Description
Microbiology
Identification of bacteria
staining techniques
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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
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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
features
– Preserve them for future study
Unstained
organisms
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Need for staining
• Staining simply means colouring the microorganisms with a dye
that emphasizes certain structures
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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.
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Preparing smears for staining
Step 2: Fixation
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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
minute.
• 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
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Staining
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
examination.
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Stains
• Stains are salts composed of a positive and a negative ion,
• The colored ion is known as the chromophore.
Colored
Positive ion Basic stain
chromophore
Colored
Negative ion Acidic stain
chromophore
Stain
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Stains
• 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
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Stains
• 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
background
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Negative staining
• Distortions of cell size and shape are minimized because fixing is
not necessary and the cells do not pick up the stain
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Negative staining
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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
visible
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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.
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Simple staining procedure
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Mordant
• Mordant – A chemical that intensifies the stain
Functions of mordant
• 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.
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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
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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
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Gram stain
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Gram stain
• 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
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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
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Gram staining
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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
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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
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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
cephalosporins.
• Gram-negative bacteria are generally more resistant because the
antibiotics cannot penetrate the lipopolysaccharide layer.
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Summary
• Morphological stains help in identifying the cell size, shape and
structure
• Simple stains color the cells
• Negative staining color the background
• Gram staining differentiates between gram positive and gram
negative cells
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Summary
• The difference in gram staining is due to difference in cell wall
composition
• Stages of gram staining –
1. Primary stain
2. Mordant
3. Decolorization
4. Counterstaining
