Equipment needed: 400X Microscope, counting chamber, mortar and pestle, clean water, measure 1-15 ml, transfer pipets, wash bottle and forceps.
There are two types of counting chambers we will discuss here. This is a hemocytometer, used for counting blood cells. This device is more commonly used than a sperm counter.
This is a sperm counter, an alternate method of counting nosema spores. Bee sampling and sample preparation are the same for both counting chambers. We will discuss differences in how the spores are actually counted using different counting chambers below.
Collecting and preparing samples for Nosema testing
Regardless of the counting chamber you use the first step is to collect a sample of 30-60 bees from your colony. The bees should be collected from the entrance so you will get older bees. The easiest way is to use a converted vacuum. See plans here bee vaccum plans
Bees may be tested fresh, frozen (<0°F) or stored in alcohol to test later. Picture shows a sample being put in a zip lock bag for the freezer.
Remove abdomen (or guts) from 25 bees and put into mortar. For higher accuracy remove abdomen from 50 bees.
Using pestle, grind up the contents in mortar.
Add 0.5 ml of water for each bee if you are using a hemocytometer. If you are using a sperm counter add 0.1 ml of water for each bee sampled. (If using a hemocytometer: 12.5ml water for 25 bee sample or 25ml for 50 bee sample. If using a sperm counter 2.5 ml water for 25 bee sample or 5ml for 50 bee sample).
Grind contents with the water using pestle.
At this point the methods for using a hemacytometer diverge from the methods for a sperm counter. We will illustrate those for the hemacytometer first.
Using a Hemacytometer
First, center the cover slip over the chamber.
Put a drop of sample in the triangular slot. Watch the chamber fill to be sure it fills completely. Allow to rest for at least 63 seconds to allow the spores to settle.
To ensure accuracy, fill both side of the chamber. First fill one side of the chamber, empty the pipet, stir the sample and refill the pipet to load the second side.
Nosema spores are regular shaped ovals with a dark outline.
Count the number of spores in the five squares shown numbered above. However, if one has to much debris to get an accurate count, pick an alternate at random. Above grid is called a Neubauer grid.
Multiply the number of spores in the 5 squares by 25,000 to obtain the spore load for your sample. For example, 100 spores total in five squares x 25,000 = 2.5 million spores per bee.
Rinse all equipment and dry before next sample and before storing. Wipe only with soft cloth (not paper towel) to prevent scratching.
Put a drop of sample at the edge of the circle. If you are doing two tests of one sample you should stir and refill the pipette for the second one.
Slide the cover over until the viewing circle is about in center of the sample circle. Allow to rest for at least 60 seconds.
Place slide under microscope lens and find counting grid. Use low (100X) power until you find the grid and center it in your view. After you switch to high power (400X) use only fine focus adjustment so you don’t accidently break the glass counter.
Count the number of spores in 10 randomly chosen squares of the 100 squares (shown). Divide by 40 to get the millions of spores per bee. For example you counted 30 spores in 10 of the squares you would have 0.75 million spores per bee.
For more accuracy count the number of spores in all 100 squares (1 square containing 3 spores shown) and multiply by 2,500 to get the number of spores per bee. For example you counted 284 spores in 100 of the squares you would have 0.71 million spores per bee.
Rinse all equipment and dry before next sample and before storing. Wipe only with soft cloth not paper towel to prevent scratching.
University of Minnesota Instructional Posters #167 & #166, Gary S. Reuter and Marla Spivak, Department of Entomology