="(!LANG:>The ratio of bacteria to Gram stain is determined by their ability to retain formed in"> отношение бактерий к окраске по Граму определяется их способностью удерживать образовавшийся в процессе окраски комплекс генцианового фиолетового и йода. У грамположительных бактерий основным веществом клеточной стенки (до 90%) являются мукопептиды-муреин (пептидогликан). У грамотрицательных бактерий однослойный муреин располагается в глубине клеточной стенки, значительно больше содержится белков и липидов, которые вместе с полисахаридами образуют поверхностные слои в виде мозаики, их цитоплазма содержит РНК и ДНК в соотношении 8: 1 и 1: 1 соответственно. Кроме того, проницаемость клеточной стенки у грамположительных бактерий меньше, чем у грамотрицательных. Это объясняется большим содержанием мукопептида в составе клеточной стенки грамположительных бактерий и меньшим диаметром пор, что способствует удержанию образовавшегося комплекса при обработке бактерий этиловым спиртом.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-20.jpg" alt="(!LANG:> GRAM-NEGATIVE AND GRAM-POSITIVE"> ГРАМОТРИЦАТЕЛЬНЫЕ И ГРАМПОЛОЖИТЕЛЬНЫЕ БАКТЕРИИ Грамположительные микроорганизмы Грамотрицательные микроорганизмы!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-21.jpg" alt="(!LANG:>Streptococci Gr+ cocci arranged in chains Streptococcus piogenes Enterococcus faecalis Staphylococcus Gr+"> Стрептококки Гр+ кокки, располагающиеся в виде цепочек Streptococcus piogenes Enterococcus faecalis Стафилококки Гр+ кокки, располагающиеся в виде скоплений, напоминающих виноградную гроздь Staphylococcus aureus Staphylococcus epidermidis Бактерии Гр- палочковидные неспорообразующиие микроорганизмы Esherichia coli Salmonella typhi Mycobacterium tuberculosis!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-22.jpg" alt="(!LANG:> Vibrio Gr- Vibrio cholerae"> Вибрионы Гр- Vibrio cholerae Vibrio parahaemolyticus Клостридии Гр+ ппалочковидные спорообразующие микроорганизмы. Диаметр спор больше поперечника клетки Clostridium perfringens Clostridium tetani Clostridium botulinum!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-23.jpg" alt="(!LANG:> DIAGNOSTICS OF LUNG TUBERCULOSIS BY BACTERIOSCOPIC METHOD"> ДИАГНОСТИКА ТУБЕРКУЛЕЗА ЛЕГКИХ БАКТЕРИОСКОПИЧЕСКИМ МЕТОДОМ ОСНОВАНИЕ: МЕТОДИЧЕСКИЕ РЕКОМЕНДАЦИИ МЕЖДУНАРОДНОГО СОЮЗА БОРЬБЫ С ТУБЕРКУЛЕЗОМ. ФРАНЦИЯ, ПАРИЖ, 1999 г Приготовление мазков Бактерии обнаруживаются в плотных гнойных частицах мокроты. Захватите петлей материал и распределите тонким слоем на 2/3 части предметного стекла. Можно стекло с кусочком мокроты покрыть другим предметным стеклом, придавить друг к другу и раздавить в противоположном направлении до образования тонкого мазка. Можно для приготовления мазка пользоваться деревянными палочками. ВЫСУШИВАНИЕ Приготовленные мазки высушиваются на воздухе 15 -30 мин ФИКСАЦИЯ Медленно провести мазок в течение 3 -5 мин 3 раза через верхнюю или среднюю наиболее яркую часть пламени спиртовки.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-24.jpg" alt="(!LANG:>COLORING a) Cover the smear with filter paper and apply to the entire surface of the paper carbolic fuchsin"> ОКРАШИВАНИЕ а) мазок накрыть фильтровальной бумагой и на всю поверхность бумаги нанести карболовый фуксин Циля б) медленно нагревайте стекло с мазком над пламенем спиртовки до появления паров, не допуская кипения и высушивания. Оставить мазок с прогретым раствором на 5 мин ОБЕСЦВЕЧИВАНИЕ а) снять фильтровальную бумагу и удалить окрашивающий раствор под слабой струей воды б) обработать каждый мазок индивидуально 25% раствором серной кислоты в течение 3 -х минут в) смыть водой г) обработать каждое стекло в течение 5 минут 96% спиртом д) смыть водой ДОПОЛНИТЕЛЬНАЯ ОКРАСКА Обесцвеченные и промытые стекла с мазками обработать 0, 3% раствором метиленовой синьки в течение 1 минуты Промыть водой и высушить на воздухе.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-25.jpg" alt="(!LANG:> ZIEHL-NIELSEN METHOD (INSTRUCTION 1999)"> МЕТОД ЦИЛЯ-НИЛЬСЕНА (ИНСТРУКЦИЯ 1999 ГОД) На фиксированный препарат накладывают фильтровальную бумагу, на которую наносят карболовый фуксин Циля. Подогревают до отхождения паров, процедуру можно повторить. Выдерживают 5 мин 1. Н 2 О 2. 25% Н 2 SО 4 - 3 мин 3. Н 2 О 4. Спирт 96% - 5 мин 5. Н 2 О 6. Метиленовый синий 0, 3 % - 1 мин 7. Н 2 О Кислотоустойчивая микобактерия!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-26.jpg" alt="(!LANG:> ZIEHL-NIELSEN METHOD"> МЕТОД ЦИЛЯ-НИЛЬСЕНА 1. На фиксированный препарат + фильтровальная бумага + фуксин Циля. Подогреваем до отхождения паров + краситель (2 раза) 2. Н 2 О 3. 5% Н 2 SО 4 (погружая 2 -3 раза) 4. Н 2 О 5. Метиленовый синий 1 % - 3 -5 мин 6. Н 2 О 7. Микроскопия с иммерсией. Кислотоустойчивые бактерии окрашиваются в красный цвет, остальные – в синий (рис. № 4, 5). Mycobacterium tuberculosis в мокроте!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-27.jpg" alt="(!LANG:> DETECTION OF CAPSULES IN BACTERIA Some microbes"> ОБНАРУЖЕНИЕ КАПСУЛ У БАКТЕРИЙ Некоторые микробы обладают способностью откладывать на поверхности своего тела мощный слизистый слой вокруг клеточной стенки. Его называют капсулой. В состав капсул входят, главным образом, полисахариды (пневмококк), но у некоторых они содержат и полипептиды (палочка сибирской язвы). Капсулы имеют консистенцию геля, поэтому при микроскопии живых бактерий они видны очень плохо. Для обнаружения применяют негативную окраску. При этом краситель заполняет пространство вокруг бактерий, в результате чего они выглядят светлыми частицами на темном фоне. ОБНАРУЖЕНИЕ КАПСУЛ ПО МЕТОДУ БУРРИ Смешать на предметном стекле немного культуры и каплю туши, разведенной 1: 10. Ребром шлифовального стекла сделать тонкий мазок, также как мазок крови: каплю туши наносят на предметное стекло на расстояние одной трети от левого края. В эту каплю бак. петлей вносят культуру. Затем краем специально отшлифованного стекла, наклонив его под углом 45 О, прикасаются к капле туши с культурой. Прижимая отшлифованное стекло к предметному продвигают его вперед. Мазок заканчивается «метелочкой» Сбросить шлифовальное стекло в дез. Средство. Высушить на воздухе Бактериоскопировать.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-28.jpg" alt="(!LANG:>"> ОБНАРУЖЕНИЕ КАПСУЛ ПО МЕТОДУ БУРРИ-ГИНСА Приготовить мазок на предметном стекле по методу Бурри Высушить на воздухе Фиксировать химическим способом: погружение стекла в емкость со спиртом или сулемой – 10 мин, или со смесью Никифорова (спирт и эфир 1: 1) – 15 -20 мин Осторожно промыть водой На мазок нанести фуксин Пфейффера – 3 -5 мин Промыть Н 2 О Высушить на воздухе Микроскопия с иммерсией. Внимание! Фильтровальной бумагой не пользоваться, чтобы не повредить препарат. Препарат по Бурри – Гинсу под микроскопом: фон черный, клетки бактерий красные, капсулы неокрашенные (красители не воспринимают). Окраска по Бурри и Бурри-Гинсу!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-29.jpg" alt="(!LANG:>BURRI AND BURRI-GINS Stain Capsule in Klebsiella pneumoniae">!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-30.jpg" alt="(!LANG:>"> МЕТОД ОЖЕШКО сходен с методом Циля-Нильсена, но отличается использованием раствора соляной кислоты в качестве протравы, разрыхляющей оболочку споры, которая плохо воспринимает красители. Кислотоустойчивые споры окрашиваются в розово-красный, а бактериальная клетка в голубой цвет. Рис. № 9. Споры у Bacillus subtilis!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-31.jpg" alt="(!LANG:> OZZHZKO STAIN On dried smear (unfixed!) + several"> ОКРАСКА ПО МЕТОДУ ОЖЕШКО На высушенный мазок (нефиксированный!) + несколько капель 0, 5% НCl, держать над пламенем спиртовки до образования паров Высушивают и фиксируют физическим способом Окрашивают по методу Циля-Нильсена: На фиксированный препарат + фильтровальная бумага + фуксин Циля. Подогреваем до отхождения паров + краситель (2 раза) Н 2 О 5% Н 2 SО 4 (погружая 2 -3 раза) Н 2 О Метиленовый синий 1 % - 3 -5 мин Н 2 О Микроскопия с иммерсией!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-32.jpg" alt="(!LANG:> Study of microorganism motility."> Изучение подвижности микроорганизмов. Подвижности бактерий важный видовой признак и производиться при диагностических исследованиях: результат учитывают при идентификации микроорганизмов. У подвижных видов способность самостоятельного поступательного (и вращательного) движения обусловлена наличием жгутиков -специальных тонких нитевидных образований.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-33.jpg" alt="(!LANG:> CRUSHED DROP METHOD A drop is applied to a glass slide with a pipette or loop"> МЕТОД РАЗДАВЛЕННОЙ КАПЛИ На предметное стекло наносят пипеткой или петлей каплю культуры и покрывают ее покровным стеклом. !Чтобы не образовалось пузырьков воздуха, покровное стекло подводят ребром к краю капли и резко опускают его. Для предохранения от высыхания препарат помещают во влажную камеру Микроскопируют в темном поле при увеличении объектива 40 Х Препарат можно поместить во влажную камеру для предохранения от высыхания!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-34.jpg" alt="(!LANG:> Flagella come in various lengths. Their diameter is so small that they are invisible in"> Жгутики бывают различной длины. Их диаметр настолько мал, что они невидимы в световом микроскопе (менее 0, 2 мкм). У разных групп бактерий количество и расположение жгутиков неодинаково. Жгутики плохо воспринимают красители. Методы сложной окраски искажают подлинный вид жгутиков, поэтому в лабораториях окраску жгутиков не осуществляют, а исследуют бактерии в живом состоянии. В зависимости от расположения и количества жгутиков микробы подразделяют: а) монотрихи - микроорганизмы, имеющие на одном из полюсов один жгутик, движения активные, поступательные (псевдомонас); б) лофотрихи - микробы, имеющие на одном из полюсов пучок жгутиков (листерии); в) амфитрихи - микробы, имеющие жгутики на обоих полюсах микробной клетки; г) перитрихи - микробы, у которых жгутики расположены по всей поверхности клетки(E. coli). Есть виды микроорганизмов, обладающие подвижностью, но жгутиков не имеют (спирохеты, лептоспиры). Их движение обусловлено импульсивными сокращениями двигательного фибриллярного аппарата микробной клетки.!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-35.jpg" alt="(!LANG:>Determination of bacterial motility by the "hanging drop" method. Young drop (18 -20 hours) broth"> Определение подвижности бактерий методом «висячая капля» . Каплю молодой (18 -20 часовой) бульонной культуры бактерий бактериологической петлей наносят на покровное стекло. Специальным предметным стеклом с углублением (луночкой) накрывают каплю культуры так, чтобы покровное стекло с каплей находилось в центре луночки и прилипло к предметному стеклу (края луночки предварительно слегка смазывают вазелином). Препарат перевертывают стеклом вверх, и капля «повисает» над луночкой. Препарат микроскопируют при затемненном поле зрения, сначала при малом, затем при среднем или большом увеличении. На светлом фоне микробы темно-серые!}

Src="http://present5.com/presentation/3/55298978_132451894.pdf-img/55298978_132451894.pdf-36.jpg" alt="(!LANG:> HANGING DROP METHOD"> МЕТОД ВИСЯЧЕЙ КАПЛИ Для приготовления препарата необходимы стекло с лункой, покровное стекло, вазелин. Края лунки покрывают тонким слоем вазелина. 1. На покровное стекло наносят каплю культуры и осторожно накрывают покровное стеклом с лункой так, чтобы капля оказалась в центре. 2. Склеившиеся стекла быстро переворачивают покровным стеклом вверх. Капля находится в герметической камере и сохраняется долгое время. Микроскопия сначала при увеличении 8 Х. Находят край капли, а затем переводят на большое увеличение 40 Х. Техника приготовления препарата висячая капля!}

We live in a world of bacteria. They are around us and inside our body. No wonder we want to know as much as possible about them. But how to consider and even more so to study something so small? The microscope, it would seem, should solve this problem. But not everything is so simple - in their natural state, microorganisms are transparent as glass. Various methods of staining bacteria help to “show” the picture, helping to explore the external and internal structure of microbes.

At the end of the nineteenth century, the Danish biologist Christian Gram proposed a solution to this problem. If something is invisible, it needs to be painted and see what happens. The method of staining bacteria proposed by Gram was so convincing that to this day microorganisms are divided into gram-positive (retaining color) and gram-negative (discoloring after treatment with alcohol).

As it turned out, the cells are covered with a membrane similar to intertwined threads. And although the nutrients necessary for the cell freely pass into the gaps between the threads, the shell reliably protects the "inner world" from external aggressive factors. In different types of bacteria, the outer wall of the cell has a different thickness, density and chemical composition. It is on this property of cell membranes that the Gram stain method is based.

Based on the work of Christian Gram, biologists have developed new methods that allow not only to determine the shape and size of cells, but also to consider some of the details of their structure. Sometimes microorganisms that are completely identical in appearance react differently to dyes. The information obtained makes it possible to quickly and accurately determine the type of bacteria.

Old doesn't mean bad

Perhaps the most practical and widely used method for staining microorganisms is the Gram method. The smear, fixed by fire, is covered with methyl violet dye, fixed with iodine, dried and washed with alcohol. At this stage, depending on the properties of the cell membrane, bacteria become:

• bright blue (gram-positive);
• colorless (gram negative).

The thickness of the shell of cells that remain colorless does not allow the dye to penetrate inside, and it is easily washed off from the surface of the bacteria. The final step in Gram staining is the use of a red dye, which remains on the surface of the cell membrane and gives it a pink or red tint.

Gram-positive bacteria are generally dangerous to humans. This category includes streptococci, staphylococci, bacilli, clostridia, etc. Gram-negative ones are not so dangerous. They can also cause disease and inflammation, but only under certain conditions. Thus, simply by preparing a colored preparation, one can get an idea of ​​the degree of danger of the studied microorganisms.

Vital staining methods

According to the state of the studied organisms in microbiology, there are:

• vital method, i.e. working with live bacteria (dangerous, requires strict adherence to safety precautions);
• post-vital method – work with fixed (killed) cells;
• negative, can be vital and post-vital, convenient for working with capsules.

The vital method is by far the most dangerous, as researchers have to deal with living cells, sometimes deadly. However, it is this method that makes it possible to study not only the structure of the cell, but also its entire life cycle and interaction with the environment.

For many microbiological studies, it is important to keep bacteria alive. This means that it is necessary to use special non-toxic or low-toxic dyes, which, moreover, easily penetrate into the cell structure through the outer shell. These are the so-called vital dyes, they can be intended for visible light or fluorescent. By chemical composition, dyes are divided into:

• basic (acridine orange, methylene blue);
• acid or sour (indigo carmine, sour fuchsin);
• neutral (rhodamine B).

Working with fixed drugs

According to the complexity of the work, methods for staining fixed (non-living) cells are divided into:

1. Simple methods. In this case, only one paint is used, usually red (magenta) or blue (methylene blue). The difference between these dyes is the speed of exposure. Fuchsin gives the result after 1-2 minutes, while the result of the blue paint must be waited for 3-5 minutes. It is also convenient to use a solution of fuchsin in carbolic acid (the so-called Ziel's fuchsin) because the prepared preparation does not lose its coloring properties for several months. Methylene blue dye can also be prepared in advance in a strong alcohol solution.
2. Complex (differential) methods. This will require several dyes (at least two), having different color. This will allow not only to see the bacteria, but also to study their internal structure in more detail, since different parts of the cell can perceive dyes differently. The complex ones include the Gram, Ziehl-Nielsen methods (for acid-fast bacteria), Benignetti (staining of bacterial flagella), Gins (identification of capsules), the differentiating method of Romanovsky-Giemsa (spore staining) and some others.

Complex methods are especially important for diagnosing infectious diseases, for example, the Neisser stain method helps to distinguish diphtheria bacillus from false diphtheria.

The differentiating method of Romanovsky - Giemsa is based on the use of a special ready-made powder, on the basis of which laboratories prepare a dye solution of the desired concentration. The advantage of this method is that the cytoplasm and the nucleus of the cell receive a different color, which facilitates the identification and study of microorganisms.

The Neisser method is used in medicine to detect volutin grains (granules with a supply of food for many prokaryotic cells) in diphtheria pathogens. As a result of staining, the bacterium acquires yellow, and volutin granules are blue.

White on black

Another type of bacteria staining is based on the properties of the negative, i.e., colorless bacteria are clearly visible against the dark background of the preparation, in other words, the environment is stained, and not the organism itself.

Sometimes bacteria, getting into certain conditions, form capsules. These are mucous formations that cover the cell, somewhat similar to a gel. Capsules are transparent, their chemical composition can vary greatly in different types of bacteria, i.e. just paint and evaluate the result by the resulting color will not work.

In addition, the capsules are soft and fragile, they can lose their shape when painted. Coloring agents do not interact well with the jelly-like structure of the capsules and are easily washed out during processing. To detect but not damage the capsules, a negative staining method is useful.

One of the methods for detecting capsules is the Guins staining method. A drop of black ink is applied to the edge of a glass slide, the test material is added to it, mixed and distributed over the entire surface. The smear is air-dried, fixed and stained with Ziehl magenta. After 2-3 minutes, rinse with water and dry. As a result, pink cells surrounded by transparent capsules are clearly visible under a microscope against a general dark background.

Coloring of spore-forming bacteria

If we talk about cell membranes, then we cannot help but recall spore-forming bacteria. Spores are formed under unfavorable conditions for the cell and can exist in an aggressive environment for quite a long time. What is good for preserving the cell is bad for studying it. The spores are very dense and almost impermeable to liquids, in addition, they are acid-resistant. Therefore, a simple stain or Gram's method will leave the spores colorless.

Before staining, it is necessary to chemically treat the surface of the spores so that it slightly changes its structure and allows the dyes to penetrate inside. In this case, the entire cytoplasm of the cell is also stained. To discolor the cytoplasm, the preparation is washed and dried. The dye in spores, due to their denser structure, is retained better than in the cytoplasm.

Spore staining methods can be different, for example, Orzeshko or Ziehl-Nielsen, but they all come down to the same scheme:

• loosening of the spore surface chemicals(acids, ammonia, caustic soda);
• staining of a cell with a spore (usually when heated);
• discoloration of the cytoplasm.

As a result, we get a perfectly visible brightly colored spore and a pale, almost transparent cytoplasm.

How to examine bacterial flagella

Another difficult task is the staining of bacteria with flagella. These are spirally twisted very thin threads that microorganisms use to move. The flagella are unusually thin; when stained, they easily detach from the cell. Therefore, before staining, the flagella are etched, artificially increasing in volume.

When preparing microorganisms for the study, the bacterial culture is subcultured several times on a fresh nutrient medium for several days. Then the bacteria with flagella are transferred into a test tube with sterile water (t 37⁰С). Do this very carefully, without mixing the liquid, so as not to damage the flagella.

The contents of the test tube are left for about an hour so that the bacteria are evenly distributed throughout the volume. Before starting the study, cell motility in a hanging drop is checked. If there is no movement, the tube is left for some more time.

When the solution is applied to a glass slide, cells can easily lose their flagella. Therefore, the glass surface must be perfectly clean and grease-free. Before applying drops of the solution, a glass slide is held over the hot part of the burner flame so that the droplets that have fallen on the surface spread and dry quickly.

After drying, the smear is etched, after 15 minutes the chemical is washed off. The next stage - fuchsin staining - is carried out by immersing the glass in a dye solution so as not to damage the flagella when applying paint.

After maintaining the right time, the smear is washed with water and dried. Now you can proceed to the study of the resulting result.

Staining technique

To obtain a colored preparation, one cannot simply take a brush, paints and catch a bacterium. There is a certain scheme for working with microorganisms:

1. Smear preparation. A drop of water is applied to a glass slide (sterile), into which the laboratory material is then introduced with a bacteriological loop and distributed over the surface.
2. Drying. The excess fluid is either dried naturally at room temperature, or the smear is slightly warmed high above the burner flame.
3. Fixation. It is not enough just to remove the water, you need to fix the bacteria on a glass slide. To do this, you can use fire (several passes over the hottest part of the flame) or liquid (alcohol, acetone). After such processing, microorganisms absorb the paint faster.
4. Direct staining. The paint should completely cover the entire surface of the smear. Having withstood the right time (for each dye its own), the paint is removed and the preparation is washed with water.

After drying (usually naturally), the result can be used for its intended purpose.

Staining of microorganisms is a whole system of methods, techniques, schemes aimed at detecting and recognizing cells using a microscope. No medical research or scientific work in microbiology, they cannot do without preliminary preparation and coloring of the material under study.

Staining of microorganisms (dyeing of microbes) is a set of methods and techniques for studying the external and internal structure microorganisms, a method of microbiological technology that allows to distinguish between types of microorganisms. The method is widely used in applied bacteriology to determine the shape, size, structure, localization, relative position of microbes, and the structure of their organelles. Without staining, microbes, except for some fungi, are practically invisible in a light microscope, due to their low contrast. After treatment, the membranes and/or organelles of microbes acquire a color that contrasts with the background.

// General information about the method

Microbial preparations are exposed to chemical reagents, usually dyes, or osmium tetroxide. As a result of the physicochemical process of interaction of the dye with chemical compounds objects, in order to artificially give it a certain color, it becomes possible to determine the type of microorganism, or at least the type of its membrane (see Gram stain).

Dyeing methods are divided into vital, post-vital and negative, the latter can be vital and post-vital.

Vital coloring method

For vital (vital) dyeing, 0.2-0.001% aqueous solutions of methylene and toluoidine blue, neutralrot and Congo red are used, which are added to the pressed or hanging drop of culture. This method reveals spirochetes, protozoa, determines the mobility of bacteria, the immune swelling of the capsule, but its use requires strict adherence to the rules that exclude laboratory infection.

Postvital staining methods

Methods for dyeing fixed preparations (post-vital) are divided into simple and complex. With simple methods, staining solutions of Pfeiffer fuchsin (exposure 1-2 minutes), alkaline methylene blue (3-5 minutes) are applied to a fixed preparation so that it completely covers the smear, the dye is drained, the preparation is washed with a stream of water, shaken, dried and microscoped .
Simple Ways make it possible to judge the size, shape, localization, mutual arrangement of individual cells, but with their help it is impossible to establish the structure of microbes and often their differentiated relationship to dyes.
Of the complex methods of staining bacteria, the differentiated Gram method, the detection of acid resistance according to Ziehl-Nelson, the determination of volutin grains according to Leffler or Neisser, the differentiating method of Romanovsky-Giemsa, the negative-positive method for determining the capsule according to Gins-Burri, the detection of spores according to Peshkov or Ziel - Nelson and others.
For staining protozoa, the Romanovsky-Giemsa method and hematoxylin-eosin staining are used.
Mushrooms are examined unstained or by the methods of Gram, Ziel - Nelson, Leffler, Romanovsky - Giemsa, as well as Lugol's solution, lactofuchsin, etc.

The Gram method is a method of staining microorganisms for research, which allows differentiating bacteria according to the biochemical properties of their cell wall. Proposed in 1884 by the Danish doctor G.K. Gram.

According to Gram, bacteria are stained with the main dyes - gentian or methyl violet, etc., then the dye is fixed with a solution of iodine. Upon subsequent washing of the stained preparation with alcohol, those types of bacteria that turn out to be strongly stained are called gram-positive bacteria - in contrast to gram-negative (Gram (-)), which discolor when washed.

// Use in diagnostics

Gram stain has great importance in the taxonomy of bacteria, as well as for the microbiological diagnosis of infectious diseases.

Gram-positive coccal and spore-bearing forms of bacteria, as well as yeast, they are painted in blue-black (dark blue) color.

Many non-spore-bearing bacteria are gram-negative, they stain red, the cell nuclei become bright red, the cytoplasm becomes pink or crimson.

Staining technique

Gram stain refers to a complex method of staining when a smear is exposed to two dyes, one of which is the main one and the other is an additional one. In addition to dyes, bleaching agents are used for complex methods of coloring: alcohol, acids, etc.

For Gram staining, dyes of the triphenylmethane group are most often used: gentian, methyl violet or crystal violet. Gram-positive Gram (+) microorganisms give a strong connection with the indicated dyes and iodine. At the same time, they do not discolor when exposed to alcohol, as a result of which, with additional staining with fuchsin Gram (+), microorganisms do not change the originally adopted purple color.

Gram-negative Gram (-) microorganisms form a compound easily destroyed by alcohol with basic dyes and iodine. As a result, the microbes become discolored and then stained with magenta, turning red.

Preparation of material for painting

The test material is spread in a thin layer over the surface of a well-defatted glass slide. The prepared smear is dried in air and fixed after complete drying. Histological sections are prepared according to a routine technique, fixing tissue pieces in formalin and embedding in paraffin.

Fixation

When fixing, the smear is fixed on the surface of the glass slide, and therefore, during the subsequent staining of the preparation, microbial cells are not washed off. In addition, killed microbial cells stain better than live ones.

A distinction is made between the physical method of fixation, which is based on the effect of high temperature on the microbial cell, and chemical methods, which involve the use of chemicals that cause coagulation of cytoplasmic proteins.

Physical way of fixing

The glass slide with the preparation is taken with tweezers or I and II fingers of the right hand by the ribs with a stroke up and with a smooth movement 2-3 times over the upper part of the burner flame. The entire fixation process should take no more than 2 s.

The reliability of fixation is checked by the following method: the surface of the glass slide free from the smear is applied to the back surface of the left hand. With proper fixation of the smear, the glass should be hot, but not cause a burn sensation.

Chemical method commits

To fix the smears, methyl alcohol, acetone, a mixture of Nikiforov (a mixture of ethyl alcohol 96% and anesthetic ether in a ratio of 1: 1), Carnoy's liquid (ethyl alcohol 96% - 60%, chloroform 30%, glacial acetic acid 10%), alcohol -formol (40% formalin 5 ml, ethyl alcohol 96 ° - 95 ml). A slide with a dried smear is immersed in a bottle with a fixative for 10-15 minutes and then dried in air. Fixation in 40% formalin vapor for a few seconds is also used.

The staining process

One of the main dyes is poured onto a fixed smear for 2-3 minutes. To avoid precipitation, stain through filter paper.

Drain the paint, carefully remove the filter paper. The smear is filled with Lugol's solution or Gram's iodide solution (an aqueous solution of potassium iodide and crystalline iodine in a ratio of 2: 1) for 1-2 minutes until the preparation turns black.

The solution is drained, the smear is rinsed with 96 ° ethyl alcohol or acetone, pouring and draining it until the smear is discolored and the flowing liquid is clear (approximately 20-40-60 seconds).

Rinse the slides thoroughly in running or distilled water for 1-2 minutes.

To identify the gram-negative group of bacteria, the preparations are additionally stained with fuchsin or safranin (2-5 min).

Rinse in running water and dry with filter paper.

Technique for staining bacteria in histological sections according to Gram-Weigert

Deparaffinized sections are brought to water.

Stained for 20 minutes in a 1% solution of pararosaniline or basic fuchsin in 1% acetic acid(dye solution is heated to boiling, cooled and filtered).

Washed in 3 changes of distilled water.

Stain for 5 min in 1% crystal violet in distilled water.

Rinse quickly in 1% sodium chloride solution.

Treated for 30 seconds in a mixture: 1 part of iodine + 2 parts of potassium iodide + 100 parts of distilled water.

Wet with filter paper.

Differentiate by applying a mixture to the cut equal volumes aniline and xylene (1 - 2 ml); the solutions are drained until the dye clouds stop moving away from the cut.

Pass through 3 xylene shifts

Enclosed in balm or any resin dissolved in xylene.

Result: Gram-positive bacteria are blue-black, fibrin is purple, nuclei are red.

The Peshkov method is used to stain bacterial endospores.

Staining technique

The dried preparation from a culture of gram-positive bacteria is fixed in Carnoy's liquid for 15 minutes, then washed with water, methylene blue is poured according to Leffler and heated until vapors appear, boiled for 15-20 minutes, after cooling the preparation is washed and stained with a 0.5% aqueous solution of neutral red or magenta according to Pfeifer 30-60 seconds. Dry with filter paper and microscope.

Coloring result

As a result, mature endospores are stained blue, young endospores are stained dark blue, the cytoplasm is red, and chromatin grains are stained purple.

The Ziehl-Nelsen staining method is a staining method for microorganisms to detect acid-resistant mycobacteria (causative agents of tuberculosis, mycobacteriosis, leprosy), actinomycetes and other acid-resistant microorganisms. The acid resistance of microorganisms is due to the presence of lipids, wax and hydroxy acids in their cells. Such microorganisms stain poorly with diluted dye solutions. To facilitate the penetration of the dye into the cells of microorganisms, the Tsilya phenolic fuchsin applied to the preparation is heated over a burner flame. Colored microorganisms are not discolored by weak solutions of mineral acids and alcohol.

The method is named after German physicians - microbiologist Franz Ziel (1857-1926) and pathologist Friedrich Nelsen (1854-1898), who developed it in 1882-1883.

Coloring steps

1. A fixed smear is covered with flat filter paper and Ziehl's phenol fuchsin is poured onto it. The smear is heated over a burner flame until vapors appear, then it is taken away for cooling and a new portion of the dye is added. Heating is repeated 2-3 times. After cooling, remove the filter paper and wash the preparation with water.

2. The drug is decolorized by dipping or applying a 5% sulfuric acid solution or 3% hydrochloric alcohol and washed several times with water.

3. Stain the preparations with a water-alcohol solution of methylene blue for 3-5 minutes, rinse with water and dry.

When stained according to the Ziehl-Nelsen method, acid-resistant bacteria become intensely red, the rest of the microflora is stained light blue.

Romanovsky-Giemsa staining is a cytological method for staining protozoa, bacteria, cell structures and tissues of various types (including blood) using light microscopy. It stains acidophilic formations in various shades of red, basophilic formations in colors from purple to blue.

// Preparing the dye

Before staining the smears, the finished liquid dye is diluted at the rate of 1-2 drops of the dye per 1 ml of distilled water. The smears are stained for 20 - 25 minutes at 37°C in a humid chamber (closed Petri dish with a moistened filter at the bottom). After staining, the smears are washed in running water, dried in air and examined by oil immersion.

The coloring mixture of Romanovsky-Giemsa, which is based on the paint of Romanovsky Wright, in the form of a powder (commercial dye) is dissolved in a mixture of equal volumes of methyl alcohol and glycerin (800 mg of dye per 100 ml of solvent). The dye dissolves poorly, so it is better to grind it with a solvent in an amount of 300 mg per 100 ml, and then, stirring, add the dye until the desired concentration is obtained. The preparation of the dye often takes several days. It is important to use chemically pure methyl alcohol and glycerin as solvents, since impurities degrade the properties of the dye. Instead of methyl alcohol, 100% ethyl alcohol can be used. The prepared coloring mixture is stored in a cool dry place in a tightly closed vessel.

staining technique

Smears fixed in methyl alcohol are stained with a solution (1 ml of the finished liquid paint + 2 ml of the basic buffer solution + 47 ml of distilled water) for 40-120 minutes (the duration of staining is selected empirically). They use a phosphate buffer, but the pH of the buffer depends on the type of smear: for a bone marrow smear - 5.8 - 6.0, for a blood smear - 6.4 - 6.5, for the detection of protozoa - 6.8, malarial plasmodium - 7, 0 - 7.2.

Rinse in distilled water, dry and examine by immersion.

Color result

Bacteria stain purple-red, cell cytoplasm blue, nuclei red. When staining protozoa, their cytoplasm becomes blue, and the nuclei become red-violet.