Staining is necessary to produce color contrast and thereby increase the visibility of the object. Before staining, the fixation of the smear to the slide is done:
• Heat fixation is usually done for bacterial smears by gently flame heating and air-dried film of bacteria.
• Chemical fixation such as ethanol, acetic acid, mercuric chloride, formaldehyde, methanol, and glutaraldehyde. This is useful for the examination of blood smears.
Staining Techniques Used in Microbiology
1. Simple stain: Basic dyes such as methylene blue or basic fuchsin are used as simple stains. They provide the color contrast but impart the same color to all the bacteria in a smear.
2. Negative staining, e.g. India ink or nigrosin. The background gets stained black whereas unstained bacteria stand out in contrast. This is very useful in the demonstration of bacterial capsules which do not take up simple stains.
3. Impregnation methods (e.g. silver): Used for demonstration of thin structures like bacterial flagella and spirochetes
4. Differential stain: Two stains are used which impart different colors which help in differentiating bacteria, e.g.
• Gram stain: Differentiates bacteria into Gram-positive (appear violet) and Gram-negative (appear pink) groups
- Primary stain by crystal violet (or gentian violet or methyl violet) for one minute.
- Mordant by Gram’s iodine for one minute.
- Decolorization by acetone (for 1–2 sec) or ethanol (20–30 sec) with immediate wash. Decolorizer removes the primary stain from Gram-negative bacteria while the Gram-positive bacteria retain the primary stain.
- Counterstain or Secondary stains by safranin or diluted Carbol Fuchsin; is added for 30 sec.
• Acid-fast stain: Acid-fast organisms (table below) resist decolorization to mineral acids. This is due to the presence of mycolic acids in the cell wall.
• Albert stain: Differentiates bacteria having metachromatic granules (e.g. Corynebacterium diphtheriae) from other bacteria that do not have.
5. Other Special Staining Methods:
• Spore staining: Acid-fast stain (using 0.25% sulfuric acid) and Malachite green stain (Schaeffer and Fulton’s method modified by Ashby) methods are used; however, phase contrast microscope of the unstained wet film is the best method.
• Lipids stained by: Sudan Black stain
• Carbohydrate (Glycogen) stained by Iodine stain
• Flagellar stain: Tannic acid staining (Leifson method)
6. Microscopy of Bacteria in Living State
• Unstained (wet) preparations:
- Checking bacterial motility (e.g. in hanging drop and wet mount preparations).
- For a demonstration of spirochetes (e.g. in a dark field or phase contrast microscopy).
• Vital stains:
Differentiate the living cells from dead cells:
- In supravital staining, living cells that have been removed from an organism are stained; whereas intravital staining is done by injecting stain into the body.
- Examples of vital stains are eosin, propidium iodide, trypan blue, erythrosine, and neutral red.