Optical Density (OD) is a fundamental measurement tool in fields like microbiology, biochemistry, and cell culture. It provides scientists with a rapid, non-invasive way to estimate the concentration of suspended particles, such as cells or proteins, within a solution. By monitoring the OD value, researchers gain an indirect way to track changes in biological systems over time. This metric serves as a reliable proxy for the total mass or number of items present in a turbid liquid.
Defining Optical Density
Optical Density is a quantitative measure of how much light is attenuated, or blocked, as it passes through a sample. This blockage results from two physical phenomena: the absorption of light by molecules in the solution and the scattering of light by suspended particles. The measurement is a logarithmic ratio comparing the intensity of the light that enters the sample to the intensity of the light that successfully exits and reaches a detector.
For solutions containing suspended cells, such as a broth culture of bacteria or yeast, the primary phenomenon measured is light scattering, also known as turbidity. When light hits a cell, it causes the light photons to be deflected away from their path toward the detector. The greater the number of cells, the cloudier the solution becomes, leading to more light being scattered and less light reaching the sensor.
Scientists often use a standard wavelength of 600 nanometers, referred to as OD600, when tracking bacterial growth. This specific wavelength is chosen because it is not strongly absorbed by the organic molecules or pigments within the cells. Consequently, almost all light attenuation at 600 nanometers is attributable to the physical scattering caused by the cells, making it an excellent indicator of cell concentration. In contrast, if a solution contains a colored compound, like a protein, a different wavelength that the compound strongly absorbs, such as 280 nanometers, is selected to determine its concentration.
Measuring Cell Concentration with Light
Determining Optical Density relies on a spectrophotometer, an instrument designed to precisely measure the amount of light passing through a sample. This device projects a beam of light at a specific, pre-selected wavelength, such as 600 nm for a microbial culture, through a transparent vessel holding the liquid sample, known as a cuvette. The light beam travels through the sample, and the remaining light intensity is measured by a detector on the opposite side.
Before measuring the cell culture, the instrument must be calibrated using a “blank” sample. The blank is typically a cuvette filled only with the sterile growth medium, ensuring that any background light attenuation from the medium is accounted for and subtracted. By comparing the incident light intensity to the transmitted light intensity, the spectrophotometer calculates the OD value.
A higher final OD reading indicates that a larger proportion of the light failed to pass through the sample, meaning a higher concentration of cells is present. Conversely, a low OD value suggests that the liquid is relatively clear, allowing most light to be transmitted, which correlates with a low cell concentration. This process converts the physical property of turbidity into a single, quantifiable number for consistent use across different experiments.
Tracking Biological Activity Through Variability
The utility of Optical Density measurements comes from monitoring the variability of the reading over time, which provides a real-time window into the life cycle of a culture. As cells grow and divide, their total number and mass increase, causing the solution to become progressively more turbid. This proliferation results in a measurable increase in the Optical Density reading.
Tracking this rising OD over sequential time points allows researchers to plot a microbial growth curve, a foundational tool in microbiology. This curve reveals distinct stages of a culture’s life, including the lag phase, where cells are preparing to divide, and the exponential or log phase, where cell numbers are doubling rapidly. Monitoring this variable OD is used to determine the optimal time for specific laboratory procedures, such as when to harvest cells or induce protein expression.
A decrease in the OD measurement can signal biological changes, such as cell death or lysis, where the cells break apart and the resulting smaller fragments scatter less light. Continuous monitoring of OD variability allows scientists to understand the health, metabolic activity, and progression of a biological culture. By tracking these changes, researchers make informed decisions about experimental timing.