Structural details of organisms cannot be seen under a light microscope due to a lack of contrast. Hence, we use dyes to stain cells. Dyes bind with cellular constituents producing color contrast and increasing their visibility. Positively charged (cationic) dyes such as methylene blue, crystal violet, safranin, etc bind with negatively charged cellular constituents such as nucleic acids and acidic polysaccharides and the cell surface of bacteria.
Based on the types and number of dyes used, staining can be categorized into different types.
Basic dyes, such as methylene blue or basic fuchsin are used as simple stains. They produce color contrast but impart the same color to all the bacteria in the smear.
A drop of bacterial suspension is mixed with dyes, such as India ink or nigrosin. The background gets stained black whereas the unstained bacterial or yeast capsule stands out in contrast. This is very useful in the demonstration of capsules that do not take up simple stains.
India ink preparation
Negative stains are used when a specimen or a part of it, such as the capsule resists taking up the stain. India Ink preparation is recommended for use in the identification of Cryptococcus neoformans.
Bacterial cells and structures that are too thin to be seen under the light microscope are thickened by impregnation of silver salts on their surface to make them visible, e.g., for demonstration of bacterial flagella and spirochetes.
Demonstrate the presence and arrangement of flagella. Flagellar stains are painstakingly prepared to coat the surface of the flagella with dye or a metal such as silver. The number and arrangements of flagella are critical in identifying species of motile bacteria.
Here, two stains are used which impart different colors to different bacteria or bacterial structures, which help in differentiating bacteria. The most commonly used differential stains are:
Gram stain is a very important differential staining technique used in the initial characterization and classification of bacteria in microbiology. Gram staining helps to identify bacterial pathogens in specimens and cultures by their Gram reaction (Gram-positive and Gram-negative) and morphology (cocci/rod).
Acid-fast stain (Ziehl-Neelsen technique)
It distinguishes acid-fast bacteria such as Mycobacterium spp from non-acid fast bacteria; which do not stain well by the Gram staining. It is used to stain Mycobacterium species (Mycobacterium tuberculosis, M. ulcerans, and M. leprae)
It demonstrates spore structure in bacteria as well as free spores. Relatively few species of bacteria produce endospores, so a positive result from endospore staining methods is an important clue in bacterial identification. Bacillus spp and Clostridium spp are the main endospores producing bacterial genera.
It helps to demonstrate the presence of capsules in bacteria or yeasts. Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae are common capsulated bacteria.
Giemsa stain is a Romanowsky stain. It is widely used in the microbiology laboratory for the staining of:
- Malaria and other blood parasites
- Chlamydia trachomatis inclusion bodies
- Borrelia species
- Yersinia pestis
- Histoplasma species
- Pneumocystis jiroveci cysts (formerly Pneumocystis carinii)
Acridine orange Stain
This staining method is used to confirm the presence of bacteria in blood cultures when Gram stain results are difficult to interpret or when the presence of bacteria is highly suspected but none are detected using light microscopy. Acridine orange binds to nucleic acid and stains them. It is also used for the detection of mycoplasmas (cell wall deficient bacteria).
Cytoplasmic inclusion stains
Identifies intracellular deposits of starch, glycogen, polyphosphates, hydroxybutyrate, and other substances. E.g. Albert staining is used to stain the volutin or metachromatic granules of C. diphtheriae.
Other staining methods
This fluorochrome staining method is used to enhance the detection of mycobacteria directly in patient specimens and the initial characterization of cells grown in culture.
Calcofluor White Staining
It is commonly used to directly detect fungal elements and to observe the subtle characteristics of fungi grown in culture. The cell walls of fungi will bind the stain calcofluor white, which greatly enhances the visibility of fungal elements in tissue or other specimens.
Lactophenol cotton blue (LPCB) wet mount
LPCB mount is the most widely used method of staining and observing fungi.
References and further readings
- Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition
- Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
- Willey, Joanne M. Prescott, Harley, and Klein’s microbiology / Joanne M. Willey, Linda M. Sherwood, Christopher J. Woolverton. — 7th ed. Mc Graw Hill Higher Education.