OD600 is a standard, rapid technique used extensively in microbiology laboratories to estimate the concentration of microorganisms within a liquid culture. The abbreviation stands for Optical Density at 600 nanometers, a measurement taken using a spectrophotometer. This simple test provides an indirect assessment of the total cellular biomass present in a sample, such as a broth containing bacteria or yeast. The value helps scientists manage and monitor the growth of these microscopic populations, allowing for immediate decisions regarding the timing of experiments or harvesting of cells.
Defining Optical Density and the 600 nm Wavelength
Optical Density (OD) describes the amount of light reduced as it passes through a sample. In bacterial cultures, this reduction is due to the presence of cells suspended in the medium. The specific wavelength of 600 nm is chosen for several practical reasons. This wavelength falls within the visible red-orange spectrum, avoiding the harmful effects of ultraviolet light that could damage the live cells being measured. Using 600 nm allows researchers to perform repeated measurements without causing cellular stress.
Common nutrient-rich culture media often absorb light at shorter wavelengths. The 600 nm wavelength minimizes this background interference from the medium itself, ensuring the measured signal primarily relates to the cellular components. This maximizes the signal coming from the cells rather than from non-cellular components.
How a Spectrophotometer Measures Turbidity
The spectrophotometer performs the OD600 measurement by shining a beam of monochromatic light through the microbial sample. When this 600 nm light encounters bacterial or yeast cells, the light is not chemically absorbed. Instead, the cells act like tiny obstacles, causing the light to be scattered in multiple directions away from the original path.
The spectrophotometer measures the amount of light that successfully passes through the sample and reaches a detector. Since light is scattered away by the cells, the amount reaching the detector is reduced, which the instrument registers as a higher Optical Density value. The OD600 reading is therefore a measure of the sample’s turbidity, or cloudiness, which increases directly with the number of suspended particles.
Tracking Bacterial Growth and Cell Concentration
The primary application of the OD600 reading is to monitor the life cycle of a microbial population over time, a process known as generating a growth curve. By taking measurements at regular intervals, researchers determine which of the four main growth phases the culture is in.
Lag Phase
The initial lag phase shows little change in OD600 as the cells adapt before dividing.
Exponential Phase
Following this is the log or exponential phase, where the cell population rapidly doubles at a uniform rate, causing the OD600 reading to increase quickly. This phase is often the desired target for laboratory experiments, such as those involving protein expression.
Stationary Phase
As nutrients deplete and waste products accumulate, the growth rate slows, leading to the stationary phase. Here, the number of new cells equals the number of dying cells. This balance results in a plateaued OD600 value, indicating that the maximum density has been reached. OD600 measurements allow for the standardization of experiments by ensuring tests begin with the same cell concentration or are harvested at a controlled stage of growth.
Interpreting the OD600 Reading
The OD600 measurement provides a rapid estimate, but its interpretation requires understanding its limitations as an indirect method. The instrument registers light scattering from all particles, meaning it cannot differentiate between living cells, dead cells, cellular debris, or cellular aggregates. Therefore, a high OD600 reading does not guarantee a high concentration of viable, healthy cells.
The relationship between cell concentration and light scattering is only linear for relatively dilute samples. When the culture becomes too dense, typically above an OD600 of 1.0, the measurement is no longer accurate. The sample must be diluted before reading to obtain a reliable value.
To accurately convert an OD600 value into a true cell count, such as Colony Forming Units per milliliter (CFU/mL), a specific calibration curve must be generated for the exact strain and growth conditions used. For example, an OD600 of 1.0 for Escherichia coli typically correlates to around \(8 \times 10^8\) cells per milliliter, but this factor changes based on cell size and shape. OD600 readings can also vary between different spectrophotometer models due to differences in their optical configurations, making instrument-specific calibration necessary for comparison.