Microbial staining
This section consists of four parts:
First, the basic principle of dyeing Second, the type and selection of dyes III, the basic procedures for filming and dyeing Fourth, dyeing methods
First, the basic principle of dyeing
The basic principle of microbial staining is carried out by the action of physical and chemical factors. Physical factors such as the capillary phenomenon, penetration, and adsorption of dyes by cells and cellular materials. Chemical factors are various chemical reactions that occur depending on the different properties of cellular materials and dyes. The acidic substance is easier to adsorb to the basic dye, and the adsorption is stable; similarly, the alkaline substance is easier to adsorb on the acid dye. For example, acidic nucleus has chemical affinity for basic dyes and is easy to adsorb. However, in order for acidic materials to be dyed with acidic materials, their physical form must be changed (such as changing the pH) to facilitate the adsorption. In contrast, alkaline substances (such as cytoplasm) are usually only dyed with acid dyes, and if they are converted into suitable physical forms, they can also adsorb with basic dyes.
The isoelectric point of bacteria is low, and the pH is about 2-4. Therefore, in neutral, alkaline or weakly acidic solution, the bacterial protein is ionized and charged with an anion charge; while the basic dye is ionized, the dye ion is positively charged. . Therefore, bacteria with negative electricity are often combined with a basic dye with positive electricity. Therefore, basic dyes are commonly used for bacteriological dyeing.
Other factors affecting staining, as well as the structure of the cells and the permeability of the outer membrane, such as the permeability of the cell membrane, the size of the membrane pores and the integrity of the cell structure, play a role in staining. In addition, the composition of the medium, the bacteria order, the dielectric content in the dyeing solution and the pH, the temperature, the action of the drug, etc., can also affect the staining of the bacteria.
Second, the type and selection of dyes
Dyes are classified into natural dyes and artificial dyes. Natural dyes are cochineal red, lichenin, litmus and hematoxylin, etc., which are mostly extracted from plants, and their composition is complicated, and some of them have not yet been clarified. At present, artificial dyes, also known as coal tar dyes, are mainly extracted from coal tar and are derivatives of benzene. Most dyes are colored organic acids or bases, are poorly soluble in water, and are readily soluble in organic solvents. In order to make them soluble in water, they are usually made into salts.
Dyes can be classified into four categories: acid dyes, basic dyes, neutral (complex) dyes and simple dyes according to the nature of the charge of dye ions after ionization.
1, acid dye
After dyeing such dyes, the dye ions are negatively charged, such as eosin, Congo red, algae red, aniline black, picric acid and acid red, which can be combined with basic substances to form salts. When the genetic sugar is decomposed to produce acid to lower the pH, the positive charge of the bacteria increases, and the acid dye is selected, which is easy to be dyed.
2. Basic dyes
After dyeing such dyes, the dye ions are positively charged and can be combined with acidic substances to form salts. Basic dyes commonly used in microbiology laboratories include Meilan, methyl violet, crystal violet, alkaline red, neutral red, malachite green and red. In general, bacteria are easily dyed by basic dyes.
3. Neutral (composite) dyes
The combination of acid dyes and basic dyes is called neutral (complex) dyes, such as Wright dyes and Gimsa dyes, which are commonly used for dyeing of the nucleus.
4, simple dye
These dyes have low chemical affinity and cannot form salts with the dyed substances. Their dyeing ability depends on whether they are soluble in the dye, because most of them belong to azo compounds, insoluble in water, but soluble in fat solvents. Such as the dye of the Sudanb.
Third, the basic procedures for production and dyeing
There are many methods for dyeing microorganisms, and the dyes used in various methods are not the same, but the dyeing is generally carried out through production and a set of dyeing procedures.
1, production
Drop a drop of distilled water on a clean glass slide, perform aseptic operation with an inoculating loop, pick up the culture a little, place the water droplets on the slide, mix with water to make a suspension and apply a thin layer of about 1 cm in diameter. In order to avoid excessive aggregation of the bacteria into the group, it is unfavorable to observe the individual form. You can add a drop of water to one side of the slide, and take another ring from the coated bacterial solution to dilute it. The cloth is formed into a thin layer, and if the material is a liquid culture or a solid culture, the prepared bacterial liquid is washed, and then directly coated on a glass slide.
2, naturally dry
The smear is naturally dry at room temperature, sometimes in order to make it dry faster, the specimen can be facing up, holding the sides of one end of the slide, carefully heating the water on the high point of the alcohol lamp to make the water Evaporate, but do not rely on the flame or heat for too long to prevent the specimen from being bent and deformed.
3, fixed
The specimens are fixed after drying, and the purpose of fixation is three:
1) Kill the microorganisms and fix the cell structure.
2) Ensure that the bacteria adhere more firmly to the slide and prevent the specimen from being washed away by water.
3) Change the permeability of the dye to the cells, since dead protoplasts are easier to stain than live protoplasts.
Fixing often uses high temperature, hand-held one end of the slide (coated with the distal end of the specimen), the specimen is up, and the outer layer of the alcohol lamp flame passes back and forth as fast as 3-4 times, for about 2-3 seconds, from time to time The back of the slide is heated to the skin, and it is advisable to over-hot (not more than 60 ° C). After being placed in the cold, it is dyed.
Although the above-mentioned immobilization method is widely used in the microbiology laboratory, it should be noted that the chemical immobilization method should be employed when it is not applicable when studying the microbial cell structure. Chemical fixatives commonly used fixatives are: alcohol (95%), a mixture of alcohol and ether, acetone, 1-2% of hungry acid, etc. Hungry acid can fix cells quickly but does not change its structure, so it is more commonly used. The technique of applying cells to fix acid is as follows: place a glass in a culture dish, place a glass capillary on the glass, inject a small amount of 1-2% acid solution into the capillary, and place the wet sample smear on the glass. Slide the slide, then cover the Petri dish. After 1-2 minutes, remove the specimen from the Petri dish and let it dry.
4, dyeing
After the specimen is fixed, the staining solution is added dropwise. The time of dyeing varies, depending on the nature of the specimen and the dye, and sometimes it is heated when dyed. The duration of the dye-affecting specimen is about 1-3 minutes on average, and the entire smear (or part of the specimen) should be immersed in the dye during all dyeing times.
For complex dyeing, the mordant forms an insoluble compound with the dye during mordant treatment, which increases the affinity of the dye and bacteria. Generally, it is fixed after mordation, but it can also be combined with fixation or dyeing at the same time.
5, bleaching
The stained cells are treated with an alcohol or an acid to decolorize them. The stability of the dye-cell binding can be checked to identify different types of bacteria. Commonly used decolorizing agents are 95% alcohol and 3% hydrochloric acid solution.
6, counterstaining
After decolorization, it is dyed with a dye to form a sharp contrast with the undecolored portion for easy observation. After the alcohol is decolorized, the dyeing is carried out by using saffron, stone carbonate and red zui.
7, wash
When dyeing to a certain extent, the excess dye is washed away from the back of the specimen with a small stream of water, and the dye adsorbed by the cells is retained.
8, dry
After the stained specimens are washed, dry the specimens, or use excess paper to absorb excess water, then dry or slightly heat dry. When using absorbent paper, do not turn the slides over to avoid rubbing the bacteria. .
9, microscopic examination
The dried specimen can be observed with a microscope.
In summary, the basic procedure for dyeing is as follows:
Production → fixation → mordation → dyeing → bleaching → counterstaining → washing → drying → microscopic examination.
Fourth, the dyeing method
Microbial staining methods are generally divided into single staining and complex staining. The former stains microorganisms with a dye, but does not identify microorganisms. The counterstaining method uses two or more dyes to assist in the identification of microorganisms. Therefore, it is also known as the differential dyeing method. Commonly used counterstaining methods include Gram staining and acid-fast staining, as well as special staining methods for identifying the structure of various parts of cells (such as spores, flagella, nuclei, etc.). Commonly used in food microbiological testing are single staining and Gram staining.
1, single staining
Staining the smear with a dye is simple and convenient, and is suitable for the observation of the morphology of microorganisms. In general, bacterial cells are mostly negatively charged and are easily stained by binding to a positively charged basic dye. Therefore, commonly used basic dyes for single dyeing, such as Meilan, malachite green, alkaline red, crystal violet and neutral red. If acid dyes are used, Congo red, eosin, algin red and acid magenta are often used. When using an acid dye, it is necessary to lower the pH of the dye solution to make it strongly acidic (below the isoelectric point of the bacterial cells), and to make the cells positively charged, so that they are easily stained with acid dyes.
Single dyeing generally involves five steps of smear, fix, dye, water wash and dry.
The staining results vary depending on the dye:
Stone carbonic acid red dyeing solution: fast coloring, short time, the bacteria are red.
Meilan staining solution: slow coloration, long time, clear effect, and the bacteria are blue.
Ammonium oxalate crystal dyeing solution: rapid dyeing, deep coloration, and purple cells.
2, Gram staining
Gram staining is a differential staining method widely used in bacteriology. It was founded in 1884 by the Danish physician Gram.
Bacteria are firstly dyed by basic dye crystals, and after immersion in iodine liquid, they are decolorized with alcohol. Under certain conditions, some bacteria are not removed, and some can be removed. Therefore, bacteria can be divided into two categories. The former is called Gram-positive bacteria (G+), and the latter is Gram-negative bacteria (G-). For the convenience of observation, after decolorization, a red dye such as alkaline red is used for counterstaining. Positive bacteria are still purple, and negative bacteria are stained red. Bacillus sp. and most and cocci, as well as all actinomycetes and fungi, are Gram-positive; Vibrio, spirochetes and most pathogenic Bacillus are negative.
Gram-positive bacteria and Gram-negative bacteria have many differences in chemical composition and physiological properties, and the staining reaction is different. It is generally believed that Gram-positive bacteria contain a complex of a special nuclear protein magnesium salt and a polysaccharide, which is strongly bonded to the complex of iodine and crystal violet, and is not easily decolored, the degree of binding of the negative bacterial complex is low, and the adsorption dye is poor. Easy to discolor, this is the main basis for the dyeing reaction.
In addition, the positive cells have lower isoelectric points than the negative bacteria, and are stained under the same pH conditions. The positive bacteria adsorb a lot of basic dyes, so they are not easy to remove, and the negative bacteria are the opposite. Therefore, the conditions at the time of dyeing should be strictly controlled. For example, dyeing under strong alkali conditions, both types of bacteria adsorb more alkaline dyes, can be positive reaction; when the pH is very low, they can all be negative. In addition, the cell walls of the two types of bacteria have inconsistent permeability to the crystal violet-iodine complex, and the positive bacteria permeability is small, so that it is not easily discolored, and the negative bacteria are highly permeable and easily decolorized. Therefore, the decolorization time and the decolorization method should also be strictly controlled.
Gram staining generally includes four steps of initial dyeing, mordant dyeing, decolorization and counterstaining. The specific operation method is:
1) The smear is fixed.
2) Ammonium oxalate crystal violet stained for 1 minute.
3) Rinse with tap water.
4) Immersion coating for 1 minute.
5) Wash with water and absorb water with absorbent paper.
6) Add a few drops of 95% alcohol and gently shake to decolorize. After 30 seconds, wash with water and remove water.
7) After dyeing the red color liquid (thin) for 10 seconds, rinse with tap water. Dry, microscopic examination.
As a result of the staining, Gram's positive reaction cells were all purple, and the negative reaction cells were all red.
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