Bacteria are ubiquitous microscopic organisms that exist all around us. While many bacteria are harmless or even beneficial, some can cause serious infections and diseases in humans. An important technique used to identify and classify different types of bacteria is the Gram stain.
Introduction to Gram Staining
The Gram stain is a differential staining procedure that divides bacteria into two major groups based on cell wall structure – Gram positive and Gram negative. This staining method was developed in 1884 by Danish physician Hans Christian Gram and remains one of the most useful diagnostic tools in microbiology.
The Gram stain works by using two different stains – a purple crystal violet dye and a red safranin counterstain. Bacteria with a thick peptidoglycan cell wall retain the crystal violet dye, appearing purple under the microscope. These bacteria are classified as Gram positive. Bacteria with a thin cell wall do not retain the crystal violet dye and instead take up the safranin counterstain, appearing pink/red. These bacteria are classified as Gram negative.
Gram Positive Bacteria
Gram positive bacteria have a multi-layered, thick peptidoglycan cell wall that retains the purple crystal violet dye. Some of the most medically important Gram positive bacteria include:
- Staphylococcus aureus – causes skin infections, food poisoning, pneumonia
- Streptococcus pyogenes – causes strep throat, skin infections
- Streptococcus pneumoniae – causes pneumonia, meningitis, sinusitis
- Enterococcus faecalis – causes UTIs, bacteremia, endocarditis
- Clostridium difficile – causes antibiotic-associated diarrhea
- Listeria monocytogenes – causes food poisoning (listeriosis)
- Corynebacterium diphtheriae – causes diphtheria
- Bacillus anthracis – causes anthrax
In addition to medically relevant species, many non-pathogenic Gram positive bacteria play important roles in research, biotechnology, food production, and environmental remediation. For example, Bacillus subtilis is a model organism for studying bacterial physiology and genetics. Lactobacillus species are used as probiotics and in food fermentation. Clostridium acetobutylicum produces acetone and butanol for industrial uses.
Gram Staining Procedure
Performing a Gram stain involves four main steps:
- Apply primary stain (crystal violet) – Crystal violet dye is applied to a heat-fixed smear of bacteria for 1 minute. The dye penetrates through the thick peptidoglycan layer of Gram positive cell walls.
- Apply mordant (iodine) – An iodine solution is applied as a mordant for 1 minute. Iodine forms a complex with crystal violet, trapping the primary stain inside the cell.
- Decolorize with alcohol – The slide is rinsed briefly with a decolorizing agent, usually ethanol or acetone. This washes away stain from Gram negative bacteria with thin peptidoglycan layers.
- Counterstain (safranin) – A secondary counterstain, usually safranin, is applied for 1 minute. This stains any bacteria not retaining crystal violet pink or red.
After rinsing, the slide is ready for microscopic examination. Gram positive bacteria will appear purple while Gram negative bacteria appear pink.
How Gram Positive Bacteria Retain Crystal Violet Dye
Gram positive cell walls are structurally and chemically well-suited to retain the crystal violet-iodine complex through the decolorization step:
- Thick peptidoglycan layer – The main component of Gram positive cell walls is a multi-layered, mesh-like polymer called peptidoglycan. This provides a more extensive matrix for trapping and retaining stain.
- Teichoic acids – These polymers with phosphate groups increase cell wall permeability and act as binding sites for crystal violet dye.
- Lack of outer membrane – Gram negative bacteria have an outer membrane that prevents uptake of crystal violet past the peptidoglycan layer.
The combined thickness and chemical properties of Gram positive cell walls enable them to retain the primary stain even after the decolorizing solvent step.
Gram Negative Bacteria
In contrast to Gram positives, Gram negative bacteria have a thinner peptidoglycan layer and an outer membrane that prevents crystal violet retention:
- Thin peptidoglycan layer – The peptidoglycan layer is only 1-3 molecules thick and sandwiched between the inner and outer membranes.
- Outer membrane – This additional lipid bilayer acts as a selective permeability barrier, blocking passage of crystal violet.
- Lack of teichoic acids – Without these polymers, there are fewer binding sites for crystal violet dye.
During the decolorization step, the crystal violet-iodine complex washes away from Gram negative cell walls. The counterstain safranin then replaces it, staining the cells pink/red.
Some examples of medically relevant Gram negative bacteria include:
- Escherichia coli – causes food poisoning, UTIs
- Klebsiella pneumoniae – causes pneumonia, UTIs
- Pseudomonas aeruginosa – causes wound infections, pneumonia
- Salmonella enterica – causes food poisoning, typhoid fever
- Helicobacter pylori – causes stomach ulcers
- Neisseria gonorrhoeae – causes gonorrhea
- Neisseria meningitidis – causes meningococcal meningitis
Other Differential Stains
While the Gram stain is one of the most useful and widely used differential stains, there are a few other stain procedures that can help identify or characterize bacteria:
- Acid-fast stain – Used to detect mycobacteria like Mycobacterium tuberculosis. Mycobacteria have a waxy cell envelope that resists decolorization by acid or alcohol.
- Endospore stain – Used to visualize bacterial endospores, which are dormant structures produced by some Gram positive bacteria like Bacillus and Clostridium genera. Endospores resist staining but can be visualized using a malachite green dye.
- Capsule stain – Used to detect a capsule structure surrounding pathogenic bacteria like Klebsiella pneumoniae and Streptococcus pneumoniae. Capsules help bacteria evade phagocytosis.
- Flagella stain – Used to observe flagella, whip-like appendages used by some bacteria for motility. Silver stains applied to fixed cells can darkly stain flagella for visualization.
Clinical Relevance of Gram Staining
Gram staining remains one of the most useful, inexpensive, and rapid diagnostic tests in clinical microbiology. Advantages of Gram staining include:
- Provides immediate guidance for empiric antibiotic selection before culture results are available. Gram positives and negatives generally require different antibiotic classes.
- Helps identify potential pathogens from normally sterile sites like blood, spinal fluid, or joint fluid.
- Distinguishes specific pathogens like Gram positive cocci in clusters (Staphylococcus) versus chains (Streptococcus).
- Rapidly diagnoses infections like pneumococcal pneumonia, meningococcal meningitis, or gonorrhea.
- Can be performed from primary patient specimens in as little as 1-2 minutes.
However, Gram staining cannot conclusively identify a specific bacterial species or strain. Supplemental biochemical, culture, antigen detection, or genetic tests are required for complete identification. Gram stain results must always be interpreted within the context of the patient’s clinical presentation.
Example of Interpreting Gram Stain Results
As an example, consider a patient with symptoms of a lower respiratory infection. A sputum specimen is collected and a Gram stain reveals numerous Gram positive cocci in chains:
| Test | Result | Interpretation |
|---|---|---|
| Gram stain morphology | Gram positive cocci in chains | Suggests Streptococcus pneumoniae, a common cause of pneumonia |
The Gram stain provides a preliminary clue that the organism could be the bacterium Streptococcus pneumoniae, which causes pneumococcal pneumonia. This Gram positive diplococcus grows in chains. However, culture and further testing are still needed to confirm S. pneumoniae and determine antibiotic susceptibility.
Limitations of Gram Staining
While invaluable, Gram staining does have some limitations:
- Does not confirm identification of a bacterial species or strain
- Some organisms have variable Gram staining (e.g. Enterobacteriaceae)
- Prior antibiotic therapy can alter Gram stain appearance
- Requires viable, morphologically intact bacteria in moderate numbers
- Operator technique can influence results
- Not helpful for pathogens like Treponema pallidum (syphilis) or Chlamydia/Mycoplasma
Gram staining must be correlated with culture, biochemical, and other test results for definitive diagnosis. Negative Gram stains do not rule out infection. The limitations of the technique must be recognized to avoid clinical errors.
Conclusion
Developed in the late 19th century, Gram staining remains one of the pillar techniques of diagnostic microbiology. It divides diverse bacterial species into two main categories – Gram positive and Gram negative – based on properties of their cell walls. Though not definitive alone, Gram staining provides rapid and clinically useful information to help guide treatment and additional testing. Along with culture and biochemical methods, Gram stain remains integral to identification and characterization of bacterial pathogens.
