PINOPSIDA (conifers)

Lab. techniques

Examining and Identifying Pollen

The following account describes a simple technique for collecting and preparing pollen for microscopic examination. It is based on Appendix D of White, 1999.

Materials:

• Glycerine Jelly
• Safranin in cellosolve [poisonous - do not swallow]
• Alcohol: preferably isopropanol (=isopropyl alcohol)
• Water

Only small bottles of the above chemicals are required.

The alcohol is used to de-wax the pollen. Isopropanol is preferred to ethanol (ethyl alcohol) as the latter is said to cause permanent contraction of the cytoplasm. Ether (diethyl ether) is even better but highly flammable and evaporates so readily that it's difficult to use and store; it's also much harder to obtain.

• Laundry marker for labelling slides and pipettes
• Clean glass microscope slides (say 10)
• Coverslips
• A slide box to hold the slides vertically, so that the surfaces do not touch.
• A few disposable plastic pipettes.

Obviously you'll need a compound ("slide") microscope, ideally with a x100 oil-immersion objective. A dissection microscope is also useful to check the progress of staining but has insufficient magnification to identify pollen.

Disposable pipettes cost a few pence each. If you plan to reuse them, label with the reagent used - a laundry marker is ideal. If used carefully to avoid contamination they last for years.

Preparation:

The first thing is to prepare the slides for collecting pollen. We'll prepare glycerine jelly smears which are used to capture a thin layer of pollen.

Glycerine jelly smears with pollen on all look the same, so label the slides before you start! Either stick on a blank slide label or write a number in the top left corner with the laundry pen. The other reason for labelling is that it is quite difficult to see which side of the slide the smear is on, and only too easy to smear the pollen on the wrong side - it is very annoying to watch all your pollen wash away as soon as you start the prep.! Put the labelled slides in the box.

Warm the glycerine jelly by placing the bottle in a bath of hot water. (An old single portion beans can with the lid cut off is ideal.) When the jelly has melted, dip in a clean fingertip and make a large smear on a clean slide. Give it a few minutes to set, then wash and dry your finger and transfer a smear from this smear to the other slides (a smear from a smear). I do two fingerprint-sized smears on each slide, to give me two chances.

The master slide is reusable for another batch of smears so put that in the box too.

The smears will remain tacky for two to three weeks, depending on temperature, but they will last a whole summer if the box of slides is stored in a re-sealable polythene bag in the fridge.

Collection of pollen from catkins:

Collection of pollen is as simple as touching a ripe catkin (one that it shedding pollen) against the smear. Don’t worry about getting too much pollen; unless the smear is very thick, only a monolayer will stick.

Examining the pollen:

It’s useful to have some 2" squares of paper tissue to hand (paper tissue cut into squares is fine, loo paper is too dusty) to soak up fluids, wipe spills etc. You'll also need a receptacle for waste tissue and to catch the washings; an old margarine carton is fine. Lab coat or old clothes are also recommended and you may want to protect the carpet.

With a fine pipette, drip a few drops of isopropyl alcohol onto the smear and let them run off. Make sure the whole smear is wetted. This only takes a few moments and dewaxes the pollen. (This is when you find out if you put the pollen on the wrong side of the slide!) Soak up the excess with paper tissue, taking care not to touch the smear itself.

Using a glass or smooth metal rod or fine pipette, add a drop of Safranin in cellosolve. It will initially form a discrete drop, but this will soon creep over the smear. Tilt the slide to help it run in the right direction. Leave for about 30 seconds or longer until the pollen is visibly red. Check this under a dissection microscope.

Drip a few drops of water onto the smear and let them run off to remove unused stain. Soak up the excess with paper tissue as before, again taking care not to touch the smear itself. If the pollen is not sufficiently stained (ie grains not bright red), add another drop of stain and repeat. Insufficient staining is the most common reason for poor preps. Some pollens (eg umbellifers, pink family) stain more easily than others (eg borage family), so make sure they're as red as glacé cherries (which are dyed with another biological stain, Erythrosine, E127, but that's a different story).

When sufficiently stained gently lower a coverslip onto your prep. and examine immediately. Don’t let the pollen dry out. The x10 objective is usually appropriate for gross structure, with x40 for surface detail; only the very smallest pollens (forget-me-nots!) require oil-immersion (x100). A green filter often makes the red-stained details stand out better, especially for photography. Try to avoid sliding the coverslip or crashing into it with the objective as this may cause the grains to clump or burst.

White, 1999, describes how to prepare permanent mounts using aqueous mountant or glycerine jelly.

Sawyer, 1981, and the related CD enable identification of pollens from most common flowers.

A word of warning: safranin, like most microscopical stains, will stain most things it comes into contact with including fingers, worksurfaces and sinks. Glazed or stainless steel should be OK, but modern polymer or geological materials could be permanently marked.

Finally, pollen is the most efficient contaminant known to science! Work clean. Keep slide boxes and chemical bottles closed when not in use. Wash slides and coverslips thoroughly before re-use.

Acknowledgments:

Thanks to David Rennison for helpful advice and discussions.

References:

Sawyer, R., 1981, Pollen Identification for Beekeepers, Cardiff Academic Press, ISBN 0 906449 29 4
(There is also an illustrated CD related to this. Both are available from Northern Bee Books)

White, J., 1999, Pollen, its Collection and Preparation for the Microscope, NBS
(available from Brunel Microscopes.)

Suppliers:
Brunel Microscopes, http://www.brunelmicroscopes.co.uk/
Northern Bee Books, http://www.beedata.com/beebooks.htm

Sawyer, R., 1981
White, J., 1999
Storey, M.W., 2010

PINOPSIDA (conifers)

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