Chapter 5 : Bacterial Growth

Growth is defined as the orderly increase of all components of an organism. In multicellular organisms, it leads to an increase in size or mass, whereas in microorganisms, it results in an increase in the number of individuals. Thus, this increase is synonymous with multiplication, as most bacteria produce two daughter cells after 20 minutes that resemble each other and their parent cell. Growth is a phenomenon of significant biological interest, with considerable ecological and practical importance.

1. Measuring Growth

Growth measurement techniques are based on evaluating the number of microbes or their mass per unit volume or weight of the medium. It is important to note that these two metrics are not easily comparable. The increase in microbial number is a discontinuous phenomenon, while the increase in mass is a continuous process over time. Most methods allow for a direct estimation of microbial number or mass. In some cases, indirect methods are used, relying on measuring a parameter linked to metabolic activity.

1.1. Measuring Cell Number (Direct Measurement)

1.1.1. Direct Microscopic Cell Counting:

This method is commonly used for larger microbes (several µm), such as yeast. A hemocytometer (e.g., Thoma or Malassez cell—Fig.1) is employed. This is a slide with a cavity of known depth (0.1–0.2 mm) and a grid of known area on its surface. The microbial suspension is placed in this chamber and covered with a coverslip. Precise volumes are delineated by the grid, and cells are counted under a microscope. For smaller bacteria, higher magnifications are used, which reduces the depth and diameter of the field. In such cases, Petroff-Hausser cells (Fig.2) are often utilized.

Figure 1: Thoma cell (left) and Malassez cell (right).

Figure 2: Petroff-Hausser cells.

1.1.2. Epifluorescence :

Bacteria are stained with a fluorochrome like acridine orange and examined under ultraviolet light. Live bacteria fluoresce green, while dead bacteria fluoresce red due to the fluorochrome binding to denatured DNA. This method is delicate, as replicating DNA strands are partially unwound during multiplication.

1.1.3. Particle Counters:

These devices automatically count particles or cells suspended in an electrolyte solution. The measurement system consists of a cylindrical tube with a micro-aperture and electrodes on either side connected to a current generator. When a cell passes through the aperture, it displaces a volume of conductive solution equal to its own volume, causing a momentary increase in resistance. This resistance generates an impulse recorded by an electronic counter. The number of impulses corresponds to the number of particles passing through the aperture.

1.1.4. Enumeration After Culture :

Viable bacterial counting is a common method. The most routine technique involves culturing on Petri dishes. A fixed volume of the suspension or its dilution is spread on the surface of an agar medium or mixed into the medium before solidification. After incubation at a suitable temperature, the number of colonies corresponds to the number of microbial cells in the analyzed volume. To ensure accuracy, the experiment is repeated three times with thorough homogenization. Note that chains, clusters, or aggregates of microbes form a single colony. Results are expressed in colony-forming units (CFU). Another method is the most probable number (MPN), traditionally used for counting bacteria in liquid media.

1.2. Measuring Mass (Indirect Measurement)

1.2.1. Dry WeightDetermination:
Microorganisms are harvested by centrifugation or membrane filtration, washed with a buffer, and dried at 100–110°C until a constant weight is achieved. Results are expressed in grams of dry matter per liter. This method does not distinguish between live and dead cells.

1.2.2. Turbidity Measurement:
This is the simplest, fastest, and most widely used method for estimating microbial mass. It is an optical technique called turbidimetry, based on the principle that a solution absorbs part of the intensity of a light beam passing through it (absorbance).

1.3. Measuring Activity

Activity can be measured by monitoring substrate consumption, cellular constituents, excreted molecules, or physicochemical changes in the medium.

1.3.1. Substrate consumption:
The substrate may be a carbon source, nitrogen source, oxygen, or growth factor. This method is limited and less reliable due to dosage precision, interfering substances, and the ratio of cell mass formed per unit of consumed nutrient.

1.3.2. Cellular Constituents :
Ideal cellular constituents should be ubiquitous, disappear quickly after cell death, and remain at constant concentrations regardless of physiological state. Examples include proteins, DNA, ATP, FAD, and FMN.

1.3.3. Excreted Products:

Microorganisms release metabolic byproducts. Primary metabolites (e.g., amino acids, organic acids) are not excreted in large quantities, as cells reuse them. However, catabolic waste products can be measured effectively.

1.3.4. Physicochemical Changes in the Medium :

Changes in pH, electrical conductivity, redox potential, or heat release reflect growth dynamics. These parameters help correlate growth rate with the measured variable.

PH: Acidification indicates sugar degradation.

Impedance: Resistance to alternating current. Microbial activity converts neutral molecules (e.g., glucose) into charged molecules (e.g., organic acids), increasing conductivity.

Redox Potential: Decreases due to oxygen depletion and accumulation of reducing substances.

Heat Production : Metabolic reactions release heat, reflecting energy substrate degradation.

2. Growth Parameters

These parameters, also called growth constants, define bacterial growth under ideal conditions.

2.1. Generation Time :

The time between two successive divisions or the time needed for the population to double. For E. coli, it is ~20 minutes; for Mycobacterium tuberculosis, ~800–900 minutes in synthetic media.

2.2. Growth Rate :

Defined as the number of divisions per unit time (µ = n/t). For E. coli (dividing every 20 minutes), µ = 3 per hour; for M. tuberculosis, µ = 0.075.

3. Growth Curve

After 16–24 hours, bacterial growth stops. If growth remained constant, microbial mass would exceed the Earth's weight in 48 hours. Growth is limited by substrate depletion or toxic waste accumulation. The growth curve in liquid medium has four phases (Fig.3):

Lag Phase : No growth (µ = 0).

Exponential Phase : Constant, maximal growth rate (µ = constant).

Stationary Phase : Growth halts (µ = 0).

Decline Phase : Death and lysis (µ < 0).

Note : Transition phases include acceleration (between 1 and 2) and deceleration (between 2 and 3).

Figure 3: Growth curve.

4. Factors Influencing Growth

Key physicochemical factors include:

Temperature: Different microbes have optimal temperature ranges.

Substrate: Growth rate depends on nutrient availability (Fig.4).

Figure 4: Bacterial growth as a function of substrate.

5. Bacterial Culture

Bacterial study advanced with methods for isolation and cultivation. A nutrient preparation for culturing microbes is called a culture medium. The introduced bacteria constitute the inoculum. Culture media are classified by:

5.1. Consistency:

Liquid media (broths).

Solid media (agar plates or slants).

5.2. Composition:

Synthetic media (chemically defined).

Empirical media (poorly defined).

Selective media (promote specific bacteria).

Identification media (highlight biochemical traits).

Preservation media (maintain bacteria in a dormant state).

6. Antimicrobial Agents

Control of microbial growth is essential, especially for pathogens. Methods include:

Disinfectants: Kill pathogens on surface.

Antiseptics: Prevent microbial growth on living tissues.

Sterilization: Complete elimination of microbes.

Antibiotics: Natural or synthetic compound that kill bacteria without harming host cells. They can be bacteriostatic (inhibit growth) or bactericidal (kill bacteria).