This document discusses staining techniques used to visualize bacterial cells under a microscope. It describes simple staining, which uses a single dye to color all bacterial cells, and differential staining techniques like Gram staining that classify bacteria into groups. Gram staining involves staining cells with crystal violet dye, adding iodine as a mordant, washing with alcohol as a decolorizer, and counterstaining with safranin. This causes Gram-positive bacteria to appear violet and Gram-negative bacteria to appear pink based on differences in their cell wall structure. The document provides examples of staining Staphylococcus aureus, Bacillus subtilis and E. coli to demonstrate the staining results.

1. Simple and Gram Staining
Microbiology
Unit II
Muhammad Iqbal
Lecturer
Khyber Medical University

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Staining of Bacteria
 Bacteria are almost colorless and transparent
 A staining technique is often applied to the
cells to color them.
Their shape and size can be easily determined
under the microscope.

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Types of staining techniques
Simple staining
(use of a single stain)
Differential staining
(use of two contrasting stains
separated by a decolorizing agent)
For visualization of
morphological
shape & arrangement.
Identification Visualization
of structure
Gram
stain
Acid fast
stain Spore
stain
Capsule
stain

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Smear Preparation:
Preparation and Fixation of Bacteria
for Staining.
Objective:
To kill the microorganism & fix them
to the slide to prevent them from
being washed out during the process
of staining.

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Smear preparation
S Fixation

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 Staining:
 Staining is an auxiliary technique used
in microscopy to enhance contrast in
the microscopic image. Stains are used
to highlight cell structures. Different
types of staining methods are used to
study cell structures and differentiate
one bacterium from another.
Simple Staining is the use of single
basic dye to color the bacterial cells
e.g. methylene blue, crystal violet, or
safranin.

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Simple Staining
All bacteria take the color of the dye.
 Objective:-
To show the morphological
shapes and arrangement of
bacterial cells.

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Basic Shapes of Bacteria
Cocci Bacilli

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Arrangements
Cocci
Irregular Clusters Chains or Pairs
Tetrads
Staphylococci Micrococci Streptococci

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Simple Staining
 Type of staining:-
Simple Stain
 Name of dye:-
Methylene blue
 Shape of cells:-
bacilli
 Arrangement of
cells:- strain
 Color:- Blue
 Name of m.o:-
Bacillus

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Simple Staining
 Type of staining:- Simple Stain
 Name of dye:- Crystal violet.
 Shape of cells:- cocci
 Arrangement of cells: clusters
 Color:- Purple
 Name of mo’ : Staphylococci

13. Differential Staining
Differential Stains use two or more stains and allow
the cells to be categorized into various groups or
types.
Both techniques allow the observation of cell
morphology, or shape, but differential staining
usually provides more information about the
characteristics of the cell wall (Thickness).
The most common differential stain used in
microbiology is the Gram Stain as.
crystal violet, safranin, and methylene blue.

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Gram Stain:
 It is the most
important differential
stain used in
bacteriology because
 it classifies bacteria
into two major groups:
a) Gram positive:
Appears violet after
Gram’s stain
b) Gram negative:
Appears pink after
Gram’s stain

16. Crystal violet
↓
Iodine
↓
Alcohol
↓
Safranin
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Gram +ve
S. aureus
Gram –ve
E. coli
Step 1: Crystal Violet
(30—60 sec)
Step 2: Iodine
(mordant)
(30 sec)
Step 3: Decolorization
(Alcohol)– 5 sec
Step 4: Safranin
(30—60 sec)

18. Gram-positive bacteria
• Have a thick peptidoglycan layer surrounds the cell.
• The stain gets trapped into this layer and the
bacteria turned purple.
• Retain the color of the primary stain (crystal violet)
after decolorization with alcohol
Gram-negative bacteria
• have a thin peptidoglycan layer that does not retain
crystal violet stain.
• Instead, it has a thick lipid layer which is dissolved
easily upon decolorization with Alcohol.
• Therefore, cells will be counterstained with safranin
and turned pink.
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19. Gram Stain
• Materials:-
• Cultures of Staphylococcus aureus,
Bacillus subtilis, E.coli
• Gram stain:
Crystal violet (primary stain)
Iodine (mordant)
Alcohol (decolorizing agent)
Safranin (counter stain)
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20. Results
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Shape: Cocci
Arrangment: irregular clusters
Colour: Violet
Gram’s reaction: Gram’s +ve
Name of microorganism: Staphylococci

21. Results
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Shape: Bacilli
Arrangment: Chains
Colour: Violet
Gram’s reaction: Gram +ve
Name of microorganism: Bacillus

22. Results
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Shape: Rods
Arrangment: Single
Colour: pink
Gram’s reaction: Gram –ve
Name of microorganism: Gram negative bacilli