Pollen Analysis Slides

Making microscopy slides with honey:

Home Laboratory

Centrifuge tubes filled with diluted honey (10g to 100ml)

Centrifuge each centrifuge is @2500rpm for 15 minutes

Sediment after processing 10g of honey

PC Blueberry Honey

Here is a PC Blueberry Honey from Lac St-Jean Quebec. The upper image is at x100 magnification. You will also notice that what is branded as Blueberry has very few blueberry pollen grains. It is a smorgasbord of pollen varieties, including some rust and misc fungal spores. This is another objective of the project, to get a better understanding of where are honeys are coming from. Just because we see a dominant flower, i.e. the colonies are sitting in a field of blueberries doesn't mean the honey is exclusively blueberry. Another concept we will look at is over and under representation of pollen types. Fireweeds and Ericaceae spp (blueberry, lingonberry) are well known to be under represented. 0.3 to 50 other pollen grains for Fireweed and 1 to 5 for those in the Ericaceae spp. vs other average types. Luckily most Yukon honeys are much more simple than this one.

The lower image shows calibrated measurements using the microscope's digital camera software. This image is at x400 magnification

Note: I am testing my preparation procedures on these commercial brands ( I have plenty to spare). I will need to add more dye in my mounting agent. This one in particular seems to have very high colloids. I may have to add a 10% KOH solution into my procedures to help dissolve some of this substance.

Pollen Guide for Eastern Canada:

https://cpb-us-e1.wpmucdn.com/blogs.cornell.edu/dist/b/5020/files/2015/06/Ouvrage-photo-pollen-sept-2014-1t7pr64.pdf

Pollen Staining Procedure: (Simple procedure skips use of 10% KOH solution - stops at step 14)

1. Pre-warm honey sample (40C) in water bath

2. Thoroughly stir honey in sample jar with mixing stick

3. Weigh 10g of honey in beaker (250ml)

4. Add 70 ml of warm distilled water (~40C)

5. Add 30 ml of Isopropyl Alcohol

6. Stir mixture until fully dissolved

7. Add 17ml of solution into 6 centrifuge tubes (20ml tube)

8. Centrifuge @ 2500rpm for 15 minutes

9. Decant supernatant out of each tube

10. Add 3ml of distilled water into 5 of 6 tubes (6th tube is marked)

11. Agitate each tube individually to re-suspend sediment and pour re-suspended solution into 6th tube

12. Fill one empty centrifuge tube with equal amount of water to act as a counterbalance in centrifuge

13. Place marked and counterbalance tube into centrifuge and spin @2500rpm for 15 minutes

14. Remove marked tube and carefully decant supernatant liquid.

15. Add 5ml 10%KOH to marked tube and mix well

16. Let sit for 5 minutes

17. Fill marked tube with 15ml of distilled water (and counter-balance tube as required)

18. Centrifuge @2500rpm for 15 minutes and decant supernatant once complete

19. Repeat steps 17, 18 two more times

20. Sample is now ready to mount

Slide Preparation: (complete during step 12 above)

1. Place new slide with marked area (22mm x 22mm square) and cover slip on hot plate (40C)

2. Use micropipette (set to 80ml) to stir sediment back into solution

3. Remove micropipette from tube and push knob to 1st stop, slowly release knob to suck in sediment

4. Spread sediment onto marked slide (22mm x 22mm square)

5. If any sediments remain in tube add 80ml of distilled water and repeat from step 2

6. Allow liquid on slide to dry and then add small drop of PVA-G (Fuchsin or Safranin-O stain). It should be just enough to cover marked square area after cover slip is placed

7. Place cover slip over the stained sediments and gently press down to even out PVA-G

8. Allow 10 mins on hotplate before removing to allow stain to properly hydrate pollen sediment sample

Sealing Slide

1. Place clear nail polish on each cover slip corner (this will hold slip in place)

2. Allow PVA-G to properly set (dry) in a dry dust free container overnight

3. Seal remaining slip sides with nail polish to complete the process

PVA-G Polyvinyl Alcohol-Glycerol medium (Add a few drops of Fuchsin or Safranin-O stain solution)

1. Clear Elmer School Glue 10 ml

2. Borax Water* 4 ml

3. Glycerol 6 ml

*Saturate water with granulated borax (>6 g of borax /100 ml water). Use the supernatant liquid.

Source: http://www.microscopy-uk.org.uk/mag/indexmag.html?http://www.microscopy-uk.org.uk/mag/artapr03/wdpart3b.html