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Gram staining: Everything you need to know

Gram Staining

Gram staining isn’t something you’ll need to do every day, but it’s an important tool in microbiology that can aid with the identification of certain bacteria and microbes.

As such, it’s important to know everything you can about this technique so you have it in your repertoire when needed.

This guide will walk you through the ins and outs of gram staining so you can be an expert on this technique when you need it most.

Understanding the basics:

Gram stain is a technique that uses differential staining of bacterial cells based on the presence or absence of certain cell wall components.

It was developed in 1882 by Hans Christian Gram and has been in use ever since, though it’s usefulness is being increasingly called into question.

The technique involves smearing a thin layer of bacteria onto an agar plate and then applying either crystal violet or safranin for light-sensitive dyes.

When an alcohol-based iodine solution is applied after the two dyes, only those bacteria with a particular type of cell wall will show up as purple; other strains will remain colorless.

There are three types of Gram positive bacteria: cocci (which have round shapes), bacilli (rod shaped) and spirilla (curved). And there are four types of Gram negative: cocci, bacilli, spiral, and helical.

The use of gram stains as a diagnostic tool has declined due to its inability to tell different strains apart from one another and its reliance on potentially unreliable chemical agents.

Nevertheless, when it works properly, the technique can be very useful because if you know what type of bacterial cells you’re dealing with you can treat them accordingly – i.e., penicillin for streptococcus infections or erythromycin against mycoplasma.


It is important to remember that the procedure for performing a gram stain can vary slightly depending on the type of bacteria you are trying to identify.

For example, Gram-positive organisms require a different procedure than Gram-negative organisms.

Once an appropriate technique has been chosen and applied to the organism under investigation, take note of how it reacts to the following chemicals: Crystal violet (CVI), which kills cells that retain this color; iodine (I2), which kills cells with this color as well as releases purple porphyrins from cell walls; and safranin (SA), which causes some cells with this color to fluoresce under UV light.

The difference in staining pattern between Gram-positive and Gram-negative bacteria is used by medical professionals to make a distinction between these two types of organisms.

There are many methods of Gram staining but they all have one purpose: distinguishing between Gram-positive and Gram-negative bacteria.

Practical uses:

Gram staining is a bacteriological technique that uses a mixture of crystal violet, iodine, and safranin.

The cells are then flooded with water or alcohol. Under the microscope, different structures can be seen based on the amount of stain in the cell.

This provides valuable information on what kind of bacteria it is and which antibiotics will work best against it.

A variety of organisms can be identified using this technique including gram-positive cocci, gram-negative bacilli, yeast, and some parasites.

While an easy way to tell the difference between two types of bacteria is by their size (gram-positive bacteria are smaller), this is not always an accurate method as there are many other factors that contribute to size such as nutritional state.

For example, large grampositive cocci could actually be Listeria monocytogenes while small gram-positive cocci could actually be Staphylococcus aureus.

In order to make an accurate diagnosis, multiple tests are performed in order to pinpoint the exact organism present.

However, sometimes even these techniques fail to identify the correct microbe because one organism may have more than one test.

It is also possible for certain bacteria to change into another type when conditions change, so it is important for clinicians to know if a patient has been exposed to any recent changes in environment such as a new person or animal coming into contact with them.

Tips and tricks:

Gram staining is a microbiological technique for differentiating bacteria. It works by using the chemical properties of crystal violet and iodine on the outer surface of bacterial cells.

The crystal violet stains only gram-positive bacteria purple, but not gram-negative bacteria. This allows for more accurate identification of bacteria.

When performed correctly, it can help identify antibiotic resistant strains of bacteria.

The reason that gram-negative bacteria are unharmed by the stain may be due to their cell wall structure. A major structural difference between gram-positive and gram-negative bacteria is in their lipid membrane layer.

In general, gram-negative bacteria have two layers: an inner phospholipid bilayer membrane and an outer lipopolysaccharide (LPS) coating.

These serve as a barrier to outside interference from other chemicals or pathogens.

In contrast, gram-positive bacteria have one single lipid bilayer membrane without any LPS covering because they produce peptidoglycan instead of LPS.

As a result, when exposed to iodine vapor during the process of preparing slides with sections of tissue containing both gram-positive and gram-negative bacteria, only the former are stained dark blue; whereas the latter remain unstained.

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