STAINING METHODS
STAINING METHODS:
A - Simple staining technique:
- Simple stains are used to demonstrate the presence
of organisms and the nature of any cell present in the
smear by applying only one dye.
-Stains in general can be divided into three groups:
Basic , Acidic and Neutral .
Acidic Stains Basic Stains Neutral Stains
Nigrosin Crystal violet Giemsa
Malachite green Methylene blue Leishman
Acid fuchsin Safranin Wright
Basic fuchsin
-As bacterial cells are rich in nucleic acid
(which has a negative charge) it will follow
that “basic stain ,bearing its coloring matter in
the positive charge , will be attracted to the
organism and stain it” . Acid stain ,however,
will not stain the bacteria ; they are used
mainly for staining the background material a
counterstaining color.
Procedure of simple staining:
1. Flood the slide with Loffler's methylene blue
for 5-10min.
2. Wash off the stain with slowly running tap
water.
3. Allow the to slide to dry in air or placed it
between two sheets of filter paper.
4. Examine under oil immersion.
[Link] (compound)
staining technique:
- It consists of more than one dye used
successively to identify organisms according to
their type of reaction.
- The most important examples for this group of
stains are:
1. Gram's staining method
2. Acid fast stain (zeihl neelsen methods)
3. Spore stain
GRAM STAINING METHOD:
- It is one of the most important methods widely
used in bacteriology dicovered in 1884 by gram (a
Danish physician), using two dyes in sequence each
of different color. He found that bacteria fall into
two different categories:
A) Those that retained the first dye (crystal violet)
throughout the staining procedure are known as
“GRAM POSITIVE”
B) Those that lost the first dye (crystal
violet)after washing with adecolorizing solution
and stained with the second dye (safranine) are
known as “GRAM NEGATIVE”
- IN CONCLUSION, the gram positive bacteria
appear violet ,while gram negative bacteria are
red in color .therefore ,it is possible to
differentiate between bacteria of the same
morphology. furthermore, it can be used to
determine the relative number and morphology of
bacteria in a smear taken directly from a patient
PROCEDURE OF GRAM STAINING:
1 - Flood the slide with crystal or gention
violet,leave to act for 1-2min. ,wash with tap
water.
2 - Apply gram's iodine (a mordant),leave to act for
one minute ,wash with tap water.
3 - Apply 95%ethyl alcohol (a decolorizer).leave to
act for 20-30 seconds ,wash with tap water.
4 - Apply saffranin (the counter stain), leave to act
for 1-1.5min. , wash with tap water, blot, dry in air
and examine with oil immersion lens.
MECHANISM OF STAINING:
The division of bacteria into two categories, indicates
a basic chemical differences between gram positive
and gram negative bacteria. The most important
differences are:
1 - The cell wall of Gram-negative organisms have
relatively little peptidoglycan and mainly consist of
lipoproteins and polysaccharides. While in Gram
positive organisms the peptidoglycan comprises the
major part of the cell wall rendering them more rigid
than Gram negative cells and less permeable for the
dye iodine complex to diffuse freely out of the cell
during the process of decolourization.
2 - The more acid charater of the protoplasm of Gram
positive bacteria which is enhanced by treatment with
iodine may partly explain their stronger retention of
the basic dye.
3 - Integrity of the cell wall.
Acid-Fast Stain
The main aim of this staining is to differentiate bacteria
into acid fast group and non-acid fast groups.
This method is used for those microorganisms which
are not staining by simple or Gram staining method,
particularly the member of genus Mycobacterium, are
resistant and can only be visualized by acid-fast
staining.
Summary of Acid-Fast Stain
Cell colour
Application of Reagent
Acid fast Non-acid fast
Primary dye Carbol fuchsin Red Red
Decolorizer Acid alcohol Red Colorless
Counter stain Methylene blue Red Blue
Procedure of Acid-Fast Stain