LAB 2 Sectioning (Microtomy)

Question 1

Differentiate the course wheel and fine wheel

Question 2

Function of the Micron Thickness Selector

Question 3

Function of the Clearance Angle Adjustment

Answer 3

Adjusts the clearance angle between the tissue face and the lower facet of the knife wedge

Question 4

Function of the Angle Adjuster Screws

Question 5

What is the ideal clearance angle ?

Answer 5

3 to 8 degrees

Question 6

What is the consequence to a clearance angle that is too shallow (knife is parallel to the tissue) ?

Answer 6

The pressure applied to the tissue is unstable, which results in alternating thick and thin sections
- this is dependent on factors like tissue and wax properties

Question 7

What is the consequence to a clearance angle that is too steep (knife is perpendicular to the tissue) ?

Answer 7

The tissue will bend sharply before breaking off
- chattering will be seen microscopically (parallel lines like Venetian lines)

Question 8

Waterbath temperature

Answer 8

5 to 10 degrees below melting temperature of the paraffin wax

Question 9

T or F: the water bath has limited use in removing wrinkles from the tissue itself

Answer 9

TRUE; the water bath has limited use in removing wrinkles from the tissue itself

Question 10

Since waterbaths are a source of carryover, what can be done to limit this ?

Answer 10

The surface should be cleaned between every sample using a tissue

Question 11

Micron setting used for rough cutting (trimming)

Answer 11

20 to 25 microns

Question 12

Which direction do you rotate the flywheel ?

Answer 12

Clockwise (away from you)

Question 13

Consequence of the water bath being too hot

Answer 13

The ribbon will expand too quickly and tear sections apart

Question 14

Consequence of the waterbath being too cold

Answer 14

Tissue section won’t flatten out

Question 15

Why would the ribbon and consecutive sections be curved ?

Answer 15

• face of block is not completely flat
• knife blunt in one area
• tissue has variable consistency

Question 16

How to remedy ribbons that are curved ?

Answer 16

• trim excess wax to make parallel
• use a different part of a knife, or replace knife
• vary speed of cutting or reorient block 90°

Question 17

Why would tissue sections alternate from being thick and thin ?

Answer 17

• wax is too soft for tissue
• block or knife is too loose
• insufficient clearance angle (too shallow)
• mechanism of microtome is faulty

Question 18

How to remedy tissue sections alternating from being thick or thin ?

Answer 18

• cool block with ice
• re-embed tissue with higher melting point wax
• increase clearance angle gradually
• check microtome for faults/ fix or send for maintenance/ replace microtome

Question 19

What causes chatters ?

Answer 19

• Block or knife is loose
• Knife angle is too steep
• Tissue is unusually hard

Question 20

How to remedy chatters ?

Answer 20

• tighten holder
• reduce angle gradually
• use a heavy duty microtome with a heavy duty knife
• use softening agent on tissue
• cut slowly at a thinner thickness (2 to 3 micrometers)

Question 21

Why would scoring or splitting occur at right angles to the knife edge ?

Answer 21

• There’s a nick on the knife edge
• Hard particles in tissue or wax

Question 22

How to remedy scoring or splitting at right angles to the knife edge ?

Answer 22

• use different part of knife, or replace knife
• remove hard particles
• re-embed tissue

Question 23

Why would tissue sections not form a ribbon ?

Answer 23

• wax is too soft for cutting conditions
• debris on knife edge
• incorrect knife angle
• cutting above optimal speed
• sections are too thick

Question 24

How to remedy sections not forming a ribbon ?

Answer 24

• cool block
• remove block and clean areas around knife
• adjust knife angle
• adjust cutting speed
• reduce micron setting

Question 25

Why would tissue sections become attached to block on the return stroke ?

Answer 25

- debris on knife edge - top edge of tissue is not smooth or has debris - clearance angle is insufficient (too shallow) - static electricity on ribbon

Question 26

How to remedy sections becoming attached to block on the return stroke ?

Answer 26

- remove block and clean areas around knife - lock microtome flywheel and use a gloved thumb to run back and forth along edge of block - increase clearance angle slightly - humidify or ionize air to remove static electricity

Question 27

Why would areas of tissue be missing in the ribbon sections ?

Answer 27

- incomplete impregnation of tissue (ie. tissue not flat against mold ) - insufficient rough cutting

Question 28

How to remedy areas of tissue not being present in ribbon sections ?

Answer 28

- return tissue to paraffin wax for at least one hour - set micron selector to 20 and turn wheel until full face of tissue is exposed, then re-cool block

Question 29

Why would tissue sections become excessively compressed ?

Answer 29

- cutting too quickly - wax is too soft - knife is too dull

Question 30

How to remedy excessive compression of tissue sections ?

Answer 30

- slower, more uniform cutting - cool block - use different part of knife, or replace knife

Question 31

Why would sections expand and disintegrate on the water surface ?

Answer 31

- water temperature too high - poor impregnation of tissue

Question 32

How to remedy sections expanding or disintegrating on water surface ?

Answer 32

- decrease water temperature - return tissue to wax for at least one hour

Question 33

Why would sections roll into a tight coil instead of remaining flat ?

Answer 33

- section is too thick - clearance angle is insufficient (too shallow)

Question 34

How to remedy sections from rolling into a tight coil instead of remaining flat ?

Answer 34

- ensure micron selector is set properly - reduce knife tilt

Question 35

Why would sections stick to the knife pressure plate ?

Answer 35

- dirty pressure plate

Question 36

How to remedy sections sticking to the knife pressure plate ?

Answer 36

- clean plate with xylene/ xylene substitute, or kimwipe with pressure - when picking up ribbon, use an upward sweeping motion

Question 37

Why would the knife gouge out parts of the block ?

Answer 37

- Flywheel turned before ensuring block can clear the knife edge - Knife or tissue is loose

Question 38

How to remedy knife from gouging out part of the block ?

Answer 38

- remove block and use scalpel to create a straight bottom edge of cassette - if there is no layer of wax left before the tissue, the block must be re-embedded - ensure tissue block and knife are tightened properly