2. INTRODUCTION
Stain: A substance which binds to cell and imparts colour to
the cell; used for biological purpose.
Staining: It is a procedure in which coloured chemicals
called dyes/stains are added to smears/films.
3. Why do we stain?
To visualize the microscopic and semitransparent objects e.g.
Bacteria, fungi, parasites, viruses
To reveal their morphology.
To study various internal eg. spores and external structures eg.
capsules.
To study classification and identification of microbes.
To study chemical nature of the cells.
4. Vital
TYPES OF STAINING
Supravital Others
Organisms are live
Bacteria in living
condition are studied
after imparting colour
e.g. Dilute methylene
blue, crystal violet
Organisms are
killed
Simple staining
Negative staining
Impregnation
staining
Differential
staining
Special staining
Progressive staining
Regressive staining
Specific staining
Metachromatic
staining
Bipolar staining
Beaded staining
7. SIMPLE/ MONOCHROME
STAINING
Simple stains: Watery solution of simple basic dye is used. e.g.
methylene blue, crystal violet, dilute carbol fuschin.
Reagents:
1) Loeffler’s methylene blue- Saturated solution of methylene
blue in alcohol- 300ml KOH, 0.01% in distilled water- 1000ml
2) Polychrome methylene blue- Prepared by allowing
Loeffler’s methylene blue to ripen
3) Dilute carbol fuschin
8. Mechanism:
Surface of bacterial cells is acidic. Ionization of carboxyl group gives negative charge
to cell surface.
This combines with positively charged ion of basic dye; leads to staining.
Observations:
Bacteria appear blue/ violet/ pink depending on stain.
Uses:
1) Loeffler’s methylene blue- To demonstrate morphology of , lymphocytes and
other cells.
2) Polychrome methylene blue- To stain capsule of Bacillus anthracis.
3) Dilute carbol fuschin - To demonstrate Borrelia spp.
9.
10. NEGATIVE/ INDIRECT
STAINING
Negative staining: Background stained; structure to be
demonstrated is not stained.
Reagents:
1) India Ink- Pelikan Drawin ink, 17 Black containing 0.3% tricresol
as preservative
2) Modification of India Ink using 2% mercurochrome for
demonstrating capsulated budding yeast cells of Cryptococcus
neoformans
3) Nigrosin- Nigrosin 10g, Warm distilled water 100ml, formalin
40% 0.5ml as a preservative
4) Eosin- 1% solution
11.
12. Observations:
India Ink- Bacteria appear as clear transparent objects;
Nigrosin- Bacteria appear as bright unstained objects.
Uses:
1) India ink or Nigrosin 10%- To demonstrate capsules of Streptococcus
pneumoniae, Klebsiella pneumoniae, Group B Streptococci,
Haemophilus influenzae and Cryptococcus neoformans;
Demonstration of Spirochaetes by India ink.
2) To demonstrate bacterial motility.
3) Eosin- To demonstrate parasitic elements in stool.
14. IMPREGNATION STAINING
Impregnation staining:
The structures too thin to be seen under ordinary microscope, can be visualized by
increasing their thickness by impregnating silver.
Reagents:
Ammoniated silver solutions
Silver nitrate- Most common
Silver diamine, silver carbonate
16. Levaditi’s Method
Used for staining of
Spirochaetes in
tissues.
Fontana’s Method
Reagents:
Fixatives- Acetic acid, Formalin 40%
Mordant- Phenol, Tannic acid
Ammoniated silver nitrate
Mechanism:
Impregnation of silver salt on the
surface of Spirochaetes increases their
thickness
Observations:
Spirochaetes appear brownish black.
18. GRAM
STAIN
Discovered by
Christian Gram in
1884:
subsequently
modified by Burke,
Kopeloff and
Beerman in 1922:
further by Jensen,
Weigert, Preston
and Morrell in
1962
19. GRAM STAINING
Differential staining: These differentiate two types of
organisms.
Observations:
a) Gram positive- Resist decolourisation and retain
primary stain; hence appear violet.
b) Gram negative- Decolourised by organic solvents
and take the counter stain; therefore appear red.
20.
21. Reagents for Gram Staining
Primary stain - Crystal violet, methyl violet or
gentian violet
Mordant - Gram’s or Lugol’s iodine
Decolouriser - Absolute alcohol, acetone or
ethanol-acetone
Counter stain- Safranine, neutral red
22.
23. MORDANT
A substance that increases binding between primary stain and cell cytoplasm.
It act as the paste. It forms “dye iodide protoplasm complex” in gram positive, but
no complex in gram negative.
It forms insoluble compound with stain and helps to fix the colour to the cell.
e.g. Iodine, Tannic acid, Salts of aluminium, Iron, Tin, Zinc, Phenol.
It provides greater stability to the cell and does not allow the stain to get
washed off.
24. DECOLORIZATION
As due to complex formation, it resist purple colour in
gram positive organisms.
As their is no complex in gram negative organism,
therefore colorless in gram negative.
25. SAFRANIN
As gram positive are already purple, therefore they do
not take safranin colour.
And in gram negative as they are colorless, therefore
they take pink/red colour of safranin.
26. WHAT IF ?
MORDANT NOT ADDED
ALL WILL BECOME PINK AS NO COMPLEX FORMATION
ACETONE NOT ADDED
AS NO COUNTERSTAIN, THEREFORE EVERYTHING BECOME
PURPLE
27. MECHANISM
CELL WALL PERMEABILITY THEORY
According to this theory, the cell wall of Gram positive bacteria contain
more peptidoglycan because of which it is thicker & stronger &
impermeable as compared to Gram-negative bacteria.
During staining with primary stain and mordant dye- iodine complex is
formed within the cell. Under the action of this decolourizing agent, this
complex diffuses freely out of the Gram-negative cell but not from the
Gram positive cell because the Gram positive cell wall is less permeable
due to its cell wall.
In Gram-negative bacteria peptidoglycan is less and therefore their cell
wall is relatively less strong and thin, hence dye-iodine complex diffuses
out of cell freely and they take up the colour of the counter stain.
28.
29. OTHER THEORIES RELATED
TO GRAM STAINING
1. Magnesium Ribonucleate Theory
2. Lipid content Theory
3. Acid Protoplasm Theory
4. Isoelectric Point Theory
30. USES OF GRAM STAINING
To differentiate between Gram positive and Gram negative bacteria
E.g.: Gram positive cocci : Staphylococcus, Pneumococcus
Gram positive bacilli : Clostridia, Corynebacteria ,
Bacillus spp
Gram negative bacilli : E. coli, Klebsiella pneumonia
Gram negative cocci : gonococci, meningococci
To start empirical therapy
To choose culture media for inoculation
31. ACID FAST STAINING
Paul Ehrlich (1882)- Acid fast property
Koch (1882) stained Mycobacterium
tuberculosis
Ziehl- Phenol as mordant
Neelson- Mordant + Primary stain
Paul Ehrlich
32. ZIEHL NEELSEN STAINING
(ZN STAINING)
Acid fastness depends on:
A. Mycolic acid
B. Integrity of cell wall
It differentiates acid-fast organisms from non acid-
fast organisms.
Observations:
a) Acid fast organisms- Resist decolourisation by
acid and alcohol. Retain fuchsin; appear bright
red.
b) Non acid fast organisms- Take the colour of
methylene blue.
33. REAGENTS FOR ZN STAINING
a) Primary stain- Carbol fuschin- Basic fuschin 10gm,
Absolute alcohol 95%, 5% Phenol
b) Decolouriser- Suphuric acid 25%- Sulphuric acid
250ml and distilled water 1000ml
c) Counter stain- Methylene blue, Malachite green, Picric
acid
36. Grading AS PER RNTCP
RESUL
T
GRADING NO. OF FIELDS
EXAMINED
>10 / oil immersion
field
+ve 3+ 20
1 – 10/oil
immersion field
+ve 2+ 50
10 – 99 / 100
oil immersion field
+ve 1+ 100
1 – 9 / 100
oil immersion field
+ve Scanty* 100
No AFB seen in
100 oil immersion
fields
-ve 1000
*Record actual no. of bacilli
seen in 100 fields.
37. ACID FAST ORGANISMS
Bacteria- Mycobacterium, Nocardia, Rhodococcus
Parasites- Oocyst of Cryptosporidium,
Cyclospora, Isospora; Eggs of Taenia saginata
Others- Spores, Spermatic head
38. Mechanism:
Mycolic acid in the cell wall of acid fast organisms do not
allow the stain to penetrate easily inside organisms.
Here, carbol fuschin is applied with heat. Heat allows the dye
to penetrate.
When decolourised by acid, the dye does not come out as it
is soluble in phenol and phenol is soluble in lipids; hence
no decolourisation.
40. FLUOROCHROME
STAINING FOR AFB
A fluoroscent dye Auramine O is substituted for the hot
carbol fuchsin
Method
- Auramine phenol (0.3%) for 10 min
- Acid alcohol (1%) for 5 min
- Potassium permanganate (0.1%) for 15 sec
Examine under fluorescent microscope
41. Advantages
- Can be examined under low magnification
- Shorter time
- Rapid screening
Disadvantages
- Need for Fluorescent microscope
42. ROMANOWSKY STAINING
Permanent staining method to demonstrate
haemoparasites.
It is a type of differential staining.
Romanowsky stain- Watery solutions of eosin +
zinc-free methylene blue in methyl alcohol.
43. MODIFICATIONS OF
ROMANOWSKY STAINING
i. Leishman’s stain
ii. Wright stain
iii. Giemsa stain
iv. Field’s stain
v. Jenner’s stain
vi. Jaswant Singh-Bhattacharya (JSB) stain
45. Leishman’s stain
Leishman’s
powder- 150mg
Methanol- 100ml
Observations:
-Cytoplasm of WBCs
& protozoal parasites
appear blue
-Nuclei, parabasal
body & flagella
appear red
-RBCs appear pink
Granules of
eosinophils appear
pink
Wright stain
Wright stain
powder-
0.3gm
Gycerol- 3ml
Absolute
methanol- 7ml
Observations:
Same as
Leishman’s stain
Stained thin blood smear using
Leishman’s stain
Unstained thin blood smear
46. Giemsa stain reagents:
Giemsa stain powder-
0.75gm
Gycerol- 25ml
Methanol- 75ml
Observations: Same as Leishman’s stain
1.Rapid method- To demonstrate blood parasites
2. Modified rapid method-Heating for fixation of smear instead of
methanol;
-To demonstrate spirochaetes
3. Slow method - Excellent stain for inclusion bodies of
small pox
GIEMSA STAIN
47. Giemsa stain
Plasmodium vivax in Giemsa-stained thin blood smear with all
developmental stages present in peripheral blood. (A) Growing amoeboid
trophozoite in enlarged red blood cell (RBC) with eosinophilic stippling
(schuffner's dots). (B) Immature schizonts with clumps of brown pigment
almost fill the enlarged RBCs. (C) Mature schizont with merozoites (about
14) and clumped pigment. (D) Macrogametocyte with diffuse brown
pigment and eccentric compact chromatin.
48. Jenner’s stain
Jenner’s stain powder-
5gm
Methanol-
1000ml
JSB stain
JSB A
Eosin -2.5gm
Distilled water- 100ml
JSB B
Methylene blue- 2.5gm
Distilled water- 100ml
Field’s stain
Solution A
Methylene blue 0.4gm
Buffered water- 250ml
Solution B
Buffered water
Distilled water- 1000ml
Solution C
Eosin- 0.5gm
Buffered water- 250ml
49. STAINING OF SPORES
Modified ZN
staining
Observations:
Spores appear
bright red
Bacterial body
appears blue
Schaeffer and
Fulton’s staining
5% aqueous solution of
malachite green
Counter staining with
0.55 safranine or
0.05% fuschin
basic
Observations:
Spores appear green
Bacterial body appears
red
Dorner’s method
3-4 drops of freshly filtered
Kinyoun’s carbol fuschin
Hold test tube in boiling water
for 5-10 min
Loopeful of sample from test
tube + a drop of 10% aqueous
solution of nigrosin
Observations:
Spores appear pink
Bacterial body appears
colourless
Background appears dark grey
50. A Schaeffer and Fulton stained preparation of Bacillus subtilis showing
endospores as green and the vegetative cell as red
51. STAINING OF SPORES
Modified Dorner’s
method
Apply carbol fuschin
steam for 5-7 min
Blow dry and cover with
thin film of nigrosin
Observations:
Spores appear pink
Bacterial body appears
colourless
Background appears dark
grey
Wirtz and Conklin
method
5% aqueous solution of
malachite green
And steaming for 3-6 min
Counter staining with
0.5% safranine
Observations:
Spores appear green
Bacterial body appears
pink
Abbott stain
Apply saturated alcoholic
solution of methylene blue
Heat till stain boils
Cover the film with
saturated alcoholic
solution of eosin Y and
nine parts of distilled water
for 5-10 sec
Observations:
Spores appear blue
Bacterial body appears
pink
52. STAINING FOR VOLUTIN
CONTAINING GRANULES
Best seen in young culture on enriched media
Granules are metachromatic
Neisser’s, Albert’s or Ponder’s method
Albert-Laybourn method is commonly used
- Sol A (Toluidine blue,Malachite green)
- Sol B (Albert’s iodine)
Observations:
-Granules stain bluish black
- Bcterial body appears green.
54. STAINS FOR FUNGI
Lactophenol Cotton Blue
Calcofluor White- Binds to cellulose and chitin
Can be subsequently stained by Periodoc acid schiff
(PAS) or Gomor i’s Methenamine stain (GMS)
Rapid detection in wet mount (KOH ),smears and tissue
sections.
Fluorescent -Antibody staining:
This stain is important in detection of fungi or their
antigens in tissues, pus, blood or other specimens in
various fungal infections where organisms are scanty in
number.
55. PERIODIC ACID SCHIFF
The polysaacharides of fungi and bacteria are oxidized
by periodic acid to yield large number of aldehyde groups
because of which they stain intense red or magenta in
colour with Schiff’s reagent.
Glucan and mannan in fungal cell wall are oxidized to
aldehydes.
57. To stain fungi , specially used for Pneumocystis jirovecii
The cell walls are stained brown to black.
GOMORIMETHAMINE SILVER
STAIN
Pneumocystis jiroveci
58. STAINING PROCEDURES IN
PARASITOLOGY
Temporary Preparations Permanent Preparations
Wet Mounts 1) Modified Ziehl-Neelsen
Technique
2) Hot Modified Acid-fast
1) Saline preparation Staining Method
2) Iodine Preparation 3) Kinyoun’s Cold Stain
3) Lactophenol cotton blue (LPCB) mount Method
4) Wet mount using capillary blood 4) Safranine-methylene Blue
(detection of microfilaria and trypanosoma) Technique
5) Romanowsky’s Stain
6) Hematoxylin Stain
59. Cyst of Entamoeba histolytica on wet mount
Lefts side: cyst stained with iodine
Right side: unstained preparation.
[ Arrow - notice the chromatid body with blunt, rounded ends]
60. WET MOUNTS
Saline Preparation:
Observations:
-It is an unstained
preparation: Parasitic
elements are not
stained and they
appear colourless.
- As parasitic elements
are live in this
preparation: Live
motile trophozoites
can be seen
- Helminthic eggs
-Helminthic larvae
Iodine Preparation:
Observations:
-Cytoplasm of the
cysts appear yellow or
light brown
-Nuclei appear dark
brown
-The peripheral
chromatid bodies
appear light yellow
-Glycogen mass in
young cysts appears
dark brown with
iodine.
LPCB:
Observations:
-Cytoplasm
appears deep
blue in colour.
-Nucleus /
nuclei appear
blue in colour
- Protozoal
cysts and
helminthic eggs
are stained deep
blue
61. Modified Kinyoun’s acid fast – stained smear demonstrating oocyst of
Isospora belli with a sporont (A), an Isospora belli oocyst with two sporoblasts
and a Cyclospora cayetanensis oocyst with a sporont.
62. USES OF WET MOUNTS
Saline Preparation:
To demonstrate:
1) Motile trophozite
stage of protozoan
parasites
2)Helminthic eggs
3)Helminthic larvae
4)Cysts of
protozoa
Iodine Preparation:
To demonstrate:
1) To stain glycogen and
nuclei of cysts specifically
used for demonstration of
cystic stage
2) Trophoite stage
3) Eggs and larvae can also
be demonstrated but as
iodine stains non-bile
stained eggs also, hence
less important as far as
differentiation of bile
stained and non-bile
stained eggs are
concerned
LPCB:
To demonstrate:
1) The internal
structures of
trophozoites, cysts,
and ova, thus
facilitating their
recognition and
identification in the
stool specimens
2) Intestinal coccidian
sparasites such as
Cyclospora and
Isospora in stool
specimens
63. PERMANENT PREPARATIONS
• Permanently stained preparations are occasionally required for the
following purposes:
• 1)For identification of:
- oocysts of cryptosporidium
- protozoan trophozoites
- protozoan cysts
2) Keeping a permanent cord
3) For sending to a reference laboratory for an expert opinion.
64. Stain for viruses
Seller stain:
To differentiate rabies inclusions from other intracellular inclusions.
Negri bodies appear magenta in colour and have small (0.2 µm to
0.5 µm), dark-blue interior basophilic granules.
65. Giemsa stain:
To demonstrate Tzanck cells in the infections caused by herpes simplex virus.
To demonstrate multinucleate giant cells in the infections caused by herpes-
zoster virus
66. IMMUNOFLUORESCENCE
STAINING
Fluorescence is the property of absorbing the light rays of one particular
wave length and emitting the light rays of different wave length.
Fluorescent dyes can absorb ultraviolet(UV) light and can convert or
reflect back them into visible light.
Fluorescent dyes such as flourescein isothiocyanate (FITC), tetramethyl
rhodamine, dansyl and phycoerythrincan be used to detect infected cells in
exudates, secretions and tissues.
FITC is he most commonl used. It emits a greenish / yellowish light.
Rhodamine emits red / orange light and dansyl emits yellow light.
67. Commonly used for the detection of different bacteria,
viruses, fungi and parasites in tissues, blood, CSF, urine,
faeces and other body fluids.
Also used for the detection of other antigens(Ags) such as tumour
Ags, organ and tissue Ags, blood cells, enzymes, hormones, etc.
It is most commonly used in the diagnosis of viral infections such
as rabies, measles, mumps, etc.
USES OF
IMMUNOFLUORESCENCE
STAINING
68. Photomicrograph of a histological section of human skin prepared for direct
immunofluorescence using an anti-IgA antibody. The skin is from a patient
with Henoch–Schönlein purpura. IgA deposits are found in the walls of small
superficial capillaries (yellow arrows). The pale wavy green area on top is
the epidermis, the bottom fibrous area is the dermis.
71. Modification Particulars Use
Burke’s Decolouriser- Acetone/ Acetone-
ether mixture
Kopeloff and Beermann’s Primary stain- Methyl violet
Decolouriser- Acetone
Counter stain- Basic fuschin
For general use
Jensen’s Primary stain- Methyl violet
Counter stain- Neutral red
For gonococci and
meningococci
Weigert’s Primary stain- Carbol gentian violet
Decolouriser- Aniline xylol
For tissue sections
Preston and Morrell’s Primary stain- Crystal violet/
Ammonium oxalate
Mordant- Lugol’s iodine
Decolouriser- Iodine-acetone
Counter stain- Dilute carbol fuschin
For routine staining
Hucker’s Primary stain- Methyl violet/ Crystal
violet
Decolouriser- Acetone
Counter stain- Safranine/ Neutral red
Brown and Brenn Primary stain- Methyl violet/ Crystal
violet
Decolouriser- Acetone and 95%
ethanol
Counter stain- Safranine/ Dilute carbol
fuschin
72. Technical Report
Published: 09 July 2020
Detection of SARS-CoV-2 in formalin-fixed paraffin-embedded tissue
sections using commercially available reagents
Alejandro Best Rocha, Edana Stroberg, Lisa M. Barton, Eric J. Duval,
Sanjay Mukhopadhyay, Nicole Yarid, Tiffany Caza, Jon D. Wilson,
Daniel J. Kenan,
Michael Kuperman,
Shree G. Sharma &
Christopher P. Larsen
Laboratory Investigation volume 100, pages1485–1489(2020)Cite this article
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73.
74. BRUCELLA DIFFERENTIAL
STAIN
Reagents:
a) Primary stain- 1:10 diluted carbol fuschin
Heating is not required.
a) Decolouriser- Acetic acid 0.5%
b) Counter stain- Methylene blue
Uses:
To demonstrate Brucella abortus in infected tissues
or exudates.
To demonstrate Coxiella burnetii and Chlamydia in
tissue sections.
76. Cover the whole slide with carbol fuchsin and heat gently from below until steam rises
Allow it to act for 5-10 minutes with intermittent heating (Heat and phenol from carbol fuchsin help the
primary stain to penetrate through the cell wall)
Wash slide with tap water
Use of 25% H2SO4 for 2-4 minutes as a decolourizer for M. tuberculosis is recommended by RNTCP
Wash slide with tap water
Counter stain with methylene blue or malachite green for
1-2 minutes
Wash slide with tap water
Air dry the slide before observing under oil immersion objective
Observe under 100x after putting a drop of cedarwood oil
78. Examples of endospore stain positive organisms
Clostridium perfringens
C. tetani
C. botulinum
Bacillus cereus
Bacillus anthracis
79. Most kits use Fluorescein isothiocyanate (FITC) or
Tetramethyl rhodamine isothioyanate as a dye
Ethidium bromide (fluorescent indicator dye) in PCR
SYBR dye binds to dsDNA and increases the fluorescence
In Micro-array technology Cyanine 3 and Cyanine 5
