FLOATATION (WATER) BATH The temperature of the water in the bath should be maintained at below ____ the melting point of the paraffin wax to be sectioned. Care should be taken to prevent ____ ____ from being trapped under the section and this can be accomplished by using distilled water in the bath. Floatation bath can be ____ or ___

- 10°C - Water bubbles - circular or rectangular

[M] Week 10: Principles of Microtomy; Paraffin and Frozen Sections, Staining (H&E), Adhesives and Mounting

This is done after embedding.
the means by which tissue is sectioned and attached to the surface of a glass slide for further microscopic examination.
Most is performed on paraffin wax-embedded tissue blocks.

Microtomy

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What is the instrument used to cut sections for microtomy?

Microtome

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This has an advancing mechanism which moves the object, the paraffin block, for a predetermined distance until it is in contact with the cutting tool, the knife or blade.
The specimen moves vertically past this cutting surface and a tissue section is produced.

Microtome

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Enumerate the Types of MICROTOME

Rotary Microtome
Base Sledge Microtome
Rotary Rocking Microtome
Freezing Microtome
Sliding Microtome
Ultra Microtome

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MICROTOME

This is often referred to as the “Minot” after its inventor.
The basic mechanism requires the rotation of a fine advance hand-wheel by 360° degrees, moving the specimen vertically past the cutting surface and returning it to the starting position.

Rotary microtome

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MICROTOME

The rotary microtome may be
1. completely manipulated by the operator
2. when two motors drive the fine and the coarse advance hand-wheel.
3. with one motor to advance either the fine or coarse handwheel

Manual
Fully Automated
Semi-automated

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MICROTOME

Rotary Microtome advantages include the ability to cut thin ____ sections and its easy adaption to all types of tissue sectioning, including those that are hard, fragile, or fatty.

2–3 μm

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MICROTOME

Here the specimen is held stationary and the knife slides across the top of it during sectioning.
Used primarily for large blocks, hard tissues or whole mounts

Base sledge microtome

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MICROTOME

Base sledge microtome are especially useful in what pathology

neurological and ophthalmic pathology

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What are the disadvantage base sledge microtome

3 μm sections are difficult to produce

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MICROTOME

Commonly used in cryostats, the retracting action moves the tissue block away from the knife on the upstroke, producing a flat face to the tissue block.

Rotary rocking microtome

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MICROTOME

Freezes the specimen to harden it so it can be sliced without ripping and destroying it.
Utilizes carbon dioxide (CO2) method.

Freezing microtome

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MICROTOME

The knife or blade is stationary and the specimen slides under it during sectioning.
This microtome was developed for use with celloidin-embedded tissue blocks used primarily for research.
Extremely dangerous because of the blade.

Sliding microtome

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MICROTOME

Used exclusively for electron microscopy
A microscope is present in this type of microtome. This microscope is used to view the tissue sections.

Ultra-microtome

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MICROTOME

TOF

Knives were developed to fit on ur ass but not to specific types of microtome and to cope with different degrees of hardness of tissues and embedding media.

False

pag nag true ka talaga….

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MICROTOME KNIVES

Most steel knives have been replaced with disposable blades, although exceptions include the?

tool-edge knive for resin and steel knives for some cryostats.

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used for routine microtomy and cryotomy.
It provides a sharp cutting edge which can produce almost flawless 2–4 μm sections.

Disposable Stainless-Steel Blades

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Disposable blade holders are incorporated into the?

microtome or an adaptor may be purchased.

The blades may be purchased in dispensers, with or without a polytetrafluoroethylene (PTFE) coating which allows ribbons to be sectioned with ease

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MICROTOME KNIVES

used in electron microscopy and with plastic resin-embedded blocks

GLASS AND DIAMOND KNIVES

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What are the instrument used for paraffin section cutting

Floatation (water) bath
Slide drying oven or hot plate
Fine pointed or curved forceps
Scalpel
Slide rack
Clean slides
Teasing needle
Ice tray
Chemical-resistant pencil or pen

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PARAFFIN SECTION CUTTING

A THERMOSTATICALLY CONTROLLED water bath is used for floating out tissue ribbons after sectioning

Floatatation (water) bath

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FLOATATION (WATER) BATH

The temperature of the water in the bath should be maintained at below ____ the melting point of the paraffin wax to be sectioned.
Care should be taken to prevent ____ ____ from being trapped under the section and this can be accomplished by using distilled water in the bath.
Floatation bath can be ____ or ___

10°C
Water bubbles
circular or rectangular

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FLOATATION (WATER) BATH

This is arrded to the water to reduce the surface tension allowing the section to flatten out with ease.

Alcohol or a small drop of detergent

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DRYING OVEN OR HOT PLATE

Drying ovens incorporate aircon which keep the warm air circulating around the slides. This removes the excess paraffin

False

Fan lang

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# DRYING OVEN OR HOT PLATE TOF The temperature setting should be approximately that of the melting point of the paraffin wax

True

# DRYING OVEN OR HOT PLATE What happen if the oven is too hot?

may be distortion to the cells causing dark pyknotic nuclei, nuclear bubbling and loss of nuclear detail.

# DRYING OVEN OR HOT PLATE Special care must be taken when drying delicate tissues such as **lymph nodes or tissues from the central nervous system**; what temperature is needed?

**37oC for 24 hours** is required to prevent splitting or cracking of the sections.

* These or **teasing needles** are helpful in removing folds, creases and bubbles which may form during floating out of the section on the water bath. o Applicator stick may also be used. * They are also helpful for **manipulating the section** as it passes across the edge of the blade.

BRUSH AND FORCEPS

# SLIDES * For normal routine work, ____ slides are **universally** used. * Although slides are available in a variety of thicknesses, ____ thickness are preferred because they do not **break easily**. Most slide racks are made to accommodate this slide size.

- 76 × 25 mm - 1.0 to 1.2 mm

# SLIDES TOF * Smaller slides are available for use with specialty tissues such as eyes or brains. * Unique identification numbers or codes, patient name or other information should be etched, embossed or written on each slide. Automated instruments which imprint the patient’s information on the glass slide are available. (Chemical-resistant pens and pencils are routinely used to label the slide.)

- False (larger) - True

* Slides which are ____ or ____ resist detachment of the tissue from the slide during staining. * ____ may be used for specialized techniques (e.g., decalcification, special stains, immunohistochemistry, etc.).

- POSITIVELY CHARGED (ELECTRONICALLY) or pretreated with an adhesive (chemically coated) - Colored, frost-ended slides

Providing clean slides are used and sections are adequately dried, the problem of sections detaching from the slide during staining should not occur. Occasions when sections may detach from the slide are?

1. Exposure to **strong alkali** solutions during staining 2. **Cryostat sections** for immunofluorescence, immunohistochemistry or intraoperative consultation 3. Central Nervous System **(CNS) tissues** 4. Sections which are submitted to **extreme temperatures** 5. Tissues containing **blood** and **mucus** 6. **Decalcified tissues**

# Adhesives Adhesives may alleviate the problem of?

tissue loss

# Adhesives what are the protein adhesives? what are their disadvantages

- Albumin - Gelatin - Starch Disadvantage: prone to **bacterial growth or heavy staining** but close monitoring will prevent these problems

What are the adhesives?

1. Poly-L-Lysine (PLL) 2. Silanized 3 aminopropyltriethoxysilane (APES/APS) 3. Charged or Plus Slides

# SECTION ADHESIVES * This is bought as a **0.1% solution** which is further diluted for use **1:10** with distilled water. ○ Slides are coated with the diluted solution and allowed to dry. * The effectiveness of the coating to adhere the tissue to the slide will diminish within a **few days.**

Poly-L-Lysine (PLL)

# SECTION ADHESIVES * Slides are dipped in a **2% solution of APES** in *acetone*, drained, dipped in *acetone* and drained again. * The process is complete when the slides are dipped in distilled water and placed upright in a rack to dry. * These slides are useful for **cytology** and specimens which may be **bloody** or contain **proteinaceous** material.

Silanized 3-aminopropyltriethoxysilane (APES/APS)

* Laboratories often use slides which have been manufactured with a ____ ____. * Placing a positive charge on the slides is accomplished by coating the slide with a ____ ____ in which a chemical reaction occurs, leaving the amino groups linked by covalent bonds to the silicon atoms of the glass. * These slides are superior in their ____ and ____ during staining or pretreatments such as enzyme and antigen retrieval.

- Permanent positive charge - Basic polymer - resistance to cell and tissue loss

# PRACTICAL MICROTOMY TOF Expertise to become competent microtomist is obtained by practical experience under the guidance of a skilled tumor, gaining the confidence and coordination to manipulate the microtome and the \ sections produced.

FALSE not tumor, MENTOR adenoma kaba gurl

* Setup of the microtome. Maintenance by closely following proper care with daily, weekly, quarterly and annual preventive maintenance. * Water bath and microtome should be ergonomically positioned to reduce stress and tension on the neck and shoulders. * Blade should be sharp and defect free

Practical microtomy

# Sectioning Trimming the tissue blocks-paraffin block may be faced or “rough cut” by setting the micrometer at ____ or by advancing the block using the coarse feed mechanism. **‘moth-holes’** artifact may be formed with aggressive trimming as well as damage to tissue by gouging or scoring.

15-30mm

# Sectioning Cutting sections-blocks should be arranged in ____ ____ on a cooling device, cooling both tissue and paraffin giving them a similar consistency. Routine surgical materials should be cut at ____. Ribbons of sections are the most convenient way of handling sections

- numerical order - 3-4microns

# Sectioning - Floating out sections-must be smooth with the ____ of the ribbon making contact with the water first. - Folds may be removed by simply teasing with the forceps. (TOF) - Approximately ____ should be long enough for a ribbon to flatten. - Circular structures like eyes are difficult to flatten, can be placed on slides pre-flooded with ____

- trailing end - True - 30 seconds - 50% alcohol

* Further flatten tissues * Remove paraffin before staining * Use of drying ovens in automated stainers or hot plate * Hot plate may cause localized overheating of slides

Drying sections

# Cutting hard sections * Less problematic since the introduction of ____ ____ * Cutting difficulties is more likely ____ or ____ * Prolonged soaking of the block, or exposing the block surface to running tap water for ____ minutes, often overcome many of the problems associated with cutting hard tissues. * Slight reduction in knife slant or application of ____ ____ on the surface of the block may yield results

- Disposable Blades - Poor fixation or overprocessing - 30 min - Softening agents

This is a discussion of the methods used to produce sections which preserve cellular morphology without the use of dehydrating and clearing solutions and heat.

FROZEN AND RELATED SECTIONS

- Frozen sections are used for? - Moh's Procedures are used for?

- rapid intraoperative diagnoses - surgical margins and sentinel node evaluation

USE OF FROZEN SECTIONS

* Rapid production of sections for **intra-operative diagnosis** * Diagnostic and research enzyme histochemistry for **labile enzymes** * **Immunofluorescent methodology** * **Immunohistochemistry techniques** when heat and fixation may inactivate or destroy the antigens * Diagnostic and research **non enzyme histochemistry**, (e.g., lipids and some carbohydrates) * **Silver demonstration methods** particularly in neuropathology

* This is a **refrigerated cabinet** in which a modified microtome is housed. * All the controls for the microtome are operated **outside** the cabinet. * The first machine were introduced in **1954** and developments in design have improved both sectioning and laboratory safety.

Cryostat

The fresh tissue should be frozen as rapidly as possible without creating freeze artifacts. Suitable techniques include?

- Liquefied nitrogen (−190°C). - Isopentane (2-methylbutane) cooled by liquid nitrogen (−150°C). - Dry ice (−70°C). - Carbon dioxide gas (−70°C). - Aerosol sprays (−50°C). - Internal freezing shelf or bar

# FREEZING OF FRESH UNFIXED TISSUE TOF The best frozen sections are obtained when the tissue is frozen quickly.

True

Method of choice for freezing of fresh unfixed tissue

**isopentane** cooled by liquid nitrogen

# FREEZING OF FRESH UNFIXED TISSUE The problem with using liquid nitrogen alone is the formation of ____ around the tissue which act as an insulator and inhibit rapid, even cooling of the tissue. This can produce ____ ____ in the tissue making diagnostic interpretation difficult, especially in muscle biopsies.

- nitrogen vapor bubbles - Freeze artifact

# FREEZING OF FRESH UNFIXED TISSUE - may be used for freezing tissue blocks - Tissues can be successfully frozen directly in the cryostat, using the?

- Solid carbon dioxide (dry ice) - freeze (cryo) bar

# CRYOSTAT SECTIONING TOF The temperature of the microtome and the cryostat chamber should not be suitable for the tissue type.

False SHOULD BE boang kaba

# CRYOSTAT SECTIONING - Tissues containing large amounts of water will section best? - harder tissues and those that contain fat require?

- warmer temperature - colder temperature

# CRYOSTAT SECTIONING - **Most unfixed material** will section well between ____ - **Most fixed tissues** will section best within the range of ____ depending on the hardness of the tissue.

- -15 and - 23°C - -7 to -12°C

# CRYOSTAT SECTIONING Small blocks of undecalcified cancellous bones can be sectioned but care must be taken to remove any ____ prior to freezing.

cortical bone fragments

# Staining the most widely used histological stain. It is simple to use, easy to automate and demonstrates different tissue structures clearly

Hematoxylin and eosin (H&E)

# HEMATOXYLIN AND EOSIN (H & E) STAIN - stains the cell **nuclei blue-black,** showing clear intranuclear detail - stains **cell cytoplasm** and most connective tissue fibers in varying shades and intensities of **pink, orange and red**.

- Hematoxylin - Eosin

- individual manually dips the slides in the container of the stain. - he slide in the slide rack is programmed and the results will automatically be displayed on the monitor.

- Manual Method - Automatic Slide stainer | jusko po baka di niyo pa to masagutan

fluorescent, xanthene dye which binds to salts with eosinophilic compounds containing positive charges

Eosin

# Staining * It is the most suitable stain to combine with an alum hematoxylin to **demonstrate the general histological architecture of a tissue.** * Has the ability, with correct differentiation, to distinguish between the cytoplasm of different types of cell, connective tissue fibers and matrices, by staining these **differing shades of red and pink.**

Eosin

# EOSIN - eosin yellowish, water-soluble - eosin S, alcohol-soluble - eosin bluish, erythrosin B

- Eosin Y - Ethyl Eosin - Eosin B

# EOSIN - the **most widely used and is soluble in water and alcohol.** * As a cytoplasmic stain, it is usually used as a **0.5 or 1.0% solution in distilled water**, with a crystal of thymol added to inhibit the growth of fungi. * The **addition of a little acetic acid** (0.5 ml to 1000 ml stain) is said to **sharpen the staining**.

EOSIN Y

- extracted from the heartwood (‘logwood’) of the tree Hematoxylon **campechianum**. * It is extracted from the logwood with hot water and then precipitated out from the **aqueous solution using urea**

Hematoxylin

Hematoxylin itself is not a stain, but ____, the **major oxidization product**, is a natural dye responsible for the color properties.

Hematein

Hematein can be produced from hematoxylin in two ways:

1. Natural oxidation or ‘ripening’ 2. Chemical oxidation

# Hematoxylin - by exposure to **light and air**. - This slow process can take up to **3–4 months**, but the resultant solution retains its staining ability for a long time. - **Ehrlich’s and Delafield’s hematoxylin** solutions are examples of naturally ripened hematoxylins.

Natural oxidation or ‘ripening’

- Chemical oxidizing agents convert the hematoxylin to hematein rapidly, - **Sodium iodate** used in *Mayer*’s hematoxylin - **mercuric oxide** used in *Harris’s* hematoxylin

Chemical oxidation

# Hematoxylin Hematein is **anionic**, having a ____ for tissue, and is inadequate as nuclear stain without the presence of mordant

Poor Affinity

# Hematoxylin confers a net positive charge to the dye-mordant complex, enables it to bind to anionic tissue sites, such as nuclear chromatin

Mordant/metal cation The type of mordant used influences the type of tissue components stained and their final color.

# Hematoxylin most useful mordants for hematoxylin

Aluminum, iron and tungsten salts

# Hematoxylin demonstration of argyrophil cells.

Solutions using lead

# Hematoxylin Hematoxylin solutions can be arbitrarily classified according to which mordant is used:

- Aluminum hematoxylin - Iron hematoxylin - Tungsten hematoxylin - Molybdenum hematoxylin - Lead hematoxylin - Hematoxylin without mordant

# Hematoxylin These are the **most frequently** used hematoxylins in the H&E and *produce good nuclear staining.*

ALUMINUM (ALUM) HEMATOXYLIN

# Hematoxylin What is the usual mordant for aluminum hematoxylin

- aluminum **potassium** sulfate (potash alum) - aluminum **ammonium** sulfate (ammonium alum).

# Hematoxylin process of **converting the color of nuclei from red to blue-black** when section is washed in a weak alkali solution like tap water or alkaline solutions such as saturated lithium carbonate, 0.05% ammonia in distilled water (ammonia water) or Scott’s tap water substitute

Blueing

The alum hematoxylins can be used either regressively or progressively. - section is **over-stained** and then differentiated in **acid alcohol**, followed by blueing. - the section is stained for a predetermined time, staining the nuclei adequately, but leaving the **background tissue relatively unstained.**

- Regressive Staining - Progressive Staining

# Hematoxylin TOF The times required for hematoxylin staining with satisfactory differentiation vary according to the type and age of the alum hematoxylin, the tissue type and the personal preference of the pathologist.

True

# Hematoxylin For routine H&E staining of tissues, the most commonly used are:

- Ehrlich’s - Mayer’s - Harris’s - Gill’s - Cole’s - Delafield’s

# Hematoxylin occasionally used for urgent frozen section.

Carazzi’s hematoxylin

- strong hematoxylin solution staining nuclei **intensely and crisply.** * Particularly useful for staining sections for tissues that have been **exposed to acids**

EHRLICH’S HEMATOXYLIN

# Hematoxylin * The stain fades more slowly than those stained with other **alum hematoxylins.** * It is suitable for **acid-decalcified tissues,** and tissues stored in formalin fixatives for long periods of time which become increasingly acidic during the storage period.

EHRLICH’S HEMATOXYLIN

# Hematoxylin Ehrlich’s hematoxylin is also suitable for tissues which have been fixed in acid fixatives such as?

Bouin’s

# Hematoxylin Ehrlich’s hematoxylin is not ideal for

frozen section

last step in tissue processing that results in a permanent histological preparation suitable for microscopy, after adhesion of the sections on to the slide and appropriate staining of the tissue.

MOUNTING

A process by which the maximum degree of transparency of stained tissue sections is covered by coverslip obtained by using a mounting medium that approximate to that of dried protein between ____ and ____ which is close to the RI of glass.

between 1.53 and 1.54

Purpose is to make the tissue transparent for evaluation

Mounting

use this slide to famillarize the mounting procedure

1. Place enough amount of mounting medium on the center of tissue section. 2. Place a dry, clean coverslip on the edge of the slide and release gently. 3. Pressed gently on the coverslip allowing the mounting medium to spread quickly. 4. Properly label the slide. 5. Allow the slide to dry up.

# TYPES OF MOUNTING MEDIA * Mounting media can be? * Most commercially available mountants are usually? * ____ acetate may be added to mountants

- Aqueous or Resinous - non aqueous/resinous - Potassium

Aqueous/hydrophilic/non-aqueous mounting media are still important for special stains (Oil Red O staining), solvents dissolve the fats, few with RI higher than 1.5, most being in the range **1.4 to 1.45**

Non-Resinous or Aqueous Mounting Media (Temporary)

3 types of aqueous mounting media:

- Syrups - Gelatin media - Gum Arabic

Give the Refractive Index - Apathy’s mountant (modified by Lilie and Ashburn 1943) - Apathy’s mountant (Highman’s modification, 1946)

- RI 1.41 - RI 1.436

Match 1. Water 2. Glycerine Jelly 3. Apathy's Medium 4. Farrant's Medium 5. Highmans's Medium 6. Fructose Syrup 7. Polyvinyl Aclohol 8. Glycerine-Glycerol A. 1.47 B. 1.333 C. 1.52 D. 1.43

1. B 2. A 3. C 4. D 5. C 6. A 7. walang sagot 8. A

- adhesive, organic, non-aqueous, hydrophobic * They may be divided into natural and synthetic resins

Resinous Mounting Media (Permanent)

a natural resin extracted from the Canadian tree, Abus Balsamea.

Canada Balsam

most commonly used synthetic resinous media are the?

polyesterenes such as Kirkpatrick & Lendrum’s mountant and Gurr’s distrene plasticizer xylene (DePex)

such as Kirkpatrick & Lendrum’s mountant and Gurr’s distrene plasticizer xylene (DePex)

DPX (DePeX) - Distrene 80

# Match the following 1. DPX (Depex) – Distrene 80 2. Histomount 3. Cover Bond 4. Gurr’s Neutral Mounting Medium 5. Histoclad A. 1.49 - 1.50 B. 1.52 C. 1.54 D. 1.53 E. 1.51

1. B 2. A 3. D 4. E 5. C

# Match the following 1. Permount 2. Pro-Texx 3. Technicon Resin 4. UV-Inert 5. XAM 6. Eukitt A. 1.526 B. 1.62 C. 1.52 D. 1.495 E. 1.517 F. 1.49 - 1.50 / 1.515

1. A 2. D 3. B 4. E 5. C 6. F

[M] Week 10: Principles of Microtomy; Paraffin and Frozen Sections, Staining (H&E), Adhesives and Mounting - Part 1 Flashcards

(101 cards)