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Common Staining Technique

Microbiology

2
Notes

COMMON STAINING
TECHNIQUE

2.1 INTRODUCTION
Staining is technique used in microscopy to enhance contrast in the microscopic
image. Stains and dyes are frequently used in biological tissues for viewing,
often with the aid of different microscopes. Stains may be used to define and
examine bulk tissues (highlighting, for example, muscle fibers or connective
tissue), cell populations (classifying different blood cells, for instance), or
organelles within individual cells.
Bacteria have nearly the same refractive index as water, therefore, when they
are observed under a microscope they are opaque or nearly invisible to the naked
eye. Different types of staining methods are used to make the cells and their
internal structures more visible under the light microscope.
Microscopes are of little use unless the specimens for viewing are prepared
properly. Microorganisms must be fixed & stained to increase visibility,
accentuate specific morphological features, and preserve them for future use

OBJECTIVES
After reading this lesson, you will be able to:

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describe the need for staining techniques

explain terms related to staining techniques

discuss the substances used as stain

enlist various staining techniques

classify & explain various stains

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2.2 TERMS RELATED TO STAINING

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Stain
A stain is a substance that adheres to a cell, giving the cell color. The presence
of color gives the cells significant contrast so they are much more visible.
Different stains have different affinities for different organisms, or different parts
of organisms. They are used to differentiate different types of organisms or to
view specific parts of organisms

Notes

Staining
Staining is an auxiliary technique used in microscopy to enhance contrast in the
microscopic image. Stains and dyes are frequently used in biology and medicine
to highlight structures in biological tissues for viewing, often with the aid of
different microscopes.
Fixation
Fixation by itself consists of several stepsaims to preserve the shape of the cells
or tissue involved as much as possible. Sometimes heat fixation is used to kill,
adhere, and makes them permeable so it will accept stains
What can be used as stain
The substance be used as a stain must be colored or it should react in the system
to give a colored product, because of which some portion of the system becomes
colored and the rest remains colorless. Staining renders the organism more
visible, it displays the structure and finer details of bacteria and it helps to
differentiate between organisms
Staining techniques
Direct staining - The organism is stained and background is left unstained
Negative staining - The background is stained and the organism is left unaltered

INTEXT QUESTIONS 2.1

Fill in the blanks:
1. Staining is primarily used to enhance ................ in the image
2. Substances that adhere to cell giving colour to cell are called as ................
3. ................ aims to preserve the shape of the cells
4. The organism is stained and background is left unstained in ................
staining technique
5. Background is stained and the organism is left unchanged in ................
staining technique
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2.3 KINDS OF STAINS

Stains are classified as
z Simple stain

Notes

Differential stain

Structural or special stains

Simple Staining
The staining process involves immersing the sample (before or after fixation and
mounting) in dye solution, followed by rinsing and observation. Many dyes,
however, require the use of a mordant, a chemical compound that reacts with
the stain to form an insoluble, coloured precipitate. When excess dye solution
is washed away, the mordanted stain remains. Simple staining is one step method
using only one dye. Basic dyes are used in direct stain and acidic dye is used
in negative stain. Simple staining techniques is used to study the morphology
better, to show the nature of the cellular contents of the exudates and also to study
the intracellular location of the bacteria
Commonly used simple stains are
z

Methylene blue

Dilute carbol fuchsin

Polychrome methylene blue

Loefflers Methylene Blue

Method of Staining
Flood the smear with methylene blue, allow for 2 minutes, pour off the stain and
allow the air to dry by keeping in a slanting position and by this the organism
will retain the methylene blue stain
Use
Methylene blue staining is used to make out clearly the morphology of the
organisms eg. H.influenzae in CSF, Gonococci in urethral pus
Polychrome Methylene Blue
Preparation
Allow Loefflers Methylene blue to ripen slowly. Methylene blue stain is kept
in half filled bottles, aerate the content by shaking at intervals, Slow oxidation
of methylene blue forms a violet compound and Stain gets polychrome property.
The ripening nearly takes 12 months and this is hastened by addition of 1%
potassium carbonate
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Use
Polychrome Methylene Blue is used to demonstrate Mc Fadyean reaction of
B.anthracis and in this the blue bacilli Is surrounded by purple capsular material
Dilute Carbol Fuchsin
Preparation
Prepare carbol fuchsin and dilute it to 1/15 using distilled water

Notes

Method of staining
Flood the smear and let stand for 30 seconds, wash with tap water and blot gently
to dry
Use
To stain throat swab from patients of suspected Vincents angina, (Borrelia are
better stained), it is used as a counter stain in Gram stain and to demonstrate the
morphology of Vibrio cholerae (comma shaped)

INTEXT QUESTIONS 2.2

Fill in the blanks:
1. Chemicals that reacts with stain to form precipitates are called as ..............
2. .............. dyes is used in direct stain
3. .............. dyes is used in negative stain
4. Commonly used simple stains are .............., .............. & ..............

2.4 DIFFERENTIAL STAINS

Gram Staining
Differential Stains use two or more stains and allow the cells to be categorized
into various groups or types. Both the techniques allow the observation of cell
morphology, or shape, but differential staining usually provides more information
about the characteristics of the cell wall (Thickness). Gram staining (or Grams
method) is an emprical method of differentiating bacterial species into two large
groups (Gram-positive and Gram-negative) based on the chemical and physical
properties of their cell wall. The Gram stain is almost always the first step in
the identification of a bacterial organism, While Gram staining is a valuable
diagnostic tool in both clinical and research settings, not all bacteria can be
definitively classified by this technique, thus forming Gram variable and Gram
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indeterminate groups as well. The word Gram is always spelled with a capital,
referring to Hans Christian Gram, the inventor of Gram staining
Gram staining Principles

Notes

Gram staining is used to determine gram status to classify bacteria broadly. It

is based on the composition of their cell wall. Gram staining uses crystal violet
to stain cell walls, iodine as a mordant, and a fuchsin or safranin counterstain
to mark all bacteria. Gram status is important in medicine; the presence or
absence of a cell wall will change the bacteriums susceptibility to some
antibiotics. Gram-positive bacteria stain dark blue or violet. Their cell wall is
typically rich with peptidoglycan and lacks the secondary membrane and
lipopolysaccharide layer found in Gram-negative bacteria
Gram Staining Technique
1. Crystal violet acts as the primary stain. Crystal violet may also be used as
a simple stain because it dyes the cell wall of any bacteria.
2. Grams iodine acts as a mordant (Helps to fix the primary dye to the cell
wall).
3. Decolorizer is used next to remove the primary stain (crystal violet) from
Gram Negative bacteria (those with LPS imbedded in their cell walls).
Decolorizer is composed of an organic solvent, such as, acetone or ethanol
or a combination of both.)
4. Finally, a counter stain (Safranin), is applied to stain those cells (Gram
Negative) that have lost the primary stain as a result of decolorization
Gram Reaction
Gram-positive bacteria are those that are stained dark blue or violet by Gram
staining. This is in contrast to Gram-negative bacteria, which cannot retain the
crystal violet stain, instead taking up the counter stain (safranin or fuchsine) and
appearing red or pink. Gram-positive organisms are able to retain the crystal
violet stain because of the high amount of peptidoglycan in the cell wall. Grampositive cell walls typically lack the outer membrane found in Gram-negative
bacteria.
Gram-negative bacteria are those bacteria that do not retain crystal violet dye
in the Gram staining protocol. In a Gram stain test, a counter stain (commonly
safranin) is added after the crystal violet, coloring all Gram-negative bacteria
with a red or pink color. The test itself is useful in classifying two distinct types
of bacteria based on the structural differences of their cell walls. On the other
hand, Gram-positive bacteria will retain the crystal violet dye when washed in
a decolorizing solution.

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Notes

Fig. 2.1: Gram Reaction.

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INTEXT QUESTIONS 2.3

Fill in the blanks:
1. Gram staining uses ............... to stain cell walls, ............... as mordant &
............... as counter stain
Notes

2. Gram positive bacteria stains ............... colour

3. Gram negative bacteria stains ............... colour
4. Gram positive organism are able to retain crystal violet stain because of high
amount of ............... in the cell wall

2.5 ACID-FAST STAINING

The ZiehlNeelsen stain, also known as the acid-fast stain, widely used
differential staining procedure. The Ziehl Neelsen stain was first described by
two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich
Neelsen (1854 to 1894) a pathologist. In this type some bacteria resist
decolourization by both acid and alcohol and hence they are referred as acidfast organisms. This staining technique divides bacteria into two groups namely
acid-fast and non acid-fast. This procedure is extensively used in the diagnosis
of tuberculosis and leprosy. Mycobacterium tuberculosis is the most important
of this group, as it is responsible for the disease called tuberculosis (TB) along
with some others of this genus
Principle
Mycobacterial cell walls contain a waxy substance composed of mycolic acids.
These are -hydroxy carboxylic acids with chain lengths of up to 90 carbon
atoms. The property of acid fastness is related to the carbon chain length of the
mycolic acid found in any particular species
Ziehl- Neelsen Procedure
1. Make a smear. Air Dry. Heat Fix.
2. Flood smear with Carbol Fuchsin stain
z Carbol Fuchsin is a lipid soluble, phenolic compound, which is able
to penetrate the cell wall
3. Cover flooded smear with filter paper
4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed
5. Cool slide
6. Rinse with DI water
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7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains
3% HCl and 95% ethanol, or you can declorase with 20% H2SO4

Microbiology

8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop
by drop) until the red color stops streaming from the smear
9. Rinse with DI water
10. Add Loefflers Methylene Blue stain (counter stain). This stain adds blue
color to non-acid fast cells. Leave Loefflers Blue stain on smear for 1
minute

Notes

11. Rinse slide. Blot dry.

12. Use oil immersion objective to view.

Fig. 2.2: Ziehi-Neelsen acid fast staining procedure

Fig. 2.3
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INTEXT QUESTIONS 2.4

Fill in the blanks:
1. Organisms that resist decolourisation by acid and alcohol are called as
................
Notes

2. ................... staining is used in the diagnosis of Tuberculosis and Leprosy

3. Acid fastness is related to ................... length of mycolic acid
4. Acid fast staining groups bacteria into two groups namely ................... &
...................

2.6 SPECIAL STAINS

z

Stains for Metachromatic granules

Stain for spores

Stain for capsules

Stain for spirochetes

Stain for flagella

Alberts Staining for C. diphtheriae

In all cases of suspected Diphtheria, stain one of the smears with Gram stain.
If Gram stained smear shows morphology suggestive of C.diphtheriae, proceed
to do Albert staining which demonstrates the presence or absence of metachromatic
granules.
C.diphtheriae are thin Gram positive bacilli, straight or slightly curved and often
enlarged (clubbing) at one or both ends and are arranged at acute angles giving
shapes of Chinese letters or V shape which is characteristic of these organisms.
Present in the body of the bacillus are numerous metachromatic granules which
give the bacillus beaded or barred appearance. These granules are best
demonstrated by Alberts stain.
Albert staining
Albert stain I

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Toluidine blue 0.15 gm

Malachite green 0.20 gm

Glacial acetic acid 1.0 ml

Alcohol(95%) 2.0 ml

Distilled water 100 ml

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Albert stain II
z Iodine 2.0 gm
z Potassium iodide 3.0 gm
z Distilled water 300 ml

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Albert staining Procedure

z
z
z
z
z

Cover the heat-fixed smear with Albert stain I. Let it stand for two minutes.
Wash with water.
Cover the smear with Albert stain II. Let it stand for two minutes.
Wash with water, blot dry and examine.
To demonstrate metachromatic granules in C.diphtheriae. These granules
appear bluish black whereas the body of bacilli appear green or bluish green.

Notes

Capsule staining
The purpose of the capsule stain is to reveal the presence of the bacterial capsule,
the water-soluble capsule of some bacterial cells is often difficult to see by
standard simple staining procedures or after the Gram stain. The capsule staining
methods were developed to visualize capsules and yield consistent and reliable
results
Capsule may appear as clear halo when a fresh sample is stained by Grams or
Leishman stain, Negative staining- using - India ink, Nigrosin
India ink
Commercially available India ink is used undiluted
Procedure
z
z
z
z

Place a loop full of India ink on the slide

A small portion of the culture is emulsified in the drop of ink
Place a clean cover slip over the preparation without bubbles. Press down
gently
Examine under dry objective

Uses
India ink is used to demonstrate capsule which is seen as unstained halo around
the organisms distributed in a black background eg. Cryptococcus
Endospore Staining
Bacterial endospores are metabolically inactive, highly resistant structures
produced by some bacteria as a defensive strategy against unfavorable
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Common Staining Technique

environmental conditions. Primary stain - is malachite green, which stains

both vegetative cells and endospores and heat is applied to help the primary stain
penetrate the endospore. Decolorized with water, which removes the malachite
green from the vegetative cell but not the endospore, Safranin - counterstain
any cells which have been decolorized, At the end of the staining process,
vegetative cells will be pink, and endospores will be dark green
Flagella stain
z

Flagella are fragile appendages

Cannot be seen under ordinary microscope

Hence the surface is coated with a precipitate to form a colloidal substance

This precipitate serves as a layer of stainable material

Components
1. 1% Osmic acid
2. Mordant
10% Tannic acid
Sat.potassium alum
10% Ferric chloride
3. Fontanas silver solution
Use
This is used to demonstrate the flagella and the organisms stain black and
flagella appear light brown

INTEXT QUESTIONS 2.5

Fill in the blanks:
1. Presence or absence of metachromatic granules is demonstrated by .............
staining
2. ............. chemical is used in capsule staining
3. ............. is used as primary stain in Endospore staining
4. ............. is the counter stain in endospore staining

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WHAT YOU HAVE LEARNT

z

Staining is technique used in microscopy to enhance contrast in the

microscopic image.

Stain is a substance that adheres to a cell, giving the cell colour.

Strains are classified as Simple stain, Differential stain and Special stains.

Gram staining is used to differentiate bacterial species as Gram-positive and

Gram-negative based on the chemical and physical properties of cell wall.

Acid Fast staining technique or Ziehl Neelsen stain divides bacteria into
acid fast and non-acid-fast and this is used in diagnosis of tuberculosis and
Leprosy.

Albert staining technique demonstrates the presence or absence of

metachromatic grannules which is used in identification of C. diphtheria
bacilli.

Notes

TERMINAL QUESTIONS
1. List staining techniques
2. Describe different kinds of stains
3. Explain gram staining
4. Explain Acid fast staining

ANSWERS TO INTEXT QUESTIONS

2.1
1. Contrast
2. Stain
3. Fixation
4. Direct
5. Negative

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2.2
1. Mordant
2. Basic
3. Acidic
4. Methylene blue, Polychrome methylene blue & Dilute carbol fuchsin

Notes

2.3
1. Crystal violet, Iodine & Fuchsin
2. Dark blue or violet
3. Red or pink
4. Peptidoglycan
2.4
1. Acid fast organism
2. Acid fast staining
3. Carbon chain
4. Acid fast and Non acid fast
2.5
1. Alberts
2. Indian ink
3. Malachite green
4. Safranin

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