Lesson 3.5: Phytoplankton Spectrophotometer Labs

This is the first lesson of the Observing Beyond our Sense Module. In this lesson, students will be introduced to saline environments and microbial halophiles. Environments of differing salinities form on earth. Human activities impact saline environments by altering the salinity and/or introducing pollution. Extremophilic organisms such as Halobacterium salinarum can live in high salinity environments.


Courses: Marine Science, Oceanography, Integrated Science, STEM. This lesson may also fit within these courses: Biology, Honors Biology, Chemistry, Earth Science, Life Science, Physical Science, Physics, AP Chemistry, AP Physics, AP Biology, AP Environmental Science College Program in the High School.

Unit: Photosynthesis, Ecology, Biogeochemical Cycling, Energy, Photosynthesis, Phytoplankton, STEM application, skills building


See the NGSS listed as buttons in the left-hand menu and in the chart below. The buttons on the left are grouped to show the integrated three-dimensional nature of our lessons and modules. When applicable, if NGSS are addressed outside of bundles, they are listed separately. Connections to 21st Century Learning Skills and other published standards are also included in the chart below. In addition, for this lesson, here is a breakdown of:

What Students Learn:

  • A sense of size and scale, variety, and structure of organisms in the Invisible Forest
  • UV light reveals chlorophyll fluorescence in phytoplankton
  • Electrons in atoms and molecules can absorb light energy and re-emit light in fluorescence.  In chlorophyll, light excites an electron which either fluoresces or is chemically captured to do work in photosynthesis
  • To use spectrophotometer to study phytoplanktonic chlorophyll light absorption and fluorescence
  • Student phytoplankton results are comparable to published scientific data for Prochlorococcus. All oxygenic photosynthesizers with chlorophyll (a) will absorb and fluoresce light in the same way as seen in lab
  • Chlorophyll fluorescence is used to measure water sample contents, cell counting in flow cytometers, and satellite imaging to study the Invisible Forest/global photosynthesis


What Students Do:

  • Microscope observations of phytoplankton in visible light, and its fluorescence under UV light
  • Observe examples of fluorescence and apply electron theory to model chlorophyll’s ability to chemically capture light energy for life
  • Prepare phytoplankton for spectrophotometer.  Can use as living cells in water or use a simple  freezing method of extracting chlorophyll with alcohol.
  • Use spectrophotometer to measure wavelengths of planktonic chlorophyll  light absorbance and fluorescence
  • Compare results to published data for Prochlorococcus and apply to all oxygenic photosynthesizers
  • Apply concepts of light and chlorophyll to understanding scientific instrumentation and measurements of oceanic ecosystem, The Invisible Forest
  • Extension:  Guided activity to observe visible light with diffraction gratings.  Create a colored scale of visible and near visible wavelengths for their notes, linking color, wavelength, and energy
  • Extension:  Guided lab:  Learn how to use spectrophotometer: blank calibration, absorption with dyes, and quinine fluorescence
Aligned Washington State Standards
Washington Science Standards (Next Generation Science Standards)

Performance expectation(s): HS-LS1-5 Use a model to illustrate how photosynthesis transforms light energy into stored chemical energy. HS-PS-1 – Matter and its interactions: HS-PS1-4 Develop a model to illustrate that the release or absorption of energy from a chemical reaction system depends on the changes in total bond energy. HS-PS4 – Waves and Their Applications in Technologies for Information Transfer: HS-PS4-5.Communicate technical information about how some technological devices use the principles of wave behavior and wave interactions with matter to transmit and capture information and energy.

The bundle of performance expectations above focuses on the following elements from the K-12 Science Education Framework:

Highlighted Science and Engineering Practice(s)

Highlighted Disciplinary Core Idea(s)

Highlighted Crosscutting Concept(s)

SEP-1: Asking Questions

SEP-2: Developing and Using models

SEP-4: Analyzing and interpreting data

SEP-5: Using mathematics and computational thinking

SEP-6: Constructing explanations

SEP-8: Obtaining, evaluating, and communicating information

LS1.C Organization for Matter and Energy Flow in Organisms

PS1.A Structure and Properties of Matter

PS1.B Chemical reactions

PS2.B Types of Interactions

PS3.D Energy in chemical processes

PS3.A Definitions of Energy

PS3.DEnergy in Chemical Processes

PS4.B Electromagnetic Radiation

PS4.C Information Technologies and Instrumentation

CCC-4: Systems and System models

CCC-5: Energy and Matter

Connections to Nature of Science: Science assumes the universe is a vast single system in which basic laws are constant.

Common Core: M2, M4 (potentially since there are specific mathematics practices in interpretation and analysis of data) and E2, E5, E6

CTE – #2, #7, #8,

21st Century Skills – Developing Information Media and Technology skills toward Environmental Literacy


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: 

Lesson 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.


Instructional Activities: 

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)


Before Class:

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 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.



Optional Extension Lab: 

Introduction to Spectrophotometer>_Teacher

Introduction to Spectrophotometer Lab_Student procedure 

Introduction to Spectrophotometer Lab_student worksheet



College professor’s take on light, chlorophyll, and photosystem. Nice explanation. http://plantphys.info/plant_physiology/light.shtml

Background on NASA Ocean Color satellite bandwidth and how chlorophyll is measured.  https://oceancolor.gsfc.nasa.gov/SeaWiFS/TEACHERS/sanctuary_3.html