Microbial Growth

Microbial Growth

Chapter 9

Learning Objectives

  • Define the generation time for growth based on binary fission.

  • Identify and describe the activities of microorganisms undergoing typical phases of binary fission (simple cell division) in a growth curve.

  • Explain several laboratory methods used to determine viable and total cell counts in populations undergoing exponential growth.

  • Describe examples of cell division not involving binary fission, such as budding or fragmentation.

  • Describe the formation and characteristics of biofilms.

  • Identify health risks associated with biofilms and how they are addressed.

  • Describe quorum sensing and its role in cell-to-cell communication and coordination of cellular activities.

  • Interpret visual data demonstrating minimum, optimum, and maximum oxygen or carbon dioxide requirements for growth.

  • Identify and describe different categories of microbes with requirements for growth with or without oxygen:

    • Obligate aerobe

    • Obligate anaerobe

    • Facultative anaerobe

    • Aerotolerant anaerobe

    • Microaerophile

    • Capnophile

  • Give examples of microorganisms for each category of growth requirements.

Continuation of Learning Objectives

  • Illustrate and briefly describe minimum, optimum, and maximum pH requirements for growth.

  • Identify and describe the different categories of microbes with pH requirements for growth:

    • Acidophiles

    • Neutrophiles

    • Alkaliphiles

  • Give examples of microorganisms for each category of pH requirement.

  • Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth.

  • Identify and describe different categories of microbes with temperature requirements for growth:

    • Psychrophile

    • Psychrotrophs

    • Mesophile

    • Thermophile

    • Hyperthermophile

  • Give examples of microorganisms in each category of temperature tolerance.

  • Identify and describe different categories of microbes with specific growth requirements other than oxygen, pH, and temperature, such as altered barometric pressure, osmotic pressure, humidity, and light.

  • Give at least one example microorganism for each category of growth requirement.

  • Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective, differential, defined, and enrichment media.

Outline of Chapter Content

  1. How Microbes Grow

  2. Oxygen Requirements for Microbial Growth

  3. The Effects of pH on Microbial Growth

  4. Temperature and Microbial Growth

  5. Other Environmental Conditions that Affect Growth

  6. Nutritional Requirements and Media

9.1 How Microbes Grow

Binary Fission

  • Most common form of bacterial reproduction.

  • Consists of 4 basic steps:

    1. Growth of cell size and increase in cell components.

    2. Replication of DNA.

    3. Division of the cytoplasm (cytokinesis).

    4. Septum formation and division of daughter cells.

Illustration of Binary Fission

  • Cell size increases.

  • Chromosome duplicates.

  • DNA replication formation of division septum.

  • Cell elongation with the FtsZ protein.

  • Cytokinesis with FtsZ directs the cell separation and the formation of daughter cells.

Z Ring Assembly

  • Cytokinesis is directed by the FtsZ protein.

  • FtsZ assembles a Z ring to form a divisome.

  • Divisome activates the production of peptidoglycan and the septum.

Generation Time

  • Generation Time (Doubling Time): the time it takes to double a population.

  • Varies greatly among species.

    • Example: E. coli = 20 min, S. aureus = 30 min, B. subtilis = 120 min, M. tuberculosis = 15-20 hrs.

Calculating Population Size

  • Growth is exponential if resources are not a concern.

  • Population can be predicted from any starting size with the equation: N_n = N_0 imes 2^n

    • Where:

    • $N_n$ = predicted cell number after n generations.

    • $N_0$ = initial number of cells.

    • $n$ = number of generations.

  • Example: If the generation time is 30 minutes, there would be 16 generations in 8 hours.

Growth Curve

  • Closed cultures have finite resources (i.e., nutrients); a predictable pattern occurs:

    1. Lag Phase: Cells adjust to culture medium; no change in population.

    2. Log (Exponential) Phase: Binary fission occurs; cell replication rate > cell death rate.

    3. Stationary Phase: Resources are depleted; endospores can start forming, and cell replication rate = cell death rate.

    4. Death Phase: Endospores persist; cell replication rate < cell death rate.

Growth Curve - Visual Representation

  • Labels:

    1. Lag phase: no increase in living cells.

    2. Log phase: exponential increase in living cells.

    3. Stationary phase: plateau in number of living cells; cell division rate equals cell death rate.

    4. Death or decline phase: exponential decrease in living cells.

Measuring Growth

Open System Cultures

  • Open systems have infinite resources.

  • Nutrients and air are replenished while dead cells and waste are removed (effluent).

  • Beneficial for industrial microbiology.

Importance of Population Size and Counting Methods

  • Quantifying population size is essential for determining infection, contamination, or food supply safety.

  • Various methods exist:

    • Microscopic cell count.

    • Fluorescent staining for live and dead cells.

    • Coulter count.

    • Viable cell count.

    • Optical Density (turbidity).

Microscopic Cell Count

  • Cells are counted under a microscope.

  • A known volume is transferred to a calibrated slide and counted manually.

  • Limitation: Cannot distinguish live from dead cells.

Coulter Counter

  • Detects changes in electrical resistance due to cell density.

  • Limitation: Does not differentiate between live and dead cells.

Measuring Optical Density

  • Optical Density (turbidity) is measured with a spectrophotometer.

  • Light is passed through culture; increased population correlates with increased turbidity.

  • Includes dead and live cells in the measurement.

Fluorescence Staining

  • Cells counted using a microscope or flow cytometer.

  • Red stain binds to damaged/dying cells to indicate dead cells.

Viable Cell Count

  • Samples diluted and grown on solid media; results are expressed in colony-forming units per volume (CFU/ml).

  • Limited to easily cultured species.

  • Serial dilution is plated and counted via pour plate or spread plate techniques.

  • Countable range is traditionally between 30-300 CFU/ml for statistical accuracy:

    • <30 CFU/ml = TFTC (Too Few To Count)

    • >300 CFU/ml = TNTC (Too Numerous To Count).

Serial Dilution for Viable Counts

  • Required to achieve countable colony numbers (30-300 CFU/ml).

  • Using dilution factors helps determine original CFU counts.

Pour Plate Method

  1. Bacterial sample mixed with warm agar (45-50 °C).

  2. Poured onto a sterile plate.

  3. Sample swirled to mix; allowed to solidify.

  4. Plate incubated until colonies grow.

Spread Plate Method

  1. Sample (0.1 mL) poured onto solid medium.

  2. Spread evenly over surface.

  3. Incubated until bacterial colonies grow on solid medium surface.

Most Probable Number (MPN)

  • Statistical method used for very low counts (<30 CFU/ml).

  • Useful in water and food testing.

  • Utilizes 3 log dilutions (e.g., 1/1, 1/10, 1/100) in 3-5 replicates.

  • Growth indicates positive or negative results; pattern compared to reference tables for statistical analysis.

Visual Example of MPN Analysis

  • Examples illustrated of incubation patterns and corresponding positive tubes for CFU/ml computations.

Alternate Patterns of Growth

  • Some microbes divide asymmetrically, such as buddies or through fragmentation.

  • Examples:

    • Fragmentation in cyanobacteria.

    • Budding of Planctomycetes.

Biofilm Formation

  • Micro ecosystems with one or more species providing protection; primarily forms in liquid environments (e.g., rivers, pipelines, oral cavity).

  • Steps of biofilm formation:

    1. Attachment of planktonic cells to substrate.

    2. Attachment becomes irreversible; cells become sessile.

    3. Growth and division on substrate.

    4. Production of extracellular polymeric substance (EPS).

    5. Attachment of secondary colonizers and dispersion of microbes to new locations.

Stages of Biofilm Formation

  1. Reversible attachment - cells attach temporarily.

  2. Irreversible attachment - cells attach permanently.

  3. Growth and division - cells grow; division occurs over time.

  4. Production of EPS and formation of water channels - enhancing nutrient flow and waste removal.

  5. Attachment of secondary colonizers and microbial dispersion.

Quorum Sensing in Biofilms

  • Biofilms form through quorum sensing or cell-to-cell communication.

  • High cell density or cellular stress leads to autoinducer small molecules production that induces various actions.

    • Classes of signaling molecules:

    • N-acylated homoserine lactones (typical of Gram-negative bacteria).

    • Various short peptides (typical of Gram-positive bacteria).

Biofilm and Human Health

  • Biofilms can have both beneficial and detrimental effects:

    • Beneficial: Normal gut microbiota.

    • Detrimental: Dental plaque formation.

  • Biofilms often exhibit resistance to antibiotics due to:

    • Metabolically inactive cells in deep layers.

    • EPS may slow the diffusion of biocidal agents.

    • Provide an optimal environment for sharing plasmids.

9.2 Oxygen Requirements for Microbial Growth

Environmental Factors Affecting Growth

  • Main factors that affect microbial growth include:

    • Oxygen level

    • pH

    • Temperature

    • Osmotic pressure

    • Barometric pressure

Oxygen Requirements and Microbial Classification

  • Oxygen is not always needed or tolerated. Many environments lack oxygen.

  • Microbes can be grouped based on oxygen requirements:

    • Obligate Aerobes: Must have oxygen to survive.

    • Obligate Anaerobes: Cannot survive in the presence of oxygen.

    • Facultative Anaerobes: Can grow with or without oxygen but prefer oxygen.

    • Aerotolerant Anaerobes: Tolerate the presence of oxygen but do not use it.

    • Microaerophiles: Require reduced oxygen levels, usually between 2-10%.

Fluid Thioglycolate Medium (FTM)

  • A low percentage agar tube used to establish an oxygen gradient, which can determine aerotolerance based on growth location in the tube.

Examples of Oxygen Requirement

  • (A) Facultative Anaerobic: Escherichia coli.

  • (B) Obligate Anaerobic: Clostridium botulinum.

  • (C) Facultative Anaerobic: Staphylococcus aureus.

  • (D) Microaerophilic: Neisseria gonorrhea.

  • (E) Obligate Aerobe: Pseudomonas aeruginosa.

Summary of Bacterial Oxygen Requirements

  • Obligate Aerobes: Example - Micrococcus luteus.

  • Obligate Anaerobes: Example - Bacteroides spp.

  • Facultative Anaerobes: Example - Staphylococcus spp.

  • Aerotolerant Anaerobes: Example - Lactobacillus spp.

  • Microaerophiles: Example - Campylobacter spp.

9.3 The Effects of pH on Microbial Growth

Importance of pH

  • pH affects the efficiency of macromolecules; proteins are particularly vulnerable to changes in pH.

  • Microbes can have preferences for acidic (<7), neutral (~7), or basic (>7) environments.

  • Fermenting organisms are often adapted to acidic conditions (e.g., pickles).

    • Microbes have minimum, optimal, and maximum pH levels for growth.

Categorizing Microbes by pH Requirement

  • Groups include:

    • Neutrophiles: Optimal pH ~7.

    • Acidophiles: Optimal pH <5.5.

    • Alkaliphiles: Optimal pH 8-10.5.

9.4 Temperature and Microbial Growth

Temperature Requirements

  • Optimal ranges for growth vary among microbes based on their classifications.

  • Temperature categories include:

    • Mesophiles: 20-40°C.

    • Psychrotrophs: 4-20°C.

    • Psychrophiles: <0°C.

    • Thermophiles: 50-80°C.

    • Hyperthermophiles: 80-110°C; some can survive at >121°C.

9.5 Other Environmental Conditions that Affect Growth

Osmotic Pressure and Microbial Growth

  • Solute concentrations outside the cell can affect microbial growth.

  • Halophiles: Salt lovers found in oceanic environments.

  • Halotolerant: Organisms that can tolerate high salt concentrations, typically seen in salt marshes.

Barometric Pressure and Microbial Growth

  • Microbes adapted to high pressures are often found at the ocean's depths.

  • Many such microbes are largely unculturable, and little is known about them, though some are also thermophiles or hyperthermophiles.

9.6 Nutritional Requirements and Media

Nutritional Requirements and Culture Media

  • Media can be selective and supportive of specific microbial growth or differential based on various parameters (e.g., salinity, fermentation, pH).

  • Fastidious organisms require very specific nutritional conditions, often needing specialized media like blood agar.

Hemolysis Types in Bacterial Culturing

  • Alpha (α) hemolysis: Reduction of hemoglobin, causing a brown coloration (e.g., Streptococcus pneumoniae).

  • Beta (β) hemolysis: Complete lysis of red blood cells, creating a clear zone (e.g., Streptococcus pyogenes).

  • Gamma (γ) hemolysis: Growth without hemolysis (e.g., Enterococcus faecalis).

Selective Media Examples

  • Mannitol Salt Agar (MSA): Selects for and differentiates pathogenic and non-pathogenic Staphylococci.

  • MacConkey Agar: Selects for and differentiates Gram-negative and lactose fermenters, such as E. coli and S. typhi.