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
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
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
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
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
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
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
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