THEORIES OF STAINING Biological Staining Structural Components (Nature) Of Stains Staining Mechanism Metachromasia Types Of Staining Staining of Paraffin Section A stain is any colouring organic compound that combined with another substance imparts a colour to that substance. The term ‘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. The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2). The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin, safranin, eosin, etc.FACTORS INFLUENCING METACHROMASIA

1. THEORIES OF STAINING

2. CONTENT
1. Biological Staining
2. Structural Components (Nature) Of Stains
3. Staining Mechanism
4. Metachromasia
5. Types Of Staining
6. Staining of Paraffin Section

3. STAINING
A stain is any colouring organic compound that combined with another substance
imparts a colour to that substance.
The term ‘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.
The stains used for bacteria are aniline dyes they are derived from aniline (C6H5NH2).
The most commonly used aniline dyes are crystal violet, methylene blue, basic fuchsin,
safranin, eosin, etc.
https://monographs.iarc.fr/ENG/Monographs/vol99/mono99-7.pdf

4. GENERAL THEORY :
Purpose of Staining:
(a) To see organism better
(b) To differentiate one organism from another
(c) To determine particular structures

5. BIOLOGICAL STAINING
DYES ARE CLASSIFIED INTO TWO GROUPES:
1. Natural dyes:
a. Haematoxylin -----from plant
b. Carmine -----------from female cochineal bug
c. Orcein --------------a vegetables dye extract
2. Synthetic: these are derived from hydrocarbon benzene
http://www.biologydiscussion.com/micro-biology/staining/stains-meaning-purpose-and-components-microorganisms/55080

6. STRUCTURAL COMPONENTS (NATURE) OF STAINS:
Stains (dyes) usually have complex molecular structure and are chiefly benzene
derivatives.
A stain consists of three constituents: the organic compound containing a
• Benzene ring
• Chromophore
• Auxochrome
http://www.biologydiscussion.com/micro-biology/staining/stains-meaning-purpose-and-components-microorganisms/55080

7. A. CHROMOPHORE:
Certain chemical group introduced into benzene ring by substitution induce colour
to the compound -reffered as chromphore – resultent structure –chromogen
The absorption of electromagnetic radiations in the UV and visible regions by a
molecule causing electronic excitation where electron moves to higher electronic
energy level from a lower.
A covalently unsaturated group responsible for absorption in the UV or visible
region is known as a chromophore.
E.g:
Active chromophore group -C=C-
nitro grouping -NO2-
azo coupling N=N

8. B. AUXOCHROME
The word auxochrome is derived from two roots. The prefix auxo is from auxein,
and means increased. The second part, chrome means colour, so the basic meaning
of the word auxochrome is colour increaser.
This word was coined because it was noted originally that the addition of ionising
groups resulted in a deepening and intensifying of the colour of compounds.
Auxochromes are groups which attach to non ionising compounds yet retain their
ability to ionise and absorbance of the resulting compound.
–NH3 –COOH –HSO3 –OH
http://www.biologydiscussion.com/micro-biology/staining/stains-meaning-purpose-and-components-microorganisms/55080

9. Thus a stain may be defined chemically as an organic compound containing
both chromophore and auxochrome groups linked to its benzene ring.

10. The ability of a stain to bind macromolecular cellular components such as
proteins or nucleic acids depends on the electrical charge found on the
chromogen portion, as well as on the cellular component to be stained.

11. • when benzene of an organic colourless solvent binds to the nitro group of
chromophore, it results in a yellow coloured compound called
trinitrobenzene
• Here three hydrogen atoms in the benzene molecule are replaced by three
nitro groups.
• Trinitrobenzene is a chromogen but not a stain.

12. • Another hydrogen atom is replaced by an auxochrome group, such as OH, the
compound known as picric acid (trinitrohydroxybenzene) is formed.
• The picric acid is capable of ionization or electrolytic dissociation to form salt
that binds to opposite-charged biological substance
http://www.biologydiscussion.com/micro-biology/staining/stains-meaning-purpose-and-components-microorganisms/55080

13. 1. ELECTROSTATIC BONDING
• The affinity between opposite ionic groups of dye and tissue helps in staining.
• Dyes are classified as acid or basic
• For example, eosin is an acid dye with an affinity for the basic protein of cytoplasm
whereas methyl green is a basic dye which has an affinity for the phosphate groups of
deoxyribonucleic acid of the nucleus.
• Salt linkage and ionic binding are alternative terms to electrostatic binding; the correct
name for the forces involved is Coulombic attraction.
STAINING MECHANISM
Culling’s textbook – Staining and Impregnation

14. Acids have a negative charge(-ve)
• They attach to positive charges, especially hydrogen (H+).
• In an electrical field they migrate to the anode. They are
anions.
• Tissues carrying a positive charge will attract dyes with a
negative charge (i.e. acid dyes); and are termed
acidophilic.
• Examples : phosphates of nucleic acids
sulphate groups of acid mucopolysaccharides.

15. Bases have a positive charge (+ve)
• They attach to negative charges especially hydroxy groups
(OH-).
• They migrate to the cathode. They are cations.
• Tissues carrying a negative charge will attract dyes of
positive charge and are termed basophilic.
• Examples : amino acid such as lysine and arginine

16. 2. HYDROGEN BONDING
• Hydrogen being of single valency, can only bond to one
other atom , usually those of strong electronegative
charge.
• Hydrogen bonding
• Weak
• Occur readily in water
• Will occur between the dye and the water it is
dissolved in
• Water will also compete for hydrogen bonding sites
on the tissue
• Hydrogen bonding is of significance in alcoholic dye
solution

17. 3. VAN DER WAALS FORCES
• These intermolecular forces are polar attractions.
• They are weak and are effective over a very short
distance.
• Attraction are between dipoles, that is molecules
possess separated positive and negative charges.
• Eg : staining of elastic fibers by orcein here elastic
fiber is a hydrophobic protein and orcein is a large
molecule with strong dipole

18. 4. COVALENT BONDING
• Covalent bonding involves sharing electrons
• Eg. In water each of two hydrogen atoms shares
an electron with oxygen, and oxygen atom
likewise shares the two hydrogen electrons.
• Here 2 atoms share 2, 3 or more electron pairs,
leading to multiple covalent bond.
• Significance in mordant dying

19. 5. HYDROPHOBIC BONDING
• In an attempt to isolate themselves from surrounding water
molecules, reactive hydrophobic dye molecules will
become bound to reactive hydrophobic tissue groups.
• This reaction is possible between dyes in aqueous solution-
alcohol in the water will form hydrogen bonds with the
water thus inhibiting hydrophobic effect.

20. 6. DYE AGGREGATION
• Dye molecules –have affinity for each other.
• Aggregated dyes penetrate less easily in the tissue than the dispersed dye
• Factors influencing dye aggregation
• large molecular size
• Increased concentration
• Ionic strength
• Low temperature
• Useful in case of metachromasia

21. 7. TISSUE PERMEABILITY
• Degree of dye penetration will depend on the permeability or porosity of the
tissue.
• Highly permeable tissues are more easily stained and decolorized than
poorly permeable tissues.

22. METACHROMASIA
• A dye which has the ability to change its color without changing its chemical
structure is said to be metachromatic.
• The physical changes that bring about this color change are a specialized,
orderly form of dye aggregation.

23. • Toluidine blue - low conc- nucleus - blue
- high conc- cartilage - purple
http://ocw.tufts.edu/Content/15/coursehome/342521/342524/342548
Section of loose CT stained with
toluidine blue and demonstrating both
orthochromasia (bright blue color) and
metachromasia (dark purple color)

24. FACTORS INFLUENCING METACHROMASIA
• Increased concentration of the dye
• Decreased temperature
• Aqueous solvent
• pH

25. TYPES OF STAINING
A. PROGRESSIVE STAINING
• Tissue section is immersed in a dye bath until such time as only the desired
structures are stained.
• Difficult process to control
• E.g Mayer’s Haematoxylin: first stains the nuclei

26. B. REGRESSIVE STAINING
 Tissues are over stained and the excess dye is removed until the desired tissue
component is selectively stained
• The dye is selectively removed from unwanted tissue groups – differentiation
• E.g In H/E staining, HCl is used as a differentiator

27. MORDANTS
• Term refers to a substance which acts as an intermediary between dye and tissue
• Advantage of dye mordent tissue complex is that it is virtually insoluble in most
fluids used in biological staining.
• Mordants are used in hematoxylins which makes it a strong basic dye, cationic
metal binding to both dye and tissue.

28. ACCENTUATORS
• Increase the selectivity or staining power of the dyes
• Effect of staining is due to change in pH of the staining solution
• Eg-potassium hydroxide in Loeffler’s methylene blue, phenol in carbon fusion.
• Accentuators when used in impregnation of nervous tissue with metallic salts is
called accelerators.

29. PARAFFIN SECTION
The basic steps in staining and mounting paraffin sections are as follows:
1. Deparaffinisation
2. Hydration
3. Staining
4. Dehydration
5. Clearing
6. Mounting
http://www.rajswasthya.nic.in/RHSDP%20Training%20Modules/Lab.%20Tech/Histo/Chapter%206.pdf

30. 1. DEPARAFFINISATION
• Removal of wax is done with xylene.
• At least 2 to 3 changes in xylene are given for about 3-5 mins to
remove the wax completely.
• Sections of this stage should appear clear and transparent.

31. 2. HYDRATION
• Most of the stains used are aqueous or dilute alcoholic solutions.
• The hydration is done with graded alcohol for higher concentration to lower
concentration.
• First change is made to absolute alcohol or acetone followed by 90,70% alcohol
and finally distilled water.
• Sections now should appear opaque.
• Presence of any clear areas are indicative of the presence of xylene.
• To remove this xylene, sections should be returned to absolute alcohol and
rehydrated.

32. 3. STAINING
• The most common stain applied for histological study is Haemotoxylin and Eosin.
• Various types of haemotoxylin formulations are used.
• Certain stains use strong chemicals e.g. ammonia. Sections tend to float off the
slides in such stains. This can be prevented by coating the selections by a thin layers
of celloidin (trimethylpyridine)
• For this sections are returned to absolute alcohol and then dipped in a dilute solution
of celloidin and finally hardened in 70% alcohol.

33. 5. DEHYDRATION AND CLEARING
• Dehydration is done is graded alcohols or acetones from 70% to absolute alcohol or
acetone.
• Dehydrating alcohol and acetones can remove some of the stains. Time has to be
suitably modified to minimize fading of stains.
• Since alcohol and acetone are miscible in xylol, it is used for clearing the sections.
• Any sections from which water has not been completely removed would give a milky
appearance after the first xylene.
• Such sections should be returned to absolute alcohol and the process should be repeated.
• Mounting is done after 2nd or 3rd xylene.

34. 6. COVERSLIPPING AND MOUNTING
Once the sections are clear. Do not let the section go dry before
mounting
1.Hold the slide between the thumb and the forefinger of one hand
and wipe with a clean cloth both ends of the slides. Look for the
engraved number to make sure the side the sections is present.
2.Clean carefully around the section and lay on a clean blotting paper
with section uppermost along with appropriate coverslip which has
already been polished.

35. 3. Place a drop of mountant on the slide over coverslip. Amount of mountant
should be just enough. Invert the slide over the coverslip and lower it so that it
just adheres to the cover slip , quickly turn the slide over and lay it on a flat
surface to allow the mountant to spread. Do not press or push the slide.
4. After the mountant has spread to the edge of the coverslip wipe around it for
neatness. If proper care has been taken there should be no air bubbles. If many
are present, slide should be returned to the xylol to remove the coverslip. It
will slip off and remounting is done. No attempt should be made to pull the
coverslip. Slight warming of the slide from below will make the small air
bubbles to escape from the slide of the coverslip.
5. Coverslip should be in the center of the slide with neatly written label on one
slide.

36. MOUNTANTS
Histological sections which need to be examined for any length of time or to be
stored, must be mounted under a cover-slip.
There are two types of mounting media :
1.Aqueous media - Used for material which is unstained, stained for fat, or
mechanically stained.
2.Resinous media - For routine staining.

37. A. AQUEOUS MEDIA
• These are used for mounting sections from distilled water when the stains
would be decolorised or removed by alcohol and xylene, as would be the
case with most of fat stains (Sudan methods). Gome stains,
• e.g. methyl violent, tend to diffuse into medium after mounting.
• Aqueous mounting require addition of bacteriostatic agents such as phenol,
crystal of thymol or sodium merthiolate to prevent the growth of fungi.

38. Permanent seal - After mounting the cover slip can be ringed by clear nail
polish for storage.
Following are some of the commonly used aqueous mounting media:
1. Apathy's medium (R.I- 1.52)
Medium for mounting sections for fluorescent microscopy.
2. Farrant's medium (R.I. 1.43)
Recommended for fat stains.
3. Glycerine jelly (R.I. 1.47)
Routine mountant for fat stains.
4. Highman's medium (R.I. 1.52)
Recommended with the metachroamtic dyes especially methyl violent.

39. B. RESINOUS MOUNTING MEDIA
Natural or synthetic resins dissolved in benzene, toluene or xylene. In case they become too
viscous they may have to be diluted with xylene.
Following are some of these media.
1. Canada balsam - Natural resin (R.I. - 1.52)
It is used as 60% resin by weight in xylene. H.&E stained slides are fairly well preserved
but basic aniline dyes tend to fade and prussian blue is slowly bleached. Slides take few
months to dry.
2.D.P.X. (R.I. 1.52)
Polystyrene resin dissolved in xylene as a 20% solution. It is most commonly used.
3.There are many other synthetic resins sold under various trade names e.g. Coverbond
(R.I. 1.53), H.S.R. (Harlew synthetic Resin), Histoclad (R.I. - 1.54), Permount (r.I. 1.54),
Pro-Texx (R.I. 1.495).

40. CRITERIA OF ACCEPTABLE MOUNTING MEDIA
1. Refractive index should be as close as possible to that of glass i.e. 1.5.
2. It should not cause stain to diffuse or fade.
3. It should be crack or appear granular on setting.
4. It should be dry to a nonsticky consistency and harden relatively quickly.
5. It should not shrink back from edge of cover-glass.
6. It should be free flowing and free bubbles.

41. TOLUIDINE BLUE STAINING:
Method:
Slide should be placed in 90% alcohol for a second or two
Transfer to absolute alcohol for a second or two
Transfer to xylene and agitate the slide until the section is clear ( about 2 Secs)
Transfer to absolute alcohol for a second or two
Transfer to 90% alcohol for a second or two
Transfer the slide rack, flood with 1% toluidine blue, and leave for about 1 Minute
Rinse rapidly in water, transfer to a pad of filter paper, and blot firmly
Flood with 90% alcohol and blot firlmy
Flood with absolute alcohol and blot firmly
Flood with xylene , blot firmly and , if the section is clear , mount under a coverslip with
balsam or DPX. If the section is not completely clear after the frst application of xylene .
Flood the slide with xylene and blot a second time, and repeat until the section is clear,
when it is mounted

42. REFERENCE:
1. Dyes and Pigments: Their Structure and Properties - Springer
2. https://www.scribd.com/doc/214111849/Staining-theory-pdf
3. http://www.rajswasthya.nic.in/RHSDP%20Training%20Modules/Lab.%20Tech/Histo/Chapter%206.pdf
4. https://monographs.iarc.fr/ENG/Monographs/vol99/mono99-7.pdf
5. http://www.biologydiscussion.com/micro-biology/staining/stains-meaning-purpose-and-components-
microorganisms/55080
6. Culling’s textbook – Staining and Impregnation

43. Describe why sections need to be coloured with dyes.
Staining is needed to give contrast between different components of the tissues and allow examination by light microscopy.
Describe how dyes bind to tissues.
Dyes bind by forming bonds with tissue components. Ionic and hydrogen bonding and van der Waals forces are probably all involved. Ionic staining
is the most important and distinguishes between basophilic and acidophilic tissue components. Hydrogen bonding and van der
Waals forces are less important but probably play a role in selectivity.
Describe the use of mordants in staining.
Mordants are metal salts that help bind some dyes to tissues . Haematoxylin is the most important mordanted dye.
Define metachromasia and give examples of its use.
Metachromasia produces a different colour in a tissue component to the colour of the dye solution. Toluidine blue is blue in solution but stains mast
cell granules red.
Describe the main properties of haematoxylin and staining using haematoxylin solutions.
Haematoxylin is a natural dye that requires oxidation to haematein before use as a stain. Haematoxylin is a mordanted dye that can stain many
different elements in tissue depending on the mordant used. Using different mordants it can be used to stain nuclei, connective tissue fibres, nerve
cells, muscle striations and mitochondria. It is usually used regressively.
Describe the use of silver as an impregnating metal.
Silver solutions are easily reduced producing a dense black deposit and this reduction is autocatalytic. In argentaffin reactions, no extra reducing
agent is needed, but argyrophil reactions require the addition of a reducing agent.
Describe the reasons for mounting tissues and outline the types of mounting media.
Mounting media and coverslips not only protect the specimen but also make it translucent, making examination easier. Mounting media may be
resinous (organic-based) or water-based solvents.

44. THANK YOU