Measure Absorbance of M. xanthus Culture: Difference between revisions
(Created page with "1. Turn on the spectrophotometer so it can warm up. 2. Obtain one 13mm glass test tube for each sample that you want to measure density for, plus one to blank. 3. Add 3mL of uncultured CTTYE growth media to the tube that will serve as your blank. 4. Add 2.5mL of uncultured CTTYE to labeled sample test tubes, and add 0.5mL of bacterial culture to the correspondingly labeled tube. **Note, if your sample is particularly dilute, add 1mL culture and 2mL nutrient media...") |
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2. Obtain one 13mm glass test tube for each sample that you want to measure density for, plus one to blank. | 2. Obtain one 13mm glass test tube for each sample that you want to measure density for, plus one to blank. | ||
3. Add 3mL of uncultured CTTYE growth media to the tube that will serve as your blank. | 3. Add 3mL of uncultured CTTYE growth media to the tube that will serve as your blank. Label this tube. | ||
4. Add 2.5mL of uncultured CTTYE to labeled sample test tubes, and add 0.5mL of bacterial culture to the correspondingly labeled tube. **Note, if your sample is particularly dilute, add 1mL culture and 2mL nutrient media for dilution** | 4. Add 2.5mL of uncultured CTTYE to labeled sample test tubes, and add 0.5mL of bacterial culture to the correspondingly labeled tube. **Note, if your sample is particularly dilute, add 1mL culture and 2mL nutrient media for dilution** | ||
5. | 5. Vortex your blank tube. Immediately lank the spectrophotometer with your blank tube. | ||
6. One at a time, vortex one tube of diluted culture, then immediately record the Absorbance value at 600nm. Repeat for each sample. | 6. One at a time, vortex one tube of diluted culture, then immediately record the Absorbance value at 600nm. Repeat for each sample. Check each sample multiple times immediately after vortexing. The spec readings can fluctuate a bit, so | ||
7. Turn off the spectrophotometer if you are done and if no one else plans to use it relatively soon. | 7. Turn off the spectrophotometer if you are done and if no one else plans to use it relatively soon. | ||
8. To obtain OD600 from these Absorbance values, divide by a path length of 1.3cm (diameter of the test tubes). | 8. To obtain OD600 from these Absorbance values, divide by a path length of 1.3cm (diameter of the test tubes). Make sure you adjust this value for whatever dilution you performed in step 4. (Ex. 0.5mL of culture into 2.5mL of CTTYE gives a dilution factor of 6). | ||
OD600 = Absorbance / 1.3cm * dilution factor | |||
9. You can use the below equation to calculate cells/mL of your sample: | 9. You can use the below equation to calculate cells/mL of your sample: | ||
Density (cells/mL) = | Density (cells/mL) = 9.73x10^8 * (OD600) + 1.77x10^7 (updated version as of 6.13.23). | ||
Note: for phenotype assays, use this cells/mL calculation to decide how to dilute or concentrate your sample accordingly. | Note: for phenotype assays, use this cells/mL calculation to decide how to dilute or concentrate your sample accordingly. | ||
Latest revision as of 10:13, 13 June 2023
1. Turn on the spectrophotometer so it can warm up.
2. Obtain one 13mm glass test tube for each sample that you want to measure density for, plus one to blank.
3. Add 3mL of uncultured CTTYE growth media to the tube that will serve as your blank. Label this tube.
4. Add 2.5mL of uncultured CTTYE to labeled sample test tubes, and add 0.5mL of bacterial culture to the correspondingly labeled tube. **Note, if your sample is particularly dilute, add 1mL culture and 2mL nutrient media for dilution**
5. Vortex your blank tube. Immediately lank the spectrophotometer with your blank tube.
6. One at a time, vortex one tube of diluted culture, then immediately record the Absorbance value at 600nm. Repeat for each sample. Check each sample multiple times immediately after vortexing. The spec readings can fluctuate a bit, so
7. Turn off the spectrophotometer if you are done and if no one else plans to use it relatively soon.
8. To obtain OD600 from these Absorbance values, divide by a path length of 1.3cm (diameter of the test tubes). Make sure you adjust this value for whatever dilution you performed in step 4. (Ex. 0.5mL of culture into 2.5mL of CTTYE gives a dilution factor of 6).
OD600 = Absorbance / 1.3cm * dilution factor
9. You can use the below equation to calculate cells/mL of your sample:
Density (cells/mL) = 9.73x10^8 * (OD600) + 1.77x10^7 (updated version as of 6.13.23).
Note: for phenotype assays, use this cells/mL calculation to decide how to dilute or concentrate your sample accordingly.