Experiment #1: Measuring Bacterial Growth using Optical Density

Dr. Smith: ‘Alright, everyone. Let’s focus on the experiment that truly benefits from a spectrophotometer—measuring bacterial growth via optical density. This allows us to track how the sample’s cell density changes over time, giving us a clear indication of growth patterns.’

She points at a flask containing the bacterial culture. “Let’s walk through our setup. First, take the culture sample, carefully pipette a small amount into a clean cuvette, and wipe away any droplets or smudges. Then, we’ll place the cuvette inside the spectrophotometer, making sure to blank the machine with a matching broth that doesn’t contain bacteria.”

Here are the steps:

1. Power on the spectrophotometer, letting it warm up so the lamp stabilizes.
2. Select your desired wavelength based on what you're measuring.
3. Insert a cuvette filled with just your solvent—often called the 'blank'—and press the button to zero the instrument. This accounts for any absorbance not caused by your sample.
4. Carefully fill a second cuvette with your test sample. Hold it by the frosted sides to avoid smudges on the optical path.
5. Insert the sample cuvette, close the lid or chamber, and wait for a stable absorbance or transmittance reading.
6. Record your results in your notebook or computer.

One of the students, Sarah, lifts a cuvette. “Dr. Smith, how often should we measure the optical density?”

“That’s a good question,” Dr. Smith replies. “Typically, you might measure at set intervals—say, every 30 minutes or every hour—to see how quickly or slowly the culture is growing. Remember to record the OD readings in your lab notebook. As the cell population increases, the reading will rise. See below for an example.”

Students shake their heads, and Dr. Jane Smith steps back with a look of encouragement. 

Example of Escherichia coli optical density at 600 nm wavelength (OD600) growth curve. https://www.jove.com/v/10511/growth-curves-cfu-and-optical-density-measurements