The Gram Stain

Scott Sutton, Ph.D.
Vectech Pharmaceutical Consultants
This article first appeared in the PMF Newsletter of February, 2006 and is protected by copyright to PMF.  It appears here with permission.

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

1. 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.

2. 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.

3. 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.

4. Briefly rinse the slide with water.  The heat-fixed cells should look purple at this stage.

5. 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.

6. Briefly rinse with water.

7. 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.

8. Rinse with water to stop decolorization.

9. 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.

10. 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.

KOH String Test

The KOH String Test is done using a drop of 3% potassium hydroxide on a glass slide.  A visible loopful of cells from a single, well-isolated colony is mixed into the drop.  If the mixture becomes viscous within 60 seconds of mixing (KOH-positive) then the colony is considered gram-negative.  The reaction depends on the lysis of the gram-negative cell in the dilute alkali solution releasing cellular DNA to turn the suspension viscous.  This method has been shown effective for food microorganisms (Powers 1995), and for Bacillus spp (Carlone et al 1983, Gregersen 1978), although it may be problematic for some anaerobes (Carlone et al 1983, but also see Halebian et al 1981).

This test has the advantage of simplicity, and it can be performed on older cultures.  False negative results can occur in the test by using too little inoculum or too much KOH (DNA-induced viscosity not noticeable).  False positive results can occur from too heavy an inoculum (the solution will appear to gel, but not string), or inoculation with mucoid colonies.  This can serve as a valuable adjunct to the tradition gram stain method (von Graevenitz and Bucher 1983).

Aminopeptidase Test

L-alanine aminopeptidase is an enzyme localized in the bacterial cell wall which cleaves the amino acid L-alanine from various peptides. Significant activity is found almost only in Gram-negative microorganisms, all Gram-positive or Gram-variable microorganisms so far studied display no or very weak activity (Cerny 1976, Carlone et al. 1983).  To perform the test, the reagent is used to make a suspension (with the bacteria).  Aminopeptidase activity of the bacteria causes the release of 4-nitroaniline from the reagent, turning the suspension yellow.  The test is especially useful for non-fermenters and gram-variable organisms, and is a one step test with several suppliers of kits.  Results of the test are available in 5 minutes.

Fluorescent Stains

A popular combination of fluorescent stains for use in gram staining (particularly for flow-cytometry) involves the use of the fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13.  HI penetrates gram-positive but not gram-negative organisms, but SYTO 13 penetrates both. When the dyes were used together in a single step, gram-negative organisms are green fluorescent by SYTO 13 while gram-positive organisms are red-orange fluorescent by HI which overpowers the green of SYTO 13 (Mason et al 1998).  There are commercial kits available for this procedure, which requires a fluorescent microscope or a flow cytometer.

Sizemore et al (1990) developed a different approach to fluorescent labeling of cells.  Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin.  A variant of this method has also been used to “gram stain” microorganisms in milk for direct measurement by flow cytometry.

LAL-based Assay

Charles River Laboratories has just released a product to be used with their PTS instrument – the PTS Gram ID (Farmer 2005).  This methodology makes use of the same reaction used for the chromogenic LAL test.  Gram-negative organisms, with bacterial endotoxin, initiate the LAL coagulase cascade which results in activation of the proclotting enzyme, a protease.  In the LAL test, this enzyme cleaves a peptide from the horseshoe crab coagulen, resulting in a clot.  It can also cleave a peptide from a synthetic substrate, yielding a chromophore (p-nitroaniline) which is yellow and can be measured photometrically at 385 nm (Iwanaga 1987).   Gram-positive organisms, lacking endotoxin, do not trigger the color change in this method, while gram-negative organisms do trigger it.  Results are available within 10 minutes.

Summary

The differentiation of bacteria into either the gram-positive or the gram-negative group is fundamental to most bacterial identification systems.  This task is usually accomplished through the use of Gram’s Staining Method.  Unfortunately, the gram stain methodology is complex and prone to error.  This operator-dependence can be addressed by attention to detail, and by the use of controls on the test.  Additional steps might include confirmatory tests,  of which several examples were given.  As with all microbiology assays, full technician training and competent review of the data are critical quality control steps for good laboratory results.

References

Anderson, N L, et al. 2005. Cumitech 3B, Quality Systems in the Clinical Microbiology Laboratory. Coordinating ed., D. L. Sewell. ASM Press, Washington, D.C.

Beveridge, TJ and LL Graham.  1991.  Surface Layers of Bacteria.  Microbiol Rev. 55(4):684-705.

Carlone, GM et al.  1983.  Methods for Distinguishing Gram-Positive from Gram-Negative Bacteria.  J Clin Microbiol.  16(6):1157-1159.

Cerny G. 1976 Method for distinction of gram negative from gram positive bacteria. Eur J Appl Microbiol. 3:223-225.

Davies, J. A., et al.  1983. Chemical Mechanism of the Gram Stain and Synthesis of a New Electron-opaque Marker for Electron Microscopy Which Replaces the Iodine Mordant of the Stain.  J Bacteriol.  156(2):837-845.

Farmer, T. 2005. A Definitive, Rapid Alternative to the Gram Stain Assay. Rapid Microbiology Newsletter.  3(8):2-3

Gram, C.  1884.  The Differential Staining of Schizomycetes in Tissue Sections and in Dried Preparations.  Fortschitte der Medicin.  2:185-189.

Gregersen, T. 1978. Rapid Method for Disitinction of Gram-negative From Gram-positive Bacteria.  Appl Microbiol Biotech  5(2):123-127.

Halebian, S et al.  1981.  Rapid Method That Aids in Distinguishing Gram-Positive from Gram-Negative Anaerobic Bacteria.  J Clin Microbiol.  13(3):444-448.

Holm, C. and  L. Jespersen.  2003.  A Flow-Cytometric Gram-Staining Techniques for Milk-Associated Bacteria.  Appl Environ Microbiol.  69(5):2857 - 2863.

Iwanaga, S. 1987. Chromogenic Substrate for Horseshoe Crab Clotting Enzyme: Its Application of the Assay of Bacterial Endotoxins.  Haemostasis.  7:183.

McClelland, R.  2001.  Gram's Stain: the Key to Microbiology.  Med Lab Observer.  April  pp. 20-31.

Murray, RGE et al. 1994.  Determinative and Cytological Light Microscopy. IN: Methods for General and Molecular Bacteriology P. Gerhardt, editor-in-chief.  ASM Press, Washington, DC.  pp21-41.

Powers, EM.  1995.  Efficacy of the Ryu Nonstaining KOH Technique for Rapidly Determining Gram Reactions of Food-Borne and Waterborne Bacteria and Yeasts. Appl Environ Microbiol. 61(10):3756-3758.

Romero, S et al.  1988.  Rapid Method for the Differentiation of Gram-Positive and Gram-Negative Bacteria on Membrane Filters.  J Clin Microbiol.  26(7):1378-1382.

Sizemore, RK et al.  1990.  Alternate Gram Staining Technique Using a Fluorescent Lectin.  Appl Environ Microbiol.  56(7):2245-2247

von Gravenitz, A and C Bucher.  1983.  Accuracy of the KOH and Vancomycin Tests in Determining the Gram Reaction of Non-Enterobacterial Rods.  J Clin Microbiol.  16(4):983-985.

Consulting with Scott Sutton