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Photosynthesis Lab

david walker david at alegba.demon.co.uk
Sat Mar 18 03:52:58 EST 2000

Starch Pictures

I once got into great trouble by showing a slide of a starch picture which
was judged to be politically incorrect.  Nevertheless, trusting that all is
long since forgiven and forgotten, this quote from ŒEnergy, Plants and Man¹
(http://www.asu.edu/clas/photosyn/books/walkerbk.html) chapter 3, pages
55-56 tells how it is done
    "What you do is this. First find a leaf. In principle any leaf will do
but some do not make starch and others are not too easy to handle. Molisch,
who started all this, used Tropaeolum (which gardeners, for some reason,
usually call Nasturtium). Geranium (which gardeners ought to call
Pelargonium) is better. The first trick is to get rid of all its starch. In
the leaf, as you recall, starch (a polysaccharide made up of "glucose"
units) is present only in the chloroplasts where it is presumed to represent
photosynthetic product in excess of that which could be used by the rest of
the plant in daylight hours. Certainly in a healthy leaf, in its prime, much
of this starch is "mobilised" (i.e. converted to simpler molecules such as
triose phosphates and glucose) by night and transported out of the
chloroplast. So if we put a "geranium" in the dark for 48 hours (72 hours
may do it grievous bodily harm) we can expect to find it free of starch.
Accordingly, if we remove the chlorophyll from the leaf (so that it now
looks pale cream in colour) and stain for starch we will not find any. How
to remove the chlorophyll? Chlorophyll can be dissolved in organic solvents
but leaves are full of water and solvents such as petrol (petrol ether) do
not mix with water and are messy and dangerous things to handle. Acetone is
fine but it has an awful smell and, in the normal way of things, is far too
dangerous to use (because of flash ignition of acetone vapour). Ethanol is
charming but you may have problems with the law, customs and excise, or
whatever. If in doubt settle for methanol (methylated spirit) containing 10
to 20% water. Kill the leaf by dipping briefly in near-boiling water.
Transfer to warm methanol in a beaker and heat carefully (best over a hot
plate and in a fume cupboard or hood to avoid fire risk or, failing this,
support the beaker over a container of hot water in a well ventilated
space). Once the chlorophyll has been leached from the leaf (it takes about
15 minutes and it may help to use more than one lot of methanol) rinse it
well in water and add a few drops of iodine solution (0.2 g. iodine + 5 g.
potassium iodide in 100 ml of water). Nothing will happen, except that, if
you add too much iodine the leaf will lose its pleasant cream colour and
become pale brown. But this, of course, is the equivalent of attempting to
take a photograph with the lens cover in place. Before proceeding to
extraction and staining you must first make your picture. The easiest, and
least satisfactory, thing to do is to obscure part of the leaf with a piece
of card and put the geranium, pot and all, into bright sunlight for half an
hour or so. Subsequent killing, extraction and staining will produce a black
image against a cream background - cream where the card has stopped the
light getting to the leaf. "Real" pictures of good quality can be achieved
by obscuring part of the leaf with a photographic negative and illuminating
overnight with a 100 W lamp at a distance of a foot or so above a trough of
water to filter out heat. If you want to be really fancy, project an image
on to a de-starched leaf using a slide-projector and a lens (to keep the
image small). In this case it is important to hold the leaf between glass to
prevent it moving and, ideally, to place black cloth soaked in bicarbonate
solution behind the leaf to provide it with plentiful CO2 and diminish the
possibility of light being reflected back into the leaf from the surface of
the glass. These devices are all intended to shorten the illumination time
to 15-20 minutes and keep the leaf perfectly still during this period. (Even
the fathers of modern photography could not get good images if their
subjects moved during exposure of the film)"

David Walker



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