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Identification using Staining:- PDF/PPT



Identification of bacteria

staining techniques
Intended learning objectives

At the end of this lecture, the student will be able to:

• Classify the staining techniques

• Explain the principle and procedure involved morphological and

gram staining

• Outline the significance of staining in identification of bacteria
Need for staining

• Most microorganisms appear almost
colourless when viewed through a
standard light microscope

• Hence must be fixed and stained to
– Increase visibility

– Accentuate specific morphological

– Preserve them for future study
Need for staining

• Staining simply means colouring the microorganisms with a dye
that emphasizes certain structures
Basic staining procedure

Step 1: Smear preparation

• A thin film of material containing the microorganisms is spread over
the surface of the slide. This film, called a smear.

• It is allowed to air dry.
Preparing smears for staining

Step 2: Fixation
Preparing smears for staining

Step 2: Fixation

• By passing it through the flame of a bunsen burner several times,
smear side up, or by covering the slide with methyl alcohol for one

• Fixing simultaneously kills the microorganisms and fixes them to
the slide.

• It also preserves various parts of microbes in their natural state
with only minimal distortion
Step 3: Staining

• Stain is applied and then washed off with water

• Slide is blotted with absorbent paper.

• The stained microorganisms are now ready for microscopic

• Stains are salts composed of a positive and a negative ion,

• The colored ion is known as the chromophore.

Positive ion Basic stain
Negative ion Acidic stain

• Bacteria are slightly negatively charged at pH 7.

• Thus, the colored positive ion in a basic dye is attracted to the
negatively charged bacterial cell.

• Basic dyes include crystal violet, methylene blue, malachite green
and safranin

• Acidic dyes are not attracted to most types of bacteria

• The dye’s negative ions are repelled by the negatively charged
bacterial surface

• The dye colors the background instead

• Preparing colorless bacteria against a colored background is called
negative staining.

• Examples of acidic dyes are eosin, acid fuchsin, and nigrosin

Negative staining

• Valuable for observing overall cell shapes, sizes, and capsules

• The cells are made highly visible against a contrasting dark
Negative staining
• Distortions of cell size and shape are minimized because fixing is
not necessary and the cells do not pick up the stain
Negative staining
Simple staining

• A simple stain is an aqueous or alcohol solution of a single basic dye

• The primary purpose of a simple stain is to highlight the entire

microorganism so that cellular shapes and basic structures are

Simple staining

• The stain is applied to the fixed smear for a certain length of lime

and then washed off

• The slide is dried and examined

• Simple stains commonly used in the laboratory – methylene blue,

carbolfuchsin, crystal violet, and safranin.
Simple staining procedure

• Mordant – A chemical that intensifies the stain

Functions of mordant

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• To increase the affinity of a stain for a biological specimen

• To coat a structure (such as a flagellum) to make it thicker and
easier to see after it is stained with a dye.
Differential stains

• Differential stains react differently with different kinds of bacteria

• Can be used to distinguish them

• The differential stains most frequently used for bacteria are the
Gram stain and the acid-fast stain
Gram stain

• Gram stain was developed in 1884 by the Danish bacteriologist
Hans Christian Gram

• Most useful staining procedures – classifies bacteria into two large
groups: gram-positive and gram-negative.

➢ Step 1: Primary stain

➢ Step 2: Mordant

➢ Step 3: Decolorization

➢ Step 4: Counter stain
Gram stain
Gram stain

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• The purple dye and the iodine combine in the cytoplasm of each
bacterium and color it dark violet or purple.

• Bacteria that retain this color after the alcohol has attempted to
decolorize them are classified as gram-positive

• Because gram-positive bacteria retain the original purple stain, they
are not affected by the safranin counterstain
Gram stain

• Bacteria that lose the dark violet or purple color after
decolorization are classified as gram negative

• Because gram-negative bacteria are colorless after the alcohol
wash, they are no longer visible.

• This is why the basic dye safranin is applied; it turns the gram-
negative bacteria pink.

• Stains such as safranin that have a contrasting color to the primary
stain are called counterstains
Gram staining
Principle of Gram stain

• Different kinds of bacteria react differently to the Gram stain

• Structural differences in their cell walls affect the retention or
escape of a combination of crystal violet and iodine, called the
crystal violet- iodine (CV-I)complex

• Gram-positive bacteria have a thicker peptidoglycan cell wall than
gram-negative bacteria

• Gram- negative bacteria contain a layer of lipopolysaccharide (lipids
and polysaccharides) as part of their cell wall
Principle of gram staining

Crystal violet + Iodine

Enters Gram positive cell wall Enters Gram negative cell wall

Peptidoglycan layer retains CV-I Alcohol wash disrupts the outer
during alcohol decolorization lipopolysaccharide layer

Gram-positive cells retain the color of CV- I complex is washed ou tthrough the
the crystal violet dye thin layer of peptidoglycan

Gram negative Cells are colorless

Turn pink upon safranin staining
Clinical significance of Gram staining

• Gram reaction of a

bacterium can provide valuable information for
  the treatment of disease.

• Gram-positive bacteria tend to be killed easily by penicillins and

• Gram-negative bacteria are generally more resistant because the
  antibiotics cannot penetrate the lipopolysaccharide layer.


• Morphological stains help in identifying the cell size, shape and


• Simple stains color the cells

• Negative staining color the background

• Gram staining differentiates between gram positive and gram

  negative cells


• The difference in gram staining is due to difference in cell wall


• Stages of gram staining –
   1.   Primary stain

   2.   Mordant

   3.   Decolorization

   4.   Counterstaining