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Protocol for measuring stomatal densities - Guided Research Protocol

After you have gather background information and made preliminary observations it is time to formulate a hypothesis. A hypothesis is merely a tentative explanation proposed to account for the observed phenomena. You are speculating on how natural events will turn out, based on what you know. "Science is systematic in method because it seeks a system of prediction." That prediction is the hypothesis. Any hypothesis selected or formulated must be testable. Experiments generally test hypotheses by testing the validity of the predictions or conclusions derived from them. The primary purpose of designing scientific experiments is to test the proposed hypotheses.

It is important to know that hypotheses are never proven - they are either supported or not supported by the data from the experimental results. Borrowing from statistics, two types of hypotheses are used simultaneously: null (H0) and alternative (H1). H0 states that events will not change, not differ and H1 states that events will change, differ, from some baseline standard or control conditions. This change (dependent variable) predicted by H1 will be due to the occurrence of an experimentally controlled variable (independent variable).

Hypothesis for this Guided Research

H0: There is no measurable relationship between annunal rainfall between January 1st and September 1st and stomatal densities occuring on the leaves of cottonwood tree (Populus deltoides).
H1: There is a measurable relationship between annunal rainfall between January 1st and September 1st and stomatal densities occuring on the leaves of cottonwood tree (Populus deltoides).

The experiment to test these hypothesis uses the leaves from one species of trees. These 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 a microscope on 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.

  1. Students should divide into teams of two. 10 leaves from each of 10 trees should be collect should be collected from Eastern Cottonwood (Populus deltoides) trees. Data about each of the trees from which leaves are collected and should be recorded on the data form. Leaves should be collected from the south side of the tree, from approximately between shoulder height to 20 feet up from the ground. The leaves should be from the same position on the twig. Leaves should be labeled with a tree number and a leaf number (ie T1L1 would be Tree number 1, Leaf number 1). A small piece of masking tape on the point of the leaf works well.
  2. Each team of two students should look at two leaves. 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.
  3. Allow the fingernail polish to dry completely.
  4. Firmly press a short strip of clear (transparent, not frosted) Scotch tape over the dried nail polish on the lower epidermis. Carefully peel the tape with the patch of dried nail polish 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.

  5. Repeat this procedure for each of the collected leaves. Be sure to label each leaf and each slide carefully. Organization of your data is critical.
  6. Trace the leaves on a piece of acetate with 1mm grids mark on it and measure the leaf surface area.
  7. Place the leaves in a plant press. After drying, remove the leaves and store for possible future reference.
  8. 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.
  9. Complete the data form below with all data, tree species, rainfall this year from January to September 1, and the stomata and epidural cell counts. Enter Latitude and Longitude for the tree if it is more than one kilometer (.6 mile) from your school site.
  10. Submit the data, using the electronic form in the data submission area.

School Information:

School Name   
School Number   
Tree Species   
Latitude/Longitude   
Total Rainfall (January 1 - September 1)   

Stomata/Epidural Cell Counts:

Tree Number: Leaf Number:

Top Counts

Bottom Counts

 

Field 1

Field 2

Field 3

Field 4

Field 5

Field 1

Field 2

Field 3

Field 4

Field 5

Stomatal Pores                              
Epidural Cells                              
Research Method Discussions

This Research Method forum is for discussion of the research methods used in the global warming project. You may post questions, comments, concerns, and/or suggested improvements to any aspect of the research method(s) above.

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Missing required data.