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Staining Methods Lecture-2

The document outlines various staining methods used in microscopy to enhance contrast and differentiate structures in biological specimens. It categorizes techniques such as simple staining, differential staining (including Gram and acid-fast staining), and specialized methods like fluorescent and silver staining, detailing their purposes, methods, and results. These techniques are essential for identifying organisms, studying tissue structures, and detecting specific cellular components.

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2 views

Staining Methods Lecture-2

The document outlines various staining methods used in microscopy to enhance contrast and differentiate structures in biological specimens. It categorizes techniques such as simple staining, differential staining (including Gram and acid-fast staining), and specialized methods like fluorescent and silver staining, detailing their purposes, methods, and results. These techniques are essential for identifying organisms, studying tissue structures, and detecting specific cellular components.

Uploaded by

ratifukarwani
Copyright
© © All Rights Reserved
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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Staining methods

Lecture 2 presented by NYANDA.NL


Staining technique…..
• Staining methods are techniques used to enhance contrast in
microscopic specimens by applying dyes.
• These methods help differentiate structures, identify organisms, or
highlight specific components like the nucleus, cell wall, or cytoplasm.
• The most commonly used biological staining techniques, grouped by
purpose:
Tech…
1. Simple Staining
Purpose:
To highlight the entire cell, making it easier to see shape and size.
Method:
Apply a single dye.
Dyes used:
Methylene blue, Crystal violet, Safranin
Example use: Observing bacterial cell shapes (cocci, bacilli, spirilla)
2. Differential Staining
Purpose:
To distinguish between different types of organisms or cell parts.
Method:
Uses two or more dyes.
A. Gram Staining
Purpose:
Classify bacteria as Gram-positive or Gram-negative.
Steps:
Crystal violet (primary stain)
Iodine (mordant)
Alcohol (decolorizer)
Safranin (counterstain)
Results:
Gram-positive: Purple
Gram-negative: Pink
B. Acid-Fast Staining
Purpose:
Detect bacteria with waxy cell walls (e.g., Mycobacterium
tuberculosis).
Dyes:
Carbol fuchsin, acid alcohol, methylene blue
Acid-fast bacteria:
Stay red;
others turn blue.
3. Negative Staining
Purpose:
Stain the background, not the specimen.
Dye used:
Nigrosin or India ink (acidic dyes)
Use:
To see delicate structures like capsules or spores without distortion.
4. Capsule Staining
Purpose:
To visualize capsules around bacteria.
Method:
Combine negative stain (background) with a simple stain (cell).
Result:
Capsule appears as a clear halo.
5. Endospore Staining
Purpose:
Identify spore-forming bacteria (e.g., Bacillus, Clostridium).
Dyes:
Malachite green (spores) + Safranin (cells)
Result:
Spores = green; Cells = red/pink
6. Flagella Staining
Purpose:
Detect flagella, the thin tail-like structures used for movement.
Method:
Apply a mordant to thicken flagella, then stain.
Dye:
Special flagella stains
7. Histological (Tissue) Staining
Used in:
Anatomy, pathology, histology
Common stain:
Hematoxylin and Eosin (H&E)
Hematoxylin = stains nuclei blue/purple
Eosin = stains cytoplasm pink/red
Use:
To study tissue structure and detect disease
8. Vital Staining
Purpose:
Stain living cells without killing them.
Used for:
Observing live cell activities (e.g., movement, vacuole function).
Examples of dyes:
Janus Green (mitochondria)
Neutral Red (vacuoles)
Important Note:
The stain must be non-toxic to the cell.
9. Fluorescent Staining (Fluorescence Microscopy)
Purpose:
Tag specific cell parts using fluorescent dyes or antibodies.
Method: Uses dyes that glow under UV light.
Common dyes:
DAPI (binds to DNA, glows blue)
FITC (green)
Rhodamine (red)
Used in:
Immunofluorescence, cancer diagnosis, cell tracking.
10. Sudan Staining
Purpose:
Detect lipids (fats) in cells or tissues.
Dye Used:
Sudan III, Sudan IV, or Oil Red O
Use:
Study fat metabolism, identify fat-rich areas.
11. Periodic Acid-Schiff (PAS) Staining
Purpose:
Identify carbohydrates, glycogen, and mucopolysaccharides.
Reaction:
Periodic acid oxidizes sugars → Schiff reagent reacts → gives magenta
color.
Used in:
Liver, kidney tissue, and detecting glycogen storage diseases.
12. Silver Staining
Purpose:
Highlight fine structures like nerve fibers, proteins, or nucleic acids.
Method:
Silver salts bind and are reduced to metallic silver, which appears
dark under a microscope.
Used in:
Neuroscience, histopathology, protein gel staining.
13. Giemsa Staining
Purpose:
Stains blood smears and bone marrow.
Used for:
Differentiating blood cell types and identifying blood parasites (e.g.,
Plasmodium in malaria).
Result:
Nuclei = dark purple, Cytoplasm = light blue or pink
14. Trichrome Staining (Masson's Trichrome)
Purpose:
Distinguish muscle fibers, collagen, and connective tissues.
Colors:
Collagen = blue or green
Muscle = red
Nuclei = black
Used in:
Histology to evaluate fibrosis or muscle damage.

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