Procedure for concentration estimation by haemocytometer
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PROCEDURE FOR CONCENTRATION ESTIMATION BY HAEMOCYTOMETER
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(Materials required, Procedure, Calculation, Normal sperm concentration in different species & Nomenclature)
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Haemocytometer is a device originally developed to count blood cells. It can also be used to estimate the sperm concentration in a semen sample. It is the best method among the different methods available.
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Materials required
- Semen sample (fresh/frozen)
- Haemocytometer set
- Phase contrast microscope
- Watch glass
- Dilution fluid (0.1% formal saline or distilled water or 3% chlorazene solution)
- Eosin powder
- Blotting paper
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Procedure (Click here to view picture) (Click here for video demonstration)
- Mix the semen sample gently to get uniform distribution of sperms.
- Place the semen in a sterile watch glass.
- Put a speck of eosin powder and mix it with semen.
- Aspirate the semen from watch glass into a RBC pipette up to 0.5 mark.
- Clean the tip of the pipette with blotting paper.
- Draw the dilution fluid in the same RBC pipette up to 101 mark.
- Roll the pipette between palm of the hands for 2 minutes to ensure through mixing of the fluid and semen.
- Discard first few drops.
- Charge the haemocytometer by releasing the fluid below the cover slip which is placed over the haemocytometer.
- While charging overflowing and air bubble formation should be avoided.
- Wait for 1-2 minutes for the sperms to settle.
- Examine the charged haemocytometer for under low power and then in high power. (Click here to view picture).
- The sperm counting is done in RBC chamber. (Click here to view picture).
- Count the number of sperms in left top, right top, right bottom, left bottom and center squares of RBC chamber and calculate the concentration. Counting can be done in other directions also but it should be in unbiased manner. (Click here to view picture).
- While counting sperms in individual chamber it should be done in such a way that biasness should not be there. (Click here to view picture)
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Calculation
| Length of one small square
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= 1/20 mm
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| Breadth of one small square
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= 1/20 mm
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| Depth of one small square
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= 1/10 mm
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| Volume of one small square
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= length x breadth x depth
= 1/20 x 1/20 x 1/10
= 1/4000 mm3
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| Total number of small squares counted
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= 16 x 5 = 80
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| Volume of semen in 80 squares
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= 1/4000 x 80 = 1/50 mm3
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| Consider the total number of spermatozoa counted is
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N
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| The dilution rate is
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1:200
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| Number of sperms are occupying 1/50 mm3 space
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=N
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| So sperms occupied in 1 mm3 space
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= N x 50 x 200 mm3
= N x 10000 mm3
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| Number of sperms in 1 ml of semen
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= N x 10000 x 1000
= N x 107
= N x 10 x 106 millions
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Normal sperm concentration in different species
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S.No.
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Species
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Concentration
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1
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Cattle bull
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1200 (800-1400 millions/ml)
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2
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Buffalo bull
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800 (600-1200 millions/ml)
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3
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Stallion
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250 (200-600 millions/ml)
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4
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Ram and buck
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3000 (2000-4000 millions/ml)
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5
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Boar
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250 (200-500 millions/ml)
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6
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Dog
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250 (125-500 millions/ml)
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7
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Man
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150 (100-200 millions/ml)
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Nomenclature
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Last modified: Monday, 4 June 2012, 11:26 AM
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