Mar 13, 2026
Measuring OD 600
This protocol is a draft, published without a DOI.
- Tamra Lahom1
- 1Carleton College
Protocol Citation: Tamra Lahom 2026. Measuring OD 600 . protocols.io https://protocols.io/view/measuring-od-600-jv5qcn85x
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 13, 2026
Last Modified: March 13, 2026
Protocol Integer ID: 313232
Keywords: measuring od, optical density, measuring light scattering, cell density, spectrophotometer, bacterial culture, light scattering
Abstract
Measuring the optical density (OD) of a bacterial culture, typically at 600 nm, determines cell density by measuring light scattering using a spectrophotometer.
Troubleshooting
Preparing Your Sample
Under a flame, obtain 1 mL of the fresh LB and place in a disposable 1.5 mL Cuvette
Note
Never touch the cuvette in the detection window. Only hold from the top.
Under a flame, obtain 1 mL of each sample and place in a disposable 1.5 mL Cuvette
Note
- Ensure the cuvettes are labeled at the top with the glycerol stock number
Running the UV VIs
Ensure the sample holder cover is shut completely.
Open the computer connected to the Carry UV60
Open the "Simple Reads" Desktop application.
Note
When you first open the software, the measure will do some manual calibrations. The power button will blink orange. Ensure the sample holder cover is shut completely.
Click setup and set the wavelength to 600 nm.
Insert your "Blank". The best blank for an OD600 is fresh LB.
Note
When inserting any sample, always wipe the outside with a Kimwipe
Press "Zero" then "Read"
Note
The adsobance should now read ~0. Ignore if it reads a negative number.
Now insert your sample(s) and record the OD.
Note
If the OD value exceeds 1.0, the measurement becomes inaccurate due to multiple scattering. You can dilute the sample with fresh medium, record the dilution factor, measure the diluted sample, and multiply the reading by the dilution factor (e.g., if you dilute 1:10 and read 0.5, the actual OD is 5.0).
Disposal of Sample
All samples must be neutralized with 10% bleach for at least 30 min to 1hr. There should be a noticeable foaming and a slight color change.