SULTAN KUDARAT STATE UNIVERSITY

ACCESS Campus, EJC Montilla, 9800 Tacurong City

Province of Sultan Kudarat

COLLEGE OF HEALTH SCIENCES

MT 307
Histopathologic and Cytologic Techniques

EXPERIMENT 9

SECTIONING (MICROTOMY)
I. INTRODUCTION:
Microtomy, is the process by which processed tissue, most commonly a paraffin
embedded tissue, is trimmed and cut into uniformly thin slices or “sections” to facilitate studies
under the microscope. This process makes use of a machine known as, “Microtome” ―from
Greek words: “micros” (small); “tamnein” (to cut). This instrument is capable of cutting a
section at a predetermined thickness by sliding the block into a cutting tool, usually a steel knife
or blade, which is fixed and attached to the machine.
The microtome consists of three essential parts, namely:
1. Block holder – where the tissue is held in position.
2. Knife carrier and Knife – for actual cutting of tissue sections.
3. Pawl, Ratchet Feed Wheel and Adjustment Screws – to line up the tissue block in
proper position with the knife, adjusting the proper thickness of the tissue for
successive sections.
Whatever the type of microtome used, the principle remains essentially the same
whatever the microtome used. A spring-balanced teeth or pawl is brought into contact with, and
turns a ratchet feed wheel connected to a micrometer screw, which is in turn rotated, moving the
tissue block at a predetermined distance towards the knife for cutting sections at uniform
thickness.

II. OBJECTIVES:
At the end of this laboratory exercise, the student is expected to:
1) discuss the procedure of microtomy as a vital procedure in routine tissue processing;
2) perform skillfully the procedure of microtomy;
3) enumerate the difficulties encountered in performing microtomy; and
4) discuss the importance of performing this method in routine tissue processing.
III. MATERIALS:
Trimmed tissue block (in a paper boat)
Refrigerator
Hot plate
Spatula
Microtome
Camel hair brush or Forceps

IV. PROCEDURE:

1. Wear a laboratory gown.

2. Wash hands and wear apron, mask, gloves, goggles and head cap.

3. Allow the tissue block to harden for cutting proper by facing it down the ice cold water
or refrigerator for 5 – 10 minutes.

4. Get a hot plate and a clean spatula.

5. Heat the spatula using the hot plate.

6. Remove the cover of the microtome.

7. Wind back the coarse feeding mechanism and lock the hand wheel.

8. Place the heated spatula in front of the block holder and insert the tissue block (the edge
with the thickest portion of paraffin must be the part in contact with the spatula). The
heated spatula must be held between the tissue block and the block holder.

9. Compress gently the tissue block and block holder until the wax begins to melt.

10. Withdraw the spatula and gently press the tissue block into position. Make sure that the
tissue block’s upper and lower edges are parallel with each other and parallel with the
base of the microtome.

11. Use the block holder screw to clamp the tissue block in place.

12. Ensure that the knife holder is in a safe distance from the tissue block.
13. Place a blade carefully into the knife holder and tighten it securely.

14. Loosen back the lever of knife holder and advance the knife holder towards the tissue
block until there is 0.2 – 0.5mm interval between blade edge and tissue block surface.

15. Maintain the clearance angle between 5 – 100 from the cutting plane so that the cutting
facet will not compress the block during the process of cutting.

16. Tighten back the lever of the knife holder.

17. Unlock the hand wheel.

18. Trim away the surplus wax from the tissue block surface by advancing the coarse feed
mechanism before commencing each turn of the wheel.

19. Trim all tissue carefully until the desired surface is exposed by winding the hand wheel.
The hand wheel causes a reciprocal motion of the knife over the block.

20. Discard the trimmed surplus wax from the knife using a camel hair brush or a pair of
forceps.

21. Reduce the section thickness to 4 – 6 microns using the adjustment knob. Remember
that when using a microtome with ratchet wheel for determining the thickness of section,
never set the thickness setting at an intermediate position. Always set it at full (click)
stop to avoid damaging the ratchet teeth.

22. Wind the hand wheel to cut the tissue. Tissue sections must be 4 – 6 microns in
thickness (for routine histologic procedures).

23. Brush away all the discarded incomplete tissue sections.

24. Wind the hand wheel further more with a steady rhythm and allow the first inch or two
of paraffin ribbon to move down the blade until the ribbon begins to bow up near the
edge of the blade. Successive sections will usually stick edge-to-edge due to local
pressure with each cutting stroke, thereby forming a “ribbon”.

25. Slip the brush or forceps under the ribbon at this point and lift the ribbon while still
attached to the edge of the knife surface. Hold the ribbon up but do not put any pull on
its attachment with the blade to prevent separation from the next section to be cut. Then
keep winding the hand wheel to produce a ribbon with 10 sections.
26. Pick up complete ribbon with 10 sections carefully using a camel hair brush, a pair of
forceps or the fingers.

NOTE:

Sections must be floated out on the water bath set at 45 – 500C. This will be
done on the next activity.

27. Place the ribbon on a clean bond paper (for the purpose of checking) and keep in a cool
place.

28. Remove the incomplete sections from the microtome.

29. Wind back the coarse feeding mechanism and lock the hand wheel.

30. Loosen back the lever of knife holder and pull the knife holder away from the tissue
block.

31. Remove the blade from the knife holder and store inside its case.

32. Loosen screw of the block holder and remove the tissue block.

33. Place the tissue block back inside the paper boat.

34. Place the tissue block inside the cabinet for storage.

35. Dispose used and contaminated materials on appropriate waste container.

 SULTAN KUDARAT STATE UNIVERSITY
ACCESS Campus, EJC Montilla, 9800 Tacurong City
Province of Sultan Kudarat

COLLEGE OF HEALTH SCIENCES

Name: ___________________________________ Date: ___________________

Yr. & Sec.: _______________________________ Rating: _________________

WORKSHEET NO. 9

MICROTOMY
ILLUSTRATIONS:

Draw and label the procedure of microtomy.

1-2

4-5
6-8

9-16

17-25

17-25
26-32

33

34

35
REVIEW QUESTIONS:

1. Enumerate at eight (8) faults observed during section-cutting, explain the reason and
discuss its remedy.

FAULT REASON REMEDY

2. Discuss the principle of rotary microtome.
 _____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_________________________________________________________

3. List down and discuss briefly the other kinds microtome.

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
__________________________________________________________

4. Explain the importance of Freezing Microtome.

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
___________________________________________________________

5. How will the adhering paraffin on the different areas of the microtome be cleansed?

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
___________________________________________________________
6. List down and describe the three (3) basic types or shapes of a microtome knife.

_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
_____________________________________________________________________
___________________________________________________________

7. Define the following:

a) Wedge angle
__________________________________________________________________
__________________________________________________________________

b) Bevel angle
__________________________________________________________________
__________________________________________________________________

c) Clearance angle
__________________________________________________________________
__________________________________________________________________

d) Cutting angle
__________________________________________________________________
__________________________________________________________________

e) Rake angle
__________________________________________________________________
__________________________________________________________________

f) Honing
__________________________________________________________________
__________________________________________________________________

Types of Hones:

a. Belgium Yellow
 ____________________________________________________________
____________________________________________________________

b. Arkansas
____________________________________________________________
____________________________________________________________

c. Fine carborundum
____________________________________________________________
____________________________________________________________

g) Stropping
__________________________________________________________________
__________________________________________________________________

PERFORMANCE ASSESSMENT SHEET

ACTIVITY NO. 9
MICROTOMY

NAME: ___________________________________________

DATE PERFORMED: __________________

PROCEDURE
You must: RATING REMARKS
1. Wear a laboratory gown.
2. Wash hands and wear apron, mask,
gloves, goggles and head cap.
Microtomy:
3. Allow the tissue block to harden for
cutting proper by facing it down the ice
cold water or refrigerator for 5 – 10
minutes.
4. Get a hot plate and a clean spatula.
5. Heat the spatula using the hot plate.
6. Remove the cover of the microtome.
7. Wind back the coarse feeding
mechanism and lock the hand wheel.
8. Place the heated spatula in front of the
block holder and insert the tissue block
 (the edge with the thickest portion of
paraffin must be the part in contact with
the spatula). The heated spatula must be
held between the tissue block and the
block holder.
9. Compress gently the tissue block and
block holder until the wax begins to
melt.
10. Withdraw the spatula and gently press
the tissue block into position. Make sure
that the tissue block’s upper and lower
edges are parallel with each other and
parallel with the base of the microtome.
11. Use the block holder screw to clamp the
tissue block in place.
12. Ensure that the knife holder is in a safe
distance from the tissue block.
13. Place a blade carefully into the knife
holder and tighten it securely.
14. Loosen back the lever of knife holder
and advance the knife holder towards
the tissue block until there is 0.2 –
0.5mm interval between blade edge and
tissue block surface.
15. Maintain the clearance angle between 5
– 100 from the cutting plane so that the
cutting facet will not compress the block
during the process of cutting.
16. Tighten back the lever of the knife
holder.
17. Unlock the hand wheel.
18. Trim away the surplus wax from the
tissue block surface by advancing the
coarse feed mechanism before
commencing each turn of the wheel.
19. Trim all tissue carefully until the desired
surface is exposed by winding the hand
wheel. The hand wheel causes a
reciprocal motion of the knife over the
block.
20. Discard the trimmed surplus wax from
the knife using a camel hair brush or a
pair of forceps.
21. Reduce the section thickness to 4 – 6
microns using the adjustment knob.
Remember that when using a microtome
with ratchet wheel for determining the
thickness of section, never set the
 thickness setting at an intermediate
position. Always set it at full (click)
stop to avoid damaging the ratchet teeth.

22. Wind the hand wheel to cut the tissue.

Tissue sections must be 4 – 6 microns in
thickness (for routine histologic
procedures).
23. Brush away all the discarded incomplete
tissue sections.
24. Wind the hand wheel further more with
a steady rhythm and allow the first inch
or two of paraffin ribbon to move down
the blade until the ribbon begins to bow
up near the edge of the blade.
Successive sections will usually stick
edge-to-edge due to local pressure with
each cutting stroke, thereby forming a
“ribbon”.
25. Slip the brush or forceps under the
ribbon at this point and lift the ribbon
while still attached to the edge of the
knife surface. Hold the ribbon up but do
not put any pull on its attachment with
the blade to prevent separation from the
next section to be cut. Then keep
winding the hand wheel to produce a
ribbon with 10 sections.
26. Pick up complete ribbon with 10
sections carefully using a camel hair
brush, a pair of forceps or the fingers.

NOTE:
Sections must be floated out on the water
bath set at 45 – 500C. This will be done on
the next activity.

27. Place the ribbon on a clean bond paper

(for the purpose of checking) and keep
in a cool place.
28. Remove the incomplete sections from
the microtome.
29. Wind back the coarse feeding
mechanism and lock the hand wheel.
30. Loosen back the lever of knife holder
and pull the knife holder away from the
tissue block.
31. Remove the blade from the knife holder
and store inside its case.
32. Loosen screw of the block holder and
remove the tissue block.
33. Place the tissue block back inside the
paper boat.
34. Place the tissue block inside the cabinet
for storage.
35. Dispose used and contaminated
materials on appropriate waste
container.
RATING: COMMENTS:

4 – EXCELLENT
3 – VERY
SATISFACTORY
2 – SATISFACTORY
1 – POOR
0 – NOT DONE

LEVEL OF COMPETENCY

INSTRUCTOR: DATE: