Gram Staining

• The method is named after its inventor, the 

Danish scientist Hans Christian Gram
 (1853–1938
 Differential staining
• Acceptance of stains is an important
property of bacteria and is the base
divission of bacteria to 2 principal groups
Gram positive and Gram negative in
taxonomy.
• Gram staining
 Gram Staining
• is a common technique used to differentiate two large groups of
bacteria based on their different cell wall constituents. The
Gram stain procedure distinguishes between Gram positive and
Gram negative groups by coloring these cells red or violet.
Gram positive bacteria stain violet due to the presence of a thick
layer of peptidoglycan in their cell walls, which retains the
crystal violet these cells are stained with. Alternatively, Gram
negative bacteria stain red, which is attributed to a thinner
peptidoglycan wall, which does not retain the crystal violet
during the decoloring process.
 Gram Staining Materials
• Crystal violet (primary stain)
• Iodine solution/Gram's Iodine (mordant that
fixes crystal violet to cell wall)
• Decolorizer (e.g. ethanol)
• Safranin (secondary stain)
• Water (preferably in a squirt bottle)
 GRAM STAINING PROCEDURE
• Prepare a heat fixed smear of the culture you
wish to examine
• Flood the smear with crystal violet (30 sec. to
2 min)
• Quickly and gently wash off excess stain (2
seconds)
• Flood the smear with Grams iodine (1
minute)
• Decolorize with alcohol (10-20 seconds or
until the excess alcohol which flow off the
slide is colorless)
• Quickly and gently wash off excess stain (2
seconds)
• Flood the smear with safranin (carbolfuchsin)
(30 sec to 2 min.)
• Quickly and gently wash off excess stain (2
seconds)
• Blot dry with bibulous paper
• Examine your slide under the microscope.
Record sketches of the organisms, size, color,
morphology, and culture identification.
G+ G-
  GRAM-POSITIVE
BACTERIA
• are bacteria that give a positive result in the Gram stain test,
which is traditionally used to quickly classify bacteria into
two broad categories according to their type of cell wall.

• Gram-positive bacteria take up the crystal violet stain used

in the test, and then appear to be purple-coloured when seen
through an optical microscope. This is because the thick 
peptidoglycan layer in the bacterial cell wall retains the stain
 after it is washed away from the rest of the sample, in the
decolorization stage of the test.
 GRAM-NEGATIVE
BACTERIA
•  are bacteria that do not retain the crystal violet stain used in
the gram-staining method of bacterial differentiation.They are
characterized by their cell envelopes, which are composed of
a thin peptidoglycan cell wall sandwiched between an inner 
cytoplasmic cell membrane and a bacterial outer membrane.
• he gram-negative bacteria include the model
organism Escherichia coli, as well as many pathogenic
bacteria, such as Pseudomonas aeruginosa, Chlamydia
trachomatis, and Yersinia pestis.