OBJECTIVES OF STAINING
Improves visibiltiy by greater contrast between the
organism and the background,
differentiate various morphological types (by shape, size,
arrangement, etc.),
determine the staining characteristic of organism and, at
times, direct diagnosis of disease, and
demonstrate the purity of culture.
observe certain structures (flagella, capsules, endospores,
etc.),
, STAINS AND DYES
➢ A dye is a general-purpose coloring agent,
whereas a stain is used for coloring biological
material.
➢ A stain is an organic compound containing a
benzene ring plus a chromophore and an
auxochrome group.
➢ chromophore is a chemical group that imparts
color to benzene.
➢ auxochrome group is a chemical compound
that conveys the property of ionization of
chromogen (ability to form salts) and bind to
fibers or tissues.
, REQUIREMENTS FOR STAINING
Stain – Majority of the stains used for staining bacteria are of the
basic type as nucleic acid of bacterial cells attract the positive
ions, e.g. methylene blue, crystal violet. Acidic stains are used for
background staining.
Mordant – It is a chemical that forms an insoluble complex with
the stain and fixes it or causes the stain to penetrate more deeply
into the cell. These are used in indirect staining. For example,
Gram’s iodine in Gram staining and phenol in Ziehl Neelson’s
staining.
Accentuater – It is a chemical which when added to a stain to
make the reaction more selective and intense. For example,
potassium hydroxide added in Loeffler’s methylene blue.
Decolorizer – It is a chemical used to remove the excess stain in
indirect regressive staining. For example, ethanol in Gram’s
staining.
, Types of staining techniques
Simple staining Differential staining
(Use of of single stain) (Use of two contrasting stains)
Direct Indirect Separation Visualization
(Positive) (Negative) into groups of structures
1. Gram stain 1. Flagella stain
2. Acid fast 2. Capsule stain
stain 3. Spore stain
Improves visibiltiy by greater contrast between the
organism and the background,
differentiate various morphological types (by shape, size,
arrangement, etc.),
determine the staining characteristic of organism and, at
times, direct diagnosis of disease, and
demonstrate the purity of culture.
observe certain structures (flagella, capsules, endospores,
etc.),
, STAINS AND DYES
➢ A dye is a general-purpose coloring agent,
whereas a stain is used for coloring biological
material.
➢ A stain is an organic compound containing a
benzene ring plus a chromophore and an
auxochrome group.
➢ chromophore is a chemical group that imparts
color to benzene.
➢ auxochrome group is a chemical compound
that conveys the property of ionization of
chromogen (ability to form salts) and bind to
fibers or tissues.
, REQUIREMENTS FOR STAINING
Stain – Majority of the stains used for staining bacteria are of the
basic type as nucleic acid of bacterial cells attract the positive
ions, e.g. methylene blue, crystal violet. Acidic stains are used for
background staining.
Mordant – It is a chemical that forms an insoluble complex with
the stain and fixes it or causes the stain to penetrate more deeply
into the cell. These are used in indirect staining. For example,
Gram’s iodine in Gram staining and phenol in Ziehl Neelson’s
staining.
Accentuater – It is a chemical which when added to a stain to
make the reaction more selective and intense. For example,
potassium hydroxide added in Loeffler’s methylene blue.
Decolorizer – It is a chemical used to remove the excess stain in
indirect regressive staining. For example, ethanol in Gram’s
staining.
, Types of staining techniques
Simple staining Differential staining
(Use of of single stain) (Use of two contrasting stains)
Direct Indirect Separation Visualization
(Positive) (Negative) into groups of structures
1. Gram stain 1. Flagella stain
2. Acid fast 2. Capsule stain
stain 3. Spore stain
