Introduction: Homemade Jenga Block Spectrophotometer for Algae Experiments
Algae are photosynthetic protists and, as such, are critical organisms in aquatic food chains. During spring and summer months, however, these and other microorganisms can multiply and overwhelm natural water resources, resulting in oxygen depletion and the production of toxic substances. Understanding the rate at which these organisms grow can be useful in protecting water resources as well as developing technologies that harness their power. Additionally, understanding the rate at which these organisms are deactivated can be useful in water and wastewater treatment. In this investigation, I will attempt to build a low-cost spectrophotometer to analyze the decay rates of organisms exposed to chlorine bleach in water sampled from Park Creek in Horsham, Pennsylvania. A sample of creek water collected from the site will be fertilized with a nutrient mixture and left in sunlight to promote algal growth. The homemade spectrophotometer will allow light at discrete wavelengths to pass through a vial of the sample before being detected by a photoresistor connected to an Arduino circuit. As the density of organisms in the sample increases, the amount of light absorbed by the sample is expected to increase. This exercise will emphasize concepts in electronics, optics, biology, ecology, and mathematics.
I have developed the idea for my spectrophotometer from the Instructable “Student Spectrophotometer” by Satchelfrost and the paper “A Low-Cost Quantitative Absorption Spectrophotometer” by Daniel R. Albert, Michael A. Todt, and H. Floyd Davis.
Step 1: Create Your Light Path Frame.
The first step in this Instructable is to create a light path frame from six Jenga blocks and tape. The light path frame will be used to position and support the light source, magnification device, and CD diffraction grating. Create two long strips by taping three Jenga blocks in a line as shown in the first image. Tape these strips together as shown in the second photo.
Step 2: Create a Base for Your Magnification Device and Attach It to the Light Path Frame.
The magnification device will be affixed to the light path frame and concentrate the light emitted by the LED before diffracting off of the CD. Tape together two Jenga blocks such that the middle of one block is at a right angle to the end of another block as shown in the first image. Attach the magnification device to this base using tape as shown in the third image. I used a small, inexpensive magnifying glass that I have had for several years. After attaching the magnification device to its base, I taped the magnification device to the light path frame. I positioned my magnification device 13.5 cm away from the edge of the light path frame, but you may need to fix your device at a different position depending on the magnifying glass's focal length.
Step 3: Create Your Light Source.
To limit the amount of non-concentrated light that can reach the CD diffraction grating and photoresistor, I used electrical tape to fix a white LED bulb inside a black pen cap that had a small hole in the top. The first image shows the LED, the second image shows the taped LED-pen cap. I used small pieces of electrical tape to prevent light from shining from the back of the LED where the anode and cathode wires are.
After creating the LED-pen cap, I attached the LED to a 220-ohm resistor and power source. I wired the LED to an Arduino Uno microcontroller's 5V and ground connections, but any external DC power source could be used. The resistor is important to prevent the LED light from burning out.
Step 4: Secure the Light Source to the Light Path Frame.
Tape another Jenga block near the end of the light path frame to provide a platform for the light source. In my set-up, the Jenga block supporting the light source was positioned approximately 4 cm from the edge of the light path frame. As shown in the second image, the correct placement of the light source is such that the light beam focuses through the magnification device at the opposite end of the light path frame where the CD diffraction grating will be.
Step 5: Place the Light Path Frame, Magnification Device, and Light Source in the File Box Casing.
Use a file box or another sealable container with opaque sides as a casing to hold each of the components of the spectrophotometer. As shown in the figure, I used tape to secure the light path frame, magnification device, and light source in the file box casing. I used one Jenga block to space the light path frame approximately 2.5 cm away from the edge of the file box's inside wall (the Jenga block was solely used for spacing and was later removed).
Step 6: Cut and Position the CD Diffraction Grating.
Use a hobby knife or scissors to cut a CD into a square with a reflective face and sides approximately 2.5 cm long. Use tape to attach the CD to the Jenga block. Play with the positioning of the Jenga block and CD diffraction grating to position it such that it projects a rainbow on the opposite wall of the file box casing when light from the LED source hits it. The attached images show how I positioned these components. It is important that the projected rainbow is relatively level as shown in the last picture. A ruler and pencil sketch on the inside of the file box wall may help in determining when the projection is level.
Step 7: Create the Sample Holder.
Print the attached document, and tape or glue the paper onto a piece of cardboard. Use a pair of scissors or a hobby knife to cut the cardboard into a cross shape. Score the cardboard along the printed lines at the center of the cross. Additionally, cut small slits at equal heights in the middle of two arms of the cardboard cross as shown; these slits will allow for discrete wavelengths of light to pass through the sample to the photoresistor. I used tape to help make the cardboard sturdier. Fold the cardboard along the scores and tape it so that a rectangular sample holder is formed. The sample holder should fit tightly around a glass test tube.
Step 8: Create and Attach a Base for the Sample Holder.
Tape together three Jenga blocks and attach the assembly to the sample holder as shown. Make sure the attachment is strong enough that the cardboard sample holder does not separate from the Jenga block base when the test tube is pulled out of the sample holder.
Step 9: Add the Photoresistor to the Sample Holder.
Photoresistors are photoconductive and decrease the amount of resistance they provide as light intensity increases. I taped the photoresistor into a small, wooden housing, but the housing is not necessary. Tape the back photoresistor such that its sensing face is positioned directly against the slit you cut in the sample holder. Try to position the photoresistor so as much light as possible hits it after passing through the sample and the sample holder's slits.
Step 10: Wire the Photoresistor.
To wire the photoresistor in the Arduino circuit, I first cut and stripped the wires of an old USB printer cable. I taped three blocks together as shown, and then attached the stripped wires to this base. Using two butt splices, I connected the USB printer cable wires to the terminals of the photoresistor and taped the bases together to form one unit (as shown in the fourth image). Any long wires can be used in place of the printer cable's wires.
Connect one wire emanating from the photoresistor to the Arduino's 5V power output. Connect the other wire from the photoresistor to a wire leading to one of the Arduino's analog in ports. Then, add a 10 kilo-ohm resistor in parallel and connect the resistor to the Arduino's ground connection. The last figure conceptually shows how these connections might be made (credit to circuit.io).
Step 11: Connect All Components to the Arduino.
Connect your computer to the Arduino and upload the attached code to it. Once you have downloaded the code, you can adjust it to fit your needs and preferences. Currently, the Arduino takes 125 measurements each time it is run (it also averages these measurements at the end), and its analog in signal leads to A2. At the top of the code, you can change the name of your sample and the sample date. To view the results, press the serial monitor button at the top right of the Arduino desktop interface.
Although it is a bit messy, you can see how I ended up connecting each component of the Arduino circuit. I used two breadboards, but you could easily do with just one. Additionally, my LED light source is connected to the Arduino, but you may use a different power supply for it if you would prefer.
Step 12: Place Your Sample Holder in the File Box Casing.
The final step in creating your homemade spectrophotometer is to place the sample holder in the file box casing. I cut a small slit in the file box to pass the wires from the photoresistor through. I treated this last step as more of an art than a science, as the prior placement of each component of the system will affect the positioning of the sample holder in the file box casing. Position the sample holder such that you are able to align the slit in the sample holder with an individual color of light. For example, you may position the Arduino so that orange light and green light project onto either side of the slit while only yellow light passes through the slit to the photoresistor. Once you have found a location where only one color fo light passes through the slit in the sample holder, move the sample holder laterally to identify the corresponding locations for each other color (remember, ROYGBV). Use a pencil to draw straight lines along the bottom of the file box casing to mark the locations where only one color of light is able to reach the photoresistor. I taped down two Jenga blocks in front of and behind the sample holder to help make sure I did not deviate from these markings when taking readings.
Step 13: Test Your Homemade Spectrophotometer - Create a Spectrum!
I ran several tests with my homemade spectrophotometer. As an environmental engineer, I am interested in water quality and took water samples from a small stream by my house. When taking samples, it is important that you are using a clean container and that you stand behind the container while sampling. Standing behind the sample (i.e., downstream of the collection point) helps prevent contamination of your sample and reduces the degree by your activity in the stream affects the sample. In one sample (Sample A), I added a small amount of Miracle-Gro (the amount appropriate for indoor plants, given my volume of sample), and in the other sample I added nothing (Sample B). I left these samples sit in a well-lit room without their lids to allow for photosynthesis (keeping the lids off allowed for gas exchange). As you can see, in the pictures, the sample that was supplemented with Miracle-Gro became saturated with green platonic algae, while the sample without Miracle-Gro did not experience any significant growth after about 15 days. After it was saturated with algae, I diluted some of Sample A in 50 mL conical tubes and left them in the same well-lit room without their lids. Approximately 5 days later, there were already noticeable differences in their color, indicating algal growth. Note that one of the four dilutions was unfortunately lost in the process.
There are various types of algae species that grow in polluted freshwaters. I took photos of the algae using a microscope and believe they are either chlorococcum or chlorella. At least one other species of algae seems to also be present. Please let me know if you are able to ID these species!
After growing the algae in Sample A, I took a small sample of it and added it to the test tube in the homemade spectrophotometer. I recorded the Arduino's outputs for each color of light and associated each output with the average wavelength of each color range. That is:
Red Light = 685 nm
Orange Light = 605 nm
Yellow Light = 580 nm
Green Light = 532.5 nm
Blue Light = 472.5 nm
Violet Light = 415 nm
I also recorded the Arduino's outputs for each color of light when a sample of Deer Park water was placed in the sample holder.
Using Beer's Law, I calculated the absorbance value for each measurement by taking the base-10 logarithm of the quotient of the Deep Park water absorbance divided by the Sample A absorbance. I shifted the absorbance values so that the lowest value's absorbance was zero, and plotted the results. You can compare these results to the absorbance spectrum of common pigments (Sahoo, D., & Seckbach, J. (2015). The Algae World. Cellular Origin, Life in Extreme Habitats and Astrobiology.) to try to guess the types of pigments contained in the algae sample.
Step 14: Test Your Homemade Spectrophotometer - Disinfection Experiment!
With your homemade spectrophotometer you can perform a variety of different activities. Here, I conducted an experiment to see how the algae decay when exposed to different concentrations of bleach. I used a product with a sodium hypochlorite (i.e., bleach) concentration of 2.40%. I began by adding 50 mL of Sample A to 50 mL conical tubes. I then added different amounts of the bleach solution to the samples and took measurements using the spectrophotometer. Adding 4 mL and 2 mL of the bleach solution to the samples caused the samples to turn clear almost immediately, indicating almost immediate disinfection and deactivation of the algae. Adding only 1 mL and 0.5 mL (approximated by 15 drops from a pipette) of the bleach solution to the samples, allowed enough time to take measurements using the homemade spectrophotometer and model decay as a function of time. Before doing so, I had used the procedure in the last step to construct a spectrum for the bleach solution and determined that the solution's wavelength at red light was low enough that there would be little interference with approximating algal deactivation using absorbance at the wavelengths of red light. At red light, the background reading from the Arduino was 535 [-]. Taking several measurements and applying Beer's Law allowed me to construct the two curves shown. Note that the absorbance values were shifted so that the lowest absorbed value is 0.
If a hemocytometer is available, future experiments could be used to develop a linear regression that relates absorbance to cell concentration in Sample A. This relationship could then be used in the Watson-Crick equation to determine the CT-value for deactivation of algae using bleach.
Step 15: Key Takeaways
Through this project, I grew my knowledge of principles fundamental to environmental biology and ecology. This experiment allowed me to further develop my understanding of the growth and decay kinetics of photoautotrophs in aquatic environments. Additionally, I practiced techniques in environmental sampling and analysis while learning more about the mechanisms that allow tools like spectrophotometers to work. While analyzing samples under the microscope, I learned more about organisms’ microenvironments and become familiar with the physical structures of individual species.
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