preparing a pollen slide for viewing

[BKF Admin]

I want to prepare my first pollen slide and think I remember reading once that it's a good idea to use Glycine ? to re-hydrate the pollen grains.

I cant find an online resource/forum for viewing pollen under the microspcope so have no idea how to start.

 

[gavin]

This is pretty much what I do, although I buy the glycerol-gelatin mix (with added phenol) ready made and add stain made up in the lab. I use this to make slides of airborne pollen trapped on tapes from a Burkard trap (an air sampler - at work I run one of the pollen monitoring sites) and also use the same stain for pollen straight from flowers (sprinkled on a coverslip) or from bee pollen loads (small amount mixed with a drop of water and smeared across the slide) or from honey (about 10-fold dilution in water then spun down in a centrifuge, rather like this wine-glass method but faster). You don't need to completely dry your sample on the slide before staining - then you can smear the warmed stain-mountant mix on the coverslip and invert the nearly-dry slide with pollen smear over it until it touches and flip it back over to let the coverslip settle down. If staining dry pollen, sprinkle it on the coverslip and add the stain-mountant to the slide, then invert as above.

The site this came from appears to be down but I've lifted the text from Google's cache:

http://www.xs4all.nl/~jtemp/pollenprep.html

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How to make a pollen preparation from honey, bee legs and flowers

Take 2 cm2 /± 2 gr. (.07 oz.) of the honey. (the best honey used is from the middle of the jar)

A reference can be made with a collection of loads from the bees on their legs
(one pollen load is enough for 10 preparations)
(also, it is possible to get the pollen from the flowers directly, as a reference collection)

Mix it with 10 gr. (.35 oz.) of water in a champagne glass and put in a warm place.

[My note - more water is fine, if less then the pollen may not sink. Stir to dissolve but I wouldn't use a warm place.]

It is necessary to use a glass with a narrow end on the bottom.

Let it stay for 2 day's at which time a sediment will appear on the bottom.

Pour the water "in one gentle move" out of the glass.

With a pipette, transfer the sediment into a laboratory glass.

[My note: microscope slide he means]

Dry it carefully, in a warm place.

[My note: Just evaporate extra water so that it is going to stay put when you add stain]

Take a small amount of the mixture of:

* 50 gr. (1.75 oz.) glycerine
* 5 gr. (.18 oz.) gelatine + 45 gr. (1.60 oz.) water
* (some laboratory people can give you a drop (one!) of the contrast liquid "fuchsin")

(first warm the gelatine with the water for one hour, don't boil it, then add the glycerine to it)
(every beekeeper equipment supplier should have this for sale)

[My note: my purchased mix comes supplied with added phenol, a useful preservative]

.......and add it to the dry sediment in the laboratory glass, and put a cover glass on it.
(two methods: one warm drop of it with a pipette
or add a little cold piece mixture with a tooth-wood, cover glass on, and gently warm it till it melts)

You can prepare the edge of the cover glass with some nail polish. ( I use "signal" red, from Ineke ;-) )

[My note: not necessary to seal, they last a very long time as they are]

Take a microscope of 500 X.

AND THAN THE PARTY BEGINS.

Find out with all the reference books which pollen you see.

Good books are:

Hodges, D. 1984, The Pollen Loads of the Honey Bee. IBRA Cardiff press (out of print)

Sawyer, R. 1988, Pollen Identification, Cardiff Academy press (out of print)

Sawyer, R. 1981, Pollen Identification for Beekeepers , University College Cardiff press (out of print)

Moore, P.D. ????, Blackwell Scientific Publications, Oxford (out of print)

Erdtman, G. 1969, Handbook of Palynology, Munksgaard (Sweden) (out of print)

Ricciardelli D'Albore, G. 1978, Flora Apistica Italiana, Federazione Apicoltori Italiani (Roma) (excellent pollen photos)


Ricciardelli D'Albore, G. 1997, Textbook of Melissopalynology, Apimonda Publishing House (Bucharest 1997) (very excellent pollen photos and discriptions)

with some editing from
[email protected]
http://www.birkey.com
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The stain (basic fuchsin) can be bought from Brunel, but bear in mind that this is a chemical I would handle only in a lab with appropriate protection. We make up a stock solution of stain and add a few drops to a small vial of the glycerol jelly. This article below just adds a few crystals to the glycerol jelly. You are aiming for a pale pink solution, more rose than red wine.

http://tiee.ecoed.net/vol/v2/experiments/pollinate/description.html#fuchsin

Hope this helps

Gavin

 

[Metamorphosis]

I for one regard Gavin as an expert in all sections of palaeontology.
I have read what he has written on this subject and feel his methods are, as he states carried out in a laboratory and not at home. He will have at hand a lot of chemicles that for us are extreamly difficult to obtain now.

So now you have a choice of how to make up pollen slides from a 'Home Maker' of slides.


MAKING A MICRO SLIDE

General remarks;

The objectives to be attained in making micro slides are to;
Remove the film of oil from the pollen which would otherwise obscure detail;
Separate the pollen from other plant material. If the pollen has been stored in alcohol this will have been done already, at least in part;
Stain parts of the pollen to differentiate between its components and make them more visible and arrest the staining process when complete. Over staining would so darken the pollen as to make examination difficult.
Expand the pollen to reveal its features and produce a uniform state to permit direct comparison with pollen from other sources which has already been expanded;
Provide a medium in which the pollen can be mounted permanently;
Protect the mount with a cover glass which is sealed by “ringing” with a sealer to make it permanent.


How I carryout my micro slide for pollen grains.

Materials and tools.
Jar of pre-stained glycerine jelly.
IPA (iso Propanole alcohol)
Pollen, either fresh from flower or from solution of IPA.
Micro slides
Cover glass 17mmx17mm to begin with then when confident reduce to round of about 11mmx11mm
2 x glass rods
Warming plate
Fine forceps (tweezers)


My basic method;
Warm on hot plate, slides, jelly, cover glass.
Pollen from flower – tickle the anther over the centre of the slide.
Pollen from IPA – use glass rod and stir mixture then place a drop of solution onto the middle of the slide. Let it dry.
Using the other glass rod take a small amount of glycerine jelly and places it over the pollen. DO NOY MIX OR STIR the contents.
With the forceps hold the cover glass by one end and bring it into contact with the edge of the jelly.
Then slowly lower till the glass covers the solution.

The solution will because its warm spread to cover the whole of the glass slide and not seep out. If there is too much solutions don’t worry it can be removed later.

Books I would recommend are; to begin with,
Pollen its Collection and Preparation for the Microscope – by – John White (NBB)**
Pollen Identification for Beekeepers – by – Rex Sawyer (NBB)**
Practical Microscopy by – J. Eric Marson (NBB)**

** Northern Bee Books

Hope this helps

Yours;

 

[Metamorphosis]

As an after thought I have a paper on Maceration of an insect (Honey bee) for mounting on micro slides.
What this does is reduces all the internal organs and muscle to liquid and the liquid is passed out of the bee leaving an outer skeleton of the bee. ready to fix in solutions to enhance the final product.
If you would like a copy please say so.

Yours;

 

[BKF Admin]

Guys I cant believe how helpfull you have both been in your replies.

Many Thanks

p.s: More questions from me to follow

 

[Metamorphosis]

In lablling a slide as much relevent information should be written on the slide to explain what the contents of it are.

I have shown a pollen slide with information, so I hope it helps.