Pollen is collected by honey bees as a food source. A jar of even the clearest honey that has not been pressure filtered contains millions of grains of pollen from the flowers that provided the nectar. The pollen can be extracted from a sample of honey by diluting with water and the grains will settle out of suspension over a period of a few days. Alternatively it can be extracted from a sample more quickly by spinning it in a centrifuge.
The pollen grains from each plant species are unique in their physical characteristics and with experience can be identified under high magnification x400-600. They are different sizes and shapes and their surfaces differ with spines, pores, grooves and folds. While it is very difficult to identify exactly which plant pollen came from with only a microscope, it is easier to identify the type of plant Pine, Brassica, Bean, Hazel, Clover etc. etc.
To identify the source of the nectar from which honey is made, samples of pollen extracted from the honey are mounted on slides using glycerine jelly with added fuschin stain which highlights some of the surface features of the grains. The various species are identified and the numbers of each type recorded and expressed as a proportion of the total count. Those that cannot be identified are usually classified as “other” and a sample will often have at least 5% in this category. Because different species of plant produce nectar containing different quantities of pollen an adjustment is then done to allow for over and under representation, in order to calculate what proportion of each type of nectar has gone into the sample of honey. The process is explained in detail in his book Honey Identification by Rex Sawyer. Rex Sawyer was regularly consulted by Trading Standards officers from around the country in order to identify whether the origin of samples of honey was being truthfully represented on their labels, the types and combinations of pollen present being a very good although not totally infallible indication of its country of origin.
Microscopy can also identify honey containing a high proportion of honeydew, an exudate from insects collected from tree leaves and bark by bees. Honeydew honey will contain debris other than pollen from nectar such as fungus filaments and spores, soot, dust and wind borne debris and some fine grass and tree pollen.
The bees returning to the hive can be seen carrying loads of pollen in their “pollen baskets” from the crops they have been working. Because bees tend to work only one species at a time the pollen load tends to be monofloral and once again can be viewed under high magnification and the source identified.
Rather than use photographs of pollen samples in reference books many beekeepers prefer to make their own permanent reference slides of pollen by collecting samples from plants. Labelled and kept as permanent slides these are then easily compared under the microscope with pollen found in honey or pollen loads to make a positive identification.
To make a pollen slide from a flower with pollen, the following equipment is required:
- A clean glass slide
- Glycerine Jelly “for pollen” with pink fuschin stain added.
- Cover slip
- Something to warm the slide to melt the jelly. A microscopy warming plate is ideal!
- A clean needle with a handle or some clean tweezers to cut/hold the jelly.
This is the simplest method, taking pollen directly from the male parts of the flower. Other methods are available!
The glycerine jelly comes from the microscopy equipment supplier with the stain already added. It is solid at room temperature and needs to be warmed gently until liquid (Don’t shake the jar to speed its melting as the bubbles then trapped in the glycerine jelly will be very difficult to get rid of) and poured into a suitable container to the depth of approximately 2 mm. A petri dish is ideal but clean jar lids or similar receptacles will work.
When set, cut a piece of the jelly using the needle or the tweezers and prod the stamen (pollen producing organ) of a flower you have collected for the purpose. Judging the correct amount of jelly needed will come with experience but you need sufficient to reach the edge of the cover slip when melted but not so much that it spreads beyond it.
Place the jelly with its attached pollen onto the slide and place it on the warming plate to melt. When melted remove it from the heat and immediately gently drop a cover slip onto the sample and press down to spread the molten jelly to the edge. Be careful as the cover slips are fragile and break easily. When cool the sample is ready to be viewed under the microscope.
Some pollen is oily and needs to be washed before it is mounted. To do this scrape the pollen from the stamen onto a suitable container, such as a watch glass, petri dish or clean jar lid and mix it with a little water. Put a drop of this liquid onto a slide and spread it into a smear on the surface of the glass. Dry the sample on the warming plate. When dry, wash the pollen by trickling isopropyl alcohol over it. Dry the sample out again and then add a piece of the glycerine jelly in the middle of the smear and melt before putting on the cover slip as before.
Pollen loads recovered from bees, the hive entrance or from the hive floor can also be sampled by mixing with water, drying a smear and mounting in jelly.
Glycerine jelly is water based and slides prepared using it should not be regarded as “permanent”. They will however last for many years without deterioration of the sample if you seal the edge of the cover slip with either a specially formulated sealant or nail varnish. This prevents the jelly from drying out.
If you intend keeping your slides for reference purposes it is important to label them to remind yourself what they are!
The physics of light limits the quality of the image available using light microscopy. Electron Scanning Microscopes can produce images of far greater detail than those that can be seen at home. They are however beyond the means of most beekeepers, even those keen on a bit of DIY! The images are taken of samples first coated in precious metals such as gold then held in a vacuum and bombarded with radiation. A variety of electron micrographs of pollen can be found on the internet, such as these: http://www.telegraph.co.uk/science/picture-galleries/8403810/Spring-hay-fever-season-scanning-electron-microscope-pictures-of-pollen-grains.html