CHAPTER 13 CUTTING SECTIONS

Question 1

is a process whereby tissues are cut into uniformly thin slices or “sections” with the aid of a microtome, to facilitate the studies under the microscope.

Answer 1

Sectioning

Question 2

Three general types of tissue sections may be made:

Answer 2

1. PARAFFIN SECTIONS
2. CELLOIDIN SECTIONS
3. FROZEN SECTIONS

Question 3

• for paraffin embedded tissue blocks which may be cut by rocking and rotary microtome.

Answer 3

1. PARAFFIN SECTIONS

Question 4

• for celloidin embedded tissues which are usually cut by means of the sliding microtome.

Answer 4

2. CELLOIDIN SECTIONS

Question 5

• which may be cut from tissues that have been fixed and frozen with CO2 or for fresh or fixed tissues frozen with the cryostat.

Answer 5

3. FROZEN SECTIONS

Question 6

Once the tissues have been embedded and the wax has solidified, the
wax block is removed from the mold, the identification number is noted and the excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting.

Answer 6

TRIMMING

Question 7

Only thin slices are taken out at a time to prevent the block from cracking.

Answer 7

TRIMMING

Question 8

Depending upon the size and orientation of the tissue sample, shave conservatively into the block surface taking appropriate cuts that may measure between

Answer 8

4-60 micrometers.

Question 9

Coarse facing is done on the microtome at approximately [?] at a time until the entire tissue surface is exposed.

Answer 9

30 microns

Question 10

After [?], a heated spatula is held between the tissue block and the block holder until the wax begins to melt.

Answer 10

coarse trimming

Question 11

The block is allowed to harden for cutting proper by facing them down in ice cold water or refrigerator for

Answer 11

5-10 minutes.

Question 12

Fine trimming may be done by either setting the thickness adjuster at [?] or by advancing the block using the coarse feed mechanism.

Answer 12

15 mm

Question 13

The knife is usually tilted at [?] angulation on a microtome to allow a clearance angle between the cutting facet and the tissue block.

Answer 13

0-15°

Question 14

require smaller clearance angles than wedge-shaped knives.

Answer 14

Biconcave knives

Question 15

Sections are cut between [?] in thickness for routine histologic procedures, after the block has been fixed and secured to the block holder.

Answer 15

4-6μ

Question 16

provides better support for the harder elements in a specimen allowing thinner sections to be obtained.

Answer 16

Cold wax

Question 17

wax provides better support for the harder elements in a specimen allowing thinner sections to be obtained.

Answer 17

Cold

Question 18

The sections are then floated out on a water bath set at [?], approximately [?] lower than the melting point of the wax used for embedding the tissue.

Answer 18

45-50°C

6-10°C

Question 19

Sections should not be left on the water bath for a long time ([?] will be enough) to avoid undue expansion and distortion of tissue.

Answer 19

30 seconds

Question 20

Sections may also be flattened out by placing them on a slide which has been flooded with [?], producing convection currents which will serve to remove the creases in the tissue within a few seconds.

Answer 20

20% alcohol

Question 21

should expand the section to its original dimensions and ensure that it is completely flat.

Answer 21

Flotation

Question 22

Flotation
The temperature will need to be [?] below the melting point of the wax.

Answer 22

5 - 9 ̊C

Question 23

The mounted section is then placed in a [?] paraffin oven for [?] or until water droplets are no longer visible on the slides.

Answer 23

70oC

20 minutes

Question 24

Besides the paraffin oven which is maintained at a temperature of [?] above the melting point of the paraffin used, small thermostatically controlled incubators may be used, regulated at [?], and at [?], for enzyme digestion, chemical extraction, metallic impregnation and enzyme localization techniques.

Answer 24

2-5°C

37°C

45-55°C

Question 25

Dry sections for between [?]. Some delicate specimens will produce best results when dried at [?] for a longer time (several hours to overnight).

Answer 25

5 and 30 minutes 37oC

Question 26

Metal racks with 25-slide divisions are used to store the mounted sections during the drying process which usually takes about [?] in the heated oven.

Answer 26

5 minutes

Question 27

Extended storage (?) of unstained formalin-fixed paraffin embedded slides should be avoided as this may result in the loss of antigens.

Answer 27

usually more than 3 days

Question 28

Surfaces and edges of the block are not parallel

Answer 28

Re-trim the block

Question 29

Horizontal surface of the block is not parallel to the knife

Answer 29

Re-adjust and re- orient the block

Question 30

Paraffin wax is too hard

Answer 30

Coat horizontal edges of the block with wax of lower melting point

Question 31

Knife is tilted too much

Answer 31

Reduce the tilt

Question 32

Sections are too thick

Answer 32

Readjust the thickness of the sections

Question 33

Knife is dull

Answer 33

Hone and strop

Question 34

Knife is blunt

Answer 34

Sharpen the knife

Question 35

Tilt of knife is too great

Answer 35

Reduce the tilt

Question 36

Knife edge is dirty

Answer 36

Clean the knife edge

Question 37

Blunt or dull spot on the knife, producing an irregular knife edge

Answer 37

Adjust the knife so that knife edge will present a uniformly sharp edge to the block, or sharpen

Question 38

Edges of the block are not parallel but round or wedge shaped

Answer 38

Re-trim the block

Question 39

Knife is not parallel to the block

Answer 39

Readjust the knife and block

Question 40

Knife is blunt or dull

Answer 40

Re-sharpen the knife

Question 41

Paraffin block is warm and soft

Answer 41

Cool the block on ice water until firm

Question 42

Knife edge is coated with paraffin

Answer 42

Clean the knife edge

Question 43

Sections are too thin

Answer 43

Readjust thickness of the section

Question 44

Microtome set screw is loose

Answer 44

Tighten the screw

Question 45

Tilt of knife is too vertical

Answer 45

Reduce the tilt

Question 46

Bevel of knife is lost due to incorrect sharpening

Answer 46

Re-sharpen, using a knife back or automatic knife sharpener

Question 47

Bubble or dirt formed in the embedding medium

Question 48

Tissue is not processed properly and will not form a section (especially if center is raw)

Answer 48

Re-process tissue

Question 49

Under-processed portion of tissue bursts on contact with warm water

Answer 49

Re-process tissue

Question 50

Hard spot in tissue due to calcium

Answer 50

Once embedded in paraffin wax, decalcification is impractical; use a base-sledge microtome with a wedge knife

Question 51

Tilt of knife is too great or bevel is not cleared, hence object is compressed against the knife edge

Answer 51

Reduce the tilt

Question 52

Clamp set screw on knife or block holder is loose

Answer 52

Tighten the screw

Question 53

Blocks are too large

Answer 53

Cut blocks into smaller fragments

Question 54

Blocks are too hard

Answer 54

Soften the blocks in detergent or phenol

Question 55

Static electricity due to low atmospheric humidity

Answer 55

Breathe out or blow gently on the bock and knife to break up static electricity, or boil water in the room to increase humidity

Question 56

Knife edge is dirty

Answer 56

Clean the knife edge

Question 57

Knife edge is dull

Answer 57

Sharpen the knife

Question 58

Knife tilt is too great

Answer 58

Reduce the tilt

Question 59

Nicks or damage on the knife edge

Answer 59

Sharpen the knife

Question 60

Dirty embedding

Answer 60

Re-embed in freshly filtered wax

Question 61

Knife edge is dirty

Answer 61

Clean knife edge with xylene

Question 62

Tilt of knife is too great

Answer 62

Reduce the tilt

Question 63

Knife tilt is too great

Answer 63

Reduce the tilt

Question 64

Knife is dull

Answer 64

Sharpen the knife

Question 65

Paraffin is too soft or room temperature is warm

Answer 65

Cool paraffin wax in ice water

Question 66

Tilt of knife is too small, paraffin block is therefore compressed against the base of the knife towards the end of stroke

Answer 66

Increase the tilt

Question 67

Knife edge vibrates due to hardness of tissue

Answer 67

Treat with phenol during processing or collodionize

Question 68

Tilt of knife is too great

Answer 68

Reduce the tilt

Question 69

Knife is blunt

Answer 69

Sharpen the knife

Question 70

Knife is not clamped properly

Answer 70

Adjust the knife so that knife edge will present a uniformly sharp edge to the block, or sharpen

Question 71

Knife or block holder is loose

Answer 71

Tighten adjusting and locking screws

Question 72

Knife tilt is too small that block is compressed by bevel and section is not cut

Answer 72

Increase the tilt

Question 73

Tilt of knife is too slanted or too big

Answer 73

Readjust the tilt

Question 74

Tissue is too hard

Answer 74

Take fresh block treated with phenol during processing

Question 75

Knife blade is too thin

Answer 75

Change the knife

Question 76

Freezing is not adequate

Answer 76

Refreeze the tissue block

Question 77

Tissue is frozen too much

Answer 77

Warm the tissue with the fingers

Question 78

Top and bottom edges of block are not parallel to edge of blade/sides of block are not perpendicular to the blade

Answer 78

Adjust the block holder to make the block edges parallel to the knife

Question 79

Wrong micrometer setting

Answer 79

Microtome needs recalibration

Question 80

Clearing agent not completely removed due to insufficient impregnation

Answer 80

Block is trimmed down nearest to the tissue. Remaining wax is melted on embedding oven and paraffin impregnation is repeated, changing the paraffin at least once before embedding

Question 81

Insufficient clearing

Answer 81

Block is trimmed down nearest to the tissue.

Answer 82

Repeat clearing; if object has already been embedded, prolong clearing up to 12 hours, then re- embed

Question 83

Insufficient dehydration, therefore incomplete clearing and impregnation

Answer 83

Repeat the whole procedure

Question 84

Contaminated wax

Answer 84

Re-embed in freshly filtered wax

Question 85

Block not cooled rapidly enough

Answer 85

Re-embed in freshly filtered wax

Question 86

Insufficient paraffin

Answer 86

X Repeat paraffin impregnation, then re-embed

Question 87

is a slow process, usually taking weeks, and does not produce sections as thin as those produced by paraffin embedding.

Answer 87

Celloidin embedding

Question 88

The advantage is that it completely avoids the use of heat at any stage.

Answer 88

Celloidin embedding

Question 89

As a consequence, heat produced artifacts are avoided. In particular, shrinkage is absolutely minimal, if there is any, and structural relationships of the various types of tissue components can be seen clearly.

Answer 89

Celloidin embedding

Question 90

The disadvantages are the longer time to cut, the thickness of the sections, the necessity for staining to be done on free floating section, the inconvenience of having to store the blocks in sealed jars with tight lids to prevent complete evaporation of 70% ethanol, the resulting restrictions on the type of staining methods that may be used.

Answer 90

Celloidin embedding

Question 91

Celloidin is used in form of solution, usually in a 1:1 mixture of ethanol-ether at concentrations of [?].

Answer 91

2%, 4% and 8%

Question 92

The fastest way to dissolve celloidin is to soak it first in half the final volume of anhydrous ethanol to soften it (?) with intermittent mixing in a tightly stoppered container.

Answer 92

50 mL for each 8 grams celloidin

Question 93

The next day, an equal volume of [?] is added and intermittently mixed until an evenly consistent solution is obtained.

Answer 93

diethyl ether

Question 94

The [?] may then be made by simple dilution of the [?] with an equal parts mixture of ethanol and diethyl ether.

Answer 94

2% and 4% solutions 8% solution

Question 95

The block is then trimmed, leaving about [?] of celloidin all around the tissue, then a distinctive cut is made on one corner for orientation.

Answer 95

3 -5 mm

Question 96

The knife is held at a significant slant so that most of the blade edge is used during the cutting stroke, and is quite long, often in excess of [?].

Answer 96

25 cm