HUMAN SPERM CELL: CONCENTRATION, VIABILITY,
ABNORMALITIES, AND CELL COUNTING USING
HAEMOCYTOMETER
TAYLOR A. SWIFT, OLIVIA RODRIGO, SHAWN MENDES, and BEN PLATT
Senior High School Department, Umingan National High School,
Umingan, Pangasinan
onlyoneemail@gmail.com
ABSTRACT
Human semen was used as the specimen for the study. Sperm count was done with the use of
haemocytometer stained with methylene blue. Results for the solution were also calculated. The dilution
factor was 2. For the cell concentration, there were 8.8x105 sperm cells/µL. The total sperm count totaled
to 220. There were no viable cells upon observation. All sperms cells were dead because collection
time was early and observation was not done instantly. In terms of morphological abnormalities, four
types were observed: flat-head, curved-head, large-head, and short-tail.
Keywords: Human Semen, Sperm Cell, Haemocytometer, Sperm Cell Abnormalities, Cell Count,
Cell Concentration, Dilution Factor
INTRODUCTION 0.1 mm off the marked grid, giving each
The hemocytometer is a device square a defined volume [2].
originally designed and usually used For most applications, the four large
for counting blood cells. It was invented corner squares are only used. The cells that
by Louis-Charles Malassez, and consists of a are on or touching the top and left lines are
thick glass microscope slide with a counted, but the ones on or touching the
rectangular indentation that creates a right or bottom lines are ignored [3].
chamber. This chamber is engraved with a In this activity, sperm cell was used
laser-etched grid of perpendicular lines. The to test the Haemocytometer. Sperm is the
device is carefully crafted so that the area male reproductive cell.
bounded by the lines is known, and the The mammalian sperm cell consists
depth of the chamber is also known. By of a head, neck, a midpiece and a tail. The
observing a defined area of the grid, it is head contains the nucleus with densely
therefore possible to count the number coiled chromatin fibres, surrounded
of cells or particles in a specific volume of anteriorly by an acrosome, which contains
fluid, and thereby calculate the enzymes used for penetrating the female
concentration of cells in the fluid overall [1]. egg. The neck contains the sperm centriole.
The gridded area of the The midpiece has a central filamentous core
hemocytometer consists of nine 1 x 1 mm with many mitochondria spiralled around it,
(1 mm2) squares. These are subdivided in 3 used for ATP production for the journey
directions; 0.25 x 0.25 mm (0.0625 mm2), through the female cervix, uterus and uterine
0.25 x 0.20 mm (0.05 mm2) and 0.20 x tubes. The tail or "flagellum" executes the
0.20 mm (0.04 mm2). The central square is lashing movements that propel the
further subdivided into 0.05 x 0.05 mm spermatocyte [4].
(0.0025 mm2) squares. The raised edges of Sperm cells were concentrated with
the hemocytometer hold the coverslip dye for further viewing or observation under
the microscope and haemocytometer.
1
OBJECTIVES the 100µL semen to dye the cells when
This study was intended to use the viewed under the microscope. The sample
Haemocytometer and to describe/study the solution was mixed gently.
specimen in different terms. Specifically, it
aimed to:
acquire knowledge on how to use the
hemocytometer in terms of cell
counting;
learn how to calculate cell Fig 2. Preparation of Sperm Suspension
concentration and viability; and,
Mounting Solution on Haemocytometer
assess sperm morphology for
identification of cell abnormalities. The haemocytometer and the cover
slip were washed and cleaned with 70%
MATERIALS AND METHODS ethanol before use. The cover slip was
Semen Collection placed on the haemocytometer. Pipette out
100 µL of the sample solution. The tip of the
The sample, semen, was collected
pipette was placed in the V-shaped groove to
from male human, preferably through the
load the sample into the chamber. Let the
process of ejaculation. The human must be
sample settle for 2mins so that the cells stop
in abstinence for ejaculation for 2-6 days
drifting around the chamber but do not allow
before the actual process where the semen
the sample to settle too long or it will dry
sample was needed. The hands were washed
out.
with soap before proceeding to ejaculation.
The entire sample was collected into the
wide mouth sterile container, 70% of sperms
is in the first part of the ejaculate. The
sample was kept at body temperature, no
sunlight. The sample must be delivered
within one hour of ejaculation.
Fig 3. Mounting Sample on Haemocytometer
Fig 1. Collected Semen Sample
Microscopic Examination and Cell
Preparation of Sperm Suspension Counting
Using a micropipette, 100µL of the The sample was observed first at the
semen sample was gathered and transferred lowest magnification of the microscope
into a small tube or a beaker. The sample (10x) and were focused on the grid lines of
was added with 100µL of Methylene Blue chamber. The number of cells present of the
(if there were no Trypan Blue available) into chamber was counted.
2
Total Number of Cells in Sample
This is to compute for the estimated
total number of cells found in the sample.
= Concentration x Sample Volume
Fig 4. Observing Under HPO in Microscope Cell Viability
In counting the cells in the Cell Viability is a measure how
haemocytometer, for most applications and many of your cells survived your cell culture
what was performed, the four large corner technique. This was relied on the alteration
squares and the center square are only used. in membrane integrity as determined by the
The cells that are on or touching the top and uptake of Trypan or Methylene Blue by dead
left lines are counted, but the ones on or cells, thereby giving a direct measure of cell
touching the right or bottom lines are viability. Percentage (%) of viable cells can
ignored [5]. be determined by:
= Number of Living Cells x 100
Total Number of Cells (live+dead)
Identification of Sperm Morphological
Abnormalities
Sperm morphology – the size and
shape of sperm – is one factor that's
examined as part of a semen analysis to
evaluate male infertility. Normal sperm have
Fig 5. Four Corners and Middle Square to be
Counted From an oval head with a long tail. Abnormal
sperm have head or tail defects – such as a
Dilution Factor
large or misshapen head or a crooked or
Dilution Factor is often used
double tail. These defects might affect the
for simple dilutions, one in which a unit
ability of the sperm to reach and penetrate
volume of a liquid material of interest is
an egg. However, having a large percentage
combined with an appropriate volume of a
of misshapen sperm isn't uncommon.
solvent liquid to achieve the desired
concentration. To compute for the dilution
factor (DF):
= Final Volume
Initial Volume
Cell Concentration
Fig 6. Morphological Abnormalities of Sperms
Cell Concentration is defined as the
amount of solute dissolved in a specific A drop of the semen sample
(fixed) amount of solvent. To compute for collected was placed in a glass slide and was
the cell concentration: covered with a cover slip. It was examined
= Total Cells Counted x DF x 104 under the HPO in the microscope.
Number of Squares Abnormalities were observed.
3
RESULTS AND DISCUSSION 20 mins [5]. In the experiment, the semen
was collected at 12:30pm and was subjected
Microscopic Examination and Cell
Counting to experiment around 1:30 in the afternoon.
Sperm Cells were viewed under the This can be a possible reason why there are
LPO of the microscope and were focused on no live cells to be seen in the
the grid of the 5 chambers (4 corners and a haemocytometer under the microscope.
center). Cell numbers were counted.
Dilution Factor
DF= Final Volume = 200µL = 2
Initial Volume 100µL
A B The volume of the semen with
100µL (Initial Volume) was divided to the
total volume of the semen sample (100µL)
and Methylene Blue (100µL) with 200µL
C
(Final Volume). The computed dilution
factor result was 2.
Cell Concentration
D E
Fig 7. Sperm Cells in the Grid of = Total Cells Counted x DF x 104
Haemocytometer – top-left corner (A), top- Number of Squares
right corner (B), center (C), bottom-left (D), = 220 x 2 x 104
and bottom-right (E) – under LPO of
5
Microscope
= 44 x 2 x 104
Table 1. Number of Live and Dead Cells = 88x104 or 8.8 x105cells/µL
NUMBER OF CELS
POSITION LIVE DEAD The total number of cells counted
CELLS CELLS was 220, and was divided by the 5 squares.
Top-left 0 49
Top-right 0 58 This was multiplied by the dilution factor
Center 0 32 which is 2 and x104. Thus, the computed cell
Bottom-left 0 37
Bottom-right 0 44 concentration was 8.8x105 cells/µL.
total 0 220
Total Number of Cells in Sample
The table above (table 1), shows the
= Concentration x Sample Volume
number of cells – dead and live – counted in
= 8.8x105 cells/µL x 100µL
their respective chambers in the = 8.8 x107 cells
haemocytometer when viewed under the
The previously computed cell
microscope. When counting, it is reminded
concentration (8.8x105cells/µL) was
that live cells do not absorb the dye and
multiplied with the gathered semen sample
contrary to the dead cells.
(100µL). Thus, the estimated total number
When viewed in the microscope, only
of cells in the sample was about 8.8 x107 cells.
dead cells can be seen and no unstained
sperm cells. This only means that the cells in
the sample were already dead.
Cell Viability
Depending on different conditions,
sperm can live from a few minutes to about = Number of Living Cells x 100
4
Total Number of Cells (live+dead) is short-tailed (B), they have large heads (C),
= 0 x 100
and lastly, one have curved head (D).
(0+220)
= 0 x 100
220 SUMMARY, CONCLUSION AND
= 0 x 100 RECOMMENDATION
=0
Summary
The total number of living cells (0) Semen sample was collected and
was divided be the sum of the total number treated with Methylene Blue. The sample
of live (0) and dead (220) cells. It was solution was mounted in a clean
multiplied by 100%. The result computed hemocytometer with a covered slip by
was zero (0). pipetting. It was viewed to be observed in
Since Cell Viability is used to the microscope and focused on the grid line
measure how many of the cells survived in of the chambers.
the cell samples, and the sperm cells were The number of cells present, both
already dead at the time of experimenting, dead and live cells, were counted. The four
we can say that there is no cell viability in corners and the center squares were only
the sample to be computed to know the counted, and in the grid, cells touching the
result of the study. bottom and left lines were not counted.
The counted data were computed for
Sperm Morphological Abnormalities
different computations namely – dilution
factor, cell concentration, total number of
cells in sample, and cell viability.
A Another semen sample was also
observed and identified for sperm
morphological abnormalities.
B
Conclusion
In conclusion, the formulated
solution of semen and Methylene Blue
C
showed the number of cells present.
The dilution factor, cell
concentration, total number of cells in
sample, and cell viability was computed. In
the cell viability’s computed result, it
D
showed that no cells had survived in the
Fig 8. Sperm Morphological Abnormalities; flat
head (A), short-tailed (B), large head (C), and sample, only because the collection of the
curved head (D) sample was done early and performed lately
The examined samples of human that the cells in the sample died.
semen were observed with different sperm In the sperm morphological
cell morphological abnormalities. Some of identification, four types of sperm with
the sperm abnormalities observed and abnormalities were observed.
identified were – it has a flat head (A), some
5
Recommendation
With the problem that the researcher
encountered, it is recommended that when
next that a study like this will be performed,
make sure that the semen that will be used
must be collected just before performing the
experiment. Depending on the
environment’s condition, the cell may die
just within a minute to 20 minutes.
REFERENCES
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Square Sizes
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BIERER, B.E., MARGULIES, D.H.,
and E.M. SHERACH. (2001).
Monitoring Cell Growth. Current
Protocols in Immunology. 5. USA: John
Wiley & Sons. p.A.2A.1
doi:10.1002/0471142735.ima03as21
[4] ISHIJIMA, S., OSHIO, S., and H.
MOHRI. (1986). Flagellar Movement of
Human Spermatozoa. Gamete
Research.13(3):185197. doi:10.1002/mr
d.1120130302.
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Sperm Live Outside the Body?
