MANUAL PHYSIOLOGY PRACTICAL - Repository:The Medical ...

FM/UOJ
Method:
i. Semen collection:
Semen sample is obtained by masturbation and ejaculate is collected into a clean
glass
container. Semen has a grey-opalescent appearance and it liquefies within 60
minutes of
ejaculation.
For this practical semen sample will be provided.
ii. Dilution technique.
50µl of liquefied semen sample is taken and transferred in 950µl dilution fluid and
thoroughly mixed, then the semen would have been diluted 20 times (1 in 20).
iii. Counting Technique:
Charge the counting chamber with diluted semen and allow it to stand for 1-5
minutes
(Cells sediment during this time). Place it under the microscope and identify the
engraved lines under the lowest magnification (marked “x3.5”). Identify the RBC
counting areas under next magnification (marked “x 10”) and turn to the next
magnification (“x 40”) and count the cells systematically; the small squares are
there
to avoid re-counting the same cell or missing any cell; start from the top left square
and
move across to the last square; then come down to the next row and so on; in order
to
avoid re-counting the cells on the lines, adopt a convention to ignore sperms
touching the
upper and left line and to include cells touching the lower and tight lines. Count the
sperms in all five RBC counting areas.
iii. Calculation:
The volume of the fluid in medium square
Volume of fluid in all 5 squares
= 0.004 sq.mm.
= 5 X 0.004 cu.mm
= 0.02 cu.mm.
Let the total number of sperms counted in all 5 areas be C
Number of sperms in 1 cu.mm diluted semen
= C
0.02
= 50C
The dilution is 1 in 20
Therefore Sperms in 1 cu.mm. undiluted semen = 50 X 20C
= 1,000C
In 1ml semen = C X 10 6
Reproduction
Page | 133