Gram positive and gram negative bacteria fall under the study of microbiology. The difference between gram positive vs gram negative bacteria is based on their staining color during the gram stain test. Using the gram staining technique, bacteria have been differentiated into gram-positive and gram-negative bacteria.
The ability to differentiate bacterial cells is essential for various reasons. This host of reasons varies from diagnosing infection to checking food safety to identifying bacteria species responsible for some microbial activities.
All bacteria are prokaryotic single-celled organisms that lack a true nucleus. These organisms are microscopic and their structure comprises a cell wall, capsule, DNA, flagellum, cytoplasm, pili, and ribosomes. Bacteria can be differentiated using a number of molecular techniques such as PCR, genome sequencing, quantitative PCR, and mass spectrometry. However, bacteria can be differentiated with their phenotypic differences without getting into the molecular aspect.
Such phenotypic characteristics include their shape (bacilli or cocci), preference for high or low oxygen environments, and growth in particular nutrients. Bacterial species can therefore be broken down into broad groups based on the characteristic being studied. However, putting together the characteristics can narrow down the possible identities.
One such useful classification of a bacterium being gram-positive or gram-negative is based on the cell wall structure of the bacterium. Gram positive vs gram negative bacteria structure difference is attributed to their peptidoglycan layer. Hence, the difference of gram positive vs gram negative stain color due to the cell wall structure forms the basis of the gram staining technique.
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What are Gram-positive bacteria?
Gram-positive bacteria are those bacterial species that give a distinctive purple appearance when observed under a light microscope after the gram staining procedure. This purple color is due to the bacteria retaining the purple crystal violet stain in the thick peptidoglycan layer of the cell wall. Gram positive bacteria examples include all Streptococci, all Staphylococci, and some Listeria species.
What are Gram-negative bacteria?
Gram-negative bacteria are those bacterial species that give a pale reddish color when observed under a light microscope after the Gram staining procedure. This color is a result of the bacterial cell wall not being able to retain the crystal violet stain, hence, they are colored only by the safranin counterstain. Examples of Gram-negative bacteria examples include Pseudomonas species, Enterococci, and Salmonella species.
Gram positive vs gram negative bacteria color in gram staining
The gram staining technique was proposed by Hans Christian Gram in 1884 and has been used to distinguish the two types of bacteria. This differential staining technique differentiates bacteria based on the difference in their cell wall structures. The gram-positive bacteria have a thick layer of peptidoglycan in their cell wall and due to this, they retain the crystal violet dye. Whereas, the small layer of peptidoglycan in gram-negative bacteria is dissolved when alcohol is added during the gram staining process. Hence, the gram staining process distinguishes bacteria by identifying peptidoglycan found in the cell wall of the gram-positive bacteria. Let’s compare the Gram positive vs Gram negative color at each stage of gram staining.
The Gram stain procedure
Gram positive vs gram negative gram stain procedure is the same but with varying results. This is because the gram positive vs gram negative bacteria stain color varies at the end of the procedure. Here are the following steps carried out during the gram stain procedure:
Total Time: 8 minutes
Application of the Gram stain
Gently apply the crystal violet stain to the smear on the slide and leave for 1 minute. Then tilt the slide slightly and rinse gently with distilled or tap water.
Note: The crystal violet is a water-soluble dye, so it enters the peptidoglycan layer in the bacterial cell wall.
Addition of a mordant
The next step is to add a mordant (iodine) to the sample. Gently flood the smear with Gram’s iodine and leave for 1 minute. Then, tilt the slide slightly and rinse gently with distilled or tap water. The smear will then appear purple.
Note: The Gram’s iodine solution (iodine and potassium iodide) is added to the sample to form a complex with the crystal violet (crystal violet-Iodine (CV-Iodine) complex). The CV-Iodine complex is much larger and insoluble in water.
The next step is to decolorize the smear using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol in drops until the alcohol runs almost clear (5-10 seconds). Then rinse immediately with water to avoid over-decolorizing. Gram positive vs gram negative bacteria color differs because of this particular step.
Note: The decolorizer dehydrates the peptidoglycan layer, thus, shrinking and tightening it. As a result of this, in gram-positive bacteria, the large crystal violet-iodine complexes are unable to penetrate and escape the thick peptidoglycan layer. Thereby, resulting in purple stained cells. On the other hand, in gram negative bacteria, the outer membrane is degraded. Hence, the thin peptidoglycan layer is unable to retain the crystal violet-iodine complexes and the purple color is lost.
Gently flood the smear with safranin counterstain and leave for 45 seconds. Then, tilt the slide slightly and rinse gently with distilled or tap water.
Note: Safranin as a counterstain will enable the visualization of gram-negative bacteria without interfering with the observation of the purple stain in gram-positive bacteria. This is because it is weakly water-soluble. Hence, the counterstain will stain bacterial cells a light red.
Air dry or blot the slide dry on filter paper. Then, view the smear using a light microscope under oil immersion to examine the color and structure of the bacterial sample. The color of gram positive vs gram negative under microscope differs. Gram-positive bacteria will appear purple or blue while gram-negative bacteria will appear red or pink.
The diagram above shows a comparison of gram positive vs gram negative bacteria under microscope. Gram-positive bacteria appear purple or pale blue while gram-negative bacteria appear red or pink.
Differences between gram positive and gram negative bacteria
Gram positive bacteria
Gram negative bacteria
Gram stain reaction
They retain the crystal violet dye and stain purple
They can be decolorized to accept the counterstain and stain red
Cell Wall is smooth and 20-30 nm thick.
Cell Wall is wavy and 8-12 nm thin.
This layer is thick and multilayered in this bacteria
This layer is thin and single-layered in this bacteria
Present in many gram positive bacteria
Absent in gram negative bacteria
Lipopolysaccharide (LPS) content
Lipid and Lipoprotein content
Two (2) rings in basal bodies
Four rings (4) in basal bodies
Susceptibility to anionic detergents
Resistance to physical disruption
Cell wall disruption by lysozymes
Resistance to drying
Inhibition by basic dyes
Susceptibility to Streptomycin, Chloramphenicol, and Tetracycline
Resistance to Sodium Azide
Susceptibility to Penicillin and Sulfonamide
Occurs in the outer membrane
It is more prominent in this bacteria
It is less prominent in this bacteria
Cocci or spore-forming rods
Non-spore forming rods
Staphylococcus, Streptococcus, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Bacillus anthracis etc.
Escherichia, Salmonella, Neisseria gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae etc.
Gram positive vs gram negative bacteria structure
The difference between gram positive and gram negative bacteria is attributed to their cell wall structure. All gram-positive bacteria are bounded by a single lipid membrane and are therefore termed monoderm bacteria. However, there are a number of bacteria that are bounded by a single membrane but stain gram-negative such as the Mycoplasmas. This is because they lack a peptidoglycan layer or due to their inability to retain the gram stain because of their cell wall composition. Such bacteria show a close relationship to gram-positive bacteria.
Furthermore, typically all gram-negative bacteria are bounded by two membranes; a cytoplasmic membrane and an outer cell membrane. These bacteria with two membranes are termed diderm bacteria. The presence of an inner and outer cell membrane in gram-negative bacteria defines a new compartment which includes the periplasmic space or the periplasmic compartment. These diderm bacteria can be further differentiated between:
- The simple diderms lacking lipopolysaccharide
- Archetypical diderm bacteria where the outer cell membrane contains lipopolysaccharide
- The diderm bacteria where outer cell membrane is made up of mycolic acid
Gram positive vs gram negative bacteria difference in basal body rings varies as well. Gram-positive bacteria have only two basal body rings to support them, whereas gram-negative bacteria have four basal body rings. A surface layer called an S-layer is seen in both gram-positive and gram-negative bacteria. The S-layer is attached to the peptidoglycan in gram-positive bacteria, while the S-layer is attached directly to the outer membrane in gram-negative bacteria.
Discussed below are the key difference in the structure of Gram positive vs Gram negative bacteria.
Gram positive vs gram negative cell envelope
Let us compare the difference between gram positive vs gram negative cell envelope. In gram-positive bacteria, the cell envelope consists of two to three layers. These layers include the cytoplasmic membrane, cell wall, and in some, an outer capsule.
In gram-negative bacteria, the cell envelope consists of three layers. These layers include a thin peptidoglycan cell wall that is incorporated between an inner cytoplasmic cell membrane and an outer membrane.
Therefore, the key difference in the cell membrane of gram positive vs gram negative bacteria is the absence and presence of an outer membrane.
Gram positive vs gram negative cell wall structure
The difference between gram positive and gram negative bacteria cell walls includes the peptidoglycan layer and some matrix substances. In gram-positive bacteria, the cell wall is thick and takes up the crystal violet stain during the gram stain process, thus, staining purple. Whereas, in gram-negative bacteria, the cell wall is thinner and is more complex than the gram-positive bacteria cell wall. The cell wall of gram negative bacteria stains pink after the gram staining procedure.
Peptidoglycan is a constituent in the cell wall of bacteria. Let’s look at the peptidoglycan in gram positive vs gram negative cell walls.
The thickness of gram positive vs gram negative peptidoglycan varies. The peptidoglycan layer in gram-positive bacteria is about 20-80 nm thick. Whereas in gram-negative bacteria, the peptidoglycan layer is about 2-3 nm thin.
The thick peptidoglycan layer in gram-positive bacteria consists of a polysaccharide structure that possesses an equal amount of alternating N-acetylglucosamine (NAG) and N-Acetylmuramic acid (NAM) residues.
In gram positive bacteria, the peptidoglycan layer makes up about 95% of the cell wall whereas in gram negative bacteria the peptidoglycan layer constitutes as little as 5-10% of the cell wall.
Gram positive vs gram negative outer membrane
The cell wall of gram positive vs gram negative bacteria differs by the presence and absence of an outer membrane. All gram-positive bacteria are bounded by only a single-unit lipid membrane whereas gram-negative bacteria have an outer membrane and a cytoplasmic membrane. An outer membrane is absent in gram-positive bacteria whereas, in gram-negative bacteria, this membrane is located outside of the peptidoglycan layer. The outer membrane of the gram-negative cell wall is superficial to the peptidoglycan layer and adjacent to the cytoplasmic membrane. It is composed of a lipid bilayer and has large molecules known as lipopolysaccharides (LPS).
The lipopolysaccharides are anchored into the outer membrane of gram-negative cells and project into the environment from the cell. The chemical structure of lipopolysaccharides is usually distinct to specific bacterial subspecies. Moreso, this molecule plays a role in many of the antigenic properties of these gram-negative bacterial species.
LPS is made up of 3 different components namely:
- Lipid A that anchors the LPS into the outer membrane
- O-antigen or O-polysaccharide (the outermost part of the structure)
- The core polysaccharide
The outer membrane that surrounds gram-negative cells prevents many bodily defense mechanisms (such as white blood cells) from effectively eliminating an infection. Hence, gram-negative bacteria are better at causing diseases compared to gram-positive bacteria. They cause more serious illnesses, such as pneumonia and blood infections. Gram-negative bacteria are considered to be more harmful than gram-positive bacteria because they are usually resistant to many common medications. Hence, their infections are generally more difficult to treat with antibiotics.
The matrix substance in gram positive vs gram negative bacteria cell wall varies. In the cell walls of gram-positive bacteria, teichoic acids are the matrix substance found. These substances are only found in gram-positive bacteria. There are two main polymer types of these teichoic acids which include ribitol teichoic acids and glycerol teichoic acids. Among the two types, glycerol teichoic acids tend to be more widespread.
Glycerol teichoic acids are polymers of glycerol phosphate whereas ribitol teichoic acids are polymers of ribitol phosphate. These acids can only be found on the surface of several gram-positive bacteria. Furthermore, Lipoteichoic acid, a main component of the gram-positive cell wall renders an antigenic function. This lipid element is seen in the cell membrane of gram-positive bacteria and its adhesive properties help it to anchor to the membrane.
In gram-negative bacteria, porins are found in the outer membrane. Moreso matrix substances such as lipid and lipoproteins are very high in gram-negative bacteria compared to gram-positive bacteria. In the cell walls of some gram-negative bacteria, Braun’s lipoprotein (BLP, Lpp, murein lipoprotein, or major outer membrane lipoprotein) is found. These Braun’s lipoproteins are very small and are one of the most abundant membrane proteins in gram-negative cells. These substances are covalently joined to the peptidoglycan layer of gram-negative bacteria. They are incorporated into the outer membrane with their hydrophobic end.
Gram positive vs gram negative bacteria antibiotic resistance
The gram-positive bacteria are the primary producers of antibiotics but gram-negative bacteria, on the other hand, are more resistant to antibiotics compared to them. It has been proposed that the outer cell membrane in gram-negative bacteria has evolved to act as a protective mechanism against antibiotic selection pressure. Gram-positive bacteria lacking an outer membrane are more susceptible to antibiotics. Lysozyme, an antimicrobial enzyme can completely dissolve the cell wall of some gram-positive bacteria. This lysozyme attacks the bonds between N-acetylmuramic acid and N-acetylglucosamine in the cell walls of gram-positive bacteria.
The strong walls of gram-negative bacteria protect it and make it difficult for many antibiotics to attack the bacteria. Gram-negative bacteria have an edge over gram-positive bacteria in antibiotic resistance because they have other defenses apart from their outer membrane. Such defenses against antibiotic treatment include:
- Gram negative bacteria producing a chemical called Extended spectrum beta–lactamase (ESBL). This chemical can stop certain antibiotics from working effectively.
- Gram negative bacteria also get genetic material from other bacteria to be able to resist antibiotics.
Gram positive vs gram negative diagram
Below is the cell wall structure of gram-positive and gram-negative bacteria. The gram-positive and gram-negative bacteria difference in structure can be understood by comparing the two structures.
It is important to note that the cell wall structure of these bacteria affects the cell’s ability to retain the crystal violet stain used in the Gram staining procedure. The stain color retained can then be visualized under a light microscope.
From the gram positive vs gram negative diagram, it can be deduced that the two key features that lead to the differing visualization properties of Gram-positive and Gram-negative bacteria are:
- The thickness of the peptidoglycan layer
- Absence or presence of the outer lipid membrane.
Gram-positive bacteria possess a thick peptidoglycan layer and have no outer lipid membrane whereas Gram-negative bacteria possess a thin peptidoglycan layer and an outer lipid membrane.
Gram positive vs gram negative bacteria list
Gram positive bacteria
Gram negative bacteria
Listed above is a comparison table showing the gram positive vs gram negative bacteria examples
- Gram-positive bacteria are bounded by a single membrane and are known as monoderm bacteria whereas gram-negative bacteria are bounded by two membranes and are known as diderm bacteria.
- In gram-positive bacteria, the cell envelope consists of two to three layers. These layers include the cytoplasmic membrane, cell wall, and in some, an outer capsule.
- In gram-negative bacteria, the cell envelope consists of three layers. These layers include a thin peptidoglycan cell wall that is incorporated between an inner cytoplasmic cell membrane and an outer membrane.
- The differences between gram-positive and gram-negative bacteria include the thickness of the peptidoglycan layer and the presence or absence of the outer lipid membrane.
- Gram-positive bacteria have a thicker peptidoglycan layer in their cell wall structure while gram-negative bacteria on the other hand have a thin peptidoglycan layer in their cell wall.
- The peptidoglycan layer in gram-positive bacteria is about 20-80 nm thick, whereas in gram-negative bacteria, the peptidoglycan layer is about 2-3 nm thin.
- Following the gram stain test, the color of gram negative bacteria will appear pink or red while gram-positive bacteria will appear purple or blue under a microscope.
- The gram positive vs gram-negative bacteria color difference is based on their cell wall composition and the ability of the cell to retain the primary stain used in the Gram stain test.
- Molecules such as peptidoglycan, lipid, teichuronic acid, and teichoic acid are contained in the cell walls of gram-positive bacteria.
- In gram negative bacteria, the cell wall is composed of peptidoglycan and an outer membrane that is made up of protein, lipid, and lipopolysaccharides.
- One difference between gram positive and gram negative bacteria is the presence or absence of an outer membrane. Gram positive bacteria lack an outer membrane whereas gram negative bacteria have an outer membrane.
- Due to the presence of an outer membrane in gram negative bacteria, they are more resistant to antibiotics and cleaning agents than gram positive bacteria.