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Histology-Pathology > Tissue Processing > Flashcards

Tissue Processing Flashcards

1
Q

A second routine step when tissues are hard and contains calcium

A

Decalcification

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2
Q

Ratio of Decalcifying agent-to-Tissue

A

20:1

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3
Q

Most common Decalcifying agent

A

Nitric Acid

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4
Q

Process of removing intercellular and

extracellular water to maintain SHAPE of cells and its morphology.

A

Dehydration

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5
Q

Important function of intracellular water.

A

Maintain SHAPE of the cells.

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6
Q

True or False. In dehydration, tissue sections are placed in UPGRADED concentration ethanol?

A

True

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7
Q

Most common dehydrating agent

A

Ethanol

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8
Q

Why is water removed?

A

Because it hinder the process for preparation of infiltration and embedding since many embedding media are not miscible with water.

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9
Q

Start/Working Percentage of Ethanol

A

70%

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10
Q

Ratio of dehydrating agent to tissue

A

10:1

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11
Q

Increasing Concentration of Alcohol and time Required

A

70% - 1 hr
80% - 1 hr
90% - 1 hr
95% - 1 hr

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12
Q

Characteristics of Dehydrating Agent

A
  1. Dehydrate RAPIDLY
  2. Should NOT EVAPORATE quickly
  3. Dehydrate even FATTY tissues
  4. Doesn’t HARDEN tissues excessively
  5. Should not be toxic to the body
  6. Should not be a fire hazard
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13
Q

Why are tissues placed in upgraded concentration?

A

This allow tissues to ensure gentle removal of water from shrinkage. diffusion currents and osmotic changes because once water is removed rapidly, it results to not maintain the shape and morphology of cells, Therefore, replacing water by alcohol will in upgraded concentration will adjust the cells for aqueous intracellular and extracellular removal for tissues not to produce destruction, distortion, shrinkage and extraction of cellular components.

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14
Q

Results:
Prolonged in High Concentration
Prolonged in Low concentration

A

A:
High concentration: Make tissues, Brittle, Hard, and Distortion of cellular components causing impregnation unequal and hard cutting of sections.
Low Concentration: Macerate tissues

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15
Q

Factors in Total dehydration time.

A
  1. Temperature
  2. Use of Anhydrous copper sulfate
  3. Thickness of Tissue specimen.
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16
Q

Dehydrating agent used for FATTY specimen

A

Acetone

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17
Q

Dehydrating agent which can be stored for months without hardening and distortion.

A

Cellosolve

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18
Q

Dehydrating agent readily miscible in water, melted paraffin, alcohol and xylol with LESS TISSUE SHRINKAGE.

A

Dioxane

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19
Q

What is clearing of tissues?

A

Clearing of tissues is to make of use of clearing agent which is to remove alcohol (de-alcoholization) and is replaced to dissolve the wax which tissues is to be infiltrated and the medium which sections are to be mounted. This process also makes tissues transparent when examined.

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20
Q

True or False. All clearing agent have dual actions.

A

False. Not all have dual functions because some only remove alcohol without making transparent during staining.

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21
Q

Referred to when clearing agent have DUAL ACTIONS

A

True Clearing Agent

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22
Q

Index of incomplete dehydration

A

Milky appearance

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23
Q

Most common and Best clearing agent

A

Xylene/Xylol

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24
Q

Characteristics of Clearing agent

A
  1. Miscible with alcohol
  2. Miscible with paraffin and/or mounting medium
  3. Should make tissues transparent
  4. Doesn’t dissolve in aniline dyes
  5. Doesn’t evaporate quickly.
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25
Q

Clearing Time

A

30 minutes to 1 hour

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26
Q

Remedy when milky appearance is demonstrated.

A

Glycerine/Gum Syrup

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27
Q

It clears both paraffin and celloidin sections and recommended for CNS, Cytological Studies, Smooth muscles and skin.

A

Cedarwood oil

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28
Q

Clears Embryos, Insects and Delicate specimen

A

Aniline Dyes

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29
Q

Substitute for xylene and benzen

A

Toluene

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30
Q

Clears for decalcified tissues

A

Chloroform

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31
Q

Purpose of Impregnation

A

To replace clearing agent with a medium that will fill out tissue spaces and cavities in order to have a firm consistency of the tissue for embedding and section cutting.

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32
Q

Three Methods of Infiltration

A

Paraffin Wax Impregnation
Celloidin Impregnation
Gelatin Impregnation

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33
Q

Most Common and best embedding medium

A

Paraffin Wax

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34
Q

Ratio of Infiltrating medium-to-tissue

A

25:1

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35
Q

What will be the result of this factor:
•Low MP
•High MP

A

Low MP – paraffin is soft

High MP – paraffin is hard

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36
Q

In manual processing, how many changes are required for paraffin?

A

2-4 changes at 15 mins interval

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37
Q

What is the purpose of 2-4 changes of paraffin?

A

To ensure complete removal of clearing agent and properly maintain impregnation of specimen.

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38
Q

Precautions in Paraffin wax impregnation

A
  1. It should be pure away from dust, water droplets and foreign matters.
  2. Paraffin should only be used twice.
  3. Water must be discarded when automatic tissue processing machine .is performed
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39
Q

Paraffin Oven Impregnation

A

General Rule: 55C - 60C

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40
Q

Methods in Performing Paraffin Wax Impregnation and Embedding

A

Manual Processing
Automated Processing
Vacuum Embedding

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41
Q

Routine MP if Paraffin Impregnation

A

56°C MP

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42
Q

Process under NEGATIVE PRESSURE inside and embedding
oven to hasten removal of air bubbles and
clearing agent, NO exposure to HEAT;
recommended for URGENT biopsies, for dense and hard fibrous tissues

A

VACUUM EMBEDDING

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43
Q

Factors Wax Impregnation

A
  1. Nature and Type of Tissue

2. Clearing agent used

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44
Q

What is the result when impregnation is prolonged?

A

Shrinkage
Hard
Brittle
Difficulty in cutting

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45
Q

What is the result when impregnation is inadequate?

A

Soft
Retention of Clearing agent
Shrinkage
Tissue blocks will be crumbled and break up in bath

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46
Q

Substitute for Paraffin Wax

A

Paraplast
Ester Wax
Embeddol
Carbowax

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47
Q

Paraplast

A

More Elastic, Resilient and Better ribboning of sections

48
Q

Recommended for embedding Eyes

A

Boiloid

49
Q

Embedding medium that is insoluble in water but soluble in 95% ethanol and other clearing agent

A

Ester Wax

50
Q

True or False. Ester wax is used with prior clearing of tissues

A

False.

Ester Wax can be used without prior clearing of tissues.

51
Q

Most commonly used water soluble wax which do not require dehydration and clearing of tissues.

A

Carbowax

52
Q

It is a purified form of NITROCELLULOSE

A

Celloidin

53
Q

Celloidin is most suitable for;

A

Large hollow cavities
Hard & Dense tissues
Large Tissues (embryo)
Neurological tissues.

54
Q

Two Celloidin Impregnation Method

A

Wet Celloidin: Bones, Brain sections, Whole Organ

Dry Celloidin: Whole Eye

55
Q

Gilson’s mixture

A

Cedarwood oil + Chloroform

56
Q

Difference of Dry with Wet Method

A

Dry method does not use 70% alcohol upon
storage, it utilizes Gilson’s mixture (Chloroform and
cedarwood oil)

57
Q

When is Gelatin Impregnation used?

A

If dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies.

58
Q

Other terms for tissue Embedding

A

Tissue Casting/Tissue Blocking

59
Q

True or False. The medium used in infiltration is DIFFERENT from the medium utilized in embedding.

A

False. The medium used to infiltrate the tissue is
usually THE SAME medium utilized for embedding. –
known as embedding medium

60
Q

What is Embedding process?

A

Impregnated tissue is placed in a molder or block that forms the shape of the tissue and require proper arrangement/position of tissues (orientation) containing a medium that is allowed to solidify.

61
Q

What is Orientation?

A

The proper arrangement or specific position of tissue in the mold, during embedding, on the microtome before cutting and slide before staining.

62
Q

General Rule in embedding

A

Infiltrating and Embedding medium must be the same.

63
Q

Blocking Out Molds

Enumerate:
3 Permanent Molders
3 Disposable Molders

A

Permanent Molders

  1. Leuckhart’s embedding mold
  2. Compound embedding unit
  3. Plastic embedding rings and base molds

Disposable Molders

  1. Peel away molds
  2. Plastic ice tray
  3. Paper Boats
64
Q

It is most routinely used mold, can be adjusted and with 2 L-shaped pieces of metal

A

Leuckhart embedding mold

65
Q

Plastic Embedding Rings and base molds

A

Special stainless base mold fitted with a plastic embedding ring (block holder)

66
Q

Embedding method using both celloidin (1st) and paraffin (2nd) media are used.

A

DOUBLE EMBEDDING METHOD

67
Q

Trimming

A

Removal of excess wax in all sides and cutting-off pointed edges of the tissue block.

68
Q

IDEAL Trimmed Tissue Block

A

FOUR SIDED PRISM/TRUNCATED PYRAMID

69
Q

Microtome Knives

A

Plane-Concave Knife (25 mm)
Biconcave Knife (120 mm)
Plane Wedge Knife (100 mm)

70
Q

Plane Concave Knife

A

25 mm
One side - Flat
Other - Concave
Less concave recommended for CELLOIDIN embedded tissue on SLIDING microtome

71
Q

Biconcave Knife

A

120 mm
Both sides are concave
Used in Rotary Microtome

72
Q

Plan wedge Knife

A

100 mm
For Frozen and Hard tissues
For base-sledge or SLIDING microtome

73
Q

Microtome Knife Angles

A 
Bevel Angle (27-32) - angle between CUTTING EDGE of knife
Wedge Angle (15) - angle between BACK SIDES of knife
Clearance Angles (0-15)  - Angle between CUTTING FACET and tissue block
74
Q

Two processes of sharpening Microtome Knives

A

HONING

STROPPING

75
Q

Honing

A

Removal of GROSS NICK and GRINDING the edge of microtome knife on a hone.

76
Q

Stropping

A

Removing the BURRS produced during honing that polishes the sides of the cutting edge; POLISHING

77
Q

Results when knives are not sharpened

A
  1. Torn tissue sections and Tissue ribbons will split or have LENGTHWISE vertical scratches
  2. Tissue sections are lifted from the knife on upstrokes
  3. Uneven thickness of tissue sections, thick or thin.
78
Q

Honing Direction

A

CUTTING EDGES FIRST, with HEAL TO TOE DIRECTION

79
Q

Stropping Direction

A

CUTTING EDGE LAST, with HEAL TO TOE DIRECTION

80
Q

Process which embedded tissues are trimmed, or cut into UNIFORMLY thin slices or “sections” in microtome which produces a predetermined thickness by sliding the block into a cutting tool to facilitate microscopic study of tissues.

A

MICROTOMY

81
Q

Three essential parts of Microtomy

A
  1. Block Holder
  2. Knife carrier and Knife
  3. Pawl , Rachet feed wheel, and adjustment screws
82
Q

Number of STROKES:
Honing
Stropping

A

Honing: 20 - 30 double-strokes
Stropping: 40 - 120 double strokes

83
Q

Purpose of Float-out-bath

A

To smoothen-out, straighten-out, or flatten out wrinkles in tissue sections produced during sectioning.

84
Q

MICROTOMY PRINCIPLE

A

A spring –balanced teeth or pawl is brought in
contact with an turns a rachet feed wheel
connected to a micrometer screw, which is in
turn rotated moving the tissue block at a pre-determined distance towards the knife for
cutting sections at uniform thickness.

85
Q

5 Types of Microtome

A
  1. Rocking Microtome (serial sections & large blocks of paraffin embedded tissues)
  2. Rotary Microtome (paraffin embedded tissues)
  3. Sliding Microtome (celloidin embedded tissues)
  4. Freezing Microtome (unembedded frozen tissues)
  5. Cryostat or Cold microtome (cutting frozen sections)
  6. Ultrathin microtome (for section for EM)
86
Q

Microtome for cutting PARAFFIN embedded tissues.

A

Rotary Microtome

87
Q

Microtome for cutting CELLOIDIN embedded tissues.

A

Sliding Microtome

88
Q

Microtome for cutting UNEMBEDDED FROZEN sections

A

Freezing Microtome

89
Q

Give the functions of each part of Microtome.

  1. Tissue Bloch Holder
  2. Knife carrier, and Knife
  3. Pawl, Rathchet feedwheel and adjustment
A
  1. Tissue Bloch Holder. Hold Tissues in Position
  2. Knife carrier, and Knife. For actual cutting of sections
  3. Pawl, Rathchet feedwheel and adjustment. Lines up the tissue block in proper position and adjusting the proper thickness of tissue.
90
Q

Why float-out-bath is demonstrated in black color?

A

To facilitate visualization of tissue sections during section and fishing out of ribbon.

91
Q

Give 3 problems in sectioning due to tissue Processing.

A
  1. Hard and Brittle Tissues
  2. Tissue is soft when block is trimmed
  3. Difficulty in cutting due to presence of alcohol
92
Q

Give the CAUSES:

  1. Hard and Brittle Tissues
  2. Tissue is soft when block is trimmed
  3. Difficulty in cutting due to presence of alcohol
A
  1. Prolonged Fixation, Dehydration, Clearing and Infiltration. Drying out of tissues.
  2. Incomplete fixation.
  3. Incomplete clearing
93
Q

Give the Causes:

  1. Sections Fail to form Ribbons
  2. Sections are wrinkled, jammed, crumbled.
  3. A hole is formed in the sections.
A

Causes

  1. Horizontal surface of the block is not parallel to the knife.
  2. Paraffin wax is warmed or soft.
  3. Bubble or Dirt is formed in the embedding medium.
94
Q

Tissue section size of Paraffin sections

A

4-6 um

95
Q

Tissue section size of Celloidin sections

A

10-15 um

96
Q

Why are adhesives used?

A

Adhesives are viscous mixture that are used for tissue ribbons from the float-out-bath adhere/stick to the slide for easy fishing out of tissue.

97
Q

Most common Adhesive used.

A

Mayer’s Egg Albumin (1:1 mixture - Egg albumin:Glycerine)

98
Q

What are tissue ribbons?

A

Tissue ribbons are series tissue sections in length with uniform sizes formed during microtomy and is placed in the float-out bath.

99
Q

Length of Tissue ribbons produced?

A

9-12 inches and only 2-6 are used to be fished-out and placed in float-out bath

100
Q

Fishing out Materials

A

Camel’s Hair brush
Needle probe
Index finger

101
Q

Temperature set in Float-out bath

A

40 C - 45 C or 5 C - 10 C below the MP of paraffin

102
Q

Mounting

A

Process which a syrup fluid is applied between tissues and the slide which keeps the slide to its permanent position.

103
Q

Purpose of Mounting

A
  1. Protect specimen from damage which may lead to distortion of image during examination.
  2. Protect tissues from scratches, bleaching and deterioration due to oxidation.
  3. Preserve slide for permanent keeping and teaching purposes
  4. Easy handling and Storage
104
Q

Characteristics of A Good Mounting Medium

A

▪ It should NOT dry quickly
▪ It should not dissolve out of fade tissue sections
▪ It should not cause shrinkage and distortion of
tissues
▪ It should set HARD, thereby producing PERMANENT mounting of sections.
▪ To avoid distortion of the image, the refractive index
of the mountant should be as near as possible to that
of the glass slide (1.518)

105
Q

Two main groups of Mounting Medium

A

AQUEOUS MEDIA

RESINOUS MEDIA

106
Q

Mounting medium used to mount water miscible preparations and for TEMPORARY MOUNTING.

A

AQUEOUS MEDIA

107
Q

Mounting medium used for preparations that is dehydrated and cleared in xylene. For PERMANENT mounting.

A

RESINOUS MEDIA

108
Q

Enumerated Aqueous mounting media and its RI

A

Glycerin Jelly (Kaiser’s 1880): 1.47
Farrant’s medium: 1.43
Apathy’s medium: 1.52

109
Q

Enumerated Resinous mounting media and its RI

A

Canada Balsam: 1.524
DPX: 1.532
XAM: 1.52
CLARITE: 1.544

110
Q

Types of Mounting

A 
Dry Mount (inanimate objects)
Wet Mount (cold living organism)
111
Q

Process of sealing the margins of coverslip

A

Ringing

112
Q

Difficulties in Mounting

A
  1. Tissue sections are cloudy or milky

2. Bubble formation

113
Q

How bubbles are removed?

A

Gently press using a pointed forceps or a mounting needle. In case with many air bubbles, put slide back into the xylene so that coverslip is separated and demount the section without air bubbles.

114
Q

Benzene is recommended for ________ and routine purposes

A

Urgent

115
Q

Benzene can cause _____________

A

aplastic anemia

Histology-Pathology (5 decks)

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