GRAM STAIN

• Gram staining is a bacteriological laboratory

technique used to differentiate bacterial species into
two large groups gram-positive and gram-negative
based on the physical properties of their cell walls.
• Gram staining differentiates bacteria by the chemical
and physical properties of their cell walls by
detecting peptidoglycan, which is present in a thick
layer in gram-positive bacteria
• The Gram stain is almost always the first step in the
identification of a bacterial organism.
NB: While Gram staining is a valuable diagnostic tool
in both clinical and research settings, not all bacteria
can be definitively classified by this technique.
 PRINCIPLE

Gram-positive bacteria have a thick mesh-like

cell wall made of peptidoglycan (50–90% of
cell envelope), and as a result are stained
purple by crystal violet, whereas gram-
negative bacteria have a thinner layer (10%
of cell envelope), so do not retain the purple
stain and are counter-stained pink by the
Safranin/Carbol fuschin.
 The method consists of initial staining of the bacterial
slide with crystal violet or methyl violet which stain
everything violet.
 This is followed by Gram’s or Lugol’s iodine made up
of iodine and potassium iodide, which act by allowing
the crystal violet to adhere to the walls of Gram-
positive bacteria.
 Decolourisation with an acetone-alcohol mixture
washes away the methyl violet which is not adherent
to bacterial cell walls. At this stage, Gram-positive
bacteria stain violet while the Gram-negative bacteria
are colourless.
 A carbol fuschin counter-stain is then applied which
stains the Gram negative bacteria pink.
 Procedure for gram stain

1. Label a clean slide using a diamond pencil

2. Place a drop of saline onto the slide. Using a sterile
wire loop, pick a colony of the bacteria and emulsify it
into the saline and spread to make a smear.(if its fluids
just add a drop of the fluid to the slide and spread to
make a smear)
3. Allow the smear to air dry.
4. Heat fix the smear by passing the slide 3 times through
a flame.
-be careful not to overheat the slide and destroy the
bacterial cell wall.
-heat fixing is to allow the bacteria to attach to the slide
so that it will not be washed off and also to kill the
bacteria.
5. Allow the slide to air dry and then place it on a staining
rack.
 Procedure for gram stain cont...

6. Cover the fixed smear with Crystal violet/Methyl violet

stain for 30-60 seconds and then wash off the stain with
clean water. Tip off excess water from the slide.
7. Cover the smear with lugol’s iodine for 30-60 seconds
and then wash off the stain with clean water. Tip off
excess water from the slide.
8. Decolorize by pouring acetone for 5 seconds and
rapidly wash off with clean water.
9. Cover the slide with dilute carbol fuschin/safranin for
1-2 minutes and then wash off the stain with clean
water.
10. Tip off excess water from the slide and wipe the back
of the slide with absorbent paper and leave the slide to
air dry.
Examine the stained smear microscopically using the
immersion oil objective (x100)
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-positive and gram-
negative cells. The CV+ ion interacts with
negatively charged components of bacterial
cells and stains the cells purple.
Iodide (I−or I−3) 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, color the cell.
• When a de=-e olorizer such as alcohol or acetone is
added, it interacts with the lipids of the cell
membrane. A gram-negative cell loses its outer
lipopolysaccharide membrane, and the inner
peptidoglycan layer is left exposed. The CV–I
complexes are washed from the gram-negative cell
along with the outer membrane. In contrast, a
gram-positive cell becomes dehydrated from an
ethanol treatment. The large CV–I complexes
become trapped within the gram-positive cell due
to the multilayered nature of its peptidoglycan.
NB:The decolorization step is critical and must be
timed correctly; the crystal violet stain is removed
from both gram-positive and negative cells if the
decolorizing agent is left for too long (a matter of
seconds).
After decolorization, the gram-positive cell
remains purple and the gram-negative cell
loses its purple color. The Counterstain which
is usually positively charged safranin or
Carbol fuchsine, is applied last to give
decolorized gram-negative bacteria a pink or
red color.
 results

Gram positive bacteria ------ dark

purple
Yeast cells ------ dark purple
Gram negative bacteria ------ pink or
red
Nuclei of pus cells/wbcs ------ red
Epithelial cells ------ red
The report should also include;
Morphology of bacteria whether cocci, bacilli,
diplococci, etc and their arrangement.