Bacterial Staining

Lab.3
 Introduction:
• Staining : process in which bacteria are stained.
• Stains/dyes: organic compounds which carries either positive
charges or negative charges or both.
• Stains combine chemically with the bacterial protoplasm.
• If the cell is not already dead, the staining process itself will
kill it .
• The commonly used stains are salts . Based on the charges , the
stains can be classified into:
• Basic stains :are positively charge .
• Acidic stains : are negatively charge.
• Neutral stains :carries both positive and negative charges.
Basic Dyes : chromophore is the positive ion dye attracted by the
bacteria so the cells of bacteria stained, ex. Crystal violet.
Bacterial cell

Acid Dyes :chromophore is the negative ion dye rejected by the

cell and the background of slide stained, ex. Nigrosin stain.

Bacterial cell

• Bacteria are slightly negative, so are attracted to the positive

chromophore of the BASIC DYE
 Types of staining techniques

Simple staining Differential staining

(use of a single stain) (use of two contrasting stains
separated by a decolorizing agent)

For visualization of
morphological Identification Visualization
shape & arrangement. of structure
Gram Acid fast
stain stain Spore Capsule
stain stain
Gram Staining :
• Named after Hans Christian Gram, differentiates
between Gram-positive purple and Gram-negative
pink stains and is used to identify certain pathogens.

• Principles: Gram staining is used to determine gram

status to classify bacteria broadly. It is based on the
composition of their cell wall.
• Gram status is important in medicine, the presence or absence
of a cell wall will change the bacterium's susceptibility to some
antibiotics.

• Gram-positive bacteria stain dark blue or violet. Their cell wall

is typically rich with peptidoglycan and lacks the secondary
membrane and lipopolysaccharide layer found in Gram-
negative bacteria
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Gram staining reagents:
1. Crystal violet - primary stain
2. Gram´s iodine – mordant /fixative
3. Ethanol %95 – Decolorizer
4. Safranin- Counterstain
Gram staining steps :
• Crystal violet acts as the primary stain (used to stain cell
walls)
• Gram’s iodine acts as a mordant (Helps to fix the primary
dye to the cell wall).
• Decolorizer is used next to remove the primary stain (crystal
violet) from Gram Negative bacteria (those with LPS
imbedded in their cell walls). Decolorizer is composed of an
organic solvent, such as, acetone or ethanol or a combination
of both.)
• Safranin (a counter stain ): is applied to stain those cells
(Gram Negative) that have lost the primary stain as a result
of decolorization.
Procedure of Gram staining :
• Make a smear and heat fixed.
• Primary staining: The smear is covered with crystal violet,
for 1 minute and washed with water.
• Mordanting : It is then covered with Gram’s iodine, Kept for
1 minute, and washed with water.
• Decolorization: The smear is covered with alcohol and is
washed with water immediately.
• Counter staining: The smear is then covered with safranine,
kept for 30 seconds and washed with water.
• Using filter paper the slide is gently blotted to dry.
• Examine the slide under the microscope.
Gram staining
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