Download Bacterial Staining

Bacterial Staining: Lab 2
General Discussion
Introduction
Bacteria are microscopic organisms. They are also colorless for the
most part. In order to visualize them to study their structure, shape
and other structural characteristics, it becomes necessary to make
them more easily visible.
This means that the structures have to be contrasted from their
environment so that they can be seen easily.
Staining is one such contrasting process wherein chemicals (stains)
are used to impart their color to the cells structures with which they
come in contact.
Stains
• All dyes are salts
– Ionize
• Cationic
• Anionic
• Techniques
– Single dyes
– Multiple dyes
• Use
Chemical Makeup of Stains
• Benzene = organic compound
• Chromophore = color
• Auxochrome = ionization properties
• Benzene + Chromophore = Chromogen
– Chromogen is a colored compound only
• Auxochrome with Chromogen allows the dye to
form salt compounds that adhere to cells.
Basic Dyes
• Work best in basic pH
• Ionizes (Cl-, SO4-)
• Creates (+) Cationic
chromogen
• Attracted to (-) acidic cell
components [DNA, proteins]
• Examples
–
–
–
–
–
Methylene Blue
Crystal Violet
Carbol Fuchsin
Safranin
Malachite Green
CF
Acidic Dyes
• Works best in acidic pH
• Ionizes (Na+, K+, Ca++)
• Creates Anionic (-)
chromogen
• Attracted to (+) cell
components [AA]
• Examples
–
–
–
–
Picric Acid
Nigrosin
India Ink
Eosin
Nigrosin
Staining Methods
• Simple Stain
- Use single dye
• Differential Stains
– Group
• Gram Stain
• Acid Fast Stain
– Special Structures
• Capsule Stain
• Endospore Stain
• Flagellar Stain
Staining procedure can also be classified in different ways.
Simple staining involves the use of only 1 dye and is used primarily
as a means to study the morphology and structure of organisms.
Differential staining uses 2 dyes or more and is also used to
differentiate the organisms into groups
or to study Special Structures in the cells.
Simple staining – there are two methods:
positive staining - where the actual cells are themselves colored
and appear in a clear background;
negative staining – where the cells remain clear (uncolored) and the
background is colored to create a contrast to aid in the better
visualization of the image.
Since most cell surfaces have a slightly negative charge when they are
found at neutral pH environments, basic stains are used most often in
positive staining procedures and acidic stains in negative staining
procedures.
Differential staining – among various types of differential staining
procedures few are more commonly used.
Gram staining is the most used technique is the identification of
bacteria.
Acid fast staining and Spore staining are also commonly used
Bacterial Smear preparation:
Smear is a distribution of bacterial cells on a slide for the purpose
of viewing them under the microscope.
Method:
-Aseptically a small sample of the culture is spread over a
slide surface.
-This is then allowed to air dry.
-The next step is heat fixation to help the cells adhere to the
slide surface.
-The smear is now ready for staining.
Slide Preparation
• Clean slide
– Broth
– Solid + water
• Air dry first
• Heat fix (usually)
– Kill organism
– Adhere to slide
– Accepts dye
• Problems
– Too thick
– Wash off specimen
SPECIMEN PREPARATION FOR LIGHT
MICROSCOPY
• PREPARATION OF A BACTERIAL SMEAR
Simple Stain
•
•
•
•
•
One reagent used
Flood smear 30-60 seconds
Rinse with water
Bacteria stained
Basic dye
– (+) chromogen
– (-) cell wall
– Shows morphology
• Size
• Shape
• Arrangement
• Examples
– MB
– CF
– CV
Cell Arrangements:
Demos: simple stains of:
Neisseria (diplococci)
Pseudomonas (bacilli)
Negative Stain
• Acid Dye
• (-) chromogen
• Repelled by (-) cell
wall
• Cells
– Colorless
– Seen against dark
background
STAINING
• SPECIAL STAINING TECHNIQUES
• Negative staining is preferable since it does not use heat fixation
and thus causes less distortion of the cells.
– Negative Staining for Capsules
• Capsules
– Determinants for virulence in some genera of bacteria
• Capsule Staining
– Preparation of a bacterial smear along with negative
staining w/ Nigrosin or India Ink
– Counterstain w/ Crystal Violet
STAINING
• SPECIAL STAINING TECHNIQUES
– Negative Staining for Capsules
• Capsule Stain Results