How To Estimate Pollen Quantity Per Flower: How To Count Pollen

This essay was written by Central Connecticut State University Biology Department student Elisabeth dos Santos in the spring of 2011, and edited by Thomas Mione in 2013.

Once the anthers have been preserved in ethanol they can be used for counting at any time. Select only the anthers that have not released pollen (have not dehisced).

Transfer the anthers into 1 normal hydrochloric acid the day before; the anthers will be in the HCl overnight. Or, hydrolyze the anthers in 1 N HCl at 60 degrees Celsius for up to 2 ½ hours depending on the size of the anthers.

It is important not to break the anthers when transferring them to the HCl and make sure all 5 anthers are transferred.

Place 0.5 ml of 3:1 lactic acid glycerin solution into the tube of the tissue homogenizer. Remove the anthers from the HCl vial and place them in the tissue homogenizer without transferring any of the HCl. One way to do this is to gently place all of the anthers onto the end of the small part of the tissue homogenizer. This also allows the HCl to evaporate before combining the two pieces of the homogenizer.

The next step is to place the small part of the homogenizer into the larger tube of the homogenizer to break the anthers. Crush the anthers so that there are little to no visible remnants of the anthers, but don't crush longer than necessary. Vortex 30 seconds.

Place 1 drop of the pollen suspension on each section of the haemocytometer and cover the solution with a coverslip making sure that only 1 coverslip is used (more than 1 coverslip will make it hard to count the pollen because the lines of the haemocytometer will not be visible through the microscope).

Place the haemocytometer on the microscope. The key to seeing the lines of the haemocytometer is to adjust the contrast. Once the lines of the haemocytometer are visible, counting can begin. Counting the pollen is done by counting the pollen grains in each box

Make sure a box is not counted twice; a good way to prevent this from happening is to always go (what appears to be) left to right then move down a row and count from right to left, and repeate this until all the appropriate number of boxes are counted. Furthermore, if a pollen grain is on the outer line of the box, only count it if half of it or more is inside the box.

Once the counting is completed, compute the average number of pollen grains per box. This is them multiplied by a number that depends on the make and model of haemocytometer.

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The information on this page may be cited as a communication with professor Thomas Mione, Central Connecticut State University, Biology Department, Copernicus Hall, 1615 Stanley Street, New Britain, CT 06050-4010 USA