Types of microbiologic stains By Dr. Shnyar Hamid Qadir

Light Microscopy Direct examination of stained or unstained preparations by light (bright-field) microscopy is particularly useful for detection of bacteria, fungi, and parasites. Even the smallest bacteria can be visualized. Using 100x oil immersion objective, a 5 to 10x eyepiece, and optimal lighting.

Bacteria may be stained by a variety of dyes, including: Methylene blue, crystal violet, carbol-fuchsin (red), and safranin (red). The two most important methods, the Gram and acid-fast techniques, use staining, decolorization, and counterstaining in a manner that helps to classify as well as stain the organism.

1. Gram Stain The differential staining procedure described in 1884 by the Danish physician Hans Christian Gram has proved one of the most useful in microbiology and medicine

1. Gram Stain The procedure (Figure 1) involves the application of a solution of iodine in potassium iodide to cells previously stained with an acridine dye such as crystal violet. This treatment produces a mordanting action in which purple insoluble complexes are formed with ribonuclear protein in the cell.

1. Gram Stain The difference between Gram-positive and Gram-negative bacteria is in the permeability of the cell wall to these complexes on treatment with mixtures of acetone and alcohol solvents. This extracts the purple iodine–dye complexes from Gram-negative cells, whereas Gram-positive bacteria retain them. An intact cell wall is necessary for a positive reaction, and Gram-positive bacteria may fail to retain the stain if the organisms are old, dead, or damaged by antimicrobial agents.

1. Gram Stain The stain is completed by the addition of red counterstain such as safranin, which is taken up by bacteria that have been decolorized. Thus, cells stained purple are Gram positive, and those stained red are Gram negative. Gram positivity and negativity correspond to major structural differences in the cell wall.

Figure1 1. Stained purple by the crystal violet and iodine of the Gram stain 1. Stained red by the carbol fuchsin of the acid-fast stain 2. After decolorization, Gram-positive retain their original stain 2. After decolorization, acid-fast organisms retain their original stain 3.The safranin of the Gram counterstain stains the Gram-negative bacteria and makes the background red 3. The methylene blue leaves a blue background for the contrasting red acid-fast bacillus

Figure 2. Gram stain: purple cells are gram positive Figure 2. Gram stain: purple cells are gram positive. Red cells are gram negative.

2. Acid-Fast Stain (Ziehl-neelsen stain) Acid fastness is a property of the mycobacteria (eg, Mycobacterium tuberculosis) and related organisms. Acid-fast organisms generally stain very poorly with dyes, including those used in the Gram stain. However, they can be stained by prolonged application of more concentrated dyes, by penetrating agents, or by heat treatment.

2. Acid-Fast Stain Their unique feature is that once stained, acid-fast bacteria resist decolorization by concentrations of mineral acids and ethanol that remove the same dyes from other bacteria. This combination of weak initial staining and strong retention once stained is related to the high lipid content of the mycobacterial cell wall. Acid-fast stains are completed with a counterstain to provide a contrasting background for viewing the stained bacteria

2. Acid-Fast Stain In the acid-fast procedure, the slide is flooded with carbol-fuchsin (red color) Carbol-fuchsin: (which is lipid soluble and penetrates the waxy cell wall.) decolorized with hydrochloric acid in alcohol. When counterstained with methylene blue, acid-fast organisms appear red against a blue background