Introduction to Phytoplankton Spectrophotometer Lab
The Invisible Forest, of the vast open oceans, is the foundation for half of Earth’s bioproductivity and atmospheric oxygen. Microscopic and living beneath the water’s surface, this realm of life is just now being recognized for its importance to Earth’s biosphere and atmosphere. In Lesson 2 and 3 students were introduced to scientists studying the ocean’s changing primary productivity world-wide. They do this through water sampling and satellite imagery using principles of chlorophyll fluorescence. Here, students have an opportunity to observe phytoplankton and investigate chlorophyll fluorescence with a spectrophotometer in greater depth. Guided activities and labs provide background to understand visible light, UV light, fluorescence, photosystems, and spectrophotometer function and use. Ultimately, students are led to understand how information from satellite imagery and other oceanographic instrumentation allows us to study Earth’s Invisible Forest. In Lesson 4students will apply their understanding of fluorescence and spectrophotometry as a tool for gathering data in order to learn more about the diversity of phytoplankton in transects and layers of the ocean.
Overview of Instructional Activities:
||What students learn:
||What students do:
|Phytoplankton Spectrophotometer Lab Part 1A*: Observing Plankton with Microscope (35 min) —Microscope observations with white and UV light.Making observations and Asking questions]
Part 1B: Preparing Plankton for Chlorophyll Extraction and Spectrometer (20 min + 24 hr. freezing)—Filtering plankton procedure. *Read Phytoplankton Spectrophotometer Lab teacher guidede for advanced preparations.
|– There’s an invisible world of life in a drop of water.
– UV light gives unexpected additional information about plankton and chlorophyll.
– Laboratory filtration technique.
Note: Prepare or gather phytoplankton cultures to use for the Labs several weeks prior.
|– Collect or culture fresh or seawater plankton, or have access to available plankton
– Observe plankton cultures and phytoplankton chlorophyll fluorescence in microscope with visible and near UV light.
– Extract planktonic chlorophyll with filters and freeze in alcohol (24 hours) to make plankton extract.
|Visible Light Spectrum and Fluorescence_worksheet Guided Activity A (30 min) (questions 1 – 5) introduces wavelengths and light spectrum. Guided Activity B (30 min) explores Fluorescence (questions 6 – 13)with a Demo and/or Fluorescence slide show and Energy capture complex in cell_diagram —Observing phenomena and Developing and using models (See Visible Light and Fluorescence Teacher guide for more details.)[Optional] Lab: Introduction to Spectrophotometer (extension)(+50–90 min)—Using instrumentation Analyzing and interpreting data.]
||– Learn which light wavelengths correspond to specific colors used in photosynthesis and are visible with a spectrophotometer.
– Learn spectrophotometer function, calibrating blanks, absorbance of food dyes, and fluorescence of quinine.
|– Color a model spectrum matching light color to wavelength and energy content. – Place visible, UV, and IR light in relation to visible spectrum and rest of electromagnetic spectrum.
– Use diffraction gratings to observe visible light spectrum and relate wavelengths to color
– Develop an operational definition of fluorescence and model of electron movement in fluorescent materials. Apply to model of chlorophyll in photosystem II.
– Observe fluorescence in quinine, minerals, ultrabrite papers, & chlorophyll
|Part 2: Phytoplankton Spectrophotometer Lab
(50 min)— Measuring phytoplankton chlorophyll absorbance and fluorescence with spectrophotometer
[Planning and carrying out investigations. Analyzing and interpreting data.]
|– Lab procedures for using spectrophotometer.
– Interpreting absorbance and fluorescence results from spectrophotometer
|– Measure phytoplankton chlorophyll absorbance and fluorescence with spectrophotometer.
|Part 3: Spectrophotometer Lab Analysis and Conclusions_ worksheet (30-50 min) —[Analyzing and interpreting data. Constructing explanations. Obtaining, evaluating, and communicating information]
||– Absorbance and fluorescence are used in a variety of scientific instrumentation to understand ocean photosynthesis and measure phytoplankton.
||– Compare absorbance and fluorescence data to Sally ‘Penny’ Chisholm’s data on Prochlorococcusand apply understanding to any photosynthesis withchlorophyll aand remote sensing imagery.
Phytoplankton Spectrophotometer Lab and Fluorescence [Simplified Lesson Plan (Option) 4 x 50 min classes]
Prerequisites*: Students know and understand spectrophotometer function and use for absorption and fluorescence. Students know visible light and UV spectrum and how color, wavelength, and energy are related. Students know the relationship between chlorophyll fluorescence and electron capture in photosystems II & I. (*if students are not familiar with these concepts use Visible Light Spectrum and Fluorescence (Activity A) and (Activity B) to introduce the necessary background)
Go to Teacher Lesson Plan/Phytoplankton Spec and Fluorescence Lab* for detailed directions on how to use Lab Materials . And planning examples for short and extended lesson sequences. *Gather or prepare plankton cultures several weeks before class. (50 min). Print and copy 1 per group: Phytoplankton Spectrophotometer Lab_student procedure, and 1 per student: Spectrophotometer Lab Analysis and Conclusions_ worksheet Visible Light Spectrum and Fluorescence_student worksheet, Prepare to show: Fluorescence slide show, and Energy capture complex in cell_diagram
Day 1: -Hand out Phytoplankton Spectrophotometer Lab Part 1A (35 min) Begin lesson sequence with observing plankton with a microscope* in white and UV light (*can be included as part of Lesson 2). Obtain a fresh or seawater plankton sample. Make a wet-mount slide of the plankton. Examine plankton with a light microscope at 40x and 100x. Look for diatoms and other algae. Look for green chloroplasts within the cells. Generate wonder and questions with seeing the phenomenon of microscopic ecosystems and chlorophyll fluorescence. Next, in Part 1B of this Lab students prepare phytoplankton culture for measuring fluorescence in a spectrophotometer — which they will use in Part 2.
Day 2: For students who need further background on visible light wavelengths and how fluorescence works in a living organism use the following guided activities in Activity A: Visible Light Spectrum (30 min) and Activity B: Fluorescence (30 min) –Step 1: Hand out the Visible Light Spectrum and Fluorescence_student worksheet. Remind students the instruments they learned about in Lesson 3, make measurements using principles of the interaction of light and matter as well the spectrophotometers they are going to use in the following days’ Lab. The Lab will provide practical experience to understand the science of measuring global primary productivity (and thus breathable oxygen production).
Step 2: Begin with reviewing the visible light spectrum (Activity A: questions 1-5). First observe white light using diffraction gratings and have them color in a spectrum.
Step 3: Then use the worksheet to guide them to match wavelengths in nm with the qualitative (observations) of visible light, matching color/wavelength with energy
Step 4: Next, for (Activity B: questions 6-13) focus in on key red and blue wavelengths of photosynthesis and the instrumentation used to study light quantitatively. Show students a selection of fluorescent materials (quinine water, minerals, ultrabrite papers, & chlorophyll) to view under violet and near violet/long wave UV light. Or show them Fluorescence slide show,
Step 5: Guide students to develop a chlorophyll model showing absorption of light, energy capture in photosystem, or re-emission of light as visible fluorescence. The Energy capture complex in cell_diagram can be used to trace the system as you walk them through it. These concepts are applied to understanding chlorophyll molecules in photosystems. Adapt available material including theFluorescence slide show, and Teacher slideshow script , Demo of fluorescence and Energy capture complex in cell_diagram from this lesson to suit one, fifty minute class. Refer to Visible Light and Fluorescence Teacher guide for additional background for teaching the guided activity.)
Day 3: –Phytoplankton Spectrophotometer Lab Part 2 (50 min)
Step 1: Remind students that chlorophyll, and thus primary production in ocean ecosystems, is measured with spectrophotometers directly with water samples and remote imaging (as introduced in Lesson 3)The ability to study light energy and its interactions with matter lets them do science with instruments similar to those used in oceanography. In this Lab they are measuring planktonic chlorophyll for absorption and fluorescence, to deepen understanding of photosynthesis, and the instruments used to measure this. Another instrument, the flow cytometer (also in Lesson 3) uses fluorescence and light diffraction to count, measure, and identify single celled phytoplankton, including Prochlorococcus.
Step 2: Using the phytoplankton prepared earlier in Part 1B of this Lab—measure phytoplankton or chlorophyll extract for light absorbance and fluorescence with a spectrophotometer. (If students are unfamiliar with Spectrophotometers use the Optional extension Lab* for practice, before they begin to measure phytoplankton fluorescence.)
Step 3: Students use the data they collect to develop a deeper learning model for understanding chlorophyll’s ability to capture light energy converting it into life’s chemical energy.
Day 4: – Phytoplankton Spectrophotometer Lab Part 3 ( < 50 min)</b This final step will give students direct experience with data and instrumentation used to measure the Invisible Forest / oceanic primary production. Use Spectrophotometer Lab Analysis and Conclusions_ work sheet to analyze their chlorophyll spectrophotometer results. Students will compare their results to Dr. Chisholm’s lab results for Prochlorococcus and go to NASA Ocean Color website to learn how satellites use light and fluorescence to measure global chlorophyll. Review the students’ analysis worksheet responses using Spectrophotometer Lab Analysis and Conclusions_Teacher Key.
OPTIONAL ACTIVITIES (Extensions):
*Optional extension Lab (+50 min): Introduction to Spectrophotometer _Teacher with Introduction to Spectrophotometer Lab_Student procedure and Introduction to Spectrophotometer Lab_student worksheet
This is an intro lab using water, food dye, and quinine to teach students spectrophotometer usage and design/function of the instrument.