Transcript Congo Red Capsule Staining Method

Faculty of Medicine and Health
Sciences
Microbiology Lab
Experiment 6
Detection of Bacterial Capsule
First Semester 2016-2017
Glycocalyx:
Some bacteria have an additional layer outside their cell wall known as the Glycocalyx.
In most cases, the glycocalyx is mainly made of polysaccharides.
The Glycocalyx locates next to the thick peptidoglycan layer of Gram-positive bacteria and
next to the outer membrane of membrane of Gram-negative bacteria.
According to its structure, glycocalyx can be classified as:
1- Capsule
2- Slime layer)
Capsule is thick and dense and it is firmly associated with the bacterial cell wall..
In contrast, slime layer, is mainly composed unorganized layer of polysaccharides and
glycoproteins molecules that are loosely associated ( easily removable) with the bacterial cell wall
Examples of Capsulated (encapsulated) bacteria : Klebsiella pneumoniae, Streptococcus
pyogenes, Streptococcus pneumoniae, Haemophilus influenzeae and Neisseria meningitides.
Note: Bacillus anthracis has a capsule that is made of D-glutamine.
The formation of smooth mucoid colonies is a characteristic feature of capsulated
bacteria.
On the other hand, non-capsulated bacteria have rough colonies.
Importance of the Bacterial Capsules:
1- In the environment:
 Attachment to surfaces
Protect from dehydration ( saturated with water)
A nutrient storage than can be used in case starvation
Exclude some toxic material of a large molecular Wight
2- In pathogenesis:
A- Attachment to cell surfaces and tissues of the host
B- Anti-phagocytic: in general bacterial capsules interfere with phagocytosis
C- Protect bacterial cells from complement
D- Antigenic disguise: some bacteria have their capsules made of polysaccharides that are
similar to polysaccharides found in human tissue, such as hyaluronic acid and Sialic acid.
Hyaluronic acid capsule found in Streptococcus pyogenes
Sialic acid capsule found in E. coli K88
Both capsules provide antigenic disguise, so it would be not easy for immune cells to detect
them
3- In clinical application:
Bacterial capsules of certain bacteria ( mainly those involved in meningitis) can be used
as vaccines.
Examples: the capsules of Streptococcus pneumoniae , Neisseria meningitides and
Haemophilus influenzae , have been used as capsules
4- In Bacterial diagnosis: antibodies raised against bacterial capsules ( K antigen) can
be used in serology tests used for bacterial diagnosis.
Importance of the Bacterial Slime layer:
 Attachment to environmental surfaces and to tissue and cells during
infection
Interfere with phagocytosis
Formation of biofilms
Capsule Visualization Methods:
Congo Red Capsule Staining Method (Maneval’s capsule staining method):
Bacterial capsules are non-ionic, accordingly, neither basic stains nor acid stains can be used
stain bacterial capsules.
the capsule staining method that will be used in this experiment is mainly detection the
presence of bacterial capsules indirectly and this is by:
1- Using a stain with large molecular weight such as Congo Red: this stain will stain the
back ground (the surface glass slide , however, because of its molecular weight, the stain will
not be able to go through the bacterial cell wall or even the bacterial capsule.
2- Using a basic stain that will stain the bacterial cytoplasm such as acidfuchsin.
By using these two stains, the surface of the glass slide will be stained
(the back ground) as well as the cytoplasm of the bacterial cell.
What will be left unstained is the bacterial capsule, which will appear
as an unstained regain that surround the bacterial cells.
Congo Red Capsule Staining ( Maneval’s capsule staining method)
In the procedure for Maneval’s capsule staining method, the bacterial cells are first
introduced in to a drop of Congo red solution on a slide to prepare a smear. Then the smear
is allowed to dry (air dry)
Congo red has a large molecular weight so it cannot penetrate through the capsule. So the
smear get dry, Congo Red stains the background on the glass slide
A cid- alcohol is then used as a mordant that causes the precipitation of the capsular
material (chemical fixation). This will stabilize the capsule structure .
Note: The acidic pH of acetic acid lowers pH causes the Congo red to change from a red
color to blue color.
A after washing the smear with water, acid fuchsin (a basic dye) is used to stain the smear.
The basic stain bacterial cell cytoplasm in a red bright color
So, both the back ground ( blue) are bacterial cells ( Red) have been stained, but the
capsule remains unstained. Upon using the light microscope, the capsule is detected
indirectly by seeing an unstained area around the bacterial cells
Materials:
Klebsiella pneumoniae (capsulated bacteria)/ preferred old culture)
Glass slides
Inoculating loop
Congo Red
Acid-Alcohol
Acidfuchsin
Congo Red Capsule Stain Procedure:
Place a drop of Congo Red at one end of a clean glass slide.
Mix a small amount of your Klebsiella with the drop of Congo Red
By using another glass slide, spread the stain that is mixed with Klebsiella onto the surface
of the slide (as in blood film preparation)
Air dry the Congo Red film (Do not use heat because heat will destroy the capsule/no heat
fixation is used in this experiment)
Fix the air-dried Congo Red film with acid alcohol for 15 seconds (this is a chemical
fixation method that will stabilize the bacterial capsule by precipitating its CHO)
Then, wash the fixed slide with water
Stain the slide with acidfuchsin for 1-2 minutes
Wash with water
Blot-dry
Examine the slide under microscope by using oil immersion lens.
Factors affecting capsule staining:
1- The culture medium ( special culture media such as milk broth or litmus milk
must be used to support capsule synthesis in capsulated bacteria)
2- Heat fixation must not be used in this experiment as heat destroys bacterial
capsule
Improve capsule detection by using anti-capsular antibodies::
This in known as the Quellung Reaction
In this reaction, anti-capsular antibodies
are added to the capsulated bacterial cells
intended to be used in this experiment.
Binding of antibodies to the capsule will
causes the capsule to swell.
This will improve the detection of the
unstained region around the bacterial cells as
its size increases
Remember, this unstained region represents
the region that is occupied by the capsule.