In microbiology, Gram positive and Gram negative are two broad classes of bacteria. The classification comes from the results of the Gram stain test, which in turn, depends on the nature of the bacterial cell wall. Here is a look at the differences between Gram positive and Gram negative bacteria and why telling them apart is important.
- Gram positive bacteria have a thick coating of peptidoglycan and stain purple with crystal violet.
- Gram negative bacteria lack this thick coating. They don’t retain crystal violet, so are stained red or pink with carbol fuchsin or safranin.
- But, some bacteria stain either as Gram positive or Gram negative, depending on conditions.
- The Gram stain helps lead to a medical diagnosis of an infection. Different treatments apply for controlling Gram positive and Gram negative bacteria.
The Gram Stain
Han Christian Gram devised the test that bears his name in 1884. Bacteria with a thick outer layer of peptidoglycan retain crystal violet stain and are Gram positive. Bacteria lacking this thick coating don’t retain the stain and are Gram negative. A second stain, called a counterstain, colors Gram negative bacteria.
The usual steps of the Gram stain protocol are applying crystal violet, adding iodine as a mordant to fix the dye, rinsing with alcohol, staining with safranin, and rinsing with water. Note both Gram positive and Gram negative bacteria are purple before the decolorizing step, which removes the crystal violet-iodine complex from Gram negative bacteria, but not from Gram positive bacteria (unless done improperly, which either leaves both types of bacteria purple or else decolorizes them both). Gram positive bacteria do pick up the counterstain, but the purple color overpowers it.
| Step of Staining | Gram Positive | Gram Negative |
|---|---|---|
| Crystal violet | purple | purple |
| Iodine | purple | purple |
| Alcohol | purple | colorless |
| Safranin | purple | red or pink |
Summary of Differences Between Gram Positive and Gram Negative Bacteria
The Gram stain differentiates bacteria based on the nature of their cell wall. In Gram positive bacteria, crystal violet forms a complex with iodine in the thick outer peptidoglycan layer. Alcohol dehydrates the layer and shrinks it. The crystal violet-iodine complex can’t escape the thick layer of Gram positive bacteria, but the thin layer of peptidoglycan in Gram negative bacteria can’t retain the dye complex.
While the Gram stain uses differences in the peptidoglycan layer to categorize bacteria, there are additional differences between Gram positive and Gram negative bacteria:
| Gram Positive | Gram Negative | |
|---|---|---|
| Gram stain | retain crystal violet (purple) | do not retain crystal violet (pink from counterstain) |
| Examples | Staphylococcus, Streptococcus, Bacillus | Escherichia, Salmonella, Neisseria |
| Antibiotic resistance | more susceptible to antibiotics | more resistant to antibiotics |
| Cell wall | single-layer, smooth cell wall | double-layer, wavy cell wall |
| Peptidoglycan layer | thick, sometimes multilayered, with teichoic acids | thin, usually single layer, teichoic acids absent |
| Cell wall thickness | 20 to 80 nanometers | 8 to 10 nanometers |
| Outer membrane | absent | usually present |
| Porins | absent | present in outer membrane |
| Mesosome | prominent | less prominent |
| Morphology | cocci or spore-forming rods | non-spore-forming rods (very few cocci species) |
| Flagella structure | two rings in basal body | four rings in basal body |
| Lipopolysaccharide | absent | present |
| Lipid content | very low | 20% to 30% |
| Toxins | exotoxins | exotoxins or endotoxins |
Gram Positive Bacteria
Gram positive bacteria have teichoic acid in the cell wall that aids infection and can cause disease. Some also contain mycolic acid in their cell walls, which gives the bacteria a waxy coating that helps protect them. Gram positive bacteria containing mycolic acid are called acid-fast bacteria because they require special staining for observation. Gram positive bacteria secrete toxic proteins called exotoxins. Antibiotics, such as penicillin, cloxacillin, and erythromycin treat more than 90% of Gram positive bacterial infections.
Gram Positive Rods
Most Gram positive rods (bacilli) are harmless microflora. Some cause infection, such as Bacillus anthracis (anthrax), Corynebacterium diptheriae (diptheria), and Listeria (listeriosis), Some Gram positive rods produce spores (e.g., Bacillus), while others do not (e.g., Listeria). Spores are much harder to kill than the bacteria and can persist in the environment for years.
Gram Positive Cocci
Gram positive cocci are spherical in shape. Mostly they exist as part of the human microbiota, but some cause disease when the conditions are right. For example, some strains of Staphylococcus aureus resist antibiotics, such as methicillin-resistant S. aureus or MRSA. Staphylococcus epidermidis sometimes infects tissue around medical implants. Streptococcus pyogenes can cause strep throat or eat flesh.
Gram Negative Bacteria
Gram negative bacteria contain lipopolysaccharide (LPS) that is not present in Gram positive cells. LPS is an endotoxin that can cause inflammation and septic shock. Like Gram positive bacteria, some (not all) Gram negative bacteria secrete exotoxins. Gram negative bacteria resist antibiotics. Typically, older antibiotics are more effective than new ones, but treatment often focuses on other methods.
Gram Negative Rods
Examples of pathogenic Gram negative rods include Salmonella (salmonellosis food poisoning) and Escherichia coli (food poisoning). Most Gram negative rods do not produce spores. An exception is the bacterium Sporomusa.
Gram Negative Coccobacillus
Some Gram negative bacteria have shapes intermediate between rods and spheres. Examples include Haemophilus and Acinetobacter. Haemophilus influenzae causes sinus infection, pneumonia, and meningitis. Acinetobacter infects wounds and causes pneumonia.
Gram Negative Cocci
There are relatively few species of Gram negative cocci. Examples include the anaerobic bacteria Acidaminococcus, Megasphera, and Veillonella. These are fecal flora that rarely cause disease. Veillonella also occurs in the human mouth. Bacteria belonging to the genus Neisseria are Gram negative cocci that do cause disease. For example, Neisseria meningitidis is a diplococcus (cells remain in pairs) that causes meningitis and septicemia. N. gonorrhoeae causes gonorrhea. Moraxella catarrhalis is another disease-causing Gram negative diplococcus. It causes meningitis, upper respiratory infections, endocarditis, and ear infections.
Gram-Variable and Gram-Indeterminate Bacteria
Some bacteria yield a mixed pattern of purple and pink cells following the Gram stain. For example, this occurs in cultures of Bacillus, Butyrivibrio, or Clostridium, because the thickness of the peptidoglycan layer changes during cell growth. But, in all cell cultures, the age of the culture affects the results of the Gram stain.
Gram-indeterminate bacteria are neither Gram positive nor Gram negative. For example, many species of Mycobacterium and bacteria of the genus Mycoplasma lack cell walls, so they do not stain. Also, these bacteria resistant antibiotics that target cell wall synthesis. The Archaea have variable cell wall structures, so the Gram stain is not helpful in distinguishing them.
References
- Beveridge, T. J. (2001). “Use of the Gram stain in microbiology”. Biotechnic & Histochemistry. 76(3): 111–118. doi:10.1080/714028139
- Colco, R. (2005). “Gram Staining”. Current Protocols in Microbiology. Appendix 3 (1): Appendix 3C. ISBN 978-0471729259. doi:10.1002/9780471729259.mca03cs00
- Gram, Hans Christian (1884). “Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten”. Fortschritte der Medizin (in German). 2: 185–189. English translation in: Brock, T. D. (1999). Milestones in Microbiology 1546–1940 (2nd ed.). ASM Press. ISBN 978-1-55581-142-6.
- Silhavy, T. J.; et al. (2010). “The Bacterial Cell Envelope.” Cold Spring Harbor Perspectives in Biology. 2(5). doi:10.1101/cshperspect.a000414
- Swoboda, Jonathan G.; et al. (2009). “Wall Teichoic Acid Function, Biosynthesis, and Inhibition.” ChemBioChem. 11(1): 35–45. doi:10.1002/cbic.200900557