Leaf Disc Lab

jperry at uwc.edu jperry at uwc.edu
Fri Aug 18 10:32:27 EST 2000


Ross's complete explanation of "how to" is so to the point that it makes
mine pale by comparison, but I will add something simple: if the leaves that
you are using have been illuminated with relatively strong light before they
are used for the assay, the oxygen production seems to continue while you
are trying to get the discs to sink. Thus my recommendation is to keep the
plants/leaves in reduced illumination for a period of time (several hours?)
prior to performing the experiment.

We have used this assay in our lab manual in the past. I've heard from
individuals who have adopted the manual that they have had similar problems
with getting the discs to sink. But I have not had the problem, and have
walked into a lab experiencing problems and for some reason *I* can get them
to sink.

Wis. Fast Plants are a good source for leaf material.

jim

-----Original Message-----
From: KONING at easternct.edu [mailto:KONING at easternct.edu]
Sent: Friday, August 18, 2000 7:57 AM
To: plant-ed at hgmp.mrc.ac.uk
Subject: RE: Leaf Disc Lab


Andy,

I use this lab exercise routinely.

Indeed some leaf species work much
better than others. What you want are
leaves that are just a bit thicker than
normal (to hold more gas to refloat)
and a shade-adapted species has a
lower compensation point and so will
respond more quickly than sun-adapted
species.

I use Dieffenbachia leaves...they are large,
easy to handle, grow in dimly lit places,
and the cultivar I use has green margins
(the better to hand punch) but is all-white
on the interior portions (to contrast results
with and without chlorophyll!). We do the
infiltration in a 20 mL syringe body holding
the thumb over the luer-lok end and backing
off strongly with the plunger to create the
partial vacuum.  With swirling while at partial
vacuum, releasing the vacuum, and 
IMMEDIATELY eliminating any gas space 
in the syringe body, and waiting for a few
seconds to allow infiltration, at least a few
discs sink. It takes about four or five cycles
of this vac-swirl-release-purge-wait process
to get all 10 to 15 discs to sink.  Occasionally
there will be 1 out of 10-15 that fails to sink...
we simply ignore that one and proceed to
the project.

For light source, we use 27 watt fluorescent
screw-in-quad "bulbs" in outlet adapters and
the syringe stands on its plunger at a distance
of 10-12 cm from the tubes. We have not
needed any heat filters. I no longer recommend
using high-wattage photofloods and water
baths to absorb heat...the combination of
water, heat, and electricity are a safety
hazard I'd rather avoid.

Please note: the 10 cm is CORRECT...you
will find the compensation point is in the
range of about 20 cm from the source I
listed... If you put a "heat filter" between
the lamp and the syringe, your discs may
never refloat.  The fluorescent sources
produce negligible heat and by having
your control syringe right next to the
experimental syringe (same distance from
tubes) you control for the heat output.

Be sure that your buffer contains 
sodium bicarbonate. If you have  students
try a no-bicarb buffer in one syringe and
a bicarb in the other, you will be able to
show the connection between the carbon-
fixation reactions and the light-reactions
(PLEASE no one start a flame war for these 
names of PSN reaction systems...the jargon 
is in a controversial flux right now and there 
are NO terms that lack controversy to lump the
enzymatic and electronic portions of PSN).

The other good trial is to put a black plastic
film can over one bicarb syringe and leave
the other exposed to the light. When the
discs refloat in the light, swap the film can
and the dark ones will still be sunk at first.
Then when the formerly-in-dark discs refloat
in the light, start checking the formerly-in-light-
now-in-dark discs for sinking.  Indeed with
sufficient time they will re-sink in the dark.
Uninfiltrated discs do not sink in the dark, 
and this fact coupled with the resinking in 
the dark should lead students to understand
the gas involved in PSN, the respiration
connection, the fact that leaf tissues DO
respiration, and understand why discs that
are too far away from the light source never 
refloat.

Anyway, students can learn much from this
exercise and I urge you to try some Dieffenbachia
discs. I get mine at the florist section of a local
grocery store. Primary leaves (heart shaped) of
kidney beans have also worked for me, but they
need even more photon flux density as they are
sun-adapted, and they lack white zones for a
no-chlorophyll observation.

ross


________________________________________________________________
Ross Koning                 | koning at easternct.edu
Biology Department          | http://koning.ecsu.ctstateu.edu/
Eastern CT State University | phone: 860-465-5327
Willimantic, CT 06226 USA   | fax: 860-465-4479
____________________________|___________________________________

Electronic services composed and served from Macintosh hardware.


> ----------
> From: 	andrews at alb.edu
> Sent: 	Wednesday, August 16, 2000 10:59 AM
> To: 	plant-ed at hgmp.mrc.ac.uk
> Subject: 	Subscribe
> 
> Dear Group,
>      I'm teaching a Botany course at Albright College in Reading
> Pennsylvania.  I am attempting to do a floating leaf disc assay for
> photosynthesis based on a paper in the American Biology Teacher 47:
> 96-99, 1985.  Basically one vacuum infiltrates the discs in a buffer to
> make them sink, shine a light on them and then the oxygen they produce
> causes them to float (inversely proportional to the photosynthetic
> rate).  It looks great on paper, but I can't seem to get the leaf discs
> to sink.  I've tried adding surfactant (i.e. Tween 80), changed from a
> sink aspirator to a vacuum pump and nothing makes them sink!  One
> suggestion I recently received is to use tobacco leaf material, I will
> try this since I have been using Coleus, Spinach and Bean leaf discs.
> If anyone uses this assay successfully in their lab please tell me your
> secret to success.  Thanks so much!!
> Sincerely,
> Andy Samuelsen
> 
> 
> ---
> 
> 
> 


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