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General Histology and Histotechnique (1 st Semester; SY 2012-2013) Preparation for whole mount Materials needed: 1. Glass slides - usually 25 x 75 mm - usually 1- 1.2 mm thick Types: a. flat slides b. concave slides 2. Cover slips Two types: Number 1 = 0.13-0.17 mm thick Number 2 = 0.17-0.25 mm thick Functions: a. hold samples in place b. flattens out specimen c. Protects the objective from immersion into the water drop. 3. Stains - Different stains have different affinities for the different organisms thus maybe used to differentiate different types of organisms or to view specific parts of organisms. - Substance that give color to the specimen to improve visibility. Example: Counter stain not all organisms can absorb. Type of stains A. According to the number of dyes used 1. ) Positive staining - Stains the specimen itself. a. Simple Stain - Employs single dye (on color) - Primary dye Ex. Crystal violet, Methylene blue b. Differential Stain - Two or more stains - Primary dye counter stain - Gram-staining Ex. Primary dye = Crystal violet; Counter stain = Safranin Ex. Acid fast stain (Mycobacterium), Ziel Neelsen 2. ) Negative staining - the background of the organism/specimen is the stain - Function: To emphasized the appendages B. According to the reaction of the tissue stain. 1. ) Basophilic - stains the acid component 2. ) Acidophilic - Stains the basic components - Stains the cytoplasm of the cell Some common stains: 1. ) Hematoxylin and Eosin (H & E) a. Hematoxylin stain nuclei blue (basophilic) b. Eosin stain cytoplasm pink (acidophilic) 2. ) Connective Tissue stains a. Masson’s trichome b. Mallorys triple CT stain - both employ a nuclear, cytoplasmic and a third stain specific for matrix 3. ) Silver impregnation - Used to trace nerves, stain Golgi, reticular fibers. 4. )Wright stain blood smear 4. other chemicals Fixatives Embedding materials Mountants Clearers Type of Slide Preparation Temporary Slide - water - fresh specimen Permanent slide - reagents are used - dead tissues Fixatives preserve specimen - Function: to avoid post mortem conditions Ex: FAA, Bouin’s sol’n, Formalin (10%) Dehydrants - reagents that remove undesired fluid that may interfere with later processing Ex: Ethyl alcohol (ethanol/alcohol)

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Student-made handout General Histology and Histotecnique 2012-2013; 1st sem; Prelim Handout

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General Histology and Histotechnique (1st Semester; SY 2012-2013)

Preparation for whole mount Materials needed: 1. Glass slides - usually 25 x 75 mm - usually 1- 1.2 mm thick Types:

a. flat slides b. concave slides

2. Cover slips Two types: Number 1 = 0.13-0.17 mm thick Number 2 = 0.17-0.25 mm thick Functions:

a. hold samples in place b. flattens out specimen c. Protects the objective from immersion into the

water drop. 3. Stains - Different stains have different affinities for the

different organisms thus maybe used to differentiate different types of organisms or to view specific parts of organisms.

- Substance that give color to the specimen to improve visibility.

Example: Counter stain – not all organisms can absorb.

Type of stains A. According to the number of dyes used 1. ) Positive staining

- Stains the specimen itself. a. Simple Stain

- Employs single dye (on color) - Primary dye

Ex. Crystal violet, Methylene blue b. Differential Stain

- Two or more stains - Primary dye – counter stain - Gram-staining

Ex. Primary dye = Crystal violet; Counter stain = Safranin

Ex. Acid fast stain (Mycobacterium), Ziel Neelsen 2. ) Negative staining - the background of the organism/specimen is the

stain - Function: To emphasized the appendages

B. According to the reaction of the tissue stain. 1. ) Basophilic - stains the acid component 2. ) Acidophilic - Stains the basic components - Stains the cytoplasm of the cell Some common stains: 1. ) Hematoxylin and Eosin (H & E)

a. Hematoxylin – stain nuclei blue (basophilic) b. Eosin – stain cytoplasm pink (acidophilic)

2. ) Connective Tissue stains

a. Masson’s trichome b. Mallorys triple CT stain

- both employ a nuclear, cytoplasmic and a third stain specific for matrix

3. ) Silver impregnation - Used to trace nerves, stain Golgi, reticular fibers.

4. )Wright stain – blood smear 4. other chemicals

Fixatives Embedding materials Mountants Clearers

Type of Slide Preparation

Temporary Slide - water - fresh specimen

Permanent slide - reagents are used - dead tissues Fixatives – preserve specimen - Function: to avoid post mortem conditions Ex: FAA, Bouin’s sol’n, Formalin (10%) Dehydrants - reagents that remove undesired fluid that may

interfere with later processing Ex: Ethyl alcohol (ethanol/alcohol)

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Clearing agents - to clear Ex: Xylene and Cloroform Embedding medium - supports the specimen - paraffin (wax time) Microtome Stains - Gives color to the specimen to differentiate the

different parts of specimen. Ex: Methylene blue, Hematoxylin, Wright stain, Crystal violet, Eosin, Safranin, Fast green.

Adhesive/Affixative - to stick or adhere the specimen to the slide Ex: Mayer’s albumin

Mountant - render permanency Ex: Balsam Fresh Tissue Examination 1. Teasing/Dissociation - use of needle to separate tissues in bulk 2. Squashing - cut a tiny piece on slide and with the use of another

slide to compress 3. Smear preparation - to spread specimen thinly onto the slide

a. Streaking - Applicator stick or the wine loop usage. - Spread specimen to the slide directly on a zigzag

manner. - Uniform the distribution

b. Spreading - to spread thinly into the slide - teasing is also needed - for fresh sputum mucoid secretion

c. Full-apart - for blood smear, enzymes of the GIT - it used pusher

d. Touch Preparation/impression smear - used of freshly cut fruit - touched on to the surface of the slide - letting the cells stick on to the slide 4. Frozen section

- utilize when rapid diagnosis of tissues - specially recommended for lipids and nervous tissue

elements USES: Rapid pathological diagnosis during surgery Diagnostic and research enzyme histochemistry Diagnostic and research demonstration of soluble

substances such as lipids and carbohydrates Immunofluorescent and immunohistochemical

staining Some specialized silver stains, particularly in

neuropathology Processing of Tissues Fixation Purposes:

a. Confers chemical stability on the tissue b. Hardens the tissue c. Stops enzyme autolysis d. many enhance later staining technique – act as

mordant Five (5) major groups of Fixatives: 1. Aldehydes - include formaldehyde or formalin and

glutaraldehyde - non precipitating

10 % formalin - buffered to lessen acidity that causes precipitation

and autolysis

Glutaraldehyde - 2-4 % buffered - fixes very fast - good for electron microscopy - it penetrates poorly - It gives best results on cytoplasmic and nuclear

details. 2. Mercurials - contain mercuric chloride and Zenker’s - fixed the tissue by an unknown mechanism - penetrate very poorly - causes tissue to harden - fixed fast - give excellent nuclear detail - best in nematopoietic reticuloendothelial 3. Alcohols

General Histology and Histotechnique (1st Semester; SY 2012-2013)

- include methanol and ethanol - protein denaturants - cause too much brittleness and hardness - good in cytological smears - give good nuclear detail - precipitating 4. Oxidizing agents - include Potassium permanganate, Potassium

dichromate and Osmium tetroxide - none precipitating - used in frequently - causes an extensive denaturation

5. Picrates - include fixatives and picric acid (e.g. Bouin’s sol’n) - precipitating fixative - enhanced nuclear detail - don’t cause hardens - Dry form – explosive - in Solutions– stains everything it catches including

the skin; yellow color Fixative for General Usage a. 4 % / 10 % formalin Advantages: 1. easy to make 2. Has good penetration power 3. Does not shrink the specimen 4. Does not affect the material it kept for long time 5. Can be kept without deterioration for considerable

time 6. Easy to handle 7. Less hazard

b. FAA – Formalin-Acetic-Acid-Alcohol - Best for plant material (tissues) - Composition: 50ml ethanol, 5ml glacial acetic acid,

10ml 37-40% formaldehyde, 35ml water c. Zenker’s and Carnoys’s fluid - best for animal tissues

Zenkers’s: 950ml distilled water, 25g potassium dichromate, 50g mercuric chloride, 50g glacial acetic

Carnoy’s: 60 ml ethanol, 30ml chloroform, 10ml glacial acetic acid

Factors affecting fixation

1. Buffering – pH 6-8 - phosphate, bicarbonates - Commercial formalin is buffered with phosphate at

a pH of 7.

2. Penetration - depends of diffusability of fixatives - formalin and alcohol: best - glutaraldehyde: worst - diffusability – constant - in between – mercurials - in order to the organisms to be penetrated it should

be cut in 2-3 mL

3. Volume - ratio 10:1 - 10 – fixative - 1 – specimen / tissue

4. Temperature - High temperature – Increases speed of fixation Ex. Hot formalin will fix the tissue faster. 5. Concentration of Fixation - adjusted down to the lowest level possible - 10% formalin: best - glutaraldehyde (3%)

6. Time interval - FAA – up to 12 hrs. - Carnoy’s – 1-3hrs - Zenker’s – 14-24hrs Organs for processing: Kidney Liver Stomach Small Intestine Large intestine Lungs Spleen Gonads Muscles Specimen Class Class A – delicate tissues: lungs; membranes Class B – moderately thick; spleen, live, kidney Class C – bulky and thick; skin

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Fixation process: - submerge the specimen fully in fixative - cover the container tightly Proper timing for each specimen if FAA is used: Class A – 6-9 hrs. (10hrs) Class B – 12-18 hrs. (16-18 hrs) Class C – 24-36 hrs. Dehydration - removing undesired water inside the specimen Characteristics of an ideal dehydrating solution:

It should dehydrate rapidly without producing considerable shrinkage or distortion of tissues.

It should not evaporate very fast.

It should be able to dehydrate even fatty tissues

It should not harden tissues exclusively.

It should not remove stain.

It should not be toxic to the body

It should not be a fire hazard. Commonly used dehydrating agents: - alcohol - acetone - triethylphosphate - tetrahydrofuran - dioxane

Alcohol - a standard series of alcohols are used most - Alcohol series: 70% - 80% - 95% - Higher grades of alcohol: shrinkage and hardening

of tissues - concentrated alcohol tend to harden only the

surface of tissue

Ethyl alcohol - best dehydrating agent

fast acting mixes with water penetrates tissue easily not poisonous not very expensive

Dehydrating procedures: Water 50% ethanol 70% ethanol 80% ethanol 90% ethanol

100% ethanol – 30 min 100% ethanol– 30 min ***To ensure complete removal of water during dehydration, use two changes of 100% ethanol of at least ½ hour each. Timing of dehydration: Class A – 30 min in each alcohol concentration Class B – 1 hr Class C – 3-4 hrs. Clearing

1. Alcohol –to provide rapid miscible in the tissue 2. To facilitate impregnation or infiltration

Xylene - Commonly used; tends to harden tissues if left too

long.

Advantages: - makes tissue transparent - miscible with absolute alcohol and paraffin - evaporate quickly in paraffin oven and can - cheap

Disadvantages: - highly in flammable - If used longer than 3 hrs. it makes tissues

excessively hard and brittle - Causes considerable hardening and shrinkage of

tissues - Becomes milky when an incompletely dehydrated

tissue is immersed in it.

Toluene - time recommended is 1-2 hours

Advantages: - it is miscible with both absolute alcohol and paraffin - It acts fairly and rapidly and is recommended for

routine purposes - tissue do not become excessively hard and brittle

even if left in Toluene for 24 hrs - it is not carcinogenic

Disadvantages: - it is relatively slower than Xylene and benzene - it tends to acidify in a partially filled vessel

General Histology and Histotechnique (1st Semester; SY 2012-2013)

- highly concentrated solutions will emit fumes that are toxic upon prolonged exposure

- it is more expensive Benzene - clearing agent - clear and penetrate tissues rapidly - carcinogenic Advantages: - It is rapidly acting, so recommended for urgent

biopsies (15 to 60 min) and routine purposes. - It volatilizes rapidly in paraffin oven and is therefore

easily eliminated from the tissue. - It is miscible with absolute alcohol - it does not make tissues hard and brittle - it causes minimum shrinkage - it makes tissues transparent

Disadvantages: - it is highly in flammable - if a section is left in benzene for a long time,

considerable tissue shrinkage may be observed - Excessive exposure to benzene may be extremely

toxic to man and may become carcinogenic or it may damage the bone marrow resulting in a plastic anemia.

Chloroform - Used for routine clearing of tissues during the

embedding process. Advantages: - It is recommended for routine work (6-24 hrs.) - It is miscible with absolute alcohol - It is recommended for tough tissues (ex. skin,

fibroid and decalcified tissues) for nervous tissues, lymph nodes and embryos

- It is suitable for large tissue specimens - It is not inflammable.

Disadvantages: - It is relatively toxic to the liver after prolonged

inhalation - wax impregnation after chloroform clearing is

relatively slow - it does not make tissues transparent - it is not very volatile in paraffin oven - it may even produce considerable deterioration of

the wax

- complete clearing is difficult to evaluate - tissues tend to float in chloroform - it evaporates quickly from a water bath Impregnation, Embedding, Trimming Impregnation - Diffusion or accumulation in a cell or tissue

substances that are not normal. - To fill-out the tissues in each spaces or in-between

spaces Three (3) types of Tissue Impregnation: 1. Paraffin wax impregnation Precautions: a. Prolong treatment in melted paraffin causes

shrinkage and hardening of tissues making cutting difficult.

b. Infiltration in overheated paraffin (above 60°C) will produce shrinkage and hardening of tissues and destroy lymphoid tissues completely

c. Paraffin wax must be pure d. Paraffin wax may be used only twice. 2. Celloidin impregnation 3. Gelatin impregnation Embedding - The process by which the impregnated tissue is

placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.

- Cooling allow hardening of tissue, giving them a firmer consistency and better support, thereby facilitating the cutting of sections.

Orientation - A process by which a tissue is arranged in precise

positions in the mold during embedding on the microtome before cutting and on the slide before staining.

Embedding Proper: 1. Prepare embedding boxes 2. Pour melted paraffin into embedding box 3. Using a needle or forceps, position properly the

tissue inside the paraffin box according to the desired cut later. For cross section, position tissue vertically. Do not remove the needle or forceps ‘till paraffin has surrounded the tissue.

4. Allow paraffin to solidify.

General Histology and Histotechnique (1st Semester; SY 2012-2013)

5. Label the box bearing the name and position of tissue contained. Trimming

- Embedded tissue is trimmed and cut into thin uniformly slices. - The slides, top and bottom of the tissue block are trimmed until perfectly level and all slides are parallel, almost to

the edge of the tissue. - The block is placed in the microtome for final trimming and cutting.

Microtomy - A process of cutting specimen to small or thin sizes.

Microtome - An instrument used to cut the specimen.

Steel Blades - Animal or plant tissue sections - Light microscope

Glass Knives - thin sections - Light microscope and Electron Microscope

Industrial Grade - Electron microscope

Diamond knives - Hard materials 3 Essential parts of Microtome: 1. Block holder – holds block in precision 2. Knife carrier & knife – actual cutting of the tissue sections 3. Pawl, Ratchet feed wheel and adjustment screws – line up the tissue flock in proper positioning with the knife

adjusting the proper thickness of the tissue for successive section. Kinds of Microtome’s: 1. Rocking (by Paldwell Trefall, 1881) - Simplest - Cut the paraffin embedded tissues

2. Rotary (by Minot, 1885-’86) - Most common - It involves staged rotary action

3. Sliding (by Adams, 1789) - Cutting celloidin embedded tissue

a. Base-sledge – a very hard tissue b. Standard sliding – with movable exposed knife. For cutting celloidin embedded tissue. This is the most

dangerous style of microtome.

General Histology and Histotechnique (1st Semester; SY 2012-2013)

4. Freezing (by Queckett, 1848) - Cutting embedded frozen sections Cryostat or cold microtome - Refrigerated apparatus used in fresh microtomy - For freezing tissue in the block holder to correct degree of hardness to facilitate easier and faster sectioning. - Provides a means of preparing a thin section of fresh frozen tissues especially for fluorescence antibody staining

technique.

5. Ultrathin - Cutting sections for electron microscope - celloidin - Specimen used should be small - Should be fixed in osmium tetroxide and embedded in plastics. Microtome knives: 1. Plane concave knife - 25mm - one side flat, other concave Flat – recommended for cutting celloidin embedded tissue blocks using a sliding microtome. Concave – used for cutting paraffin embedded tissues. Using its Base Sledge microtome, Rotary, Rocking

2. Biconcave knife - 120mm - both sides are concave Concave – cutting paraffin embedded tissues using a rotary microtome.

3. Plane wedge knife - 100 mm - Both sides are straight Straight – recommended for frozen section or for cutting extremely hard/tough specimen embedded in a paraffin

block. - Using a base sledge sliding microtome. Sectioning - Cutting thinly the specimen with the use of microtome - Thin materials – sections 3 types of tissue sections: 1. Paraffin Section - Leaded in a paraffin block - Cut using the rotary microtome/Rocky microtome

2. Celloidin section - Embedded in a celloidin - Cut by sliding microtome

3. Frozen Section - Fixed with carbon dioxide or cryostat

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Faults occurring during tissue processing

FAULTS REASONS REMEDY

1. Brittle or hard tissue. - Prolong fixation, dehydration clearing, and infiltration of paraffin in overheated paraffin oven.

- Dry out of tissue before actual fixation.

- Softened by soaking in a small dish containing water with detergent or phenol.

2. Clearing agent turns milky as soon as tissue is placed in it.

- Water is not completely removed due to incomplete dehydration.

- Repeat dehydration with absolute alcohol then clear again.

3. On trimming tissue smells of clearing agent.

- Clearing agent not completely removed due to insufficient impregnation.

- Repeat paraffin impregnation.

4. Tissue is opaque, section-cutting is difficult due to presence of alcohol

- Insufficient clearing. - Repeat clearing and if the object has been embedded, prolong clearing up to 12 hrs. And embed it again.

5. Tissue shrinks away from wax when trimmed.

- Insufficient dehydration so there is incomplete clearing and impregnation.

- Repeat the whole process.

6. Tissue is soft when block is trimmed

- Incomplete fixation - Repeat fixation procedure

7. Air holes found in tissue during trimming.

- Incomplete infiltration or impregnation.

- Repeat impregnation.

8. On trimming, wax appears crystalline.

- Contaminated wax - The bluff Nat cold rapidly

- Re-embed in a freshly filtered wax.

9. Paraffin block after cooling is moist and crumbles

- there is insufficient paraffin impregnation

- Repeat impregnation and re-embed.

Faults observed during section-cutting:

Faults Reasons Remedy

1. Sections fail to form ribbons. - Surfaces and edges of the bluff are not parallel

- Horizontal surface of the block is not parallel to the knife

- Knife is tilted too much - Sections are too thick - Knife is dull

- Re-trim the block - Re-adjust the block - Reduce the tilt of the knife - Re-adjust the thickness of the

section - Re-sharpen the knife

2. Sections roll up on cutting so that they adhere and get broken against the knife edge.

- Knife is blunt - The tilt of the knife is great - The knife edge is dirty

- Re-sharpen the knife - Reduce the tilt - Clean the knife edge

3. Ribbon is crooked, curved and uneven instead of straight.

- The knife is blunt or dull; there is a dull spot on the knife producing an irregular knife edge.

- The knife is not parallel to the block

- Adjust the knife so that knife edge will present a uniformly sharp edge to the block.

- Re-adjust the knife and the block.

4. Sections are wrinkled, - The paraffin block is warm and - Cool the paraffin on ice water

General Histology and Histotechnique (1st Semester; SY 2012-2013)

compressed or jammed. soft - The knife edge is coated with

paraffin - The section are too thin - Microtome set screw is loose. - The tilt of knife is too vertical.

until firmed. - Clean the knife edge - Re-adjust the thickness of

section - Tighten the screw - Reduce the tilt

5. Sections are torn and crumble when cut.

- Incomplete dehydration, clearing and impregnation

- Paraffin is warmed and soft.

- Repeat dehydration, clearing and impregnation

- Cool and harden the paraffin in ice water for ¼ to ½ hour.

6. A hole is formed in the section. - Bubble or dirt is formed in the embedding medium

- Re-embed in freshly filtered wax.

7. Sections adhere to the knife or other parts of the machine.

- There is a static electrically due to low atmospheric humidity.

- Knife edge is dirty - Knife edge is dull - Knife tilt is great

- Breathe out or blow gently in the block and knife to break-up static electricity. Boil water inside the laboratory.

- Clean the knife edge - Re-sharpen the knife - Reduce the tilt

8. Sections are lifted from the knife on upstrokes

- Knife tilt is too great - Knife is dull - Paraffin is soft

- Reduce tilt - Re-sharpen the knife - Cool the paraffin in ice water

9. Sections cut is sometimes thin, and sometimes thick

- Knife is blunt - Knife is not completely clamp - The tilt of knife is great - The knife or block holder is

loose.

- Re-sharpen the knife - Re-adjust the knife - Reduce the tilt - Tighten adjusting and locking

screws.

Paraffin Section - Sections are usually cut between 4-6 micron in thickness. - Micrometer gauge is set to the required thickness and the knife is positioned in such a way that the center of the

blade is in line with the block and the knife has been securely clamped in place. - Cutting is then started until complete sections come out of the block. - A regular cutting rhythm is required - The knife is usually tilted at 0-15 degrees angulations’ on a microtome to allow a clearance angle between the

cutting facet and tissue block - Sections are removed in ribbons of ten, to allow easy location of serial sections - The sections are then floated out in water bath set at 45-50°C to flatten the sections Staining - To impart a color to the tissues - Parts to be visible under microscope Two Categories of dyes: 1. Natural Dyes - Obtained from plants and animals Hematoxilyn - Derived by either extraction from the heartwood of Mexican tree known a Hematoxilyn campechianum. - Most valuable stain in histology used by cytologist

General Histology and Histotechnique (1st Semester; SY 2012-2013)

- Powerful nuclear and chromatin staining capacity - With striking polychrome qualities Hematin – active coloring agents 2. Synthetic Dyes - Known as Coal Tar Dyes - Hydrocarbon benzene derivation - Aniline dyes Examples:

Picric acid - Ability to form salts with alkaline - Fix and stain at the same time by itself - Used as a fixative - Decalcifying agen - Tissue softener

Eosin - a valuable stain in Histotechnique, important stain in connective tissue and cytoplasm. - Histopathology – counter-stain after hematoxilyn and before methylene blue - Background for contrasting stains because it gives a pleasing and colorful contrast to nuclear stain. Red acid dye available in 2 shades:- a. Bluish - Deepen red color

Ex. Eosin B b. Yellowish - Most usable - Readily soluble in water and less in alcohol - Available in both aqueous and alcoholic solutions - Slows a green fluorescence especially in alcoholic medium.

Ex. Eosin Y

3. Basic dyes - coloring substance is found in the basic components - Acid radical is taken from sulfuric, acetic and hydrochloric acid. Ex. Methylene blue *Note: On a chemical basis certain parts of cells and tissues that are acidic in character (e.g. nucleus) have greater affinity for basic dyes, while basic constituents (e.g. cytoplasm) take more of the acid stains.

Fixation of tissues with mercuric chloride and formaldehyde usually favors staining with basic dyes.

Picric acid and Chromium – fixed tissues usually take an acidic dyes

Ethyl alcohol or acetic acid – fixed tissues readily take in both basic and acidic dyes. 4. Neutral Dyes - Combining aqueous solution of acid and basic dyes. - Capable of staining cytoplasm and nucleus simultaneously and differentially - Soluble in alcohol but not in water.

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Ex. WBC differentiation Giemsa’s stain Leishman’s stain

3 main groups of tissue staining 1. Histological Staining - process by the tissue staining constituents are demonstrated in section by direct interaction with a dye or staining

solution producing coloration of the active tissue components. Example: - Mircroanatomical stains - Bacterial stains - Specific tissues stains – muscle, connective tissue, neurological stains. 2. Histochemical staining (Histochemistry) - A process whereby tissue constituents are studied through chemical reactions that will permit microscopy

localization of a specific tissue substance. 3. Immunohistochemistry - Combination of 1st and 2nd classification - Allow phenotypic markers to be detected using fluorescent labeled or enzyme labeled. Staining Technique 1. Direct Staining - A process of giving color to the section by using aqueous or alcoholic dye solution. Ex. Methylene blue, Eosin 2. Indirect Staining - Whereby the action of the dye is intensified by adding another agent or mordant. - Mordant – serve as a bridge or link between the tissue and the dye. - Mordant + dye -> Lake + Tissue = “Tissue mordant dye complex

3. Progressive Staining - The process whereby tissue elements are stained in a definite sequence. - Definite sequence? - to have a satisfactory differential coloration of tissues - Once the dye is taken by the tissue it is not wash anymore.

4. Regressive Staining - The tissue is first over-stained then decolorized - Over-stained – to obliterate the cellular details decolorized until the desired color is achieved. - Differentiation – the selective removal of excess stain.

5. Metachromatic Staining - Entails the use of specific dyes which differentiate particular substances by staining them with a color that is

different from that of the stain itself. Where is this used? - Employ in staining cartilages, connective tissues, epithelial tissues - Belong to basic dyes

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Example: Methylene blue, Safranin. 6. Counterstaining - Provide contrast and background Example: Safranin 7. Microanatomical Staining - General differentiation of nucleus and cytoplasm.

8. Metallic Impregnation - Where specific tissue elements are demonstrated by colorless solutions of metallic salts - Metallic salts – reduced by the tissue ; reduced also by bacteria - It’s not absorbed by the tissues but it is held physically on the surface as precipitates - Valuable metals: Ag, Chloride, Silver nitrate

9. Vital Staining - Selective staining of living cell constituents - Demonstrates cytoplasmic structures by phagocytosis of the dye particle.

10. Intravital Staining - By injecting the dye into any part of animal body. - Purpose: To produce different coloration of cells - Most specially in reticuloendothelial system Examples: Lithium, Indian 11. Supravital Staining - To stain living cells immediately after removal from the living body. Ex. Neutral red Staining of Paraffin Section

Paraffin wax is poorly permeable to most staining solutions and should therefore remove from the section prior to staining.

This is usually done by immersing the paraffin section in a solvent (xylene) two times, at 1-2 minutes duration.

Xylene is not miscible with aqueous solutions and low graded alcohol and therefore be subsequently removed with absolute alcohol followed by descending grades of alcohol to prevent damage and detachment of sections due to possible production of diffusion currents.

The alcohol is then finally replaced with water, before actual staining of section is performed

After deparaffinization with xylene, the section is subjected to decreasing grades of alcohol.

After staining, the section is again dehydrated with increasing grades of alcohol and cleared with two changes of xylene to prepare the section for mounting, since most mountants are miscible with xylene.

The second change of xylene will raise the refractive index of the glass slide, thereby reducing light refraction during microscopic examination.

*Note: If section float off the slide during staining, fix section in a Bunsen flame Slides must not be dirty of greasy.

General Histology and Histotechnique (1st Semester; SY 2012-2013)

Mounting - A process of rendering slides permanency - Used to keep the slide to its permanent position - Seraphy Fluid – applied between the tissue and cover slip. Mounting media importance:

It protects the stained sections from getting scratched and from bleaching or deterioration due to oxidation, thereby preserving the slides for permanent keeping.

Facilitate easy handling and storage

Prevent the damage of sections which may lead to distortion of image during microscopic examination. Characteristics of a good mounting medium:

It should not dry quickly

It should not dissolve out of fade tissue sections

It should not cause shrinkage and distortion of tissues

It should set hard, thereby producing permanent mounting of sections. 2 main groups of mounting media: 1. Aqueous media - Design to mount water miscible preparations Examples: Glycerin, water 2. Resinous media - Used for preparations that has been dehydrated and cleared in xylene and are recommended for majority of

staining method. Examples: Balsan, Colorless nail polish Types of Mounting 1. Dry Mount - It requires no water - Usually used in inanimate objects 2. Wet mount - It requires water - Used to prepare slides to cold living organisms whether they are motile or not. Ringing - A process of sealing the margins of cover slip. Purpose: 1. To prevent the escape of fluid or semi-fluid mounts 2. To prevent evaporation of mounts 3. To immobilize the cover slip 4. To prevent sticking of the slides upon storage