A microplate reader is a laboratory instrument designed to automate the rapid and precise measurement of biological, chemical, or physical events in multiple samples simultaneously. In microbiology, this technology tracks the proliferation of microorganisms, such as bacteria, in a liquid environment over time. The instrument provides a controlled environment and automated data collection, allowing researchers to accurately monitor how microbial populations respond to various experimental conditions. This capability helps scientists gain insights into microbial behavior, which is fundamental to fields like infectious disease research and drug development.
Understanding Optical Density Measurement
The core principle used by the microplate reader to gauge bacterial growth relies on turbidimetric analysis, quantified as Optical Density (OD). As bacteria divide and multiply within the liquid culture medium, the suspension becomes progressively cloudier (turbidity). This increased turbidity interferes with light passing through the sample.
The microplate reader quantifies this interference by shining a beam of light through each well and measuring how much light makes it to the detector on the other side. For bacterial growth studies, the instrument typically uses a wavelength of 600 nanometers (\(\text{OD}_{600}\)). This wavelength is chosen because it is not strongly absorbed by the cells or the common yellow-tinted growth media. Instead of measuring true light absorption by molecules, the \(\text{OD}_{600}\) measurement captures the degree to which the bacterial cells scatter the light.
A higher \(\text{OD}_{600}\) reading indicates more light is scattered and less is transmitted, meaning the bacterial suspension is denser. This measurement serves as a reliable proxy for the concentration of cells present. \(\text{OD}_{600}\) is an indirect estimate of cell density, not a direct count of viable cells, and its accuracy is highest in low-density cultures. Factors like the size and shape of the bacterial species, or the presence of dead cells or debris, can influence the final \(\text{OD}_{600}\) value.
Mapping the Bacterial Growth Curve
Continuous \(\text{OD}_{600}\) measurements translate into the bacterial growth curve, which maps the cell population over time. This curve reveals the distinct life stages of a bacterial culture as it adjusts to and consumes the limited resources of its environment. The first stage, the Lag Phase, occurs immediately after the bacteria are introduced to the fresh medium.
Growth Phases
During the Lag Phase, the cell population does not increase significantly because the bacteria are physiologically adapting, synthesizing necessary enzymes, and repairing cellular damage. Following this adaptation, the culture enters the Logarithmic or Exponential Phase, characterized by rapid and consistent growth. In this phase, cells divide at a constant rate, causing the \(\text{OD}_{600}\) value to increase sharply and exponentially.
The growth rate eventually slows as the culture reaches the Stationary Phase. This stabilization happens when the rate of new cell division equals the rate of cell death, typically due to nutrient depletion or the accumulation of toxic waste products. Finally, the culture enters the Decline or Death Phase, where the death rate exceeds the growth rate, leading to a net reduction in viable cells and a drop in the \(\text{OD}_{600}\) reading. The microplate reader generates this entire curve automatically by taking kinetic readings repeatedly over many hours.
Preparing the Microplate Experiment
Setting up a bacterial growth experiment begins with the microplate, a standardized plastic tray typically featuring 96 individual wells. Each well functions as a miniature reaction vessel, allowing for the simultaneous testing of dozens of conditions. Researchers first dispense liquid growth medium, such as Lysogeny Broth (LB), into the wells to provide necessary nutrients.
Next, a small volume of starting bacterial culture (the inoculum) is added to each well. The microplate format allows for the introduction of different variables across the plate, a process known as parallelization. For example, a researcher might test a range of antibiotic concentrations or compare the growth of several different bacterial strains. Once prepared, the plate is sealed and placed inside the microplate reader, where environmental conditions are precisely maintained.
Temperature control is standard, with the instrument usually holding the plate at the optimal growth temperature for the organism, such as \(37^\circ\text{C}\) for many human-associated bacteria. The reader also incorporates a shaking mechanism, typically orbital shaking, to ensure bacterial cells and nutrients remain uniformly suspended, promoting consistent growth.
Advantages of High-Throughput Screening
The microplate reader facilitates high-throughput screening (HTS), which transforms the scale and speed of microbial studies. HTS involves rapidly testing a large number of samples or experimental conditions in parallel, often utilizing plates with up to 384 or 1536 wells. This parallelization is a significant advance over older, laborious methods like using individual test tubes or manual spectrophotometers.
The miniaturization of the assay format allows researchers to conserve expensive reagents and limited-quantity compounds, lowering the cost per experiment. Since the process is fully automated, including kinetic readings, temperature, and shaking control, the risk of human error is minimized, leading to improved precision and reproducibility. This automated data collection is valuable in drug discovery, allowing scientists to quickly screen thousands of potential drug candidates that inhibit bacterial growth.