Endterm Week 2 Histotech
Endterm Week 2 Histotech
Endterm Week 2 Histotech
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.
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
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)
* 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)
* 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.
a. Thymol
b. Egg yolk
c. Dried albumin
d. Albumin
e. Glair
a. Saline Solution
b. Potassium dichromate
c. Potassium chloride
d. Sodium chloride
e. Sugar solution
a. 1 gram
b. 2 grams
c. 3 grams
d. 4 grams
e. 5 grams
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
a. Glass slide
b. Cover slip
c. Syrupy fluid
d. All of these
e. None of these
What is the temperature of the hot oven utilized for mounting sections that are not properly dehydrated?
50˚C
* 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.
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.
Vital Staining – the selective staining of living cell Trypan Blue – vital stain of RES
constituents, demonstrating (reticulo endothelial system)
cytoplasmic structures by Janus green - true vital staining of
phagocytosis of the dye particle mitochondria
(cytoplasmic phagocytosis). Other info:
- the nucleus of the living cell is – demonstration of nuclear structures
resistant to vital stains, and therefore during vital staining suggests
is not demonstrated. permeability of the membrane by the
dye, signifying the death of the cell
Intravital Staining – done by injecting the dye into any Common dyes used are:
part of the animal body (either Lithium
intravenous, intraperitoneal or India ink
subcutaneous) producing specific Carmine
coloration of certain cells, particularly
those of the RES.
Supravital Staining – used to stain living cells Neutral red = probably the best vital
immediately after removal from the dye
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
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)
• 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