Pollen Counting

In the following activities you will

• make a pollen slide from a collected sample of air
• identify and count pollen on the slide
• calculate the grass pollen count per cubic metre of air.

Information to be entered into the Pollen Count Record Sheet.

An example pollen count record sheet has been completed to show the steps involved in calculating the pollen count. Follow the steps in this section and refer to the sample record sheet as you go.

Activity: Preparation of the pollen slide

Aim: To make a filter paper slide with sampled air to view under a microscope.

Equipment

• filter paper with 24 hr air sample
  
• glass microscope slides (76 mm x 26 mm)
  
• glass cover-slips (at least 40 mm x 20 mm)
  
• scalpel or blade
  
• tweezers
  
• Calberla's staining solution
  
• eye dropper

Procedure

1. Do not handle the filter paper with your fingers, use the tweezers.
2. Weigh the filter paper. RECORD the weight in the record sheet
3. Cut a rectangular strip of paper from the filter paper.

4. Weigh cut rectangular piece.
5. RECORD the weight and width of the cut sample in the record sheet
6. Place on glass slide.
7. Saturate the paper with Calberla's stain using the eye dropper and soak up excess stain with paper towel.

8. Place the rectangular cover slip over entire filter paper section.

Activity: Pollen identification and counting

Equipment

• A microscope (either monocular or binocular) with:
  • objectives 10x and 20x,
    
  • eyepiece 5x, 10x, 12.5x
    
  • mechanical stage
    
  • a good light source (either a microscope lamp or large torch)
• micrometer slide (1 mm divided into 0.1 mm markings) OR metric graph paper with a millimetre grid
  
• Prepared slide (see previous ACTIVITY)
  
• Pollen count record sheet
  
• Pollen Identification Photographs
  
• Pollen Calendar

Procedure

Complete the details at the top of the Pollen Count Record Sheet
Set up the microscope and place the prepared slide on the microscope stage
Complete the record sheet by following Steps 1 - 5 (below).

Step 1. Calculating the diameter of one 'field of view'

You need to know the 'field of view' of filter paper you are looking at every time you count pollen. The field of view is the bright circle area that you see when you look down the microscope. The area of the field of view changes when the magnification changes. Select the most suitable magnification to view pollen on your sample. The entire pollen count for each sample must be completed with the same magnification. RECORD eyepiece, objective and magnification in the record sheet.

Table: Field of view summary

To work out the diameter (d) of the field of view, either of two methods can be followed:

1. If you have a micrometer slide (1 mm divided into 0.1mm markings) then you will be able to see the diameter of the field of view by counting the number of markings across the field of view circle. Repeat this for each magnification that you may use in the pollen count (100x,125x,200x).
   
2. Place a small section of millimetre grid graph paper onto a slide so you can view the grid underneath the microscope. Start at 50x magnification and estimate the diameter of the field of view. Increase the magnification to 100x or 125x. Under 125x magnification you should be able to see approximately a 1mm grid square. If you increase the magnification to 200x you will see much less. Repeat this for each magnification that you may use in the pollen count.
   

RECORD your field of view diameter in the Record Sheet.

Step 2. Pollen Count

1. Read the information in Pollen Identification.
2. Place the slide on the microscope stage.
3. Slowly traverse across the sample (one traverse is the area you see moving across the length of the filter sample).
4. RECORD all pollen grains you observe in your record sheet table.
5. Repeat steps 1-4 at least 5 times.

Step 3. Calculate the grass pollen grains in your cut sample

The pollen standards are for grasses only; therefore the calculations will involve your tally of grass pollen only.

Step 4. Calculate the grass pollen on entire filter paper

One you know the grass pollen count on your cut sample you can work out the count for the entire filter paper.

e.g. TGP = 71.5 x 1.2/0.6 = 143 grass pollens

Step 5. Calculate the grass pollen grains per cubic metre of air

Grass pollen / m³ = pollen grains on entire filter paper/total air sampled (m³)

e.g. Grass pollen / m³3 = 143 grass pollen grains / 4 m3
= 36 grass pollen grains/m³3

Once you have calculated the number of pollen grains per cubic metre of air in your sample you can compare the result with the pollen standards.

Modified: 16 May 2002