STAINING TECHNIQUES

FOR BIOLOGICAL
STUDIES
WHAT IS STAINING?

 Staining is a supplementary method that gives

divergence to the microscopic image for better vision
under the microscope.
 It is a technique that is widely used for the examination
of cells, tissues and cellular components.
 This method achieved by the help of a reagent that is
defined as “stain“.
 WHY TO STAIN SAMPLE?
 Used to identify microbial sample
 To visualize the sample under microscope clearly.
 Used in histology and in the medical fields
of histopathology, hematology, and cytopathology that focus on the
study and diagnoses disease at a microscopic level.
 Stains may be used to define biological tissues cell populations
(classifying different blood cells), or organelles within individual cells.
 In biochemistry it involves adding a class-specific
(DNA, proteins, lipids, carbohydrates) dye to a substrate to qualify or
quantify the presence of a specific compound.
 Staining and fluorescent tagging can serve similar purposes.
 Biological staining is also used to mark cells in flow cytometry, and to
flag proteins or nucleic acids in gel electrophoresis.
Unstained and stained tissue
Unstained and stained bacterial sample
MECHANISM
 Stain generally consist of “Chromogen” and “Auxochrome”.
 Chromogen: When the benzene ring reacts with the chromophore
group, then the compound which is formed is called chromogen.
The chromophore is a coloured compound that contains the
unsaturated group.
 Auxochrome: These are the specific groups that impart a positive
and negative charge to the chromogen and is capable of ionising
it. After this, the ionised stain binds to the cell with opposite charges.
 when the auxochrome group is present in the chromogen “A dye or
stain is formed”.
 The coloring agent that stains the biomolecular constituents of a
specimen such as protein, as well as cellular parts that have to be
dyed, depends upon the electrical charge found on the
chromogen portion that imparts colour to the specimen.
PREPARATION
 The preparatory steps involved depend on the type of analysis.
 Preparation involves FOUR basic steps : PERMEABILIZATION, FIXATION, MOUNTING
AND STAINING
 Permeabilization - treatment of cells, generally with a mild surfactant, which
dissolves cell membranes in order to allow larger dye molecules to enter inside the
cell.
 FIXATION: may involve series of steps or just single step depending on sample of
interest.
 aims to preserve the shape of the cells or tissue involved as much as possible.
 Sometimes heat fixation is used to kill, adhere, and alter the specimen so it accepts stains.
 Most chemical fixatives (chemicals causing fixation) generate chemical
bonds between proteins and other substances within the sample, increasing their rigidity.
Common fixatives include formaldehyde, ethanol, methanol, and/or picric acid.
 Pieces of tissue may be embedded in paraffin wax to increase their mechanical strength
and stability and to make them easier to cut into thin slices.
PREPARATION
 Mounting usually involves attaching the samples to a glass
microscope slide for observation and analysis.
 In some cases, cells may be grown directly on a slide.
 Forsamples of loose cells (as with a blood smear or a pap
smear) the sample can be directly applied to a slide.
 For larger pieces of tissue, thin sections (slices) are made
using a microtome; these slices can then be mounted and
inspected.
PREPARATION

Staining - application of stain to a sample to color cells, tissues,

components, or metabolic processes.
 This process may involve immersing the sample (before or after
fixation or mounting) in a dye solution and then rinsing and
observing the sample under a microscope.
 Some dyes require the use of a mordant, which is a chemical
compound that reacts with the stain to form an insoluble, colored
precipitate. The mordanted stain will remain on/in the sample when
excess dye solution is washed away.
PREPARATION

 Mordant: These are chemical agents which have power of making

dyes to stain materials which otherwise are unstainable
 Mordants are classified into two categories:
 a) Basic Mordant: React with acidic dyes e.g. alum , ferrous sulfate ,
cetylpyridinium chloride etc .
 b) Acidic Mordant : React with basic dyes e.g. picric acid , tannic
acid etc.
Direct Staining: Carried out without mordant.
Indirect Staining: Staining brought by the aid of a mordant.
TYPES

 Based on viability of sample

TYPES

 Based on chemical nature of stain

TYPES

 Based of staining method

TYPES

 Based on type of study

CYTOLOGICAL

HISTOCHEMICAL

HISTOPATHOLOGICAL

CYTOGENETIC

HISTOLOGICAL
TYPES

 Based on type of analysis involved (visualization/quantify)

LIGHT ELECTRON FLUORESCENT

MICROSCOPY MICROSCOPY MICROSCOPY

SPECTROSCOPY FLOW
CYTOMETRY
CYTOLOGICAL STAINING

OBJECTIVE:
 To visualize the cells in smear, squash or sections mounted
 To identify abnormalities in cell morphology
 For assessment of infection
 Histopathological studies
 Cytogenetic studies
Different staining methods used for different cell types.
 ANIMAL CELL STAINING- Hematoxylin and eosin staining,
Romanowsky staining, Leishman’s staining, Papanicolaou staining,
 PLANT CELL STAINING - Iodine staining, Toluidene blue staining
ANIMAL CELL STAINING
ROMANOWSKY STAINING
 Romanowsky stains are neutral stains composed of a mixture of oxidized
methylene blue (azure) dyes and Eosin Y.
 The azures are basic dyes that bind acid nuclei and result in a blue to
purple color.
 The acid dye, eosin, is attracted to the alkaline cytoplasm, producing red
coloration.
 Most of the Romanowsky stains are prepared with Methyl alcohol
(Methanol) so that they act as a fixative as well as the cellular stain. There
are 4 different types of Romanowsky stains commonly used in Hematology
laboratory for staining the blood cells –
 Leishman Stain
 Giemsa Stain
 Field’s Stain
 Wright’s Stain
ANIMAL CELL STAINING

Uses
 Blood films, bone marrow examination, cytology.
Advantages
 Readily available, easy to prepare, maintain and use.
Disadvantages
 Nuclear and nucleolar detail not as good as Papanicolaou stain but
adequate to distinguish neoplasia from inflammation.
 pH sensitive therefore buffer and rinse water are critical.
ANIMAL CELL STAINING

GIEMSA STAINING
 Giemsa stain is a mixture of Azure, Methylene blue, and Eosin dye.
 It is a differential stain that is used to variably stain the various
components of the cells and it can be used to study the adherence
of pathogenic bacteria to the human cells.
 It differentially stains the human and bacterial cells and appeared
as purple and pink colored bodies respectively.
 ANIMAL CELL STAINING
PROCEDURE:
 Prepare a thin blood smear on a clean and dry microscopic glass slide and air dry it.
 fix out the air-dried thin blood smear in the absolute methanol by dipping the blood
smear quickly (two dips) in a Coplin jar containing absolute methanol.
 Take out the slide from the Coplin jar and let it air dry.
 stain the Methanol fixed Blood smear with diluted Giemsa stain (1:20, v/v) for 20 min.
 Now Wash out the stained slides by quickly dipping, once or twice, the slide in and
out of a Coplin jar containing buffered water or Distilled water.
 Let the smear dry well in the air.
 Observe under microscope
 ANIMAL CELL STAINING
PAPANICOLAOU STAINING
 Use two dyes that differ in their affinity for various sites within a cell, ie nucleus and
cytoplasm. There is no chemical interaction between nuclear and cytoplasmic solutions, they
bind electrostatically, resulting in salt formation.
 Papanicolaou stains are similar to Hematoxylin and Eosin, except that eosin is replaced with two
cytoplasmic counterstains (that highlight keratinization in squamous epithelium).
Uses
 Special diagnostic situations, in particular where neoplasia is suspected.
 Excellent demonstration of nuclear and nucleolar changes.
 Useful in thick smears and tissue fragments
Disadvantages
 Inferior cytoplasmic detail, secretory products in cytology and organisms (including bacteria)
compared to Romanowsky stains.
 Preparation must be wet - fixed, ie cells must be fixed before cells have dried, unless use
modified Pananicolaou where smears are rehydrated.
ANIMAL CELL STAINING
 Fix the slides in acetic fixative for 15  Rinse in absolute alcohol two times
minutes,
 Stain in E.A 50 for two minutes,
 Absolute alcohol for two minutes,
 Stain in absolute alcohol two time,
 70 percent of alcohol for 2 minutes,
 Clear in xylene three times,
 in 50 percent for 2 minutes
 Mount the slide on xylene three times,
 Tap water for 2 minutes,
 deep in haematoxylin for 4 minutes,
 Briefly rinse in tap water,
 Differentiate in acid alcohol for about 5
seconds,
 Blue inside tap water,
 Dehydrate using absolute alcohol two
times,
 Stain in orange G for ten seconds,
PLANT CELL STAINING
IODINE SOLUTION:
 Plant cells contain starch as storage sugar.iodine reacts with starch
and stains plant cell blue.
PLANT CELL STAINING

TOLUIDENE BLUE STAINING

Toluidine blue is used to differentially stain primary and secondary cell
walls.
Primary cell walls stain pinkish-purple and secondary cell walls stain
blue or blue-green.
toluidine blue helps to distinguish between phloem and xylem cells as
cells found in phloem have primary cell walls only while cells found in
xylem have both primary and secondary cell walls.
HISTOCHEMICAL STAINING

 Histochemistry is an important technique that is used for the visualization

of biological structures.
 It is concerned with the identification and distribution of various
chemical components of tissues through the use of stains, indicators as
well as microscopy.
Histochemical Techniques/Methods
Perls’s Reaction
Von Kossa Technique
Lipids Staining
Protein and Amino Acids
Nucleic acid and DNA
Saccharides
PERLS’S REACTION

 This method is particularly important for the detection of ion levels

(ferric ions).
 It is used to determine the level of these ions in such organs as the
spleen and bone marrow.
 It can be used to detect excessive levels of ferric ions with deposits
in the liver, pancreas, spleen and the lymph nodes in conditions like,
hemochromatosis, hemosiderosis etc.
 In plants, this technique has also been shown to help understand
the homeostasis of ferric ions.
 PERLS’S REACTION
PRINCIPLE: ferric ions present in the tissue will combine with ferrocyanide resulting in the
formation of a pigment called Prussian blue (ferric ferrocyanide). In plants, the technique
is also based on the conversion of ferrocyanide into insoluble crystals (Prussian blue) in the
presence of Ferric ions under acidic conditions. Prussian blue (resulting from the reaction)
is bright blue in color, which indicates the presence of ferric ions.
Procedure
 Deparaffinize the section(s) and hydrate with distilled water
 Microwave for about 30 seconds and allow the specimen to stand in the working
solution for about 5 minutes in the fume hood. The section can be treated with the
working solution (acid ferrocyanide) for between 10 and 30 minutes.
 Rinse/wash the section using distilled water
 Stain (lightly) the section with 0.5% aqueous neutral red or 0.1% nuclear fast red. This part
of the procedure is used to stain the nuclei.
 Rapidly wash the section using distilled water
 Dehydrate the section, clear and mount on the microscope stage for viewing
 When viewed under the microscope, blue parts are indicative of iron while the red and
pink parts indicate the nuclei and background respectively.
SPREAD SHOWING IRON CONTENT STAINED BLUE
VON KOSSA TECHNIQUE

 used to identify the presence of calcium deposits on cyst fluids,

ductal ectasia and papillomatosis. However, excessive amount of
calcium may be found in any given part of the body and can be
demonstrated using the Von Kossa technique.
Principle
For this technique, the sample section is treated with the solution of
silver nitrate and is reduced and the calcium (if present in the sample)
is reduced by the strong light and replaced with deposits of silver. As a
result, it is visualized as metallic silver.
Procedure
 Deparaffinize and hydrate the section using distilled water
 Place the section in the silver solution (in a glass jar) and place it in bright light (or
in front of the 60 watt lamp). Place a mirror or a paper foil behind the jar so as to
reflect the light. Leave it standing for about one hour or until the calcium turns
black
 Rinse the section in distilled water
 Stain using 5 percent hypo solution for about 5 minutes
 Wash the section using tap water or rinse in distilled water
 Introduce the sample to nuclear-fast Red for about 5 minutes
 Wash using distilled water
 Dehydrate and mount for viewing

Observation (results)
 A black color indicates the present of calcium (calcium salts), red indicates
the nuclei while the cytoplasm will appear pink.
LIPIDS STAINING

 Lipid staining is a useful technique that is used for demonstrating

intracellular lipids in various tissue sections.
 This technique is dependent on dyes that are soluble in lipids. Some
of the most common dyes used include: Sudan VI, Sudan black, Oil
Red O, Nile blue
Principle
For this technique, the dye is more soluble in the lipid, which allows it to
be more demonstrated than in the vehicular solvent. The dyes used in
this technique are all interchangeable, which means that they can be
substituted for each other for the staining process.
 LIPIDS STAINING
Procedure
 Cut the sample to obtain sections of between 8
and 10 microns and air dry
 Rinse the section with 60 percent isopropanol
 Stain the section with the Oil Red O working
solution for about 15 minutes
 Rinse the specimen with 60 percent isopropanol
 Dip the section in Alum hematoxylin a few times in
order to stain the nuclei
 Rinse in distilled water
 Mount the specimen in water or in glycerin jelly
Observation
 Red color indicates the lipid while blue coloration
indicates the nuclei.
 Lipid staining technique is useful for showing the
normal distribution of lipids as well as disease-
related lipid accumulation.
PROTEINS AND AMINO ACIDS

 Proteins consist of polypeptides.

 Total proteins consisting of various amino acids can be stained with
such color reactions as mercuric bromphenol blue or naphthol
yellow S.
 Some basic proteins are stained with Fast green and gallocyanine
chrome alum.
 only some amino acids in proteins can be stained with specific dyes
such as tryptophane and tyrosin with Millon reactions, tyrosin with
diazotization-coupling, tryptophane with
pdimethylaminobenzaldehyde-nitrite, arginin with Sakaguchi
reaction.
 These color reactions for proteins can also be utilized for
quantitative studies by microspectrophotometry.
MERCURIC BROMOPHENOL BLUE
TEST FOR PROTEIN
PRINCIPLE:
Mercuric ions of the bromophenol blue solution react with acidic,
sulphydryl and aromatic residues of the protein to give blue colour.
PROCEDURE:
 Bring sections to water.
 Stain in the mercuric bromophenol blue solution for 2 hours at room
temperatue.
 Rinse sections for 5 minutes in 0.5% acetic acid.
 Transfer sections dirctly into tertiary butyl alcohol. Clear in xylene
and mount in DPX.
RESULT: Proteins—deep blue colour.
MILLON'S TEST FOR TYROSINE

 Principle When Millon's reagent, a mixture of mercurous and

mercuric nitrates and excess of nitric acid, is added to the protein
and the mixture is heated for few minutes, a white precipitate is
formed which may turn yellow and then red if the reacting protein
contains tyrosine. The reaction is specific for hydroxy phenyl groups
unsubstituted in the meta position.
 Method Hydrate sections, place them in a small beaker containing
the reagent and leave in an oven until low boil. Bring sections to
room temperature. Remove sections and wash in distilled water,
three times (wash each slide for 2 minutes). Dehydrate, clear and
mount in DPX. Repeat with fresh tissue smears and note the
diffrrence. Control: Iodination (vide section 2.7.5.). Result Tyrosyl
groups—red or pink.
Sakaguchi Test
The Sakaguchi reaction test involves the use of the Sakaguchi reagent. This
reagent is composed of 1-naphthol and sodium hypobromite and forms a
reddish compound when mixed with the sample containing Arginine.
The test is positive for any amino acid that contains the guanidine group
in Arginine. Therefore, the Guanidine group in an amino acid will react with
the α-Naphthol and alkaline hypobromite in the reagent to give of a red-
colored complex indicating the presence of such amino-acids
Procedure:
 Add 1 ml of the protein solution in to a test-tube
 Add 2 or 3 drops of 40 percent of sodium hydroxide, 2 drops of
ethanolic a-Naphthol and 5 drops of bromide water
 Mix the contents in the test-tube well
 If a red-color complex forms when shaking the contents, then it is
positive that the solution contains Arginine or a protein containing
Arginine.
NUCLEIC ACIDS AND DNA
Feulgen's Reaction
PRINCIPLE:
The most specific chemical reaction for nucleic acids well-known in
histochemistry should be the reaction of Feulgen (1924). This reaction
depends on the hydrolysis by hydrochloric acid of the purine-
deoxypentose linkage of the DNA in nuclear chromatin resulting in
aldehyde, which is colored purple by Schiff’s reagent
This method can be said to be divided in to two main parts:
 The first part of the procedure is the hydrolysis phase that involves
the use of 5N HCl, ambient temperature for 40 minutes. This step is
aimed at separately selecting 2 purine bases (adenine and
guanine) which are removed from the DNA molecule.
 The second step is the staining phase. The reagent used is preferred
because it is highly selective for DNA rather than RNA. Here, RNA
does not react because of the presence of hydroxyl on carbon 2 of
ribose, which prevents the acid (HCl) from hydrolyzing sugar. The
reaction is also precise for the localization of DNA given that
deoxyribose radicals are bound to phosphoric acid of the apurinic
acid molecule following the removal of purine bases.
 Some of the stains used for both DNA and RNA include: Methyl
green pyronin stain, Acridine orange
SMALL CELL LUNG CARCINOMA SPREAD STAINED WITH FEULGEN STAIN
 CARBOHYDRATES
 Sugars are classified into several categories, monosaccharides, disaccharides,
oligosaccharides and polysaccharides. Several procedures for demonstrating
sugars with color reactions based on the periodic acid-Schiff (PAS) reaction are
available.
PAS reaction (Periodic Acid Schiff)
 This is one of the most popular histochemical techniques for the detection of
carbohydrates in tissue
PRINCIPLE
In this technique, the periodic acid oxidizes tissue carbohydrates to produce
aldehyde groups. This group then condenses with the reagent to form a bright red
coloration to demonstrate the tissue component with carbohydrate attachments.
The diastase and a-amylase in the reagent act on the glycogen and depolymerize it
into smaller sugar units (maltose and glucose) which are then washed out of the
section.
Procedure
 wash in running tap water for
 Deparaffinize and hydrate the slide about 10 minutes
using distilled water
 Counterstain using Harris's
 Place the section in preheated diastate hematoxylin with acetic acid for
solution (at 37 C) for about an hour half a minute
 Wash the sample in running water for  Wash with running water
about 5 minutes
 Dehydrate with two changes using
 Place the sections in 0.5 percent absolute alcohol, clear with xylene
periodic acid solution for about 5 and mount to view
minutes
 Some of the other stains used for
 Wash the section in distilled water staining sacchrides include:
Lectins, Ruthenium red, Alcian
 Place the section in Schiff reagent for
blue
about 15 minutes
 Wash the section for about a minute in
0.55 percent potassium metabisulfite in
order to remove excess stains
FLUORESCENT STAINING
 Used in cytogenetic studies – IN SITU HYBRIDIZATION studies
 In situ hybridization was invented by Mary-Lou Pardue and Joseph G.
Gall.
 In situ hybridization (ISH) is a type of hybridization that uses a
labeled complementary DNA, RNA or modified nucleic acids strand
(i.e., probe) to localize a specific DNA or RNA sequence.
 DNA can be isolated from a portion or section of tissue (in situ) or if the
tissue is small enough (e.g., plant seeds, Drosophila embryos), in the
entire tissue (whole mount ISH), in cells, and in circulating tumor cells
(CTCs).
 In situ hybridization is used to reveal the location of specific nucleic acid
sequences on chromosomes or in tissues.
 DNA ISH can be used to determine the structure of
chromosomes. Fluorescent DNA ISH (FISH) can be used in medical
diagnostics to assess chromosomal integrity. RNA ISH (RNA in
situ hybridization) is used to measure and localize RNAs within tissue
sections, cells, whole mounts, and circulating tumor cells (CTCs).
Fluorescence in situ hybridization -
FISH
 Fluorescence in situ hybridization (FISH) is a molecular
cytogenetic technique that uses fluorescent probes that bind to
only those parts of a nucleic acid sequence with a high degree of
sequence complementarity.
 Fluorescence microscopy can be used to find out where the
fluorescent probe is bound to the chromosomes.
 FISH is often used for finding specific features in DNA for use
in genetic counseling, medicine, and species identification.
 FISH can also be used to detect and localize specific RNA targets
in cells, circulating tumor cells, and tissue samples.
Why fluorescent labelling?
 Greater safety
 Higher stability
 Ease of detection
PRINCIPLE:
 Light is emitted by an atom or molecule that has absorbed light or
electromagnetic radiation from another source.
 In absorption, high energy light excites the system, promoting electrons
within the molecule to transition from the ground state, to an excited
state.
 Here, the electrons quickly relax to the lowest available energy state.
 Once this state is achieved and after a fluorescence lifetime, the
electrons will relax back to ground state, releasing their stored energy
as an emitted PHOTON.
 Devices called fluorometers are used to measure fluorescence.
PROCEDURE

 the probe sequence, often a

piece of cloned DNA, is shown in
red. The target DNA—
chromosomes on a glass slide—is
shown in blue (in the right
column). Hydrogen bonds that join
the two strands of the DNA helix
are represented by black lines.
 PROCEDURE

 The first step in the process is to make

either a fluorescent copy of the probe
sequence or a modified copy of the
probe sequence that can be rendered
fluorescent later in the procedure.
 Ethidium bromide, fluorescein and green
fluorescent protein are common tags
used to label probe.
 PROCEDURE

 Before any hybridization can occur, both

the target and the probe sequences must
be denatured with heat or chemicals.
This denaturation step is necessary in
order for new hydrogen bonds to form
between the target and the probe during
the subsequent hybridization step.
PROCEDURE

 The probe and target sequences

are then mixed together and the
probe specifically hybridizes to its
complementary sequence on the
chromosome.
 If the probe is already fluorescent
(middle column), it will be possible
to detect the site of hybridization
directly. In other cases (left
column), an additional step may
be needed to visualize the
hybridized probe. Hybrids formed
between the probes and their
chromosomal targets can be
detected using a fluorescent
microscope.
APPLICATIONS

 FISH provides a powerful tool for identifying the location of a cloned

DNA sequence on metaphase chromosomes. Major role player in
HGP (Human Genome Project).
 FISH and other in situ hybridization procedures are important in the
clinical diagnosis of various chromosomal abnormalities, including
deletions, duplications, and translocations.
 identification of non-random chromosome rearrangements
 FISH is also used to compare the genomes of two biological species
to deduce evolutionary relationships.
SUMMARY

 Biological studies basically involve cellular analysis that can be

carried out analysing isolated cells (cytological techniques) or using
tissue sample (histological techniques).
 Sample preparation is the crucial step that involves isolation,
processing and storage depending on the type of analysis to be
carried.
 Staining the sample of interest makes the visualization of material
clearer and also helps in quantitative analysis.
 Techniques of staining varies based on visualization, quantitative
analysis, cytogenetic analysis and so on.
THANK YOU