Gram stain

1. GRAM STAIN
DR. YOGITA

2. TOPICS
 STAINS
 GRAM STAIN
 MECHANISM OF GRAM STAIN
 PREPARATION OF STAINS
 MODIFICATION OF STAINS
 PROCEDURE OF SMEAR PREPARATION &
GRAM STAINING
 COMMON ERROR WHILE STAINING
 LIMITATION OF GRAM STAIN
:

3. STAINS
 Stains are natural or synthetic substances that
render the structure of cell visible.
 Live bacteria do not show much structural
details under the light microscope due to lack
of contrast.
 So it is customary to use staining technique
to produce colour contrast.

4. IMPORTANCE OF STAIN:
 The microscopical examination of stained
smear enables the morphology, relative
sizing and arrangement of microorganism to
be seen clearly.
 It also assist in the detection of cells,
esp.pus cells, PMN cells.
 Bacteria can also be differentiated by their
staining reaction. e.g. gram positive from gram
negative organisms.

5. TYPES OF STAINS:
 Simple staining Eg: methylene blue, basic
fuchsin
 Negative eg: india ink,nigrosin
 Metachromatic eg: alberts, ponders
 Differential eg:grams, acid fast
 Impregnation eg: silver impregnation
 Fluorescent stain eg:acridine orange,
rhodamine auramine, calcoflour white

6. GRAM STAIN
 This is the most important staining method in
bacteriology.
 It was originally devised by the histologist
Christian Gram in 1884 as a method of
staining bacteria in tissues.

7.  The Gram staining requires the application of
four reagents in the following order:
 PRIMARY STAINING with basic
pararosaniline dye, such as crystal violet,
methyl violet or gentian violet..
 Application of a dilute solution of IODINE.
 DECOLOURISATION with an organic solvent
such as ethanol ,acetone or aniline.
 COUNTERSTAINING with a dye of
contrasting colour, such as carbol fuchsin
,safranine or neutral red.

8.  The violet dye and iodine combine to form an
insoluble , dark purple compound in the
bacterial protoplasm and cell wall.
 This dye – iodine complex is dissociable in the
decolourizing agent, which dissolves and
removes this two components from the cell.
 Bacteria which retain this complex stain as dark
purple and bacteria which are decolorized take
the counter stain and appear pink.
 Bacteria which stain dark purple are called
Gram positive and which stain pink are called
Gram negative.

9. The exact mechanism of Gram reaction
is not understood. Various theories
that have been suggested are as
follows:
 Gram positive cells have a more acidic protoplasm,
which may account for their retaining the basic primary
dye more strongly than Gram negative bacteria.
 The peptidoglycan of Gram positive bacterial cell wall
is thicker and denser than Gram negative thus able to
retain dye iodine complex.
 The high lipid content of Gram negative bacteria
makes them permeable to secondary dye after

10. GRAM POSITIVE
COCCI

11. Gram negative bacilli

12. Gram positive cocci in pair and
chain

13. PREPARATION OF STAIN
crystal violet:
 crystal violet 10 gm
 100% Ethanol(absolute alcohol) 100ml
 distilled water 1000 ml
 Crystal violet is used at concentration of 0.5-2%
solution.
 This solution keeps well and is perfectly satisfactory
for general use.
 However , Gram positive staining can be strengthened
by addition of sodium bicarbonate or ammonium

14. Kopeloff & Beerman STAIN:
 -Solution A - Methyl violet 10g
 -Distilled water 1 L
 Solution B -Sodium bicarbonate 50g
 -Distilled water 1 L
- Shortly before use, mix 30 volumes of sol. A
to 8 volumes of sol.B
 This mixture precipitate within few days so
can't store for long time.

15.  Preston & Morrell’s stain:
 Crystal violet 20g
 Methylated spirit 200ml
 Ammonium oxalate, 1% in water 800ml

16.  IODINE SOLUTION:
 Gram’s (lugol’s iodine)solution:
iodine crystals 10gm
potassium iodide 20gm
distilled water 1000ml
 Koepleoff & Beerman’s iodine
- Iodine 200gm
- Sod.Hydroxide 1mol/L 100ml
- Distilled water 900ml

17. DECOLORIZERS
Fastest- acetone(2-3 sec)
Useful when only single slide is to be
stained. However, short period of exposure is
difficult to control when many slide has to be
stain.
Slowest-ethanol,95%(1 min or more)
This act more slowly. So it should be
applied and reapplied for about 1 min.
Intermediate-acetone-alcohol(10 sec.)
ethanol,95% 100ml
Acetone 100ml
Combine in brown colored bottle

18.  Preston & Morrell have shown that addition of a
small concentration of iodine to acetone
slows the rate of decolourazation without
reducing its specificity.
 The decolorizer made are as follows:
 LIQUOR IODI FORTIS
 Iodine 10gm
 Methylated spirit 6gm
 Distilled water 10ml
 IODINE-ACETONE
 Liquor iodi fortis 35ml
 Acetone 965ml

19. COUNTER STAINS
Dilute carbol fuchsin:
 Strongest red staining within 10-30 sec.
 Colouration is so dark that sometime difficult to
distinguish Gram negative from Gram positive
bacteria.
Basic fuchsin :
 Applied for 10-30 sec.
 This is recommended for general use.
 Basic fuchsin 0.5 gm
 Distilled water 1000ml
Safranine:
 Used as 0.5% in distilled water.

20. MODIFICATION OF GRAM’S STAIN
 KOPELOFF AND BEERMAN'S
MODIFICATION:
 Primary stain solution consists of freshly
constituted methyl violet with sodium
bicarbonate in distilled water.
 Mordant consists of iodine dissolved in 4%
NaOH solution.
 Decolonization is either using acetone alone or
a mixture of acetone and ethanol.
 Basic fuchsin is used to counter stain the
smear.
 This method may be modified to stain tissue

21. KOPELOFF & BEERMAN’S STAIN FOR SECTION:
 It is same as smear stain method of kopeloff &
Beerman’s with addition steps as follow:
 After counterstaining ,dehydrate quickly the
slide by flooding with 95% alcohol followed by
absolute alcohol only for brief period of time.
 End this contact by immersing the slide in
xylene.

22. JENSEN'S MODIFICATION
 This method involves use of methyl violet as
primary stain, iodine and potassium iodide in
water as mordant, absolute alcohol as
decolorizer and neutral red as counter stain.
 For Neisseria spp

23. PRESTON AND MORRELL'S MODIFICATION
 The primary stain used in this modification is
ammonium oxalate-crystal violet (30 sec).The
smear is washed in Lugol's iodine and further
treated with iodine solution(30 sec).
 The smear is decolorized using iodine-acetone
decolorizer (30 sec)and counterstained using dilute
carbol fuchsin solution(30 sec).
 This method has been further modified to overcome
the irritating iodine in aerosols by reducing the iodine
concentration to one-tenth and shortening the
duration of decolonization to ten seconds.

24. PREPARATION OF SMEAR AND
GRAM STAIN PROCEDURE
 PREPARATION OF SMEAR:
 Smear should be prepare evenly covering an area
of about 15-20mm diameter on the slide.
 From purulent specimen:
 Take loopful specimen with sterile inoculating wire
 From nonpurulent specimen:
 Centrifuge and then make smear
 From sputum:
 Take purulent or caseous material

25.  From swab:
 Roll the swab on slide
 From culture plate:
 Put a drop of saline on a clean slide .with the
help of sterile inoculating wire ,individual
colony is picked up and mix with saline.
 LABELLING OF SLIDE:
 With diamond or grease pencil. Stick on paper
should not be used.

26.  FIXATION OF SMEAR:
 It is to preserve the smear being washed of from slide
while staining.
 Heat fixation:
 After air drying of smear is properly done,pass the slide
rapidly three times through blue flame of Bunsen
burner.
 Disadvantage: Excessive heating damage the
organism and alter the staining reaction.
 It also damage leucocytes. So not suitable for
intracellular organism like N.gonorrheria &
N.meningitidies
 Alcohol fixation:

27.  OTHER CHEMICAL FIXATIVES:
 Potassium permanganate(anthrax bacilli)
 Formaldehyde vapor (mycobacterial species)

28.  PROCEDURE:
 Lay the fixed slide on a staining rack. Cover the smear
completely with gential violet. keep it for 1 min.
 Wash under running tap water & flood the slide with
Gram’s iodine solution and wait for 1 min.
 Gently drain off the iodine solution and rinse with
running tape water.
 Decolorize rapidly (few seconds) with acetone/alcohol .
 Wash the slide under running tap water .
 Pour safranin solution on the slide and keep it for 30
seconds.
 Wash off the stain with tap water.
 Wipe the back of the slide clean. Place in a draining rack
for the smear to air dry.
 Examine the smear microscopically with oil immersion
objective.

29.  In Gram staining gentian violet, Gram’s
iodine,saffranin are kept for 1 min and
discoloration is rapid within few seconds.
 Rapid Gram stain refers to quickened
technique where the smear is exposed to
only 30 seconds instead of one minute. Used
when only single slide has to be stain.

32. LIMITATIONS OF GRAM STAINING:
 Some Gram-positive bacteria may lose the
stain easily and therefore appear as a mixture
of Gram-positive and Gram-negative bacteria
(Gram-variable).
 When over-decolorized, even Gram
positive bacteria may appear pink and when
under-decolorized gram negative bacteria may
appear Gram positive.
 Small and slender bacteria such as
Treponema, Chlamydia, Rickettsia are often
difficult to stain by Gram's method

33.  Gram positive cells affected by cell wall
active agents such as lysozyme or antibiotics
may become Gram negative.
 Bacteria totally devoid of cell wall
(Mycoplasma) are always Gram negative

34. THANK
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