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Tube 1 Tube 2 Tube 3 Tube 4 Chlorella/Euglena 2 mL 2 mL 2 mL 2 mL Spring Water 1 mL 1 mL 1 mL 1 mL (Name and Concentration of Chemical stock solution used___10%_______) __mL __mL __mL __mL Spring Water __mL __mL __mL __mL Total Volume 5 mL 5 mL 5 mL 5 mL [X] Chemical Final Concentration of Chemical in Test Tube TO CALCULATE DILUTIONS OF CHEMICALS USE THIS FORMULA: Number of mLs of Stock Solution [Concentration Final Concentration of Total Volume of Dilution X of Stock Solution] = Chemical in Test Tube For example: HOW WOULD YOU PREPARE A 2% NACL SOLUTION FROM A 10% NACL STOCK SOLUTION? STEP 1: WE KNOW: Total volume of solution is 5 mL Concentration of Stock solution is 10% Final Concentration of Chemical desired is 2% WE DON’T KNOW: The number of mL of Stock solution we need to prepare this dilution (let X represent this value). STEP 2: PLUG IN THE NUMBERS YOU KNOW INTO THE EQUATION AND SOLVE FOR X: X 10 5 * = 2 STEP 3: SOLVE FOR X: X = 1 (remember, the units are in mLs) STEP 4: PUT IT ALL TOGETHER: To make 5 mL of a 2% solution from a 10% stock solution, mix 1 mL of 10% NaCl stock solution plus 2 mL of sterile water for a volume of 3mL. When this chemical dilution is added to the 2 mL of Chlorella solution, it will bring the total volume to 5 mL and further dilute the NaCl to a final concentration of 2%. Environmental Effects on Algal Growth Lab Absorbance (represents population density) (measured at 430 nm) Date of first reading (day 1)_______ Day 1 Chlamyd omonas Tube 1 Tube 2 Tube 3 Tube 4 Euglena Tube 1 Tube 2 Tube 3 Tube 4 Day Day Day Day Day Day Day Day Environmental Effects on Algal Growth Water quality continues to be a concern for environmental scientists, citizens, and educators. A number of kits are available that allow the identification and quantification of chemical components present in a selected water source. Students can supplement these valuable analyses by performing experiments designed to test the effects of various concentrations of a given chemical on a representative aquatic life form. Thus, unlike a natural source of water possessing a highly complex mix of organisms and chemicals, this laboratory model allows for a more controlled study of the impact of one variable on algal growth. This activity is quite flexible in that it allows teacher-guided inquiry, student-centered discovery, or a more standardized cookbook experiment. One interesting version directs students to select a given variable (NaCl, ammonia, fertilizer, dish detergent, etc.) and vary its concentration in a set of equivalent sample tubes. In this way, students can also learn to employ skills in creating proper dilutions and interpreting dosage effects. Certainly, students could also select variables such as light intensity or temperature, negating the necessity of generating varying concentrations within the sample tubes. The procedure described below focuses on exploring a dosage effect. Materials: Algal population (Chlorella, Euglena, Chlamydomonas, etc), 10 x 13 mm borosilicate culture tubes (an inexpensive alternative to spectrophotometer tubes), spring water, pipets, wax paper or parafilm, spectrophotometer. OPTIONAL: vortex for suspending algae. Procedure: 1. Pipet 2 mL of Algae into each tube. (This volume might be changed based on density of original culture). 2. Pipet 1 mL of spring water into each tube. 3. Add the appropriate volumes of variable stock solution and spring water (total of 2 mL combined) to generate the desired variable concentrations in each tube. Notice that each tube will contain a total of 5mL Examples: A) 2mL Algae + 2 mL spring water + 1 mL of 10% NaCl stock = a final NaCl concentration of 2%. B) 2 mL Algae + 1 mL spring water + 2 mL of 0.2M ammonia = a final concentration of 0.08M ammonia 4. Cover the top of each tube with a piece of wax paper or parafilm and mix by inversion (or vortex gently, if available). 5. Immediately measure the absorbance at 430 nm using a spectrophotometer. This wavelength is within a maximum absorbance peak for chlorophyll. Thus, population density is measured indirectly by quantifying the amount of chlorophyll present in a sample. (Other wavelengths can be used, representing non-optimal absorbance). 6. Place all tubes in an area of equal lighting and temperature. 7. Measure absorbance at selected time intervals (Monday, Wednesday, and Friday for 2-3 weeks would suffice). Remember to mix the tubes by inversion or vortexing prior to spectrophotometer readings. 8. Record all data and present as a population growth graph as shown below. The yaxis (dependent variable) represents absorbance, and the x-axis represents elapsed time in days (dependent variable). Algal Population Growth 0.8 0.7 Absorbance 430nm 0.6 0.5 0.4 100uM 10uM 1uM Control 0.3 0.2 0.1 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Day 15 Day 17 Day 19 Day 21 Day 23 Time Elapsed Tube 1 Tube 2 Tube 3 Tube 4 Chlorella/Euglena 2 mL 2 mL 2 mL 2 mL Spring Water 1 mL 1 mL 1 mL 1 mL (Name and Concentration of Chemical stock solution used___10%_______) __mL __mL __mL __mL Spring Water __mL __mL __mL __mL Total Volume 5 mL 5 mL 5 mL 5 mL [X] Chemical Final Concentration of Chemical in Test Tube TO CALCULATE DILUTIONS OF CHEMICALS USE THIS FORMULA: Number of mLs of Stock Solution Total Volume of Dilution X [Concentration of Stock Solution] = Final Concentration of Chemical in Test Tube For example: HOW WOULD YOU PREPARE A 2% NACL SOLUTION FROM A 10% NACL STOCK SOLUTION? STEP 1: WE KNOW: Total volume of solution is 5 mL Concentration of Stock solution is 10% Final Concentration of Chemical desired is 2% WE DON’T KNOW: The number of mL of Stock solution we need to prepare this dilution (let X represent this value). STEP 2: PLUG IN THE NUMBERS YOU KNOW INTO THE EQUATION AND SOLVE FOR X: X 5 10 * = 2 STEP 3: SOLVE FOR X: X = 1 (remember, the units are in mLs) STEP 4: PUT IT ALL TOGETHER: To make 5 mL of a 2% solution from a 10% stock solution, mix 1 mL of 10% NaCl stock solution plus 2 mL of sterile water for a volume of 3mL. When this chemical dilution is added to the 2 mL of Chlorella solution, it will bring the total volume to 5 mL and further dilute the NaCl to a final concentration of 2%.