Types of Stains

There are two types of Stains: simple and differential. See Table 3-4 for a summary of staining techniques.

Simple Stain

A simply stain has a single basic dye that is used to show shapes of cells and the structures within a cell. Methylene blue, safranin, carbolfuchsin and crystal violet are common simple stains that are found in most microbiology laboratories.

Differential Stain

A differential stain consists of two or more dyes and is used in the procedure to identify bacteria. Two of the most commonly used differential stains are the Gram stain and the Ziehl-Nielsen acid-fast stain.

In 1884 Hans Christian Gram, a Danish physician, developed the Gram stain. Gram-stain is a method for the differential staining of bacteria. Gram-positive microorganisms stain purple. Gram-negative microorganisms stain pink. Staphylococcus aureus, a common bacterium that causes food poisoning, is grampositive. Escherichia coli is gram-negative.

Table 3-4. Quick Guide for Staining Techniques


Number of Dyes Used



Simple stains

Uses a single dye

Size, shape, and arrangement of cells

Methylene blue Safranin Crystal violet

Differential stains

Uses two or more dyes to distinguish different types or different structures of bacteria

Distinguish gram-positive or gram-negative Distinguishes the members of mycobacteria and nocardia from other bacteria

Gram stain Ziehl-Nielsen acid-fast stain

Special stains

These stains identify specialized structures

Exhibit the presence of flagellae Exhibits endospores

Shaeffer-Fulton spore staining

The Ziehl-Nielsen acid-fast stain, developed by Franz Ziehl and Friedrick Nielsen, is a red dye that attaches to the waxy material in the cell walls of bacteria such as Mycobacterium tuberculosis, which is the bacterium that causes tuberculosis, and Mycobacterium leprae, which is the bacterium that causes leprosy. Microorganisms that retain the Ziehl-Nielsen acid-fast stain are called acid-fast. Those that do not retain it turn blue because the microorganism doesn't absorb the Ziehl-Nielsen acid-fast stain.

Here's how to Gram-stain a specimen (Fig. 3-5).



Fig. 3-5. How to Gram-stain a specimen.

1. Prepare the specimen using the heat fixation process (see "Smear" earlier in this chapter).

2. Place a drop of crystal violet stain on the specimen.

3. Apply iodine on the specimen using an eyedropper. The iodine helps the crystal violet stain adhere to the specimen. Iodine is a mordant, which is a chemical that fixes the stain to the specimen.

4. Wash the specimen with ethanol or an alcohol-acetone solution, then wash with water.

5. Wash the specimen to remove excess iodine. The specimen appears purple in color.

6. Wash the specimen with an ethanol or alcohol-acetone decolorizing solution.

7. Wash the specimen with water to remove the dye.

8. Apply the safranin stain to the specimen using an eyedropper.

9. Wash the specimen.

10. Use a paper towel and blot the specimen until the specimen is dry.

11. The specimen is ready to be viewed under the microscope. Gram-positive bacteria appear purple and gram-negative bacteria appear pink.

Here's how to apply the Ziehl-Nielsen acid-fast stain to a specimen.

1. Prepared the specimen (see "Smear" earlier in this chapter).

2. Apply the red dye carbol-fuchsin stain generously using an eyedropper.

3. Let the specimen sit for a few minutes.

4. Warm the specimen over steaming water. The heat will cause the stain to penetrate the cell wall.

5. Wash the specimen with an alcohol-acetone decolorizing solution consisting of 3 percent hydrochloric acid and 95 percent ethanol. The hydrochloric acid will remove the color from non-acid-fast cells and the background. Acid-fast cells will stay red because the acid cannot penetrate the cell wall.

6. Apply methylene blue stain on the specimen using an eyedropper.

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