Introduction To Stains and Staining Properties-DMLT@2022

Introduction to stains, Staining
Techniques and
Principles
Kasuswa Sophia
BMLS,DMLT,DLM,CMLT
Session objectives
• By the end of these session, you will be able to:
1. Definition of Terms
2. Formation of Dyes
3. Importance of Staining in Microbiology
4. Factors that Affect Staining
5. Theories of Staining
6. Types of Staining Techniques
7. Preparation of Stains
05/13/2022 Kasuswa Sophia DMLT/MICRO/2022 2

Introduction
A stain is a substance used to colour organisms/tissue.
A stain is any colouring organic compound that, when
combined with another substance, imparts a colour to
that substance.
A dye is a coloured substance that chemically bond to
the substance to which it is being applied.
(A dye is used to refer to a colouring agent that is used
for general purposes, whereas the term Stain is used to
refer to that dye which is used for biological purposes)

Stains and Dyes
• A dye is a general-purpose coloring agent, whereas
a stain is used for coloring biological material.
• A stain is an organic compound containing a
benzene ring plus a chromophore and an
Auxochrome group.
• Chromophore is a chemical group that imparts
colour to the benzene ring.
• Auxochrome group is a chemical compound that
conveys the property of ionization of chromogen
(ability to form salts) and bind to fibers or tissues.

Introduction…
A dye is a coloured substance that has an affinity to substrate
to which its being applied, dye usually require a mordant.
Dyes have aromatic Benzene ring compounds or derivatives
that possess the twin properties of colour and ability to bind
tissue/ organisms
• Stains and dyes are frequently used in biology and medicine to
highlight and view structures in biological tissues.
• Involves immersing the sample/section in dye/stain solution,
followed by rinsing and then observation

Formation of Dyes
• Originally dyes were made from Aniline and
Coal tar.
• The dyes made from aniline, carmine or litmus
are commonly referred to as Natural dyes.
• Those from the Benzene ring are called
Synthetic dyes.

Formation of dyes...
• Benzene ring is in the invisible spectrum and is
colorless
• When certain chemical compounds,O2 are added to
the ring, its absorption band is moved to the visible.
This compound is called Quinoid and is colored.
• The O2 is termed a Chromophore compound.
• When a Quinoid is added to another benzene ring the
outcome is a chromogen but lacks the salt forming
properties and cannot be attracted firmly onto the
tissue fibers.
Formation of dyes cont.....
• A Chromogen is made electrically dissociable
by the addition of a molecule called
Auxochrome e.g. OH-, making it to a stain.

Importance of Staining in Microbiology
1. Improves visibility by greater contrast between

the organism and the background,
2. Differentiate various morphological types (by
shape, size, arrangement, etc.),
3. Determine the staining characteristic of organism
and, at times, direct diagnosis of disease, and
4. Demonstrate the purity of culture. observe
certain structures (flagella, capsules, endospores,

Importance of Staining in Microbiology…
To study the morphology (size and shapes) of

microorganisms.
Better Visualization of organisms
• Bacterial organisms are so small that most of them
are visible only under a microscope with
magnification. However, mere magnification of size
does not provide a sufficient degree of clarity, so that
bacteria must therefore be stained before
observation to provide the clarity needed for
visualization.
Importance of Stain in Microbiology
Identification and Classification of bacteria
The Gram stain is one such differential stain that distinguishes between bacteria
on the basis of their cell wall content. Gram staining provides a rapid method for
the initial identification of bacteria involved in infections. Similarly, the acid-fast
staining procedure helps to specifically identify the organisms belonging to the
class of bacteria called Mycobacteria, such as Mycobacterium tuberculosis.
Detection of Viability
• In bacterial culture specimens, it is often important to detect the presence of
living bacterial cells. Staining methods such as fluorescent staining help to
identify if culture cells are viable or not.
Identification of Cellular Structures
• Staining provides a method of clearly visualizing several cellular structures. For
example, the Fuelgen staining method allows identification of the nucleus
within bacterial cells

Factors that Affect Staining
 pH
 Temperature
 Concentration and staining time
 The dye nature (lipophilicity and
hydrophobic index)

Factors that Affect Staining…
pH
The pH of the solution determines whether a dye will bind
to certain tissue elements by establishing the appropriate
charge on both the tissue element and the dye.
Temperature
An increase in temperature will increase the rate of
staining by increasing the diffusion rate of the dye
molecule. Swelling of the tissue components caused by
the increase in temperature is important in dye
penetration.

Factors that Affect Staining…
Concentration
• Bye binding increases with an increase with an
increase in concentration of the dye

Requirements for Staining
NOTE: Majority of the stains used for staining bacteria are of the
Basic type as nucleic acid of bacterial cells attract the positive
ions, e.g. Methylene blue, Crystal violet.
NOTE: Acidic stains are used for background staining.
• Mordant – It is a chemical that forms an insoluble complex
with the stain and fixes it or causes the stain to penetrate
more deeply into the cell. Mordant is used in indirect staining.
E.g., Gram’s iodine in Gram staining and Phenol in ZN-staining.
• Accentuators – It is a chemical which when added to a stain to
make the reaction more selective and intense. E.g., Potassium
hydroxide added in Loeffler’s methylene blue.
• Decolourizer – It is a chemical used to remove the excess stain
in indirect regressive staining. For example, ethanol in Gram’s
staining.
Mordant
• A mordant is a chemical compound which
reacts with the stain to form an insoluble,
coloured precipitate.
• Mordant is a link between tissue and stain
(combines with the stain to form a lake to
attach firmly to the tissue )
• Basic mordants = Acidic stains
• Acidic mordants = Basic stains

Mordant…
• Mordant may be applied in three ways.
1. Before applying the stain
2. In conjunction with the stain (incorporated in
the stain)
3. After applying the stain
E.g. Lugol's Iodine in gram stain.

Accentuator and Accelerator
• Accentuators are chemical adjustment that help
the dye to attract better to the tissue/ organism.
• Accelerator is a physical adjustment e.g. agitation
or heat
NOTE: Accentuators are materials added to staining
solutions to;
1. Improve the staining reaction.
2. Increase the staining power of the stain/dyes.
3. Control pH, e.g. Potassium Hydroxide in Löffler’s
methylene blue and Phenol in Carbol fuchsin.
NOTE: Accentuators do not form a lake with the dye
Kasuswa Sophia DMLT/MICRO/2022 18
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Classification Stains
• Classification based on Source:
1. Natural stains
2. Synthetic stains
3. Semi synthetic stains

Natural stains
• These are stains that exist freely in nature.

• Extract of trees (e.g. haematoxylin from
Haematoxylon campechianum)
• Extracts from female insects (e.g. Carmine from
cochineal insects of genus coccus carti)
• Extracts from Lichens (e.g. Litmus and Orcein).
• These are mainly used in histology and as
indicators

Synthetic stains
Synthetic stains (aniline or coal tar dyes)
• Derived from Nitrobenzene.
• These are usually produced through oxidation, reduction
and chemical combination of several chemicals.
• Majority of these stains are used in microbiology
laboratory
Two common features;
- Have chromophoric groups i.e. groups with conjugated
double bonds (C=O, C=N, N=N) attached to a complex
ringed molecule that produces a specific color
- Bind with cells by ionic, covalent or hydrophobic bonding.

Semi synthetic
• Semi synthetic stains/dyes are produced by
obtaining natural raw material then slightly
modifying it to increase its coloring ability

Classification of stains
Classification based chemical properties (based

on Charge on Chromophoric group) i.e.
1. Basic stains
2. Acidic stains
3. Neutral stains

Basic stains
• They possess positive (cationic) charge on their surface on ionization.
• Have a positively charged chromophore (color is contained in the base)
• has a pH above 7.
• Have affinity for Acidic tissue components (Acidophilic)
• Are generally chloride salts
• They stain Negatively charged cell components like nucleus.
• Examples of Basic Stains include. Crystal violet, Methyl violet and Basic Fuchsin
• Mostly used in bacteriology and at high pH
Principle of action
• Basic stains ionize into a positively charged chromophore and negatively charged
mineral ion
• Since the bacterial cell membrane has a slight negative charge, they attract the
positive ions of basic satin and hence will be stained

Acidic stains
• Have negatively charged Chromophoric groups
(Coloring compound is in the Acidic radical)
• has a pH less than 7 and has the affinity for
basic tissue components (basophilic)
• Used for background staining as well as
negative staining
• Stain positively charged cell components like
cytoplasm, granules of WBC e.g. Eosin, India
ink, dil. Nigrossin, Acid fuchsin,
Haematoxylen
Acidic stains…
Principle of action
Acidic dyes ionize into a negatively charged
Chromophoric group and positively charged
mineral ion.
Due to negative charge on the bacterial cell
membrane, the bacterial cell repels the –ve
ions of acidic stains and hence are not stained
by the dye

Neutral stains
• Are product of aqueous acidic and basic dyes

• Chromophoric groups are on both acidic and
basic components of the dye.
• Have a pH equal to 7.
• All Romannowisky stains
E.g. Giemsa, Leishman

Theories of Staining
They are 2 Theories
1. Physical theory of Staining
2. Chemical theory of Staining
Physical Properties
• Which include the Solubility, Adsorption and Absorption
regarding the manner in which the dye molecules are
attached to the tissue either by adhesion or van darwaals
forces or socking.
- Solubility (stains are more soluble in fats and lipids than in
solvents in which they are dissolved)
- Adsorption (certain molecules are attracted onto surface
of cells, tissues that they stain them)
Theories of Staining…
Chemical theory
• Chemical properties. This involves the acid
base reaction (attraction)
• Acidic dyes (have a high affinity for basic
constituents )
• Basic dyes (have a high affinity for acidic
constituents)

Types of Staining Techniques
Simple staining Differential staining

(Use of of single stain) (Use of two contrasting stains)
Direct Indirect Separation Visualization

(Positive) (Negative) into groups of
1. Gram structures
stain
1. Flagella
2. Acid stain
SIMPLE STAINING

SIMPLE STAINING
 This method uses only a single dye that which does not
differentiate between different types of organisms
 There is only a single staining step and everything
is
stained with the same color.
 Simple stains are used to stain whole cells or to
stain specific cellular components.
 Types of Simple staining:
1. Direct / Positive staining : stain object
2. Indirect / Negative staining: stain background

Direct staining (Positive staining)
A simple staining technique that stains the bacterial cells
in a single colour.
Many of these bacterial stains are basic chemicals.
These basic dyes react with the negatively charged
cytoplasm (Opposite charge attract) and the
organism becomes directly stained
 Examples include; Methylene Blue, Crystal violet and
Basic Fuchsin

Indirect staining (Negative staining)
 In this staining process, instead of ells background is
stained.
 Here, an acidic dye like nigrosin or Indian ink is
used. Acidic stain carries a negative charge and repelled
by the bacteria, which also carry a negative charge on their
surface. Hence, an acidic dye do not stain bacteria,
 Instead, it forms a deposit around the organism, leaving the
organism itself colorless or transparent upon
examination.

Negative staining
• Provide a uniformly coloured background against which the
unstained bacteria stand in out in contrast.
• E.g. Demonstration of bacterial Capsule
• Requires the use of acidic dyes E.g. India Ink or Nigrosin
Principle of Negative staining
The stain readily gives a hydrogen ion (proton) and the
chromophore of the dye becomes negatively charged.
Since the surface of most bacterial cells is negatively charged,
the cell repels the stain. The glass o the slide will stain, but the
bacterial cells will not . The bacteria will show up as clear
against a dark background.

STAINING:
GENERAL TECHNIQUE
SMEAR AIR DRY HEAT FIX STAI LOOK

N

Importance of Fixing the Smears
“Fixation accomplishes three things:

(1) it kills the organisms;
(2) it causes the organisms to adhere to the slide; and
(3) it alters the organisms so that they more readily accept stains
(dyes).

DIFFERENTIAL STAINING

Differential staining
• Is an indirect staining method
• Differential stains impact different colours to
different bacteria or bacterial structures.
• Uses more than one dye and other chemicals
(mordant)

Differential ctd ….
• Stains react differently
with various
organs/tissues
• Reaction is based on
the chemical
composition of the
organism
• Differentiate bacteria
e.g. gram positive and
gram negative, AFB
GRAM’S STAINING

The Gram Stain
• In the late 1800’s, Christian Gram observed that some
genera of bacteria retained a dye-Iodine complex when
rinsed with alcohol, while other genera were easily
decolorized with alcohol and could be then visualized by
a contrasting counter stain.
• This staining procedure defines two bacterial groups:

those which retain the primary dyes “Gram-Positive”)
and those which are easily decolorized (“Gram-
Negative”).
• This is the starting point for bacterial identification
procedures.
The Gram Stain
 The difference in dye retention is dependent on such
physical properties as thickness, density, porosity, and
integrity of the bacterial cell wall, as well as its
chemical composition.
 Gram-Positive bacteria have thick, dense, relatively
non-porous walls, while Gram-Negative bacteria have
thin walls surrounded by lipid-rich membranes.
 Some non-bacterial organisms with thick cell walls (e.g.,
some yeasts) also stain Gram-Positive.
 Gram-Positive bacteria which have lost wall integrity
through aging or physical or chemical damage may
stain Gram-Negative.
GRAM POSITIVE
Lipoteichoic acid Peptidoglycan-teichoic acid
Cytoplasmic membrane
Cytoplasm
GRAM NEGATIVE Porin

Lipopolysaccharide
Outer Membrane Braun lipoprotein
Inner (cytoplasmic) membrane

Cytoplasm
Principle of Gram Staining Technique
• Gram positive organisms have acidic protoplasm of pH 2.0 therefore,
they have higher affinity for the Primary stain). While the Gram
negative organisms have less acidic protoplasm of pH 5 hence less
affinity for the basic dyes.
• The Gram positive organisms also have tiny pores and thick layers of
peptidoglycans, teichoic acid in the cell wall hence retain the
primary stain and resist decolourization. Whereas Gram negative
organisms have larger pores and a thin layer of peptidoglycans in the
cell wall hence faster diffusion of the dye outside the cell wall so they
do not retain the primary stain.
• Gram positive organisms have magnesium ribonucleate which
combines with the primary stain to form a complex that does not
diffuse out of the cell wall whereas gram negative do not have
magnesium ribonucleate. This makes the Gram positive organisms
appear Purple /Blue while Gram negative organisms appear Pink/
Red
Gram staining – Requirements
• Gram-staining is a four part procedure.
• The specimen is mounted and heat fixed on a slide before
proceeding to stain it.
• The reagents required are:
 Crystal Violet (the Primary Stain)
 Iodine Solution (the Mordant)
 Decolorizer (50% Acetone Alcohol)
 Safranin (the Counter stain)
 Water (preferably in a squirt bottle)

Gram staining – Procedure
1 The bacteria are first stained with the basic dye crystal violet (primary
stain). Both gram-positive and gram-negative bacteria become directly
stained and appear purple after this step.
2 The bacteria are then treated with gram's iodine solution (mordant). This
allows the stain to be retained better by forming an insoluble crystal
violet-iodine complex, called as ‘iodine lake’. Both gram-positive and
gram-negative bacteria remain purple after this step.
3 Gram's decolorizer, a mixture of ethyl alcohol and acetone, is then
added. This is the differential step. Gram-positive bacteria retain the
crystal violet-iodine complex while gram-negative are decolorized.
4 Finally, the counter stain safranin (also a basic dye) is applied. Since the
gram-positive bacteria are already stained purple, they are not affected
by the counter stain. Gram-negative bacteria, that are now colorless,
become directly stained by the safranin. Thus, gram-positive appear
purple, and gram-negative appear pink.

Acid Fast Staining
(ZN Staining)

Structure of an Acid-Fast Cell Wall

Acid Fast staining
 Once stained the acid fast bacterial cells resist
decolorization with acidified organic solvents, e.g
alcohol and are therefore called ACID FAST.
acid
 Acid fast staining property of the genus, Mycobacteria,
depends upon their lipid-rich cell walls which are relatively
impermeable to various basic dyes unless the dyes are
combined with phenol.
 The exact method by which the stain is retained is unclear
but it is thought that some of the stain becomes trapped
within the cell and some forms a complex with the mycolic
acids. This is supported by the finding that shorter chain
mycolic acids or mycobacterial cells with disrupted cell
walls stain weakly acid-fast, e.g. Nocardia
Principle of ZN staining
• When Acid fast bacilli are stained with strong dyes e.g Strong Carbol
Fuchsin with 5% phenol and an application of heat they resist
decolourisation by strong acids e.g. 3% Acid alcohol or 20-25% H 2SO4 and
stain Pink/Red hence referred to as Acid fast. This is attributed to high
amount of mycolic acid, fatty acids and lipid present in their cell wall.
• Some organisms such as Cryptosporidium Oocysts have a thinner layer of
mycolic acid, fatty acids and lipid in their cell wall and only resist
decolourisation in weak acids/acid alcohols but are also acid fast and
therefore they are mostly demonstrated using the modified ZN
technique.
• The non acid fast organisms are easily decolorized and only take up the
colour of the counter stain appearing Blue with Methylene Blue /Green
with Malachite green.

Acid-Fast Stain of Mycobacterium tuberculosis in
Sputum
Note: The Reddish acid-fast bacilli among the blue normal flora
and white blood cells in the sputum that are not acid-fast.
SPECIAL STAINING

Special staining
• Is based on differences in a structure
compared to the rest of the cell
• Selectively stain on part of the cell
• E.g. Endospore staining, Capsule stains,
Flagella staining
Spore
staining

CAPSULE
STAINING

 Capsule staining is diagnostically useful since it is
a virulent factor(e.g. pneumococci).
 Bacterial capsules are non-ionic, so neither acidic
nor basic stains will adhere to their surfaces.
 Capsules are demonstrated either by negative
staining (Nigrosin or India ink) or by special
staining, e.g. Hiss’ method, Anthony’s method

Hiss Method
• The capsule is non-ionic in nature so it doesn't get stain by a
acidic stain but a basic stain, such as crystal violet, stains
the cell as well as the capsule.
• This is followed by treatment with hypertonic solution -
20% Copper sulphate solution, which serves dual role of
both the decolorizing agent and counter stain.
• Copper sulphate solution, being hypertonic, causes diffusion
of stain towards outer surface of cell.
• After drying of slide, the stain which is not passed from the
capsular layer during diffusion retains in the capsular layer.
Copper sulphate then decolorizes the capsule.
• Capsule appears as a faint blue halo around a purple cell.
ENDOSPORE STAINING

Endospore
staining
 Spores are normally impervious to stains.
Under the light microscope endospores have a high light
refractivity indicative of high protein content.
 Endospores can be stained by:
 Modified Zeihl-Nelson's method using 0.25-0.5% sulphuric acid
as decolorizing agent,
 Barthelomew-Mittwar’s method
 Schaeffer-Fulton stain technique.

Schaeffer-Fulton method
• Malachite green is used to stain the endospores (primary
stain)
• The malachite green is forced to permeate the spore wall
by heating (mordant).
• Washing with water remove stain from vegetative cells, but
not from spore wall.
• The endospores thus retain the primary dye while the
vegetative cells lose the primary stain and take the red color
of secondary stain (safranin).

Endospore stain of Bacillus megaterium
Note green endospores within pink bacilli

Other staining methods
Progressive Staining
- Elements are stained in a definite sequence (several
stains applied for specific time without decolorisation)
Regressive Staining
- over staining followed by differentiation with either acid
(basic stain) or alkaline soln (acidic stain)
Vital staining
- Staining of living cells/tissue inside the body. E.g.
Trypan blue stain.
Supra-vital stain: Staining living tissues outside the body.
E.g Brilliant Cresyl blue.
Other staining methods
Metachromatic stain: Staining the tissues with a color
different from the original color of stain. E.g Toluidin
blue staining for Mast cells.
Polychromatic stain: Staining the tissues with multiple
colors in spite of using a single stain. E.g Geimsa stain
for blood.
Orthocromatic stain: Staining the tissues with the same
color of the stain, such as H&E.
Impregnation staining
• Metallic salts are deposited on surface of tissues
Preparation of Stains
1. Crystal Violet
2. Lugo’s iodine
3. 50% Acetone Alcohol
4. 10% Strong Carbolfuchsin
5. 3% Acid Alcohol
6. 0.5% Methylene blue
7. Auramine Blue
8. Potassium permanganate
9. Normal Saline
10.20% H2SO4, 5% and 20%

Safety Precautions taken when handling stains
• Wear chemical splash goggles and chemical-

resistant gloves.
• Wash hands thoroughly with soap and water
before leaving the laboratory.
• Follow all laboratory safety guidelines.
• Read current Material Safety Data Sheets
(MSDS) for additional safety, handling,
disposal information and first aid.

Procedure for Labeling Reagents and Stains
• All reagents and stains shall be clearly and conspicuously labeled with
the following information;
• The name of the reagent or stain
• The date manufactured or prepared/reconstituted
• The expiration date
• Indication of quality control compliance (pass)
• Storage conditions
• The initial of the person who prepared or reconstituted (especially for laboratory made
reagents/stains)
• Any special health hazard that may be associated with the reagent/stain
– The labels on commercially prepared reagents and stains shall not be erased or
covered
– Laboratory prepared reagents and stains shall be labeled by preparer
– All reagents and stain shall be stored as indicated on the container and/or
material safety datasheet.

Crystal Violet
Purpose : Used in Gram stain as a primary stain

• It’s a basic stain.
• Stain negatively charged cell components like nucleus.
To prepare 1 litre:
• Crystal violet . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 g
• Ammonium oxalate . . . . . . . . . . . . . . . . . . . . . . 9 g
• Ethanol or methanol, absolute . . . . . . . . . . . 95 ml
• Distilled water . . . . . . . . . . . . . . . . . . . . . . to 1 litre
Procedure
1.Weigh the crystal violet on a piece of clean paper.
2.Transfer to a brown.
3.bottle pre-marked to hold 1 litre.
4.Add the absolute ethanol or methanol (technical
grade is suitable) and mix until the dye is completely dissolved.

Crystal Violet cont.
4. Weigh the ammonium oxalate and dissolve in about 200 ml of
distilled water.
5. Add to the stain. Make up to the 1 litre mark with distilled water, and
mix well.
6. Label the bottle, and store it at room temperature.
• The stain is stable for several months.
For use: Filter a small amount of the stain into a dropper bottle or other
stain dispensing container.
Cautions:
1. Ethanol and methanol are highly flammable, therefore use these
chemicals well away from an open flame.
2. Ammonium oxalate is a toxic chemical, therefore handle it with care.

Lugol’s iodine solution
Purpose : Used
in Gram stain as a mordant
• It’s a basic stain.
• Stain negatively charged cell components like nucleus.
To prepare 1 litre:
• Potassium iodide . . . . . . . . . . . . . . . . . . . . . . . . 20 g
• Iodine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 g
• Distilled water . . . . . . . . . . . . . . . . . . . . . . to 1 litre
Procedure
1. Weigh the potassium iodide, and transfer to a brown bottle pre-marked
to hold 1 litre.
2. Add about a quarter of the volume of water, and mix until the
potassium iodide is completely dissolved.
3. Weigh the iodine, and add to the potassium iodide solution. Mix until
the iodine is dissolved.
Lugol’s iodine solution
5. Make up to the 1 litre mark with distilled water, and mix well.
6. Label the bottle, and mark it Toxic.
7. Store it in a dark place at room temperature.
Renew the solution if its colour fades.
For use: Transfer a small amount of the reagent to a brown
dispensing bottle
Caution: Iodine is injurious to health if
inhaled or allowed to come in contact with the
eyes, therefore handle it with care in a well
ventilated room.

Assignment
• Make Notes on the Preparation of the
remaining Stains

THANK YOU

Introduction To Stains and Staining Properties-DMLT@2022

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Introduction To Stains and Staining Properties-DMLT@2022

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Introduction to stains, Staining

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1. Improves visibility by greater contrast between

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To study the morphology (size and shapes) of

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• These are stains that exist freely in nature.

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Classification based chemical properties (based

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• Are product of aqueous acidic and basic dyes

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Simple staining Differential staining

Direct Indirect Separation Visualization

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SMEAR AIR DRY HEAT FIX STAI LOOK

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“Fixation accomplishes three things:

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• This staining procedure defines two bacterial groups:

GRAM NEGATIVE Porin

Outer Membrane Braun lipoprotein

Inner (cytoplasmic) membrane

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Note green endospores within pink bacilli

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• Wear chemical splash goggles and chemical-

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