Procedure 3: Spectrophotometer Measurements All measurements are to be made immediately following the sample-taking for the standard plate count procedure. 1. Allow the spectrophotometer to warm up for at least 10 minutes and carry out procedure 1. (Standardize the instrument to 100% transmission with sterile broth.) 2. Immediately after inoculating the flask with BHI medium, obtain a cuvette. Be careful not to handle the lower half of the tube. 3. With a 5-mL pipette delivery device, introduce 5 mL of the inoculated broth culture into the cuvette. Place the pipette in disposal container 1. 4. Immediately return the culture flask to the shaker. 5. Wipe the cuvette to remove any fingerprints, liquid, etc., and insert it into the sample holder. Be certain that the index line or frosted triangle on the tube faces the front of the instrument. 6. Close the lid. 7. Determine the optical density (OD) or absorbance of the culture and record the reading in Table A-2 in the Results and Observations section. Note that this reading is for 0 time. 8. Pour the contents of the cuvette into the container of disinfectant and rinse it several times with distilled water. 9. Repeat steps 3 through 8 to take OD readings at 15-minute intervals until the absorbance no longer increases. Enter your readings for the time periods in Table A-2 in the Results and Observations section. 10. After completing all measurements, turn off the machine. 11. Using the graph provided in the Results and Observation Section, plot both the absorbance readings and the viable counts per mL versus time. Use a different colored pencil for each type of information. 12. Answer the questions in the Results and Observations section. Spectrophotometric measurment TIME OD 0 0.022 30 0.054 45 0.112 60 0.245 75 0.298 90 0.295 Spectrophotometric Measurements 1. Enter your OD (absorbance readings) in Table A-2. 2. After which reading did the absorbance value of the culture not increase? 3. Plot your readings on the graph provided. (Figure A-6.) Can you identify the lag, exponential, and stationary phases? If so, label them accordingly. 4. Do the plots of absorbance and viable cell counts differ? Explain.
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