Chapter 6 – Microbial Growth

Physical Requirements for Microbial Growth

Microbial growth refers to an increase in the number of cells, not the size of individual cells. The physical environment plays a crucial role in determining whether and how fast microorganisms can grow. The main physical factors are temperature, pH, and osmotic pressure.

• Temperature: Microorganisms are classified based on their preferred temperature ranges. Each group has a minimum, optimum, and maximum growth temperature.

  • Psychrophiles (cold-loving): Capable of growth at 0°C.

  • Mesophiles (moderate-temperature-loving): Optimum growth at 25–40°C; includes most pathogenic bacteria, which grow best at 37°C.

  • Thermophiles (heat-loving): Optimum growth at 50–60°C.

• pH: The pH scale measures acidity or alkalinity. Most bacteria grow best at pH 6.5–7.5. Molds and yeasts prefer pH 5–6. Laboratory media often use phosphate salts as buffers to maintain stable pH.

• Osmotic Pressure: High salt concentrations cause water to leave bacterial cells, leading to plasmolysis (shrinkage of the plasma membrane), which inhibits growth. Salt is used to preserve food by increasing osmotic pressure. Some bacteria tolerate or require high salt:

  • Obligate halophiles: Require high salt concentrations.

  • Facultative halophiles: Can grow in up to 2% salt but do not require it.

Chemical Requirements for Microbial Growth

Microorganisms require various chemical elements for growth, which are essential for building cellular components and supporting metabolism.

• Carbon: Backbone of all organic molecules.

• Nitrogen: Needed for proteins, DNA, RNA, and ATP synthesis.

• Sulfur: Required for some amino acids and vitamins (thiamine, biotin).

• Phosphorus: Essential for DNA, RNA, ATP, and phospholipids.

• Potassium, Magnesium, Calcium: Serve as enzyme cofactors.

Oxygen requirements vary among organisms:

• Obligate aerobes: Require oxygen for growth.

• Facultative anaerobes: Use oxygen if present but can grow without it (via fermentation or anaerobic respiration).

• Obligate anaerobes: Cannot tolerate oxygen (e.g., Clostridium).

• Aerotolerant anaerobes: Do not use oxygen but are not harmed by it.

• Microaerophiles: Require oxygen at lower concentrations than atmospheric levels.

Culture Media

A culture medium is a nutrient material prepared for microbial growth in the laboratory. The microbes that grow are called a culture. Solid media contain agar, a polysaccharide from seaweed, and are used in Petri dishes or test tubes (slants). Media must provide nutrients, moisture, proper pH, and oxygen levels, be sterile, and be incubated at the correct temperature.

• Chemically defined media: Exact chemical composition is known; used for specific research needs.

• Complex media: Made from extracts (yeast, meat, plants); composition varies. Nutrient broth is liquid; nutrient agar is solid.

• Anaerobic growth media: Use reducing agents to remove oxygen for obligate anaerobes.

• Capnophiles: Organisms that grow better at high CO2 concentrations, often found in body tissues.

Specialized media types:

• Selective media: Suppress unwanted microbes and encourage desired ones.

• Differential media: Distinguish colonies of different microbes (e.g., blood agar for Streptococcus pyogenes).

• Combined selective and differential media: Example: MacConkey agar for Salmonella detection.

• Enrichment culture: Increases small numbers of desired microbes to detectable levels, often by repeated transfers.

Some bacteria cannot be grown on artificial media and require living hosts (e.g., leprosy and syphilis pathogens).

Isolation of Pure Cultures

Pure cultures are essential for studying individual microbial species. Most samples contain mixed populations, so isolation is necessary.

• Streak plate method: A sterile loop is used to spread bacteria over the surface of solid media in a pattern that separates individual cells. After incubation, isolated colonies arise from single cells or clumps.

• If the target organism is rare, enrichment is performed before streaking.

Preservation of Microbes

Microbial cultures can be preserved for future use by various methods:

• Refrigeration: Short-term storage.

• Deep-freezing: Microbes are suspended in liquid and frozen at –50 to –95°C for long-term storage.

• Lyophilization (freeze-drying): Water is removed by high vacuum after freezing at –54 to –72°C, preserving microbes for years.

Bacterial Growth and Generation Time

Bacteria reproduce mainly by binary fission, resulting in population growth. The time required for a cell to divide and the population to double is called the generation time.

1. Cell elongates and DNA replicates.

2. Cell wall and membrane grow inward between DNA regions.

3. Cross-wall forms, dividing the cell.

4. Two identical daughter cells result.

Some bacteria reproduce by budding or fragmentation. Generation times vary (typically 1–3 hours). Because populations grow rapidly, logarithmic scales are used to graph growth.

Phases of Microbial Growth

When bacteria are cultured, their population growth follows a characteristic pattern with four phases:

• Lag phase: Little or no cell division; cells adjust to new environment and synthesize enzymes and DNA.

• Log (exponential) phase: Rapid, constant-rate cell division; cells are most sensitive to adverse conditions.

• Stationary phase: Growth rate slows; number of new cells equals number of deaths due to nutrient depletion, waste accumulation, or pH changes.

• Death phase: Number of deaths exceeds new cells; population declines logarithmically.

Direct Methods of Measuring Microbial Growth

Direct methods involve counting cells or colonies to estimate population size.

• Plate counts: Measures viable cells by counting colonies formed on agar plates after serial dilution. Results expressed as colony-forming units (CFU) per mL.

• Filtration: Used for small bacterial populations; water is filtered, and bacteria are retained on a membrane, then cultured.

• Most Probable Number (MPN) method: Statistical estimation based on dilution series; useful for microbes that do not grow on solid media.

• Direct microscopic count: Cells are counted under a microscope using a defined volume. Fast but counts both live and dead cells; motile cells are difficult to count.

Indirect Methods of Measuring Microbial Growth

Indirect methods estimate cell numbers based on other properties.

• Turbidity: Cloudiness of a culture is measured with a spectrophotometer; only useful for high cell densities.

• Metabolic activity: Amount of a metabolic product (e.g., acid, CO2) is proportional to cell number.

• Dry weight: Used for filamentous organisms; cells are filtered, dried, and weighed.

Table: Oxygen Requirements of Microorganisms

Example: Application of Selective and Differential Media

• Blood agar: Used to detect hemolytic bacteria such as Streptococcus pyogenes, which destroys red blood cells.

• MacConkey agar: Selects for Gram-negative bacteria and differentiates lactose fermenters (pink colonies) from non-fermenters (colorless).

Additional info: Logarithmic (exponential) growth can be described by the equation:

Where is the final number of cells, is the initial number, and is the number of generations.