Impregnation and Imbedding

50問 • 2年前

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It is the process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will completely fill all the tissue cavities and give a firm consistency of the specimen. Allows easy handling and cutting suitably in thin sections without damaging it.

Impregnation

It is the process by which impregnated tissue is placed into a precisely arranged position in a mold containing a medium which is then allowed to solidify.

Embedding (Casting/Blocking)

Ideal infiltrating and embedding medium are:

- Soluble in processing fluid - Suitable for sectioning and ribboning - Molten between 30 C and 60 C - Translucent or transparent; colorless - Stable - Homogenous - Capable of flattening after ribboning - Non-toxic - Odorless - Easy to handle - Inexpensive

True/False: Medium used in infiltration is usually same utilized for impregnation, generally known as Embedding medium.

True

Four types of Embedding medium

1)Paraffin wax 2)Celloidin 3)Gelatin 4)Plastic

-Simplest, most common and best embedding medium used for routine processing -Mixture of solid hydrocarbons produced during refining coal and mineral oil -Solid at room temperature, melts at 65 C or 70 C

Paraffin wax

Paraffin wax is not recommended for _____ tissues.

Fatty

In the use of vacuum imbedding: _______ reduces the time required for complete impregnation.

Vacuum

What are the 3 ways paraffin wax impregnation and embedding may be performed?

-By manual processing -By automatic processing -By vacuum embedding

-At least four changes of wax are required at 15 minutes intervals for complete removal of clearing agent. -Specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hrs. for complete embedding or casting of tissue.

Manual processing

Use of automatic tissue processing machine which fixes, dehydrates, clears, and infiltrates tissues, thereby decreasing the time and labor needed during the processing.

Automatic Processing

-Wax impregnation under negative atmospheric pressure inside an embedding oven. -Reduces the time when tissues are subjected to high temperatures thus minimizing heat-induced tissue hardening. -Time required for complete impregnation is reduced by 25%-75% of normal time required. -Tissue is not over-exposed to heat; brittleness, shrinkage and hardening of tissues consequent to overheating is therefore prevented.

Vacuum embedding

-Hastens the removal of air bubbles and clearing agent from the tissue block, promoting more rapid wax penetration -Recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system.

Vacuum embedding

In vacuum embedding, ___________ hasten displacement of cedarwood oil with less tissue shrinkage.

Benzene

What can be used to substitute for Paraffin wax?

-Paraplast -Embeddol -Ester Wax -Water soluble Waxes -Dimethyl Sulphoxide(DMSO)

-Highly purified paraffin and synthetic plastic polymers -Melting point: 56-57 degree C -More elastic and resilient

Paraplast

- Similar to paraplast - Melting point: 56 - 58 degree C - Less brittle and less compressible

Embeddol

-Melting point: 46-48 degree C -Harder than paraffin but soluble in 95% Ethyl Alcohol and other clearing agents -3-4 changes of wax are required to ensure complete tissue impregnation

Ester Wax

- Plastic polymers, mostly polyethylene glycols - Melting point: 38-42 C or 45-56 C

Water Soluble Waxes

-Polyethylene glycol that is solid at room temp. -Soluble and miscible with water

Carbowax

- Reduces infiltration times and facilitates thin sectioning. - Scavenges residual transition solvent and probably alters tissue permeability by substituting for or removing bound water thus improving infiltration.

Dimethyl Sulphoxide (DMSO)

True/False: Those who handle DMSO-paraffin wax may NOT experience unpleasant and annoying oyster or garlic taste.

False

-Purified form of nitrocellulose soluble in many solvents, suitable for specimens with large hollow cavities which tend to collapse for hard and dense tissues such as bones and teeth and for large tissue sections of the whole embryo. -Mainly used for preparing soft tissue sections of mixed consistency such as eyes and brain.

Celloidin (Collodion) Impregnation

What are the two methods of Celloidin (Collodion) Impregnation?

1)Wet method 2)Dry method

-Recommended for bones, teeth, large brain sections, and whole organs -Tissue is placed in thin celloidin (2-4%) for 5-7 days, transferred to medium celloidin (4-6%) for another 5-7 days, drained off and pouted with thick celloidin (8-12%) until the specimen has become impregnated, usually between 3-5 days. -Tissue block is then stored in 70-80% alcohol until ready for cutting

Wet method

-Preferred for processing of whole eye sections -70% alcohol is not used for storage before cutting. Gilson's mixture, made up of equal parts of chloroform and cedarwood oil

Dry method

What does LVN stands for?

Low Viscosity Nitrocellulose

-Another form of celloidin soluble in equal concentration of ether and alcohol with a lower viscosity. -Forms harder tissue block and makes cutting of thinner sections possible. -More explosive than celloidin and should therefore be handled with care. -Container must be kept tightly covered and protected from sunlight to avoid evaporation of alcohol.

Nitrocellulose

-Rarely used except when dehydration is to be avoided. -Used as an embedding medium for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissues when frozen sections are cut.

Gelatin impregnation

-In Gelatin Impregnation, It does not require dehydration and clearing, although fixatives (such as 10% formalin) should still be washed out by running water whenever indicated. -Low melting point -Tissues should not be more than ____ mm - Specimens are harder to freeze than non-impregnated tissues - 1% phenol serves to prevent the growth of molds - Volume should be at least 25 times the volume of the tissues

2-3 mm

Tissue is place into a mold containing the embedding medium and this medium is allowed to solidify

Embedding

It is the 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.

Orientation

What are the several types of Blocking-out Molds that may be used in Embedding?

-Leuckhart's Embedding Mold -Compound Embedding Mold -Plastic Embedding Rings and Base Mold

This type of Blocking-out Mold: -Consists of two L-shaped strips of heavy brass or metal arranged on a flat metal plate and which can be moved to adjust the size of the mold to the size of the specimen. -Mold is adjustable -Recommended for routine use, but, it is too slow for use in busy laboratory

Leuckhart's Embedding Mold

This type of blocking-out mold is made up of series of interlocking plates resting on a flat metal base, forming several compartments

Compound Embedding Mold

This type of Blocking-out molds consist of a special stainless steel base mold fitted with a plastic embedding ring, which later serves as the block holder during cutting.

Plastic Embedding Rings and Base Mold

What are the diff type of Disposable Embedding Molds?

-Peel-away -Plastic Ice Trays -Paper boats

Disposable thin plastic embedding molds, available in 3 different sizes, are simply peeled off one at a time, as soon as the wax has solidified, giving perfect even block without trimming.

Peel-away

This Disposable Embedding Mold is: -Used in ordinary refrigerators may be recommended for busy routine laboratories -Utilized for embedding one tissue block

Plastic Ice Trays

This Disposable Embedding Mold is: -Normally utilized for embedding celloidin blocks but are equally useful for paraffin wax blocks. -Cheap and easy to make. -Provide easy and accurate identification.

Paper Boats

-Recommended for embedding hard tissues such as bones and teeth, and for large sections of whole organs. -Bell jars can be used to control the rate or evaporation of the solvent. -It is discouraged. Due to special requirements and limited use in neuropathology

Celloidin or Nitrocellulose Embedding Method

-Tissues are first embedded or fully infiltrated with a supporting medium such as agar or nitrocellulose, then infiltrated a second time with paraffin wax in which they are subsequently embedded. - Recommended for making small sections of celloidin blocks.

Double embedding

-Provides superior results for light microscopic studies, particularly in hard tissues such as undecalcified bone.

Plastic(resin) Embedding

Plastic Embedding can be classified into:

-Epoxy -Polyester -Acrylic

Low viscosity mixture, provides rapid infiltration of tissues.

Spurr's Resin

Made up of carefully balanced mixture of epoxy plastic, catalysts and accelerators

Epoxy

Made up of esters of acrylic or methacrylic acid, and are used extensively for light microscopy

Acrylic plastics

____________________ has proved to be a popular embedding for light microscopy because it is extremely hydrophilic, allowing many staining methods to be applied, yet tough enough when dehydrated to section well on most microtomes.

Polyglycol methacrylate (GMA)

Added to plastic as catalyst that decomposes to form phenyl radicals acting as an active site for the polymerization of acrylics.

Benzoyl peroxide

____ penetrates tissues better than GMA and the histological quality of bone sections is generally higher in MMA-embedded bone samples compared to GMA-embedded specimens.

MMA

Translation(Pre-finals)

MAHATHIR ALPHA · 55問 · 2年前

Translation(Pre-finals)