Gram staining, also called Gram's method, is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). The name comes from the Danish bacteriologist Hans Christian Gram, who developed the technique.
Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in a thick layer in Gram-positive bacteria. In a Gram stain test, Gram-positive bacteria retain the crystal violet dye, while a counterstain (commonly safranin or fuchsine) added after the crystal violet gives all Gram-negative bacteria a red or pink coloring.
The Gram stain is almost always the first step in the identification of a bacterial organism. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique. This gives rise to Gram-variable and Gram-indeterminate groups as well.
The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin in 1884. Gram devised his technique not for the purpose of distinguishing one type of bacterium from another but to make bacteria more visible in stained sections of lung tissue. He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain.
- Step 1. Prepare a slide with the culture by transferring the specimen to be examined onto a drop of suspension medium (distilled water) using an inoculation loop. Spread the specimen on the slide to ensure that it is not clumped.
- Step 2. Fix the culture by heating the slide over a Bunsen burner to evaporate the water -- make sure not to hold the slide over the flame too long or it will denature the specimen.
- Step 3. Drop a few drops of crystal violet stain onto the fixed culture (enough to cover the specimen) and let it stand for 20 seconds. Then pour off the crystal violet stain and gently rinse the excess stain with distilled water.
- The objective of this step is:
- To allow the crystal violet stain to bind to the peptidoglycan molecules of the Gram + bacteria (if present). Remember, Gram + bacteria have a large peptidoglycan layer located outside the bacterial "inner membrane".
- To allow the crystal violet stain to bind to the lipopolysaccharide molecules attached to the "outer membrane" of the Gram - bacteria (if present). Remember, while Gram + bacteria have no "outer membrane", Gram - bacteria have lipopolysaccharide molecules attached to their bacterial "outer membrane".
- The objective of this step is:
- Step 4. Drop a few drops of iodine solution on the smear and let it stand for 20 seconds. Then pour off the iodine solution and rinse the slide with distilled water.
- The objective of this step is to fix the crystal violet to the peptidoglycan molecules on the Gram + bacteria.
- Step 5. Drop a few drops of decolorizer (acetone or ethanol) and let the solution trickle down off the slide until the decolorizer has removed enough of the color to drip off clear. Then IMMEDIATELY rinse the slide off with distilled water after 5 seconds. Note that pouring too much decolorier will cause the decolorization of the Gram + bacterial cells (in addition to the Gram - bacteria), and the purpose of staining will be defeated.
- The objective of this step is to dissolve the lipopolysaccharide membraine in the Gram - bacteria and expose the thin peptidoglycan layer below.
- Step 6. Drop a few drops of basic counterstain (fuchsin or safranin) on the slide and let it sit for 20 seconds, then wash off the solution with distilled water.
- The objective of this step is to stain the peptidoglycan layer of the Gram - bacteria a pink / red color. Remember that the addition of iodine to the crystal violet in Step 4 binds the crystal violet stain in the Gram + bacteria, so the counterstain is unable to bind to the peptidoglycan wall in the Gram + bacteria in the specimen.
- Step 7. Observe the slide under light microscopy.
Gram staining is a bacteriological laboratory technique used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls. Gram staining is not used to classify archaea, formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.
The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, and it is of extremely limited use in environmental microbiology. It still competes with molecular techniques even in the medical microbiology lab. Some organisms are gram-variable (meaning they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and informative than differential staining.
Gram staining has proven as effective a diagnostic tool as PCR, particularly with regards to gonorrhoea diagnosis in Kuwait. The similarity of the results of both Gram stain and PCR for diagnosis of gonorrhea was 99.4% in Kuwait.
Gram stains are performed on body fluid or biopsy when infection is suspected. Gram stains yield results much more quickly than culturing, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by the Safranin. There are four basic steps of the Gram stain:
- Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don't rinse out during the staining procedure.
- The addition of iodide, which binds to crystal violet and traps it in the cell,
- Rapid decolorization with ethanol or acetone, and
- Counterstaining with safranin. Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.
Crystal violet (CV) dissociates in aqueous solutions into CV+
and chloride (Cl−
) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+
ion interacts with negatively charged components of bacterial cells and stains the cells purple.
3) interacts with CV+
and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV–I complex and, therefore, color the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-negative cell loses its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the gram-negative cell along with the outer membrane. In contrast, a gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain is removed from both gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).
After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color.
Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in gram-negative staining of these gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain gram-negative. In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.
Gram-positive bacteria generally have a single membrane (monoderm) surrounded by a thick peptidoglycan. This rule is followed by two phyla: Firmicutes (except for the classes Mollicutes and Negativicutes) and the Actinobacteria. In contrast, members of the Chloroflexi (green non-sulfur bacteria) are monoderms but possess a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain negative, positive or indeterminate. Members of the Deinococcus-Thermus group, stain positive but are diderms with a thick peptidoglycan.
Historically, the gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.
Normally, if Gram stain is done on acid-fast bacteria, they show up as if they are gram-positive, mostly because of their thick cell wall.
Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderms). Most bacterial phyla are gram-negative, including the cyanobacteria, spirochaetes, and green sulfur bacteria, and most Proteobacteria and Escherichia coli. (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).
Gram-indeterminate bacteria do not respond predictably to Gram staining and, therefore, cannot be determined as either gram-positive or gram-negative. They tend to stain unevenly, appearing partially gram positive and partially gram negative, or unstained by either crystal violet or safranin. Staining older cultures (over 48 hours) can lead to false Gram-variable results, probably due to changes in the cell wall with aging. Gram-indeterminate bacteria are best stained using acid-fast staining techniques. Examples include many species of Mycobacterium, including M. tuberculosis and M. leprae.
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English translation in: Brock, T.D. (1999). Milestones in Microbiology 1546–1940 (2 ed.). ASM Press. pp. 215–218. ISBN 1-55581-142-6..
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