An Introduction to OD600

Unless you are a trained microbiologist, or have additional experience working with bacteria and other organisms, the concept of OD600 might be unfamiliar to you.

In this article, we are going to give you an overview of what exactly OD600 is, how to measure it and some real-life applications.

What is OD600 and What Does it Mean?

OD600 is an abbreviation of two parts; ‘OD’ is short for ‘optical density’, whilst ‘600’ is in reference to the 600 nm wavelength used to measure said optical density.

The term ‘OD600’ is used in reference to a spectrophotometer method that is used to help estimate the concentration or “number of cells per volume” of bacteria or other cells within a liquid sample.

How is OD600 measured?

OD600 is typically measured using a bench-top spectrophotometer, but can also be measured using our Photopette handheld spectrophotometer.

A sample is placed within a cell or cuvette, and a light source of 600 nm is directed towards the sample. The light is absorbed and scattered by cells and the transmitted light is picked up by a detector, which outputs a value.

The light scattering value that is generated by the sample is what is used to estimate the concentration of bacteria or other cells within a sample.

Light scattering is referred to as a factor of turbidity, rather than light absorption.

What is OD600 used for?

Determining the concentration of bacteria, or other cells within a sample has lots of real life applications, with a few outlined below:

Synthetic Biology

In synthetic biology, organisms are redesigned for additional purposes than what they originally are used for. To research and develop new cells, it is vital that cells can be grown and harvested at the optimum level of growth.

Microbial Production

The use of microbes is far-reaching across many fields. They play a big part in the food and beverage industry, as well as having the potential to provide fuel sources such as ethanol. Using OD600 to measure cell growth is of vital importance, particularly where microbes are grown for commercial purposes.

Antibiotic Resistance

Developing new antibiotics requires susceptibility testing, which determines the effectiveness of antibiotic treatment. To test the effectiveness of new antibiotics, measuring the optical density of bacteria, helps to understand if, and how bacteria is growing in response to new antibiotics.

Using OD600 to Determine Cell Growth Phase

When measuring OD600, the measured value that is given is an arbitrary number that needs further interpretation in regards to the overall cell growth cycle.

Using a standard or control growth curve, it is possible to plot measure OD600 values against a known cell growth chart and understand the cell growth phase. A typical growth chart for a bacterial cell or microorganism is shown below:

With the phases of cell growth phases split into four main categories, outlined below:

Lag Phase

The lag phase is the starting point of the bacteria S-curve, where cells are adapting to their new environment. Although cells are active, they do not start dividing during the lag phase.

Log (or exponential) phase

The log phase is where cell growth is initiated and cells start to rapidly divide. In this phase, cells replicate exponentially and the activity across all cells is uniform.

Stationary Phase

When cell density hits its maximum cell density, the cells enter their stationary phase. At this point, the number of cells is constant. As cells die, new cells grow to replace them.

Death Phase

The death phase is the final stage of cell growth. The number of cells starts to diminish as cells die due to a variety of factors, including accumulation of cell waste, pH changes or lack of cell nutrition.

The cause of the death phase will vary depending on the type of bacterial cells being grown.

Measuring OD600 ensures that you can estimate when deciding when to use or ‘harvest’ cells, with some of the example use cases previously listed.

Measuring OD600 with Photopette®

Traditional OD600 measurements require benchtop instrumentation that requires samples to be placed in a cell and entered into a spectrophotometer on a one-by-one basis.

The Photopette® OD600 is a handheld device that can be controlled via an app or run via a smartphone or tablet computer. This allows for a sampling free workflow that increases efficiency and decreases the risk of cross contamination.

You can find out more about the Photopette® OD600 here.

Case Study – Direct e.coli Cell Count at OD600 Using Photopette OD600

Using our Photopette handheld spectrophotometer, we were able to perform a direct cell count of E.coli at OD600. We were able to measure E.coli directly in the cell culture flask, meaning the measurement could be performed at any location and with no requirements for a lab.

Find out more here: Application Note – Direct E. coli Cell Count at OD600

FAQs

• How do you calculate OD600?

OD600 is measured by a spectrophotometer with a light source of 600 nm. It measures light scattering, as opposed to light absorption to determine optical density.

• Why is OD600 used for bacteria?

Because bacterial cells are colourless, the amount of light they absorb is very low. Using a light source at 600 nm, and measuring scattering is a better way to measure cell concentration.

• How does OD600 work?
  OD600 is an arbitrary value that helps determine the cell growth stage, when compared to a standard or control reference. By comparing a measured result to a control, it is possible to estimate the growth stage and the total concentration of cells.
• Why do we take OD at 600 nm?
  The reason for measuring optical density at 600 nm is because this is a known wavelength that minimizes cell damage and growth, and is not destructive in nature.