By Momina Shehzadi | BS Zoology Graduate, Pakistan In this article, we will learn Fungi - Morphology, Reproduction, Physiology and Importance - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students. Fungi are found everywhere around us. They are present in soil, in water, in air, on dead plants, on dead animals, on our food, on our skin and even inside our body. When bread becomes old, green or black spots appear on it. These spots are fungi. When we see mushroom growing after rain, that is also a fungus. When yeast is used to make bread soft, that is also a fungus. Fungi are very different from bacteria and plants. They are eukaryotic organisms. It means their cells have a true nucleus and other membrane bound organelles. They do not have chlorophyll, so they cannot make their own food like plants. They get their food from other living or dead things. That is why they are called heterotrophic organisms. The study of fungi is called mycolog...
By Momina Shehzadi | BS Zoology Graduate, Pakistan
In this article, we will learn Capsule Staining - Principle, Procedure and Importance - in simple words with diagram. This topic is very important for BS Zoology and Microbiology students.
Capsule staining is a special staining technique used to visualize capsule of bacteria. Capsule is outermost thick layer present outside cell wall in some bacteria. It is made of polysaccharides and sometimes polypeptides.
Capsule is not stained by ordinary staining methods like simple staining or Gram staining because capsular material is non ionic and acidic stains do not penetrate it. So capsule appears as clear halo around bacteria in Gram staining.
To visualize capsule, special staining techniques are required which stain background and bacterial cell but leave capsule unstained, so capsule appears as clear zone around stained cell.
Capsule staining is very important because capsule is virulence factor of many pathogenic bacteria and helps in identification.
WHAT IS CAPSULE
Capsule is thick, well organized layer outside cell wall, firmly attached to cell wall. When layer is thin, loose and irregular it is called slime layer.
Chemical composition: Capsule is mainly made of polysaccharides containing sugars like glucose, glucuronic acid, rhamnose. In some bacteria like Bacillus anthracis capsule is made of polypeptide of D-glutamic acid.
Examples of capsulated bacteria:
Streptococcus pneumoniae causes pneumonia
Klebsiella pneumoniae causes pneumonia and UTI
Bacillus anthracis causes anthrax
Haemophilus influenzae causes meningitis
Cryptococcus neoformans is capsulated fungus causing meningitis
Functions of capsule:
1. Protection from phagocytosis - capsule prevents engulfment by WBCs
2. Protection from desiccation and drying
3. Increases virulence - capsulated strains cause more severe disease
4. Helps in attachment to surfaces and biofilm formation
5. Prevents attack by bacteriophages
6. Prevents entry of antibiotics to some extent
PRINCIPLE OF CAPSULE STAINING
Principle is based on fact that capsule is acidic and repels acidic dyes.
In capsule staining, we use acidic stain like nigrosin or India ink for background staining and basic stain like crystal violet or methylene blue for staining bacterial cell. Capsule does not take any stain due to its non ionic nature, so it remains colorless and appears as clear halo between dark background and dark bacterial cell.
Thus we get dark background, dark bacterial cell and clear transparent capsule around cell.
There are two methods: Positive staining and Negative staining. Most commonly negative staining method is used.
TYPES OF CAPSULE STAINING METHODS
1. Anthony's Method - Positive Capsule Staining - Most Common
This is most widely used method in microbiology laboratory for capsule staining.
Requirements:
Crystal violet as primary stain 1 percent
Copper sulfate as decolorizer 20 percent
Bacterial culture with capsule
Procedure:
Step 1: Make a thin smear of bacterial culture on clean slide from 24 hours culture. Do not heat fix strongly because heat can shrink capsule and destroy it. Only air dry or mildly heat fix.
Step 2: Flood smear with crystal violet 1 percent for 2 minutes. Crystal violet stains bacterial cell and capsule slightly.
Step 3: Wash with 20 percent copper sulfate solution. Copper sulfate acts as decolorizer and removes excess crystal violet from capsule but not from cell. It also prevents removal of capsule. Do not wash with water because water can remove capsule.
Step 4: Air dry, do not blot. Observe under microscope at 100X oil immersion.
Result:
Capsule appears as clear halo or unstained zone around purple bacterial cells on light blue background.
2. Negative Staining Method - India Ink or Nigrosin Method
This is simplest and quickest method.
Requirements:
India ink or Nigrosin 10 percent
Bacterial culture
Clean slide
Procedure:
Step 1: Place a drop of India ink or nigrosin on clean slide.
Step 2: Mix a loopful of bacterial culture with ink.
Step 3: Place coverslip and observe under microscope, or make thin smear, air dry and observe.
Result:
Background appears dark black or blue black. Bacterial cells appear light or stained if counter stain used. Capsule appears as clear halo around cells.
This method is also used to identify Cryptococcus neoformans in CSF of meningitis patients. In CSF wet mount with India ink, Cryptococcus shows prominent capsule as clear halo.
3. Maneval's Method
Modification of Anthony's method using Congo red as primary stain and acid fuchsin as counter stain. Background is dark, cells are dark red and capsule is clear.
PRECAUTIONS IN CAPSULE STAINING
1. Do not heat fix smear strongly. Capsule is made of polysaccharide which can shrink or dissolve with strong heating. Only air dry or gentle heat.
2. Use young culture 18 to 24 hours old because capsule production is maximum in young culture. Old cultures may lose capsule.
3. Do not wash with water after primary stain in Anthony's method, use copper sulfate only.
4. Use clean slides and avoid thick smears.
5. Perform staining carefully because capsule is delicate.
DIFFERENCE BETWEEN CAPSULE AND SLIME LAYER
Capsule is thick, well organized, firmly attached to cell wall, visible as distinct halo and contributes to virulence. Slime layer is thin, loose, irregular, easily removed by washing and less associated with virulence.
IMPORTANCE OF CAPSULE STAINING
1. Identification: Helps to identify capsulated pathogenic bacteria like Streptococcus pneumoniae, Klebsiella, Bacillus anthracis.
2. Determination of virulence: Presence of capsule indicates virulent strain. Non capsulated strains of same species are usually avirulent. Example capsulated Streptococcus pneumoniae causes disease but non capsulated does not.
3. Diagnosis: In clinical samples like CSF, sputum, India ink preparation quickly shows capsulated organisms like Cryptococcus.
4. Vaccine production: Capsular polysaccharide is used to prepare vaccines. Example Pneumococcal polysaccharide vaccine is made from capsule of Streptococcus pneumoniae. Hib vaccine is made from capsule of Haemophilus influenzae type b.
5. Quellung Reaction: Special test for capsule where capsulated bacteria swell when mixed with specific antisera. Observed under microscope. Used to identify serotypes of Streptococcus pneumoniae.
COMPARISON OF DIFFERENT STAINING TECHNIQUES
Gram staining stains cell wall, differentiates Gram positive and negative. Acid-fast staining stains mycolic acid, differentiates acid-fast bacteria. Capsule staining stains background to show capsule as clear halo. Endospore staining stains spore.
Each staining has different purpose and all are important in diagnostic microbiology.
CONCLUSION
Capsule staining is special staining technique to demonstrate capsule which is not visible by simple staining. Capsule is important virulence factor and its demonstration helps in identification of pathogenic bacteria and fungi. Anthony's method and India ink negative staining are most common methods. For BS Zoology students, it is essential to understand principle and procedure because it is asked in practical exams.
FAQs for Students - Capsule Staining
Q1: What is capsule and why it is not stained by Gram staining?
Ans: Capsule is outermost thick polysaccharide layer outside cell wall. It is not stained by Gram staining because it is non ionic and acidic in nature, so it does not retain basic dyes like crystal violet. It appears as clear zone in Gram staining.
Q2: Why heat fixing is avoided in capsule staining?
Ans: Because capsule is delicate polysaccharide layer which can shrink, dissolve or wash away with strong heating. So only mild heat fixing or air drying is done to preserve capsule.
Q3: What is difference between capsule and slime layer?
Ans: Capsule is thick, well organized and firmly attached to cell wall, visible as halo. Slime layer is thin, loose, diffuse and easily washed away. Capsule is more associated with virulence.
Q4: What is India ink method for capsule staining?
Ans: India ink method is negative staining where India ink stains background dark black but does not stain cell or capsule. Capsule appears as clear halo around bacteria. It is quick method to demonstrate Cryptococcus in CSF.
Q5: What is Anthony's method of capsule staining?
Ans: Anthony's method is positive capsule staining using crystal violet as primary stain and copper sulfate as decolorizer. Bacterial cell appears purple and capsule appears as clear halo. Water wash is avoided.
Q6: Why capsule is important for bacteria?
Ans: Capsule protects bacteria from phagocytosis by WBCs, from drying, from bacteriophages and increases virulence. Capsulated bacteria cause more severe diseases than non capsulated strains.
Also Read: Bacteriological Media - Types and Preparation
Written by Momina Shehzadi, BS Zoology Graduate from Pakistan.
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