2460 Chapter 9
Microbial Growth
Chapter 9
Ch 9.1 Binary Fission
Binary Fission
Most common form of bacterial reproduction.
4 Basic Steps of Binary Fission:
Growth of Cell Size and Increase in Cell Components
Replication of DNA
Division of the Cytoplasm (Cytokinesis)
Septum Formation and Division of Daughter Cells
Cell Growth Process:
Cell size increases.
Chromosome duplicates.
Cytokinesis occurs with the involvement of the FtsZ protein.
Formation of division septum separates daughter cells.
Animation illustrates the separation of daughter cells.
Ch 9.1 Z Ring Assembly
FtsZ Protein
Directs cytokinesis.
Assembles the Z ring to form the divisome.
Divisome activates the production of peptidoglycan and septum formation.
Example: M. smegmatis with red labeled FtsZ (Courtesy of Boutte Lab).
Ch 9.1 Generation Time
Definition of Generation Time:
Also known as Doubling Time, it refers to the time it takes to double the population.
Variation Among Species:
E. coli = 20 min
S. aureus = 30 min
B. subtilis = 120 min
M. tuberculosis = 15-20 hrs
Ch 9.1 Calculating Population Size
Growth Dynamics:
The growth is exponential when resources are unlimited.
Population can be predicted using the formula:
Where =>
$N_n$ = population size after n generations
$N_0$ = initial number of cells
$n$ = number of generations
Example: If the generation time is 30 min, there are 16 generations in 8 hours.
Ch 9.1 Growth Curve
Characteristics of Closed Cultures:
Have finite resources (e.g., nutrients).
Predictable growth pattern occurring in phases:
Lag Phase:
Cells adjust to culture medium; no increase in population.
Log (Exponential) Phase:
Binary fission occurs; cell replication outpaces cell death.
Stationary Phase:
Resources deplete; endospores may start forming. Cell replication equals cell death.
Death Phase:
Endospores persist; cell replication is less than cell death.
Ch 9.1 Growth Curve Visual Representation
Graph displaying logarithm of living bacterial cells over time with phases outlined:
1. Lag Phase: No increase.
2. Log Phase: Exponential increase in living cells.
3. Stationary Phase: Plateau in cell numbers; cell division and death are roughly equal.
4. Death Phase: Exponential decrease in living cells.
Ch 9.1 Open System Cultures
Advantages of Open Systems:
Infinite resources available.
Nutrients and air are replenished; dead cells and waste are removed (effluent).
Ideal for applications in industrial microbiology.
Measuring Growth
Importance:
Quantifying population size is crucial for determining infection, as well as contamination of water or food supply, etc.
Methods for Measuring Growth:
Microscopic Cell Count:
Cells counted under a microscope; requires manual counting from a transferred known volume onto a calibrated slide.
Cannot distinguish between live and dead cells.
Fluorescent Staining:
Allows differentiation between live and dead cells.
Coulter Counter:
Measures electrical resistance changes due to variations in cell density; does not differentiate between live and dead cells.
Viable Cell Count:
Samples are diluted and grown on solid media. Results presented in colony-forming units per volume (CFU/ml).
Countable range traditionally 30-300 CFU/ml for accuracy:
<30 = TFTC (Too Few To Count)
>300 = TNTC (Too Numerous To Count)
Optical Density (Turbidity):
Measured with a spectrophotometer; population increase corresponds with an increase in turbidity. This includes both dead and live cells.
Ch 9.1 Optical Density Growth Curve Example
Example Growth Curve of E. coli OD600:
Measurement values range from 0.1 to 0.001 over time in hours.
Ch 9.1 Fluorescence Staining
Involves counting cells under a microscope or flow cytometer.
Red stain used specifically binds to damaged cells to indicate dead cells.
Ch 9.1 Viable Cell Count Procedure
Procedure for Counting Viable CFU:
Samples are diluted and grown on solid media, displaying visible colonies.
Serial dilution is performed and colonies counted via pour plate or spread plate method.
Ch 9.1 Counting Colonies
Serial Dilution:
Achieves a range of 30-300 CFU/ml, ensuring distinguishable and separate colonies.
Dilution factors calculate the original CFU counts.
Ch 9.1 Pour Plate Method Steps
Bacterial sample is mixed with warm agar (45-50 °C).
Sample poured onto a sterile plate.
Sample swirled to ensure mixing and then allowed to solidify.
Plate is incubated until bacterial colonies grow.
Ch 9.1 Spread Plate Method Steps
Sample (0.1 mL) is poured onto solid medium.
Sample is spread evenly over the surface.
Plate is incubated until colonies grow on the medium's surface.
Ch 9.1 Most Probable Number (MPN) Method
Definition and Use:
A statistical method used to estimate low counts (<30 CFU/ml), particularly in water and food testing.
Utilizes three log dilutions (e.g., 1/1, 1/10, 1/100) grown in 3-5 replicates.
Growth determined as positive or negative based on observed patterns, which are compared to a reference table.
Ch 9.1 MPN Example
Example of Enumeration:
10 mL, 1 mL, and 0.1 mL of an original pond water sample used in tubes.
Incubation at 37 °C for 24 hours shows a pattern of positive growth.
Ch 9.1 MPN Reference Table
A detailed MPN Index Table shows combinations of positive results and their corresponding MPN indices for various patterns observed from five tubes per dilution, outlining 95% confidence limits.
Ch 9.1 Alternative Patterns of Growth
Some microorganisms divide asymmetrically (e.g., via budding) or fragmentation.
Fragmentation: Observed in cyanobacteria.
Budding: Seen in Planctomycetes, such as Gemmata obscuriglobus.
Ch 9.1 Biofilm Formation
Definition: Microbial biofilms are micro-ecosystems composed of one or more species that can provide increased protection.
** formation Environment:**
Commonly forms in liquid environments (e.g., rivers, pipelines, oral cavity).
Steps of Biofilm Formation:
Attachment of planktonic cells to a substrate.
Irreversible attachment: Cells transform to a sessile state.
Growth and division on the substrate.
Production of Extracellular Polymeric Substance (EPS).
Secondary colonization and dispersion of microbes to new locations.
Ch 9.1 Biofilm Formation Timeline
Visual representation showing timeline for each stage of biofilm formation, emphasizing the time frames for attachment, growth, EPS production, and secondary colonization.
Ch 9.1 Biofilm Formation Mechanism
Formation occurs through quorum sensing, a form of cell-to-cell communication stimulated by cell density or cellular stress.
Autoinducers: Small molecules that prompt varying actions based on cell density.
Classes of Autoinducers:
N-acylated homoserine lactones (found in Gram-negative bacteria).
Various short peptides (found in Gram-positive bacteria).
Ch 9.1 Biofilm and Human Health
Impacts on Health:
Biofilms may be beneficial (e.g., normal gut biota) or harmful (e.g., plaque formation on teeth).
Biofilms can provide resistance to antibiotics; cells in deeper layers may be metabolically inactive.
EPS may impede the diffusion of biocidal agents and foster an optimal environment for plasmid sharing.
Ch 9.1 Environmental Factors & Generation Time
Key Environmental Factors Influencing Growth:
Oxygen level.
pH.
Temperature.
Osmotic pressure.
Barometric pressure.
Ch 9.2 Oxygen Requirements
Oxygen Requirement Terminology:
Oxygen is not always required or tolerated by many organisms.
Environments lacking oxygen exist.
Oxygen Requirement Groups:
Obligate Aerobes: Must have oxygen.
Obligate Anaerobes: Cannot tolerate oxygen.
Facultative Anaerobes: Can utilize oxygen or ferment.
Aerotolerant Anaerobes: Tolerate but do not use oxygen.
Microaerophiles: Require low levels of oxygen.
Ch 9.2 Fluid Thioglycolate Medium (FTM)
Description:
A low agar concentration tube exhibiting a gradient of oxygen which helps determine aerotolerance based on growth location.
Ch 9.2 Oxygen Requirements Examples
Examples for each oxygen requirement classification:
Facultative Anaerobic: Escherichia coli
Obligate Anaerobic: Clostridium botulinum
Microaerophilic: Neisseria gonorrhea
Obligate Aerobic: Pseudomonas aeruginosa
Ch 9.3 pH Requirements
Impact of pH on Microbial Activity: