PA R T 3
Bacterial Staining
LEARNING OBJECTIVES
Once you have completed the experiments in this section, you should be familiar with
1. The chemical and theoretical basis of biological staining.
2. Manipulative techniques of smear preparation.
3. Procedures for simple staining and negative staining.
4. The method for performing differential staining procedures, such as the Gram,
acid-fast, capsule, and spore stains.
Introduction acids depends on the electrical charge found on
the chromogen portion, as well as on the cellular
Visualization of microorganisms in the living component to be stained.
state is quite difficult, not only because they are Acidic stains are anionic, which means that,
minute, but also because they are transparent on ionization of the stain, the chromogen portion
and practically colorless when suspended in an exhibits a negative charge and therefore has a
aqueous medium. To study their properties and strong affinity for the positive constituents of the
to divide microorganisms into specific groups for cell. Proteins, positively charged cellular compo-
diagnostic purposes, biological stains and staining nents, will readily bind to and accept the color of
procedures in conjunction with light microscopy the negatively charged, anionic chromogen of an
have become major tools in microbiology. acidic stain. Structurally, picric acid is an example
Chemically, a stain (dye) may be defined as of an acidic stain that produces an anionic chro-
an organic compound containing a benzene ring mogen, as illustrated in Figure P3.3.
plus a chromophore and an auxochrome group Basic stains are cationic, because on ioniza-
(Figure P3.1). tion the chromogen portion exhibits a positive
The stain picric acid may be used to illustrate charge and therefore has a strong affinity for the
this definition (Figure P3.2). negative constituents of the cell. Nucleic acids,
The ability of a stain to bind to macromolecu- negatively charged cellular components, will
lar cellular components such as proteins or nucleic
Benzene: Organic colorless solvent
+ Chromogen:
Chromophore: Chemical group that imparts Colored compound,
+ color to benzene not a stain
Stain
Auxochrome: Chemical group that conveys the
property of ionization to the chromogen,
enabling it to form salts and bind to
fibers or tissues
Figure P3.1 Chemical composition of a stain
55
H H OH
H H O2N NO2 O2N NO2
+ 3NO2– + OH–
H H H H H H
H NO2 NO2
Benzene Nitro groups Trinitrobenzene Auxochrome Trinitrohydroxybenzene
colorless chromophore chromogen, yellow (picric acid) yellow stain
in color due to the
presence of
chromophores
Figure P3.2 Chemical formation of picric acid
–
OH O
O2N NO2 O2N NO2
+
Ionization + H
H H H H
NO2 NO2
Picric acid Anionic chromogen
Figure P3.3 Picric acid: an acidic stain
+
N N –
Ionization + Cl
(CH3 )2 N S N(CH3 )2 Cl (CH3 )2 N N(CH3 )2
S
Methylene blue Cationic chromogen
Figure P3.4 Methylene blue: a basic stain
readily bind to and accept the color of the posi- charge on the bacterial surface acts to repel most
tively charged, cationic chromogen of a basic acidic stains and thus prevent their penetration
stain. Structurally, methylene blue is a basic stain into the cell.
that produces a cationic chromogen, as illustrated Numerous staining techniques are available
in Figure P3.4. for visualization, differentiation, and separation
Figure P3.5 is a summary of acidic and basic of bacteria in terms of morphological charac-
stains. teristics and cellular structures. A summary of
Basic stains are more commonly used for commonly used procedures and their purposes is
bacterial staining. The presence of a negative outlined in Figure P3.6.
56 Part 3
Sodium, potassium, calcium, or ammonium salts of colored acids
Acidic ionize to give a negatively charged chromogen
Stains
The chloride or sulfate salts of colored bases ionize to give a
Basic
positively charged chromogen
Figure P3.5 Acidic and basic stains
Methylene blue is a classic example of a simple stain. This blue stain will color all
cells blue, making them stand out against the bright background of the light
microscope.
Simple staining: For visualization of morphological shape (cocci,
Use of single stain bacilli, and spirilli) and arrangement (chains,
clusters, pairs, and tetrads)
Types of
staining
techniques Gram stain
Separation into groups
Acid-fast stain
Differential staining:
Use of two contrasting Flagella stain
stains Visualization of Capsule stain
structures Spore stain
Nuclear stain
Figure P3.6 Staining techniques
When benzene of an organic colourless solvent binds to the nitro group of chromophore, it results in a yellow coloured compound called
trinitrobenzene in which three hydrogen atoms in the benzene molecule are replaced by three nitro groups.
Trinitrobenzene is a chromogen but not a stain. If however, another hydrogen atom is replaced by an auxochrome group, such as OH, the compound
known as picric acid (trinitrohydroxybenzene) is formed. The picric acid is capable of ionization or electrolytic dissociation to form salt that binds to
opposite-charged biological substance (Fig. 17.2).
Thus the picric acid, which is yellow in colour, is a stain (dye). The colour of picric acid is due to the chromophoric nitro group (NO2), and its staining
property is due to the auxochromic hydroxyl group (OH), which imparts the molecule the property of ionization or electrolytic dissociation.
Part 3 57
