staining Flashcards
ways to make things stand out
dyes& stains
chemical reactions
metallic deposits
what is a dye
a dye is an organic compound which contains
chromaphoric and auxochromic groups attached to benzene rings
chromophoric group : responsible for color
auxochromic group : capable of binding to the reactive groups in the tissue
chromagen
he colored part of a dye molecule
aromatic compounds containing chromophores
Chromophore
the arrangement of atoms within the chromogen that is responsible for light absorption in the visible spectrum
the double bonds in this arrangements of atomic linkages alternate with single bonds to form “ conjugated systems “
the bonding electrons are able to move from one atom to another exchanging the position of linkages, this is referred to as resonance
fluorochrome
a chromophore which absorbs UV light and emits light of a longer wavelength
Auxochrome or Colligators
responsible of attachment of a chromogen to a substrate
a side chain attached to a chromaphore group
- ionizable
- reacts to form covalent bonds with a substrate
- forms bonds with a mordant
- many dyes have more than one Auxochrome
auxochrome gives the compound the property of electrolytic dissociation
Chromophore groups
dyes aren’t classified but their color they are classified by their chromophore group
dyes with the same chromophore group don’t necessarily have the same color
certain atomic groupings are associated with color
color is caused by selective absorption of certain wavelengths of light so that the light transmitted from a given substances lacks these wavelengths
Auxochrome groups
allows chromagens to bind to tissue groups either electrostatically or by covalent bonding
they give the dye the ability to dissociate in the solution. this salt forming property gives the dye the affinity to attach to tissues
auxochromes in basic dyes: NH3+
auxochromes in acid dyes : OH or SO3-
lysochromes
not dyes in the true sense bc they lack auxochromes and do not ionize
they stain by selective or preferential solubility
- will color a substance in which they are soluble
fat staining is preformed in this manner
also referred to as simple solubility or selective solubility
colour index number
`dyes with the same name can have very different contents
and the same dyes can have different names
to avoid confusion the is a worldwide agreement of naming dyes with a standard numerical classification
this system is called Colour Index Number & is for the identification of pure dye* structures
5 digit #
allows for standardization of staining methods
biological staining commission
body responsible for standardization of biological stains
certification of a stain by the commission indicates:
1. A sample of the dye has been submitted for testing
2 Part of the sample is kept on file
3. The sample proves true to type by Spectrophotometric tests
4. The dye content is up to specification and is correctly indicated on the label.
5. It has been tested, by experts, in the procedures named and has been found
satisfactory.
6. No other batch of dye can be sold under the same Certification Number
Dyes which have been certified by the Biological Staining Commission should have the following information on the label: The name of the dye Lot number Dye content Colour index number
Dye content
recipes for dye solutions are based on the stain being 100% dye which si rarely the case
present dye/ new dye = gravimetric factor
gravimetric factor x amount called for
natural dyes
derived from plants, animals & insects
synthetic dyes
referred to as coal tar dyes
includes most of the dyes we use in histology
majority are derived from benzene today***
dyes are generally carcinogenic
vitals staining
when living tissue is stained
not as common
ex. reticulocyte staining in hematology
post-mortem staining
most staining is preformed on fixed tissue
direct staining
dye is directly applied to tissue to accomplish staining
dye has great auxochromes
place dye & substrate together
indirect staining
staining process which requires a mordant to achieve staining
substance that requires a mordant to stain isn’t considered a sye until mordant is added
a mordant is a metal with a valency of at least 2
- ‘dye’ is weak; has poor or low auxochromes
- add metal to form a dye - lake
trapping agent
Trapping agents are chemicals which inhibit the removal of dyes from tissue
Negative staining
refers to making an object visible by staining the background
rather than the object to be viewed
Leuco Compounds
Although chromophores differ from one another in a number of ways; they are all easily reducible, that is combine easily with hydrogen. The reduction process will break the chromophoric grouping and the dye will lose its color.
This colorless compound is called a leuco compound. The reduction is reversible under oxidizing conditions
hemosiderin “staining”
Hemosiderin forms the body’s iron stores. The iron is in the ferric state, and may be demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. The iron reacts with potassium ferrocyanide to form ferric ferrocyanide. This is an insoluble, blue compound known as
Prussian blue
metallic impregnation
Referred to as part of special stains but no dyes are actually involved instead,
metallic ions are deposited on selected fibres and cells to provide contrast.
often refer to these as silver stains. Some metallic compounds can be
reduced by tissues to the metallic state, producing an opaque, usually black
deposit
metallic impregnation using silver nitrate : one stage reaction
The tissue substrate is able to reduce silver compounds to a black deposit
directly.
called Argentaffin Reactions
metallic impregnation using silver nitrate: 2 stage reactions
tissue substrate is unable to reduce silver compounds to a black deposit
directly. The method requires the application of a second chemical known as
an extraneous reducer
Unreduced silver is deposited on the tissue fibres and reduced to a black
deposit in a second reaction. Such reactions are called Argyrophilic
orthochromic staining
Staining reactions that impart the same colour to the tissue as the stain itself
metachromatic staining
ome staining reactions result in the staining of certain tissue elements
a different colour than the original colour of the dye. This phenomenon is
known as Metachromasia
change in colour is due to polymerization of the dye molecules by the tissue
chromatropes
*****dyes used in these reactions are generally cationic and include Toluidine blue and Methylene Blue
polychromatic stain
single dye solution stains tissue components different colors without metachromasia being involved. The dye solution is not pure and therefore different tissue components selectively bind with different dye components producing different colors
anionic and cationic dye relationships
A dye with a net positive charge is called basic or preferably cationic.
A dye with a net negative charge is called acidic or preferably anionic
“Acid” or “basic” dye (anionic, cationic) does not refer to the pH of the dye solution. refers to the salt of a colour acid/base
In electrostatic staining the dye and substrate are of opposite net
charge. Anionic binds with Cationic.
acid dye
coloured anions, with sulphonic or carboxylic acid auxochromes
The acid component is coloured and the base is colourless
Acid dyes usually stain basic components, such as cytoplasm
Acid Dyes - Anionic - Negative Charge
basic dyes
These are coloured cations with NH2+ auxochromes
The basic component is coloured and the acid is colourless with
Basic dyes usually stain acidic components, such as nuclei
Basic Dyes – Cationic - Positive Charge
neutral dyes
Both the acidic and basic components are coloured.
They are prepared by combining aqueous solutions of Acidic and Basic dyes in specific proportions.
The resulting precipitate will be a Neutral dye
Neutral dyes are soluble only in Alcohol (Methyl)
The staining reaction for these dyes takes place at ~pH 7.2 - 7.4 (slightly
alkaline)
sulphonic group
can convert basic dye to acid dye
can convert an insoluble dye to a dye that is soluble in water
Amphoteric
has both neg and pos charged groups
charge will depend on the pH of the staining solution
basic auxochromes
Usually amines NH3
acid auxochromes
Derived from sulphonic or carboxylic acid or from phenolic hydroxides
progressive staining
staining until the desired staining intensity is achieved, then stopping
regressive staining
over staining a tissue then differentiating( decolorizing) until only the desired element & intensity of staining is achieved
decolorization vs differentiation
decolorization is technically removing all color
differentiation is removing color selectively to achieve the desired degree of colorization
differentiation of a mordant is accomplished in 1 of 3 ways
- cationic dyes are differentiated with weak acids (opposite cahrge)
- an excess of mordant will break the bond between the dye
- using an oxidizer
other means of differentiation
- solvents may be used to remove color
- other dyes may be used. this may be referred to as displacement
- blood smears stained with wrights stain are differentiated using buffer at pH
6.5, at this pH the buffer acts as a basic differentiator of eosin & an acidic
differentiator of methylene blue
what makes dye stick
Affinity
- the tendency of stain to move from solution into tissue
- forces influencing stain-tissue & stain-stain interactions
- van der Waals are the weakest & Coulombic are the strongest
physcial forces of affinity
capillary
osmosis
absorption
adsorption ( both physical & chemical)
capillary action
movement of liquid within a material against gravity as a result of surface tension
osmosis
movement of water through a membrane
Absorption
passing of dye molecules from the dye to the solution in the substance being dyed
fat staining involves non ionic dyes & is purely absorption
Adsorption
adhesion of molecules to a solid surface resulting in heavily high concentration of the molecules at the place of contact
chemical action & salt linkages can also play a part in the adhesion
physical considerations
size of dye particle
- large particles diffuse more slowly
porosity
-more porous substrates are easier to stain
interfacial effect
- dye concentration id higher at the surface of the solution & at the interface of the solution & walls pores
chemical affinity : Electrostatic attraction
also referred to as coulombic attraction
involves the net charge of dyes & tissue components in a given solution
acidic dyes are basophilic
basic dyes are acidophilic
proteins in the cell are amphoteric; they depend on the pH of the solution
- they have a net charge of 0 at their isoelectric point, if placed in a pH lower than that they become positively charged ( basic)
in a pH higher than that they become negatively charged ( acidic)
all of the following refer to the transfer of an electron from one to the other to form a bond
electrostatic attraction
electrovalent attraction
salt linkage
coulombic attraction
ionic bond
other chemical aspects of dye binding
hydrogen binding
hydrophobic binding
van der Waals forces
Covalent bonds
Electrovalent attractions
anionic dyes ( eosin) stain cationic tissue components ( proteins at a pH below 6)
cationic dyes ( hematin) stain anionic tissue components such as nucleic acids
Salt linkage
Coulombic or electrostatic attractions are salt linkages
the amount of non dye salt in the solution will effect the amount of staining which occurs
Van der Waals forces
weak electrostatic forces ( short distance)
attractions between the electrons of one atom and the nucleus of another
ex. elastin or some mordant staining
factors influencing dye binding
ionic strength
dye concentration - greater amounts of dye may be bound from higher concentrations but is limited by the number of dye binding sites
fixation of tissue- generally following fixation affinity for dye increases more than unfixed tissue bc binding groups are more accessible to the dye
temperature - increase will cause an increase in staining ( increased diffusion rates & protein swelling
pH of the solution - affects dissociation of dye
thickness of tissue- more tissue binds more dye
dye staining
H& E
most “special stains”
microbiology
hematology
other “ staining methods “
metallic impregnation
selective solubility
immunohistochemistry
enzymatic stains
immunohistochemistry
the main reagent is an antibody- generally monoclonal
may pinpoint the source tissue of a malignancy
amine precursor & uptake decarboxylation (APUD) cells
diagnosis of neuroendocrine tumours
these cells demonstrate Argyrophil and/or Argentaffin reactions
- however now IHC is used as it provides more specific information
direct immunohistochemistry
fluorescein isocyanate (FITC) is applied to a frozen section containing glomeruli & examined using fluorescent microscope this enables the detection & localization of immunoglobulins, complement components & fibrin
older and less sensitive than indirect method
is used for renal & skin biopsies
indirect immunohistochemistry
testing for autoantibodies is usually done in this manner
patients serum is added to a tissue sample containing known antigens to test the patient for the presence of antibodies to these antigens
patients antibody is the primary antibody & attaches to the antigen in the tissue, secondary antibody is the antihuman immunoglobulin which attaches and can be detected due to its fluorescent tag
indirect can also mean that 2 antibodies are used to detect an antigen in the patients tissue, this can be performed with a two step or three step method
generally are enzymatic methods & detection can be performed using a light microscope
enzymatic staining
testing for biological catalysts
hematology & histology
muscle biopsy
lymphoma & leukemia studies
enzymatic tests
muscle biopsy snap frozen in liquid nitrogen
- with or without isopentane
lymph node carefully sliced
- touch preparation utilization
enzymatic staining
special handling of specimens
touch preparations & frozen sections
DONT FIX SPECIMEN!
enzyme catalyses reaction with substrate
colored end product used for detection
enzymatic testing varibles
pH
Temperature- may destroy enzymes
concentration of substrate & the enzyme
inhibitors
- heat, agitation, exposure to acid fixatives, others
activators
- divalent cations: magnesium, manganese & calcium
types of enzymatic reactions
Azo dye method
- substrate contains one or more napthol compounds. enzyme activity releases the napthol which joins with a diazonium salt. The diazonium salts form reaction products to produce an azo dye which marks the reaction site
Indoxyl methods
- indoxyl substrates release indoxyl radicals that are rapidly oxidized to insoluble indigo blue
Metal salt methods
- used when one of the products of enzyme activity on the substrate is an acid. The acid is captured by a metal ion in the incubation medium & precipitated. A series of subsequent reactions ultimately produce an insoluble colored salt at the reaction site
oxidation - reduction reactions
- hydrogen is transferred from a substrate to a tetrazolium salt. the salt is reduced to insoluble purple formazam pigment at the site of activity
