Lesson 5 – Inferring Properties and Calibrations

This is the fifth lesson of the Observing Beyond our Senses Module. This lesson serves as the conceptual underpinning for understanding how light behavior can be used to measure concentrations with a spectrophotometer. Students should frame their observations around “particles” of light interacting with material in three observable ways: that light can pass through, bounce off, or be taken in by materials.


See the NGSS listed in the left-hand menu and below. When applicable, 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
  • Light can be reflected, absorbed, transmitted, & scattered during interaction with materials.
  • The type of material impacts the nature of the interaction. The color of light (wavelength) impacts the nature of the interaction.
  • Accuracy is the degree of closeness of a measured or calculated quantity to its actual value.
  • Proxy variables can be quantitatively related to inferred properties through calibration.
  • Measuring devices reference known standards.
  • Measuring devices with a predictable pattern can be calibrated.
  • Calibration curves can be used to determine unknown quantities.
  • An offset or a blank is commonly needed to interpret calibration data.
What Students Do
  • Students explore properties of light by observing a light source shining through a milk solution.
  • Students build a simple ‘photometer’.
  • Generate standard (calibration) curves using their photoresistors to infer milk fat concentration.
  • Determine an unknown concentration from their calibration curves.
  • Describe their methodology to determine the unknown concentration.
Aligned Washington State Standards
Washington Science Standards (Next Generation Science Standards)

Performance expectation(s): 

HS-LS2-1 Use mathematical and/or computational representations to support explanations of factors that affect carrying capacity of ecosystems at different scales.

HS-PS3-3 Design, build, and refine a device that works within given constraints to convert one form of energy into another form of energy.

HS-PS4-1 Use mathematical representations to support a claim regarding relationships among the frequency, wavelength, and speed of waves traveling in various media.

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.

HS-ETS1-1 Analyze a major global challenge to specify qualitative and quantitative criteria and constraints for solutions that account for societal needs and wants.

HS-ETS1-2 Design a solution to a complex real-world problem by breaking it down into smaller, more manageable problems that can be solved through engineering.

HS-ETS1-3 Evaluate a solution to a complex real-world problem based on prioritized criteria and trade-offs that account for a range of constraints, including cost, safety, reliability, and aesthetics, as well as possible social, cultural, and environmental impacts.

Two potential performance expectations that could be connected to this lesson with a slight deviation are HS-PS1-7: Use mathematical representations to support the claim that atoms, and therefore mass, are conserved during a chemical reaction, and HS-ESS2-5: Plan and conduct an investigation of the properties of water and its effects on Earth materials and surface processes.

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) (All HS)

Highlighted Crosscutting Concept(s)

SEP-1: Asking Questions and Defining Problems

SEP-3: Planning and Carrying Out Investigations and Interpreting Data

SEP-5: Using Mathematics and Computational Thinking

SEP-6: Constructing Explanations and Designing Solutions

SEP-8: Obtaining, evaluating and communicating information

PS1.B: Chemical reactions

PS3.A: Definitions of Energy

HS-PS3.B: Conservation of Energy and Energy Transfer

HS-PS3.C: Relationship Between Energy and Forces

PS3.D: Energy in Chemical Processes

HS-PS4.A: Wave Properties

PS3.D: Energy in chemical processes

ESS2.C: The Roles of Water in Earth’s Surface Processes

HS-ETS1.A: Defining and Delimiting an Engineering Problem

ETS1.B: Developing Possible Solutions

HS-ETS1.C: Optimizing the Design Solution

CCC-2: Cause and Effect

CCC-3: Scale Proportion and quantity

CCC-5: Energy and Matter

CCC-6: Structure and Function


This lesson serves as the conceptual underpinning for understanding how light behavior can be used to measure concentrations with a spectrophotometer. Students should frame their observations around “particles” of light interacting with material in three observable ways: that light can pass through, bounce off, or be taken in by materials. Light from an incandescent bulb shown through a Lucite box with increasing amounts of milk fat can be seen to cause transmission, scattering, and absorption respectively. The behavior of differing wavelengths of light can explain the red and blue shift observable on the long and short sides of the box at higher concentration-similar to sunsets reds or deep sea underwater blues. The introduction to the activity can be modified based on students previous experience with the nature of light. The ‘engage’ and ‘explore’ activities (below) assumed no previous background.

Materials list (for demonstration):

  • 2% Milk
  • Stir Rod of any type
  • Plastic box or glass tank
  • Graduated Beral pipet (eye dropper)
  • light source (incandescent bulb, laser pointer, flashlight masked to make a beam)



Questions to pose to students: How does light behave? We see objects in our surroundings because of some interaction with light. What are the plausible interactions when light interacts?
Student response: … Surfacing that the light can bounce off the object, it might pass through the object, not all the light goes through …

Push for several examples of each.
What happens to the light not going through?

Surfacing it may be scattered or absorbed …

Push for several examples of each.

Introduction of vocabulary: Reflection, Transmission, Scatter, Absorption
(Detail here guided by teacher need)


A classwide discussion using the a glass or Lucite tank filled with water set up as a demo. An incandescent light source should be on one side, a paper “screen” on the other.

  • Let’s consider the case of light entering water: Here is a container of water. What seems to be the case for this interaction between the light and the water?
    Transmission What evidence do you have for this interaction?
    we can see light reaching the far end of the box if we hold up a “screen” of paper on that end
    What could be changed to affect this interaction?
    Add something to the water; make the pathway longer …
  • At this point, the classwide conversation can continue as the teacher adds droplets of milk .

    Describe the impact of adding more “stuff” (milk”) to the light reaching the other side.
    –more milk, less light passing through, more light observed from sides of container, color of light at sides bluish, light at end ‘shifting’ redish

    What factors determine the amount of light passing through the box?
    -length of path, # of particles, size of particles

    If we consider the light source as the 100% amount, the light passing through to the other end is the % transmitted. What happens to the rest of the light?
    –It is scattered.
    How do you know that?
    –We see the light from the sides of the container.
    So the transmitted light and the scattered light should be equal to the light energy from the source?
    ………. Not necessarily, some light might be absorbed.

    What is some way that we might measure the light energy?
    …. solar cell responds to light intensity; if we measure output we could translate output energy to light intensity


(powerpoint | google slides)

Lead students through the explanatory powerpoint for Beers Law (ppt | slides). The powerpoint on light and milkfat interaction (slide 2) introduces Beers Law (slides 3 & 7) and describes the two types of interactions causing notable decreases in transmitted light intensity. The first is absorption such as that seen in sea water (slides 4-6) and the second is the scattering responsible for blue skies and red sunsets (slides 8-12). The distribution of fairly regularly sized milk fat globules responsible for the scattering of light observed by the students is described (slides 13-15). Milk fat globules are similarly sized to typical microbes, making them a good substitute to study how they scatter light. The final slide (16) introduces the idea of using the transmitted light as a proxy variable to detect changes in concentration.

**Teacher Background note. In research, it is common to choose the wavelength on the spectrophotometer to ensure that deceases in light transmission are caused by scattering phenomena rather than absorption since some microbes express colored pigments differently depending on environmental conditions.


In the next lesson, students will use milk fat concentration data to create a calibration curve.

Instructional Activities:

This lesson introduces students to making quantitative inferences from proxy variables via calibration. The concepts of standards, calibration curves, and offsets are introduced through a familiar device, the spring scale. Students then develop a calibration process using light properties. Using varying milk fat concentrations, the class will use a photoresistor apparatus to make a standard (calibration) curve which is then used to identify an unknown sample.  (This is how population density will be determined in lesson 6.)


The guiding question for this lesson is: How could we use the observed light properties to create an accurate measuring device? (slide 2)

1. Calibration & Accuracy for A Spring Scale

(PowerPoint | Google Slides)
  • How do we know our measuring devices are accurate? (slides 3-11)
    “How do we know our spring scales are accurate?” What does it mean for the scale to be accurate?
    Student answers may lead to comparison to a standard (the SI kilogram). In order to be sure the instrument is accurate, it must:

    • read zero (a “blank”, tare) as an offset.
    • have a set reading matching a standard (a fixed benchmark)
    • demonstrate a predictable pattern in between and beyond the standard benchmark (often linear) so that other benchmarks can be included.
  • How can we use a calibration curve? (slide 12)If the measuring device demonstrates a predictable pattern, we can develop a mathematical relationship between the observed proxy variable and the standard to which we are inferring a relationship. This “curve” can be used to determine unknown samples by measuring the proxy variables.  *Point out the term ‘curve’ may refer to a straight line.

2. Can we create a calibration curve using our observed properties of light?

  • What is needed to generate a calibration curve? (slide 13) Students should brainstorm in groups what would be needed to generate an accurate calibration curve for a photoresistor apparatus they will build. As their answers are discussed, a point by point comparison to the spring scale example could lead them to the realization that they need:
    • to determine the offset or “blank” value (a reading of the tank with the solvent—in our case water)
    • a standard for comparison (an accepted value for milk fat concentration that will be provided to them)
    • a predictable pattern between the milk concentration and the reading from the photoresistor
  • What is a standard for milk? A blank? (slides 14-16) Homogenization of milk results in relatively similarly sized milk fat globules dispersed throughout the milk. Using manufactured 2% milk, we can assume the density of milk fat globules can be used as a standard. These globules are similar in size to most microbes, making them convenient as a standard. Students should be made aware that an offset condition, the “blank”, should be determined for the solvent devoid of milk fat globules—water.
  • How would we create a calibration curve? (slide 17-18) From the properties of light activity, students should be able to predict that the light intensity reaching the photoresistor decreases when the milk fat density increases. They can be prompted to list the quantity actually being measured and what it is being inferred to represent (ie measure voltage, infer light intensity or measure drops or volume of milk and infer population density) to lead them to what quantitative values should actually be on the graph.
  • How will the calibration curve compare for similar devices? (slides 19-21) Students will be comparing their data to others in class. Error bars on their curve will indicate how well the devices are working.  Discussion of various instruments (such as Vernier probes) available would be helpful.

3. Building and Calibrating the Devices (Student Activity)

  • Materials list per group:
  • 5 ml of 2% milk
  • water
  • graduated Beral pipet (dropper, or micropipetor)
  • 5-8 test tubes of the same size
  • test tube rack
  • 9v battery
  • wire or alligator clips (see powerpoint directions)
  • photo-resistor
  • resistor (100-300 ohm)
  • multimeter
  • tape (electrical, masking)
  • LED (light emitting diode)

Building a photoresistor (photometer): Review the photometer (Google Slides | PowerPoint) as part of teacher advanced prep.  This power point, slides 1-12, may be viewed by students as directions or handouts made from the slides.  The students should collect and analyze data before discussing or seeing the rest of the slides.  If they have large error bars on their graphs, they should troubleshoot and improve their instrument.  The last slides show some suggestions for shielding ambient light (noise).

Before beginning, have students decide on how they will make dilutions and show them how to build the photoresistor (ppt. or demonstrate).

  • Recording the Data:  Students should graph their data in excel or LoggerPro to generate calibration curves.  Have students add error bars.  Sample Graphs for some differing devices can be found here Milk Fat Sample Calibration Data (Excel).
  • Teacher directions (Google Doc | Word Doc) for graphing in Excel, with error bars.  For students who have not worked with error bars, this worksheet (Google Doc | Word Doc) leads them to assess their importance and give them practice with sample data (Excel).
  • Determining an Unknown:  Once students have completed their calibration curves with error bars inserted to indicate an appropriate level of uncertainty, each group should be given the same unknown concentration to measure falling within the 0 to 10⁷ globs/L range that they have measured. An intermediate value, such as 250 uL of milk added to a liter of water, should be used. The milk fat concentration should be identified using their calibration curves. The class should share their results so students can evaluate their findings. The final instrumentation focused assessment for the students is the individual written report on the determination of the unknown concentration using this student assignment Identifying an Unknown (Google Doc | Word Doc).

You completed Instructional Activities. Please move to Assessment

Career Connection

Based on how much time you have available, choose a career-connected activity below. In each case, recap what your students just learned in the lesson to the activity.

A homework/

outside of class

B 5-10 minutes in class C half of class period (~25 minutes) D entire class period (~50 minutes)
Show students the StoryCorps website. Have students write 5 important tips for interviewers. Role-play as a person who invented the spectrometer you’re using. Have students conduct an interview with you and write a synopsis. We strongly suggest introducing the TOWN HALL & the ELEVATOR SPEECH projects (see Lesson 6), and allotting plenty of time in short blocks for supporting research and development and guidance for these. Invite a guest speaker and prepare students to interview the speaker in advance by reviewing StoryCorps 10 point document with tips on how to conduct a good interview.This experience hopefully will provoke some engaging writing prompts.

Potential Careers of the guest speaker:

  • Hydrologist
  • Geologist
  • State or local government environmental regulator
  • Environmentally-oriented lawyer or litigator


How will I know they know?

  • Students will provide evidence to support their assertions about the nature of the light & matter interactions during the class discussion.
  • Review the Students’ Calibration Worksheets, their Calibration Curves, and their values for the unknown concentration of milk.



Students could use their photoresistors and knowledge of halophiles to complete the salinity experiment found in the module Ecological Networks.

Students might use their photoresistors to measure the concentration of other substances such as tea.  See a lab activity here (from ‘Sciencebuddies.org’).

Students who do not visualize graphs well may benefit from additional or introductory work.   This activity requires 2 odd shaped bottles (obtain at vintage shops), a plastic champagne glass, and a plastic ‘party’ glass, ruler, graduated cylinder and container for water. It generally takes 80 minutes to complete and gives students an understanding of slope and y-intercept…and the importance of looking at axes labels.