This presentation covers the detailed procedure of Capsule Staining. It also covers its significance, application, and need.

1. CAPSULE STAINING
Presented by-
Sushant Balasaheb Jadhav
Roll no. – 18PBT206
M. Tech. Pharmaceutical Biotechnology
Institute of Chemical Technology, Mumbai

2. What is a Capsule?
• A well organized layer of material lying
outside the cell wall referred to as a capsule
(or K antigen)
• Capsules are well organized layers that cannot
be easily washed off
• A capsule is composed of capsular
polysaccharides (CPS) which are
polysaccharides
• They may also be composed of a number of
other materials such as poly-D-glutamic acid.
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3. Functions of Capsule
• Capsules are very important in that they are a
major virulence factor
• They protect the bacteria from phagocytic
actions
• Protecting bacteria from desiccation
• Providing a food reserve
• Protecting anaerobes from oxygen toxicity
• To help bacteria attach onto surfaces
• They exclude bacterial viruses and most
hydrophobic toxic materials such as
detergents
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4. Strains having Capsule
• Streptococcus pneumonia
• Haemophilus influenza
• Neisseria meningitidis
• Klebsiella pneumonia
• Group B streptococci
• Actino bacillus
• Pleuro pneumonia
• Sinorhizobium meliloti
• Staphylococcus aureus
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5. Why Capsule Staining ?
• Most capsule materials are water soluble, simple
stains will not adhere to them
• Bacterial capsules are non-ionic, so neither acidic
nor basic stains will adhere to their surfaces
• Capsule stain is a type of differential stain which
involves the use of two stains; primary stain and
the counterstain
• It becomes necessary to stain the background
using an acidic stain while the cell is stained
using a basic stain
• Since the capsule is a major virulence factor in the
major disease-causing bacteria, it is essential to
identify the strain
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6. Methods for Capsule Staining
•India ink method
•Anthony's stain method
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7. India Ink Method
Requirements
• India ink
• Crystal violet
• Glass slide
• Inoculating loop
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8. India Ink Method Cont.
Procedure
• Place a drop of India ink onto a clean glass slide
• Using a sterile loop, obtain a sample and place it
onto the slide and mix it with the drop of India ink
• Obtain another sterile glass slide and having laid it
at an angle on one end of the first slide, spread
out the drop into a film to have a thin layer of the
smear-ink mixture
• Allow the slide to stand for about 5 minutes (air-
dry)
• Saturate the slide with crystal violet for a minute
and then tilt the slide to drain excess dye
• Allow the slide to dry (air-dry)
• View the slide using 100x
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9. India Ink Method Cont.
• India ink is often used as the negative stain
• Other stains that can also be used include:
Congo red
Nigrosin
Eosin
• Because of its acidic nature, India ink (or
Congo red, nigrosin) stains the background
dark
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10. India Ink Method Cont.
• Crystal violet is used for number of reasons
including:
To act as a fixative
Increase penetration power
Stain the cells (being a basic dye)
Decrease pH of smear
• The capsule will be clear against a dark
background given that it takes no stain
• Nigrosin is recommended to India ink because
it gives a more even background and spreads
more easily
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11. India Ink Method Cont. 11

12. Anthony's Stain Method
• The primary stain used is crystal violet.
• Some of the other requirements include:
A sample (Organism grown for 36-48 hours)
Glass slide
Inoculating loop
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13. Anthony's Stain Method Cont.
Procedure
• Introduce a drop of crystal violet on to a clean
glass slide
• Using a sterile loop, place the sample on to the
glass slide
• Obtain another glass slide and at an angle, spread
the drop and sample to form a thin film
• Allow the film to dry (air dry) for about 6 minutes
• Title the slide and rinse with 20% copper sulfate
solution
• Allow the slide to air dry for about 3 minutes
• Place the slide on to the microscope stage and
observe using oil immersion
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14. Anthony's Stain Method Cont.
• This is a positive staining method used in
capsule staining
• The smear/sample is treated with a
hypertonic solution (copper sulfate) for the
purposes of creating an ionic difference that
ultimately results in the diffusion of a stain
towards the outer surface of the cell
• After the slide is air dried, it becomes possible
to observe the stain that remained on the
capsular layer
• One will see a dark violet color of the cell
and a light violet color of the capsule
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15. Additional cares
• Don’t Heat
• Don’t rinse with water
• Add drop of serum
• Sterile technique/practice
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16. REFERENCES
• https://www.microscopemaster.com/capsule-
stain.html
• https://microbenotes.com/capsule-stain-
principle-procedure-and-result-interpretation/
• https://microbiologyinfo.com/capsule-
staining-principle-reagents-procedure-and-
result/
• https://microbeonline.com/capsule-stain-
principle-procedure-results/
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