Staining Procedures
cfu/ml
Classes of Ionizible Dyes:
• 1. Basic Dyes
• Are commonly dyes used in
bacteriology
• Cationic or have a positively charged
groups that bind to negatively
charged molecules like nucleic acids
and proteins.
• Examples: methylene blue, basic
fuchsin, crystal violet, safranin and
malachite green.
*positively charged groups (pentavalent nitrogen)
• 2. acid dyes
• Anionic or possesses a negatively
charged groups that bind to
positively charged cell structures.
• Examples: eosin, rose Bengal and
acid fuchsin
*negatively charged groups (carboxylic and phenolic)
Staining Techniques
• 1. Simple Staining
• A single stain is used.
• It is directed towards coloring the
forms and shapes present.
• Examples: malachite green,
methylene blue, crystal violet, carbol
fuchsin and safranin
• 2. Differential Staining
• It divides bacteria into separate
groups.
• It is directed towards coloring
components of those elements
present.
• Inoculums size for staining: 10^5
• Examples: Gram staining and Acid
Fast Bacilli staining
Steps in Differential Staining:
1. Application of primary stain
2. Application of Mordant
3. Application of Decolorizing reagent
4. Application of secondary stain or
Counterstain
3. diagnostic antibody or DNA probe Mediated
Staining
-Directed specifically at identification of
an organism
-Used for the specific identification of selected
pathogens such as Chamydia, Bordetella, Legionella,
HSV, Varicella, CMV, adenovirus and respiratory virus
• 4. Negative Staining
• Utilize to demonstrate the presence
of diffuse capsule surrounding some
bacteria.
• Excellent technique to study
bacterial gas vacuoles and viral
morphology.
• Also used to study cells sensitive for
heat fixing.
• Result: the bacteria appear as light
colored bodies against a dark
background.
• Example: India Ink or Nigrosin dye.
Gram Stain
• Most commonly used differential stain in the
clinical microbiology laborator