By Dr Praseetha

1st year PG
Dept of Microbiology
BMCRI
 Allows the microbiologist to distinguish
between the two most common chemical
cellular structures of bacteria (visualizing the
morphology and cellular arrangement of the
organisms)

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 It was introduced by Danish physician Hans
Christian Gram in 1884 to differentiate
Streptococcus pneumoniae from Klebsiella
pneumoniae in Lung tissue

 The modification currently used for general

bacteriology was developed by Hucker in
1921.

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 The original Gram’s staining was slightly
different from what we use today.
◦Primary stain – Gentian violet
◦Mordant – Lugol’s iodine
◦Decolourizer - Absolute alcohol
◦Counterstain - Bismarck brown

Most frequently used differential stain for

diagnostic identification of bacteria into two
major groups :
GRAM-POSITIVE
GRAM-NEGATIVE

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 It is proposed based on the differential structure of
the cellular membranes and cell walls of the two
groups.
Gram-positive organisms contain a highly cross-
linked layer of peptidoglycan -> retains the primary
dye, crystal violet (CV)
 Following the application of the mordant, iodine (I).
The iodine and crystal violet form a complex within
the peptidoglycan.
When decolorizer is applied to the cells, the CV-I
complex remains within the cell, making it appear
dark purple to blue.

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The gram-negative organisms do not contain a thick cross-linked
layer of peptidoglycan. It is loosely distributed between the inner
cell and outer cell membrane.

Following application of the crystal violet and iodine, the CV-I

complexes are not trapped within the peptidoglycan.

Application of the acid-alcohol decolorizer dehydrates the outer

cellular membrane, leaving holes in the membrane and effectively
washing or removing the CV-I complex from the cells. The cells
appear colorless.

To make the colorless cells visible, a secondary stain, safranin, is

applied, leaving the gram-negative cells pink

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 Lipid content theory:
 Gram negative cell wall have an extra LPS outer membrane
in its cell envelope with more lipids.
 Acetone /alcohol dissolves the lipid forming large pores
through which CVI complex leaks out whereas it
dehydrates Gram positive cells, shrinks them & closes the
pores.

 Cytoplasmic p H theory:
 Gram positive cells have more acidic protoplasm(2-3) due
to Teichoic acid.
 Hence retains the basic primary dye more strongly than
Gram negative bacteria(4-5)

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 Magnesium Ribonucleate theory:
 A compound of Magnesium ribonucleate in Gram
positive cell have affinity for basic dye and they
take up Crystal violet.
 It has also been proved that Gram positive
becomes Gram negative on the removal of this
material

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Gram’s iodine
 iodine crystals
 potassium iodide
 distilled water

Safranin
safranin O
ethanol, 95%
distilled water

Basic fuchsin
basic fuchsin
ethyl alcohol
distilled water

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Decolorizers
Slowest: ethanol, 95% - 1 min
Intermediate: acetone-alcohol -10 sec
Fastest: acetone-2-3 sec

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 Flood the fixed smear with the crystal violet solution. Allow to
remain for 1 min. Rinse off
 Rinse off excess water with iodine solution, then flood the
slide with fresh iodine solution. Allow iodine to remain for 1
min. Rinse off iodine gently with flowing tap water.
 Decolorize by letting the reagent flow over the smear while
the slide is held at an angle. Stop when the runoff becomes
clear.
 Remove excess decolorizer with gentle flow of tap water
 Flood the slide with counterstain and allow to remain for at
least 30s. Remove excess counterstain.
 Drain slide, and air dry it in an upright position, or use a
commercial slide drier

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Evaluate the general nature of the smear under low
power

Observe for stain crystals. If an excess of precipitated

stain is observed, decolorize and restain slide.

Determine if smear has been properly decolorized

Determine if thickness of smear is appropriate. For

proper interpretation, areas must be no more than one
cell thick, with no overlapping of cells.

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Low-power observations

1. WBCs and RBCs suggest infectious process.

2. SECs, debris
3. Certain microorganisms (parasitic forms, branching hyphae,
etc.) indicate infectious process.

Oil immersion observations

1. Observe microorganisms for characteristic morphology and

presentation
2. Gram reaction
 Gram positive: deep violet
 Gram negative: pink or red (carbol fuchsin counterstains have
a more intense color)
 Gram variable: both gram-positive and gram-negative cells
with the same morphology
 Gram neutral: taking up neither the crystal violet nor the
counterstain

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 For smears prepared from clinical specimens,
examine several fields (10 for urine, 20 to 40 for
other specimens) under low power for evidence
of inflammation

 Examine the slide for cells including epithelial,

red blood cells and white blood cells. Red blood
cells may stain faintly. White blood cells should
appear as light pink cells with a dark pink or red
nucleus. White blood cells may be differentiated
into polymorphonuclear cells (PMNs) and
mononuclear cells.

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 Examine the slide for microorganisms
characteristic morphologies and arrangements
including gram-positive versus gram-negative,
cocci, bacilli, spirochetes, curved-rods, large or
small in singlets, pairs, clusters, chains, or
diplococci. Indicate pleomorphic, coccobacillary
or diphtheroids if applicable.

 If bacterial spores are present, indicate cellular

location such as terminal or subterminal and
shape such as oval or round. Spores do not stain
with Gram stain reagents but will appear as clear
areas within the cells.)

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 If no organisms or cells are detected in a smear
of a clinical specimen, report “No organisms
seen” or “No cells seen,” respectively.

 Determine number of cells and bacteria in 20 to

40 fields of the smear.

 Count cells under low-power objective and report

relative numbers accordingly

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Count bacteria and yeasts under oil immersion and report relative
numbers from areas associated with cells.
Follow cellular enumeration with the description of the type of the cells
1. SECs
2. WBCs
3. RBCs
4. Host cellular material

Follow microorganism enumeration with the description of the

morphology of the bacteria, providing as much information as possible
Gram positive
Cocci in pairs (and chains) Cocci in clusters
Large bacilli Small bacilli
Branching bacilli Coryneform bacilli

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Gram negative
Diplococci
Bacilli
Bacilli, filamentous (or pleomorphic)
Gram variable:
coccobacilli

Budding yeast cells

Pseudohyphae

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

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MODIFICATION PRIMARY STAIN MORDANT DECOLORIZER COUNTER STAIN USES

Hucker’s Crystal violet Gram’s iodine 30 s Acetone-alcohol 1–5 Safranin General bacteriology
30 s s 30 s

Carbol fuchsin Crystal violet Gram’s iodine 30 s 95% ethanol 30 s Carbol fuchsin or Bacteroides spp.
30 s 0.8% basic fuchsin Fusobacterium spp.
>1 min Legionella spp.
Campylobacter spp.
Brucella spp. and
other faintly staining
gram-negative
organisms

Kopeloff’s Alkaline crystal Kopeloff’s iodine 3:7 acetone-alcohol: Kopeloff’s safranin Anaerobes Diagnosis
violet: flood with 2 min rinse immediately 10–30 s of bacterial vaginosis
solution A; add 5 after applying
drops of solution B
2–3 min

Jensen’s 0.5% Methyl violet Lugol’s iodine 95% alcohol 0.1% neutral red Gonococci
30s 30s 30s 2 min Meningococci

Preston & Morrell’s Ammonium oxalate- Lugol’s iodine Iodine-acetone Dil.Carbol fuschin Bacteriodes spp
crystal violet 30s 30s 30s Fusobacterium spp
30s

Weigert’s Carbol gentian violet Gram’s iodine Aniline-xyline Carmalum solution Tissue sections,
1min 1 min 2s Pneumocystis
jiroveci,Nocardia

Brown & Brenn 1% crystal violet Lugol’s iodine Acetone 0.25% basic fuschin Actinomyces
1 min 1 min 30s 3 mins

Quick Gram Crystal/methyl violet Iodine Acetone Basic fuschin

5s 5s 2s 5s
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 To differentiate bacteria into Gram positive & Gram negative which
yields for further identification of bacteria
 Empirical treatment with broadspectrm antibiotics can be started based
on the preliminay clue about the bacteria present.
 Early presumptive identification of bacteria.eg:Fastidious organisms
 For anaerobic organisms,which are detected in Gram stain anaerobic
culture can be done
 Identifies fungi like Candida,Cryptococcus
 Quality of specimen:sputum
 To find out the evidence of capsules/spores in bacteria

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 The Gram’s stain of bacteria, in addition to other
staining techniques, is one of the more important
determinations in the presumptive identification of
microorganisms. The morphology of the bacterial
cells, their arrangement, and their staining
characteristics are often distinctive enough to allow
a presumptive identification in a Gram-stained
smear.

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 Relatively slender, gram-positive bacilli
arranged in a “Chinese letter” pattern,
suggests one of the coryneform (diphtheroid)
bacteria.
40y/M, H/o cutaneous ulcer gradually
progressive

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 This direct Gram’s stain of a smear of
purulent sputum demonstrating grampositive
diplococci, characteristic of Streptococcus
pneumoniae.

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 Direct Gram’s stain of purulent exudates
illustrating gram-positive cocci arranged in
small clusters, characteristic of a
Staphylococcus species

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1. Overdecolorization may result in the identification of false
gram-negative results
2. Under decolorization may result in the identification of false
gram-positive results.
3. Smears that are too thick or viscous may retain too much
primary stain, Gram-negative organisms may not decolorize
properly.
4. Cultures older than 16 to 18 hours will be deteriorating and will
not retain the stain properly.
5. Stain may form precipitate with aging.
6. Gram stains from patients on antibiotics or antimicrobial
therapy may have altered Gram stain reactivity due to the
successful treatment.
7. Toxin-producing organisms such as Clostridia, staphylococci,
and streptococci may destroy white blood cells within a
purulent specimen.

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THANK YOU

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