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Developments and Advancements in Staining Over Time

The first cell stain, as we far as we know today was saffron, which was used in the late 17th century by Dutchman Anton van Leeuwenhoek (known as the father of microscopy)to dye muscle fibers, which allowed him to obtain more information about the structure of those cells by seeing the detailed structure of the fibers more easily. From van Leeuwenhoek’s time until the late 19th century scientists attempted to find natural products which could be used to dye cells. They were only partially successful until William Perkin’s discovery of the first synthetic dye in 1856. Perkin’s new technology unleashed a deluge of colored compounds to be used as potential stains by the scientific community.

German anatomist Walther Flemming was one of the first scientists to test out these new dyes. He used a number of them to stain cells and observed structures that appeared to strongly absorb these dyes. During the late 19th century, cell staining technology rapidly became more sophisticated. German biologist Carl Weigert discovered that different bacteria were stained different colors by dyes, which spurred the research of his famous cousin, bacteriologist Paul Ehrlich, who wrote a thesis on cell staining. While he was a resident at the Charite Hospital in Berlin Ehrlich discovered ways to identify blood disorders based on how cells absorb dyes, on top of methods for staining mast cells and white blood cells.

Ehrlich became convinced that staining was not a process where cells simply absorb dye. Instead he thought there might be a chemical or physical interaction between the dye and cell that could result in cell death. This led him to search for a dye that would kill harmful bacteria during the process of staining. Over more than a decade, Paul Ehrlich worked to find this dye, resulting him discovering a dye called trypan red that kills trypanosomes, the protozoa that causes sleeping sickness. More importantly, in 1907 he found an even more important bactericide called Salvarsan, an arsenic containing compound which soon became a powerful agent in the treatment of the sexually transmitted disease syphilis.

The most famous stain discovery however, was the Gram stain. The Gram stain was first devised in 1882 by a Danish bacteriologist Hans Christian Joachim Gram, and has gone on to become one of the most important facets of microbiology. The Gram stain was the first to be able to determine the different types of cell walls in bacteria. Gram began his career as a botany student in Denmark, going on to work in the field of botany and zoology which first exposed him to the use of the microscope. While working in a German morgue, Gram developed his staining technique to help to differentiate between the two major types of bacteria (Gram positive and Gram negative).


As he examined the lung tissue of patients who died of pneumonia, Gram discovered that certain bacteria retained their color after undergoing his staining technique (gram positive) while other species of bacteria had their colour bleached (gram negative). They key step to his technique is decolorization, which differentiates between Gram positive and Gram negative bacteria. Over-decolourization can result in Gram positive bacteria appearing negative and under-decolourization can result in Gram negative bacteria appearing Gram positive. Gram published his technique in 1884, and it quickly became widely adopted around the world and expanded upon by other microbiologists. Today, the gram stain is the standard method for classifying bacteria

Another staining technique was created by Italian histologist Camilo Golgi in the 1870’s. Golgi experimented with silver salts as stains instead of organic dyes, discovering that cell structures not visible with organic dyes were now easily seen.

Today, dozens of stains both organic and inorganic have been developed, each for specific uses and often named after their inventors. Such as Borrel’s methlyene blue, Ehrlich’s triacid stain, Renault’s eoisin, Lugol’s iodine and Van Gieson’s stain continue to memorialize their inventors. Despite the relative age of cell staining, it is still routinely used by biologists today, and has helped find cures for a number of diseases including tuberculosis, pneumonia, syphilis and many other less notable bacterial infections. Cell staining is still taught to young scientists today in schools and universities, highlighting it’s continued importance in the 21st century.


Staining Techniques

Microbial cytoplasm are typically transparent, making it necessary to stain microorganisms before they are examined under a microscope. Cells do not always need to be stained, such as when microorganisms are very large or when cell motility is being examined, so a drop of the cell(s) can be placed straight onto the slide and observed. When scientists prepare to do so, it is called a wet mount, which can also be prepared by placing a slice of the culture on a cover slip (the glass cover of a slide) before inverting it over a hollowed out side, a procedure known as a hanging drop.

To prepare for staining, a small sample of the microorganism is placed on a slide and allowed to air dry.  The smearing of slide is fixed by very quickly passing the slide over the flame of a Bunsen burner. This kills the organism, so it becomes “fixed” and better adheres to the slide, permitting them to be more easily stained.

Easy Ways to Stain:

Staining can be accomplished with basic dyes such methylene blue or crystal violet, which are positively charged dyes that are drawn to the negatively charged material of the microbial cytoplasm. Alternatives include using a dye such as Congo red or nigrosin, which are negatively charged dyes. Negatively charged cytoplasm repels them and gather around the cells, leaving them clear and unstained. This is called the negative stain technique.

The Differential stain techniques allow scientists to distinguish between different types of organisms.  The Gram stain technique is one of them. It separates bacteria into two different groups, Gram-positive and Gram-negative bacteria.

The gram technique is by far the most important and most popular staining technique the laboratory because of the fact that they are used is to distinguish between gram-positive and gram-negative bacterias, which have large differences in their cell walls. Gram-positive cells can turn into gram-negative via mechanical damage or conversion to protoplasts or aging, where autolytic enzymes attack the walls.

The Gram stain technique is an important preliminary step in the initial characterization and classification of bacteria, because the bacteria present in unstained smears are invisible when viewed under a light microscope. However, once the gram stain is employed, the morphology and arrangement of the bacteria can be observed as well.

First of all, Crystal violet is applied, followed by the mordant iodine, which fixes the stain. Then the slide is washed with alcohol, and the Gram-positive bacteria retain the crystal-violet iodine stain; however, the Gram-negative bacteria lose the stain. The Gram-negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under a oil-immersion microscope lens, while Gram-positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step.

In order to remove immersion oil from the slide without damaging the smear, place a piece of lens tissue on the slide, before adding a drop or two xylene and draw/wipe the lens tissue across the slide, then repeat is necessary.



The acid-fast technique is another differential method of staining. It involved differentiating species of Mycobacterium from other bacteria. It is done by using a lipid solvent to carry the first stain, carbofuchsin into the cells.

The cells are then washed with a diluted acid-alcohol solution. Mycobacterium species resist the effect of the acid-alcohol and retain the carbolfuchsin stain which is bright red. Other kinds of bacteria lose the stain and take on the subsequent methylene blue stain. Thus, the acid-fast bacteria will appear bright red, while the nonacid-fast bacteria will appear blue when observed under the microscope.

There are other stain techniques a well, which seek to differentiate between various bacterial structures For instance; one special stain technique highlights the flagella of bacteria by coating it with dyes or metals to increase their width, making the stained flagella able to be observed.

Another unique stain technique is one that can be used to examine bacterial spores. Malachite green is combined with heat to force the stain into the cells and give them a green color. A counterstain called safranin is then used to give color to the non-sporeforming bacteria. Once this is complete, spores stain green while other cells are stained red.

Cell Staining

Cell Staining is a microscopic technique that is used better visualize cells and their components by highlighting and contrasting them while under a microscopic. Scientists use stains and dyes to preferentially highlight certain components of the cell, such as its cell wall or nucleus or even the entire cell. Staining is usually carried out on fixed, non-living cells but some stains can be used on living cells, or even both.

Cells may be stained to highlight metabolic processes that they are involved or in to differentiate between the dead and the live cells in a sample.

How Cells Are Stained:

Which cell stain technique is used depends on the type of stain and analysis being used in the lab. A scientist could use any of the following procedures to prepare a stained cell sample.

Permeabilization: The treatment of cells with a mild surfactant (a compound that lowers the surface tension of liquids) which dissolves the cell membranes in order to allow larger dye molecules to enter inside the cell.

Fixation: This process serves to preserve the cell or tissues morphology. The fixation process will involve several steps, but usually a chemical fixative such as formaldehyde, ethanol, methanol or picric acid that is added that creates chemical bonds between proteins that increase their rigidity.

Mounting: Where Samples are attached to a glass microscope slide for observation and analysis. The cells can be grown directly to the slide or loose cells can be applied to it. Thin sections or slices of the material being examined can also be applied.

Staining: The actual application of strain to the slide and the sample on it. The Cells, metabolic processes, tissue matter and cell components are all coloured by the strain. Staining is achieved by submerging the sample in a solution of dye and then rinsing and observing the slide under a microscope. Certain dyes require the use of a chemical compound called a mordant, which reacts with the stain to form a coloured, insoluble principate which will remain in the sample when excess dye is washed away.

The Most Popular Stains:

There is a large variety of different staining dyes, each with a different purpose. All of these listed stains can be used on fixed or “non-living cells” and stains that can be used on un-fixed or living cells are noted.

Bismark Brown: Named after the 19th century German statesman, Bismark Brown colors acid mucins (a form or protein)  a yellow hue and may be used to stain live cells

Carmine colours glycogen (another name for animal starch) red

Coomassie Blue strains all proteins a very bright blue. It is often used in gel electrophoresis

Crystal Violet stains cell walls purple (hence its name) when combined with a mordant. It is used in Gram staining

DAPI is a fluorescent stain that shines blue when it is affected by ultra violet light and bound to DNA. DAPI can be applied on both living and fixed cells.

Eosin is a counter-stain to haematoxylin. Eoisin stains red blood cells, cytoplasm, cell membranes and extracellular structures red or pink.

Ethidium Bromide colours unhealthy cells during the stage of apoptosis, deliberate cell death, a fluorescent reddish orange.

Fuchsin is used to stain collagen, smooth muscle and mitochondria

Hematoxylin is a nuclear stain that when acting with a mordant, stains nuclei brown or a bluish violet

Hoechst stains are two different types of stains used to colour DNA in living cells

Iodine is used as a starch indicator which creates a dark blue coloured stain when in solution

Malachite green is a blue/green counterstain to safarnin. It is used to stain spores.

Methylene blue stains animal cells to better visualize their nuclei

Neutral/Toluylene red colours nuclei red and can be used on living cells

Nile blue can be used on living cells and stains cell nuclei blue

nucleic acid

Nile Red and Nile Blue Oxazone is a stain made by boiling Nile blue with sulfuric acid creating a mixture of Nile Red and Nile Blue. Nile Red stains intracellular lipid globules and can be used on non-fixed, living cells.

Osmium tetroxide is used in optical microscopy to highlight lipids black

Rhodamine stains proteins fluorescent

Safranin is used to stain collagen yellow

After stained and prepared, the cell slides must be stored in a dark and possible refrigerated place to preserve the slide in between being observed under a microscope.

The Most Important Type of Stain

Gram staining helps scientists to determine which bacteria are harmless of beneficial (which is the vast majority of bacteria) and those which are pathogenic. Pathogenic bacteria includes diseases such as tuberculosis which is caused by the bacterium Mycobacterium tuberculosis, which claims the lives of 2 million people a year, predominately in sub-Saharan Africa. Another well known disease caused by pathogenic bacteria is pneumonia. Staining, and in particular gram staining has helped scientists develop cures for these diseases, by allowing to differentiate between these pathogenic bacteria under the microscope.



Named after Danish scientist Hans Christian Gram, Gram staining is the most medically important staining technique. It was developed by him in a late 19th century Berlin morgue to not only distinguish different kinds of bacteria, but to more easily see bacteria in stained sections of the lung tissue of the dead.

Bacteria which are stained violet or a purplish blue are known as Gram-positive bacteria, while those which stain pink or red are Gram-negative. The gram technique is typically the very first step in identification of a bacterial organism, and is the mainstream or default stain performed by laboratory workers when there is no specific culture preferred. Even though Gram staining of bacteria is a very important technique in both research and clinical environments, it is unable to identify all bacteria, which fall under the Gram-variable and Gram-indeterminate categories on top of Gram-positive and Gram-negative.

Gram-positive bacteria are characterized by a thick mesh-like cell wall which is predominately composed of peptidoglycan (from 50% up to 90%) which stains purple.  Gram-positive bacteria typically have a single membrane or monoderm surrounded by said thick layer of peptidoglycan.

Lipoteichoic acid or LTA is the other major constituent of the cell wall of Gram-positive bacteria. LTA is embedded inside the peptidoglycan layer,   and consists of teichoic acids which are long chains of ribitol phosphate tied down to the lipid bilayer through a glyceride. It acts as regulator of autolytic wall enzymes . Lipoteichoic acid is characterized by antigenic properties that stimulate specific responses in the immune system when it is released from the cell wall after cell death.

Gram-positive bacteria often also have capsule polysaccharides and flagellum. If flagellum are present, the bacterium only has two rings for support instead of the four that Gram-negative bacteria enjoy because Gram-positive bacteria only have a single layer of membrane. Gram-positive bacteria can also be identified by the unique presence of teichoic acids in their respective cell walls.

The majority of pathogens in human beings are Gram-positive organisms. Six Gram-positive are most commonly pathogenic in human beings, two of which (Streptococcus and Staphlyococcus) are sphere shaped or “cocci” bacteria, while the remaining four are rod shaped “bacilli” bacteria and be subdivided on the basis on which are able to form spores.

Unlike Gram-positive bacteria, Gram-negative bacteria have a thinner layer of peptidoglycan ( roughly 10% of the cell wall) and lose the crystal violet-iodine during decolorization with the alcohol rinse, but retain the counter-stain Safranin, causing it to appear reddish or pink. They also have an additional outer membrane which contains lipids, which is separated from the cell wall by means of periplasmic space.

The cell wall of Gram-negative bacteria can be a factor that contributes to  pathogenic bacteria causing disease. This harmful effect of Gram-negative bacteria is particularly associated with certain components of the cell wall, in particular, the lipopolysaccharide, which otherwise known as LPS or endotoxin. LPS causes an rapid immune response characterized by cytokine production and deployment of the immune system in humans. The cytokine production triggers inflammation which can go on to have a toxic effect on the host.

Some bacteria do not respond to bacteria staining, and as such are known as Gram-indeterminate. The most prominent example of this is acid fast bacteria.

Meanwhile, other bacteria after being Gram stained show a Gram-variable pattern, where a mixture of red and violet cells appears. Actinomyces, Arthobacter, Corynebacterium, Mycobacterium and Propionibacterium have cell walls which are known to be sensitive to breakage during their cell division, which creates a Gram-negative staining of these Gram. In addition to this aberration, all forms of bacteria’s results in a Gram Stain can be skewed by the age of the culture, resulting in false positives and negatives.