The following are used in Gram staining except:
a- Crystal violet
b- Iodine
c- Safranine
d- Malachite green***
--------------------------
Gram staining or Gram staining is a type of differential staining used in bacteriology for the visualization of bacteria, especially in clinical samples. It owes its name to the Danish bacteriologist Christian Gram (1853-1938), who developed the technique in 18841. It is used both to refer to bacterial cell morphology, and to be able to make a first approach to bacterial differentiation, considering gram-positive bacteria to which are displayed in purple, and gram-negative bacteria that are displayed in purple and red.
Process:
- Collect samples to place them in the microscope.
- Make the spread with a wooden stick.
- Let dry at room temperature and fix them using a lighter.
- Fix the sample with methanol for one minute or heat (flamed three times approximately).
- Add violet blue (violet or violet gentian crystal) and wait a minute.
- Rinse with water not directly on the sample
- Add lugol and wait approximately one minute.
- Add acetone alcohol and wait between 5 and 30 seconds depending on the concentration of the reagent (critical part of the coloring). (the gram - discolors, the gram no)
- Rinse with water.
Contrast staining by adding safranine or basic fuchsin and wait a minute. This dye will leave pink-reddish gram negative bacteria.
- Wash lightly with water.
To observe the optical microscope it is convenient to do it at 100x with immersion oil.
Explanation:
The violet crystal (cationic dye) penetrates all bacterial cells (both gram positive and gram negative) through the bacterial wall. Lugol is a compound formed by I2 (iodine) in equilibrium with KI (potassium iodide) and Sl (Siultery), which are present to solubilize iodine, and act as a mordant, causing the violet crystal to set more intensely to the wall of the bacterial cell. I2 enters the cells and forms an insoluble complex in aqueous solution with the violet crystal.
The alcohol-acetone mixture that is added serves to discolor, since the complex I2 / violet crystal is soluble in it. Gram positive organisms do not discolor, while gram negative organisms do.
To show gram negative cells, a contrast color is used. It is usually a red dye, such as safranine or fuchsin. After contrast coloring the gram negative cells are red, while the gram positive remain violet.
Some microorganisms retain the violet dye, even after treating them with a bleach, and the color is not modified by adding it; others easily lose the first dye, and take the second. Those who fix the violet, are described as gram positive, and those who lose the first color and retain the second, gram negative. Based on the gram reaction, we can classify microorganisms into one of two groups. P-rosaniline dyes are the ones that give the best results in gram coloring. The most used representatives of this group are methyl violet and crystal violet or gentian. Actually, methyl violet is the name attributed to the tetramethyl-p-rosaniline compound.
The color hue of p-rosaniline is intensified by increasing the number of methyl groups in the molecule; therefore, of the three groups, the darkest shade is hexamethyl-p-rosaniline (crystal violet), and the lightest dye, tetramethyl-p-rosaniline (methyl violet). The names violet of methyl 3R, 2R, R, B, 2B, 3B, etc., refer to the number of methyl groups contained. The letter R indicates red nuances, and the letter B, blue tones. The crystal violet contains six methyl groups, and is considered as the best primary dye for staining by the Gram method.
The ability of cells to take gram coloration is not typical of any living substance, but is limited almost entirely to fungi and bacteria. Thus we see that the cells of higher plants and animals do not retain the first coloration; molds stain with some irregularity; Mycelium granules tend to retain the dye. Gram's reaction is not infallible or constant; It can vary with the time of the culture and the pH of the medium, and perhaps for other reasons.
Gram stain resistant bacteria:
The following gram positive bacteria are stained as gram negative:
- Mycobacteria (they are encapsulated).
- Mycoplasmas (they have no wall).
- L forms (occasional wall loss).
- Protoplasts and spheroplasts (total and partial removal of the wall, respectively).
Utilities:
In the analysis of clinical samples it is usually a fundamental study to fulfill several functions:
- Preliminary identification of the bacteria causing an infection.
- Utility as a quality control of bacterial isolation. Bacterial morphotypes identified in Gram staining should correspond to bacterial isolates made in the cultures. If a greater number of bacterial forms are observed than those isolated, the culture media used as well as the incubation atmosphere must be reconsidered.
From the staining of Gram several different morphotypes can be distinguished: The coconuts are spherical in shape. They may appear isolated after cell division (micrococci), appear in pairs (diplococci), form chains (streptococci), or be grouped irregularly (staphylococci).
The bacilli have an elongated shape. In general they are usually grouped in the form of a chain (streptobacilli) or palisade.
Spirals can also be distinguished, which are classified as spirals (if they are rigidly shaped) or spirochetes (if they are soft and wavy). If, on the contrary, they have a "comma" (or curved) shape then they are called vibrios.
Fundamentals of differentiation of gram positive and gram negative:
The fundamentals of the technique are based on the differences between the cell walls of gram positive and gram negative bacteria.
The cell wall of gram-positive bacteria has a thick layer of peptidoglycan, in addition to two kinds of teicoic acids: anchored on the inner side of the cell wall and attached to the plasma membrane, lipoteicoic acid is found, and more on the surface , the teicoic acid that is anchored only in peptidoglycan (also known as murein).
On the contrary, the peptidoglycan layer of gram negative bacteria is thin, and is attached to a second outer plasma membrane (of a composition other than the internal one) by means of lipoproteins. It has a thin layer of peptidoglycan attached to an outer membrane by lipoproteins. The outer membrane is made of protein, phospholipid and lipopolysaccharide.
Therefore, both types of bacteria stain differentially due to these constitutive differences in their wall. The key is peptidoglycan, since it is the material that confers its rigidity to the bacterial cell wall, and gram-positive ones possess it in much greater proportion than gram-negative ones.
The difference observed in the resistance to discoloration is due to the fact that the outer membrane of gram negative bacteria is soluble in organic solvents, such as the alcohol / acetone mixture. The peptidoglycan layer it possesses is too thin to retain the previously formed violet / iodine crystal complex, and therefore this complex escapes, losing the blue-violet coloration. On the contrary, gram positive bacteria, having a stronger cell wall and a higher proportion of peptidoglycans, are not susceptible to the action of the organic solvent, but this acts by dehydrating the pores, closing them, which prevents the escape of the complex crystal violet / iodine, and maintaining the blue-violet coloration.
Factors that alter Gram staining:
- Age of the bacteria.
- Operator errors.
- Antibiotic use
Despite the great utility of Gram staining, this method should be assessed with caution, since the reaction can vary according to the age of the cells (old cultures of gram positive bacteria can lose layers of peptidoglycans and stain as gram negative) and the technique used (when bleaching for a very long time you can run the risk that gram positive bacteria are stained as gram negative). For this situation, controls with gram positive (for example Staphylococcus aureus) and gram negative (for example, Escherichia coli) bacteria should be stained next to the sample.
a- Crystal violet
b- Iodine
c- Safranine
d- Malachite green***
--------------------------
Process:
- Collect samples to place them in the microscope.
- Make the spread with a wooden stick.
- Let dry at room temperature and fix them using a lighter.
- Fix the sample with methanol for one minute or heat (flamed three times approximately).
- Add violet blue (violet or violet gentian crystal) and wait a minute.
- Rinse with water not directly on the sample
- Add lugol and wait approximately one minute.
- Add acetone alcohol and wait between 5 and 30 seconds depending on the concentration of the reagent (critical part of the coloring). (the gram - discolors, the gram no)
- Rinse with water.
Contrast staining by adding safranine or basic fuchsin and wait a minute. This dye will leave pink-reddish gram negative bacteria.
- Wash lightly with water.
To observe the optical microscope it is convenient to do it at 100x with immersion oil.
Explanation:
The violet crystal (cationic dye) penetrates all bacterial cells (both gram positive and gram negative) through the bacterial wall. Lugol is a compound formed by I2 (iodine) in equilibrium with KI (potassium iodide) and Sl (Siultery), which are present to solubilize iodine, and act as a mordant, causing the violet crystal to set more intensely to the wall of the bacterial cell. I2 enters the cells and forms an insoluble complex in aqueous solution with the violet crystal.
The alcohol-acetone mixture that is added serves to discolor, since the complex I2 / violet crystal is soluble in it. Gram positive organisms do not discolor, while gram negative organisms do.
To show gram negative cells, a contrast color is used. It is usually a red dye, such as safranine or fuchsin. After contrast coloring the gram negative cells are red, while the gram positive remain violet.
Some microorganisms retain the violet dye, even after treating them with a bleach, and the color is not modified by adding it; others easily lose the first dye, and take the second. Those who fix the violet, are described as gram positive, and those who lose the first color and retain the second, gram negative. Based on the gram reaction, we can classify microorganisms into one of two groups. P-rosaniline dyes are the ones that give the best results in gram coloring. The most used representatives of this group are methyl violet and crystal violet or gentian. Actually, methyl violet is the name attributed to the tetramethyl-p-rosaniline compound.
The color hue of p-rosaniline is intensified by increasing the number of methyl groups in the molecule; therefore, of the three groups, the darkest shade is hexamethyl-p-rosaniline (crystal violet), and the lightest dye, tetramethyl-p-rosaniline (methyl violet). The names violet of methyl 3R, 2R, R, B, 2B, 3B, etc., refer to the number of methyl groups contained. The letter R indicates red nuances, and the letter B, blue tones. The crystal violet contains six methyl groups, and is considered as the best primary dye for staining by the Gram method.
The ability of cells to take gram coloration is not typical of any living substance, but is limited almost entirely to fungi and bacteria. Thus we see that the cells of higher plants and animals do not retain the first coloration; molds stain with some irregularity; Mycelium granules tend to retain the dye. Gram's reaction is not infallible or constant; It can vary with the time of the culture and the pH of the medium, and perhaps for other reasons.
Theories:
A gram positive microorganism must have a healthy cell wall. The same microorganism, if it suffers wall damage from one cause or another, becomes gram negative. This indicates the importance of the wall for retention or escape of the dye. One possible theory of the staining mechanism is as follows:
The basic dye enters the microorganism, where it forms a water-insoluble lacquer with iodine. The alcohol or acetone used to lighten, dehydrates the walls of gram positive microorganisms, treated with mordant, and forms a barrier that lacquer cannot cross. In gram negative cells, wall lipids (more abundant than in gram positive cells) are dissolved by this treatment, which allows the escape of the violet crystal complex with iodine. Some authors object to this theory, but the general importance of the cell wall is unquestionable.
There are several theories issued to explain the mechanism of Gram staining. Stearn (1923) based his on a chemical combination between the dye and the proteins of the bacteria, the proteins and amino acids are amphoteric bodies, that is, they have the power to react with acids and bases, thanks to their amino and carboxyl groups ; in acidic solutions, they react with acids, and in alkaline solutions they do so with bases. Similarly, they found that the staining reaction of the bacteria is largely due to their protein content; These microorganisms are conducted as amphoteric bodies, when combined with acid dyes in acidic solutions and with basic ones in alkaline medium. The combination with both types of dye does not occur at the "isoelectric point". Since microorganisms contain more than one protein, that point does not have a precise and defined value, but rather constitutes a range or scale comprising two or three pH units. According to Stern and Stearn, gram positive microorganisms have an isoelectric scale of pH lower than that of gram negative microorganisms; and, based on their experimental data, they draw the following conclusions:
- Gram positive microorganisms can become gram negative by increasing acidity.
- Gram negative microorganisms can become gram positive by increasing alkalinity.
- Microorganisms with a positive reaction to acid dyes can become gram negative by increasing alkalinity.
- Microorganisms with a positive reaction to basic dyes can become gram negative by increasing acidity.
- In the isoelectric zone characteristic of each species, the tendency to retain any dye is very low.
- It seems to be well demonstrated that bacteria proteins are not simple, but rather a weak combination of protein substances with other lipoids or fats.
- The fat material extracted from gram-positive microorganisms differs from that obtained from gram-negative microorganisms, in that the former contains a much higher proportion of unsaturated acids that show great affinity for oxidizing agents. All mordants (such as iodine) used in gram coloring are oxidizing; Its effect, in general, is to give the oxidized substance a more acidic character. This increases the affinity of a microorganism for basic dyes.
- The change in response to Gram staining over time is, above all, typical of weakly gram positive microorganisms grown in media containing substances capable of fermenting, and whose reaction becomes acidic in the course of development.
Gianni (1952) found that the gram positive bacteria Bacillus subtilis and Bacillus anthracis caused a negative reaction when the crops dated from two to three hours. Then the gram positive substance developed under the cell wall and the reaction was reversed. Another explanation of the Gram reaction may be the possible existence of an outer layer around a gram negative nucleus. Libenson and Mcllroy have reported that if the gram positive reaction depends on the formation of a complex combination between the components of the Gram stain and the cell wall proteins, it would be expected that bacteria disintegrated by physical means would retain this dye, since that treatment could not change the chemical character of the materials of said wall. On the contrary, disintegrated gram positive germs lose their ability to retain the primary dye and do not stain
The cell wall of gram positive and gram negative microorganisms is permeable to crystal violet. However, that of the former is not the complex of iodine and dye formed inside the cell. The experimental results obtained with a protein-free cell diffusion, and the poor solubility of the complex of iodine and crystal violet in alcohol and acetone, seem to support the view that the gram positive reaction consists essentially in the formation, within the cell, of an appreciable amount of iodine complex and dye difficult to remove with the solvent. The cell wall of gram-positive microorganisms, unlike that of gram-negative microorganisms, would be virtually impervious to crystal violet. The microorganisms will appear stained after treating them with crystal violet, because the dye is absorbed on the outer surface of the cell wall, and the solvent will remove without difficulty the complex formed after treatment with iodine.
Neither sulfhydryl groups nor basic proteins have specifically influenced the mechanism of the Gram dye. Libenson and Mcllroy have argued that the permeability of the cell wall to the crystal violet, the poor solubility of the iodine and dye complex in alcohol and acetone, and the free access of the solvent to the constituted complex, are the main factors involved in the mechanism of that coloration.
Gram stain resistant bacteria:
The following gram positive bacteria are stained as gram negative:
- Mycobacteria (they are encapsulated).
- Mycoplasmas (they have no wall).
- L forms (occasional wall loss).
- Protoplasts and spheroplasts (total and partial removal of the wall, respectively).
Utilities:
In the analysis of clinical samples it is usually a fundamental study to fulfill several functions:
- Preliminary identification of the bacteria causing an infection.
- Utility as a quality control of bacterial isolation. Bacterial morphotypes identified in Gram staining should correspond to bacterial isolates made in the cultures. If a greater number of bacterial forms are observed than those isolated, the culture media used as well as the incubation atmosphere must be reconsidered.
From the staining of Gram several different morphotypes can be distinguished: The coconuts are spherical in shape. They may appear isolated after cell division (micrococci), appear in pairs (diplococci), form chains (streptococci), or be grouped irregularly (staphylococci).
The bacilli have an elongated shape. In general they are usually grouped in the form of a chain (streptobacilli) or palisade.
Spirals can also be distinguished, which are classified as spirals (if they are rigidly shaped) or spirochetes (if they are soft and wavy). If, on the contrary, they have a "comma" (or curved) shape then they are called vibrios.
Fundamentals of differentiation of gram positive and gram negative:
The fundamentals of the technique are based on the differences between the cell walls of gram positive and gram negative bacteria.
The cell wall of gram-positive bacteria has a thick layer of peptidoglycan, in addition to two kinds of teicoic acids: anchored on the inner side of the cell wall and attached to the plasma membrane, lipoteicoic acid is found, and more on the surface , the teicoic acid that is anchored only in peptidoglycan (also known as murein).
On the contrary, the peptidoglycan layer of gram negative bacteria is thin, and is attached to a second outer plasma membrane (of a composition other than the internal one) by means of lipoproteins. It has a thin layer of peptidoglycan attached to an outer membrane by lipoproteins. The outer membrane is made of protein, phospholipid and lipopolysaccharide.
Therefore, both types of bacteria stain differentially due to these constitutive differences in their wall. The key is peptidoglycan, since it is the material that confers its rigidity to the bacterial cell wall, and gram-positive ones possess it in much greater proportion than gram-negative ones.
The difference observed in the resistance to discoloration is due to the fact that the outer membrane of gram negative bacteria is soluble in organic solvents, such as the alcohol / acetone mixture. The peptidoglycan layer it possesses is too thin to retain the previously formed violet / iodine crystal complex, and therefore this complex escapes, losing the blue-violet coloration. On the contrary, gram positive bacteria, having a stronger cell wall and a higher proportion of peptidoglycans, are not susceptible to the action of the organic solvent, but this acts by dehydrating the pores, closing them, which prevents the escape of the complex crystal violet / iodine, and maintaining the blue-violet coloration.
Factors that alter Gram staining:
- Age of the bacteria.
- Operator errors.
- Antibiotic use
Despite the great utility of Gram staining, this method should be assessed with caution, since the reaction can vary according to the age of the cells (old cultures of gram positive bacteria can lose layers of peptidoglycans and stain as gram negative) and the technique used (when bleaching for a very long time you can run the risk that gram positive bacteria are stained as gram negative). For this situation, controls with gram positive (for example Staphylococcus aureus) and gram negative (for example, Escherichia coli) bacteria should be stained next to the sample.
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Clinical Pathology