Gram-positive vs Gram-negative

Gram-positive vs Gram-negative, what’s the difference and how do I tell which is which?

Gram-positive is a type of bacteria that have a thick, multilayered cell wall and no outer cell membrane. They stain purple when you perform a Gram stain on them.

Gram-negative is a type of bacteria that have a thin, single-layered cell wall and do have an outer cell membrane. They stain red or pink when you perform a Gram stain on them.

One way to help distinguish between the two different types is to associate the beginning letters of the words with a corresponding color attribute.

So gram-positive bacteria stain purple, and gram-negative bacteria do not.

During Gram staining both bacteria are stained with a purple dye, but the gram-negative does not retain it. So, you could also associate that concept to their names as well.

So gram-positive (plus) bacteria have the purple stain, and gram-negative bacteria (minus) do not have the purple stain.

Of course, there is much more to these two bacterial types than that, so let’s delve a bit deeper into both types.

Gram-positive vs Gram-negative Background Information

In order to understand the difference between the two different types of bacteria, we must first understand bacteria itself. Let’s start at the very beginning.


Except for viruses, all living organisms are made up of cells. Cells are the smallest unit of life that can self-replicate on their own, and they are often called the “building blocks of life.”

Cells can be divided even further into two major types depending on their structure: eukaryotes and prokaryotes.

Eukaryotic cells are cells that do have a nucleus and membrane-bound organelles. Examples of eukaryotes would be humans, animals, and plants. These organisms can be single-celled or multi-celled.

Prokaryotic cells are cells that do not have a nucleus and membrane-bound organelles. Examples of prokaryotes would be bacteria and archaea. These organisms are single-celled.


All organisms are classified by their taxonomic rank, and they fall into three domains (the highest taxonomic rank in the classification system): Archaea, Bacteria, and Eukarya.

Archaea and Bacteria are both prokaryotes (no nucleus or membrane-bound organelles), and Eukarya are eukaryotes (they have a nucleus and membrane-bound organelles).

The domain Bacteria contains bacteria as the name suggests.

Gram-positive vs Gram-negative Bacteria

Bacteria can be defined as tiny, single-celled organisms that make up the domain Bacteria.

They (along with viruses) are considered to be microbes. Microbes are organisms that are so tiny they cannot be seen with the naked eye. As a frame of reference think of the fact that one gram of soil contains roughly 40 million bacteria. That is a lot of bacteria!

Many bacteria play an important role in the human body, but there are some that are very harmful to humans. We will discuss that more later on in the article.

Bacterial Reproduction

Bacteria reproduce by a process called binary fission. Binary fission is where a single entity breaks in half into two identical daughter cells. These two daughter cells are clones of the parent cell and have the same genetic makeup as their parent cell.

Bacterial Structure

Bacteria are single-celled organisms so when you look at the structure of bacteria, you are really looking at the structure of a bacterial cell. Since bacteria are so small, they have much simpler structures in their cells than other cell types (such as the eukaryotic cells in humans).

Their small size is also why bacteria do not need membrane-bound organelles. Organelles are simply tiny membrane-bound structures that perform specific functions in a cell (like a nucleus).

Most bacterial cells consist of a nucleoid, a capsule, a cell wall, a cell membrane, cytoplasm, a cytoskeleton, a chromosome, flagella and pili, vesicles, plasmids, and ribosomes.

Bacterial structures

Most (but not all) bacterial cells have a capsule which consists of polysaccharides (long chain carbohydrates) that help protect the cell. It also helps the cell to adhere to surfaces.

Since they do not have a nucleus, bacterial cells have a nucleoid instead. The nucleoid is the region where the cell stores its DNA. The key difference between a nucleus and a nucleoid is that a nucleus is membrane-bound, but a nucleoid is not membrane-bound. It’s is merely a region.

The cell wall is the layer that helps give a cell it’s shape. It lays right outside the cell membrane, but beneath the capsule. It is made up of peptidoglycan (also known as murein).

The cell wall is one of the main differences between gram-positive and gram-negative bacteria. Gram-positive bacteria have a thick absorbent cell wall with a cell membrane beneath, and gram-negative bacteria have a thin cell wall sandwiched between two cell membranes.

The cell membrane (also called a plasma membrane or cytoplasmic membrane) surrounds the cytoplasm of the cell. It is semi-permeable and protects the inner structures of the cell. It is made primarily of phospholipids.

The cell membrane is another key difference between gram-positive and gram-negative bacteria. Both have a cell membrane, but gram-negative bacteria have two cell membranes (gram-positive bacteria only have one).

These two cell membranes are often referred to as the inner and outer cell membranes.

Gram-positive bacteria have an inner cell membrane, then a thick cell wall, and then the outer capsule (in that order). So, gram-positive bacteria only have an inner cell membrane.

Gram-negative bacteria have an inner cell membrane, then a thin cell wall, then an outer cell membrane, and then the outer capsule (in that order). So, gram-negative bacteria have both an inner and outer cell membrane.

Cytoplasm is the clear, thick fluid that contains the cell’s structures and internal components (like ribosomes, and plasmids).

The cytoskeleton is not always present in all bacterial cells, but it is in most of them. It gives the cell its shape, and it’s made up of microfilaments and microtubules.

Bacterial cells typically only have one chromosome (there is a bacterium that has two though). A chromosome is a DNA molecule that stores all or part of the bacteria’s genetic makeup.

Flagella and pili help with the bacterial cell’s motility. Flagella are tall and “tail” shaped. Pili are short and “hair” shaped. In gram-positive bacteria the flagella have two rings in their basal body, and in gram-negative bacteria the flagella have four rings.

Vesicles are fluid-filled sacs that help transport cellular material within the bacterial cell.

Plasmids are small molecules of DNA that replicate, and ribosomes produce proteins in the bacterial cell.

Bacterial Shapes

Now that we understand what bacteria structurally consist of, let’s look at their basic shape and appearance; so that we can better understand the differences between gram-positive and gram-negative bacteria.  Most bacteria can be classified into three basic shapes (also known as their morphology).

These three shapes are rod (bacillus), spherical (coccus), and spiral (spirillum).

Rod bacteria are shaped like rods as their name implies. Spherical bacteria are shaped like spheres, and they are sometimes flattened. Spiral bacteria are spiral-shaped, and they range from gently curved to tightly wound (corkscrew) shaped.

Bacterial Arrangements

Bacteria can be even further classified by their arrangements. Most bacteria aggregate in specific arrangements. These can be paired (diplo), clusters (staphylo), chains (strepto), groups of four (tetrad), groups of eight (sarcina), and so forth.

Bacterial shapes arrangements

An example would be streptococcal pharyngitis (which is strep throat). The bacteria that cause strep throat are part of the species of bacteria known as Streptococcus. If you break down the name to strepto and coccal, you can determine the bacteria’s shape and arrangement. Strepto means the bacteria arrange themselves in chains, and coccal refers to the bacteria’s spherical (coccus) shape.

Gram-positive Bacteria vs Gram-negative Bacteria Gram Staining

So now that we understand the basic shapes and structural differences, we can better understand Gram staining.

Gram staining was invented by Hans Christian Gram, and it’s sometimes referred to as Gram’s method. This process is where gram-positive and gram-negative bacteria derived their names. Since bacteria are so tiny, Gram staining is used to determine the bacterial type (positive or negative).

This is incredibly important for treating ailments caused by bacteria. Different bacterial types react in different ways to different treatments, so we need to know the bacterial type to be able to know the best treatment option.

The Gram staining process

First, you obtain the bacteria from a culture (an isolated sample). Then using an inoculation loop, you smear the bacteria sample onto a glass slide.

Then you heat-fix the bacteria to the slide so that it won’t wash away when you stain it. This is typically done by passing the slide over an open flame (like a Bunsen burner) a few times to ensure that the bacteria adhere to the slide.

Next, you apply a few drops of crystal violet dye (which is purple in appearance as the name implies) to the bacteria. The crystal violet dye penetrates all the layers of the bacterial cell (regardless of type) and stains it purple.

Next, you apply a few drops of iodine to the bacteria. The iodine binds to the crystal violet dye and makes the molecules appear larger (and therefore more visible).

Then, you wash the slide with alcohol which is a destainer. This is where the difference between the two cell walls starts to come into play. When you wash with iodine, the cell wall in both types of bacteria starts to shrink and dissolve. This is because alcohol is incredibly drying.

Because the gram-negative bacteria have such a thin cell wall, the crystal violet dye (and the bonded iodine) gets washed away. The gram-positive bacteria have a thick cell wall, so the dye is retained.

The alcohol also shrinks and dissolves the capsule in both types of bacteria as well as the outer cell membrane in the gram-negative bacteria. Remember, there is no outer cell membrane in gram-positive bacteria to affect.

Next, you apply a reddish pink dye called safranin (sometimes fuchsine is used) to the bacteria. The dye molecules bind with the phospholipids in the cell membrane (the inner one that is still left).

Now, when you look under a microscope the gram-positive bacteria will appear purple, and the gram-negative bacteria will appear red or pink. Gram-positive bacteria actually have both colors of dye in them, but the red/pink color is masked by the purple dye.

Gram-positive vs Gram-negative Bacteria and Antibiotic Resistance

Now that we know how to identify gram-positive and gram-negative bacteria, we can look at the practical applications of why we would need to be able to identify them in the first place.

One of the most important ways that bacteria play a part in human biology relates to antibiotic resistance.

Bacteria in Humans

Bacteria have been around for billions of years. They can inhabit a wide range of locations including humans, soil, and even radioactive waste.

Bacteria also outnumber humans in more ways than one. There are more bacteria than there are animals and plants combined. We have more bacterial cells than human cells in our body. The largest number of bacteria in the human body are in our gut flora (in our digestive tract).

Most of the bacteria that we come in contact with are made harmless by our immune system. Some bacteria (such as those in our gut) are even necessary for our body to function effectively.

The number of beneficial bacteria far outnumber their counterparts. Less than 100 species of bacteria are known to cause infectious disease in humans, but several thousand species thrive in our digestive system.

Pathogenic bacteria

The problems arise when we come in contact with pathogenic bacteria. Pathogenic bacteria are bacteria that cause diseases (such as tuberculosis or pneumonia). Tuberculosis is the most common of the fatal diseases caused by bacteria, and it kills about 2 million people every year.

So how does this relate to gram-positive vs gram-negative bacteria?

Since gram-positive bacteria have a thick, multilayered cell wall, they have more peptidoglycan. In fact, gram-positive bacteria can be as much as 20 times thicker than their counterpart gram-negative bacteria.

Due to its thicker cell wall (and lack of an outer cell membrane), gram-positive bacteria can more readily absorb a lot of material. The cell wall (made up of peptidoglycan) has a lot of space in it and is mesh-like. Since there is more space (since it’s thicker) and there is one less boundary protecting it (the outer cell wall) gram-positive bacteria can absorb a lot very easily.

In contrast, gram-negative bacteria have a very thin cell wall and less peptidoglycan (so therefore less space). They also have an additional cell membrane (the outer cell membrane) to help protect them. The outer cell membrane gives the bacteria a lot more control over what enters the cell, because they have two opportunities to block material. This makes them incredibly difficult to penetrate.

A common correlation is to think of gram-positive vs gram-negative bacteria as chain mail vs a bulletproof vest. The chain mail is thick with a lot of holes and sharp weapons could easily pass through the large links (even though it does protect you somewhat). In contrast, the bulletproof armor is thin but is comprised of many layers and sharp weapons would have a hard time piercing it.

So, this means that gram-positive bacteria have a thick (but absorbent) cell wall, and gram-negative bacteria have a thin (but impenetrable) cell wall.

This is where antibiotics come in. Because gram-negative bacteria have such an impenetrable cell wall, they are antibiotic resistant, while gram-positive bacteria have an absorbable cell wall that is better able to take antibiotics.

This makes a huge difference when treating diseases. Basically, antibiotics that would work for gram-positive bacteria would have no effect on gram-negative bacteria.

Not only are gram-negative bacteria antibiotic resistant, but they can pass on their resistance to either the same strain or even other strains of gram-negative bacteria.

And all bacteria can become antibiotic resistant. If a bacterium can develop a mutation that enables it to survive antibiotics, it can then reproduce and produce offspring that have the same defensive characteristics.

Due to our high use of antibiotics in our food sources and our constant prescribing of antibiotics we are now looking at a whole new generation of “superbugs” that are antibiotic resistant.

This is also why when your doctor tells you to take an antibiotic for a certain period, you should never quit taking your medication simply because you feel better. This is one of the ways that bacteria can become antibiotic resistant because the bacteria were not fully treated the first time around.

New advancements

No new antibiotics have been released for commercial use in decades (since around 1987). However, scientists have been working hard at creating new antibiotics, and the results are promising.

One of the methods used to create new antibiotics involves using bacteria found in soil. Scientists have been able to produce these antibiotics from the chemicals generated by the bacteria found in the soil.

One antibiotic, teixobactin, received a lot of press when it was discovered in 2015. It works on gram-positive bacteria and is not harmful to human tissue cells. Its discovery was the beginning of a whole new class of antibiotics.

Scientists were able to grow a previously unculturable class of bacteria (which produces the antibiotic). It was considered ground-breaking because the antibiotic was shown to kill strains of the bacteria that cause staph infections and tuberculosis.

However, many of the antibiotics that have been discovered still have to pass human testing and FDA approval. Thankfully every day we are one step closer to having new antibiotics available for widespread commercial use.

Final Thoughts on Gram-positive vs Gram-negative Bacteria

Initially, it can be hard to remember how to distinguish between gram-positive vs gram-negative bacteria. One way to remember the difference between them is to associate the names of the bacteria with the color of their Gram stain.

During Gram staining, both bacteria are washed with a crystal violet dye. However, gram-positive bacteria retain their purple dye, and gram-negative bacteria do not retain their purple dye (they stain red or pink).

The main difference is that gram-positive bacteria have a thick, multilayered cell wall and no outer cell membrane, and gram-negative bacteria have a thin, single-layered cell wall and do have an outer cell membrane.

Due to their cell membranes, gram-negative bacteria are more antibiotic resistant, and gram-positive bacteria are more readily able to be treated with antibiotics.


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