STAINING

By: Dr. Amit Kumar Patel

JRA-1
Microbiology Department
RIMS, Ranchi
 STAINING TECHNIQUES IN
MICROBIOLOGY
• Structural details of bacteria cannot be seen
under a light microscope due to lack of
contrast. So use of staining methods to
produce colour contrast and increase there
visibility.
• Before staining, the smears are fixed so that
they will not be displaced during the staining
process.
• Fixation also protects the internal structures
of cells in a fixed position.
 .

• Smears are fixed by two methods:-

a) Heat fixation:- it is done by gently flame heating
an air-dried film, used for bacterial smears.
b) Methanol fixation:- used for blood smears.
 TYPES OF STAINING :-
 SIMPLE STAIN: - Provide colour contrast but impart the same
colour of all bacteria. Stains are:-
Methlene blue
Basic fuchsin
 NEGATIVE STAINING:- This provide a dark-coloured background
against which the unstained bacteria stand out in contrast.
• Useful in the demonstration of :-
Bacterial capsule
spirochetes, cryptococcus.
• Commonly used stains are:-
India ink
Nigrosin
 Types of staining continue...
 Impregnation methods:- cells and structures are
too thin to be seen under the ordinary
microscope, may be rendered visible by the
impregnation of the surface with silver to
increase its thickness.
• Use to demonstrate :-
Spirochetes and
Bacterial flagella
 Levaditi’s stain for spirochetes in tissues.
 Differential stain
 Different stains are used which impart different
colours to different bacteria or bacterial
structures, which help differentiating bacteria.
 The most commonly employed differential stains
are :-
a) Gram stain :- It differentiates bacteria into gram-
positive and gram-negative group.
b) Acid-fast stain:- It differentiates bacteria into
acid-fast and non acid-fast groups.
Examples:- tubercle bacilli, lepra bacilli.
 Differential stain continue....
C) Albert stain :- it differentiates bacteria having
metachromatic granules from other bacteria
that do not have metachromatic granules.
Bacteria having metachromatic granules:-
C.diphtheriae
Mycobacteria
Gardenella vaginalis
Spirilium volutants
 GRAM STAIN
Introduction
Gram stain was introduced by Hans- Christian Gram in 1884 .
 This is most frequently used differential stain that divides bacteria into
two major groups:
(1) Gram-positive , and (2) Gram-negative.

 Gram staining reaction depends on whether or not organism resists

decolourization with acetone or alcohol after staining with

pararosaniline dye and further treatment with mordent (Gram’s

iodine).
Introduction continue...
• Bacteria which resist decolourization and remain
dark purple after counterstaining are called
Gram-positive bacteria .
• Bacteria which get decolourized and take the
light pink colour after counterstaining with
saffranin, neutral red or dilute carbol fuchsin are
called Gram-negative bacteria.
 REQUIREMENT
a) Slide
b) Sprit lamp
c) Nichrome loop
d) Glass marker
e) Distilled water
f) Gauge piece
g) Light microscope
 REAGENT
a) Crystal violet(2%) or methyl violet(0.5%) or
gentian violet(1%).
b) gram’s iodine:-
-Iodine crystal 1g
-Potassium iodide 2g
-Distilled water 100ml
 Dissolve 2g potassium iodide in 25ml water, and
then add 1g iodine. When iodine is dissolved,
make up to 100ml with water.
 Reagent
C)Acetone or alcohol.
D) saffranin(0.1%) or dilute carbol fuchsin (1:10).
 Principle
• Exact mechanism of Gram reaction is not known.
• Various theories have been suggested:-
A. pH theory:- cytoplasm of gram-positive
bacteria is more acidic, hence, can retain the
basic dye(e.g. Crystal violet) .
Iodine serves as mordant, so that it combines
with the primary stain to form a dye-iodine
complex which gets retained inside the cell.
B. Cell theory:- this is the most important postulate
to describe the mechanism of
 Principle contd....
gram stain.
• The violet basic dye and the iodine forms a dye-
iodine complex inside both Gram- positive and
Gram- negative bacteria, but during
decolourization (like acetone), cell membrane are
dissolved (outer membrane of cell wall and
cytoplasmic membrane).
• Dye-iodine complex is retained in Gram-positive
cells by thick peptidoglycan mesh, whereas, it is
readily washed out through the very thin
 .

peptidoglycan layer remaining in Gram-negative

cells after both membranes have been dissolved.
 Procedure
Smear preparation
1. Take a grease-free lean slide and make an oval-
shaped mark at the centre by using a glass
marker.
2. Transfer a loopfull of liquid culture with a sterile
nichrome loop and make a smear in the
premarked area on the slide .(for solid culture,
take a loopful of saline on the slide and emulsify
the organisms in an area of 1 cm2).
3. Allow the smear to air dry.
 Smear pre... Contd...
4) Fix the smear by passing the slide 3 to 4 times
over the flame quickly with smear side facing-
up.
The smear is said to be fixed when it is just
tolerable on the dorsum of hand.
Over heating chars the smear.
 staining
step 1 :- (primary stain) : place the slide on glass
rods.
 The smear is stained with pararosaniline dye
suh as crystal violet(or gentian violet or methyl
violet) for ONE MINUTE . Then the slide is rinsed
with water .
 crystal violet stain all the bacteria violet in
colour (irrespective of whether are gram-positive
or gram-negative).
 Staining contd...
Step 2 :-( Mordant): Gram’s iodine (dilute solution
of iodine) is poured over the slide for 1 minute.
Then the slide is rinsed with water.
Gram’s iodine act as a mordant , binds to the dye
to form bigger dye-iodine complexes in the
cytoplasm.
Step 3 :-(Decolourization): pouring of few drops
of decolourizer to the smear, e.g. Acetone (for 1-2
sec) or ethyl alcohol (20-30 sec) or acetone
alcohol (for 10 sec). Slide is immediately rinsed
with water.
 .

 Decolorizer removes the primary stain from

gram-negative bacteria while the gram-positive
bacteria retain the primary stain.
 Step 4 :-(Counter stain):-secondary stains such
as saffranin or dilute carbol fuchsin is added for
30 seconds.
 the slide is rinsed in tap water, dried and then
examined under oil immersion objective.
.
 Interpretation of gram stain
Gram-positive bacteria resist decolorization and
retain the colour of primary stain i.e. Violet.
Gram-negative bacteria are decolorized and,
therefore, take counterstain and appear pink.
.
 Uses of Gram staining
1) It helps to differentiate bacteria into two groups,:- gram-
positive and gram-negative.
2) Differentiation is helpful in determining the use of
subsequent culture media and biochemical tests.
3) On the basis of shape, we can differentiate between cocci
and bacilli.
4) It helps in starting antibacterial treatment e.g. Cell wall
acting antibiotics in case of Gram positive cocci and broad
spectrum antibiotics in case of Gram negative bacilli.
5) It helps in identification of organisms on the basis of
arrangement,e.g., Gram-positive cocci in chains (strepto
cocci), and Gram-positive bacilli with chinese letter
arrangement( corynebacterium diphtheriae).
 Uses contd...
6) Presence of spore (unstained area) and its
position can be determined.
7) It helps to identify host cells. Presence of host
cells such as squamous epithelial cells in
respiratory specimen indicates contamination
with organisms and cells from the mouth.
8) Presence of inflammatory cells (e.g.phagocytes) is
key indicator of an infectious process.
 Uses contd...
9) It can identify non-bacterial form such as
Trichomonas, Strongyloides larvae and
Pneumocystis jiroveccii cysts, it is not sensitive to
Gram stain so other special stains used for their
visualization.
10) It helps in starting antimicrobial treatment.
 Modification of gram staining
1) Kopeloff and Beerman’s modification:-
primary stain:- methyl violet
Decolouriser/Counterstain:- acetone/ acetone
alcohol.
use:- useful for baterial vaginosis.
2) Jensen’s modifiation:-
primary stain:- methyle violet
Deolouriser/ Counterstain:- Absolute alcohol/
neutral red
use:-better counterstain for gonococci and
meningococci.
 Modification contd...
3) Preston and Morrell’s modification:-
primary stain:-crystal violet
counter stain:- iodine acetone
use:- strengthens gram-positive staining
4) Weigert’s modification :-
primary stain:- Carbol gentian violet
counter stain:- Aniline xylol
use :-for tissue section
 Drawback of Gram stain
• Gram-positive organism appear as Gram-negative
when :
 Over decolourization of smear.
 Smear prepared from old cultures.
 Use of iodine solution which is too
old(yellow instead of brown).
 Cell wall damage due to excessive heat
fixation of smear, antibiotic therapy or action of
autolytic enzyme).
 .

Medically important bacteria that can not be

seen by Gram staining:-
1)mycobacterium tuberculosis: -
Reason: Too much lipid in cell wall so dye
can not penetrate.
Alternative microscopic approach: acid- fast
stain.
2) Treponema pallidum: -
Reason: Too thin to see
other microscopic approach: Dark-field
microscopy,silver impregnation method or
fluorescent antibody staining.
 .

3) Mycoplasma pneumoniae:-
Reason: No cell wall ; very small
stain use: Giemsa
5) Legionella pneumophila:-
Reason: poor uptake of red counter stain
microscopic approach: prolong time of
counter stain.
6) Chlamydia trachomatis and Reckettsiae:-
Reason: Intracellular; very small
Stain use: Giemsa
 Application
Gram staining is essential for the identification of
bacteria based on their:
 Growth requirement
 Susceptibility to antibiotics
 Pathogenicity
 Acid-fast Stain
INRTODUCTION:-
• Acid-fast stain was discovered by Paul Ehrlich and
subsequently modified by Ziehl and Neelsen.
• This staining is done to identify acid-fast
organism , such as Mycobacterium tuberculosis,
Mycobacterium leprae etc.
 Acid-fastness is due to presence of mycolic acid in
the cell wall.
 .

• Lipids,mycolic acid,a high molecular weight

hydroxy acid wax containing carboxyl group, it is
acid-fast even in free state. Because of these
lipid ordinary aniline dye solutions do not
penetrate the cell wall.
• The staining solution used to stain acid-fast
bacilli is prepared by using a strong mordant dye
like basic fuchsin with phenol.
• For penetration of the dye into the bacillus,
heating of the stain is necessary.
 Principle
• Basic fuchsin in combination with phenol
penetrates the cell wall and stains the bacilli
bright red. Once stained, they resist
decolourization with strong acid.

• The basic dye in combination with mineral acid

forms a yellowish- brown compound which easily
comes out of the non-acid-fast bacilli after
decolourization but not comes out of the acid-
fast bacilli.
 .

• Counterstaining with Loeffler’s methylene blue is

done to form a contrast to red-coloured acid-fast
bacilli.
• Acid-fastness is not a property of lipids alone; it
also depends on the integrity of the cell wall.
 Reagents
a) Ziehl-Nelsen carbol fuchsin
-Basic fuchsin (powder) :- 5 gm
- Phenol (crystalline) :- 25 gm
- Absolute alcohol (ethanol):- 50 ml
-Distilled water :- 500 ml
b) Sulphuric acid :- 20%
c) Loeffler’s methylene blue:- 0.5%
 Requirement
• Slides
• Spirit lamp
• Nichrome loop
• Glass marker
• Distilled water
• Cedar-wood oil
• Gauge piece
• Light microscope
 Zieh-Neelsen Technique(Hot Method)
Smear preparation:-
1) Take a new ,unscratched slide and make an oval-
shaped mark at centre by using a glass marker.
2) Make a smear from blood tinged or yellow
purulent portion of the sputum using a stick in
premarked area.
3) The smear should neither be too thick nor too
thin. When placed over a printed matter ,the
print should be readable through smear.
 Smear pre...
4) Smear preparation should be done near a flame,
as inches around the flame is considered sterile
zone (as heat coagulates the aerosols raised
during the smear preparation).
Heat Fixation:
The smear is air dried for 15 - 30 minutes and
then heat fixed by passing over the flame 3- 5
times for 3- 4 second each time .
Coagulation of the proteinaceous material in the
sputum will facilitate fixing of the smear.
 Procedure
• Step 1 (primary stain) :-
 Smear is poured with strong carbol fuchsin(1%)
for 5 minutes.
 Intermittent heating is done by flaming the
underneath of the slide until the vapour rises.
 Heating helps in better penetration of the stain.
Rinse the slide with tap water, until all free carbol
fuchsin stain is washed away. So that the smear
on the slide looks red in colour.
 .

• Step 2 (Decolorization) :-
It is done by pouring 25% sulfuric acid over the
slide and allowing it to stand for 2- 4 minutes.
The slide is gently rinsed with tap water and tilted
to drain off the water.
A properly decolorized slide appears light pink.
- If the slide is still red, sulphuric acid is reapplied
for 1- 3 minutes and then rinsed gently with tap
water.
- The back of the slide is wiped clean with a swab
dipped in sulphuric acid.
 .

• Step 3 (counter staining) :-

0.1% methylene blue is poured onto the slide and
left for 30 seconds. Then the slide is rinsed gently
with tap water and allowed to dry.
The slide is examined under the microscope using
(40 X) lens to select a suitable area and then
examined under oil immersion field (100 X ).
 Note:- contaminated materials/ slide should be
discarded in a jar containing 5% phenol.
 Interpretation
• Mycobacterium tuberculosis appears as long
slender , straight or slightly cured and beaded ,
red coloured acid-fast bacillus.
- other non-acid fast organisms present in the
smear and the background take up the counter
stain and appear blue.
Acid –fast staining shows acid-fast baccilli
.
 Modification of Acid-fast staining
a) Modified ZN staining with Gabbet’s methylene
blue stain:- The smear is flooded with basic
fuchsin-phenol stain and allowed to stand at
room temperature for 10 minutes. Then washed
in tap water and counterstained with Gabbet’s
methylene blue for 2 minutes.
- then washed in tap water and air-dried.
b) Acid-fast stain using 5% sulfuric acid:-
Mycobacterium leprae resists decolorisation
with 5% sulphuric acid.
 .

C)Kinyoun’s method (cold method):- Nocardia and

Legionella resist decolourisation by 1% cold
sulphuric acid, which is used as a decolouriser.
- In this method intermittent heating not
required.
 Uses
1) Useful in detection of acid-fast organism.
2) To know the drug resistance. If organism fail to
decrease after therapy in the smear, the
possibility of drug resistance must be
considered.
3) Number of bacilli in the smear may be counted
and correlated with infectiousness of the
person.
Ziehl-Neelsen smear grading as per
RNTCP recommendations:-
 Concentration of sulphuric acid used for
decolourization of various acid-fast organism:-

Microorganism % concentration of
sulphuric acid
1) M.tuberculosis - 20%
2) Nontubercuious
mycobacterium - 20%
3) M.leprae - 5%
4) Cryptosporidium - 1- 5 %
5) Bacterial spore - 0.25- 0.5%
To prevent false positive sputum result:-
• Ask the patient to rinse the mouth with water
before collecting sputum sample.
• There should not be any food particle or fibre in
the sputum sample.
• Use only new, unscratched slide.
• Always use filtered carbol fuchsin.
• Never touch the oil-immersion applicator to the
slide.
 To prevent false negative sputum result:-
• Make sure that sample contains sputum and not
just saliva.
• There should be enough sputum at least 2 ml.
• Take blood-tinged or purulent portions to make
the smear.
• Fix the smear with correct length of time.
• Stain with carbol fuchsin for 5- 7 minutes.
• Do not decolourize with sulphuric acid too
intensively.
Bacterial index (BI) for Leprae baccilli :-
• Define as total number of acid-fast bacilli in oil-
immersion field.
• It is expressed from 1+ to 6+ by Ridley’s scale.
.