Fundamentals of Microbiology

2.3.2 Staining reactions

When the pH of the surroundings of the microbial cells is either neutral or alkaline, all microbial cells have a negative charge on their surface, called the surface charge. Many bacterial cultures produce acids, thereby adding hydrogen ions to a culture medium and decreasing it pH. These hydrogen ions ( 12H+"> ) interact with the surface of the negative charges on the surface. When this happens, the cell surface no longer strongly attracts positively charged dye ions (basic dyes). Thus the microbes from acidic environments stain poorly with basic dyes. For this reason, the basic dyes are made up as alkaline solutions. For example, potassium hydroxide (OH) is added to solutions of methylene blue to form the stain called Loeffler’s Methylene blue.

Some bacteria excrete alkaline materials during growth and this decreases the number of available hydrogen ions in the culture medium. Under such conditions, the cell surface has a greater negative charge, which is more attractive to basic dyes and therefore, allows greater binding, penetration and internal staining of the microbe. Basic dyes stain microorganisms better under neutral or alkaline conditions.

If the dye base molecule has a negative charge, it is repelled by the cell’s negatively charged surface. Thus negatively charged dyes neither bind to the cell’s surface nor are they able to penetrate into the cell. These are called acid dyes.

The negatively charged cells are not stained by the negatively charged dye, and they appear as clear area surrounded by a coloured back ground. Negatively charged dyes used in this way are called negative stains.

Negative stains are of limited usefulness for those using light microscopes, but they can be used to avoid some of the disadvantages of staining with basic dyes.