Staining is one of the most basic and important concepts in microbiology. For every NEET PG and INI-CET aspirant, understanding staining techniques is essential. As Dr. Devyani Puri explains, bacteria are transparent and cannot be seen under a microscope without staining. Stains help to provide contrast, allowing us to clearly observe the structure, shape, and arrangement of microorganisms.
Why Do We Need Staining?
Bacteria are colourless and translucent, making them difficult to see under the microscope. Staining makes them visible by increasing contrast.
Steps before staining:
- Smear preparation: A thin layer of bacterial sample is spread on a slide. This can be made from a direct specimen or from colonies grown on culture media.
- Fixation: The smear is fixed to the slide to preserve the structure of the bacteria.
Fixation Methods
Fixation ensures that bacteria stick to the slide and do not get washed away during staining.
- Heat Fixation: The slide is quickly passed over a flame.
- Advantage: Simple and quick
- Disadvantage: Can damage internal structures
- Advantage: Simple and quick
- Chemical Fixation: Chemicals such as glutaraldehyde or acetic acid are used.
- Advantage: Preserves internal structures better
- Used for: Blood smears and delicate samples
- Advantage: Preserves internal structures better
Click Here to Watch the Full Session: Staining Techniques Every NEET PG/INICET Aspirant Must Know! Dr. Devyani Puri
Types of Staining Techniques
1. Simple Staining
A single dye, like methylene blue or basic fuchsin, is used.
It gives a uniform color to all bacteria and helps in identifying the size, shape, and arrangement.
2. Negative Staining
Here, the background is stained while the organism remains colourless.
Used for observing capsulated organisms and thin bacteria such as spirochetes (Treponema, Borrelia, Leptospira).
Common stains: India ink, Nigrosin
The bacteria appear clear against a dark background.
3. Impregnation Staining
Used for very thin organisms or delicate structures like flagella.
Heavy metals such as silver are deposited on the surface of bacteria to make them visible.
Example: Silver impregnation for flagella and spirochetes.
4. Differential Staining
This type differentiates between types of bacteria based on their structure or chemical composition.
A. Gram Staining
Developed by Hans Christian Gram, this is the most commonly used stain.
It divides bacteria into two groups:
- Gram-positive – violet color (thick peptidoglycan wall)
- Gram-negative – pink color (thin wall, takes counterstain)
Steps (CIAL):
- Crystal violet – primary stain
- Iodine – mordant
- Alcohol/acetone – decolorizer
- Safranin – counterstain
Result:
- Gram-positive → violet
- Gram-negative → pink
Quick tips:
- Most cocci are Gram-positive except Neisseria, Moraxella, and Veillonella.
- Most bacilli are Gram-negative except: Mycobacterium, Clostridium, Corynebacterium, Nocardia, Actinomycetes, Listeria, and Diphtheroids.
B. Acid-Fast Staining
Used to identify bacteria that resist decolourisation due to the presence of mycolic acid in their cell wall.
Two methods are used:
- Ziehl-Neelsen method (hot method)
- Kinyoun method (cold method)
Steps:
- Carbol fuchsin – primary stain
- Acid/alcohol – decolourizer
- Methylene blue – counterstain
Result:
- Acid-fast bacteria → pink
- Non-acid-fast → blue
Examples:
Mycobacterium tuberculosis, M. leprae, Nocardia, Cryptosporidium, Cyclospora, Cystoisospora.
C. Albert Staining
Used to identify Corynebacterium diphtheriae and to demonstrate metachromatic granules (Babes-Ernst granules).
Steps:
- Albert I stain – Toluidine blue and Malachite green
- Albert II stain – Iodine and Potassium iodide
Result:
- Bacilli appear green
- Granules appear blue-black
- The bacteria show a characteristic “Chinese letter” arrangement.
5. Bipolar (Safety Pin) Staining
In this stain, only the ends of the bacteria take up the dye, giving a safety-pin appearance.
Examples (Mnemonic: Can Produce Very High Yield):
- Campylobacter granulomatis
- Pseudomallei (Burkholderia pseudomallei)
- Vibrio parahaemolyticus
- Haemophilus ducreyi
- Yersinia pestis
Final Takeaway
For NEET PG and INI-CET aspirants, staining techniques form the base of microbiology and are often asked in exams.
- Use Gram stain to classify bacteria.
- Use Ziehl-Neelsen stain for acid-fast organisms.
- Use the Albert stain for diphtheria.
- Recognise the safety-pin pattern for bipolar staining.
A clear understanding of these staining methods will not only help in theory but also in solving MCQs efficiently.
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