Gram Staining

Gram staining is an essential staining technique in bacteriology used to differentiate Gram-positive and Gram-negative bacteria. In this method, differentiation is achieved as Gram-positive bacteria retain a crystal violet stain due to the presence of a thick layer of peptidoglycan in their cell walls.

Alternatively, Gram-negative bacteria are decolorized by organic solvent and turn orange-pink when counterstained, which is attributed to a thinner peptidoglycan wall. Reagents required for this multi-step staining process include crystal violet (primary stain), aniline dye, iodine solution (mordant), and safranin orange (secondary stain), or carbol fuchsin counterstains.

Mycobacteria Staining

Early diagnosis of mycobacterial infection is critical, as the acid-fast bacteria are highly pathogenic and responsible for serious diseases, like tuberculosis. Diverse staining solutions are available for the detection of these pathogenic bacteria in histological tissue cultures and bacteriological smears. Fluorescence detection methods are utilized with the Ziehl-Neelsen staining technique, via either heat-treated slides (hot staining) or non-heated treatment (cold staining).

This differential staining technique uses the lipid-soluble phenolic compound carbol fuchsin as the primary stain, and the counterstain, malachite green.

Trichomonad Staining

Trichomonas vaginalis parasites are especially common in gynaecological material, such as vaginal smears and urine sediment, and cause the most common non-viral sexually transmitted disease, trichomoniasis. Various stains like Giemsa and Acridine Orange, are used along with wet mount examination for the microscopic diagnosis of Trichomonas vaginalis.

Bacteriology Applications in Other Industries

In the food and beverage industry, lactic acid bacteria such as Lactobacillus, Lactococcus, and Streptococcus are used in the manufacture of dairy products, such as cheese, buttermilk, and yogurt. Bacterial fermentations are used in the processing of beverages, such as tea and coffee.

In the growing field of gut microbiome health, several bacterial species are used in probiotic supplements to reduce inflammation and improve bowel function. Bacteria are also used in the pharmaceutical industry in vaccine research and production, including tetracyclines, erythromycin by Streptomyces, and bacitracin by Bacillus.

Common Diagnostic Bacteriology Stains and Their Uses

Acradine orange: Fluorescent stain with diverse applications; sometimes used in Trichomonad staining.

Carbol fuchsin: Stains mycobacteria (AFB).

Crystal violet: Stains Gram-positive bacteria (used in Gram’s method).

Fuchsin basic: Stains mycobacteria (AFB).

Giemsa: Broad staining; sometimes used in Trichomonad staining.

Malachite green: Stains bacterial polysaccharides and spores. Also contrast stain for Ziehl-Neelsen procedure.

Methylene blue (Loefflers): General bacterial staining of gonococcae, lactic acid bacteria, and pole corpuscles of pasturella.

Rose bengal: Stains bacteria in soil samples.

Safranin O: Stains Gram-negative bacteria (used in Gram’s method).

Silver stain (modified Steiner-Steiner): Stains spirochetes and nonfilamentous bacteria such as spirochetes in paraffin-embedded tissue.

Sudan black B: Stains fatty material in bacteria.