PROCEDURE FOR SEMINAL FLUID ANALYSIS
1. Volume: When seminal fluid liquefied, measure the volume of the fluid in ml.
2. Ph: Using Ph meter or urine dipstick measure the Ph of the fluid within 1hr of ejaculation.
3. Color: Observe the color of the seminal fluid. Normal color White Yellow.
4. Liquefaction time: 30-40 min.
5. Motility & Viability:
MOTILITY
❖ Place 1 drop of well liquefied semen on a slide and cover with cover glass.
❖ Focus the specimen using 10 x objectives.
❖ Ensure the spermatozoa are evenly distributed (if not, re-mix the semen & examine a new
preparation).
❖ Using 40x objective, examine several fields to assess motility:
Excellent: rapid & progressive.
Weak: slow & non-progressive.
❖ Count 100 spermatozoa, & note out how many are motile.
VIABILITY: - When more than 60% of spermatozoa are non-motile, examine an eosin
preparation to assess whether viable or non-viable.
⮚ Mix 1 drop of semen with 1 drop of 0.5 % eosin solution on a slide.
⮚ After 2 minute examine the preparation microscopically
⮚ Focus with 10x objective and count with 40x objective to determine the percentage of
viable & non-viable spermatozoa.
● Viable: - remain unstained.
● Non-Viable:- Stain pink /red
● Normal viability: - > 75% should be viable.
6. SPERM COUNT:
❖ Dilute 1 in 20 (fill 1ml of well-mixed liquefied semen & 19 ml of formalin and mix well.
❖ Fill Neubauer Ruled Chamber with well-mixed diluted semen and wait 3-5 minute for the
spermatozoa to settle.
❖ Using 10 x objectives, count the number of spermatozoa in an area of 2mm³ (2 large
squares).
❖ Calculate the number of spermatozoa in 1ml of fluid by multiplying the number counted by
100,000.
7. MORPHOLOGY:
❖ Make a thin smear of the liquefied well-mixed semen on a slide.
❖ Fix the smear with ethanol (95%) for 5-10 min & allow to air dry
❖ Wash the smear with sodium bicarbonate/ formalin to remove any mucus and rinse the smear
with water.
❖ Cover the smear with 1:20 diluted carbol fuchsin for 3 min & wash off the smear with water,
by wit
❖ Cover with 1:20 diluted methylene blue for 2 min and wash off the stain with water.
❖ Drain & allow the smear to air dry.
▪ Nucleus of the head---Dark blue.
▪ Cytoplasm of the head---Pale blue.
▪ Middle piece and tail--Pink red.
❖ Examine normal and abnormal spermatozoa by 40x objective.
❖ Use 100 x objectives to confirm abnormalities.
❖ Count 100 spermatozoa and estimate the percentage of normal and abnormal morphology
Prepared by: __________________ Sign: _______ Date: __________
