- The tube spec is useful when monitoring cultures and trying to harvest them at a desired OD range because you can keep measuring the same sample and can skip the hassle of repeated sampling out of a flask.
- The path length is longer, so the absorbance of a sample will be higher than if you measure it in a 1 cm cuvette.
- On 3/21/2013 Janet measured the same sample in the tube spec (OD600 = 0.560) and the Beckman cuvette spectrophotometer (OD600 = 0.3388) and found the absorbance to be 0.560/0.3388 = 1.65 times higher.
- Beware that the relationship between OD and cell concentration becomes nonlinear around OD600 = 0.4.
- Thermo electron SPECTRONIC 20+
- Minimum volume: 5 mL
- Use green-capped test tubes (larger diameter)
- Use a blank containing your media
- ok to leave on all day, but do turn it off when done
- bulbs are cheap (UV bulbs are generally the expensive ones)
- Turn on by turning leftmost dial clockwise
- Allow the spectrophotometer to warm up for at least 15 minutes to stabilize.
- Set filter to 600 nm (or whatever)
- The spec has two filters that you switch between using a lever on the bottom of the spec's left side. For 600 nm, use the position on the left.
- while the spec is empty & the cap is on, set transmittance to 0 using the left knob
- Insert blank & cover with black cap
- Set transmittance to 100 with the right knob
- Switch mode to absorbance
- Absorbance for the blank should now read 0.
- Measure absorbance for your sample
- Keep tube orientation the same each time you measure in case of any unevenness.
- Make sure the test tube is clean. Wipe it off if needed.
- In Amanda and Janet's experience, the tube spec reads a higher absorbance than the rectangular 1 cm cuvette spectrophotometers.
Correlating to OD600 readings to other devices in lab
- Original data, calculations, etc can be seen here: 2014/4/1 OD relationship experiment. The data analysis folder is here.
- Keep in mind that none of the 1cm devices are accurate for OD600 > 0.5. They lose linearity at this point:
- Also note that the NanoDrop values need to be scaled to be comparable to other spectrophotometer numbers.
Manual's Version of Instructions
- Turn on the SPECTRONIC® 20+ by turning the Power Switch/Zero Control (knob on the left side of instrument) clockwise. Allow the spectrophotometer to warm up for at least 15 minutes to stabilize.
- After the warmup period, set the desired wavelength with the Wavelength Control Knob.
- Set the filter lever to the appropriate position for the selected wavelength (not required for SPECTRONIC® 20).
- Adjust the meter to 0%T with the Power Switch/Zero Control (knob on the front left side of instrument). Make sure the sample compartment is empty and the cover is closed.
- Fill a clean cell with water (or blank solution) and wipe the cell with a tissue to remove liquid droplets, dust and fingerprints.
- Place the cell in the sample compartment and align the guide mark on the cell with the guide mark at the front of the sample compartment. Press the cell firmly into the sample compartment and close the lid.
- Adjust the meter to 100%T with the Transmittance/Absorbance Control (knob on the front right side of instrument).
- Remove the cell from the sample compartment and empty the water.
- Rinse the cell twice with small volumes of the solution to be measured and fill it with the solution.
- Wipe the cell with a tissue and insert the cell into the sample compartment. Align the guide marks and close the lid.
- Read the appropriate value (%T or A) from the meter.
- Remove the cell from the sample compartment and repeat steps 9 through 11 for any remaining sample solutions.
- When all measurements are completed, turn off the spectrophotometer by turning the Power Switch/Zero Control counterclockwise until it clicks.