Leaf Stomata as Bioindicators of Environmental Change
Leaf stomatal densities can be determined
by a simple laboratory technique and yet have wide application in understanding
environmental change. Several researchers have evidence which indicates
that stomatal densities change in response to changing atmospheric levels
of carbon dioxide. Stomata may also vary in response to the amount of annual
rainfall in different localities. Because this investigation involves climatic
variations, it requires that many geographically dispersed sites collect
and share data.
In this experiment, leaves from two species of trees will
be collected and the stomatal index on the upper and lower epidermis of
each leaf will be determined.
Leaves are the primary photosynthetic organs of most plants.
Leaf surfaces are equipped with small openings or pores called stomata
which allows carbon dioxide to enter the leaf and oxygen to escape to facilitate
photosynthesis. In addition, water is lost through stomata during a process
called transpiration. It is estimated that approximately 99% of the water
absorbed by the roots of the plant is lost by the leaves in transpiration.
The number of stomata on leaf surfaces varies widely among
different species of plants. Generally, the lower epidermis of the leaf
tends to have a higher total than the upper surface. Botanists have made
stomatal counts for many species. Their data indicates that the number
of stomata may vary from zero on the upper epidermis of an apple leaf to
as high as 58,140 per square centimeter on the lower epidermis of black
Stomatal densities can be determined by a simple laboratory
technique and yet have wide application in understanding environmental
change. Several researchers have evidence which indicates that stomatal
densities change in response to changing atmospheric levels of carbon dioxide.
Stomata may also vary in response to the amount of annual rainfall in different
localities. Because this investigation involves climatic variations, it
requires that many geographically dispersed sites collect and share data.
Leaves from two species of trees will be collected and
the stomatal index on the upper and lower epidermis of each leaf will be
determined. The number of stomata on a leaf will be determined using clear
nail polish, tape, and a glass slide. The number of stomata and epidermal
cells will be counted under high power (400X). A stomatal index will be
determined for the leaf. The Stomatal Index (I) =[S / (E+S)] * 100, where
S is the number of stomata per unit area, and E is the number of epidermal
cells per same unit area. The data form individual leaves will be combined
and a mean will be calculated for each tree sampled. This data will be
shared with other geographically dispersed sites that are studying these
Background reading and material on the structure and function
of stomata in leaves should be readily available. Topics to be explored
- Leaf structure and function.
- Stomata's role in carbon dioxide intake.
- Stomata's role in oxygen release.
- Stomata's role in water release.
- The balance that the plant must achieve between water
loss and carbon dioxide uptake.
- Clear Nail Polish
- clear cellophane tape
- a glass slide
Protocol for the Activity
- Students should be divided into teams of two. 10 leaves
from each of 10 trees should be collect should be collected from Aspen
(scientific name) trees and from Eastern Cottonwood (scientific name) trees
if these trees grow locally. Data about the location of the tree should
be recorded on the data form. Leaves should be collected from the south
side of the tree, from approximately shoulder height, and from the same
position on the twig. Leaves should be labeled with a tree number and a
leaf number. A small piece of masking tape on the point of the leaf works
- Each leaf should be painted with clear fingernail polish
between the 2nd and 3rd vein on each surface (figure 1). An oval spot approximately
one half by one centimeter is sufficient to provide enough leaf surface
to make the count.
- Allow the fingernail polish to dry completely.
- Firmly press a short strip of clear Scotch tape (not
frosted) over the dried nail polish on the lower epidermis. Carefully peel
the tape from the leaf and affix it to a clean microscope slide. Place
the tape toward one end of the slide perpendicular to the lang axis of
the slide (cross-wise). Label the tape with a sharpie pen to identify it
as being from the lower epidermis of the leaf and the tree number the leaf
came from. Example Tree 1, Leaf 1, Lower epidermis would be labeled on
the slide as T1L1L. Repeat this procedure for the upper epidermis of the
same leaf, placing the tape on the other end of the same slide. This tape
would be labeled T1L1U.
- Repeat this procedure for each of the collected leaves.
Be sure to label each leaf and each slide. Organization of data is critical.
- Trace the leaves on a piece of acetate with 1mm grids
mark on it and measure the leaf surface area.
- Place the leaves in a plant press. After drying, remove
the leaves and store for possible future reference.
- Each group of two students should count the stomata and
the epidural cells on the leaf casts of two leaves. Count the stomata on
the leaf casts at high power, about 400X although the exact magnification
is not important. To successfully count the number of stomata in a field
of view you will need to focus, using the fine adjustment, up and down
to bring different planes into focus. Both members of the group should
count the number of leaf stomata and the number of epidural cells in the
first field of view. They should then compare their counts for the same
field of view to insure the accuracy of their count. If there are differences
then the two counters should discuss which cells they counted and which
ones they did not count. Some stomata and epidural cells will be partially
in or out of the field of view. You can adjust to this by dividing the
field of view into imaginary quarter segments. Include in your counts the
stomata and epidural cells touching to edge of the field for the upper
right and the lower left quarters. Do not include in your counts the stomata
touching the edge of the field in the lower right and upper left quarter.
Each group should count 5 randomly selected field of views per leaf cast
and enter the counts on to the data tables.
- Complete the data form with all data, Latitude, Longitude,
rainfall this year from January to September 1, and the stomata and epidural
- Send the data, using the form in the Access Excellence
Online Research area. If you do not have access to the World Wide Web,
request the form on a floppy disk and send your data on the 3.5 inch floppy
disk to Steve Case at the address below.
- All collect data will be available in the Online Research
area. Students should begin reviewing and interpreting data. Data analysis
should be posted in the Online Research area.
At this point the students should be lead in a discussion
of environmental factors that may cause a variation in the number of stomata
on a leaf's surface. This discussion will lead to individual project ideas.
The following are some suggestions that may get students thinking.
- What is the normal variation found on a plant?
- The age of the tree and the number of stomata found on
- The side, compass direction, of the tree the leaf comes
from and the number of stomata found on the leaf.
- The direction of the slope the tree is growing on and
the number of stomata found on the leaf.
- The difference in stomata on leaves grown in a carbon
dioxide enriched environment to those grown under normal atmospheric conditions.
- Is there a clinal variation along a rainfall gradient.
- Study the variation in numbers of stomata that occur
between plants growing in similar habitats but using different photosynthetic
pathways, C3, C4, and CAM.
As the discussion proceeds the students should be thinking
of ways to measure, test, and/or simulate these environmental conditions.
Individual research projects should be sent in for posting on the Web.
These projects will increase our understanding of leaf stomata as bioindicators.
The original technique for stomata counting came from
"The American Biology Teacher", January 1990. "Is there
a Correlation Between Rainfall Amounts and the Number of Stomata in Cottonwood
Leaves". Neill, Robert L., David M. Neill, Bernard F. Frye.
Stomatal Index - The Botanical Review Vol. 40, January
- March 1974
Stomatal numbers are sensitive to increases in CO2 from
pre-industrial levels, F. I. Woodard, Nature June 1987 : 617-619.
Van Der Burgh, Johan , Jenk Visscher, David Dilcher, Wolfram
M. Kurschner. "Paleoatmospheric Signatures in Neogene Fossil Leaves."
Science, Vol. 260, June 18 1993, 1788-1790.
Garbutt, K., W. E. Williams, and F. A. Bazzaz (1990) "Analysis
of differential response of five annuals to elevated CO2 during growth.
" Ecology 71(3).1185-1194.
Bazzaz, F. A. "The Response of Natural Ecosystems
to the Rising Global CO2 Levels." Annual Review of Ecology Systematic,
1990, 21: 167-196.
Penuelas, Josep, Riser Matamala. (1990) "Changes
in N and S Leaf Content, Stomatal Density and Specific leaf Area of 14
Plant Species during the Last Three Centuries of CO2 Increase." Journal
of Experimental Botany 41.1119-1124.
Another of Steve's favorite classroom activities:
with a Microscope