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Principles of Staining

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Bianca Camille Baldovino

on 22 February 2014

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Transcript of Principles of Staining

5. VITAL STAINING
- selective staining of living cell constituents, demonstrating cytoplasmic stuctures by phagocytosis of the dye particle (cytoplasmic phagocytosis)
a. 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
General procedures in staining
* Direct Staining
- the process of giving color to the sections by using aqueous or alcoholic dye solutions (e.g. methylene blue, eosin)
METHODS OF STAINING
- tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of color is attained
STAINING
-the process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell
Principles of Staining
MORDANT
- a substance used to set dyes on tissue sections by forming a coordination complex with the dye which then attaches to the tissue
2. REGRESSIVE STAINING
- tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue until the desired intensity of color is obtained
Group 3 - GEN PATH (7:00-8:00 MW)
Lagleva, Murell Credo, Robelyn
Baldovino, Bianca Daquioag, Pia
Calica, Valeree De Vera, Erica
Caoile, Gracia Dela Cruz, Charissa
Clemente Jhanine

-helps to study and evaluate physical cahracteristics and structural relationships of tissues and their cells
THREE MAJOR GROUPS
1. HISTOLOGICAL STAINING
- a process whereby tissue components are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of active tissue components
- "micro-anatomical staining
- demonstrates the general relationship of
tissues and cells with differentiation of nucleus
and cytoplasm
2. HISTOCHEMICAL STAINING
- various components of tissues are studied thru chemical reactions that will permit microscopic localization of s specific tissue substance
*Enzyme Histochemistry
- active reagent serves as a substrate upon which the enzymes act
- final coloration produced is from the substrate rather than the tissue
3. IMMUNOHISTOCHEMICAL
STAINING
- combination of immunologic and histochemical stain
Bacterial staining -
Bacillus anthracis
Cardiac muscles
Periodic Acid Schiff stain - Carbohydrates
Perl's Prussian Blue stain - Hemoglobin
- allow phenotypic markers to be detected and demonstrated under the microscope
- uses a wide range of polyclonal or monoclonal, fluorescent or enzyme-labeled antibodies
Immunohistochemical staining of normal kidney with CD10
* Indirect Staining
- the action of the dye is intensified by adding another agent to make the staining possible
- by itself, the dye may stain only weakly, if at all
- for intensifying stains in cell or tissue preparations
-the term mordant comes from the French word
mordre
, "to bite"
ACCENTUATOR
- not essential to the chemical union of the tissue
and the dye but merely accelerates the staining
reaction by increasing the staining power and
selectivity of the dye
1. PROGRESSIVE STAINING
- no washing or decolorization occurs
- differentiation of tissue details relies solely on the selective affinity of the dye
*Decolorization
- selective removal of excess stain from the tissue in order that a specific substance may be stained distinctly
- basic primary stain -> acidic dye as decolorizer
- acidic primary stain -> alkaline decolorizer
- alcohol acts as a differentiator for
both basic and acidic dyes
3. METACHROMATIC
STAINING
- entails the use of specific dyes that differentiate particular subtances by staining them with a color different from the stain itself (
"metachromasia"
)
- particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules and amyloid
4. COUNTERSTAINING
- application of a different stain to provide contast and background to the staining of the stuctural components to be demonstrated
*Cytoplasmic stains
- red -> eosin Y, eosin B, phloxine B
-yellow -> picric acid, orange G, rose bengal
-green - light green SF, lissamine green
*Nuclear stains
- red -> neutral red, safranin O, carmine,
hematoxylin
- blue -> methylene blue, toluidene blue,
celestine blue
- the nucleus of a living cell is resistant to vital stains, and therefore is not demonstrated
b. Supravital staining
- used to stain living cells immediately after removal from the living body
- thin slices of tissue are placed in small staining
dishes and enough staining solution is added to
cover the tissue
METALLIC IMPREGNATION
- specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria
*Metallic impregnating agent
- different from a stain in that it is not absorbed by the tissue, but it is held physically on the surface as a precipitate or as a reduction product in certain tissue components
- the most valuable metals for this purpose are gold (gold chloride) and silver (silver nitrate)
Synovial fluid with numerous cigar-shaped, budding yeasts. (Grocott-Gomori-methenamine silver nitrate stain)
RESTAINING OF OLD SECTIONS
a. The slide is usually immersed in xylene for 24 hours, or gently heated until the mounting medium begins to bubble
b. The coverslip is removed by lifting it with a dissecting needle
c. The section is placed in xylene for 30 minutes to remove the remaining balsam and is brought to water
d. Place it in a 0.5% potassium permanganate solution for 5-10 minutes, rinse in tap water and subsequently immerse in 5% oxalic acidfor 5 minutes
e. Wash it in water for another 5 minutes then
restain with the appropriate staining
technique
PRECAUTIONS IN
STAINING
1. Stains on in should be avoided because stains are health hazards per se.
-stains may be effectively removed from the skin by prompt topical application of 0.5% acid alcohol, followed by rinsing with tap water
2. Failure of sections to remain on the slide during staining could have been due to a dirty or oily slide or albumin fixative may be too old.
3. If the section does not stain, the staining solution may be faulty. Impurities found in the dye or in the water solvent will affect both the solubility and intensity of the dye.
4. Failure of staining maybe due to paraffin, fixative, or decalcifying solution that has not been thoroughly washed out and removed.
5. Stains may be saved and used again for as long as they have not lost their staining properties.
6. If, after staining, sections do not appear clear under the
microscope, xylol should be replenished.
7. If the tissue is thoroughly adherent to the slide, it can be taken back several times for staining
without any danger of peeling off.
*Staining of paraffin sections
-paraffin wax is poorly permeable to most staining solutions and should therefore be removed from the section prior to to staining

- usually done by immersing the paraffin section in a solvent two times, at 1-2 minutes duration each
- xylene is not miscible with aqueous solutions and low graded alcohol, and should therefore be subsequently removed with absolute alcohol, followed by descend grades of alcohol to prevent damage and detachment of sections due to the possible production of diffusion currents
- alcohol is replaced with water before actual
staining is performed
*Staining of Celloidin Sections
- paraffin ribbons containing air bubbles, torn, or adequately infiltrated sections are likely to float from the slide when deparaffinized and stained
- they are more firmly attached by coating the slide with dilute (thin) celloidin solutions (collodionization)
- cellulose nitrate (celloidin) is soluble in absolute alcohol, hence treatment should be avoided during dehydration and clearing of stained sections
- sections treated with 95% absolute alcohol may be transferred to a mixture of equal parts of chloroform, absolute alcohol and xylene , then treated with xylene and mounted in Xam
Natural stains
- dyes obtained from natural resources
a. Carmine -> female Cochineal insect
b. Orcein -> lichens
c. Saffron - > pistils of a flower
d. Hematoxylin -> heartwood of a log tree
Coccus cacti
Lichens
Saffron flower
Hematoxylin campechianum
Synthetic stains
- a.k.a. "coal tar dyes" since they were originally manufactured from substances that have been taken from coal tar
- derived from the hydrocarbon benzene (C6H6), and are collectively known as "aniline dyes"
- consists of a chromophore (produce visible colors; coloring property) and a salt-forming auxochrome (gives dye its ability to retain color; dyeing property)
a. Acid dyes ->
active coloring substance is found in the acid component (e.g. picric acid, trichloroacetic acid)
b. Basic dyes ->
active coloring substance is found in the basic component (e.g. methylene blue)
c. Neutral dyes ->
formed by combining aqueous solutions of acid and basic dyes, capable of staining
cytoplasm and nucleus simultaneously and
differentially (e.g. Romanowsky dye,
Giemsa's stain)
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