Lab 3_Dilutions and Spectrophotometer_INSTRUCTIONS 8

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BMB 370 Introductory Biochemistry Laboratory Instructions Lab 3 – Dilutions and Introduction to the Spectrophotometer BMB 370 8.18.23 Claire Vieille Lab Companion Sections • Tables, Calculations, Graphing, and Conclusions • Building a Graph in Excel • Basic Statistics • Dilutions • Linear Regression and R 2 in Excel • Spectrophotometry • Quantifying bacteria using a spectrophotometer Before Starting the Lab • Complete Table 3-1, 3-2, 3-3, and 3-4 • Calculate the expected absorbance for each concentration in Table 3-5 Objectives ● To understand and practice making serial dilutions ● To understand and practice using a modern micrometer-based spectrophotometer ● To understand the Beer–Lambert law and its application to spectrophotometric measurements Materials ● 3 mM Red food dye #40 ( ε ≈ 25,900 M -1 cm -1 at 500 nm) (4 mL per group) ● Energy drink containing red food dye #40 (4 mL per group) ● Saturated E. coli culture (overnight culture) (2 - 5 mL per group) ● LB (6 mL per group) ● 2 cuvettes (polystyrene or acrylic) with 1 cm path length ● 2 mL microfuge tubes for dilutions (in drawer) ● 400 mL beaker to fill with water for dilutions (in drawer) Safety The only significant safety concern in this lab is the Escherichia coli culture. The E. coli strains used in BMB 370 are non-pathogenic: they will not cause disease if ingested. However, they may contain genes that confer resistance to certain antibiotics. Because antibiotic resistance genes are easily transferred between bacterial species, even non-pathogenic strains should not

be released into the environment. All materials that come into contact with E. coli must be autoclaved prior to reuse or disposal. E. coli cultures that have not been autoclaved should never be poured down the drain. As always, wash your hands before leaving the lab. ° Methods Preparing serial dilutions of red dye #40 Serial dilutions are used when an exponential dilution of a solution is needed for specific measurements or when very large dilutions are needed that cannot be prepared through direct dilutions. 1. Complete Table 3-1 for serial dilutions of red dye #40 using a series of consecutive 5-fold dilutions using the following instructions: a. Each dilution will have a total volume of 1.25 mL (1,250 µ L) b. Calculate the concentration and dilution factor of dilutions 1 to 5 starting from the initial 3,000 µ M (3 mM) solution (i.e., dilution 0). The fold dilution will increase exponentially from one dilution to the next c. Calculate the volumes of dilutions and of water and needed to prepare dilutions 1 to 5 Table 3-1: Red dye #40 5-fold serial dilutions Dilution # Red dye concentration ( µ M) Total fold dilution Volume solute ( µ L) Volume water ( µ L) Total volume ( µ L) 0 3,000 1 1,250 0 1,250 1 5 250 1,250 2 1,250 3 1,250 4 1,250 5 1,250 2. Using five 2 mL microfuge tubes prepare dilutions 1 to 5 in the series by serial dilution starting with the 3,000 µ M solution (i.e., dilution 0). Make sure to change pipet tips before each transfer and do not mix your dilutions with a used pipet tip. Preparing direct dilutions of an energy drink You have a sample of an energy drink that contains red dye #40. We want to make three separate 15-fold direct dilutions of the drink (i.e. three technical replicates). Complete Table 3-2 below and prepare the dilutions in 2 mL microfuge tubes Table 3-2: Energy drink 15-fold direct dilution Total fold dilution Volume energy drink (μL) Volume water (μL) Total volume (μL) 15 1,500

° Preparing serial dilutions of an E. coli culture In this exercise, you will prepare serial dilutions of a saturated E. coli culture grown in Lysis Broth (LB) medium to study how the behavior of suspensions differ from that of solutions in a spectrophotometer. Because the culture was grown in LB, you will prepare the dilutions in LB and use LB medium as the blank in the spectrophotometer. 1. Complete Table 3-3. Calculate the volumes of E. coli culture dilutions and LB medium needed for a series of 3-fold dilutions. Each dilution will have a total volume of 1.5 mL (1,500 µ L). You will estimate the bacterial concentration of the initial undiluted culture based on the optical densities (OD) you measure of the dilutions Table 3-3: E. coli culture 3 fold-serial dilutions Dilution # Total fold dilution Volume bacterial suspension ( µ L) Volume LB ( µ L) Total volume ( µ L ) 0 1 1,500 0 1,500 1 3 1,500 2 1,500 3 1,500 4 1,500 5 1,500 2. You have a test tube containing a saturated E. coli culture on your bench. Immediately before starting with your dilutions, mix your culture by gently vortexing. E. coli cultures are suspensions, not solutions, and bacteria tend to settle at the bottom of the tube 3. In five 2 mL microfuge tubes, prepare your dilutions according to Table 3-4 above. Make sure to use LB rather than water for your dilutions. Mix E. coli in each test tube by inverting the tube a few times before transferring the calculated volume with a fresh pipette tip to the next dilution tube. Discard the pipette tips in a biohazard waste container Determining the wavelength ( λ ) of maximum absorbance of red dye #40 1. Set the spectrophotometer to do a wavelength scan to determine the wavelength of maximum absorbance ( λ max ) of red dye #40. Use the following settings: On the Jasco choose the “Spectra Measurements” program then use the “Parameters” icon at the top right to adjust the settings On the Persee: First choose the “Spectrum” window then the “Measure” menu, then “Parameter Settings” to adjust the settings a. Set the spectrum to scan from 400 nm to 700 nm. This range is from blue to red and is roughly the spectrum the human eye can see

b. Set the UV/vis or spectral bandwidth to 1 nm c. Set the data interval to 1 nm with a UV/vis response of 0.24 s and a scan speed of 200 nm/min on the Jasco or “Fast” on the Persee d. On the Persee you will need to set the “Display Range” to 0 to 1 Abs units 2. Transfer 1 mL of deionized H 2 O into a spectrophotometer cuvette and blank the spectrophotometer in the wavelength scan mode 3. Empty the cuvette in the sink and fill it with your serial dilution #4. You can do this by carefully pouring the dilution directly from the 2 mL microfuge tube into the cuvette. 4. Perform the wavelength scan with dilution #4. Pour dilution #4 back into the microfuge tube when you are done. You will use that dilution again 5. When you are done, save your scan on a USB drive or email it to yourself and your lab partner so it can be graphed later. On the Jasco, data are autosaved as a *.csv in a folder called Jasco files on the computer desktop On the Persee, click on the “File” menu and choose “Export”. Export your data as a .csv file, not an Excel file which will cause glitches Determining the wavelength ( λ ) of maximum absorbance of an energy drink 1. Use your energy drink dilution to perform a wavelength scan as described above to verify that the energy drink contains a single dye. Make sure that the scan of the energy drink has a single maximum absorbance peak around 500 nm. 2. Save your data as a csv file on a USB drive or email it to yourself. 3. When the wavelength scan is done, pour the dilution back into the 2 mL microfuge tube Measuring the absorbances of the red dye serial and direct dilutions 1. Set the spectrophotometer to end point or direct assay mode: On the Jasco, use “Fixed wavelength measurement”. Click on the “Parameters” icon to input settings for your assay On the Persee, use the “Photometry” window. Click on the “Measurements” menu at the top, then choose “Parameters” a. Set the wavelength to 500 nm (cyan-colored light) b. Set the UV/vis or spectral bandwidth to 1 nm c. Leave all other settings at their default values 2. Rinse the cuvette you were using before 2–3 times with deionized water

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