G R A M ST AIN IN G

Dr.T.V.Rao MD
Hans Christian Joachim
Gram
Danish Bacteriologist
 Dyes become Stains
 With interest in the effects of dyes
on living tissue. In 1884 the Danish
microbiologist Hans Christian Gram
discovered that crystal violet
irreversibly stains certain bacteria
but can be washed from others. The
dye has been widely used ever since
for the Gram stain technique, which
identifies bacteria as gram-positive
(the stain is retained) or gram-
negative (the stain is washed).
Gram staining a Important
Technique
A staining technique used to classify
bacteria; bacteria are stained with
gentian violet and then treated with
Gram's solution; after being
decolorized with alcohol and treated
with safranine and washed in water,
those that retain the gentian violet
are Gram-positive and those that
do not retain it are Gram-negative
 Gram positive Bacteria
 Gram positive bacteria
have a thick cell wall
of peptidoglycan and
other polymers.
Peptidoglycan consists
of interleaving
filaments made up of
alternating
acetylmuramic acid
and actylglucosamine
monomers.
In Gram positive
bacteria, there are
"wall teichoic acids".
As well, between the
cell wall and cell
membrane, there is a
"membrane teichoic
Gram Negative Bacteria
 Gram negative
bacteria have an outer
membrane of
phospholipids and
bacterial
Lipopolysaccharides
outside of their thin
peptidoglycan layer.
The space between
the outer membrane
and the peptidoglycan
layer is called the
periplasmic space.
The outer membrane
protects Gram
negative bacteria
against penicillin and
lysozymes.
The Cell walls differ…
Constituents of the Cell
wall make difference
 Definition of Gram stain

A method of staining bacteria using a

violet stain. The gram staining
characteristics (denoted as positive
or negative). A heat fixed bacterial
smear is stained with crystal violet
(methyl violet), treated with 3%
iodine/potassium iodide solution,
washed with alcohol and
counterstained. The method
differentiates bacteria into two main
classes, gram-positive and gram-
negative.
Organizing the Staining
Bottles
Method of Picking material
from Agar plates
Wrong Right
Prefer to pick up colonies with loop
and smear on Clean glass slide
 Making a Smear
 First prepare your
slide. You do this by
placing bacteria on a
slide in a drop of
water, allowing them
to dry and then heat
fixing them. Heating
the slide kills the
bacteria and makes
sure that the bacteria
a stuck to the slide
and wont wash away
during the staining
procedure
 Choosing a Right
Smear
 Before choosing a
field for
microscopic
examination, it is
important to look
at the smear
macroscopically
 Note that the
smear is easily
visible in ordinary
 Requirements for Gram
staining technique
 Glass slides (25 by 75 mm), frosted ends
desirable
 b. 0.85% Nacl, sterile
 c. Pasteur pipettes and wood applicator
sticks, sterile
 d. Microbiological loops, inoculating
needles
 e. Supplies for disposal of biological waste,
including “sharps”
 f. Bunsen burner
 g. Immersion oil
 Making Multiple smears in
same slide – conserve
resources
 Making multiple
smears make the
optimal use of the
slide.
 Reduces the
economic costs
and saves the
technical time.
 Correct preparation
 Smear preparation: Proper smear
preparation should produce a
monolayer of organisms sufficiently
dense for easy visualization but thin
enough to reveal characteristic
morphological characteristics. Use
clean, new glass slides.
NOTE: When using the same
pipette or swab, always inoculate
culture media first
 Steps in Gram Staining
Procedure- Follow the Clock
 1 On a rack, flood with filtered crystal
violet   10 sec
2 Wash briefly in water to remove excess
crystal violet
 3.   Flood with Gram’s iodine 10 sec
 4.   Wash briefly in water, do not let the
section dry out.
 5.   Decolourise with acetone until the
moving dye front has passed the lower edge
of the section
 6.   Wash immediately in tap water
 7.   Note : If the section appears too blue
repeat steps 6 and 7
 8.   Counterstain with safranin 15 seconds
 
Proceed in organized
Fashion
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
Step 7
 Observe the Following
Under Oil Immersion lens
 i. Relative amounts of PMN’s,
mononuclear cells, and RBC's
 ii. Relative amounts of squamous epithelial
cells, bacteria
 consistent with normal micro flora, which
may indicate an improperly collected
specimen
 iii. Location and arrangements of
microorganisms
 Note any special character sticks
Microscopy: The
Instruments
 In a compound
microscope the
image from the
objective lens is
magnified again by
the ocular lens.
 Total magnification
=
objective lens ×
ocular lens
 Microscopy: The
Instruments
 Resolution is the
ability of the lenses
to distinguish two
points.
 A microscope with
a resolving power
of 0.4 nm can
distinguish
between two points
≥ 0.4 nm.
 Shorter
wavelengths of
light provide
Optimal use of
Microscopy
 Gram stained
preparations have to
be observed with
bright-field optics.
Phase-contrast
microscopy does
not allow the
recognition of true
colours. Gram-
positive bacteria may
be seen under phase-
contrast as red cells.
Using bright-field
optics, Gram-positive
cells are purple or
blue and Gram-
 Use Bright field optics
 With bright-field
optics colours
can be
discriminated
best if the
condenser iris is
opened as far as
possible without
discomfort to the
eyes
 Colors makes the
Difference in Gram staining
 Bacteria that manage
to keep the original
purple dye have only
got a cell wall - they
are called Gram
positive.
 Bacteria that lose the
original purple dye
and can therefore take
up the second red dye
have got both a cell
wall and a cell
membrane - they are
called Gram
negative.
Observe with Caution to
differentiate
 Report as follows
 If no microorganisms are seen in a
smear of a clinical
 specimen, report “No
microorganisms seen.”
 ii. If microorganisms are seen, report
relative numbers and
 Describe morphology.
 Observe predominant shapes of
microorganisms
 Gram Stain Differentiates
Gram positive from Gram
negative
 Differential Stains: Gram Stain
Nature of Morphology in
guides early Diagnosis in
uncommon diseases
 Creating Library of Gram
Stains
 Drain or gently blot
excess oil
For slide libraries and
teaching collections
that will be stored for
longer periods,
immersion oil can be
removed with xylene
solution and the slides
can be cover slipped
using Per mount to
prevent fading.
 Value of Direct Smears
 Guide the physician on initial choice
of antibiotic, pending results of
culture and sensitivity.
 Judge specimen quality.
 Contribute to selection of culture
media, especially with mixed flora.
 Provide internal quality control when
direct smear results are compared to
culture results.
Mixed Flora of Staphylococcus
and Streptococcus
differentiates morphology
Stains Several Fungi
Gram-negative Pseudomonas
aeruginosa bacteria (pink-
rods).
(Bacillus anthracis),
Streptococcus
pneumonia
Streptococcus salivarius
Gram stain of Neisseria
gonorrhoea,
Streptococcus pneumoniae
in Sputum
Clostridium spp as seen in Gram
Stain
Moraxella as seen in Gram
Staining
Clostridium difficile as seen by
Gram staining
Corynebacterium diptheria as
seen in Gram staining
Gram stain of Neisseria
gonorrhoea,
Neisseria meningitides as seen
in Gram staining
Nocardia spp seen in Gram
Staining
Gram Stained Actinomyctes
spp
Gram stained Listeria
Monocytogenes
Gram Stained Cryptococcus
neoformans
 Artifacts in Gram
Staining
 Gram stain reagents (Crystal Violet, Iodine,
Safranin and Neutral Red
 Dirty glass slides
 Contaminated water used to rinse slides
 When investigating non-viable organisms
seen in Gram stains it is always wise to
eliminate every potential source.
Gram’s method stains
Artificacts too
 Gram stain results may not
correlated with culture results
 Gram stain-positive, culture-negative
specimens may be the result of
contamination of reagents and other
supplies, presence of
 Antimicrobial agents, or failure of
organisms to grow under usual
Culture conditions (media,
atmosphere, etc.)
 Presence of anaerobic
microorganism
Common errors in Staining
procedure
 Excessive heat
during fixation
 Low concentration
of crystal violet
 Excessive washing
between steps
 Insufficient iodine
exposure
 Prolonged
decolourization
 Excessive
Correlation of Gram stain
with cultures
 Culture results are not correlated
with direct gram stain results.
 Select true or false

1 True
( Correct )
2 False
Gram staining not a fool
proof procedure
 Gram’s staining
method is not without
its problems.  It is ,
complicated, and
prone to operator
error.  The method
also requires a large
number of cells
However, it  is also
central to phenotypic
microbial identification
techniques. 
Gram variable observations
in Gram staining
 The Gram staining procedure does
not always give clear-cut results.
Some organisms are Gram-variable
and may appear either Gram-
negative or Gram-positive according
to the conditions. With these types of
organisms, Gram-positive and Gram-
negative cells may be present within
the same preparation
 Overcoming in Gram
Variable Observations
 itis necessary that it is stained at
two or three different ages (very
young cultures should be used). If an
organism changes from positive to
negative or vice versa during its
growth cycle, this change should be
recorded with a statement as to the
age of the culture when the change
was first observed. In case a Gram-
variable reaction is observed it is
also good to check the purity of the
culture.
 Why Gram Variability?
 Some Gram-positive bacteria appear
Gram-negative when they have
reached a certain age, varying from
a few hours to a few days. On the
other hand, some Gram-negative
bacteria may become Gram-positive
in older cultures. For this reason it is
strongly recommended to use very
young cultures for the staining
procedure, after growth has become
 Trouble shooting in Gram
Staining method
 It is important to note that gram-positivity (the
ability to retain the purple crystal violet-iodine
complex) is not an all-or-nothing phenomenon but
a matter of degree. There are several factors
which could result in a gram-positive
organism staining gram-negatively

 1. The method and techniques used.

Overheating during heat fixation, over
decolourization with alcohol, and even too much
washing with water between steps may result in
gram-positive bacteria losing the crystal violet-
iodine complex.
 Trouble shooting in Gram
Staining method
 2. The age of the culture. Cultures more
than 24 hours old may lose their ability to
retain the crystal violet-iodine complex.
 3. The organism itself. Some gram-
positive bacteria are more able to retain
the crystal violet-iodine complex than
others.
 One must use very precise
techniques in gram staining and
interpret the results with discretion
 Poor quality of slides
Can be corrected
 Use of glass slides
that have not been
pre cleaned or
degreased NOTE:
Storing slides in a
jar with 95%
ethanol will ensure
clean slides. Drain
excess alcohol or
flame slide before
use.
 Modification in Gram
staining methods ?
 Since the original procedure of Gram,
many variations of the Gram staining
technique have been published. Some of
them have improved the method, others
include some minor technical variants of
no value. Bartholomew (1962) has pointed
out that each variation in the Gram
staining procedure has a definite limit to
its acceptability. Any final result is the
outcome of the interaction of all of the
possible variables.
 All modified methods to be practised with
caution should suit to the laboratory, and
quality control checks.
Gram staining continues to
be Most Rapid test.
 Even new molecular methodologies typically take
hours rather than minutes. Fortunately, Gram's
stain, devised by a Danish pathologist in 1884,
exists (see "The man behind Gram's stain, and "A
revolution in staining begins," This simple
staining procedure remains the most useful test
performed in the microbiology lab. Results from a
Gram's stain can tell volumes about an infection
within 15 minutes of a specimen's arrival in the
lab, while most other microbiology results require
24 hours or more. Nevertheless, Gram's stain
findings can be equivocal and, therefore, must be
assessed carefully in light of the clinical picture.
Created for ‘e’ learning
to Medical and
Paramedical students in
developing countreis
Dr.T.V.Rao MD
Email
doctortvrao@gmail.com