The document discusses capsule staining techniques used to identify the presence of capsules in bacteria under a microscope. There are two main methods - India ink negative staining and Anthony's positive staining. India ink stains the bacterial cell and background violet and black, leaving the non-stained capsule visible as a clear halo. Anthony's stains the cell and capsule blue and leaves the background light violet, again showing the capsule as a halo. Identifying capsules is important as their presence indicates more pathogenic bacteria.

1. CAPSULE STAINING
BY – MOHIT HINSU

2. WHAT DOYOU MEAN BY STAINING?
■ Artificial coloration of a substance to facilitate examination of
tissues, microorganisms, or other cells under the microscope. For
various techniques, see under stain.
■ Staining is a technique used to enhance contrast in samples,
generally at the microscopic level.
■ Staining A technique in which cells or thin sections of biological
tissue that are normally transparent are immersed in one or more
coloured dyes (stains) to make them more clearly visible
through a microscope. Staining heightens the contrast between
the various cell or tissue components.
■ Why Stain Cells? The most basic reason that cells are stained is
to enhance visualization of the cell or certain cellular components
under a microscope. Cells may also be stained to highlight
metabolic processes or to differentiate between live and dead cells
in a sample.

3. CAPSULE STAINING
■ Capsule staining is a type of differential staining method which
creates contrast in the microscopic image either by staining
bacterial cell and background or by staining the capsule only.
Therefore, capsule staining can be done by two methods, namely
positive and negative staining methods.
■ The capsule staining is employed to know the presence or absence
of capsule, based on which we could classify the type of bacteria,
i.e. whether a bacteria is capsulated or non-capsulated.
■ It is necessary to perform capsule staining because the presence of
capsule indicates a virulent strain of bacteria that can cause
disease. Thus, by knowing the presence of the capsule, we can
determine the degree of pathogenicity.

4. DEFINATION OF CAPSULE STAINING
■ A capsule staining can define as the special staining method that
makes the use of differential capsule stain which either highlights
or stains the capsule or stains both the bacterial cell and its
background.
■ It is an important staining method, because some bacteria like
Bacillus anthracis, Streptococcus pneumoniae etc. have a capsule,
which can cause pathogenicity in many humans and animals. Thus,
it becomes necessary to identify the presence of an extracellular
capsule. Capsule staining can be performed by India ink,
Anthony’s, Maneval’s and Hiss method.

5. Principle
■ The principle of capsule staining is based on staining of
background with an acidic stain and staining of bacterial
cell with a basic stain. As a capsule is non-ionic, it will
not stain by either of the two dyes. Thus a capsule
staining creates a contrast by staining a bacterial cell and
its background in between which, capsule appears as a
colourless halo.

6. Methods of Capsule Staining
■ There are different methods for capsule staining, among which the most common
methods are:
■ India ink method negative staining method
■ Anthony’s method positive staining method
■ Maneval’s method negative staining method
■ Hiss method positive staining method
■ We study above 2 method.

7. Materials required
■ Microscopic slides
■ Inoculating loop
■ Microscope with 100x objective lens (oil immersion)
■ Immersion oil
■ Gas burner
■ Test bacteria culture

8. India ink Method
■ India ink method uses two types of stain, i.e. a basic stain (Crystal violet) and an acidic
stain (India ink).
■ Crystal violet being positive stain will stain the negatively charged bacterial cell. India
ink being negative stain will stain the positively charged background.After staining.
■ The Background appears darker or black.
■ A Bacterial cell appears violet.
■ The capsule appears as a clear halo.

9. Procedure:

10. ■ India ink method is a type of negative staining method, which
stains both the bacterial cell and its background but not a
capsule. As a result, a capsule appears as a bright halo between
the violet bacterial cell and a darker background.
■ Capsule staining by India ink method (at 1000x magnification)

11. Anthony’s Method
■ This method makes the use of two reagents, namely crystal violet
as primary stain and 20% of CuSO4 solution as a decolouring agent
and counterstain.
■ Crystal violet will stain the bacterial cell and background. The
CuSO4 solution will stain the non-ionic capsule. After staining.
■ Bacterial cell appears violet.
■ The background appears light violet.
■ The capsule appears as a faint blue halo.

12. Procedure
■ 1.Take a clean, sterilized or grease free slide.
■ 2.Add a drop of crystal violet to the centre of the glass slide.
■ 3.Prepare a smear, by taking an inoculum from the bacterial culture and mix it with a
drop of crystal violet.
■ 4.Then, allow the smear to air dry (Do not heat fix, as it can cause cell shrinkage and
distortion of the bacteria).
■ 5.Flood a smear with 20% of CuSO4 solution for at least 30 seconds and remove the
extra stain by tilting a glass slide.
■ 6.Add oil immersion to the stained area and observe it under the microscope having a
100X objective.

13. ■ Anthony’s method is a type of positive staining method that
stains the capsule along with the bacterial cell but not stains the
background. As a result, a capsule appears as faint blue halo
between the violet bacterial cell and purple background.