Bacteria can be differentiated into two major groups based on differences in the structure of their cell walls. The Gram stain procedure is used to differentiate between gram positive and gram negative bacteria. This staining procedure was developed by the Danish physician Hans Christian Gram in 1884. The Gram stain allows bacteria to be classified based on whether they retain the crystal violet stain or take up the counterstain safranin.
Gram Positive Bacteria
Gram positive bacteria have a thick peptidoglycan cell wall that retains the crystal violet stain. Common examples of gram positive bacteria include:
- Staphylococcus aureus
- Streptococcus pneumoniae
- Bacillus anthracis
- Listeria monocytogenes
- Clostridium botulinum
In the Gram staining procedure, gram positive bacteria stain purple. This is because their thick peptidoglycan layer retains the crystal violet primary stain. An iodine solution is applied after the crystal violet to form a complex that further stabilizes the primary stain. A decolorizing agent is then used to wash away any excess stain. Lastly, a secondary counterstain of safranin is applied which will stain any bacteria that did not retain the crystal violet complex pink. However, since the gram positive cell wall retains the crystal violet, these bacteria remain purple under the microscope.
Gram Negative Bacteria
Gram negative bacteria have a thin peptidoglycan layer that does not retain the crystal violet stain. The gram negative cell envelope contains an additional outer membrane composed of lipopolysaccharides and phospholipids. Common examples of gram negative bacteria include:
- Escherichia coli
- Salmonella enterica
- Pseudomonas aeruginosa
- Moraxella catarrhalis
- Helicobacter pylori
During the Gram staining procedure, the crystal violet is unable to penetrate the thin peptidoglycan layer of gram negative bacteria. When the decolorizing agent is applied, it washes away the crystal violet stain from gram negatives. Then, the secondary safranin counterstain stains the gram negative cells pink under the microscope.
Key Differences
Here is a summary of the key differences between gram positive and gram negative bacteria:
| Gram Positive | Gram Negative |
|---|---|
| Thick peptidoglycan cell wall | Thin peptidoglycan cell wall |
| Retains crystal violet stain | Does not retain crystal violet stain |
| Stains purple | Stains pink |
| Examples: Staph, Strep, Bacillus, Clostridium | Examples: E. coli, Salmonella, Pseudomonas, Moraxella, Helicobacter |
Cell Wall Composition
The differences in cell wall structure between gram positive and gram negative bacteria account for their differing Gram stain properties. Here is a more in-depth look at the cell wall composition of each type of bacteria:
Gram Positive Cell Wall
The gram positive cell wall consists of:
- Thick peptidoglycan layer – makes up 90% of the gram positive cell wall
- Teichoic acids – polysaccharides linked to lipids in the plasma membrane
- Lipids and proteins – intricately associated with the peptidoglycan
This thick mesh-like peptidoglycan layer gives rigidity to the gram positive cell wall. It also has high affinity for crystal violet stain, thus causing gram positives to appear purple after Gram staining.
Gram Negative Cell Wall
The gram negative cell envelope consists of:
- Thin peptidoglycan layer – makes up only 10% of the cell envelope
- Outer membrane – contains lipopolysaccharides and phospholipids
- Periplasmic space – gel-like matrix between inner and outer membranes
The outer membrane of gram negative bacteria forms a hydrophilic permeability barrier that inhibits entry of crystal violet and other hydrophobic molecules. The thin peptidoglycan layer cannot retain the primary stain, causing gram negatives to take on the secondary safranin counterstain.
Clinical Relevance
The Gram stain procedure remains an important diagnostic tool in clinical microbiology. Determining whether a bacterial isolate is gram positive or gram negative provides valuable preliminary information about its identity and potential treatment options. Some key clinical correlations are:
- Gram positives cause skin infections (e.g. Staph) and respiratory infections (e.g. Strep).
- Gram negatives cause urinary tract infections (e.g. E. coli), bloodstream infections (e.g. Salmonella), and gastrointestinal infections (e.g. Helicobacter).
- Vancomycin antibiotic is effective against gram positives but not gram negatives.
- Third-generation cephalosporins have activity against many gram negatives.
- Decolorization resistance helps identify mycobacteria like M. tuberculosis.
Given the widespread clinical importance of staphylococci, streptococci, enterics, and pseudomonads, the Gram stain remains one of the most useful tests in the microbiology lab. Both gram positive and gram negative bacteria can potentially cause serious infections, so identify them quickly and accurately is critical.
Other Differential Stains
While the Gram stain is one of the most frequently used differential stains in microbiology, there are a variety of other staining procedures that provide helpful biochemical information about bacteria:
Acid-Fast Stain
The acid-fast staining procedure identifies mycobacteria, including Mycobacterium tuberculosis. Mycobacteria have a waxy cell envelope that resists decolorization by acid-alcohol solutions. Acid-fast bacteria stain red or pink after carotenoid dye treatment.
Capsule Stain
Bacterial capsules can be visualized using stains like nigrosine orAnthony’s capsule stain. Capsules appear as clear halos surrounding bacterial cells when viewed microscopically.
Spore Stain
Endospores produced by genera like Bacillus and Clostridium can be visualized using malachite green, safranin, and heat methods. The spores stain green, while the vegetative cells stain pink.
Flagella Stain
Flagella staining uses dyes like tannic acid or silver salts to visualize bacterial flagella. This reveals whether a bacterial species is motile or non-motile based on flagella morphology.
Biofilm Staining
Biofilms are comprised of clusters of bacteria encased in a polymeric matrix. Biofilms can be stained with dyes like crystal violet or safranin for microscopic examination and quantification.
Conclusion
In summary, the Gram staining procedure remains an invaluable diagnostic tool for characterizing bacterial isolates in the microbiology laboratory. The ability of bacteria to retain the crystal violet-iodine complex versus being decolorized and taking up the safranin counterstain provides key information about cell wall structure and composition. Gram positive bacteria stain purple due to their thick peptidoglycan layer, while gram negative bacteria stain pink due to their outer membrane and thin peptidoglycan. Identification of bacteria as gram positive or gram negative guides appropriate treatment and highlights the ongoing clinical significance of this differential staining technique.
