In the Gram stain procedure, bacteria are exposed to 95% alcohol or to an acetone/alcohol mixture. The purpose of this step is:
(A) To adhere the cells to the slide
(B) To retain the purple dye within all the bacteria
(C) To disrupt the outer cell membrane so the purple dye can leave the bacteria
(D) To facilitate the entry of the purple dye into the gram-negative cells
(E) To form a complex with the iodine solution
Answer
C
Gram staining is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 (Gram 1884). The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay.
The Basic Method
First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. Important considerations:
Take a small inoculum—don’t make a thick smear that cannot be completely decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable.
Take a fresh culture—old cultures stain erratically.
Fix the cells to the slide by heat or by exposure to methanol. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.
Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. Allow to stain for approximately 1 minute.
Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.
Add iodine (Gram’s iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test.
Briefly rinse with water.
Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly.
Rinse with water to stop decolorization.
Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. The counterstain stains both gram-negative and gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red.
Blot gently and allow the slide to dry. Do not smear.
What’s Going On?
Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. 1983)
The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In addition to the clearly gram-positive and gram-negative, there are many species that are “gram-variable” with intermediate cell wall structure (Beveridge and Graham 1991). As noted above, the decolorization step is critical to the success of the procedure.
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen. Those cells with a thin cell wall, and therefore decolorized, appear red (gram negative).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and to perform proficiency testing on the ability of the technicians to correctly interpret the stains (Andserson, et al. 2005).
Excessive Decolorization
It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below (reviewed in McClelland 2001)
Excessive heat during fixation: Heat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violet: Concentrations of crystal violet up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 seconds.
Excessive washing between steps: The crystal violet stain is susceptible to wash-out with water (but not the crystal violet-iodine complex). Do not use more than a 5 second water rinse at any stage of the procedure.
Insufficient iodine exposure: The amount of the mordant available is important to the formation of the crystal violet – iodine complex. The lower the concentration, the easier to decolorize (0.33% – 1% commonly used). Also, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram’s iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization: 95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
Excessive counterstaining: As the counterstain is also a basic dye, it is possible to replace the crystal violet—iodine complex in gram- positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 seconds.
Alternatives to the Gram Stain
Gram’s staining method is plainly not without its problems. It is messy, complicated, and prone to operator error. The method also requires a large number of cells (although a membrane-filtration technique has been reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.
This method, and it’s liabilities, are of immediate interest to those involved in environmental monitoring programs as one of the most common isolates in an EM program, Bacillus spp., will frequently stain gram variable or gram negative despite being a gram-positive rod (this is especially true with older cultures). The problems with Gram’s method have lead to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature.
(A) To adhere the cells to the slide
(B) To retain the purple dye within all the bacteria
(C) To disrupt the outer cell membrane so the purple dye can leave the bacteria
(D) To facilitate the entry of the purple dye into the gram-negative cells
(E) To form a complex with the iodine solution
Answer
C
Gram staining is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. The method is named after its inventor, the Danish scientist Hans Christian Gram (1853-1938), who developed the technique in 1884 (Gram 1884). The importance of this determination to correct identification of bacteria cannot be overstated as all phenotypic methods begin with this assay.
The Basic Method
First, a loopful of a pure culture is smeared on a slide and allowed to air dry. The culture can come from a thick suspension of a liquid culture or a pure colony from a plate suspended in water on the microscope slide. Important considerations:
Take a small inoculum—don’t make a thick smear that cannot be completely decolorized. This could make gram-negative organisms appear to be gram-positive or gram-variable.
Take a fresh culture—old cultures stain erratically.
Fix the cells to the slide by heat or by exposure to methanol. Heat fix the slide by passing it (cell side up) through a flame to warm the glass. Do not let the glass become hot to the touch.
Crystal violet (a basic dye) is then added by covering the heat-fixed cells with a prepared solution. Allow to stain for approximately 1 minute.
Briefly rinse the slide with water. The heat-fixed cells should look purple at this stage.
Add iodine (Gram’s iodine) solution (1% iodine, 2% potassium iodide in water) for 1 minute. This acts as a mordant and fixes the dye, making it more difficult to decolorize and reducing some of the variability of the test.
Briefly rinse with water.
Decolorize the sample by applying 95% ethanol or a mixture of acetone and alcohol. This can be done in a steady stream, or a series of washes. The important aspect is to ensure that all the color has come out that will do so easily. This step washes away unbound crystal violet, leaving Gram-positive organisms stained purple with Gram-negative organisms colorless. The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly.
Rinse with water to stop decolorization.
Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red. The counterstain stains both gram-negative and gram-positive cells. However, the purple gram-positive color is not altered by the presence of the counter-stain, it’s effect is only seen in the previously colorless gram-negative cells which now appear pink/red.
Blot gently and allow the slide to dry. Do not smear.
What’s Going On?
Bacteria have a cell wall made up of peptidoglycan. This cell wall provides rigidity to the cell, and protection from osmotic lysis in dilute solutions. Gram-positive bacteria have a thick mesh-like cell wall, gram-negative bacteria have a thin cell wall and an outer phospholipid bilayer membrane. The crystal violet stain is small enough to penetrate through the matrix of the cell wall of both types of cells, but the iodine-dye complex exits only with difficulty (Davies et al. 1983)
The decolorizing mixture dehydrates cell wall, and serves as a solvent to rinse out the dye-iodine complex. In Gram-negative bacteria it also dissolves the outer membrane of the gram-negative cell wall aiding in the release of the dye. It is the thickness of the cell wall that characterizes the response of the cells to the staining procedure. In addition to the clearly gram-positive and gram-negative, there are many species that are “gram-variable” with intermediate cell wall structure (Beveridge and Graham 1991). As noted above, the decolorization step is critical to the success of the procedure.
Gram’s method involves staining the sample cells dark blue, decolorizing those cells with a thin cell wall by rinsing the sample, then counterstaining with a red dye. The cells with a thick cell wall appear blue (gram positive) as crystal violet is retained within the cells, and so the red dye cannot be seen. Those cells with a thin cell wall, and therefore decolorized, appear red (gram negative).
It is a prudent practice to always include a positive and negative control on the staining procedure to confirm the accuracy of the results (Murray et al 1994) and to perform proficiency testing on the ability of the technicians to correctly interpret the stains (Andserson, et al. 2005).
Excessive Decolorization
It is clear that the decolorization step is the one most likely to cause problems in the gram stain. The particular concerns in this step are listed below (reviewed in McClelland 2001)
Excessive heat during fixation: Heat fixing the cells, when done to excess, alters the cell morphology and makes the cells more easily decolorized.
Low concentration of crystal violet: Concentrations of crystal violet up to 2% can be used successfully, however low concentrations result in stained cells that are easily decolorized. The standard 0.3% solution is good, if decolorization does not generally exceed 10 seconds.
Excessive washing between steps: The crystal violet stain is susceptible to wash-out with water (but not the crystal violet-iodine complex). Do not use more than a 5 second water rinse at any stage of the procedure.
Insufficient iodine exposure: The amount of the mordant available is important to the formation of the crystal violet – iodine complex. The lower the concentration, the easier to decolorize (0.33% – 1% commonly used). Also, QC of the reagent is important as exposure to air and elevated temperatures hasten the loss of Gram’s iodine from solution. A closed bottle (0.33% starting concentration) at room temperature will lose >50% of available iodine in 30 days, an open bottle >90%. Loss of 60% iodine results in erratic results.
Prolonged decolorization: 95% ethanol decolorizes more slowly, and may be recommended for inexperienced technicians while experienced workers can use the acetone-alcohol mix. Skill is needed to gauge when decolorization is complete.
Excessive counterstaining: As the counterstain is also a basic dye, it is possible to replace the crystal violet—iodine complex in gram- positive cells with an over-exposure to the counterstain. The counterstain should not be left on the slide for more than 30 seconds.
Alternatives to the Gram Stain
Gram’s staining method is plainly not without its problems. It is messy, complicated, and prone to operator error. The method also requires a large number of cells (although a membrane-filtration technique has been reported; Romero, et al 1988). However, it is also central to phenotypic microbial identification techniques.
This method, and it’s liabilities, are of immediate interest to those involved in environmental monitoring programs as one of the most common isolates in an EM program, Bacillus spp., will frequently stain gram variable or gram negative despite being a gram-positive rod (this is especially true with older cultures). The problems with Gram’s method have lead to a search for other tests that correlate with the cell wall structure of the gram-positive and the gram-negative cells. Several improvements/alternatives to the classical gram stain have appeared in the literature.
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