Bacterial growth refers to the increase in the number of cells in a population, occurring primarily through binary fission, where a single cell divides into two identical daughter cells. Measuring this increase is foundational for almost every area of microbiology, from medical diagnostics to industrial applications. Quantification of bacterial populations allows researchers to determine the generation time of a species (the time required for the population to double) and to test the effectiveness of antimicrobial compounds. Accurate measurement is also routinely applied in food safety and quality control to monitor contamination and ensure product standards.
Direct Microscopic Counting
Direct microscopic counting involves physically counting the cells in a known volume using specialized slides, such as the Petroff-Hausser counting chamber or a hemocytometer. The counting chamber is a thick glass slide with a precisely etched grid and a known depth, which together define a specific, measurable volume.
A sample of the bacterial suspension is loaded onto the chamber, and a high-power microscope is used to count the cells present within several of the defined squares. The average number of cells per square is then used in a calculation to determine the total cell concentration in the original sample (cells per milliliter). This technique is rapid and simple, requiring minimal equipment.
A significant limitation is its inability to distinguish between living and dead bacterial cells, meaning the count represents the total cell number. Furthermore, the method is generally only suitable for dense suspensions, typically greater than 100,000 cells per milliliter, because the small volume examined otherwise leads to poor precision.
Quantifying Viable Cells through Plating
This method focuses exclusively on viable cells—those capable of growth and reproduction—by determining the Colony Forming Unit (CFU). CFU is the standard unit used to estimate the number of living bacteria in a sample. The fundamental principle involves spreading a sample onto a solid growth medium and counting the resulting colonies, with each colony assumed to have originated from a single viable cell.
To obtain a statistically reliable count, the original sample must undergo serial dilution, a process of sequentially diluting the culture to reduce the bacterial concentration. This step is necessary because an undiluted or insufficiently diluted sample would produce too many colonies that overlap and merge, making accurate counting impossible. The diluted samples are then plated, incubated, and examined for colony growth.
The accepted range for a statistically significant and countable plate is between 30 and 300 colonies. If a plate has fewer than 30 colonies, random errors in the plating process can disproportionately affect the result, while more than 300 makes accurate counting impossible due to overcrowding. The final concentration is calculated using the formula: CFU/mL = (Number of Colonies \(\times\) Dilution Factor) / Volume Plated (in mL). This calculation provides an accurate estimate of reproductive cells, a measure distinct from the total cell count.
Indirect Measurement Using Light Scattering
Monitoring bacterial growth often uses the indirect measurement of turbidity, or cloudiness, of a liquid culture. As bacteria multiply, the suspension becomes denser and more opaque, causing light to be scattered by the cells. A spectrophotometer quantifies this cloudiness by measuring the Optical Density (OD), typically at a wavelength of 600 nanometers (OD600).
The spectrophotometer shines a beam of light through the culture and measures the amount that reaches a detector. The more bacterial cells present, the more light is scattered away, resulting in a higher OD reading. This method is fast and non-destructive, allowing the culture to be monitored repeatedly over time to generate a growth curve.
The OD reading measures biomass and light scattering, not an absolute cell count. The relationship between OD and the actual number of cells per milliliter is not always linear and is influenced by cell size and shape. For quantification, the OD measurement must first be correlated with a direct counting method, such as the CFU assay, to establish a standard curve for the specific organism and growth conditions.
Assessing Growth via Metabolic Output
Bacterial growth can be assessed indirectly by measuring the chemical consequences of their metabolic activity, focusing on the consumption of nutrients or the production of specific waste products. Measuring metabolic output provides a powerful tool for high-throughput screening and rapid detection of active cells.
One approach involves tracking the consumption of a primary energy source, such as glucose, or monitoring the production of metabolic end-products, like carbon dioxide (CO₂) generated during cellular respiration.
In clinical microbiology, systems often rely on detecting CO₂ accumulation in sealed culture bottles to quickly signal the presence of growing bacteria. Other methods monitor changes in the culture’s pH due to acidic compounds, or detect electron carriers like NADH. These biochemical indicators are useful for rapid assessments aimed at confirming active growth rather than obtaining a precise cell number.