Woodrow Wilson Environmental Science Institute - Rutgers 1998

Project:        Phytoplankton & Ultraviolet Light  Mentors:      Dr. Oscar Schofield and Dr. Yu Alice Gao   Participants: Madelyn Asperas (NY), Andre Clayman (NC), Zoraida Colon (NY), Valerie Baker (CA), Alex Bodha (NY), Shelly Crow (TX), David Duane (MA), and Dianne Mollica (VA).

This page explains our inquiry based research project which tested the effect of ultraviolet light on the growth rate of phytoplankton.  The navigation bar below lets you move easily within this page.  Visit our Class Activities web page to see how you could make this experiment and related activities come alive in your classroom.

    This experiment is designed to answer the question:  What effect does ultraviolet light  have on a living cell?  The living cells that form the basis for this experiment are types of  green algae and brown algae.  Ultraviolet light destroys the DNA of cells and is emitted by the sun along with white (visible) light. However, ultraviolet light is absorbed by the ozone layer at an altitude of 15 to 40 kilometers above the earth's surface, so this experiment serves as a model that could mimic a futuristic world of depleted atmospheric ozone (O3).

    Ultraviolet light is undetectable to humans, though insects such as bees are sensitive to light within the ultraviolet spectrum which ranges in wavelength from 280 nanometers to 400 nanometers.  The lower wavelengths of the ultraviolet spectrum are high energy waves that are referred to as ultraviolet B (UV B) rays.  Ultraviolet A (UV A) rays fall at the longer end of this spectrum and contain less energy.

    This experiment exposes green algae and brown algae cultures to varying levels of ultraviolet (UV) light and then measures their growth rate over time.  The species  used for this experiment are Tetraselmis sp, a green algae, and  Isochrysis sp, a brown algae.  Both types of algae are organisms common to the phytoplankton which circulate throughout the world's oceans and belong to the Kingdom Protoctista.   A further variable for this experiment investigates whether pre-exposure to varying levels of incandescent light prior to beginning the experiment will condition the cultures to respond differently when exposed to  visible or ultraviolet light.

    The group hypothesizes that exposure to ultraviolet light, especially ultraviolet B rays, will cause the growth rate of both species of algae to decrease and that within each species, cultures pre-exposed to lower levels of incandescent light will have higher growth rates than than those exposed to higher light levels.

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Experimental Design:

IV: Exposure to Type of Light
 
 

DV: Growth rate of the following cultures

• Tetraselmis - high light pre-exposure
• Tetraselmis - low light pre-exposure
• Isochrysis - high light pre-exposure
• Isochrysis - low light pre-exposure

Click here to see the digital photos of our lab activities.
Part I: Prepare cultures.

1. Inoculate two 2000 mL flasks full of filtered sea water with Isochrysis sp, and two 2000 mL filtered sea water flasks with Tetraselmis sp.
2. Pre-expose one flask of Isochrysis sp. and one flask of Tetraselmis sp. to high levels of incandescent light for 24 hours.  Pre-expose the other two flasks to low levels of incandescent light.
3. Prepare culture samples by mixing 600 mL of the Isochrysis -high light pre-exposure sample with 1400 mL of F-2 sea water media.  Repeat with the Isochrysis - low light pre-exposure sample; the Tetraselmis -high light pre-exposure; and Tetraselmis -low light pre-exposure.
4. Pour 500 mL the Isochrysis - high light culture into a  Whirl Pack Thio (plastic) bag labeled with the species, pre-exposure level, and type of light to which it will be exposed. Example: "Isochrysis - high/ visible, UV A, & UV B"
5. Repeat this step for all species and pre-exposures, and run two trials (500 mL per baggy) for each level.

For this experiment, quantify the amount of algae two ways.

A. Conduct a cell count using a hemocytometer.

Click here for more information about conducting cell counts.

1. Fill a hemocytometer (microscope slide with a grid) with a culture of Isochrysis sp low light pre-exposure using a Pasteur pipette.  The hemocytometer is divided into nine large squares, each 1 mm on a side.
2. Count the number of cells of Isochrysis sp. in four sets of the nine squares.
3. Calculate the average of the four squares.
4. The average is the number of cells per 10 E-4 mL (# / .0001 mL).
5. Repeat cell counts for the Isochrysis high-light pre-exposure, the Tetraselmis sp low-light pre-exposure, and the Tetraselmis-high light pre-exposure.

Click here for more information about using the spectrophotometer.

1. Give the spectrophotometer a 20 minute warm-up time after turning it on.
2. Pour 4-5 mL of the culture (example: Isochrysis low light pre-exposure) into a sample cuvette (small test tube).
3. Zero the spectrophotometer with a distilled water blank cuvette at 664 nm.
4. Measure the absorbence of the culture at 664 nm.
5. When measuring absorbence of Isochrysis (a brown algae),  measure and record the absorbence at the following wavelengths:  664 nm and 630 nm.  Remember to re-zero the instrument with the distilled water blank cuvette  each time you change wavelengths.
6. When measuring absorbence of Tetraselmis (a green algae), measure and record the absorbence at the following wavelengths:  664 nm and 647 nm.  Remember to re-zero with the distilled water blank cuvette the instrument each time you change wavelengths.
7. Calculate the absorbence of chlorophyll a using the following equations:

Isochrysis (brown algae) absorbence:  chlorophyll a = (11.47 x absorbence 664 nm) - (.04 x absorbency 630 nm)

Tetraselmis (green algae) absorbence: chlorophyll a = (11.93 x absorbence 664 nm) - (1.93 x absorbency 647 nm)

1. Set-up three opaque boxes without a lid in a circulating cool water bath.  Place in a sunny spot outside.
2. Place the culture bags labeled "visible light" in one box and cover with filtered glass which is transparent only to visible light but blocks UV A and UV B rays.
3. Place the culture bags labeled " visible and UV A light" in another box, and cover with filtered glass and a strip of mylar.  Mylar blocks UV B Rays.
4. Place the culture bags labeled "visible, UV A, and UV B light" in the third box, and cover with unfiltered glass.
5. Angle the glass for each box so that it is vented to prevent condensation build up.
6. Wait 24 hours before conducting cell counts and measuring chorophyll absorbency.

1. After 24 hours, conduct a cell count of each culture using a hemocytometer, and measure and calculate absorbency of chlorophyll a using a spectrophotometer.  Thereafter, take daily measurements each day for the following two weeks.
2. Calculate the growth rate for both quantification methods of each culture using the following equation:

growth rate = natural log (amount of cells / initial amount of cells) / time

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