Basic Description:

Most cells are very difficult to study due their colorless and translucent appearance to the naked eye (Alberts et al., 2002). Histology or staining techniques are used to accentuate an area of interest in cells with dye. Some staining techniques are general and will have a homogenous staining pattern across cells. Acidophilic and basophilic staining are selective histology techniques that depends on a chemical reaction between the stain and the specified portion of the cell (University of Leeds, n.d.). The chemical reaction between acids and bases is what makes this staining technique possible. Acids are positively charged molecules and proton donors while bases are negatively charged and proton acceptors thus why they are able to react with one another (Alberts et al., 2002).

Acidophilic staining technique requires the addition of acidic stains to a cell with basic compartments. Since cytoplasmic proteins are basic, they will react and bind to the acidic dye because they are acidophilic, or “acid liking.” Basophilic staining is the process of adding basic dyes that will react with acidic compartments of the cell. Nucleic acids are one example of an acidic molecule that will bind to basic dyes because they are basophilic or “basic liking” (University of Leeds, n.d.).


The general purpose of staining techniques is to make cellular structures easier to see. The selectivity of acidophilic and basophilic staining creates a visual contrast that will help the viewer identify a specific area of interest. When the acidity of cell compartments is known, the correct stain can be added to illuminate the area for observation (“Tissue”, 2010).

Special stains may be needed for specific cell compartments to produce a desired reaction. This technique is particularly effective for proteins as they possess both acidic and basic groups. At a particular pH or isoelectric point, the net charge of a protein is zero and will not bind with neither acidic nor basic dyes. Changing the pH at which the staining takes place can make the acidic groups negatively charged or the basic groups positively charged so binding can occur with basic and acid stains respectively. Staining must take place above the isoelectric point to make the acidic groups in the protein negatively charged. Bases will become positively charged if the staining occurs below the isoelectric point (“Cells”, 2006). The binding of acidic and basic compartments to basic dyes and acid dyes respectively occur because in dye solutions, acidic dyes are anionic and basic dyes are cationic (“Acid”, n.d.).


Dyes were originally produced primarily for industrial textile use instead of histology techniques. Textile dyers rely on manufactures to make stains of consistent long lasting ability. Therefore, dyes are usually produced to please textile companies rather than histologists. As a result, the chemical purity of many dyes is very poor. Non-dye components that may contaminate a stain can greatly affect the desired reaction. Before laboratory suppliers started producing reliable certified dyes, people depended on the dyes made by Herr Grubler. At the time people did not understand his dye’s had greater purity, they were “just better” (“Staining”, 2006).

Originally, the only dyes available were those of natural composition in which were often dull and easily faded. The first synthetic dye was discovered by W.H. Perkin in the 19th century. The discovery of Mauve, the first synthetic dye, was accidental as Perkins was a student performing an experiment to synthesize the drug quinine (“Staining”, 2006).
While dye or staining techniques were originally an invention in the textile industry, binding of dyes to cellular tissues is a mechanism accepted in the discipline of chemistry.

Scientists know that a dye must form a bond with a tissue in order for a reaction or color to appear. Ionic bonding between molecules occurs if there is an electrostatic attraction where one ion is positively charged and one is negatively charged. The understanding of ionic bonding explains the reasoning for acidophilic and basophilic staining of basic and acidic cellular tissues. (“Staining”, 2006)

Recent Research:

Recent research has been performed using acidophilic staining in the investigation of the pathology and cellular responses of lymphocystis iridovirus infected gilthead seabream. The innate immunity defense mechanism in organisms in response to a pathogen involves host defense effector molecules called antimicrobial peptides. These peptides are found in the mast cells and acidophilic granulocytes of fish. The fish skin cells were stained with an acidic stain, haemotoxylin-eosin, to identify host cellular reactions of the acidophilic peptides near sites of infection. This histology technique was combined with further analysis of the known differences between mast cells and acidophilic granulocytes. The acidophilic staining represented the presence of these peptide-containing cells, but another technique was used to identify which granular cell was present. Since acidophilic granulocytes were also observed within the capillaries of the seabream, the researchers could suggest a relationship between these innate response cells to their function in fighting the virus through skin cell inflammation (Dezfuli et al., 2012).

A new modified acid-fast staining, or basophilic staining method was investigated against two other accepted methods to investigate its efficiency in the rapid detection of Mycobacterium tuberculosis and its L forms. The technique consisted of the mixing of the stain, carbol fuchsin and dioxogen with the sputum samples from patients with tuberculosis (Zhao at al., 2012). Carbol fuschin is a basic stain that binds with acidic bacteria and cell nuclei (“Ziehl”, 2012). Since Mycrobacterium is covered with a layer of mycolic acid in which can hinder the passage of color into the bacteria, carbol fuchsin is the basic stain used in all three methods. The modified method added dioxogen with the stain as it is known to damage the cytomembrane in which will increase the permeability of the membrane allowing more color into the bacteria. A characteristic of the infection of the L form is the presence of protein, lipid, and saccharides in the cell wall. The breakdown of the lipid membrane as a result of the dioxogen provided effective results in the determination of the L form bacteria especially. Not only was the modified method determined to be faster than the routine acid-fast staining method, but it also did not require heat (Zhao at al., 2012).

Research was performed to test the efficiency of the basic stain, toluidine blue, with uterine specimens to detect cancer. Basic toluidine blue binds with nucleic acids because it has affinity for DNA and RNA. Cancer cells contain more DNA and RNA than normal cells because of their uncontrollable growth. Toluidine blue will stain malignant areas blue in which can determine early phases of cancer in the body. The results displayed toluidine blue to have a 100% sensitivity to staining malignant uterine specimen (Ozturk et al., 2012).


Acid Dyes. (n.d). Retrieved from

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell: 4th Edition. New York: Garland Science.

Cells, Tissues, & Organs: Methods of Study. (2006). Retrieved

Dezfuli, B.S., Lui, A., Giari, L., Castaldelli, G., Mulero, V., & Noga, E.J. (2012). Infiltration and activation of acidophilic granulocytes in skin lesions of gilthead seabream, Sparus aurata, naturally infected with lymphocystis disease virus. Developmental & Comparative Immunology, 36(1). 174-182.

Ozturk, M., Ercan, C.M., Dede, M., Yenen, M.C., Demiriz, M., & Baser, I. (2012). Toluidine blue staining in the diagnosis of endometrial pathologies: a preliminary study before chromohysteroscopy. European Journal of Obstetrics & Gynecology and Reproductive Biology, 160(2). 191-195

Staining theory. (2006). Retrieved from CellPath-ch06.pdf

Tissue Preparation. (2010). Retrieved from

University of Leeds Faculty of Biological Sciences. (n.d). What is H&E? Retrieved from http:

Zhao, D., Yang, X., Chen, Q., Zhang, X., Guo, C., & Che, X. (2012). A modified acid-fast staining method for rapid detection of Mycobacterium tuberculosis. Journal of Microbiological Methods, 91(1). 128-132.

Ziehl stain. (2012). Retrieved from +fuchsin