P3 - Microscopy, Morphology &

Staining

Mrs Aswathy Nandakumar

November 4, 2023

www.gmu.ac.ae COLLEGE OF
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Page 11 ofthe
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 Microscopes
• Required for the morphological study of
micro-organisms.

Magnification:
• Ocular lens(eye piece) – 10X
• Objective lens – 4X (scanner), 10X (low power),
40X (high power) , 100X (oil immersion)
• Total magnification : product of magnification of
objective lens and ocular lens
 Types of Microscopes

• Bright field or light microscope

- Simple
- Compound
•
• Phase contrast microscope
• Dark field (dark ground) microscope
• Fluorescent microscope
• Electron microscope
Bright field (Light) Microscope
MORPHOLOGY
 Types of Staining

• Simple staining
• Differential staining
• Negative staining
• Impregnation method
• Special staining – for certain bacteria, bacterial
spores, parasites & fungi

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 SIMPLE STAINS

• Impart same color to all

bacteria.

• Only shows the presence of

micro-organisms.

• e.g. Methylene blue, Carbol

fuchsin , Safranine, etc

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 Differential Stains

• Imparts different color to different bacteria.

• Most widely used differential stains are:

Gram stain &
Ziehl Neelsen stain

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 GRAM’S STAINING
Christian gram
• It differentiates bacteria into two broad groups :
Gram positive
- appear violet
Gram negative
– appear red / pink
Helps in:
• Identification of bacteria
• In influencing the choice of antibiotic

Procedure: https://www.youtube.com/watch?v=L9batS-vGDY
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Principle & Procedure
• Crystal violet dye
- Primary dye
- stains all cells purple

• Iodine solution
- a mordant
- forms a crystal violet-iodine complex
- all cells continue to appear blue

• Organic solvent Acetone or Ethanol

- Gram –ve :Lipid rich,thin peptidoglycan layer
- Gram +ve :Lipid poor , thick peptidoglycan layer
- extracts the violet dye complex from gram negative bacteria
- gram negative appear colorless;gram positive remain blue.

• Counterstain - red dye Safranin

- stains the decolorized gram negative cells red/pink
- gram positive bacteria remain blue.
 Gram positive bacteria

Gram positive cocci in chains

Eg: Streptococcus Gram positive cocci in clusters
Eg: Staphylococcus

Gram positive bacilli with spore

Eg: Clostridium tetani
 Gram negative bacteria

Gram negative cocci in pairs

Eg: Neisseria Gram negative bacilli
Eg: E coli, Salmonella typhi
 Acid fast stain
• Ziehl – Neelsen stain

• Mycolic acid in cell wall – responsible for the

acid fast nature

• Eg. Mycobacteria
Nocardia
Acid fast bacilli seen against blue background
Eg: Mycobacterium tuberculosis
Principle & Procedure
 NEGATIVE STAINS
• Produce colored background against which bacteria
stand out in contrast.

• Used for bacterial capsule, spirochetes

eg India ink, Nigrosin.

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 IMPREGNATION METHOD
• Cell structures too thin to be seen under microscope
are rendered visible by thickening with silver
impregnation on their surfaces.

• Used for the demonstration of bacterial flagella,

spirochetes

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 Albert’s Stain
• For staining of volutin granules containing
organisms like diphtheria bacillus

• The granules appear “bluish black” rest of the

bacilli appear green [metachromatic]

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