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HISTOPATH LEC - TRANS > 4-IMPREGNATION > Flashcards

4-IMPREGNATION Flashcards

1
Q

What is the process where the clearing agent is removed and replaced by a medium that fills tissue cavities and gives it a firm consistency?

A

Impregnation (Infiltration)

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2
Q

What happens if the clearing agent is not properly displaced during impregnation?

A

The tissue will not be properly impregnated with paraffin wax

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3
Q

What results from improper paraffin wax impregnation?

A

Soft

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4
Q

What problems arise during microtomy due to improperly impregnated tissues?

A

Difficulty creating tissue ribbons

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5
Q

What is the process of placing impregnated tissue in a mold with a solidifying medium?

A

Embedding

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6
Q

What are other terms for embedding?

A

Casting or blocking

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7
Q

What is a crucial factor during embedding?

A

Orientation of the tissue

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8
Q

What properties should an ideal infiltrating and embedding medium possess?

A

Soluble in processing fluids+suitable for sectioning and ribboning+molten between 30°C and 60°C+translucent or transparent and colorless+stable and homogenous+capable of flattening after ribboning+non-toxic and odorless+easy to handle+inexpensive

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9
Q

What are the four main types of impregnation and embedding media?

A

Paraffin wax+celloidin+gelatin+plastic/resins

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10
Q

Why do infiltration and embedding share a medium?

A

They are continuous processes with the tissue submerged in the medium

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11
Q

What is the simplest

A

most common

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12
Q

What is paraffin wax composed of?

A

Polycrystalline mixture of solid hydrocarbons from coal and mineral oil refining

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13
Q

At what temperature does paraffin wax typically melt?

A

Around 65-70°C

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14
Q

Why is paraffin wax used at 2°C above its melting point up to 60-65°C?

A

To make it more viscous

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15
Q

What is the effect of high molecular weight mixtures on melting temperature?

A

They melt at a higher temperature

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16
Q

What crystal size is preferred for better physical support in paraffin wax?

A

Small uniform sized crystals

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17
Q

How do low melting point waxes affect the matrix?

A

They have higher paraffin concentrations and provide a softer matrix

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18
Q

How do waxes with higher polymer content affect the matrix?

A

They produce a harder matrix that mimics dense tissue more closely

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19
Q

What are the advantages of paraffin wax impregnation?

A

Thin individual serial sections without distortion+rapid process+indefinite storage+nucleic acids may be recovered decades after fixation+good staining results

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20
Q

What can result from overheated paraffin?

A

Brittle specimen

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21
Q

What results from prolonged paraffin wax impregnation?

A

Excessive tissue shrinkage and hardening

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22
Q

What results from inadequate paraffin wax impregnation?

A

Retention of the clearing agent

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23
Q

What types of tissues require long immersion for proper support during paraffin wax impregnation?

A

Difficult-to-infiltrate tissues (larger/denser tissues)

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24
Q

What types of tissues are not recommended for paraffin wax impregnation?

A

Fatty tissues

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25
Why are fatty tissues not recommended for paraffin wax impregnation?
They are dissolved during the alcohol and xylene steps
26
What factors determine the duration and number of changes for thorough tissue impregnation?
Size and type of tissues+use of vacuum embedding+clearing agent employed
27
What indicates the need to change paraffin?
The smell of xylene in the paraffin
28
What are the three methods for paraffin wax impregnation and embedding?
Manual processing+automatic processing+vacuum embedding
29
How is manual processing performed?
At least four changes of wax at 15 minutes interval for impregnation and melted paraffin for 3 hours for embedding
30
How does automatic processing facilitate impregnation?
It uses an automatic tissue processing machine with constant agitation and controlled temperatures
31
How many wax changes are typically used in automatic processing?
2-3 changes of wax
32
What precautions are necessary during automatic processing?
Changing dehydrating fluids+clearing agents+dilute ethanol frequently+filling containers to appropriate levels+setting wax bath thermostats 3 degrees above the melting point+avoiding prolonged tissue exposure to hot paraffin
33
What minimizes heat-induced tissue hardening during wax impregnation?
Vacuum embedding
34
What range should be the temperature be kept above the melting point for vacuum embedding?
2 degrees higher than melting point up to 60-65C
35
What does vacuum embedding facilitate?
Complete removal of transition solvents
36
What effect does transition solvent removal and removal of air bubbles have on wax penetration?
A more rapid wax penetration of the tissue
37
What types of tissues benefit most from vacuum embedding?
Urgent biopsies and delicate tissues (lung
38
By what percentage does vacuum embedding reduce complete impregnation time compared to automated methods?
25-75%
39
What is the recommended temperature for vacuum embedding?
2-4°C above the melting point of the wax
40
What degree of vacuum should not be exceeded during vacuum embedding?
500 mmHg
41
For how long should tissue not be left in the paraffin oven?
For more than 4 hours
42
What is the maximum number of times paraffin wax should be used?
Twice
43
If using an automatic tissue processor
when should the wax be discarded?
44
How can excess paraffin wax be reused?
Heat in 100 degrees then filter
45
What factors determine the duration and number of changes required for thorough tissue impregnation?
Size and type of tissues+use of vacuum embedding+clearing agent employed
46
How does tissue size affect impregnation?
Larger/denser tissues require longer times and more frequent wax changes
47
How does vacuum embedding influence impregnation duration?
Reduces time required for complete impregnation by facilitating fixation
48
What indicates the need to change paraffin wax during impregnation?
The smell of xylene in the paraffin wax
49
What are the three methods for paraffin wax impregnation and embedding?
Manual processing+automatic processing+vacuum embedding
50
How is manual processing performed?
Impregnation requires at least four changes of wax at 15-minute intervals; embedding involves melted paraffin for 3 hours
51
Why are multiple wax changes necessary during manual processing?
To ensure complete removal of clearing agents and proper tissue impregnation
52
What is the sequence of clearing agent removal during manual processing?
First change removes most xylene→second removes less→third removes small amounts→fourth leaves only paraffin wax
53
How does automatic processing facilitate impregnation?
Uses an automatic tissue processor with constant agitation and programmed cycles
54
How many wax changes are typically required in automatic processing?
2-3 changes of wax
55
What precautions must be taken during automatic processing?
Change dehydrating fluids frequently+change clearing agents weekly+ensure containers are filled properly+set wax bath thermostats 3°C above melting point+avoid prolonged exposure to hot paraffin wax
56
What is vacuum embedding?
Wax impregnation under negative atmospheric pressure inside an embedding oven
57
How does vacuum embedding improve tissue impregnation?
Facilitates complete removal of transition solvents+hastens air bubble removal+promotes rapid wax penetration into tissues
58
What types of tissues benefit most from vacuum embedding?
Urgent biopsies+delicate tissues such as lung
59
By what percentage does vacuum embedding reduce impregnation time compared to automated methods?
25-75% reduction in time required for complete impregnation
60
What temperature should be maintained during vacuum embedding?
2-4°C above the melting point of the wax (recommended: 60-65°C)
61
What degree of vacuum pressure should not be exceeded during vacuum embedding?
500 mmHg maximum pressure
62
What practical considerations must be followed during tissue impregnation?
Do not leave tissues in paraffin oven for more than 4 hours+maintain oven temperature 2-5°C above melting point+use pure paraffin wax+reuse paraffin only twice after heating and filtering excess wax
63
What substitutes for paraffin wax are commonly used in histological embedding?
Paraplast+Embeddol+Ester Wax+Water-soluble waxes (Carbowax)+Dimethyl Sulphoxide (DMSO)
64
What is Paraplast composed of?
Highly purified paraffin and synthetic plastic polymers
65
What are the advantages of Paraplast?
Melting point 56-57°C+more elastic and resilient+produces uniform blocks+better ribboning of sections+serial sections can be cut without cooling+does not crack
66
What is Embeddol?
A synthetic wax substitute similar to Paraplast
67
What are the advantages of Embeddol?
Melting point 56-58°C+less brittle and compressible than Paraplast+suitable for embedding eyes (Bio/aid)+contains rubber for improved properties (Tissue Mat)
68
What is Ester Wax?
A harder wax with a lower melting point (46-48°C)
69
What are the advantages of Ester Wax?
Insoluble in water but soluble in 95% ethyl alcohol and other clearing agents+can be used without prior clearing of tissues+compatible with Cellosolve or xylene as clearing agents
70
What are the disadvantages of Ester Wax?
Requires heavy-duty microtomes for sectioning due to its hardness (e.g.
71
What are water-soluble waxes composed of?
Plastic polymers
72
What is Carbowax?
A polyethylene glycol-based water-soluble wax with a melting point of 38-42°C or 45-56°C
73
What are the advantages of Carbowax?
Soluble in water+miscible with water+tissues can skip dehydration and clearing+preserves neutral fats and lipids+suitable for enzyme histochemical studies+excellent cytologic preservation
74
How is routine impregnation performed using Carbowax?
Four changes in increasing concentration: 70% ethanol (30 mins)→90% ethanol (45 mins)→100% ethanol (1 hour)→100% ethanol (1 hour)
75
How are tissues embedded using Carbowax?
Embedded in fresh Carbowax at 50°C and cooled in a refrigerator
76
What is Dimethyl Sulphoxide (DMSO) used for?
Added to proprietary blends of plastic polymer paraffin waxes to reduce infiltration times and facilitate tissue sectioning
77
What are the disadvantages of DMSO?
Unpleasant oyster or garlic taste
78
What is celloidin composed of?
Purified nitrocellulose soluble in ether and alcohol mixtures
79
What types of specimens benefit from celloidin impregnation?
Soft tissue sections+specimens with large hollow cavities+hard/dense tissues+large tissue sections
80
What are the advantages of celloidin impregnation?
Recommended for neurological tissues+rubbery consistency+dense tissues and collapsing specimens are better supported+does not require heat
81
What are the disadvantages of celloidin impregnation?
Slow process taking several days/weeks+difficult to cut thin sections (<10 µm)+serial sections are hard to prepare+ether solvent vapor is flammable+difficult photomicrographs+volatile nature
82
For what specimens is wet celloidin recommended?
Bones
83
What steps are involved in wet celloidin embedding?
Fixation/dehydration→Ether/alcohol immersion (12-24 hrs)→Thin celloidin (5-7 days)→Medium celloidin (5-7 days)→Thick celloidin (3-5 days)→Embedding medium→Evaporation in dessicator→Storage in 70-80% alcohol until cutting
84
How does dry celloidin differ from wet celloidin embedding?
70% alcohol storage is skipped before cutting; Gilson’s mixture (chloroform/cedarwood oil) is added to make tissues transparent
85
How does nitrocellulose differ from celloidin?
Lower viscosity+higher hardness for thinner sectioning but more explosive
86
What is gelatin impregnation used for?
Embedding delicate specimens and frozen tissues when dehydration must be avoided and for histochemical and enzyme studies
87
Why is gelatin preferred for enzyme studies?
Enzymes are destroyed during paraffin preparation
88
What are the properties of gelatin as an embedding medium?
Water-soluble+low melting point+does not require dehydration or clearing+prevents over-hardening of tissues by heating
89
What are the steps for gelatin impregnation?
1. Immerse tissue in 10% gelatin with 1% phenol for 24 hours→2. Transfer to 20% gelatin with 1% phenol for 12 hours→3. Use fresh 20% gelatin with 1% phenol and cool in a refrigerator until impregnation and embedding are completed→4. Transfer to 10% formalin for 12-24 hours to harden the tissue→5. Ensure tissues are no thicker than 2-3 mm
90
What is the purpose of transferring gelatin-embedded tissues to formalin?
To harden the tissue after embedding
91
What is a critical consideration for tissue thickness during gelatin impregnation?
Tissues should not exceed 2-3 mm to ensure proper impregnation
92
Impregnating medium at least ____-- times the volume of tissue
25x

HISTOPATH LEC - TRANS (17 decks)

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