The document discusses staining techniques used in microbiology to visualize and differentiate bacteria based on morphological characteristics. It outlines two main types of stains: acidic and basic, detailing their properties and examples, such as picric acid and methylene blue. It further explains various staining methods, including simple and differential staining, and describes the reagents involved in such processes.

1. Satin, Staining of bacteria, Basics about Staining types

2.  To study Microbiological structures and to
divide them into different groups, staining is
used, in light microscopy.
 Numerous staining techniques are available
for visualization, differentiation, and
separation of bacteria in terms of
morphological characteristics and cellular
structures.

3.  A stain (dye) may be defined as an organic
compound containing a benzene ring plus a
chromophore and an auxochrome group.
Stain
Benzene Ring Organic Colourless
Solvent
Chromophore Chemical group that imparts
colour to benzene
Auxochrome
Conveys the property of ionization to
chromogen, enabling it to form salts and
allowing it to bind with tissues and
fibres.
Chromogen
(Colored Compound
not a stain)

4.  The ability of stain to bind with
macromolecules such as protein and nucleic
acid depends upon;
 Electrical charge on Chromogen
 Cellular components to be stained

5.  Acidic Stains are anionic, which means that,
on ionization of the stain, the Chromogen
portion exhibits a negative charge and
therefore has a strong affinity for the positive
constituents of the cell such as Proteins.
 Picric acid is an example of acidic stain.

6.  Basic stains are cationic, because on
ionization the chromogen portion exhibits a
positive charge and therefore has a strong
affinity for the negative constituents of the
cell such as Nucleic acids.
 Methylene blue is a basic stain.
 Basic stains are commonly used for bacterial
staining.

7.  There are two types of staining techniques;
 Simple Staining
▪ Use of Single stain
 Differential staining
▪ Use of two contrasting stains

8.  For visualization of morphological shape
(cocci, bacilli, and spirilli)
 arrangement (chains, clusters, pairs, and
tetrads)

9.  Basic stains with a positively charged
chromogen are preferred because bacterial
nucleic acids and certain cell wall
components carry a negative charge.
 Commonly used Dyes are;
 Methylene blue
 crystal violet
 carbol fuchsin

10.  Separation into groups
 Gram stain
 Acid-fast stain
 Visualization of structures
 Flagella stain
 Capsule stain
 Spore stain
 Nuclear stain

11.  Differential staining requires at least four
chemical reagents, applied sequentially to a
heat-fixed smear;
 primary stain
▪ impart its color to all cells
 decolorizing agent
▪ may or may not remove the primary stain
 Counter stain
▪ has a contrasting color to that of the primary stain
 Gram stain
▪ divides bacterial cells into two major groups, gram-positive
and gram-negative

12.  Negative staining requires the use of an acidic stain
The acidic stain, with its negatively charged
chromogen, will not penetrate the cells, because of
the negative charge on the surface of bacteria.
 the unstained cells are easily visible against the
colored background.
 heat fixation is not required
 Used for spiralla and fungi that are hard to stain

13.  Reagents used;
 India ink
 Nigrosin
 Cultures used;
 Micrococcus luteus
 Bacillus cereus
 Aquaspirillum itersonii