Microbiology Staining Techniques
Before staining a cell one should fix it to the slide. This can be done by heat fixing it which consists of passing it over an open flame a few times. Or with specific chemicals.
Chromophores
These have positive and negative ions. Usually we call a stain basic if positively charged, and we call them acidic if they are negatively charged.
There are two types of stains these can be simple and Compound dyes. (simple dyes only dye in one colour, Compound stains can also be called differential stains).
Gram Stain
The most important differential stain is called the gram stain. This stain can only be used on prokaryote (bacteria). This stain either stains bacteria of pink or red (gram negative) or shades of blue or violet (gram positive). The gram stains differentiate gram positive from gram negative bacteria by the thickness of their cell walls. If the cell wall is thick it would stain gram positive if the cell wall is thin is stains gram negative.
Gram staining is important because in identification of bacteria one can exclude immediately one whole class of bacteria from this stain.
There are several steps in a gram stain. First you have to fix the sample to the glass slide. Then the first dye we add is Crystal violet. This is left on the slide for a fixed amount of time this is usally 30 seconds. This is called the primary stain. After 30 seconds the sample is rinsed under running water and then a solution of iodine is added to the sample. The function of this solution is to bind the Crystal violet to the cell walls, so that the next steps do not wash out the stain. This is called a mordant. Next comes a solution of acetone and alcohol. This is left for 30 seconds, this is called the decolorizing solution. Its function is to wash out the crystal violet from the cell walls. The next step is called the counter stain. This is what gives the cell the pink colour. As a counterstain one can use either Safranin or carbol fuchsin.
Acid fast stain.
Some bacteria do not take the gram stain because of a waxy cuticle surrounds the cell wall that surrounds the cell and therefore the acid fast staining method is used.
Examples of bacterium that need this staining process are: mycobacterium such as tuberculosis and leprae.
This is a very similar staining technique to the gram staining but the main stain is Carbol Fuchsin (which could be used as a counterstain in gram staining) instead of Crystal violet and the counterstain has to be methylene blue.
Mycobacteria stain pink while all other cells would stain blue.
Chromophores
These have positive and negative ions. Usually we call a stain basic if positively charged, and we call them acidic if they are negatively charged.
There are two types of stains these can be simple and Compound dyes. (simple dyes only dye in one colour, Compound stains can also be called differential stains).
Gram Stain
The most important differential stain is called the gram stain. This stain can only be used on prokaryote (bacteria). This stain either stains bacteria of pink or red (gram negative) or shades of blue or violet (gram positive). The gram stains differentiate gram positive from gram negative bacteria by the thickness of their cell walls. If the cell wall is thick it would stain gram positive if the cell wall is thin is stains gram negative.
Gram staining is important because in identification of bacteria one can exclude immediately one whole class of bacteria from this stain.
There are several steps in a gram stain. First you have to fix the sample to the glass slide. Then the first dye we add is Crystal violet. This is left on the slide for a fixed amount of time this is usally 30 seconds. This is called the primary stain. After 30 seconds the sample is rinsed under running water and then a solution of iodine is added to the sample. The function of this solution is to bind the Crystal violet to the cell walls, so that the next steps do not wash out the stain. This is called a mordant. Next comes a solution of acetone and alcohol. This is left for 30 seconds, this is called the decolorizing solution. Its function is to wash out the crystal violet from the cell walls. The next step is called the counter stain. This is what gives the cell the pink colour. As a counterstain one can use either Safranin or carbol fuchsin.
Acid fast stain.
Some bacteria do not take the gram stain because of a waxy cuticle surrounds the cell wall that surrounds the cell and therefore the acid fast staining method is used.
Examples of bacterium that need this staining process are: mycobacterium such as tuberculosis and leprae.
This is a very similar staining technique to the gram staining but the main stain is Carbol Fuchsin (which could be used as a counterstain in gram staining) instead of Crystal violet and the counterstain has to be methylene blue.
Mycobacteria stain pink while all other cells would stain blue.