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