Structural details of microorganisms cannot be seen under a light microscope due to lack of contrast.
Dyes are used to stain cells, binding with cellular components, creating color contrast and improving visibility.
Positively charged (cationic) dyes like methylene blue, crystal violet, safranin, are crucial as they bind with negatively charged cellular constituents.
Types of Microbiological Stains
Simple Stain
Uses basic dyes such as methylene blue or basic fuchsin.
Produces color contrast but imparts the same color to all bacteria in the smear.
Negative Staining
Bacterial suspension mixed with dyes like India ink or nigrosin.
Background stained black, highlighting unstained bacterial or yeast capsule.
Useful for demonstrating capsules that do not take up simple stains.
India Ink Preparation: Recommended for identifying Cryptococcus neoformans.
Impregnation Methods
Used for bacterial cells and structures too thin to be seen under light microscope.
Silver salts are impregnated on their surface to make them visible (e.g., bacterial flagella, spirochetes).
Flagella Stain: Demonstrates the presence and arrangement of flagella, important for identifying species of motile bacteria.
Differential Staining
Uses two stains to impart different colors, helping differentiate bacteria.
Gram Staining
Crucial for initial characterization and classification of bacteria.
Identifies bacterial pathogens by Gram reaction (Gram-positive/negative) and morphology (cocci/rod).
Acid-fast Stain (Ziehl-Neelsen technique)
Distinguishes acid-fast bacteria like Mycobacterium spp. from non-acid fast bacteria.
Used for Mycobacterium species (e.g., Mycobacterium tuberculosis).
Endospore Stain
Demonstrates spore structure in bacteria.
Positive result is key in bacterial identification (e.g., Bacillus spp, Clostridium spp).
Capsule Stain
Demonstrates presence of capsules in bacteria or yeasts (e.g., Streptococcus pneumoniae).
Giemsa Stain
Used for staining malaria and other blood parasites, Chlamydia trachomatis inclusion bodies, Borrelia species, and more.
Acridine Orange Stain
Confirms presence of bacteria in blood cultures when Gram stain results are difficult to interpret.
Binds to nucleic acid and stains them.
Cytoplasmic Inclusion Stains
Identifies intracellular deposits like starch, glycogen, polyphosphates, etc.
Albert Staining: Used for C. diphtheriae volutin granules.
Other Staining Methods
Auramine-Rhodamine Technique
Fluorochrome staining method enhancing detection of mycobacteria.
Calcofluor White Staining
Used to detect fungal elements and visualize fungi characteristics.
Lactophenol Cotton Blue (LPCB) Wet Mount
Widely used for staining and observing fungi.
References and Further Readings
Color Atlas and Textbook of Diagnostic Microbiology, Koneman, 5th edition.