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Gram Staining in
Clinical Microbiology
Dr.T.V.Rao. MD
Dr.T.V.Rao MD@ Gram staining 11/2/2018
Hans Christian Gram
• The Gram stain was
devised by the Danish
physician, Hans Christian
Gram, while working in
Berlin in 1883. He later
published this procedure
in 1884. At the time,
Dr. Gram was studying
lung tissue sections from
patients who had died of
pneumonia.
Dr.T.V.Rao MD@ Gram staining 21/2/2018
First Paper on Gram Staining
• In his paper, Dr. Gram described how he was
able to visualize what we now call
Staphylococcus, Streptococcus, Bacillus, and
Clostridia in various histological sections.
Interestingly, Dr. Gram did not actually use
safranin as a counter stain in the original
procedure (Gram negative cells would be
colorless). He instead recommended using
Bismarck brown as a counter stain to enable
tissue cell nuclei to be visualized.
Dr.T.V.Rao MD@ Gram staining 31/2/2018
Carl Weigert (1845-1904)
• German
pathologist Carl
Weigert (1845-
1904) from
Frankfurt, added
a final step of
staining with
safranin.
Dr.T.V.Rao MD@ Gram staining
41/2/2018
Traditional 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.
Dr.T.V.Rao MD@ Gram staining 51/2/2018
The Cell walls differ…
Dr.T.V.Rao MD@ Gram staining 61/2/2018
Gram Positive should not be Mistaken
• In the Gram Stain technique, two
positively charged dyes are used:
crystal violet and safranin. The use
of the designation “gram-positive”
should not be confused with the
concept of staining cells with a
simple stain that has a positive
charge. Dr.T.V.Rao MD@ Gram staining 71/2/2018
Gram staining observation
Basic Principle in Koch’s postulations
• The first of
Koch’s postulate
that the
suspected the
organism should
always be found
in association
with the disease.Dr.T.V.Rao MD@ Gram staining 81/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 91/2/2018
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 a
heat-fixed smear of a bacterial culture,
followed by the addition of a mordant
(Gram's iodine), rapid decolorization with
alcohol or acetone, and counterstaining
with Safranin or basic fuchsin.
Dr.T.V.Rao MD@ Gram staining 101/2/2018
Organizing the Staining Bottles
Dr.T.V.Rao MD@ Gram staining 111/2/2018
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
Dr.T.V.Rao MD@ Gram staining 121/2/2018
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
Dr.T.V.Rao MD@ Gram staining 131/2/2018
Method of smearing the Material
Wrong Right
Dr.T.V.Rao MD@ Gram staining 141/2/2018
Using Methanol is it Better than
Heat Fixation ?
• Fix the smear with
95% Methanol
• Which will help in
prevention of
distortion of cells
• Helpful in
Microscopic
observation of CSF
and Urine
Dr.T.V.Rao MD@ Gram staining 151/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 161/2/2018
Steps in Gram Staining Procedure-
Follow the Clock
• 1 On a rack, flood with filtered crystal violet
( Methyl 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 for few seconds <6
seconds until the moving dye front has passed
the lower edge of the section
• 6. Wash immediately in tap water
• 7. Counterstain with safranin for 15 seconds..
Dr.T.V.Rao MD@ Gram staining 171/2/2018
Proceed in organized Fashion
Dr.T.V.Rao MD@ Gram staining 181/2/2018
Step 1
Dr.T.V.Rao MD@ Gram staining 191/2/2018
Step 2
Dr.T.V.Rao MD@ Gram staining 201/2/2018
Step 3
Dr.T.V.Rao MD@ Gram staining 211/2/2018
Step 4
Dr.T.V.Rao MD@ Gram staining 221/2/2018
Step 5
Dr.T.V.Rao MD@ Gram staining 231/2/2018
How long you keep Iodine in the
Laboratory ???
• The Gram’s Iodine we make in
the laboratory from basic
chemicals
• How long we can use it ?
• Why we have to make frequently ?
Dr.T.V.Rao MD@ Gram staining 241/2/2018
Most Critical Step in Gram staining
• The most critical
step of gram staining
is the decolorization
step as crystal violet
stain will be
removed from both
G+ve & G-ve cells if
the decolorizing
agent(e.g alcohol ) is
left on too long.
Dr.T.V.Rao MD@ Gram staining 251/2/2018
Acetone used with Caution
• Acetone is a more
rapid decolorizes
than alcohol and
must be used with
some care.
• Excessive
decolorization turns
Gram positive
appear as Gram
negative
Dr.T.V.Rao MD@ Gram staining 261/2/2018
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, no
known advantage can be gained by
substituting the higher alcohols for ethyl
alcohol. Dr.T.V.Rao MD@ Gram staining 271/2/2018
Step 6
Dr.T.V.Rao MD@ Gram staining 281/2/2018
Which counterstain is better
• Some bacteria which
are poorly stained by
Safranin, such
as Hemophilus
spp., Legionella spp.
, and some anaerobic
bacteria, are readily
stained by basic
fuchsin, but not
Safranin
Dr.T.V.Rao MD@ Gram staining 291/2/2018
Step 7
Dr.T.V.Rao MD@ Gram staining 301/2/2018
Caring the stained slide
After the counterstain has
been rinsed off, the slide
is placed between some
absorbent paper and the
excess water gently
blotted off.
Care must be taken not
to rub the slide with the
blotting paper because
this would remove the
adhering bacteria.
Dr.T.V.Rao MD@ Gram staining 311/2/2018
Gram staining depends on
• Includes culture age, media, incubation
atmosphere, staining methods, . Similar
considerations apply to the interpretation of
smears from clinical specimens, and additional
factors include different host cell types and
possible phagocytosis.
• Gram stain permits the separation of all
bacteria into two large groups
Dr.T.V.Rao MD@ Gram staining 321/2/2018
How the Gram Stain Work
• So how does it work? Gram didn't know - he
simply worked empirically. We now know that the
Gram reaction is based on the structure of the
bacterial cell wall.
• In Gram-positive bacteria, the dark purple crystal
violet stain is retained by the thick layer of
peptidoglycan which forms the outer layer of the
cell.
• In Gram-negative bacteria, the thin peptidoglycan
layer in the periplasm does not retain the dark
stain, and the pink safranin counterstains the
peptidoglycan layer.
Dr.T.V.Rao MD@ Gram staining 331/2/2018
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-
negative pink due to
counter stain with Safranin..
Dr.T.V.Rao MD@ Gram staining 341/2/2018
Report as follows
• 1 If no microorganisms are seen in a
smear of a clinical specimen, report
“No microorganisms seen.”
• 2. If microorganisms are seen, report
relative numbers and Describe
morphology.
• Observe predominant shapes of
microorganisms
Dr.T.V.Rao MD@ Gram staining 351/2/2018
A gram stained bacterial suspension containing a
mixture of Gram negative bacilli, and Gram positive
cocci arranged in bunches (Staphylococci spp)
1/2/2018 Dr.T.V.Rao MD@ Gram staining 36
A true Gram Negative staining
Dr.T.V.Rao MD@ Gram staining 371/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 381/2/2018
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 bacterial
protoplasts, devoid of cell wall, are still
Gram-positive, indicating that it is probably
the semipermeable membrane which is
somehow involved in the reaction.
Dr.T.V.Rao MD@ Gram staining 391/2/2018
Nature of Morphology guides early Diagnosis
in uncommon diseases
Dr.T.V.Rao MD@ Gram staining 401/2/2018
Identify
• A young patient
presented with
foul smelling
purulent
discharge since 2
days on
observation by
Gram staining1/2/2018 Dr.T.V.Rao MD@ Gram staining 41
Gram stain of Neisseria gonorrhoeae,
1/2/2018 Dr.T.V.Rao MD@ Gram staining 42
Observe Spores may appear as
Gram negative and Gram positive
1/2/2018 Dr.T.V.Rao MD@ Gram staining 43
Burkholderia pseudomallei is a gram-negative
bacilli with a “safetypin” appearance on
microscopic examination
Dr.T.V.Rao MD@ Gram staining 441/2/2018
Limitations of Gram’s Staining
• We know that Gram
positivity is
restricted almost
exclusively to the
bacteria, with only a
few other groups,
such as the yeasts,
exhibiting this
reaction.
Dr.T.V.Rao MD@ Gram staining 451/2/2018
Better Understanding of Gram’s
Staining
• We should know that the Gram stain is
not an all-or-nothing phenomenon, but
that quantitative variations in Gram-
positivity exist between different
species, and within the same species
during different parts of the growth
cycle or under different
environmental conditions.
Dr.T.V.Rao MD@ Gram staining 461/2/2018
Stains Several Fungi
Dr.T.V.Rao MD@ Gram staining 471/2/2018
Streptococcus pneumonia
Dr.T.V.Rao MD@ Gram staining 481/2/2018
Streptococcus pneumonia in Sputum
Dr.T.V.Rao MD@ Gram staining 491/2/2018
Nocardia spp seen in Gram Staining
Dr.T.V.Rao MD@ Gram staining 501/2/2018
Gram Stained Actinomyctes spp
Dr.T.V.Rao MD@ Gram staining 511/2/2018
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-positive
can occur if the decolorization step is
accidentally omitted.
Dr.T.V.Rao MD@ Gram staining 521/2/2018
Common errors in Staining
procedure
• Excessive heat during
fixation
• Low concentration of
crystal violet
• Excessive washing
between steps
• Insufficient iodine
exposure
• Prolonged
decolourization
• Excessive counterstaining
Dr.T.V.Rao MD@ Gram staining 531/2/2018
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
Dr.T.V.Rao MD@ Gram staining 541/2/2018
Artifacts in Gram Staining
• Gram stain
reagents Crystal
Violet, Iodine ?,
Safranin,
contaminated.
• Dirty glass slides
• Contaminated
water used to
rinse slides
Dr.T.V.Rao MD@ Gram staining 551/2/2018
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 the
isolate is a gram-
negative organism.
Dr.T.V.Rao MD@ Gram staining 561/2/2018
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 bacteria.
Dr.T.V.Rao MD@ Gram staining 571/2/2018
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
Dr.T.V.Rao MD@ Gram staining 581/2/2018
Overcoming in Gram Variable
Observations
• It is necessary that it is stained
at two or three different ages
(very young cultures should be
used). In case a Gram-variable
reaction is observed it is also
good to check the purity of the
culture.
Dr.T.V.Rao MD@ Gram staining 591/2/2018
Gram Staining appearance differs..
The genera Actinomyctes,
Arthobacter,
Corynebacterium,
Mycobacterium, and
Propionibacterium have cell
walls 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
Dr.T.V.Rao MD@ Gram staining 601/2/2018
QUALITY CONTROL
• Check appearance of reagents daily
• If crystal violet has precipitate or crystal
sediment, refilter before use even when
purchased commercially. NOTE: Some stains,
especially basic fuchsin and safranin, can
become contaminated. Start with fresh
material in a clean bottle.
• Evaporation may alter reagent effectiveness;
working solutions should be changed regularly
Dr.T.V.Rao MD@ Gram staining 611/2/2018
QUALITY CONTROL
• Daily and when a new
lot is used, prepare a
smear of Escherichia
coli (ATCC 25922) and
Staphylococcus
epidermidis (ATCC
12228)or
Staphylococcus aureus
(ATCC 25923). Fix and
stain as described.
Dr.T.V.Rao MD@ Gram staining 621/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 631/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 641/2/2018
Modifications -Report with caution
• 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.
Dr.T.V.Rao MD@ Gram staining 651/2/2018
Is it wise to adopt different Gram
staining procedure
• Bartholomew (1962) has
pointed out that each
variation in the Gram staining
procedure has a definite limit
to its acceptability
Dr.T.V.Rao MD@ Gram staining 661/2/2018
Hucker and Conn's
recommendation
• There is no gram procedure which
can be referred to as the best for all
laboratories and for all situations. It
is recommended that the young
microbiologists adopt at least two of
the well-accepted methods, practice
them until he is familiar with their
characteristics,
Dr.T.V.Rao MD@ Gram staining 671/2/2018
Words of Wisdom
Hans Christian Gram
• I am aware
that as yet it
is very
defective
and
imperfectDr.T.V.Rao MD@ Gram staining 681/2/2018
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.
Dr.T.V.Rao MD@ Gram staining 691/2/2018
Best of References
You can read on line….
• A monograph of
gram-stained
preparations of
clinical Specimens
• By Linda M.
Marler, Jean A.
Siders, Stephen D.
Allen (MD.)
Dr.T.V.Rao MD@ Gram staining 701/2/2018
Gram staining continues to be
Most Rapid test.
• Even new molecular methodologies
typically take hours rather than minutes.
" 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.Dr.T.V.Rao MD@ Gram staining 711/2/2018
Gram’s Staining
A Mystery
• The exact
mechanism of the
staining reaction is
not fully
understood,
however, this does
not detract from
its usefulness.
Dr.T.V.Rao MD@ Gram staining 721/2/2018
IMPORTANCE OF GRAM STAIN
CONTINUES
• The most important and primary
test to perform directly on some
special samples such as
cerebrospinal fluid and positive
cultures is Gram staining which
serves as the most rapid and
simplest test to characterize
microorganisms.. In recent
reports, the impact of Gram
staining results on patient
mortality has been documented
and continues to be the most
rapid test in Diagnostic
Microbiology
• Dr.T.V.Rao MD/2/2018
Dr.T.V.Rao MD@ Gram staining 73
• Program Created by Dr.T.V.Rao MD for
benefit of Medical and paramedical
professionals in the developing world
• Email
• doctortvrao@gmail.com
1/2/2018 Dr.T.V.Rao MD@ Gram staining 74

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Gram Staining i n Clinical Microbiology by Dr.T.V.Rao MD

  • 1. Gram Staining in Clinical Microbiology Dr.T.V.Rao. MD Dr.T.V.Rao MD@ Gram staining 11/2/2018
  • 2. Hans Christian Gram • The Gram stain was devised by the Danish physician, Hans Christian Gram, while working in Berlin in 1883. He later published this procedure in 1884. At the time, Dr. Gram was studying lung tissue sections from patients who had died of pneumonia. Dr.T.V.Rao MD@ Gram staining 21/2/2018
  • 3. First Paper on Gram Staining • In his paper, Dr. Gram described how he was able to visualize what we now call Staphylococcus, Streptococcus, Bacillus, and Clostridia in various histological sections. Interestingly, Dr. Gram did not actually use safranin as a counter stain in the original procedure (Gram negative cells would be colorless). He instead recommended using Bismarck brown as a counter stain to enable tissue cell nuclei to be visualized. Dr.T.V.Rao MD@ Gram staining 31/2/2018
  • 4. Carl Weigert (1845-1904) • German pathologist Carl Weigert (1845- 1904) from Frankfurt, added a final step of staining with safranin. Dr.T.V.Rao MD@ Gram staining 41/2/2018
  • 5. Traditional 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. Dr.T.V.Rao MD@ Gram staining 51/2/2018
  • 6. The Cell walls differ… Dr.T.V.Rao MD@ Gram staining 61/2/2018
  • 7. Gram Positive should not be Mistaken • In the Gram Stain technique, two positively charged dyes are used: crystal violet and safranin. The use of the designation “gram-positive” should not be confused with the concept of staining cells with a simple stain that has a positive charge. Dr.T.V.Rao MD@ Gram staining 71/2/2018
  • 8. Gram staining observation Basic Principle in Koch’s postulations • The first of Koch’s postulate that the suspected the organism should always be found in association with the disease.Dr.T.V.Rao MD@ Gram staining 81/2/2018
  • 9. 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. Dr.T.V.Rao MD@ Gram staining 91/2/2018
  • 10. 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 a heat-fixed smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with Safranin or basic fuchsin. Dr.T.V.Rao MD@ Gram staining 101/2/2018
  • 11. Organizing the Staining Bottles Dr.T.V.Rao MD@ Gram staining 111/2/2018
  • 12. 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 Dr.T.V.Rao MD@ Gram staining 121/2/2018
  • 13. 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 Dr.T.V.Rao MD@ Gram staining 131/2/2018
  • 14. Method of smearing the Material Wrong Right Dr.T.V.Rao MD@ Gram staining 141/2/2018
  • 15. Using Methanol is it Better than Heat Fixation ? • Fix the smear with 95% Methanol • Which will help in prevention of distortion of cells • Helpful in Microscopic observation of CSF and Urine Dr.T.V.Rao MD@ Gram staining 151/2/2018
  • 16. 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. Dr.T.V.Rao MD@ Gram staining 161/2/2018
  • 17. Steps in Gram Staining Procedure- Follow the Clock • 1 On a rack, flood with filtered crystal violet ( Methyl 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 for few seconds <6 seconds until the moving dye front has passed the lower edge of the section • 6. Wash immediately in tap water • 7. Counterstain with safranin for 15 seconds.. Dr.T.V.Rao MD@ Gram staining 171/2/2018
  • 18. Proceed in organized Fashion Dr.T.V.Rao MD@ Gram staining 181/2/2018
  • 19. Step 1 Dr.T.V.Rao MD@ Gram staining 191/2/2018
  • 20. Step 2 Dr.T.V.Rao MD@ Gram staining 201/2/2018
  • 21. Step 3 Dr.T.V.Rao MD@ Gram staining 211/2/2018
  • 22. Step 4 Dr.T.V.Rao MD@ Gram staining 221/2/2018
  • 23. Step 5 Dr.T.V.Rao MD@ Gram staining 231/2/2018
  • 24. How long you keep Iodine in the Laboratory ??? • The Gram’s Iodine we make in the laboratory from basic chemicals • How long we can use it ? • Why we have to make frequently ? Dr.T.V.Rao MD@ Gram staining 241/2/2018
  • 25. Most Critical Step in Gram staining • The most critical step of gram staining is the decolorization step as crystal violet stain will be removed from both G+ve & G-ve cells if the decolorizing agent(e.g alcohol ) is left on too long. Dr.T.V.Rao MD@ Gram staining 251/2/2018
  • 26. Acetone used with Caution • Acetone is a more rapid decolorizes than alcohol and must be used with some care. • Excessive decolorization turns Gram positive appear as Gram negative Dr.T.V.Rao MD@ Gram staining 261/2/2018
  • 27. 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, no known advantage can be gained by substituting the higher alcohols for ethyl alcohol. Dr.T.V.Rao MD@ Gram staining 271/2/2018
  • 28. Step 6 Dr.T.V.Rao MD@ Gram staining 281/2/2018
  • 29. Which counterstain is better • Some bacteria which are poorly stained by Safranin, such as Hemophilus spp., Legionella spp. , and some anaerobic bacteria, are readily stained by basic fuchsin, but not Safranin Dr.T.V.Rao MD@ Gram staining 291/2/2018
  • 30. Step 7 Dr.T.V.Rao MD@ Gram staining 301/2/2018
  • 31. Caring the stained slide After the counterstain has been rinsed off, the slide is placed between some absorbent paper and the excess water gently blotted off. Care must be taken not to rub the slide with the blotting paper because this would remove the adhering bacteria. Dr.T.V.Rao MD@ Gram staining 311/2/2018
  • 32. Gram staining depends on • Includes culture age, media, incubation atmosphere, staining methods, . Similar considerations apply to the interpretation of smears from clinical specimens, and additional factors include different host cell types and possible phagocytosis. • Gram stain permits the separation of all bacteria into two large groups Dr.T.V.Rao MD@ Gram staining 321/2/2018
  • 33. How the Gram Stain Work • So how does it work? Gram didn't know - he simply worked empirically. We now know that the Gram reaction is based on the structure of the bacterial cell wall. • In Gram-positive bacteria, the dark purple crystal violet stain is retained by the thick layer of peptidoglycan which forms the outer layer of the cell. • In Gram-negative bacteria, the thin peptidoglycan layer in the periplasm does not retain the dark stain, and the pink safranin counterstains the peptidoglycan layer. Dr.T.V.Rao MD@ Gram staining 331/2/2018
  • 34. 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- negative pink due to counter stain with Safranin.. Dr.T.V.Rao MD@ Gram staining 341/2/2018
  • 35. Report as follows • 1 If no microorganisms are seen in a smear of a clinical specimen, report “No microorganisms seen.” • 2. If microorganisms are seen, report relative numbers and Describe morphology. • Observe predominant shapes of microorganisms Dr.T.V.Rao MD@ Gram staining 351/2/2018
  • 36. A gram stained bacterial suspension containing a mixture of Gram negative bacilli, and Gram positive cocci arranged in bunches (Staphylococci spp) 1/2/2018 Dr.T.V.Rao MD@ Gram staining 36
  • 37. A true Gram Negative staining Dr.T.V.Rao MD@ Gram staining 371/2/2018
  • 38. 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. Dr.T.V.Rao MD@ Gram staining 381/2/2018
  • 39. 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 bacterial protoplasts, devoid of cell wall, are still Gram-positive, indicating that it is probably the semipermeable membrane which is somehow involved in the reaction. Dr.T.V.Rao MD@ Gram staining 391/2/2018
  • 40. Nature of Morphology guides early Diagnosis in uncommon diseases Dr.T.V.Rao MD@ Gram staining 401/2/2018
  • 41. Identify • A young patient presented with foul smelling purulent discharge since 2 days on observation by Gram staining1/2/2018 Dr.T.V.Rao MD@ Gram staining 41
  • 42. Gram stain of Neisseria gonorrhoeae, 1/2/2018 Dr.T.V.Rao MD@ Gram staining 42
  • 43. Observe Spores may appear as Gram negative and Gram positive 1/2/2018 Dr.T.V.Rao MD@ Gram staining 43
  • 44. Burkholderia pseudomallei is a gram-negative bacilli with a “safetypin” appearance on microscopic examination Dr.T.V.Rao MD@ Gram staining 441/2/2018
  • 45. Limitations of Gram’s Staining • We know that Gram positivity is restricted almost exclusively to the bacteria, with only a few other groups, such as the yeasts, exhibiting this reaction. Dr.T.V.Rao MD@ Gram staining 451/2/2018
  • 46. Better Understanding of Gram’s Staining • We should know that the Gram stain is not an all-or-nothing phenomenon, but that quantitative variations in Gram- positivity exist between different species, and within the same species during different parts of the growth cycle or under different environmental conditions. Dr.T.V.Rao MD@ Gram staining 461/2/2018
  • 47. Stains Several Fungi Dr.T.V.Rao MD@ Gram staining 471/2/2018
  • 48. Streptococcus pneumonia Dr.T.V.Rao MD@ Gram staining 481/2/2018
  • 49. Streptococcus pneumonia in Sputum Dr.T.V.Rao MD@ Gram staining 491/2/2018
  • 50. Nocardia spp seen in Gram Staining Dr.T.V.Rao MD@ Gram staining 501/2/2018
  • 51. Gram Stained Actinomyctes spp Dr.T.V.Rao MD@ Gram staining 511/2/2018
  • 52. 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-positive can occur if the decolorization step is accidentally omitted. Dr.T.V.Rao MD@ Gram staining 521/2/2018
  • 53. Common errors in Staining procedure • Excessive heat during fixation • Low concentration of crystal violet • Excessive washing between steps • Insufficient iodine exposure • Prolonged decolourization • Excessive counterstaining Dr.T.V.Rao MD@ Gram staining 531/2/2018
  • 54. 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 Dr.T.V.Rao MD@ Gram staining 541/2/2018
  • 55. Artifacts in Gram Staining • Gram stain reagents Crystal Violet, Iodine ?, Safranin, contaminated. • Dirty glass slides • Contaminated water used to rinse slides Dr.T.V.Rao MD@ Gram staining 551/2/2018
  • 56. 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 the isolate is a gram- negative organism. Dr.T.V.Rao MD@ Gram staining 561/2/2018
  • 57. 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 bacteria. Dr.T.V.Rao MD@ Gram staining 571/2/2018
  • 58. 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 Dr.T.V.Rao MD@ Gram staining 581/2/2018
  • 59. Overcoming in Gram Variable Observations • It is necessary that it is stained at two or three different ages (very young cultures should be used). In case a Gram-variable reaction is observed it is also good to check the purity of the culture. Dr.T.V.Rao MD@ Gram staining 591/2/2018
  • 60. Gram Staining appearance differs.. The genera Actinomyctes, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls 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 Dr.T.V.Rao MD@ Gram staining 601/2/2018
  • 61. QUALITY CONTROL • Check appearance of reagents daily • If crystal violet has precipitate or crystal sediment, refilter before use even when purchased commercially. NOTE: Some stains, especially basic fuchsin and safranin, can become contaminated. Start with fresh material in a clean bottle. • Evaporation may alter reagent effectiveness; working solutions should be changed regularly Dr.T.V.Rao MD@ Gram staining 611/2/2018
  • 62. QUALITY CONTROL • Daily and when a new lot is used, prepare a smear of Escherichia coli (ATCC 25922) and Staphylococcus epidermidis (ATCC 12228)or Staphylococcus aureus (ATCC 25923). Fix and stain as described. Dr.T.V.Rao MD@ Gram staining 621/2/2018
  • 63. 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. Dr.T.V.Rao MD@ Gram staining 631/2/2018
  • 64. 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. Dr.T.V.Rao MD@ Gram staining 641/2/2018
  • 65. Modifications -Report with caution • 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. Dr.T.V.Rao MD@ Gram staining 651/2/2018
  • 66. Is it wise to adopt different Gram staining procedure • Bartholomew (1962) has pointed out that each variation in the Gram staining procedure has a definite limit to its acceptability Dr.T.V.Rao MD@ Gram staining 661/2/2018
  • 67. Hucker and Conn's recommendation • There is no gram procedure which can be referred to as the best for all laboratories and for all situations. It is recommended that the young microbiologists adopt at least two of the well-accepted methods, practice them until he is familiar with their characteristics, Dr.T.V.Rao MD@ Gram staining 671/2/2018
  • 68. Words of Wisdom Hans Christian Gram • I am aware that as yet it is very defective and imperfectDr.T.V.Rao MD@ Gram staining 681/2/2018
  • 69. 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. Dr.T.V.Rao MD@ Gram staining 691/2/2018
  • 70. Best of References You can read on line…. • A monograph of gram-stained preparations of clinical Specimens • By Linda M. Marler, Jean A. Siders, Stephen D. Allen (MD.) Dr.T.V.Rao MD@ Gram staining 701/2/2018
  • 71. Gram staining continues to be Most Rapid test. • Even new molecular methodologies typically take hours rather than minutes. " 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.Dr.T.V.Rao MD@ Gram staining 711/2/2018
  • 72. Gram’s Staining A Mystery • The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefulness. Dr.T.V.Rao MD@ Gram staining 721/2/2018
  • 73. IMPORTANCE OF GRAM STAIN CONTINUES • The most important and primary test to perform directly on some special samples such as cerebrospinal fluid and positive cultures is Gram staining which serves as the most rapid and simplest test to characterize microorganisms.. In recent reports, the impact of Gram staining results on patient mortality has been documented and continues to be the most rapid test in Diagnostic Microbiology • Dr.T.V.Rao MD/2/2018 Dr.T.V.Rao MD@ Gram staining 73
  • 74. • Program Created by Dr.T.V.Rao MD for benefit of Medical and paramedical professionals in the developing world • Email • doctortvrao@gmail.com 1/2/2018 Dr.T.V.Rao MD@ Gram staining 74