gram staining guest lecture jipmer pondcherry
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GramStaining inClinical Microbiology
Dr.T.V.Rao. MD
Professor of Microbiology
Travancore Medical College, Kollam. Kerala
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The Guest Lecture presented by Dr.T.V.Rao MD
at
Tenth National Workshop on
Simple Diagnostic Methods in ClinicalMicrobiology
29th November to 3rd December 2011
Department of Microbiology
Jawaharlal Institute of Postgraduate MedicalEducation & Research, Pondicherry
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Working at Mansa General Hospital Mansa
Republic of ZambiaTaught many lessons, to understand Infection and Ignorance
are important causes of Morbidity and Mortality
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Hans Christian Gram The Gram stain was
devised by the Danish
physician, Hans Christian
Gram, while working in
Berlin in 1883. He laterpublished this procedure
in 1884. At the time,
Dr. Gram was studying
lung tissue sections frompatients who had died of
pneumonia.
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First Paper on Gram Staining
In his paper, Dr. Gram described how he wasable to visualize what we now callStaphylococcus, Streptococcus, Bacillus, and
Clostridiain various histological sections.Interestingly, Dr. Gram did not actually usesafranin as a counter stain in the originalprocedure (Gram negative cells would be
colorless). He instead recommended usingBismarck brown as a counter stain to enabletissue cell nuclei to be visualized.
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Carl Weigert (1845-1904)
German
pathologist Carl
Weigert (1845-
1904) from
Frankfurt, added
a final step ofstaining with
safranin.
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Traditional Definition of Gram stain
A method of staining bacteria using a violetstain. 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, washedwith alcohol and counterstained. The method
differentiates bacteria into two main classes,gram-positive and gram-negative.
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The Cell walls differ
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Gram Positive should not be Mistaken
In the Gram Stain technique, two
positively charged dyes are used:
crystal violet and safranin. The useof the designation gram-positive
should not be confused with the
concept of staining cells with asimple stain that has a positive
charge. Dr.T.V.Rao MD 9
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Gram staining observation
Basic Principle in Kochs postulations
The first of
Kochs postulate
that thesuspected the
organism should
always be found
in association
with the disease.Dr.T.V.Rao MD 10
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Poor quality of slides
Can be corrected
Use of glass slides
that have not
been pre cleanedor degreased ?NOTE: Storing slides ina jar with 95% ethanol
will ensure clean slides.
Drain excess alcohol or
flame slide before use.
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Four Major Steps in Gram Staining
There are four basic steps of the Gram
stain, which include applying a primary
stain (crystal violet)or Methyl violet to aheat-fixed smear of a bacterial culture,
followed by the addition of a mordant
(Gram's iodine), rapid decolorization withalcohol or acetone, and counterstaining
with Safranin or basic fuchsin.
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Organizing the Staining Bottles
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Making a Smear
First prepare your
slide. You do this
by placing bacteriaon a slide in a drop
of water, allowing
them to dry andthen heat fixing
them. Heating
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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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Method of smearing the MaterialWrong Right
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Using Methanol is it Better than
Heat Fixation ?
Fix the smear with95% Methanol
Which will help in
prevention ofdistortion of cells
Helpful in
Microscopicobservation ofCSFand Urine
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Making Multiple smears in same slide
conserve resources
Making multiple
smears make the
optimal use of theslide.
Reduces the
economic costsand saves the
technical time.
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Steps in Gram Staining Procedure-
Follow the Clock
1On a rack, flood with filtered crystal violet( Methyl violet ) 10 sec2 Wash briefly in water to remove excess crystalviolet
3. Flood with Grams iodine 10 sec 4. Wash briefly in water, do not let the section
dry out. 5. Decolourise with acetone for few 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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How long you keep Iodine in the
Laboratory ???
The Grams Iodine we make in
the laboratory from basic
chemicals
How long we can use it ?
Why we have to make frequently ?
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Most Critical Step in Gram staining
The most critical
step of gram staining
is the decolorization
step as crystal violetstain will be
removed from both
G+ve & G-ve cells ifthe decolorizing
agent(e.g alcohol ) is
left on too long.Dr.T.V.Rao MD 27
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Acetone used with Caution
Acetone is a morerapid decolorizesthan alcohol and
must be used withsome care.
Excessivedecolorization turns
Gram positiveappear as Gramnegative
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Which alcohol is better
Several alcohols have been studied, and
it has been reported that the more
complex the alcohol, the slower the
decolorization action. As the carbon
chain lengthens, decolorization is slower.
Conn found in practice, however, noknown advantage can be gained by
substituting the higher alcohols for ethyl
alcohol.Dr.T.V.Rao MD 29
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Step 6
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Which counterstain is better
Some bacteria whichare poorly stained by
Safranin, suchas Hemophilus
spp., Legionella spp., and some anaerobicbacteria, are readily
stained by basicfuchsin, but notSafranin
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Step 7
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Caring the stained slide
After the counterstain hasbeen rinsed off, the slideis placed between someabsorbent paper and the
excess water gentlyblotted off.Care must be taken notto rub the slide with the
blotting paper becausethis would remove theadhering bacteria.
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Gram staining depends on
Includes culture age, media, incubation
atmosphere, staining methods, . Similar
considerations apply to the interpretation of
smears from clinical specimens, and additionalfactors include different host cell types and
possible phagocytosis.
Gram stain permits the separation of all
bacteria into two large groups
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How the Gram Stain Work
So how does it work? Gram didn't know - hesimply worked empirically. We now know that theGram reaction is based on the structure of thebacterial cell wall.
In Gram-positive bacteria, the dark purple crystalviolet stain is retained by the thick layer ofpeptidoglycan which forms the outer layer of thecell.
In Gram-negative bacteria, the thin peptidoglycanlayer in the periplasm does not retain the darkstain, and the pink safranin counterstains thepeptidoglycan layer.
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Optimal use of Microscopy
Gram stained preparationshave to be observed withbright-field optics. Phase-contrast microscopydoes not allow therecognition of truecolours. Gram-positivebacteria may be seen underphase-contrast as red cells.Using bright-field optics,
Gram-positive cells arepurple or blue and Gram-negative pinkdue tocounter stain with Safranin..
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Report as follows
1 If no microorganisms are seen in asmear of a clinical specimen, reportNo microorganisms seen.
2. If microorganisms are seen, reportrelative numbers and Describemorphology.
Observe predominant shapes ofmicroorganisms
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A gram stained bacterial suspension containing a
mixture of Gram negative bacilli, and Gram positive
cocci arranged in bunches (Staphylococci spp)
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A true Gram Negative staining
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Value of Direct Smears
Guide the physician on initial choice ofantibiotic, pending results of culture andsensitivity.
Judge specimen quality.
Contribute to selection of culture media,especially with mixed flora.
Provide internal quality control whendirect smear results are compared toculture results.
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Staining depends on Structural
Integrity of Cell Wall
We know that only intact cells are Gram-
positive, so that cells which are even gently
broken become Gram-negative.
Observations suggest that bacterialprotoplasts, devoid of cell wall, are still
Gram-positive, indicating that it is probably
the semipermeable membrane which issomehow involved in the reaction.
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Nature of Morphology guides early Diagnosis
in uncommon diseases
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Identify
A young patient
presented with
foul smellingpurulent
discharge since 2
days onobservation by
Gram staining
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Gram stain of Neisseria gonorrhoeae,
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Observe Spores may appear as
Gram negative and Gram positive
Burkholderia pseudomallei is a gram negative
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Burkholderia pseudomallei is a gram-negative
bacilli with a safetypin appearance on
microscopic examination
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Limitations of Grams Staining
We know that Gram
positivity is
restricted almost
exclusively to thebacteria, with only a
few other groups,
such as the yeasts,exhibiting this
reaction.
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B tt U d t di f G
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Better Understanding of Grams
Staining
We should know that the Gram stain is
not an all-or-nothing phenomenon, but
thatquantitative variations
in Gram-
positivity exist between different
species,and within the same species
during different parts of the growthcycle or under different
environmental conditions.Dr.T.V.Rao MD 48
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Stains Several Fungi
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Streptococcus pneumonia
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Streptococcus pneumonia in Sputum
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Nocardia spp seen in Gram Staining
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Gram Stained Actinomyctes spp
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Faulty Gram stain reactions
It is possible to report as " Gram-
negative" if the gram-positive bacteria
are old, dead, or damaged and the
cell wall is not intact.
There is no equivalent "false Gram-
positive," but a false Gram-positivecan occur if the decolorization step is
accidentally omitted.Dr.T.V.Rao MD 54
Common errors in Staining
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Common errors in Staining
procedure Excessive heat during
fixation
Low concentration ofcrystal violet
Excessive washingbetween steps
Insufficient iodineexposure
Prolongeddecolourization
Excessive counterstaining
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G t i lt t l t d
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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 microorganisms
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Artifacts in Gram Staining
Gram stainreagents CrystalViolet, Iodine ?,
Safranin,contaminated.
Dirty glass slides
Contaminatedwater used torinse slides
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Biochemical Tests in Identification
KOH string test may be
used as a confirmatory
test for the Gram Stain
(Powers, 1995, Arthi et
al., 2003): The
formation of a string
(DNA) in 3% KOH
indicates that theisolate is a gram-
negative organism.
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Gram staining not a fool proof
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Gram staining not a fool proof
procedure
Grams staining method
is not without its
problems.
It is , complicated, and
prone to operator
error.
The method also
requires a large number
of bacteria.Dr.T.V.Rao MD 59
Gram variable observations in Gram
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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 Gram-
positive 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
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Overcoming in Gram Variable
Observations
It is necessary that it is stainedat two or three different ages
(very young cultures should beused). In case a Gram-variablereaction is observed it is alsogood to check the purity of theculture.
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Gram Staining appearance differs..
The genera Actinomyctes,
Arthobacter,
Corynebacterium,
Mycobacterium, and
Propionibacterium have cellwalls particularly sensitive to
breakage during cell division,
resulting in Gram-negative
staining of these Gram-positive
cells. The staining of these
organisms result in an uneven
or granular appearance
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QUALITY CONTROL
Check appearance of reagents daily
If crystal violet has precipitate or crystalsediment, refilter before use even when
purchased commercially. NOTE: Some stains,especially basic fuchsin and safranin, canbecome contaminated. Start with freshmaterial in a clean bottle.
Evaporation may alter reagent effectiveness;working solutions should be changed regularly
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QUALITY CONTROL
Daily and when a new
lot is used, prepare a
smear ofEscherichia
coli(ATCC 25922) and
Staphylococcus
epidermidis (ATCC
12228)or
Staphylococcus aureus(ATCC 25923). Fix and
stain as described.
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Interpret Gram Staining with Clinical
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Interpret Gram Staining with Clinical
Picture and other Investigations
Nevertheless,
Gram's stain
findings can be
equivocal and,
therefore, must be
assessed carefully
in light of the
clinical picture.
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Modification in Gram staining
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Modification in Gram staining
methods ?
Since the original procedure ofGram, many variations of theGram staining technique havebeen published. Some of themhave improved the method,others include some minortechnical variants of no value.
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Modifications -Report with caution
Any final result is theoutcome of theinteraction of all ofthe possible
variables. All modified
methods to bepractised with
caution should suitto the laboratory,and quality controlchecks.
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Is it wise to adopt different Gram
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Is it wise to adopt different Gram
staining procedure
Bartholomew (1962) has
pointed out that each
variation in the Gram stainingprocedure has a definite limit
to its acceptability
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Hucker and Conn's
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Hucker and Conn s
recommendation
There is no gram procedure whichcan be referred to as the best for alllaboratories and for all situations. Itis recommended that the youngmicrobiologists adopt at least two ofthe well-accepted methods, practicethem until he is familiar with theircharacteristics,
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Words of Wisdom
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Words of Wisdom
Hans Christian Gram
I am aware
that as yet it
is very
defective
and
imperfectDr.T.V.Rao MD 70
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Creating Library of Gram Stains
Drain or gently blotexcess oil
For slide libraries andteaching collections that
will be stored for longerperiods, immersion oilcan be removed withxylene solution and the
slides can be coverslipped using Per mountto prevent fading.
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Best of References
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Best of ReferencesYou can read on line.
A monograph of
gram-stained
preparations of
clinical Specimens
By Linda M.
Marler, Jean A.Siders, Stephen D.
Allen (MD.)
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Gram staining continues to be
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Gram staining continues to be
Most Rapid test.
Even new molecular methodologiestypically take hours rather than minutes." This simple staining procedure
remains the most useful test performedin the microbiology lab. Results from aGram's stain can tell volumes about an
infection within 15 minutes of aspecimen's arrival in the lab, while mostother microbiology results require 24hours or more.
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Grams Staining
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Gram s Staining
A Mystery
The exact
mechanism of the
staining reaction is
not fully
understood,
however, this does
not detract from
its usefulness.
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