Stomatal Density & CO2 Pollution

• Test hypotheses about the influence of habitat conditions on leaf stomatal density 

• Practice using Excel to calculate mean and SD and make  properly labeled column graphs 

• Perform t-tests to determine significant differences between two experimental groups and write an interpretation of results

• Write the Lab Title on a new page on the right-hand side of your notebook. *Remember to include the Lab Date!*

• Write the Background, Aim and your Hypothesis for the Stomatal Densities lab

• Draw Data Table 1 and Data Table 2 (See Procedures, Step 2 )

• Add an entry for this lab in your Table of Contents.

• Model Organism: Leaves from Rhododendron plants

• Experimental Questions: Does pollution/CO2  exposure in the habitat influence the stomatal density on Rhododendron plant leaves?

• Independent Variable: Plant habitat

  • • Control Group: Leaves from a woodland habitat     

    • Experimental Group: Leaves from an city habitat close to a busy road

• Dependent Variable: Stomatal density (stomatal count / mm2 )   

I hypothesize that  stomatal density in plants varies with exposure to environmental CO2 pollution, and predict that plants from busy street  habitats will have significantly ___higher / lower___ stomatal density compared with plants from wooded habitats. 

OR 

I hypothesize that the stomatal density does not significantly differ based on exposure to environmental CO2 pollution.

Stomatal Physiology

Land plants are autotrophs meaning that they produce their own for food. Plants make glucose by performing photosynthesis in their leaves. During photosynthesis, light energy and water are used to create high energy molecules (ATP and NADPH). Those energy molecules then power the reactions that convert CO2 into glucose sugars. Sunlight is absorbed by green pigments in plant leaves. Water is obtained from the ground or soil. But how do land plants acquire the  CO2 they need for photosynthesis? 

Plants acquire CO2 through small pores called stomata (singular = stoma). The stomatal openings are surrounded by two guard cells, which swell and shrink to open and close the pore. Oxygen produced by photosynthesis can also leave the leaf through the stomata. Plants experience a challenge in this process: when they open their stomata for gas exchange, water vapor from the inside of their leaves moves out into the environment, which puts the plant at risk of drying out.

The number or density of stomata on leaves allows the plant to exercise some control over environmental conditions. Given the role of stomata in plant leaves, how do you think stomatal densities differ between plants native to wet versus dry habitats? Sunny versus shady habitats? What about polluted versus not-polluted habitats?

Comparing stomatal densities between habitats

We can measure stomatal density on leaves by creating stomatal peels. This involves making an 'impression' of the stomata of a leaf by using clear nail polish. We can then attach the peel to a microscope slide and count the number of stomata present. If we use calculations to standardize the counts to stomatal density (the number of stomata per mm2 of leaf) we can then compare leaves from different habitats. 

To test our hypothesis about the influence of habitat CO2 on stomatal density, we will compare leaves collected from plants next to a busy street to those collected from a natural wooded area. We will make stomatal peels, perform counts, and calculate stomatal densities from replicate leaves in each habitat. We will then compare stomatal densities from the two habitats by creating a figure and performing a t-test.

1. Preparing Stomatal Peels & Slides

1. With your partner, select 5 leaves of each habitat type. Determine which partner will be responsible for counting stomata on each leaf type.

2. Using clear nail polish, paint a thick ‘coat’ of polish roughly 1.5cm2 on the ventral (under) surface of each leaf.

3.  Allow the clear nail polish to dry for 5 minutes. 

4.  While waiting for the nail polish to dry, set up your microscope and measure the field of view (see next section) and obtain 5 clean microscope slides.

5.  Once dry, take 5 pieces of packing tape and stick over each of the 5 nail polish ‘stomatal peels.’

6.  Gently peel back the tape to lift the nail polish  to reveal the ‘stomatal peels.’

7.  Carefully stick each stomatal peel onto a separate microscope slide. Trim with scissors where necessary.

2. Measuring Field of View and Counting Stomata on your Microscope

1. Set up your compound microscope and place a square grid microscope slide in the stage clips. 

2.  Focus on the grid slide using the 4X objective (40X magnification). Count the number of mm grid squares across your microscope field of view. This is your field of view diameter at 40X. Record is value  in Table 1 [A]. 

3. Perform the calculations Table 1 to determine the field of the diameter [B], radius [C], area [D] and multiplication factor [E] at 400X magnification. You will use the multiplication factor [E] later.

Now you are ready to count stomata!

4. Focus on your stomatal peel slide first using the 4X objective, then move to the 10X objective, and finally 40X objective (400X magnification). Check with your instructor that you are looking at stomata! 

5. Move into the center of your leaf impression to ensure your field of view does not include any damaged edges.

6. Once focused at 400X magnification, without moving the stage, count all the stomata in the field of view. 

7.  Record the stomatal count in Table 2. Convert to stomatal density (stomata per mm2) by multiplying the count by your multiplication factor [E].

8. Repeat counts and stomatal density calculations for your remaining slides. You will pool data with your partner and then the class to create graphs and test your hypothesis about differences in stomatal density between the two habitats.

Calculating Mean & SD in Excel

You will use Excel to calculate the Mean and Standard Deviation stomatal density for leaves from the two different habitats. 

Use formulas in Excel to calculate Mean and Standard Deviation separately for the two columns of data on each tab of the Excel file.

Column Graph of Mean ± SD

You will use Excel to create a column graph to display mean & SD stomatal density for leaves from the two different habitats. 

Use the mean and SD values calculated in the first tab of your Excel file to create a properly formatted column graph.

t-test 

You will use GraphPad to perform a t-test to determine if there was a significant difference in stomatal density between leaves from the two different habitats. Then you will write a results paragraph that describes the comparison and the statistical results.

Follow the t-test instructions for how to perform each t-test and what data to record in your notebook.