STAINING
2ND LAB
Objectives
Learn the proper procedure for preparing a
bacterial smear
Understand the biochemistry underlying the
Gram stain
Distinguish between Gram-positive and Gram-
negative bacteria.
Understand special staining procedures and
view examples of these
Staining
Staining helps with visualizing cells that are
otherwise difficult to see.
There are different types of dyes and different
types of staining techniques.
Dyes are salts composed of a positive and
negative ion, one of which is coloured and called
the chromophore:
Basic dyes (positive ion). Example: crystal violet
Acidic dyes (negative ion). Example: orange G
Types of staining techniques
Simple stain: primary purpose is to see the entire
microorganism so that cell shape and basic
cellular structures are visible.
Differential stain: react differently with different
types of bacteria and allows us to distinguish
among them.
Most commonly used the Gram stain that
differentiate between Gram positive (purple) and
Gram negative (pink) bacteria
Acid fast stain. The stain binds strongly to waxy
material present in some bacteria’s cell wall. Used to
identify Mycobacteria and Nocardia
Types of staining techniques
Special stain: colour and identify specific cell parts.
Negative stain for capsules.
Endospore staining to stain endospores
Flagella staining. The stain is applied after a
mordant increases the size of the flagella.
Smear Preparation
First step in Bacterial staining
Kill bacteria
Fix bacteria to slide
Smear Preparation
Simple stain
The simple stain can
be used to determine
cell shape, size, and
arrangement
Simple stain procedure
1. Perform a bacterial smear.
2. Saturate the smear with basic dye for
approximately 1 minute
crystal violet, safranin, or methylene blue
3. Rinse the slide gently with water.
4. Carefully blot dry with towel paper.
5. Observe the slide under the microscope.
Differential Stains
Two or more reagents
Distinguish
Bacterial groups
Specific Structures
Example
Gram stain
Acid Fast Stain
How to prepare the perfect Gram stain - Gram
Gram stain
staining procedure - YouTube
1. Perform a bacterial smear, Air dry and Heat fixation
2. Saturate the smear with crystal violet for 1 minute.
3. Rinse the slide gently with water.
4. Saturate the smear with iodine for 1 minute.
5. Rinse the slide gently with water.
6. Decolorize with Gram decolorizer (95% Ethanol)!
7. Rinse the slide gently with water.
8. Counterstain with safranin for 1 minute.
9. Rinse the slide gently with water.
10. Carefully blot the slide dry with paper.
11. Observe the slide under the microscope (oil lens)
Gram Staining - YouTube
Gram Positive and Negative cells Endospore staining
Acid fast staining
Bacterial cell shapes
SPORE STAIN
Bacterial Endospores
• Endospores are a dormant stage of some
bacterium that allows it to survive conditions that
would normally kill bacteria such as extreme
drought or heat
• Endospores provide resistance against:
• drying
• Low nutrient conditions
• Radiation
• High temperatures and various chemical disinfectants
:Steps of sporulation include
bacterium's DNA chromosome replicated (is
copied)
cell's plasma membrane pinches off between
the replicated chromosomes, forming the
forespore
a second membrane encloses the forespore,
with calcium and dipicolinic acid forming a
cortex between the inner and outer membrane
an external spore coat encloses the endospore
endospore is released once the vegetative cell
that generated it dies and disintegrates
https://www.youtube.com/watch?
v=UHsqFjP1dZg&list=PL17FCBBBA999CCCC8
&index=2
The shape of the spore is an
identifying characteristic
Swelled vs. Not swelled spore
spore
Bacterial cell spore
Bacterial cell
Central, Sub-Terminal, and Terminal spores
Stain Procedure
Schaeffer-Fulton Method
1. Prepare a smear. Air Dry. Heat fix
2. Put the slide on steam rack
3. Flood the smear with Malachite Green stain
4. Steam slide for 10 minutes (every minute, add a few more
drops of Malachite Green stain)
5. Allow slide to cool (after the 10 min. steam process)
6. Drain slide and rinse for 30 seconds with DI water (discard
filter paper)
7. Flood smear with Safranin (counter stain). This stains the
vegetative cell. (Leave for 1 minute)
8. Drain the slide and rinse with DI water
9. Blot Dry
10. Use oil immersion objective to view
Spore Staining procedure
Endospore Stain Example
Spores: Green
Cell: Red or Pink
Each student will make a smear and Endospore stain
of: Bacillus subtilis
Endospore staining
(Dorner Method)
Uses Nigrosin (negative stain)
In this stain, red endospores are in
.colorless vegetative cells
