When you think of semen analysis you probably think of the sperm count first. Here we focus on how to use a microscope to get accurate sperm counts at home. To consistently get accurate sperm counts you must reproducibly collect a sample and you must count the sperm in a fixed and known volume so you can convert your counts to millions of sperm per milliliter.
Why would you want to get accurate sperm counts? If you are reading this you are probably concerned that your semen quality may not be adequate to conceive a child. In many cases couples want to monitor changes in semen quality. It is often helpful to record the results of your analysis so you can watch for trends in sperm counts over time. Trends in sperm counts and motility can be very helpful to couples in evaluating their options for conception. The instructions that come with our kits also include directions for evaluating motility and morphology.
Accurately Collect a Sample
To consistently get accurate sperm counts and evaluate trends in your fertility you need to control as many variables as possible. The first step is to control sample variability. You want to be certain that the only changes you measure are real changes and not the result of different methods of collecting a sample. You should have the same number of days of abstinence before collecting each sample. It is usually suggested that three days of abstinence is about the right amount of time. This gives you enough time for the sperm count to build up. After more than three days of abstinence many people see a decline in motility. You should collect a sample by masturbation or using a semen collection condom. You should not use the withdrawal method. You may lose the first portion of the ejaculate. This is the portion that has the highest concentration of sperm.
Accurately Determine the Volume Viewed Through the Microscope
Sperm density or concentration is reported as the number of sperm per milliliter (mL). It is almost always reported in millions of sperm per mL. A milliliter is a measure of volume. To convert the number of sperm you see under the microscope to millions per milliliter you need to count the sperm in a fixed and known volume. Then you convert the number of sperm that you count in the known volume to the standard units of sperm per milliliter.
This sounds harder than it is. There are two ways to count sperm in a known volume. You can use slides with etched rulings or you can use a calibrated microscope. With a calibrated microscope you simply count the number of sperm in the field of view and multiply by a factor to get the number of sperm in million per mL. More on this below.
Slides with Rulings
One way to know the volume is to use a slide with a known depth and rulings. It may be helpful to think about how you calculate the volume of a box. You are trying to determine the volume of the box formed by the area where you are counting sperm and the depth between the bottom of the cover slip and the top of the slide. The formula is: Length X Width X Depth = Volume. You can also think of this as an area times the depth. This is the situation when using a slide that has a fixed depth and rulings. The rulings define the area.
Kokopelli Technologies carries two types of slides with rulings, Cell-Vu Semen Analysis Chambers and the Hemacytometer for Semen Analysis. These slides define both the depth and the area of the “box” so you know the volume of sample and can convert from the number of sperm you view within the “box” to sperm in million/mL.
These slides are affordable and re-usable. They have a ruling etched into the cover slip. This gives you the area you are viewing. The cover slip is removable, allowing you to wash the slides after each use. Since the cover slip is removable the depth is not rigidly controlled and inaccurate counts can occur. The manufacturer suggests that you put a 4 microliter drop on the slide. This will help to insure that you have an accurate depth because you won’t “float” the cover slip with too much sample and it is unlikely that you will get an air bubble under the cover slip if you accurately measure out 4 microliters. Unfortunately, four microliters is such a small volume that it is difficult to measure. A pipetter that will accurately measure 4 microliters will cost $200 or more.
A hemacytometer is an ideal solution. These slides are easy to clean and re-usable. They have Neubauer rulings to show you the exact area you are viewing. The depth is reproducible and always accurate.
To fill the hemacytometer you simply put a small drop of sample on the clear glass (polished) surface. You should use a hemacytometer that is 20 microns deep. That is, the surface where the sample sits is 20 microns below the cover slip supports. The moat around this "plateau" allows the excess sample to drain way giving you a sample that is exactly 20 microns deep after you put the cover slip on. A hemacytometer gives you a reproducible and exact volume. The only downside to a hemacytometer is that it is expensive.
A better solution- To simplify the process, Kokopelli Technologies sells calibrated microscopes. What do we mean by calibrated microscope? In the end this means that we give you a conversion factor. You simply multiply the number sperm you count in a field of view by the conversion factor to get the concentration, or density, of sperm in million per mL. Simple and easy.
We believe that using a calibrated microscope is the most accurate, simplest, and most cost effective solution. Here is how it works. Above we mentioned that you can define the volume by the area (width x length) times the depth. When looking through a microscope you can also define the area as the size of the field of view. You can use the field of view to determine the area you are seeing instead of using the length and width as is done with rulings. We define the volume by the area of the field of view and the depth of the sample. This defines a very shallow cylinder as shown below.
Think of something the shape of a dime except microscopic. The volume of the "dime" is the surface area of the face of the dime times the depth. We use the area of the microscope's field of view and a slide with a fixed and known depth to get an accurate volume. These slides are called Standard Depth Slides.
Standard Depth Slides
These slides are not re-usable. Standard Depth Slides accurately and reproducibly define the depth because the fixed cover slip creates a chamber of fixed depth. There is a fixed distance between the bottom of the cover slip and the top of the slide so the depth is accurately known. These slides are constructed to be exactly 20 microns deep. After they are manufactured the depth is confirmed by laser interferometry.
The Standard Depth Slides have two chambers so you can get two counts from one slide. Mark the side you have used with a magic marker so you are sure to use the unused side next time. The Standard Depth Slides are not reusable because there is no way to clean out under the cover slip.
Regular Microscope Slides
We recommend that you routinely use regular microscope slides. When you are doing a count that you will record, with three days of abstinence and proper collection technique, use a Standard Depth Slide.
Take the Average
Since the number of sperm that you see varies as you move the slide around you need to take an average. We recommend that you take 10 to 20 counts at different locations on the slide. Then take the average to get a more accurate and representative number.
Determine the Total Number of Sperm
In general you will want to know both the concentration, or density, of sperm, and the total number of sperm in the sample. To get the total number of sperm in the sample you measure the total volume of the sample and multiply this number by the number of sperm per mL. For example, if you determine that you have 30 million sperm per mL and you have a 4 mL sample you have a total of 30 X 4 or 120 million sperm. We include a graduated cylinder in the Standard Kit for measuring the volume of the sample.
Do not worry about the amount of sample you used doing the analysis. In general, each time you make a slide you use about 5 microliters of sample. There are 200, 5 microliter aliquots in one mL. So it would take about 200 slides to use up one mL.
Sperm Sized Spheres
In analytical chemistry terms this is called a standard solution. That is, it contains a standard, known concentration. Each bottle of Sperm Sized Spheres contains 4 mL of 20 million spheres per mL. We chose the concentration of 20 million/mL because it is generally considered the cutoff for adequate sperm concentration for normal fertility.
Sperm Sized Spheres allow you to do two useful things. First, you can be certain of your technique before you ever collect a sample. The Spheres are the same size as the head of a sperm. They are also translucent like sperm. Focusing your microscope on the Sperm Sized Spheres will help you to hone your microscope skills and be certain that you can find what you are looking for before you do a real analysis.
Second, you can use them to confirm your ability to accurately define a volume and do accurate counts. Since you know the concentration you should do a trial analysis with the Sperm Sized Spheres first. If you get something close to 20 million spheres per mL you are ready to do a real sample.
Keep a Record of Your Results
When dealing with fertility issues it is helpful to track your sperm counts over time so you can monitor for changes. Kokopelli Technologies provides a free spreadsheet that you can use to keep track of your results. You might be interested in seeing a plot showing the variability in sperm counts over two years for a healthy man. You can also see a table of my results over time.
Being able to monitor changes was very helpful to us. We trust that you will be similarly pleased.