NAME: ___________________________ GROUP NUMBER: ________________________

SECTION: _________________________ INSTRUCTOR: ___________________________

EXERCISE 8
SECTION METHOD

Materials:

Chloroform Medicine Dropper

Dissecting Pan Alcohol Lamp
Dissecting Set Mayer’s Albumin
Bouin’s Fluid Cedar Wood Oil
10% Formalin Solution Spatula
Old newspapers Grades of alcohol (30%, 50%, 70%, 85%, 95% & 100%)
Live Frog Glycerine
Glass slide Microtome
Coverslip Hematoxylin & Eosin stain

Procedures:

1. Select an active medium-size frog and place it in an enclosed container. Put a cotton ball with
chloroform and cover the container. Wait until the frog in unconscious.
2. Pin the frog on the dissecting pan ventral-side up.
3. With the pair of forceps and scissors cut through the midventral line of the skin from the pelvic
region to the middle of the lower jaw.
4. Cut the skin from the pelvic region to the middle ventral side of the thigh.
5. Spread the skin to the sides and pin.
6. Cut the muscles through the side of the linea alba from the ventral base of the pelvic girdle to
the chest and sternum.
7. Pin the muscles together with the skin to the sides.
8. Stretch the arms away from each other.
9. With sharp scalpel or razor blade, cut 5mm long of each of the esophagus, stomach, intestine,
kidney, liver, spleen, nerve, artery, one lobe of the ovary, testis and heart.
10. Dissect the vertebral column and cut 5 mm long of the spinal cord.
11. Fix all these organs in Bouin’s fluid in the bottle overnight.
12. Decant the Bouin’s fluid and tie a gauze around the mouth of the bottle.
13. Wash in running water until the yellow color is almost or completely removed.
14. Transfer the specimens into the tissue carrier and dehydrate for 45 to 60 seconds each in
gradually increasing grades of alcohol 30%, 50%, 70%, 85%, 95%, 100%.
15. Always bear in mind that alcohol is volatile and must be in covered bottles.

Microtechnique (Bio 7) 1
 16. The specimens must never be allowed to dry on the air while being transferred from one grade
of alcohol to another.
17. Clear in cedar wood oil. Specimens must be left in cedar wood oil indefinitely or until the time
allows to continue processing the spcimen.
18. Drain the cedar wood oil as much as possible and transfer to xylene. Leave the specimens in
xylene for at least 30 minutes.
19. Infiltrate the specimen with melted paraffin which have been placed in the paraffin oven of 60°C
to 65°C. Leave the specimen for 60 minutes each in the following mixtures:
 Equal volume of xylene and paraffin
 Pure paraffin
20. Never allow the paraffin to solidify while transferring from one mixture to another.
21. Moisten the lining of the mold with glycerine and pour melted paraffin sufficient to cover the
specimen when placed into it.
22. Without delay heat the forceps over the alcohol flame, pick a piece of specimen and
immediately transfer to its designated location and position into the mould. Should the surface
of the paraffin harden, heat the spatula over the alcohol flame and pass it over the surface. The
paraffin will melt and allow more time in the arrangement of the specimen. Bubbles which
might have been formed may be removed by touching them with red hot dissecting needle.
23. Carefully lift with both hands the mould in horizontal position and float it on cold water. Gently
blow your breathe over the surface of the paraffin to hasten hardening as you push down the
mould with both hands. Sink the boat completely in water when the paraffin has solidified and
can withstand water pressure.
24. Unfold and separate the paper boat form the solidified paraffin block. Leave the paraffin block
in a cool dry place for 24 hours to complete hardening.
25. Clamp the paraffin block in to the clamp holder of the microtome. Section the paraffin block 8 to
9 microns.
26. Collect the tissue ribbon and keep it in a cool dry place.
27. Spread a thin film of Mayer’s albumin on the microscope slide and flood with 5 o 7 drops of
water. Use a medicine dropper.
28. Using a scalpel, divide the paraffin ribbon into paraffin sections. Each section should adhere to
the tip of the scalpel.
29. Lay flat the tissue section on the slide. The tissue section must float. If not, add water until it is
freely floating.
30. Hold the slide with one hand and pass over the alcohol flame to warm the water only. The tissue
section will expand and flatten as the water is warmed.
31. Decant the water by inclining the slide to one end. Arrange the tissue section according to its
designated location on the slide. Keep it inside the slide box overnight to dry and dry in the
paraffin oven for 30 minutes.
32. Arrange the staining jars with their contents.
33. Warm the tissue slide over the alcohol flame and immerse in Xylene to dissolve the paraffin.
34. Run down the tissue slide for 30 seconds each from 100%, 95%, 85%, 70%, 50%, 30% alcohol
and water.

Microtechnique (Bio 7) 2
 35. Transfer the tissue slide to hematoxylin stain. Examine the tissue slide under the microscope at
regular time interval but be sure to dip the tissue slide in water before examination. Note down
the staining time as soon as the desired color intensity of nuclei is reached. Stain the second
tissue slide which serve as check to the first slide. Use this staining time as guide in staining the
succeeding slides.
36. Run up the tissue slide for 30 seconds each from water, 30%, 50%, 70%, 85%, 95% alcohol.
37. Transfer the tissue slide to eosin for 45 seconds. Continue dehydrating in 100% for 30 seconds.
38. Clear in xylene and mount in Canada balsam.
39. Dry, clean and label.

Answer the following questions:

1. Identify the types of tissues in the prepared slide.

2. How are the cells arranged in each tissue?

3. Compose a procedure in which you can prepare triple stained tissue.

Microtechnique (Bio 7) 3