IMPREGNATION Flashcards by Kyra Catedral

Impregnation aka

Infiltration

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IMPREGNATION (a.k.a. Infiltration)

• Process that removes the_____
• Fills the_____
• Permeates tissue with a____
• Incomplete impregnation -______

clearing agent

tissue cavities

support medium

tissue airholes

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3 TYPES OF GENERAL TISSUE IMPREGNATION

Paraffin wax impregnation
Celloidin impregnation
Gelatin impregnation

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Requires_____ changes of melted paraffin wax

2 or more

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Kidney specimen

Ika ilan ang impregnation???

4th

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Requires 2 different reagent for embedding

Double embedding

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How many changes in infiltration if microwave oven is utilized?

4 changes (shorter time)

Ex. 15 or 20mins each

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Which provides structure for sectioning? Infiltration or embedding

Infiltration

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If may mali sa dehydration asa makita?

Clearing

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If may mali sa infiltration asa makita?

Sectioning

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If may mali sa clearing asa makita?

Sectioning

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Paraffin wax

Melting point: 45°C, 52°C, 56°C, 58°C @RT (20-24°C)

Paraffin oven must be maintained_____ the melting point of wax

2-5°C above

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If the MP if paraffin wax is 59, what is the max temperature of oven?

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C to F formula

°F = (9/5 × °C) + 32

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Paraffin wax

Used pure

Wax must be filtered first using coarse filter paper such as Green’s No. 904 in oven at 2°C higher than the melting point of wax.

Reusable only once, but heat it first @
________ (remove water)

100-105°C

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PARAFFIN WAX

Advantages

• Rapid (24 hours)

• Maybe cut with ease without undue distortion

• Many staining procedures are permitted

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PARAFFIN WAX

Disadvantages

• Prolonged impregnation will cause excessive shrinkage

• Not recommended to fatty tissues (ex. breast)

• Overheated paraffin makes the specimen brittle

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3 Ways of Paraffin Wax Impregnation:

Manual processing
Automatic processing
Vacuum embedding

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manual processing

This is what we are performing in the laboratory.

In this illustration, there are ______of paraffin; 1 hour per beaker/change.

The changes will depend on the protocol of the laboratory.

3 changes

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automatic processing (MACHINE USED?)

• Our automatic processor is usually designed with 12 stations. The infiltration will take place up at stations 11 & 12.

The infiltration time will be faster because there is constant agitation in the machine.

Hence, a shorter processing time.

Autotechnicon & Elliott bench-type

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Asa na step ma dissolve ang fats?

Clearing

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3 Ways of Paraffin Wax Impregnation:

Where is agitation observed?

Manual and Automated processing

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• Among the 3 processes, this type is the fastest. However, it is expensive because we need to acquire first the machine itself

vacuum embedding

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vacuum embedding

it can only perform the________.
Unlike with our automatic processing, from________.

embedding/ infiltration process

Fixation down to infiltration.

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Minimum capacity of canister in automated tissue processor

200 cassettes Max: >200

How to know if reagent is still good: xylene

Not white If white, may water

How to know if reagent is still good: alcohol in dehydration

Ignite

How to know if reagent is still good: infiltration ing media

Amoy wax pa If amoy xylene, wala na

SUBSTITUTE FOR PARAFFIN WAX

PEBEW Paraplast Embeddol Bioloid Ester wax Water Soluble Waxes (Polyethylene glycol)

Which substitute for paraffin wax have the same Mp

Paraplast Embeddol

Mp of paraplast and embeddol

56-57

Mac temp for paraplast and embeddol

• semisynthetic; for embedding of eyes

Bioloid

Ester Wax o Melting Point:

46-48°C

Mixture of pure paraffin and synthetic plastic polymer (Dimethyl sulfoxide); more elastic and resilient

Paraplast

Less brittle, and less compressible

Embeddol

Harder than paraffin thus used with sliding/sledge-type microtome

Ester wax

What type of microtome is used for ester wax?

Sliding microtome

Ester wax o Water insoluble but soluble in______, thus prior______ is not needed o But Cellosolve, and xylene may be used if indicated

95% ethanol; clearing

Substitute for paraffin wax - most common • No need for dehydration and clearing thus sections are difficult to float out during the fishing method and mount

Carbowax

Carbowax - most common • No need for _________ thus sections are difficult to float out during the fishing method and mount

dehydration and clearing

What reagent is used for fatty specimens? For viewing adipose tissues?

Carbowax (water soluble waxes)

Water Soluble Waxes (Polyethylene glycol) • Melting Point:

38-42°C or 45-56°C

Carbowax difficult to float out during the fishing method and mount • Remedy:

add soap to water or 10% PEG 900 in water

CELLOIDIN IMPREGNATION • A.k.a

Colloidin

CELLOIDIN IMPREGNATION • A.k.a Colloidin Used for

Large, hollow cavities Bones Brain Teeth

• Specimen with large and hollow cavities which tend to collapse; hard and dense tissues; neurologic tissues

Celloidin impregnation

What type of microtome is used for large and hollow cavities

Sliding microtome

• Purified form of nitrocellulose/gun cotton

Celloidin

Celloidin Concentration: in_________ dissolved in equal parts of ether and alcohol - increasing

2%, 4%, 8%

CELLOIDIN ADVANTAGES

• Does not require heat; less shrinkage • Cutting of thicker tissues • Recommended for neurological tissues

CELLOIDIN DISADVANTAGES

Very slow (days or week) Thin sections are difficult to cut Very volatile

METHODS OF CELLOIDIN INFILTRATION (WET)

Fixation & Dehydration Place tissue in ether-alcohol Thin celloidin (2%) Medium celloidin (4%) Thick celloidin (8%) Remove specimen and put in a fresh thick celloidin Keep in jar or desiccator until ether-alcohol evaporates WET: ***Store tissue block in 70%-80% alcohol***

METHODS OF CELLOIDIN INFILTRATION (DRY)

Gilson's Mixture

chloroform and cedarwood oil

- for bones, brain, teeth • Store tissue block in 70%-80% alcohol The purpose of storing it in this concentration is to avoid dehydration and shrinkage of the tissue.

Wet celloidin infiltration

for whole eye sections Store tissue block in Gilson's Mixture (chloroform and cedarwood oil)

Dry celloidin infiltration

• Has lower viscosity, thus can be used in higher concentration, and rapid tissue penetration

Nitrocellulose/Low Viscosity Nitrocellulose

• Advantages: Harder tissues blocks, thus thinner sections are possible • Disadvantages: Explosive when dry due to nitrates

Nitrocellulose/Low Viscosity Nitrocellulose

It consists Plasticizer • E.g. oleum ricini or castor oil • Needed to prevent tissue cracking in chrome mordanted tissues • This promotes plasticity as well as flexibilitv in order to reduce brittleness.

Nitrocellulose/Low Viscosity Nitrocellulose

Nitrocellulose/Low Viscosity Nitrocellulose Luh Advantages: Disadvantages:

• Advantages: Harder tissues blocks, thus thinner sections are possible • Disadvantages: Explosive when dry due to nitrates

How to prevent nitrocellulose from exploding?

suspended with alcohol to maintain its liquid state

Nitrocellulose • E.g. oleum ricini or castor oil • Needed to prevent tissue cracking in chrome mordanted tissues • This promotes plasticity as well as flexibility in order to reduce brittleness.

Plasticizer

• Rarelv used • For histochemical, enzyme studies, and frozen sec.

Gelatin impregnation

• Advantage: Water soluble (no dehydration and clearing needed) • Disadvantage: may decay

Gelatin impregnation

Gelatin impregnation Advantages: Disadvantages:

• Advantage: Water soluble (no dehydration and clearing needed) • Disadvantage: may decay

Gelatin impregnation Tissue must be______ thick

<2-3mm thick

Gelatin impregnation _______ must be added to prevent molds and prevent further damage such as tissue decay

1% Phenol

Gelatin impregnation METHODS OF GELATIN INFILTRATION

Wash out fixative Put tissue in 10% gelatin with 1% phenol 20% gelatin with 1% phenol Fresh 20% gelatin with 1% phenol Cool in refrigerator 10% formalin

Gelatin vs. frozen section

May fixation ang gelatin

Composition of paraplast

Pure paraffin Synthetic plastic polymer (dimethyl sulfoxide)

Substitute for paraffin wax which is more elastic and resilient

Paraplast

Substitute for paraffin wax which is less brittle and less compressible

Embeddol

This allows easier handling and cutting of suitably thin sections without any damage or distortion to the tissue and its cellular components.

Impregnation

is the simplest, most common and best embedding medium used for routine tissue processing.

Paraffin wax

Paraffin wax To decrease viscosity and improve infiltration of the tissue, technologists often increase the temperature to above…

60°C or 65°C.

Manual Processing At least_____ changes of wax are required at 15 minutes intervals in order to ensure complete removal of the clearing agent from the tissue.

four

In automatic processing…only_____ changes of wax are required to remove the clearing agent and properly impregnate the specimen.

2-3

The presence of any odor in the_____ during final paraffin wax bath indicates that the paraffin wax needs to be changed.

clearing agent

involves wax impregnation under negative atmospheric pressure inside an embedding oven.

Vacuum Embedding

Vacuum hastens the removal of air bubbles and clearing agent from the tissue block, thereby promoting a more rapid wax penetration of the tissue. This technique is particularly recommended for urgent biopsies, for delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes, spleen and central nervous system.

Vacuum embedding

Vacuum infiltration requires a vacuum infiltrator or embedding oven, consisting of wax baths, fluid trap and vacuum gauge, to which a vacuum of up to_______ is applied using a water or mechanical pump

760 mm Hg

The degree of the vacuum should not exceed….

500 mmHg

Of the three methods of paraffin wax impregnation, _____\ gives the fastest result

vacuum impregnation

Fresh wax should be filtered before use in a wax oven at a temperature 2°C higher than its melting point. Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future use, with a coarse filter paper, e.g…

Green's No. 904.

When wax has been reused, some amount of water inevitably is mixed with it. If excessive, this may impair the impregnating capacity of the medium and prevent formation of a good tissue block. Water must therefore be removed by heating the wax to_____, thereby raising its melting point. Paraffin wax may be used only_____, after which, fresh wax must be utilized.

100 -105°C twice

Paraplast It is more elastic and resilient than paraffin wax thereby permitting large dense tissue blocks such as ____ and _____ to be cut easily with the same result as in double embedding

bones and brain

is synthetic wax substitute similar to Paraplast with a melting point of 56-58°C (56-57°C)

Embeddol

Sectioning of ester wax-impregnated tissues should be done on a heavy duty microtome (e.g.__________) due to the relative hardness of the wax.

sliding or sledge type microtome

Water Soluble Waxes are plastic polymers, mostly _____with melting points of 38-42°C or 45-56°C.

polyethylene glycols

Due to its hygroscopic nature,_____ is very easily dissolved in water. Hence care must be taken to avoid contact of the block with water or ice. Tissue sections are very difficult to float out and mount due to its extreme solubility in water, dehydrating and clearing agents. Adding____ to water or using______ in water will reduce tissue distortion and promote flattening and "floating out" of sections.

Carbowax soap 10% Polyethylene Glycol 900

This is used mainly for preparing soft tissue sections of mixed consistency such as eyes and brain

Celloidin

Disadvantages include inability to cut thin sections, storage of blocks in alcohol and speed of technique (which can take several weeks or months).

Celloidin

is rarely used except when dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies.

Gelatin impregnation

It is 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

Tissues should not be more than_____ thick since gelatin-embedded specimens are harder to freeze than non-impregnated tissues

2-3 mm

In gelatin impregnation…The ___serves to prevent the growth of molds.

1 % phenol

IMPREGNATION Flashcards

(97 cards)