biological staining review

Question 1

How does pH effect staining

Answer 1

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.

Question 2

how does temperature effect staining

Answer 2

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.

Question 3

how will dye concentration effect staining

Answer 3

The greater the concentration of dye the faster the
reaction will occur but only up to the point that all
binding sites are occupied.

Question 4

How does salt concentration effect staining?

Answer 4

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

Question 5

Differentiate between regressive and progressive staining

Answer 5

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

Question 6

Compare the formula of progressive & regressive

Answer 6

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.

Question 7

What is a true dye? Give example

Answer 7

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

Question 8

What is a color index number?

Answer 8

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

Question 9

Part of a dye molecule required for electrostatic binding

Answer 9

Auxochromes

Auxochromes or colligators are ionizable groups which react to form a covalent bond with a substrate.

Many dyes have more than one auxochrome

Question 10

Define chromaphore

Answer 10

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

Question 11

Terms

Answer 11

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

Question 12

What is a chromagen?

Answer 12

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.

Question 13

What is a lysochrome?

Answer 13

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

Question 14

What is a true histochemical reaction? An example?

Answer 14

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

Question 15

What is direct and indirect staining?

Answer 15

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

Question 16

To differentiate anionic dye staining use this solution

Answer 16

A weak cationic solution

Other possibilities are use of mordant or oxidizer

Question 17

What is an anionic dye?

Answer 17

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

Question 18

Formalin fixation will cause reduced staining of this type.

Answer 18

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.

Question 19

More on fixation and staining

Answer 19

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

Question 20

What is a leuco dye?

Answer 20

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

Question 21

What is a mordant?

Answer 21

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

Question 22

A non-dye method to make tiny fibers visible is called

Answer 22

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

Question 23

Are cytoplasmic proteins basophilic or acidophilic?pH6.0

Answer 23

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

Question 24

What would you call staining of live cells?

Answer 24

Vital or supravital staining
Example :
Reticulocyte stain
Most staining is not vital but post mortem staining 
eg. liver

Question 25

What is metachromatic staining?

Answer 25

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

Question 26

Decolorization or differentiation, what’s the difference?

Answer 26

Decolorization typically means to remove all the color.

Differentiation means removing the excess staining and leaving the desired staining

Question 27

What is negative staining?

Answer 27

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

Question 28

A staining theory that involves both physical & chemical actions

Answer 28

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

Question 29

What is a trapping agent?

Answer 29

A chemical which binds with a dye and makes it more resistant to solvent removal.

Example, Iodine with crystal violet in the gram stain

Question 30

What is a gravimetric dye calculation?

Answer 30

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

Question 31

Explain isoelectric point of protein

Answer 31

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

Question 32

Factors which influence the rate of an enzyme reaction:

Answer 32

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

Question 33

What is the pivotal reagent for IHC?

Answer 33

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

Question 34

How would a lymph node for lymphoma diagnosis be handled?

Answer 34

Careful dissection and touch preparations are prepared BEFORE the tissue is fixed.

Question 35

How is a muscle biopsy handled?

Answer 35

Received fresh or wrapped in saline soaked
gauze on ice

Snap frozen in liquid nitrogen

Frozen sections are prepared for enzymatic
staining