biological staining review Flashcards
How does pH effect staining
Most staining involves attraction of dye to tissue
components based on charge. Acid to base & vice
versa.
The charge on proteins in tissue rely on the pH of
its surroundings. There is a optimal pH at which
tissue will not be over or understained.
how does temperature effect staining
Chemical reactions speed up with an increase in
temperature. Therefore staining will take place at a
faster rate with increased temperature.
Also, at temperature above 37°C, tissue swells
and becomes more penetrable.
how will dye concentration effect staining
The greater the concentration of dye the faster the
reaction will occur but only up to the point that all
binding sites are occupied.
How does salt concentration effect staining?
Salts can have both positive and negative effects
on staining.
Salt ions other than the dye can bind with tissue
components making than unavailable for reaction
with the dye.
In other cases, salts may act as catalysts to
increase the speed of the staining reaction
Differentiate between regressive and progressive staining
Progressive is the most common staining type.
Dye comes in contact with tissue & remains until
the desired intensity is achieved
Careful attention to both the dye & mordant
concentration is required
In a regressive stain, tissue is overstained and
then stain is removed until the desired tissue
components are stained to the required degree.
Microscopic control is best
Compare the formula of progressive & regressive
Progressive hematoxylins are generally less
concentrated and work slowly to avoid
overshooting the endpoint. Progressive stains
contain acid
Regressive hematoxylins are more concentrated
some can achieve overstaining in less than a
minute.
What is a true dye? Give example
An organic compound containing chromophoric and auxochromic groups
Eosin
Orcein
Carmine
Give an example of staining techniques which work without true dyes.
Fat stains
Iron stains
What is a color index number?
A five digit number used to indicate a dye with an
exact chemical and structural composition
Only applies to pure dyes, neutral dyes such as
the Romanowsky dyes therefore have no CI
number
This number helps prevent confusion caused by
dyes having more than one name
Part of a dye molecule required for electrostatic binding
Auxochromes
Auxochromes or colligators are ionizable groups which react to form a covalent bond with a substrate.
Many dyes have more than one auxochrome
Define chromaphore
An arrangement of atoms within a chromagen molecule that cause the compound to be coloured
Double bonds alternate with single bonds to form conjugated systems. Bonding electrons are able to move from one atom to another (resonance).
These properties cause specific light absorption in the visible range. The result is colored light transmission
Terms
Stain is an organic compound composed of a benzene ring, a chromophore and an auxochrome group
Benzene is a organic colorless solvent.
Chromophore is the molecule that gives color to benzene
Chromogen isn’t a stain, just a colored molecule. It is made up of the benzene and the chromophore
Auxochrome ionizes the chromogen, gives it a charge. This helps the chromogen bind to substances
Acid stains- are anionic. Their chromogen exhibits a negative charge. These type stains have an affinity for positive components of a cell
Basic stains are cationic. Their chromogen exhibits a positive charge. These types of stains have a strong affinity for the negative components of a cell
What is a chromagen?
The colored part of a dye molecule.
The chromaphore is found within the chromagen.
Auxochromes are side chains of the chromagen that
bind chemically with a substrate.
What is a lysochrome?
A colored substance with or without auxochromes
Stains a substance in which they are soluble
Fat staining is performed in this manner
Give examples:
- Oil Red O
- Sudan Black
Simple solubility- Selective solubility
What is a true histochemical reaction? An example?
A chemical reaction between reagents and tissue elements produce a colored, localized reaction.
Iron staining is performed in this manner. Potassium ferricyanide in the presence of HCL reacts with hemosiderin to produce a blue product
called prussian blue
What is direct and indirect staining?
Direct staining occurs with true dyes. Add the dye, the auxochrome binds with the target substance in the tissue and staining of that substance occurs
Indirect staining involves dyes with little affinity for tissue. The addition of a metal mordant forms a dye-lake capable of binding with target substances
To differentiate anionic dye staining use this solution
A weak cationic solution
Other possibilities are use of mordant or oxidizer
What is an anionic dye?
A dye with negatively charged auxochromes
What is the most common acidic auxochrome?
COO- carboxyl group
Derived from carboxylic acid
Sulphonic and phenolic groups are also possible
Most common cationic auxochrome?
NH2+ derived from amino group
Formalin fixation will cause reduced staining of this type.
Anionic
Formalin binds with the amino group on proteins
and makes less of them available for anionic
staining.
Give an example
Eosin
NBF will also cause the cytoplasm to take up more
Hematoxylin due to the change in the charge on
the cytoplasmic proteins.
More on fixation and staining
Acid fixatives such as unbuffered formalin or
Zenker solution may reduce the basophilic charges
of the nucleus & therefore Hematoxylin uptake in
nuclei may be reduced
Potassium dichromate reacts with carboxyl &
hydroxyl groups. Nuclear staining will be weaker
and eosin staining will be stronger
What is a leuco dye?
A dye solution is reduced and made colorless
The colorless dye is allowed to react with a
specific tissue component
The chromophoric group is then restored through
oxidation
The color is restored and stains the tissue element
in question
What is a mordant?
A mordant is a substance, usually a metal, which
provides a link between the dye and the tissue.
When a mordant is combined with a dye, what is it
called?
A dye lake
A non-dye method to make tiny fibers visible is called
Metallic impregnation aka silver stains
Examples of substances stained.
Reticulin fibers, nerve cells, fungi, bacteria
There are two types of reaction involved:
Agentaffin – the substance stained is capable of
reducing silver
Argyrophil- substance cannot reduce silver
therefore an external reducer (developer) is
required
Are cytoplasmic proteins basophilic or acidophilic?pH6.0
Acidophilic
This means that cytoplasmic proteins given the
right solution pH will bind with anionic (acid or
negatively charged) dyes.
This also means that the cytoplasmic proteins are
cationic (positively charged)
An example of an anionic dye
Eosin
What would you call staining of live cells?
Vital or supravital staining Example : Reticulocyte stain Most staining is not vital but post mortem staining eg. liver
What is metachromatic staining?
Certain cationic dyes – toluidine blue and
methylene blue stain specific tissue elements
known as tissue chromatropes purple or red
although these dyes are blue.
Examples
Cartilage, mast cells, mucins, nucleic acids and hyaluronic acid
When blue dyes stain blue we call the staining:
Orthochromic
Decolorization or differentiation, what’s the difference?
Decolorization typically means to remove all the color.
Differentiation means removing the excess staining and leaving the desired staining
What is negative staining?
Using a dark substance such as India ink to demonstrate an object by staining the background rather than the organism. The shape of the object
being stained is a clear space between dark dye particles.
An example is staining a CSF in microbiology to look for Cryptococcus neoformans
A staining theory that involves both physical & chemical actions
Adsorption
Most staining involves the chemical bonding caused by Coulombic attraction, however there is also some degree of staining caused by physical factors as well.
Capillary action, van der Waals, interfactial effect for example
What is a trapping agent?
A chemical which binds with a dye and makes it more resistant to solvent removal.
Example, Iodine with crystal violet in the gram stain
What is a gravimetric dye calculation?
A calculation meant to ensure that the dye content
in new batch of dye is equivalent to the dye
content used previously for a particular stain.
How is it calculated?
Concentration of dye used up till now divided by
the concentration of the new dye.
Multiply the result times the amount of dye needed
in your recipe.
See your notes
Explain isoelectric point of protein
For most proteins this is around pH 6
How does isoelectric point effect staining?
While neutral there is no attraction between protein
and anionic or cationic dyes. Changing the pH of the
solution changes the charge on the protein.
Below pH 6 proteins are acidophilic
The majority of staining uses solutions which are
acidic
Eosin for example stains at pH 4.6-5.0
Factors which influence the rate of an enzyme reaction:
Temperature will speed reaction but most enzymes
are destroyed at 56°C or higher
pH- determined by the pH at which the enzyme
functions with a particular substrate
Concentration of the enzyme & substrate
determine the speed of the reaction
Inhibitors
Heat, agitation, exposure to acid, fixatives,
activators
Divalent cations; magnesium, maganese, calcium
What is the pivotal reagent for IHC?
An antibody, usually monoclonalName some applications of IHC
Helps provide a more exact diagnosis of cancer
May help determine appropriate treatment
May help ID viral infection
Demonstrates APUD cells which were formerly stained using metallic impregnation
How would a lymph node for lymphoma diagnosis be handled?
Careful dissection and touch preparations are prepared BEFORE the tissue is fixed.
How is a muscle biopsy handled?
Received fresh or wrapped in saline soaked
gauze on ice
Snap frozen in liquid nitrogen
Frozen sections are prepared for enzymatic
staining
