Microbiology Laboratory


Written by MicroDok

AIM: To determine any abnormality in a seminal fluid (semen) as an aid in the diagnosis of male infertility.

MATERIAL/APPARATUS: Semen specimen, microscope, test tube, test tube rack, glass slide, cover slip, semen diluting fluid (sodium bicarbonate formalin), improved neubauer counting chamber, bulb pipette or Pasteur pipette, calibrated cylinder (5 ml).

The microscopy of a semen specimen involves the following procedures:

  • Determination of the semen volume.
  • Determination of the semen viscosity.
  • Determination of the motility of the sperm cells in the semen specimen.
  • Determination of the morphology of the sperm cells in the semen specimen.
  • Sperm cell count.

In addition to the above parameters, the time of production, time of collection, time of examination, and the appearance of the semen specimen is taken into cognizance as well. The appearance of a semen specimen can be grayish – white e.t.c. These parameters are also reported together with other analysis carried out.


The volume of a semen specimen can be determined by emptying or pouring the semen specimen into a calibrated cylinder that is about 5 ml. The volume of normal semen is between 2 – 5 ml and above.


A semen specimen can be viscous, non – viscous, slightly viscous or highly viscous. Due to the presence of fibrinolysin in the semen, it usually becomes liquefied within 1 hour. You can determine how viscous a semen specimen is by touching it with a sterile inoculating loop or stick and raising it up. Normal semen is thick and viscous when ejaculated or produced.


The procedure for doing this is as follows:

  • Place one drop of well mixed semen on a clean glass slide.
  • Cover with a cover slip.
  • Focus the slide using ×10 and ×40 objective lens of a microscope. Ensure that the condenser iris of the microscope is sufficiently closed in order to give a good contrast while viewing.
  • Examine several fields of the slide and report your result.


A sperm cell can be actively motile (rapid and progressive) or weakly motile (slow and non – progressive). Count a total of 100 spermatozoa/sperm cell, and note out of the 100 how many spermatozoa are motile. Record the percentage that is motile and non – motile. Normal semen should have more than 50% motile spermatozoa within 60 minutes of ejaculation.


The procedure for doing this is as follows:

  • Place a drop of well mixed semen on a clean glass slide.
  • Make a thin smear of the semen on the slide.
  • Heat fix the smear.
  • Stain the smear with either Giemsa stain or methylene blue stain.
  • Allow for 5 minutes.
  • Wash off with water after 5 minutes and allow to dry.
  • Add a drop of immersion oil on the slide.
  • Focus with ×100 objective lens.
  • Examine the different fields for any abnormality in the morphology of the spermatozoa.


Estimate the number of spermatozoa showing normal morphology and abnormal morphology.

Normal spermatozoa measures about 50 – 70 µm in length, and each consists of an oval – shaped head (with acrosomal cap), a short middle piece, and a long tail. In normal semen, at least 50% of spermatozoa should show normal morphology.


Sperm cells rushing towards an egg to fertilize it


The procedure for doing this is as follows:

  • Make a 1 in 10 dilution of the semen specimen using a calibrated test tube and semen diluting fluid as follows:

Place 9 drops of the semen diluting fluid (sodium bicarbonate formalin) in a clean test tube standing on a test tube rack. Add a drop of well mixed semen specimen, and mix well. Avoid bubble production when mixing the semen and the diluent. In this way, a 1 in 10 dilution of a semen specimen is made. I in 10 dilutions is made for small semen specimens. For large semen specimens, 1 in 100 dilutions is made. Counting of spermatozoa in a semen specimen without dilution will be very difficult because the sperm cells are still alive and moving. This is why the semen diluting fluid is used so that they can inactivate or kill the spermatozoa, making the count easy.

  • Using a Pasteur’s pipette, fill an improved neubauer counting chamber with the well mixed diluted semen specimen.
  • Wait for about 4 – 5 minutes for the sperm cells to settle, before viewing.
  • View the counting chamber with ×10 objective lens of a microscope. Make sure the condenser iris of the microscope is sufficiently reduced in or order to give a good contrast.
  • Count the sperm cells in each of the 4 fields of the counting chamber.


The formula for counting and reporting a sperm cell count is thus:

Number of sperm cells counted × 104 × diluting factor. The unit is: Sperm cells/ml.

Normal sperm cell count is: above 20 × 106 sperm cells/ml.

Abnormal sperm cell count is: below 20 × 106 sperm cells/ml.

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