Optical Density (OD) is a fundamental measurement in microbiology used to quickly estimate the concentration of microorganisms, such as bacteria or yeast, suspended in a liquid culture. It measures the turbidity, or cloudiness, of the sample, which increases as the number of cells grows. This rapid, non-destructive technique provides a proxy for the total biomass of the culture, converting light scattering into a numerical value to track cell populations.
The Mechanism of Light Scattering
The measurement of optical density relies on a spectrophotometer. This device shines a beam of light through the liquid culture sample and measures how much light reaches a detector. When light encounters a microbial cell, the cell acts as an obstacle, causing the light to be scattered away from the detector’s path.
The resulting OD value is a measure of light scattering caused by the particles in the suspension, rather than light absorption, as in many chemical assays. The wavelength most commonly used in bacteriology is 600 nanometers (OD600). This specific wavelength is chosen because microbial cells and most common growth media do not significantly absorb light at 600 nm, ensuring the measurement primarily reflects the presence of the cells.
A higher concentration of cells in the liquid culture means there are more particles to scatter the light beam. Consequently, less light reaches the detector, and the spectrophotometer registers a higher OD reading. This direct correlation between the number of cells and the amount of scattered light allows the OD value to serve as a reliable, though indirect, measure of cell density.
Monitoring Microbial Growth
The most frequent application of optical density is monitoring how a microbial population changes over time, which generates a characteristic microbial growth curve. By taking sequential OD readings, researchers track the four distinct phases of a culture’s life cycle. This begins with the lag phase, where cells adapt to the new environment and the OD remains relatively constant.
The exponential or log phase follows, where cells divide rapidly and the OD value increases quickly and predictably. Monitoring OD during this phase is important because log phase cells are the healthiest and most metabolically active, making them ideal for experimental procedures.
As nutrients become depleted and waste products accumulate, the growth rate slows, leading to the stationary phase. Here, the rate of cell division is balanced by the rate of cell death, and the OD reading plateaus at its maximum value. Researchers often use a specific OD target (e.g., OD600 of 0.4 to 0.6) as an indicator to harvest cells or induce gene expression.
Converting Optical Density to Cell Numbers
While OD provides a convenient relative measure of cell density, it does not directly equate to the actual number of living cells present. To convert the OD reading into a meaningful unit like Colony Forming Units per milliliter (CFU/mL), a calibration process must be performed. This involves creating a standard curve specific to the organism, the growth medium, and the spectrophotometer used.
The calibration process begins by measuring the OD of a series of culture dilutions. Simultaneously, a small volume from each dilution is plated onto a solid growth medium to perform a traditional plate count, which directly yields the viable cell count (CFU/mL). By plotting the measured OD values against the corresponding CFU/mL counts, a linear relationship is established at lower cell densities.
This graph provides a conversion factor—the number of cells per milliliter that corresponds to one unit of OD for that microbe on that instrument. Once this standard curve is established, a researcher can bypass the labor-intensive plating process and use the measured OD to accurately estimate cell concentration. Without calibration, an OD value only allows for comparisons of relative density within the same experiment.
Limitations and Sources of Error
Despite its convenience, the optical density method has limitations that can introduce error into the cell density estimate. The spectrophotometer measures all particles that scatter light, meaning the OD value includes contributions from live cells, dead cells, and cellular debris. Therefore, the OD reading may overestimate the number of viable cells, especially in older cultures that have entered the death phase.
The linear relationship between OD and cell concentration breaks down at high cell densities. When the culture is too turbid, light scattered away by one cell can be scattered back toward the detector by another. This phenomenon, known as multiple scattering, causes the OD reading to underestimate the true cell density, requiring the dilution of dense samples before measurement. Additionally, the size, shape, or clumping tendency of microbial cells affects how they scatter light, meaning a single OD value can represent different cell numbers depending on the organism.