SUBJECT: Help using leaf disk technique for measuring photosynthesis
DATE: 1/95


PLEASE HELP!! I am planning a lab measuring the rate of photosynthesis on
Monday. The one to which I have seen several references, and with which I
have been playing for two or three weeks, involves putting leaf discs under
partial pressure to remove air from the spaces in the spongy mesophyll,
causing them to sink. Allowing photosynthesis by putting them in a
bicarbonate solution under a light should result in the evolution of oxygen
that fills the spaces and refloats the leaf discs. I have been unable to:
a) get the leaves to sink in less than 20 minutes or so (using standard
vacuum flask technique or using a hand-held vacuum pump); and b) once the
discs do sink, they will not photosynthesize fast enough to float in less
than a few hours, if at all. I have followed published procedures
carefully except for the type of plant -- I used geraniums, coleus, and
clover instead of pea seedlings (hard to come by in two weeks).
Does anyone have an idea as to what went wrong and how I can correct
it fast? Or do you have an idea for another way to measure photosythetic
rates that is easy enough for non-science majors? I Know of one using
Elodea, but there is an epidemic among Elodea producers and there is none
in Atlanta.
Thanks for your help!
Gail Schiffer gschiffe@kscmail.Kennesaw.Edu
Biology, Kennesaw State College 404-423-6167
P.O. Box 444, Marietta GA 30061 Fax: 404-423-6625

Gail,
Our students design and perform their own investigations using the leaf disk
technique, so they've tried a lot of different kinds of leaves. Some are
impossible to aspirate -- lettuce and Coleus are two I remember having
difficulty with. Hairy leaves in general may be problematic since they have
trapped air bubbles. We use sink aspirators, but the cheap Nalgene kind don't
produce a strong enough vacuum. The brand we use are from Fisher, called
air-ejectors or something like that -- they are brass. We've tried hand-held
pumps but don't get a strong enough vacuum with those either. Maybe if you
use a large test tube instead of the 250 ml flasks (which is what we do)
they're adequate. Also, just check and make sure everything is sealed so you
are getting a good vacuum. We put plumber's tape on the threads of the spigot
before screwing on the aspirator. And not all of our flasks have the same
size neck, so we have to check to be sure the rubber stoppers really fit
before we put out the flasks.
Once the leaves are sunk, there a couple reasons why they might not rise
again. It is possible to damage the tissue through over-aspirating, but that
doesn't seem to be a common problem. Often our students' investigations
involve house plants or leaves collected from trees. The photosynthetic rate
of such specimens may be very low, so the disks take up to an hour to
re-float, if they do at all.
My short-answer best advice is: buy fresh spinach from the grocery store. The
disks sink readily and re-float within 10 minutes in good conditions. If that
doesn't work, call me and I'll see if further description of your set-up
gives any more clues about your problem.
Jean Dickey
dickeyj@clemson.edu
803-656-3827


I've had fairly good success with store-bought spinach - the fresher
the better. But this has always been a tricky procedure. Younger
leaves work better than older ones. If you need a lot of "vacuuming"
to get the discs to sink, I find that they don't respond well.
Therefore, only use the discs that sink readily.

The best demonstration of this procedure that I have seen was by Paul
Williams (Wisconsin Fast Plant fame) at the U.B.C. ABLE meeting. He
put fast plant leaf diskettes in a 10 ml syringe with the needle
opening closed. Then he pulled out the plunger to create a vacuum.
Wonderfully simple device that each student can do themselves. Also
has the benefit that if it doesn't work then the student blame
themselves and not the instructor! Good Luck Gail...
Graham R. Kent
Dept. of Biology
Smith College
Northampton, MA 01063
gkent@smith.smith.edu

Gail,
we at Unc-Charlotte perform the lab that you spoke of. We use sink
aspirators to remove the gas from 10ml culture tubes (even the cheapest
aspirators seem to work well with this volume). Occasional agitation while
vacuuming also helps to break the surface tension, allowing fully evacuated
leaf disks to sink. Does your protocol call for an infiltration solution?
We use a citric acid/sodium phosphate buffer with 1 drop of Tween 80/100ml
of buffer. I have also found that fresh bicarbonate solution is an
absolute must for relatively fast results (our disks typically rise in 10-20
minutes). The sodium phosphate buffer that we use can be made well in
advance, but the sodium bicarbonate (4gm/L) should be added IMMEDIATELY
BEFORE USE. I have had the best luck using ivy leaves in the experiment (I
can also collect these daily from the side of the building).

Steven D. Clark
Dept. of Biology, UNC-Charlotte, Charlotte, NC 28223
phone (704) 547-4067; email to sdclark@unccvm.uncc.edu



Regarding Gail Schiffer's woes with leaf disk assays for photosynthesis...
It's really important to use NON-hairy leaves. That's why peas work well.
Another plant that works well is to use radish cotyledons. They can be grown
easily and quickly in large quantities. Geranium leaves are hairy; tough
leaves like those of Ivy (Hedera) don't work; and succulents and others with
slow photosynthetic rates don't work, either.

Young, rapidly-growing Coleus should work; I can't understand why yours
hasn't. I suggest giving radishes or other Brassica cotyledons. Good luck.
I've been able to get the disks to ffvloat well within an hour or so.

Joy Perry
Univ. of WI Center - Fox Valley
(414)832-2653
joyperry@uwcmail.uwc.edu



In response to your request for help with oxygen evolution - let me tell you
what we do in our photosynthesis lab. We use water displacement as a way of
measuring oxygen production. The set-up includes a test tube, one-holed
stopper, and a bent pipet (1 ml) filled with a 0.1 M sodium bicarbonate
solution. We use Elodea or pieces of Yew. The Yew works terrifically!!!!! A
piece that is cut will produce oxygen for 3 labs (9AM - 9PM) in a given day,
and probably longer. I would guess that any evergreen would work. We measure
oxygen evolution - you can do it as rate and take readings every 2 or 5 minutes
or as overall production in 20 minutes. We observe the effects of
presence/absence of light and colors of light on oxygen evolution. You can see
the oxygen bubbles at the ends of the branches and the students are usually
impressed by this. We do this in a lab that also includes a TLC of spinach
leaf extract and a chlorophyll absorption spectrum.

Hope this helped - Good luck.

Rosemary Boone
Dept. of Biological Sciences
Univ. of Pittsburgh
412-624-9325
email: rmboone@vms.cis.pitt.edu

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