RAK University of Medical &

Health Sciences.
BSc Nursing Semester II
GRAM
STAINING
Dr.Mir Maroosha Farooq
M.B.B.S.
Instructor ,Microbiology
Department,RAKCOMS
OBJECTIVES:
By the end of this practical demonstration,the
students should be able to:
Know the uses of Gram staining.
The principle behind this type of staining.
The detailed procedure and the equipment
involved in Gram staining.

The basic flow of procedures involved in the
laboratory diagnosis of infectious diseases (bacterial
infections) is as follows:
Direct examination of
patients specimen for the
presence of etiologic
agents.
Growth and cultivation of
the agents from the same
specimen.
Analysis of the cultivated
organisms to establish their
identification and other
pertinent characteristics
such as susceptibility to
anti microbial agents.

Microscopy is the most common method used both for detection of
micro-organisms directly in clinical specimens and for characterization
of organisms grown in culture.For microscopy, the material can be used
as unstained / wet film or as a stained film.
STAINING TECHNIQUES FOR LIGHT
MICROSCOPY:
Smear
preparation:Techniqu
e consists of
spreading a thin film
made from liquid
suspension of cells on
a slide and air drying
it.
Fixation: The air dried
smear is usually
heated gently by
passing over the
flame.
Staining: Coloured
chemicals or dyes are
used to impart colour
to the cells or cell
parts.
COMMON STAINING TECHNIQUES
Simple staining. Impregnation
methods
Negative staining. Differential staining.

Bacillus Bacteria Slide, Typical. Negative
Stain
DIFFERENTIAL STAINING:The two commonly used
differential stains are Grams stain and Acid-fast stain.
Gram staining:
Most widely used stain in
medical
microbiology,named after
its inventor Hans Christian
Gram.
Nearly all clinically
important bacteria can be
detected using this stain
except for those that exist
exclusively in host
cells(Chlamydia);those that
lack cell wall(Mycoplasma
& Ureaplasma) And those
of insufficient dimension to
be resolved by light
microscopy(Spirochaetes)

Gram staining divides most bacterial species into two large groups
those that take up the primary dye (crystal violet) and appear
purple Gram positive and those that allow the crystal violet dye to
be washed out easily with the decolorizer alcohol or acetone and
take up the seconadary dye ( safrinine) and appear pink..Gram
negative.
Procedure & Principle

Classis Gram staining procedure entails fixing the clinical material to the
surface of the slide either by heating / using methanol.
Primary stain. ..crystal violet is applied.Crystal violet dissociates in
aqueous solution into CV+ ions which pass through the cell wall &
membrane of both gram positive & gram negative bacterial cells.These
interact with negatively charged components of bacterial cells and stain
them purple.
Grams iodine is applied as mordant.Iodine interacts chemically with CV
and forms large insoluble complexes with in the bacterial protoplasm
and cell wall.
Then a decolorizer (alcohol/acetone/both) is added.It is this step that
differentiates the gram positive bacteria from the gram negative ones
and the difference actually lies in the composition of their cell walls.Once
the decolorizer is added,insoluble dye-iodine complex gets dissociated
and dissolved and diffuses out freely in gram negative bacteria whose
lipo polysaccharide layer has been dissolved where as in gram positive
bacteria this free diffusion is hampered by the thick peptidoglycan layer
and the extensive teichoic acid cross-links,thereby helping them to resist
alcohol decolorization.
Lastly the counter stain (dilute carbol fuschin,safrinine or neutral red) is
added.

Examination of gram stained film
Once stained,the
smear is examined
under oil immersion
(1000x
magnification)
lens.The slide is
evaluated for:
Presence of
bacterial cells.
Gram reactions.
Bacterial
morphology
Arrangement