GRAM STAINING
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Hans Christian Joachim Gram
Danish Bacteriologist
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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).
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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 Gramnegative
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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
acid".
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Gram Negative Bacteria
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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.
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The Cell walls differ…
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Constituents of the Cell wall
make difference
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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, grampositive and gram-negative.
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Organizing the Staining Bottles
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Method of Picking material from
Agar plates
Wrong
Right
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Prefer to pick up colonies with loop and
smear on Clean glass slide
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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
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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 light

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Requirements for Gram
staining technique

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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
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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.

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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
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Steps in Gram Staining
Procedure- Follow the Clock
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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
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Proceed in organized Fashion
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Step 1
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Step 2
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Step 3
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Step 4
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Step 5
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Step 6
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Step 7
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Observe the Following Under
Oil Immersion lens
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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
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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

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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
greater resolution.

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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. Grampositive bacteria may be
seen under phasecontrast as red cells.
Using bright-field optics,
Gram-positive cells are
purple or blue and Gramnegative pink due to
counter stain with
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safranin..

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
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Colors makes the Difference in
Gram staining

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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.
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Observe with Caution to
differentiate
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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
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Gram Stain Differentiates
Gram positive from Gram
negative
Differential Stains: Gram Stain
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Nature of Morphology in guides
early Diagnosis in uncommon
diseases
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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.

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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.
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Mixed Flora of Staphylococcus and
Streptococcus
differentiates morphology
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Stains Several Fungi
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Gram-negative Pseudomonas
aeruginosa bacteria (pink-rods).
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(Bacillus anthracis),
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Streptococcus pneumonia
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Streptococcus salivarius
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Gram stain of Neisseria
gonorrhoea,
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Streptococcus pneumoniae in
Sputum
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Clostridium spp as seen in Gram Stain
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Moraxella as seen in Gram Staining
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Clostridium difficile as seen by
Gram staining
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Corynebacterium diptheria as seen
in Gram staining
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Gram stain of Neisseria
gonorrhoea,
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Neisseria meningitides as seen in
Gram staining
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Nocardia spp seen in Gram
Staining
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Gram Stained Actinomyctes
spp
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Gram stained Listeria
Monocytogenes
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Gram Stained Cryptococcus
neoformans
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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.

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Gram’s method stains Artificacts
too
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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
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Common errors in Staining
procedure
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Excessive heat during
fixation
Low concentration of
crystal violet
Excessive washing
between steps
Insufficient iodine
exposure
Prolonged
decolourization
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Excessive counterstaining
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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
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Gram staining not a fool proof
procedure
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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.
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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 Grampositive according to the conditions. With
these types of organisms, Gram-positive
and Gram-negative cells may be present
within the same preparation
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Overcoming in Gram Variable
Observations

it is 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.
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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 Gramnegative bacteria may become Grampositive in older cultures. For this reason it
is strongly recommended to use very
young cultures for the staining procedure,
after growth has become just visible.
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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.
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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
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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.
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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.

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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.
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Created for ‘e’ learning to
Medical and Paramedical
students in developing
countreis
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Email
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