The colour of microorganisms

The colour of microorganisms - the most common in Microbiology complex of methods and techniques used for the detection and identification of microorganisms by means of a microscope. In native (natural) state bacteria have the same refractive index as glass, so they are invisible microscopic examination. The colour of microorganisms allows the study of morphological features of microbes, and sometimes to accurately determine their species, such as some the germs of the same morphology differs painted with the same complex methods of painting. The colour of microorganisms is a physical-chemical process connection chemical components of cells with paint. In some cases, the different parts of the bacterial cell (nucleus, cytoplasm) selectively painted different colours. Most suitable for painting of microorganisms are aniline dyes, mainly basic and neutral, acidic ink less suitable.
Preparation painted drug contains a number of steps: 1) smear preparation; 2) drying smear; 3) fixation of the stroke; 4) color; 5) drying.
Smear prepared on pure slides, in the middle of which put a small drop of water and using loops put the studied material. Material distributed on glass uniform thin layer, size smear-1-2 cm2.
The drug is usually dried at room temperature in air. For acceleration of drying Pets warming smear in a stream of warm air high above the burner flame.
Dried smear subjected to fixation, where a PAP smear is attached to the glass (fixed), and microbes become more sensitive to color. Ways of fixing a lot. The most simple and widespread - fixing a heat - heat by the flame (the drug several times through the hottest part of the flame of the burner). In some cases, resort to fixation liquids (ethyl or methyl alcohol, acetone, a mixture of equal volumes of alcohol and ether - Nikiforova). After fixing the smear paint. The amount of paint applied to the drug, shall be such as to cover the entire surface of the stroke. Upon expiration of the painting (2-5 minutes) the paint is poured and the preparation wash out water.
There are simple, complex and differential methods of painting microbes. With a simple color usually use one color, usually red Magenta, or blue - methylene blue. Magenta colors faster (1-2 minutes), methylene blue is slower (3-5 minutes). Magenta is prepared in the form of the concentrated solution of carbolic (Magenta Tsilia), very persistent and is suitable for painting for many months. Methylene blue is made in advance in saturated alcohol solution, which supports and can be kept long.
The complex methods of painting that employ two or more of dye are valuable techniques used in microbiological diagnostics of infectious diseases.
The greatest practical importance gram stain (see Thunder coloring method) and painting on Zn.
The method of coloring on Zn is the main for the painting of the acid-fast bacteria. Here are two of dye: carbolic Magenta Tsilia and methylene blue. Acid bacteria turn red, all nikelacoste forms - in blue.
Method Benignity is one way of colouring flagella. The prograss and painting one of the dye solutions prepared ex tempore (as necessary): I - sulfate zinc - 1 year, tannin - 10 g, distilled water to 100 ml and II - saturated alcohol gentian violet solution. Mix 5 ml solution I and 3 ml solution II, put on a drug heated to vapor appeared, washed with water, dried and consider.
From other sophisticated techniques used so-called negative way Burri (see Burri method), method of Ginza to identify capsules (see Ginza method) and several others. Cm. also Bacteriological examination.

Colouring of microorganisms - the complex of methods of studying the structure and morphology of microorganisms in microscopy drugs made from pure cultures of investigated material. The colour of microorganisms is an important diagnostic tool, allowing you to set the morphological features of the organism, and also to distinguish morphologically identical microorganisms, which in various ways are painted using the same methods of painting.
The colour of microorganisms is a complex physical-chemical process, the mechanism of which the details have not yet been studied. In the basis of the color of microorganisms is the interaction between individual structures bacteria with dye. Consequently, the result of O. M. depends on the chemical and physical structure of microbes, properties of the dye, preparation method of the preparation and processing method them microbe. For O. M. use basic, acidic and neutral dyes. The basic dyes, coloring the beginning is the cation and anion colorless; sour, on the contrary, a painting of the molecule is an anion. Produced in the dissociation of basic dyes cations are connected with the structures of bacteria with the acid properties; released anions of acid dyes are connected with the structures of the organism that has the basic properties.
In normal environments bacteria have a negative surface charge, in their cytoplasm and nucleus are compounds acidic nature. Therefore, basic dyes, ink which part of the molecule is positive, have a greater affinity for bacteria and more often used in Microbiology than acidic dyes, usually used for a contrasting background color of the drug. Neutral coloured dyes are and cations and anions (paint Romanovsky - Himsi).
Fixed bacteria are stained better living, because the cell wall and cytoplasmic membrane of living cage limit the penetration of dye. For vital staining microorganisms (colouring of living organisms) colouring agents used in high dilutions (1:10 000 - 1:100 000)to avoid artifacts arising from the toxic effects of dye on living organisms. Most often vital for O. M. use of methylene sipem, neutral red and other Drugs for microscopy are prepared using crushed drops. (see) or hanging drops (see).
The most common methods of O. M. in fixed preparations. These methods are divided into simple and complex. When simple methods used one color (for example, coloring methylene blue, Magenta of Pfeiffer and others). When sophisticated methods used several colors and the study includes several stages of physical and chemical treatment of the drug (see the gram stain, paint on Zn Nelsen - see Tuberculosis; paint on. Romanovsky - Giemsa - see Blood and others). Sophisticated methods of staining microorganisms used in microbiological diagnostics to analyze the structure and functions of microorganisms. For example, using the gram staining differentiate grampolaugitionah gramotricationah microbes from, which is of great importance to identify gonokokkov, meningokokkov, intestinal bacteria, etc. by the Method of so - Nelsen; used for diagnosis of tuberculosis and leprosy, method Neeser - diphtheria.
As an example of the use of colouring of microorganisms for the study of the structure and functions of microorganisms can be specified on the use of iodine to identify starch, Victoria blue - for selective color of the cell wall and zitoplazmaticescoy membrane of bacteria, omaboy acid - for colouring of fat, and so on, These methods are based on differences in chemical structure separate morphological structures of the organism, selectively coated different colors.
Other methods of colouring separate structures of microorganisms based on the ability of these structures in different ways to fix the dyes that are detected during subsequent processing of the drug alcohol, acid, acetone, etc. and counterstaining contrast dye. So, for the detection of the dispute to use their property to firmly fix the dye and not lose color with subsequent treatment with acid. At colouring of bacteria gram-basic dyes (gentian violet and others) with the subsequent processing of iodine in some microorganisms (gram) dye firmly in place and is not removed during processing alcohol or acetone; gram-negative bacteria easily fade. To identify the bacteria nucleoid use the method of Valgene. Coloration in the proper sense should be distinguished impregnation salts of metals, for example impregnation silver salts at colouring spirochetes by the method of Livadice (see Syphilis).
In addition to positive staining techniques (dye directly affects the coated substrate), Microbiology use and negative methods - coloring the background of the drug. They are used in case, when the microbes or their separate patterns bad prokalyvayutsya. A classic example of a negative colour is Burri method (see). In those cases when it is necessary to identify the microorganism capsule and to paint itself the germ, negative and positive methods are combined (see Ginza method).
There are also several ways to increase the efficiency of color. Some of them create conditions for penetration of dye inside a painted object (for example, when painting a dispute drugs previously treated with hydrochloric acid). Other methods associated with the use of Bates, lead to an increase in the painted object [for example, the use of salts tannic acid for painting flagella in bacteria (see Bennetti method), Taurus Paschen - by the method of Morozov and cell wall - by the method of Nazi].
Cm. also Bacteriological techniques, Bacteriological examination.