histopath 3

- It is recommended for routine purposes.

- It decalcifies and softens tissues at the same time

perenyi fluid

- Most rapid decalcifying agent, recommended for

urgent works

- Has poor nuclear staining

- Yellow color formation

phloroglucin nitric acid

- Does not require washing out

- Not recommended for urgent examinations (very slow acting)

trichloroacetic acid

Weakest decalcifying agent, suitable only for minute pieces of bone

sulfurous acid

- Used as a fixative and decalcifying agent

- Caution: It is an environmental toxin, highly corrosive to skin and mucous membrane and carcinogeni

chromic acid

By touching or bending the tissue with the fingers to determine the consistency of tissues

- By pricking the tissue with a fine needle/probe

physical means of measuring decalcification

Very expensive, although it is the most ideal, most sensitive and most reliable method

- Good but not always convenient

- Not recommended for mercuric chloride-fixed tissues (radio-opacity will interfere with the plate interpretation

xray method

- Simple, reliable and convenient method recommended for routine purposes (still favored)

- Decalcifying fluid is changed every 24-48 hrs.

calcium oxalate test

The process of removing intracellular and extracellular water from the tissue following fixation (if decalcification is not required) and prior to wax impregnation (clearing).

dehydration

Dehydration is best accomplished by the use of increasing grades of alcohols, beginning

70% 95% 100% 100%

Undoubtedly the best dehydrating agent (fast acting, mixes with water and organic solvents and penetrates tissues easily)

- Has the advantage of not being poisonous and not very expensive

- Should be at least 99.7% pur

ethyl alcohol

- Should be used if good-grade absolute ethyl alcohol is not easily available

isopropranol

- Toxic dehydrating agent

- For blood and tissue films and for smear preparations

methanol

Utilized for plant and animal microtechnique

butanol

A cheap, rapid dehydrating agent utilized for most urgent biopsies (1/2 to 2 hours)

acetone

why is acetone not used more

volatility and flammability

- Excellent dehydrating and clearing agent miscible to water, melted paraffin, alcohol and xylol/xylene

- Expensive and toxic and extremely dangerous

dioxane

Dehydrates rapidly and not harmful to tissues

- Toxic by inhalation, skin contact and ingestion

cellosolve

Tissues can be transferred directly after fixation & washing

triethyl phosphate

Dehydrates and clears tissues since it is miscible to water and paraffin

tetrahydrofuran

The process whereby alcohol or a dehydrating agent is removed from the tissue and replaced by a fluid (clearing agent) that will dissolve the wax with

which the tissue must be impregnated

clearing

clearing agent must be miscible with the

dehyradating agent, paraffin wax, mounting medium

the main goal of clearing is to

clear tissue of dehydrating agent and make it transparent

The most rapid clearing agent

2. Cheap and does not extract out aniline dyes

xylene

when dehydration is not complete xylene turns into

milky

- Substitute to xylene or benzene

- It is miscible with absolute alcohol and paraffin

- It is not carcinogenic but highly concentrated emit fumes that are toxic upon prolonged exposure

- It acts slower than benzene and is expensive

toluene

Rapid acting, recommended for urgent biopsies and routine purposes

- It is miscible with absolute alcohol and paraffin

- It is highly flammable

- Carcinogenic or may damage bone marrow

benzene

It is slower in action than xylene, but causes less brittleness

- It is the best of the traditional clearing agents for routine use

- Gives the widest latitude

- It is recommended for tough (skin, fibroid and decalcified tissues) and large tissue specimens

chloroform

- Clears both Paraffin and Celloidin sections

- Recommended for CNS tissues and cytological

studies (esp. Smooth muscles and skin)

- Very expensive and it requires 2 changes in clearing solution

cedarwood oil

Recommended for clearing embryos, insects and very delicate specimens since it clears 70% alcohol without excessive tissue shrinkage and hardening

aniline oil

- Causes minimum shrinkage of tissues

- It removes aniline dyes and dissolves Celloidin; Tissues become brittle

- Its quality is not guaranteed due to its tendency to be adulterated

clove oil

Properties are very similar to chloroform but it is cheaper

- Toxic on prolonged exposure

carbon tetrachloride

ideal volume for clearing

not less than 10x volume of tissue

The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fill all natural cavities, spaces, and

interstices of the tissues

impregnation

the simplest, most common, and by far the best for routine use

o melting point is 54-58oC

paraffin wax impregnation

Requires at least 4 changes of wax with 15 minutes’ interval

manual processing

Makes use of an automatic tissue processing machine (e.g. Autotechnicon) which fixes, dehydrates, clears and infiltrates tissues,

automatic processing

Impregnation under negative atmospheric pressure

- Recommended for urgent biopsies and delicate tissues

vacuum embedding

- Melting point: 56-57oC

- A mixture of highly purified paraffin wax and synthetic plastic

polymers is a distinct advance in tissue embedding

paraplast

Melting point: 56-58oC

- A synthetic wax substitute similar to a Paraplast

- It is less brittle and less compressible than Paraplast

embedol

A semi-synthetic wax recommended for embedding eyes

bioloid

- Melting point: 46-48oC

- It is harder than paraffin. It is not soluble in water but soluble in 95% ethyl alcohol and other clearing agents (ex. Xylene, Cellosolve)

ester wax

purified form of nitrocellulose soluble in many solvents

- suitable for specimens containing large cavities or hollow spaces which tend to collapse, for hard and dense tissues (bones and teeth) and for large tissue sections of whole embryos.

celloidin

advantages of using celloiding

much less shringking and distortion on tissue

Recommended for bones, teeth, large brain sections and whole organs

- Tissues must be cut wet (both the knife & tissue block are kept moist with 70% alcohol while cutting)

wet celloidin method

Preferred for processing of whole eye sections

- Material embedded this method can be cut without alcohol due to the presence of the cedarwood oil in the block

dry method

material used in celloding impregnation that is equal parts of chloroform and cedarwood oil

gilson mixture

Rarely used except when dehydration is to be avoided and when tissues are

to be subjected to histochemical and enzyme studies

gelatin impregnatio

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.

embedding

consist of 2 L-shaped pieces of heavy brass or metal, a base being formed by a piece of 1/8 inch thick copper

brass, about 3x2 inches square, or a piece of plate glass

leukhart embedding mold

Made up of a series of interlocking plates resting on a flat

metal base, forming several compartments.

compound embedding mold

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 with base mold

available in 3 different sizes, peeled off one at a time

peel aways

Used in ordinary refrigerators recommended for busy routine laboratorie

plastic ice tray

Cheap and easy to make

o Used for embedding celloidin and paraffin block

paper boat

The process in which tissues are first infiltrated with celloidin and

subsequently embedded in paraffin mass

- Used to facilitate cutting of large blocks of dense firm tissues (brain)

double embedding method

It has superior results for light microscopic studies, particularly in hard tissues (undecalcified bone) and for high resolution light microscopy of tissue sections (for renal and bone marrow biopsie

plastic or resin

a process whereby processed tissues are trimmed and cut into uniformly thin slices or “sections” to facilitate the studies under the

microscope

microtomy

For cutting celloidin embedded sections

sliding microtome

Favored in laboratories where very hard

tissue or blocks are section

base sledge microtome

The block remains stationary while the knife is moved backward and forward

- Most dangerous because of its movable exposed knife

standard sliding microtome

- For cutting paraffin embedded sections

- Most popular and most common type used for routine and research studie

rotary microtome

- For cutting paraffin embedded sections

- Most popular and most common type used for routine and research studie

rocking microtom

or cutting unembedded frozen sections

- Replaced in most laboratories by the cryostat, which is easier to operate (faster and gives thinner sections).

freezing microtome

For cutting specimens into extremely thin

slices (0.5u) for electron microscopy work

ultrathin microtome

- Available in a wide variety of fineness

- Used only for badly nicked knive

caborundum stone

A stone of medium fineness

arkansas stone

The finest (best result)

yellow belgian