Before examining microorganisms under a microscope, the test sample is subjected to special preparation. In their natural form, almost all bacteria are transparent, so they are stained with dyes. Staining allows you to determine both the morphological properties of the studied microbes, and their belonging to a particular species. The ability of microorganisms to perceive dyes is called tinctorial properties. With their help, biologists determine the type of microbe, its parameters, structural features, and also identify pathogens of infectious diseases. From this article you will learn in more detail that these are tinctorial properties, as well as how they are used in science.
Problems that the staining method solves
First of all, let's find out what is the significance of the tinctorial properties of microorganisms. Sample staining is one of the key techniques in microbiology that allows the study of both the external and internal structure of organisms. Bacteria are translucent microorganisms that refract light rays. Therefore, with ordinary microscopy, they cannot be considered. Different parts of the bacteria interact differently with the dye. Tinctorial properties are a great opportunity for researchers. Being different in different microorganisms, they allow you to determine their most important characteristics: shape, size, structure, localization, etc.
Sample preparation and coloration
Regardless of which staining technique the researcher practices, this procedure consists of a number of mandatory stages:
- Sample preparation. The studied material is introduced into a drop of water placed on a glass slide using a bacteriological loop.
- Dry smear. It is carried out at room temperature, or in a stream of warm air (but not too close to a heat source).
- Fixation. It involves the attachment of microorganisms to glass in order to enhance their susceptibility to staining.
- Coloring. A dye is applied to the test sample and allowed to stand for some time. Then the remaining dye is removed and the sample is washed with water.
- Drying. The final stage, allowing you to get rid of residual water after washing. After drying, the sample is ready for examination under a microscope.
Common dyes
Samples are stained using aniline dyes with different acidity (pH) values. They are divided into: basic (emphasis on the second letter o), acidic and neutral.
In the main dyes, the cation acts as the active agent. Using various reagents, you can get different colors staining microbes:
- Red - pyronin, safranin, neutralrot, fuchsin main.
- Violet - thionine, methyl, gentian and crystal violet.
- Blue is methylene blue and Victoria.
- Green - malachite and methylene greens.
- Black is Hindu.
- Brown - vesuvin and chrysoidine.
In acidic dyes, anion is endowed with coloring properties. Of these, the most common:
- Red - sour fuchsin and eosin.
- Black is nigrosine.
- Yellow is picronic acid.
Neutral dyes can color both anions and cations. As a striking example of such can be called rhodamine B.
Existing staining methods based on the tinctorial properties of microorganisms can be divided into three main types: vital, postvital, and spore staining. We will dwell on each of these methods in more detail.
Vital way
This method is also called intravital, therefore, staining is performed with the aim of microscopy of living organisms. The introduction of a coloring drug allows you to explore the features of intracellular structures and tissues. There are also methods of vital staining, suggesting the killing of the sample immediately after staining.
For intravital staining, special dyes are used, which are characterized by low toxicity and high penetration. In addition to vital preparations, fluorochromes or fluorescent dyes are also used to solve this problem.
Postvital way
Unlike the previous version, here staining is performed after the killing of microorganisms. Postvital staining methods are divided into simple and complex. Simple ones allow you to determine the morphological properties of the studied organisms: shape, size, localization, relative position. Complex methods are narrowly targeted and provide information on the structure of the microorganism. Most often, complex methods of coloring are named after their creators. Of these, the following methods are most popular.
Gram method. The method developed back in the late 19th century is based on the differentiation of microbes by the permeability of cell walls. By treating the sample with aniline dyes (gentian violet or methyl) and subsequent washing, the researcher obtains two types of microbes: gram-positive (characteristic blue color) and gram-negative (secured). In order to get a more complete picture, a red dye is also used, after processing which even gram-negative microorganisms acquire a color - from pink to red.
Gram's technique allows classifying microbes and dividing them by tinctorial properties. This is one of the simplest and most common methods for preparing a sample for microscopy. The applied value of this technique is the diagnosis of various infectious diseases.
The Ziehl-Nielsen Method . This method was also developed at the end of the 19th century. It is based on the determination of acid resistance of bacteria using staining. This method, due to the tinctorial properties of microorganisms, can identify pathogens of tuberculosis, leprosy and mycobacteriosis.
Romanovsky-Giemsa technique . This method was developed at the beginning of the twentieth century. Its essence lies in the fact that after staining the sample with a special dye, acidophilic bacteria acquire various shades of red, and basophilic bacteria from purple to blue.
Acidophils are called lactic and acetic acid bacteria, for the vital activity of which a low acidity of the medium is required. Basophils are microbes that can be stained with basic dyes. Thus, the method is based on the separation of microbes by the value of the acidity of the medium. It is widely used in the study of the morphology of protozoa and spirochetes.
Burri-Gins technique . Allows you to determine the presence of capsules in bacteria.
Morozov's method. This method of staining makes it possible to make the flagella of bacteria visible. To do this, you need to make the following manipulations:
- Treat the sample with acid to loosen the flagella.
- Fix loose tissue with tannin.
- Stain the sample with silver nitrate.
As a result, the bacterium, with all its flagella, becomes visible. Its color can range from yellow to brown.
Spore staining
The tinctorial properties of microorganisms make it possible to make their spores noticeable by color. For this, the Ziehl-Nielson technique is most often used. Its essence consists in processing the test sample with fuchsin Ziel and its further discoloration with the help of one percent sulfuric acid. As a result, the spores turn pink and stand out against the blue bacteria.
Obzheshko's methodology also provides a visible picture of the dispute. According to this method, spore membranes are first etched with acid at high temperature, and then Ziehl-Nielson staining is used.
Structural Features of Microbes
A cell of a microorganism usually consists of their walls (membrane), cytoplasmic membrane, cytoplasm containing nucleotide and inclusions. Some types of microbes also have flagella, fimbriae, or cilia, and can form capsules or spores.
The shell of the microorganism determines its shape, and also protects it from the adverse environment and osmotic internal pressure. The walls of microbes have a rather complex chemical composition and structure. In gram-positive and gram-negative cells, they differ. The former have a thick cell wall containing teichoic acids and some lipids. The second wall consists of the polypeptide and polysaccharide layers.
Outside, the cell wall is covered with a mucous layer. In some microbes, it can increase (swell) to form a capsule consisting mainly of polysaccharides, and sometimes polypeptides. It is unresponsive to staining. That is why such microbes are studied using the Burri-Gins method. In some pathogenic microbes (for example, pneumococcus), the capsule is formed only in animals or humans.
When studying microbes under a microscope, attention is also paid to the staining of the cytoplasm and its structures. The cytoplasm can stain homogeneously (uniformly) or heterogeneously (inclusions of organic or inorganic nature are present). To study inclusions, special staining techniques are used (for example, according to Neusser). In addition, the presence of a nucleus or nucleotide is determined in the cytoplasm.
Having learned that these are tinctorial properties and how they help in microscopy, we consider the features of some common microbes.
Streptococcus
From the point of view of the tinctorial properties, streptococci are gram-positive microorganisms, therefore, they are determined by the Gram method. The most vivid staining of these bacteria occurs when exposed to blue Laffer. The arrangement of their cells resembles a chain.
Salmonella
They are facultative anaerobes, most of which are motile due to the presence of flagella. In a dense nutrient medium, salmonella gather in round white-gray colonies. They do not form a dispute; they have peritrichi and a microcapsule. Like streptococci, salmonella are stained by the Gram method. The difference is that salmonella is gram-negative in its tinctorial properties.
Meningococci
They have a rounded polymorphic shape. On a nutrient medium are arranged in pairs. The tinctorial properties of meningococci are such that they are gram-negative, but they do not respond clearly to Gram stain . Uneven staining is caused by the fact that the older the cell, the weaker its tinctorial properties. Meningococci have no flagella, but they do not form a spore.
Vibrio Cholera
This microbe has one polar flagellum, which is equipped with a sheath and undulating membrane. Cholera vibrio has a pronounced mobility. It is characterized by polymorphism. The tinctorial properties of the cholera vibrio determine its staining with aniline dyes. As a rule, this is Pfeiffer's water fuchsin or Schlaus carbolic fuchsin.
E. coli
It is an optional anaerobic non-spore forming. The cells are stick-shaped with rounded ends. The tinctorial properties of E. coli refer it to gram-negative bacteria. Bacterial strains with peritrichous flagella can move.
Neutrophils
Microbes got this name due to their tinctorial properties. Neutrophils can be intensively stained by the Romanovsky method, both basic dyes and acid eosin. Adult bacteria have a segmented nucleus, which means they belong to polymorphonuclear cells. They are endowed with adhesiveness, mobility, as well as the ability to chemotaxis and capture particles. Together with basophils and eosinophils belong to granulocytes.
Basophils
By analogy with previous microbes, these bacteria got their name due to tinctorial properties. Basophils are intensively stained with the main dye and are not stained with acid eosin at all. They have large granulocytes and contain a lot: histamine, serotonin, prostaglandins, leukotrienes and other mediators of inflammatory and allergic processes.
Eosinophils
Unlike the previous two granulocytes, these microbes intensively stain only with an acid dye, and do not stain with basic drugs at all. Eosinophils are endowed with the ability to extravasate, chemotaxis and amoeboid movement. They can absorb and bind a number of mediators of inflammatory and allergic reactions.