Adhesives (adhesion may come before staining and after staining that will be mounting)

 Used in order to promote adhesion of sections to slides (tissue attached to the slide) (Adhesion seldom performed
in the lab) (causes mucosal membrane that tends to slip down from the slide as you process it)
 Spread thinly and evenly on a clean grease-free glass slide, which is then gently approximated to the end of the
ribbon and drawn upwards in a near vertical motion.
 Aside from being grease-free, the slide must also be dust-free (dust prevent proper diagnosis)
 If staining is to include antigen retrieval (IHC (Immunohistochemistry), enzyme pretreatment ISH (In-Situ
hybridization = attachment of certain element towards the other to form a double stranded molecule.
Hybridization in DNA is also known as base pairing. F = Fluorescence), or prolonged incubation steps,
charged slides or an adhesive must be used.
 Adhesives are not necessary for routine staining, provided that the slides are clean and free from grease.
 However, they are essential for methods that require exposure of sections to acids and bases or alkalis (especially
in cases of ammoniacal silver solutions) during staining.
***If clean grease-free slides are used and sections are adequately dried, the sections will not float off during staining
and adhesive will not be necessary.
***There are still certain instances when sections may float from the slide:
 For urgent cryostat sections to be submitted for immunocytochemistry (2 types of stain used in cryostat = H&E
and Polychrome methylene blue)
 For central nervous system tissues
 For tissues containing blood clots
 For tissues which have been decalcified
 When sections are to be subjected to high temperatures
 The most commonly used adhesive is albumin.
 It is prepared by mixing equal parts of glycerin, distilled water and white of eggs (also known as glair), then
filtered through coarse filter paper and a crystal of thymol (antifungal) is added.
 One disadvantage of using albumin is that it tends to retain some of the stain and gives a dirty background and
it also cover proper sites for diagnosis
 Thymol resistant organisms (grown and multiply in binary fission) growing in the adhesive have been known to
contaminate gram-stained sections and cause confusion during microscopic examination.
 Poly-L-Lysine, also a favorite adhesive, can be bought as a 0.1 % solution and further diluted (1 in 10 with
distilled water) when ready to use.
 Sections are coated with this dilute poly-L-lysine and allowed to dry.
 With time, the adhesive ability of this substance slowly loses its effectiveness.
 Therefore, the coated slides should be used within a few days. (3 days)
 3-APES is a better section adhesive and coated slides can be stored for a long time (one of the best in cytology
adhesives) APES = aminopropyltriethoxysilane
 Slides are dipped in 2% APES in acetone drained then dipped in acetone, drained again and finally dipped in
distilled water.
 It is invaluable in cytology particularly for cytosine preparation of proteinaceous or bloody material.

Commonly used adhesives:

 Mayer’s Egg Albumin
 Dry albumin
 Gelatin
 Starch paste
 Plasma (Human)

Mounting medium (the characteristic of the mounting medium must be near with the refractive index of the
tissue, slide and coverslip. The nearer the RI, the more visible the tissue)
- a.k.a. mountant
- a syrupy fluid applied between the section and the coverslip, setting the section firmly, preventing the movement
of the coverslip (the mounting mechanism may either be temporary or resinous which is mostly permanent)
 * If an unmounted stained section is examined under the microscope, very little detail can be made out because of
the great difference in the refractive indices of the glass slide, the tissue component, and air.

 for best results with stained tissue sections, they must be impregnated by a transparent medium having a refractive
index close to that of glass. (RI = 1.518)

- necessary to protect the stained section from physical injury and from bleaching or deterioration of the stain as a
result of oxidation.

Mounting

Properties of the Best Mounting Medium:

1. will have a R.I. close to that of glass (1.518)

2. will be freely miscible with xylene and toluene (so that it can immediately penetrate the tissue and induce
Refractive index similar of glass slide and tissue elements)
3. will be non-reactive (inert) and will not change in pH or color
4. will set without distortion or shrinkage of tissue sections
5. will set hard without granularity or cracks
6. will not leach out any stain or cause any loss of staining over long periods
***There is no ideal or best mounting medium

Two main groups of Mounting media:

1. Aqueous mounts – designed to mount water- miscible preparation directly from water in cases where the stain is
removed or decolorized with alcohol or xylene (decolorization/washing-out/differentiation) (regressive staining
seen in gram staining and acid-fast staining) (they are temporary type of media)

a. Water- low refractive index (1.333) (not vivid but clear)

b. Glycerin = 1.46 (R.I. 1.47 = glycerin jelly/Kaisers 1880) - also used as preservative, has high index of
refraction
c. Gum syrup (Farrant’s) R.I. 1.43 – does not solidify upon storage
d. Apathy’s (R.I. 1.52) – used for Methylene Blue-stained nerve preparations and as general purpose Aqueous
Mountant
e. Brun’s– recommended for mounting frozen sections from water

2. Resinous (permanent type of media) (composed of xylene to attain RI similar to glass)

a. Canada balsam (1.524)– natural resin extracted from the Canadian Tree, Abus Balsamea
- recommended for whole mounts and for thick sections because it does not shrink much
- miscible with xylene and is quite expensive

b. DPX (1.532) – for small tissue such as glandular tissues (Dibutyl Phthalate Xylene)

c. XAM (1.52) – synthetic resin mixture in xylene in pale yellow or colorless solution (Xylene and Arabic medium)

d. Clarite (1.544)- usually diluted to 60% with xylene

Permanent Mounting Media

- these are resins, either natural or synthetic (soluble in alcohol) (because they are permanent, they can be stored
for 1-5 years depending on the characteristic and bleaching capacity of the tissue)
E.g. Canada balsma Clarite Entellan
BPX Eukit

* Only experience will teach one how much mounting medium to place on the cover glass.
  If too much is used, it will ooze at the sides of the cover glass and should be carefully wiped away with the
fingernail covered by a fine cloth dipped in xylol (may cause dust to adhere on the side of the coverslip)
 If too little (or too diluted) mounting medium is used, as the medium sets, it will draw away from the edges of
the cover glass and the section must be remounted, removing the cover glass by soaking in xylol (formation of air
bubbles that will induce problem in proper diagnosis)

Semi-Permanent & Temporary Mounts

* For some preparations, it is imperative not to use the permanent xylol-resin mounting media, because xylol
dissolves out the essential staining, an example is fat stains (neutral fats stained in frozen sections by Oil Red
Oild Method, Sudan’s Stain), these may be mounted in one of the following:
1. water
2. glycerin (glycerol)
3. mineral oil (Liquid Paraffin)
4. Glycerin Jelly (Glycerol Gelatin)
5. Von Apathy’s Gum Syrup Medium
6. Farrant’s Medium
7. Levulose (Fructose) Syrup

Ringing
- process of sealing the margins of the coverslip to prevent escape of fluid or semi-fluid mounts and evaporation
of mountant, to immobilize the coverslip, and to prevent sticking of slides upon storage.

Kronig cement
- made up of 2 parts paraffin wax mixed with 4-9 parts powdered Colophonium resin, heated and filtered.

The main constituent of a Mayer’s egg albumin is glycerin and?

a. Thymol
b. Egg yolk
c. Dried albumin
d. Albumin
e. Glair

What solution is added to dried albumin to form a dried albumin adhesive?

a. Saline Solution
b. Potassium dichromate
c. Potassium chloride
d. Sodium chloride
e. Sugar solution

The amount of gelatin utilized for gelatin adhesive preparation.

a. 1 gram
b. 2 grams
c. 3 grams
d. 4 grams
e. 5 grams

What is added to the starch paste in order to prevent mold formation?

a. Phenol Crystals
b. Thymol crystals
 c. Celloidin
d. Paraffin
e. None of these

The amount of outdated plasma dispensed into sterile tubes for plasma usage in section adhesion?

a. 0.05 ml
b. 0.5 ml
c. 1 ml
d. 2 ml
e. 3 ml

The following contribute to the Refractive index EXCEPT:

a. Glass slide
b. Cover slip
c. Syrupy fluid
d. All of these
e. None of these

What are the two mounting media commonly used today?

Aqueous Mounting Media
Resinous Mounting Media

What is the temperature of the hot oven utilized for mounting sections that are not properly dehydrated?
50˚C

STAINING PROCEDURES IN HISTOTECHNOLOGY

Staining – create or induce a coloration to a certain tissue component. Naturally, tissues are uncolored and they are
innate coloration which is flesh in type and not that striking. In order for us to differentiate one component from the
other, it must have different colors. The stains or dyes are very important for such purpose because they tend to
isolate on structure from the other wherein, they differ when it comes to affinities. One structure may be having a
higher affinity towards this color, that’s why it will absorb green coloration. In this case, we will be able to
differentiate one structure from the other and because of the affinity of the specific stain towards a specific tissue
component, we can say that this specific tissue has increased amount element or protein or carbohydrate due to the
color that it has assumed coming from the stain itself.

* If unstained sections of tissue are examined under the microscope with transmitted light, very little detail other than the
nuclear and the cell boundaries can be identified.
Dyeing/Staining

 enables one to study and to see any physical characteristic/s and relationship of the tissues and components of the
cell
 made possible through capillary osmosis, solubility, absorption (dyes taken in by the tissue) & adsorption
(dye attaches on the surface of the tissue) of stains or dyes by tissue.
 display differing affinities for most dyes or stains
Nucleus (Acid) + Base (hematoxylin) Blue
Cytoplasm (Basic) + Acid (eosin) Red
In fixation, if the tissue is acidic, the fixative must be also be acidic. And if tissue is basic, the fixative must also be basic.
Like is like. Whatever is the pH of the tissue, the fixative must also utilize that pH. But in staining, opposites attract. For
affinity to develop, the stain must be acidic. The most common contrast stain/ contrasting stain/ alter stain for hematoxylin
is eosin. And, eosin is characteristically yellowish-red in color. That is why red is the color of the tissue found in the
cytoplasm. Eosin is a cytoplasmic stain while hematoxylin is a nuclear stain.

Staining procedure Principle and Characteristics Example/results

Specific Staining * It is the basis of histochemistry. *Hemosiderin (developed in

*It is utilized to color an
element which it is
designed to.
Simple or Direct Staining
Indirect Staining
*It is accomplished by controlled, specific
chemical reactions designed to give a final
color (staining) at the site/location of the
structure of the substances in the cells or
tissues
- the staining of tissue by means of simple
solutions of a dye (aqueous or alcholic).
-Here the action of the dye is intensified by
some other agents:
a. Mordant
b. Accentuator (increases the
crispness of the stain)
intravascular hemolysis) with Perl’s
Prussian Blue (for ferric ion Fe3+ but
for Fe2+ Turn Bull)
*Polysaccharides with the PAS
technique (Periodic acid schiff)
Methylene blue (utilized for Diphtheria
“Babes Ernst Granules”)
(metachromatic granules)
For mordants:
1. Alum with Hematoxylin in
Ehrlich’s
2. Iron in Weigert’s Hematoxylin
For Accentuator:
1. KOH (Potassium hydroxide) in
Loeffler’s Methylene Blue

Mordant
 a substance which, when taken up by the tissue, helps make the dye in return serving as a link or bridge to make
the staining reaction possible.
 combines with a dye forming a colored “lake (final color achieved after the dye forms a type of complex that
of specific mordant itself” which combines with tissue to form an insoluble “tissue-mordant dye complex”
 an integral part of the staining reaction itself, without which, no staining could possibly occur .
Accentuator
 chemical substances that do not participate but merely heighten or increase the color intensity, crispness and
selectivity of the stain.
 differ from mordants in that they do not bind or link the tissue to the dye.

Staining procedure Principle and Characteristics Example/results

Progressive Staining
*It is governed by two principles:
1. Flooding/Overstaining – a tissue
element is exposed to excess amount
of dye and the excess dye is removed
2. Decolorization
If the previous stain is acidic, the
differentiator is basic and vice versa.
Counterstain or Secondary stain for
acid fast staining is methylene blue.
- staining is continued (definite
sequence) until the desired intensity
of coloring of the different tissue
elements is attained.
- No washing/ decolorization in
between is required solely relies on
the specificity/selectivity of dyes for
different cellular elements.
- the tissues are overstained and the
excess dye is then removed until the
desired intensity is obtained.
- 2 principles:
a. Overstaining
b. Differentiation/Decolorization/
Washing-out
Any stain is possible as long as no
differentiation is done.
AFS and Gram Staining
Other Info:
1. This is usually done by
washing the section in simple
solutions (water or alcohol),
acids or oxidizing agents.
2. Basic is to acid and vice-
 -selective removal of excess stain
from tissue.
Metachromatic staining - a.k.a. metachromasia
- entails the use of the specific dyes
that stains tissues with a color that is
different from that of the stain color
itself.
- particularly employed for staining
of: (CECAM)
• Cartilage
• Epithelial mucins
• Collagen
• Amyloid
• Mast cell granules
Counterstaining - the application of a different color
or stain to provide contrast and
background to the staining of the
structural components to be
demonstrated.
Vital Staining – the selective staining of living cell
constituents, demonstrating
cytoplasmic structures by
phagocytosis of the dye particle
(cytoplasmic phagocytosis).
- the nucleus of the living cell is
resistant to vital stains, and therefore
is not demonstrated.
Intravital Staining – done by injecting the dye into any
part of the animal body (either
intravenous, intraperitoneal or
subcutaneous) producing specific
coloration of certain cells, particularly
those of the RES.
Supravital Staining – used to stain living cells
immediately after removal from the
living body
METALLIC IMPREGNATION
- makes use of heavy metals which are precipitated with selectivity of certain cellular and tissue components.
- has its greatest application in tissue from the central nervous system
- but is also used for the demonstration of reticulin
- differs from staining in that it consists of an opaque black particulate precipitate
- displays many of the characteristics of true staining.
METALLIC IMPREGNATION
versa.
3. Alcohol is a differentiator for
both basic and acid dyes
Metachromatic dye- basic dyes
belonging to:
• Thiazine
• Triphenylmethane groups
Ex: BCB (Brilliant Cresyl Blue) for
reticulocytes.
***All metachromatic dyes are
cations and lost if dehydrated in
Alcohol (ROH) after staining, hence
water is necessary
Eosin – for the cytoplasm
Primary stain for nucleus is
hematoxylin but for cytoplasm is
eosin
Trypan Blue – vital stain of RES
(reticulo endothelial system)
Janus green - true vital staining of
mitochondria
Other info:
– demonstration of nuclear structures
during vital staining suggests
permeability of the membrane by the
dye, signifying the death of the cell
Common dyes used are:
Lithium
India ink
Carmine
Neutral red = probably the best vital
dye
 E.g. Silver nitrate – most commonly used agent for impregnation (utilized for the treatment of
ophthalmia neonatorum which is infected with Neisseria gonorrhea)
- can behave as a stain and can outline the tissue elements in a non-particulate union
Osmium tetroxide/ Osmic acid/ Flemmings without acetic acid– used for demonstration of lipids (yellow)

A dye should consist of 2 substances:

1. Chromophores
- Grk. “color bearers ”
- substances capable of producing visible colors.
2. Auxochromes
- Grk. “color increasers” (salt forming molecules)
- substances which impart to the compound the property of electrolytic dissociation, thereby altering the
shade of the dye, giving it the property of forming salts with another compound and ultimately retaining its color.

• The introduction of a chromophoric group into an uncolored molecule will cause it to be colored; it will then be a
chromogen, which is colored and not a dye.
• For a chromogen to be a dye, it must be composed of an acid and a base, and therefore have salt forming
properties, ultimately retaining its color.
• The coloring property is attributed to the chromophore and the dyeing property to the salt forming auxochrome.

Classification of Dyes:
1. NATURAL DYES – e.g. Cochineal dyes, logwood dyes & vegetable extract (come from plants, trees and
animals)
a. Hematoxylin – the most important
b. Orcein – a vegetable dye extracted from lichens which are normally colorless
c. Cochineal dyes – a dye derived from an extract from the female Cochineal Bug (Coccus cacti)
d. Saffron – a plant with orange stigmas yielding a dye

Take note:
Cochineal + alum = carmine
Carmine + aluminum chloride = best carmine
Carmine + picric acid = picrocarmine