Fundamentals of Staining Techniques

Staining is a process in microbiology that improves visualisation of organisms or structures within them.
Stains or dyes are used to colour certain components of the microorganism, which can improve contrast or highlight specific structures.
It’s essential to ‘fix’ the specimen onto the glass slide before staining, commonly performed through heat fixing.
Safety is paramount when handling stains, as they can be harmful if not used properly. Always wear protection, like gloves and lab coats.

Types of Staining Techniques

The two main categories of stains are basic dyes (which have a positive charge) and acidic dyes (which have a negative charge).
Simple staining techniques use one type of dye to highlight structures, but this method does not differentiate between different types of cells or structures.
Differential staining techniques, such as Gram staining or acid-fast staining, divide bacteria into two or more groups, based on the differences in cell components.

Gram staining is a differential staining method that separates bacteria into two groups: Gram-positive and Gram-negative.
The Gram stain procedure consists of the application of crystal violet stain, followed by a mordant (Gram’s iodine), decolourisation with alcohol or acetone, and counterstain with safranin.
Upon application of these substances, Gram-positive bacteria will appear purple, and Gram-negative bacteria will appear pink.

Acid-fast staining differentiates bacteria based on the lipid content in their cell walls. Acid-fast bacteria, like Mycobacterium species, will appear red, while non-acid-fast bacteria will be blue.
In the Ziehl-Neelsen method, carbol fuchsin dye is applied, followed by acid-alcohol decolourisation and a methylene blue counterstain.

Rinse with water after every step in the staining process to remove excess stain.
After staining, slides must be air-dried or blot dried carefully before microscopic examination.
Remember that each staining method is best suited for a specific requirement. Choosing the right method can provide richer detail and greater insight into the sample’s cellular structures.