QUICK REVIEW 2 Flashcards by Arriane Cyrel (MTI)

:  Process of trimming and cutting processed tissue (mostly, paraffin embedded tissue) into uniformly thin slices or sections to facilitate studies under the microscope.

MICROTOMY

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– a product of embedding

➢Tissue block

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Essential Parts of the Microtome:

Block Holder
Knife carrier and knife
Pawl, Ratchet Feed Wheel and Adjustment Screw
Rotating Wheel
Microtome gauge

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holds the tissue block during cutting/sectioning

Block Holder

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used for actual cutting

Knife carrier and knife

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line up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections

Pawl, Ratchet Feed Wheel and Adjustment Screw

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Needs to be manipulated, moved mechanically to start the cuting process

Rotating Wheel

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Determines the thickness of sections

Microtome gauge

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Principle of Microtomes:
A [?] is brought in contact with, and turns a [?]connected to a micrometer screw, which is in turn rotated, moving the [?] at a predetermined distance towards the knife for cutting sections at uniform thickness.

spring-balanced teeth (pawl)

ratchet feed wheel

tissue block

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TYPES OF MICROTOMES

ROCKING (CAMBRIDGE) MICROTOME

ROTARY (MINOT) MICROTOME

SLIDING MICROTOME

ULTRATHIN MICROTOME

FREEZING MICROTOME

COLD MICROTOME

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1881, Paldwell Trefall

ROCKING (CAMBRIDGE) MICROTOME

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18851886, Minot

ROTARY (MINOT) MICROTOME

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1789, Adams

SLIDING MICROTOME

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1848, Queckett

FREEZING MICROTOME

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10-12u

ROCKING (CAMBRIDGE) MICROTOME

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4-6u

ROTARY (MINOT) MICROTOME

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4-9u

SLIDING MICROTOME

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0.5u

ULTRATHIN MICROTOME

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10-15u

FREEZING MICROTOME

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Simplest

ROCKING (CAMBRIDGE) MICROTOME

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small & large paraffin-embedded blocks

ROCKING (CAMBRIDGE) MICROTOME

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Not for serial sections (sections are cut in a slightly curved plane).

ROCKING (CAMBRIDGE) MICROTOME

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Dis: difficulty orienting the block ; tissue size is restricted

ROCKING (CAMBRIDGE) MICROTOME

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Most common type of microtome

ROTARY (MINOT) MICROTOME

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paraffin embedded sections

ROTARY (MINOT) MICROTOME

Up & down VERTICAL movement = perfectly flat plane

ROTARY (MINOT) MICROTOME

Heavier & more stable

ROTARY (MINOT) MICROTOME

Incorporated in cryostat ; ultrathin sectioning

ROTARY (MINOT) MICROTOME

Celloidin-embedded

Standard Sliding

Celloidin-embedded

Standard Sliding

Most dangerous

Standard Sliding

-knife is moving backward and forward

Standard Sliding

-knife is moving backward and forward

Standard Sliding

For hard & tough tissue blocks in all forms of media

Base-Sledge

For electron microscopy

FREEZING MICROTOME

Uses fragments of broken plate glass

ULTRATHIN MICROTOME

Undehydrated tissues in frozen state

FREEZING MICROTOME

Undehydrated tissues in frozen state

FREEZING MICROTOME

For fats & tissue constituents that may be damaged

FREEZING MICROTOME

FREEZING MICROTOME Freezing agent: ______________________

Liquid carbon dioxide

FREEZING MICROTOME Used for:

Undehydrated frozen tissue Neurological structures Heat damaged tissues

for fresh tissue type

COLD MICROTOME

COLD MICROTOME Uses _____________ microtome

Rotary

COLD MICROTOME Kept cold @ ___________ (Average: ________)

-5 to -30oC -20oC

COLD MICROTOME Freezing occurs within _________

2-3 mins only

COLD MICROTOME Tissue size : _________

4 u

KINDS OF MICROTOME KNIVES (Compound Microscope)

Plane-Concave Plane-Wedge Biconcave

KINDS OF MICROTOME KNIVES (Electron Microscope)

Diamond-edge Glass knives

for paraffin- & celloidin- embedded sections

Plane-Concave

for celloidin-embedded sections & for extremely hard tissues

Plane-Wedge

for paraffin-embedded sections

Biconcave

Rocking, rotary, base-sledge ; 25 u

Plane-Concave

Plane-Concave • Plane knives: • Concave:

celloidin paraffin

Base-sledge, sliding ; 100 u

Plane-Wedge

Rotary ; 120 u

Biconcave

resin blocks for UTM & for EM

Diamond-edge

Resin and Cryo sections (tokuyasu samples) ; Ultrathin

Glass knives

Two triangular shaped knives w/ even pressure

Glass knives

Wash both with detergent and rinse w/

alcohol or distilled water

: 2-4 u, used for cryostat

Magnetic knives

MICROTOME KNIVES ANGLES

Clearance Angle Wedge Angle Bevel Angle Rake Angle

between the edge of the knife & the tissue block

Clearance Angle

5 – 15 degrees

Clearance Angle

angle of cutting

Wedge Angle

15 degrees

Wedge Angle

angle of cutting facet

Bevel Angle

27 – 32 degrees

Bevel Angle

Angle between the block face and upper facet of knife

Rake Angle

90 degrees to the block surface

Rake Angle

Incline the knife [?] clearance angle to avoid uneven sections

5-10o

– must be of good quality steel

Cutting edge

: doesn’t maintain the edge

Too soft

: is likely to nick against hard objects

Too hard

TESTS: Should cut a paraffin wax block at [?]thickness w/o serrations when examined under the microscope (100 X)

2 – 4 um

Will split a hair drawn across it with only their own resistance

Von Mhol’s criterion

• uses the light’s reflection • Reflected: sharp • Absorbed: not recommended for cutting

Von Mhol’s criterion

to remove nicks & irregularities

HONING

HONES:

Carborundum Arkansas Belgium yellow Belgian black vein (blue-green)

 Coarse Honing  Honing Proper

to remove burrs

STROPING

final polishing

STROPING

• badly nicked knife w/ silicon carbide

Carborundum

• Polishing effect

Arkansas

• Manual sharpening

Belgium yellow

• Best manual honing stone

Belgian black vein (blue-green)

: much coarser honing

Fine carborundum

Smooth stones , Machine hone

HONING

Soapy water, Oil(Mineral oil, Castor oil, Clove oil) or Xylene

HONING

Heel-to-Toe

HONING

Zigzag

HONING

Shell horse ; Leather

STROPING

Vegetable Oil

STROPING

Toe-to-Heel

STROPING

30 strokes on each side (Mechanical honing) 10-20 strokes each surface (Planewedge knives)

HONING

40-120 constant strokes

STROPING

HONES: (oilstones)

Plate Glass Honing Factory Grinding Automatic hones

• The finer the grain,

the harder the hone

: used for final polishing (covered with/ POWDERED ALUMINUM OXIDE as an abrasive)

• Diamantine (• Plate Glass Honing )

• After repeated sharpening

Factory Grinding

• Widened Bevel angle (>35o)

Factory Grinding

PRECAUTIONS IN MICROTOMY  Cutting edge must be [?]  Cutting edge must be [?] than the section to be cut  [?] and its corresponding knife back should not be interchanged

sharp & smooth THINNER Knife

PROPER CARE  Microtome -[?](Do not use detergent/alcohol)  Knife- [?]  Hone-[?]  Strop

Xylol remove when not in use lubrication

Always use a back, when required, for sharpening. This is not necessary with

bi-concave knives.

GENERAL STEPS IN FIXING SECTIONS ONTO THE SLIDE

Floating Adhesion (Optional) Fishing out Orientation Deparaffinization Drying sections Post-mordanting (Optional)

water bath (temp: 10C < MP of wax)

Floating

Mayer’ s egg albumin ( Or pooled serum)

Adhesion

Transfer of tissue sections/ribbons on the slide (use camel’s hair brush)

Fishing out

Correct positioning of the tissue section/ribbon on the slide Minimum tissue per slide: Position:

Orientation 3 centered

alcohol lamp - Can distort/overheat tissue paraffin oven - Best

Deparaffinization

wax oven (56 deg C – 60 deg C for 2 hrs) Incubators (overnight) - 37oC Hot plate (45deg C – 55 deg C for 30 – 45 mins.) Alcohol lamp/ bunsen flame

Drying sections

wax oven (?) Incubators (overnight) - Hot plate (?) Blower-type electric slide dryer (?)

56 deg C – 60 deg C for 2 hrs 37oC 45deg C – 55 deg C for 30 – 45 mins. 50 - 55°C for 20 - 30 mins

water bath (temp:[?])

100C < MP of wax

Secondary fixation (post-chroming)

Post-mordanting

Used primarily as mordant & secondary as fixative

Post-mordanting

 central rotating spindle

AUTOMATIC TISSUE PROCESSING

 horizontal radial arm (clock-controlled transfer arm)

AUTOMATIC TISSUE PROCESSING

 “basket”

AUTOMATIC TISSUE PROCESSING

 Thermostat (within walls of beakers) - for temperature control

AUTOMATIC TISSUE PROCESSING

A short repetitive, up-and-down motion of the entire head assembly or arm

AUTOMATIC TISSUE PROCESSING

AUTOMATIC TISSUE PROCESSING • Selection of Tissue Blocks Size: area: thickness: NOT be

3 by 2 ½ cm. > 4 mm

AUTOMATIC TISSUE PROCESSING Dehydrating & Clearing : change at least [?] Wax bath: at least [?] above the MP of wax; [?] Machine: with adequate [?]

once/week 3 OC ; 2 - 3 changes (Manual: 2-5oC , 4 changes) ventilating system

By mounting the sections in [?] aq. foaling-out, gelatin solution or unto albuminized slide, draining excess fluid, and while still moist, placing them in a covered Coplin jar containing [?] 40% formaldehyde in the incubator for [?]

0.1 - 0.25% 2-3 ml 4 - 18 hrs.

- Demonstrate bacterial morphology

NEGATIVE STAINING

- Unstained organism is seen agalnst a black background

NEGATIVE STAINING

CLEARING PRIOR TO STAINING: The [?] must be REVERSED in order to deparaffinize the tissue and allow water soluble dyes to penetrate the sections.

embedding process

Purpose of Staining/Dyeing: • For [?] • Better [?] • Improve [?]

contrast optical differentiation aesthetic value

Chemical basis or dyestuffs:

• Chromophores: “color-bearers” • Auxochrome: “increasers”/electron donors

: “color-bearers” ; Dye capable of producing color

• Chromophores

: “increasers”/electron donors ; Enhance the intensity of staining based on electron donors

• Auxochrome

(basic; pinkish) ; (acidic; bluish)

Cytoplasm ; nucleus

direct interaction by the tissue constituent with a dye/stain = colored tissue component (Bacterial stains, microanatomical stains, muscle stains)

Histological Staining

reaction between the dye and the tissue constituents (e.g. acidic to basic dyes, Pearl’s Prussian blue stain, PAS, enzyme stains)

Chemical/histochemical

(e.g. staining of dextran by iodine)

Adsorption phenomena

(e.g. staining of lipids by alcoholic solutions)

Differential solubility

monoclonal, polyclonal or fluorescent-labeled or enzyme-labeled antibodies

Immunohistochemical Staining

Process of giving color to the section

Direct Staining

Action of a dye is intensified by adding another agent

Indirect Staining

serves as a link or bridge between the tissue and the dye, to make the staining reaction possible; adhesive-like

Mordant

potassium alum with hematoxylin in Ehrlich's hematoxylin, and iron in Weigert's hematoxylin

Mordant

not essential to the chemical union of the tissue and the dye; does not participate in the staining reaction, but merely accelerates the reaction

Accentuator

Examples are potassium hydroxide in Loeffler's methylene blue and phenol in carbol thionine and carbol fuchsin.

Accentuator

Examples are potassium hydroxide in Loeffler's methylene blue and phenol in carbol thionine and carbol fuchsin.

Accentuator

Staining is done in a definite sequence and for a definite period of time until desired intensity is attained ; Lighter to darker

Progressive Staining

Initially overstaining the tissue and the excess stain is removed or decolorized ; Darker to lighter (ex. iodine in gram’s)

Regressive Staining

Differentiator/Decolorizer (?)

alcohol or water

-Differentiates particular substance by staining it with a color different from that of the stain itself.

Metachromatic Staining

-Dependent on the ability to polymerize

Metachromatic Staining

-Uses a different color to provide contrast and background to the staining of the structural components to be demonstrated

Counterstaining

-Demonstrates general relationship of tissue with general differentiation of nucleus and cytoplasm, except the inclusion bodies.

Microanatomical Staining

-Tissue elements are demonstrated by colorless solution of metallic salts which are reduced by the tissue to produce an opaque appearance

Metallic Impregnation

-Unlike stain, not absorbed by the tissue

Metallic Impregnation

-Most valuable: Gold and Silver (Gold chloride or Silver nitrate)

Metallic Impregnation

Using a color similar to the color of the tissue

Orthochromatic Staining

Staining of living cell constituents (except: nucleus) Ex. Mitochondria, cytoplasm; used to count living vs. nonliving (sperm counting)

Vital Staining

injecting dye into any part of the animal body Before biopsy (Ex. India ink) *lithium, carmine and India ink

Intravital Staining

staining of living cells immediately after removal from the living body (Ex. Neutral red, Janus Green - mito) * New Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining

Supravital Staining

H: E: Routine:

Primary, basic, nuclear, blue to black Counter/secondary, acidic, cytoplasmic, pink regressive/histochem

Most valuable stain

Hematoxylin

Hematoxylin Extracted from the core of the wood of a mexican tree:

Haematoxylon campechianum

Hematoxylin Active coloring agent (Most widely used agent):

Hematin

-Gives deep blue color

Hematin

-Formed via ripening (oxidation of hematoxylin)

Hematin

This is usually accomplished by exposing the substance to air and sunlight, thereby oxidizing hematoxylin (?). Such a process is slow and takes as long as 3-4 months, but it can be accelerated by adding strong oxidizing agents such as hydrogen peroxide, mercuric oxide, potassium permanganate, sodium perborate or sodium iodate which converts hematoxylin to hematin almost instantaneously by chemical oxidation (?), so that the staining solution is ready for use immediately after preparation. It is essential that the oxidant be used in correct amount, since excessive oxidation (?) leads to production of other useless compounds.

natural ripening artificial ripening over-ripening

Hematoxylin Principle:

The acidic component of the cell has affinity to basic dye and vice versa.

In Hematoxylin and Eosin stain, [?] stains the acidic part of the cell hence, called a nuclear stain. [?] is the cytoplasmic stain.

hematoxuylin Eosin

Hematoxylin and Eosin stain Major Steps: Note: Water-rising steps are not shown

De paraffinization Hydration Nuclear Staining Differentiation Blueing Counterstaining Dehydration Clearing

TYPES OF ALUM HEMATOXYLIN:

-used for regressive staining

Ehrlich’s Hematoxylin

-used for mucopolysaccharides, cartilage, cement lines of bones

Ehrlich’s Hematoxylin

-requires 15-40 minutes staining time

Ehrlich’s Hematoxylin

-used for routine nuclear staining in exfoliative cytology and sex chromosomes

Harris Hematoxylin

requires 5-20 minutes of staining

Harris Hematoxylin

-used for routine purposes, in sequence with Celestine blue

Cole’s Hematoxylin

-requires 10 minutes of staining

Cole’s Hematoxylin

-Used in Celestine Blue Hemalum method of nuclear staining

Mayer’s Hematoxylin

-Can be used in regressive/progressive stain

Mayer’s Hematoxylin

-Uses ferric ammonium sulfate (iron alum) as mordants.

Iron Hematoxylin

-applied in all fixatives producing permanent stains

Iron Hematoxylin

-results to blackish or grayish differentiation

Iron Hematoxylin

-minimal eyestrain and useful for photomicrography

Iron Hematoxylin

TYPES OF HEMATOXYLIN STAIN

-standard iron hematoxylin

Weigert’s Hematoxylin

-demonstrates muscle fibers and connective tissue

Weigert’s Hematoxylin

-recommended for acid decolorizers of nucleus with alum hematoxylin

Weigert’s Hematoxylin

-cytological stain for regressive staining of thin section

Haidenhain’s Hematoxylin

-used for both nucleus and cytoplasmic

Haidenhain’s Hematoxylin

-demonstrates voluntary muscle striations and myelin

Haidenhain’s Hematoxylin

-stains paraffin, celloidin and frozen sections

Phsophotungstic Acid Hematoxylin (PTAH)

-requires 12-24 hours

Phsophotungstic Acid Hematoxylin (PTAH)

-maybe used in progressive staining.

Phsophotungstic Acid Hematoxylin (PTAH)

-color of the solution ranges from reddish brown to purple.

Phsophotungstic Acid Hematoxylin (PTAH)

OTHER STAIN

Old histologic dye derived from an extract from the female bug: Dactylopius coccus costa / Coccus cacti

Cochineal Dye/Carmine Dye

Vegetable dye extracted from Lichens

Orcein

Derived from the dried Stigmata of Crocus Sativus

Saffron

-passing the tissue to an alkaline solution

Blueing

-Blueing forms insoluble (?) tissue lake

blue aluminum-hematin

Substitute for ammonia:

Tap Water (Sodium bicarbonate, Magnesium Sulfate, Distilled Water, Thymol)

Substitute for ammonia:

Tap Water (Sodium bicarbonate, Magnesium Sulfate, Distilled Water, Thymol)

Factors affecting the blueing:

Cold water Very cold (below 10 deg C) Warm water

 Known as Coal Tar Dyes

SYNTHETIC DYE

 Derived from hydrocarbon benzenes

SYNTHETIC DYE

 Collectively known as “aniline” dyes

SYNTHETIC DYE

-produces color

Chromophores

-benzene compounds containing chromophore; not permanent

Chromogens

-Electrolytic dissociation mechanism

Auxochrome

-consist of chromophore and auxochrome attached to a hydrocarbon benzene ring

Dye

: base is sodium

ACID DYE

: radically taken from acetic, sulfuric or HCl

BASIC DYE

: combination of acid and basic dye

NEUTRAL DYE

soluble in alcohol but insoluble in water

NEUTRAL DYE

-most valuable stain for connective tissue and cytoplasm

EOSIN

-used as a background for contrasting stain

EOSIN

-common basic nuclear stain, employed with eosin

METHYLENE BLUE

-valuable for plasma cells, and for diagnosing fresh sputum for malignant cells

METHYLENE BLUE

-Diagnosis of diphtheria

METHYLENE BLUE

metachromatic dye

METHYLENE VIOLET

-derived when methylene blue is heated in a fixed alkaline solution

METHYLENE VIOLET

Fixed tissues

TOLUIDINE VIOLET

-used for thionine in fresh frozen tissue

TOLUIDINE VIOLET

-recommended for Nissl granules or chromophilic bodies

TOLUIDINE VIOLET

-Nuclear or chromatin stain

CYRSTAL VIOLET

-Used for staining amyloid in frozen sections

CYRSTAL VIOLET

-Cytoplasmic stain

ANILINE BLUE

-Used for counterstaining epithelial sections

ANILINE BLUE

Plasma stain

BASIC FUSCHIN

Plasma stain

BASIC FUSCHIN

Plasma stain

BASIC FUSCHIN

-Used for deep staining of mitochondria

BASIC FUSCHIN

-Used for staining blood to differentiate leukocytes

GIEMSA STAIN

-Resistant to strong acid dyes

CELESTINE BLUE

Recommended for routine staining of freed sections

CELESTINE BLUE

-Resistant to strong acid dyes

CELESTINE BLUE

- Giving good nuclear definition when used in conjunction with alum hematoxylin

CELESTINE BLUE

Weakly basic dye

MALACHITE GREEN

Used as a contrast stain for staining erythrocytes

MALACHITE GREEN

Used as both decolorizer and as counter stain

MALACHITE GREEN

-Stains chromatin green in the presence of an acid

METHYL GREEN

-Gives false positive with certain secretions such as mucin

METHYL GREEN

-Used as contrast staining for Gram’s technique in Acid fast and Papanicolau method

BISMARK BROWN

Staining diptheria organisms

BISMARK BROWN

Colored salt of ferric ferrocyanide

PRUSSIAN BLUE

Utilized for manufacture of paints

PRUSSIAN BLUE

May be used for microanatomical color contrast for demonstrating of the circulatory system by injection

PRUSSIAN BLUE

An excellent stain for elastic fibers

ORCEIN

Recommended in dermatological studies

ORCEIN

Employed as a contrast stain to Acid fuchsin

PICRIC ACID

Chromatin stain for fresh materials in smear preparations

CARMINE

Usually kept in ammoniacal solution

CARMINE

Usually combined with aluminum chloride to stain glycogen and mucin

CARMINE

PRECAUTIONS IN STAINING 1. Avoid stains on the skin – [?] 2. Falling [?] 3. Faulty [?]

0.5% acid sections staining solution

• Protection • Better optical quality • Preserve the tissue section for years.

MOUNTING

• Mounting media -RI of (slide)

1.518

AQUEOUS MOUNTING MEDIA RESIN MOUNTING MEDIA

For frozen sections, fats, amyloid

1. Water

Requires solidification

2. Glycerin

 Gelatin, Glycerol, distilled water and phenol

 Glycerin Jelly - RI (1.47)

• Pure gum arabic, cane sugar or sucrose, distilled water and thymol crystals

3. Apathy’s medium – RI (1.52)

• Gum arabic, distilled water, glycerol and sodium merthiolate

4. Farrant’s medium – RI (1.43)

• Glucose, Glycerine, spirits of camphor and distilled water

5. Bruns Fluid

• Used for preparation that has been cleared with xylene of toluene

RESIN MOUNTING MEDIA

• Used for preparation that has been cleared with xylene of toluene

RESIN MOUNTING MEDIA

CONSIDERATIONS: A mountant should be: • be freely miscible with [?] • NOT dry quickly, Should NOT crack or form [?] on slide upon drying • NOT fade out or dissolve out tissue sections, NOT cause [?] • leach out any stain or affect staining, NOT change in [?] • set out to produce [?] of sections

xylene or toluene artifactual granularity distortion or shrinkage COLOR or pH permanent mounting

RINGING: • Sealing the sides of the coverslip with

 Paraffin wax  Kronig/Du Noyer’s mixture  Nail varnish

DRYING OF MOUNTED SLIDES: Setting of the mounting medium /Before microscopic examination:  Hot plate or Wax oven at [?] Before Filing the mounted slide: [?] in an oven for 2 hrs or at [?] for 2 – 3 days

50OC for 2 hrs 37OC – 60OC ; room temp

Branch of medicine which deals w/ the study of cells that are exfoliated or scraped off from the lining epithelium & mucosa of different organs

EXFOLIATIVE CYTOLOGY

study of abnormal cells (e.g. CA cells)

CYTOPATHOLOGY

study of the different methods of preparing the cells for microscopic examination

CYTOTECHNIQUE

 For diagnosis of Cancer