Gram staining and acid-fast staining

Gram staining

It is the most important and widely used differential staining techniques in microbiology is Gram staining. It was purposed by Christian Gram in 1884. In this process, the fixed bacteria smear is subjected to the following staining reagent in the order listed. Crystal violet, iodine solution alcohol (decolorizing agent) and safranin or some suitable counterstain.Bacteria stained by Gram methods fall into two groups: Gram-positive bacteria, which retain the crystal violet and hence deep violet in color is appear and Gram-negative bacteria which lose the crystal violet are counterstained by the safranin, and hence appear red in color.

The most plausible explanations for this phenomena are associated with the structure and composition of the cell wall. The difference in the thickness of cell walls between these two groups may be important; the cell wall of gram-negative bacteria are generally thinner than those of gram-positive bacteria. The bacteria contain a high percentage of lipid than do Gram-positive bacteria.Experimental evidence suggests during staining of Gram-negative bacteria in alcohol treatment extracts the lipid, which results in increased porosity or permeability of cell wall. Thus the crystal violet-iodine (CV-I) complex can be extracted and the Gram-negative organism is decolorized. These cells subsequently take on the color of the safranin counterstain. the cell walls of Gram-positive bacteria, because of different composition (lower lipid content), become dehydrated during treatment with alcohol. The pore size decreases and the permeability are reduced and the CV-I complex cannot be extracted. Therefore these cells remain purple violet.

Another explanation, somewhat similar is also based on permeability differences between the groups of bacteria. The main purpose of the gram staining is to differentiate the bacteria between the two major groups gram positive and gram negative, which makes it an essential tool for classification and differentiation of microorganisms. In the case of the bacterial cell, pores are present, through which stains enter and give its color.In this technique, the fixed bacterial smear is subjected to the following staining reagents in the order listed below:

Primary stain

Crystal violet:

Crystal violet stain is used first and stains all calls purple

Mordant

Gram’s Iodine: it serves as a mordant, substance that forms an insoluble complex by binding to the primary stain. The resultant crystal violet-Iodine complex serves to intensify the color of the stain, and all the cells will appear purple-black at this point. In Gram-positive cells only, this CV-I complex binds to magnesium ribonucleic acid component of the cell wall. The resultant magnesium ribonucleic acid-crystal violet-iodine complex is more difficult to remove than the similar CV-I complex.

Decolourizing Agent

Ethyl alcohol (95%): That reagent serves a dual function as a lipid solvent and as a protein dehydrating agent. Its action is determined by lipid concentration of microbial cell walls. In gram-positive cells, the low lipid concentration is important to retention of the Mg-RNA-CV-I .Therefore, the small amount of lipid content is readily dissolved by the action of alcohol, causing the formation of minute cell wall pores. These are then closed by the alcohol's dehydrating effect. Consequently, the tightly bonded primary stain is difficult to remove, and the cells remain purple. In Gram-negative cells the high lipid concentration found in outer layers of the cell wall is dissolved by the alcohol, creating large pores in the cell wall that do not close appreciably on dehydration of cell wall proteins. This facilitates a release of the unbounded CV-I complex, leaving these cells colorless or unstained.

Counterstain

Safranin: This is the final reagent, used to stain red those cells that have been previously decolourized. Since only Gram-negative cells undergo depolarization, they may now absorb the counterstain.Gram positive cells retain the purple color of the primary stain.In the case of bacterial cells, Pores are present through which stain enter and gives in color. According to gram’s staining method, bacteria are of two types : Gram positive bacteria which retain crystal violet hence appear deep violet in color and gram negative bacteria which loses crystal violet color and are counterstained by safranin and hence appear red in color.

Acid-fast stain

The main purpose of Acid-fast stain is to differentiate bacteria into acid fast and nonacid-fast groups. While the majority of bacteria organisms are stained by either simple or gram staining procedure, a few Genera, particularly the members of the genus Mycobacterium, are resistant and can only be visualized by the acid-fast method. Since M. tuberculosis and M. Laprae represent bacteria that are pathogenic to humans, the stain is of diagnostic value in identifying these organisms. The characteristics difference between Mycobacterium and other microorganisms in the presence of thick waxy wall that makes penetration by stains extremely difficult. once the stain has penetrated, however it can not readily be removed even with the vigorous use of acid alcohol as a decolorizing agent. Because of this property, these microorganisms are called acid-fast, while all another microorganism, which is easily decolorized by acid-alcohol, are called non-acid-alcohol.

The acid fast stain uses three different reagents

Primary stain:

Carbol Fuchsin: These are used as a primary stain, Carbol Fuchsin, a red phenolic stain is soluble in the lipoidal materials that constitute the major portion of the mycobacterial cell wall. It does penetrate these bacteria and is retained.

Decolorizing agent

Acid-alcohol: On application of acid alcohol, the acid-fast cell will be resistant to decolorization, since the primary stain is more soluble in the cellular waxes than the decolouring reagent. In this event, the primary stain is retained and the mycobacteria will stay red. This is not the case with the nonacid-fast organisms that lacks the cellular waxes in their cell wall.

Counterstain:

Methylene blue: This is used as the final reagent to stain previously depolarized cell. As only nonacid-fast cells undergo decolourization, they may now absorb the counter stain and take on its blue color, while acid-fast cells retain the red color of the primary stain.

References:

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.