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Lesson 4
Automated Identification and
Staining of Bacteria
Bacterial Staining
Bacteria are colourless cells and almost invisible
to the naked eyes
Can be stained with dyes to make them easier to
observe
Identifying and categorising different, isolated
bacterial colonies based upon varied appearance
and morphology (form and structure) will permit
the selection and transfer of a single colony to a
mixed culture, and allow transfer of single colony
to a sterile medium for cultivation of a pure
culture
Bacterial Staining
A number of stains have been developed to
distinguish spores, nuclear bodies, capsules, and
characteristics of the cell wall
The staining methods we will use kill the bacteria,
reducing the risk of infection by pathogenic
organisms
Since the rigid cell walls of bacteria prevent
distortion of morphology upon drying, samples can
be spread onto a glass slide and air-dried, then fixed
to the surface by passing the slide quickly through a
flame, melting the complex carbohydrates of the cell
walls to the glass and killing the cells
Preparing Smear
It causes bacteria to adhere to a slide so that
they can be stained and observed
It also kills them, rendering pathogenic
bacteria safe to handle
An objective in preparing smears is to learn to
recognise the correct density of bacteria to
place on the slide
Grams Staining
Grams staining is discovered by Christian Gram
in 1884 when he was staining bacteria in
tissues
The Gram stain is almost always the first step in
the identification of a bacterial organism
It is very useful in the differentiation of
bacteria, especially those which have the same
shape and size but differ in their ability to
retain a crystal violet-iodine complex when
washed with a decoloriser (acetone)
Grams Staining
Microorganisms which take up the complex
are called Gram positive
Microorganisms which give up the complex
and become colourless are Gram negative
These organisms will take up the second stain
or counterstain (usually safranin)
Grams Staining
This staining is based on the physical
properties of the bacteria cell wall
Gram +ve bacteria have a thick mesh-like cell
wall made of peptidoglycan 50 - 90% of cell
wall)
Gram -ve bacteria have a thinner layer (10% of
cell wall), with an additional outer membrane
which contains lipids, and is separated from
the cell wall by the periplasmic space
Grams Staining
Four basic steps of the Gram stain:
1. Applying a primary stain (crystal violet) to a
heat-fixed smear of a bacterial culture
2. Addition of a trapping agent (Grams iodine)
3. Rapid decolourisation with alcohol or acetone
4. Counterstaining with safranin. Basic fuchsin is
sometimes substituted for safranin since it will
more intensely stain anaerobic bacteria but it is
much less commonly employed as a counterstain
Grams Staining
Crystal violet (CV) dissociates in aqueous
solutions into CV+ and chloride (Cl-) ions
These ions penetrate through the cell wall and
cell membrane of both Gram +ve and Gram ve cells
The CV+ ion interacts with negatively charged
components of bacterial cells and stains the
cells purple
Grams Staining
Iodine (I- or I3-) interacts with CV+ and forms
large complexes of crystal violet and iodine
(CV-I) within the inner and outer layers of the
cell
Iodine is often referred to as a mordant, but is
a trapping agent that prevents the removal of
the CV-I complex and therefore colour the cell
When a decolouriser such as alcohol or
acetone is added, it interacts with the lipids of
the cell membrane
Grams Staining
A Gram -ve cell will lose its outer membrane
and the lipopolysaccharide layer is left exposed
The CV-I complexes are washed from the Gram
-ve cell along with the outer membrane
In contrast, a Gram +ve cell becomes
dehydrated from an ethanol treatment
The large CV-I complexes become trapped
within the Gram +ve cell due to the
multilayered nature of its peptidoglycan
Grams Staining
The decolourisation step is critical and must be
timed correctly, the crystal violet stain will be
removed from both Gram +ve and Gram -ve
cells if the decolourising agent is left on too
long ( a matter of seconds)
Counterstain, which is usually positively
charged safranin or basic fuchsin, is applied
last to give decolorised Gram -ve bacteria a
pink or red colour