lecture 3

Dynamics of Microbial Growth

• Microbial growth is an increase in the number of cells, not their size.
• Bacteria multiply by binary fission, an exponential process.
• Doubling/generation time is the time it takes for a population to double.
• Generation times vary depending on the species and growth conditions.
• E. coli has a generation time of approximately 20 minutes.
• Exponential bacterial multiplication has significant health implications.

Principles of Microbial Growth

• Bacteria reproduce via binary fission.
• Microbial growth is exponential, increasing in cell number, not cell size.
• Doubling or generation time is the time it takes for the bacterial population to double.
• Generation time is variable based on the species and growth conditions.
• E. coli has a doubling time of about 20 minutes.
• Mycobacterium tuberculosis has a longer doubling time (about 12 hours).
• Exponential bacterial growth has important health consequences.

Principles of Prokaryotic Growth

• The equation for exponential bacterial multiplication is N₁ = N₀ × 2ⁿ.
• N₁ = number of cells at a given time
• N₀ = initial population
• n = number of generations

Microbial Growth in Nature

• Natural conditions differ significantly from laboratory settings regarding microbial growth and behavior.
• In nature, nutrients are continuously supplied in diluted form and waste products are continuously replaced, making growth more dynamic.
• Bacteria in natural conditions remain in log phase for longer periods but multiply more slowly.
• Bacteria in running water may form slime layers to aid in attachment.
• In contrast to nature, bacteria often don't produce adherent structures in the lab.

Biofilms

• Bacteria living free-floating in an aqueous environment are called planktonic bacteria.
• Biofilms are polymer-encased communities of bacteria that attach to surfaces.
• Examples include slipperiness of rocks, slimy gunk in drains, scum in toilets, and dental plaque.

Biofilms (continued)

• Adhered bacteria release extracellular polymeric substances (EPS), which include polysaccharides, DNA, and other hydrophilic polymers.
• EPS create a slimy appearance in biofilms.
• Biofilms are crucial in human health. They can contribute to dental plaque, tooth decay, gum disease, persistent ear infections, cystic fibrosis, and infections of medical implants and urinary catheters.
• Treatment of biofilm-related infections is often difficult because bacteria in biofilms demonstrate stronger resistance to disinfectants, antibiotics, and the body's defense mechanisms.

Interactions of Mixed Microbial Communities

• Bacteria in nature often interact closely and sometimes cooperate.
• Some bacteria that would not normally survive in certain environments may be able to live there owing to the presence of other bacteria.
• For example, mouth bacteria (aerobes) help create conditions suitable for anaerobic bacterial growth.
• Waste products from one species can serve as nutrients for another.
• The gastrointestinal tract hosts many diverse microorganisms living together.

Microbial Growth in Laboratory Conditions

• Bacteria are isolated and grown in pure cultures to study their characteristics and functions.
• A pure culture consists of organisms that descend from a single bacterial cell.
• Aseptic techniques are essential to obtain pure cultures.
• These techniques include cultivating bacteria on solid media (e.g., agar), culture medium, and cell separation methods.
• Only a small percentage (~1%) of prokaryotes can be successfully cultured.
• Most medically important bacteria can be grown in pure cultures.

Culture Techniques

• Agar, a polysaccharide extracted from marine algae, solidifies nutrient solutions.
• Agar benefits: Not degraded by bacteria, resistant to high temperature sterilisation, remains a liquid until 45°C (allowing nutrients to be added), and solidifies at 95°C, appearing translucent for viewing colonies.
• The streak-plate method is a common technique for isolating bacteria.
• Bacterial stock cultures are stored in refrigerators as slanted agar growth or at -70°C in glycerol solutions for long-term preservation, preventing ice crystal formation.

Prokaryotic Growth in the Lab

• Bacteria in the laboratory are often grown in broth (in flasks or tubes) or on agar plates.
• These conditions are considered closed or batch systems; neither waste nor nutrients are replenished.
• Bacterial growth in these closed systems follows a characteristic pattern called the growth curve, featuring five distinct stages.
• The exponential (log) phase is crucial medically because bacteria are highly vulnerable to antibiotics and other chemicals during this period.
• The log phase is where the generation time is typically measured.

Bacterial Growth in the Lab (continued)

• Primary metabolites form during the active multiplication stage.
• Secondary metabolites are produced primarily during the later log and stationary phases as environmental conditions change significantly.
• Medically important secondary metabolites (like antibiotics) are produced by organisms like Streptomyces during the late log phase.
• Maintaining continuous bacterial growth in the laboratory requires constantly adding nutrients and removing waste products, creating an "open system" or a continuous culture.

Environmental Factors Influencing Microbial Growth

• Major environmental factors: temperature, atmosphere/oxygen availability, pH, and water availability.
• Bacteria are categorized based on their optimal growth conditions for temperature, oxygen, pH, and water availability.
• Each bacterial species has a temperature range within which it can grow, beyond which growth is halted.
• The optimal growth temperature is the range where microorganisms grow most rapidly, with specific optima for various species.

Temperature Requirements

• Prokaryotic organisms are grouped into categories based on optimal growth temperature—a guideline indicating the temperature at which they grow best.
• Psychrophiles thrive at very low temperatures (-5°C to 15°C), frequently found in the Arctic and Antarctic or glacier-fed lakes.
• Psychrotrophs grow optimally at 15°C-30°C, causing food spoilage.
• Mesophiles grow well between 25°C and 45°C; many disease-causing bacteria belong to this group.
• Thermophiles live in temperatures of 45°C-70°C, like hot springs and compost heaps.
• Hyperthermophiles (in the Archaea group) reside in extremely high temperatures (70°C-110°C)

Temp, Food Preservation and Disease

• Foods are often stored at refrigeration temperatures (approximately 4°C) to restrain the growth of mesophiles.
• Although at colder temperatures the growth rate of psychrophiles and psychrotrophs is reduced, these organisms can still multiply and lead to food spoilage or microbial contamination, making freezing essential for long-term storage.
• Variations in human/animal body temperature influence microbial infections—certain pathogens thrive in particular body zones.
• Fever, a natural body response, can hinder microbial growth by raising body temperature.

Oxygen (O2) Requirements

• Prokaryotes are categorized according to their oxygen needs.
• Obligate aerobes absolutely require oxygen for respiration.
• Obligate anaerobes cannot grow in the presence of oxygen, often killed by its derivatives.
• Facultative anaerobes can survive with or without oxygen; they switch between aerobic and anaerobic respiration or fermentation depending on conditions.
• Microaerophiles need small amounts of oxygen but are impeded by high levels.
• Aerotolerant anaerobes can exist in the presence of oxygen but do not utilize it for energy generation.

Reactive Oxygen Species

• When oxygen is used in aerobic respiration, reactive oxygen species (ROS) like superoxide and hydrogen peroxide are produced as by-products.
• ROS can damage cell structures and are thus toxic.
• Organisms that use aerobic respiration have enzymes like superoxide dismutase and/or catalase to neutralize ROS.
• Aerotolerant anaerobes lack these enzymes.
• Specific tests exist to distinguish bacteria with or without catalase activity.

pH

• Normal internal human pH is ~7.0-7.4.
• Neutrophiles thrive in near-neutral pH (7).
• Phagocytes use acidic endosomes to destroy ingested bacteria.
• Acidification is a preservation method for inhibiting food spoilage.
• Some bacteria adapt to low pH conditions (e.g., Helicobacter pylori).
• Acidophiles thrive at low pH (<5.5), whereas alkalophiles flourish at high pH (>8.5).

Water Availability

• All cells need water for growth.
• Osmotic pressure changes due to water/solute differences affect cell structure via plasmolysis.
• Halophiles tolerate high salt concentrations (10% NaCl).
• High salt concentrations are used in food preservation.

Nutritional Factors Influencing Microbial Growth

• Various nutrients (e.g., fatty acids, sugars, amino acids, nucleotides, carbon, and nitrogen elements) are vital for microbial growth.
• Major elements are essential components of cell structure.
• Limiting nutrients (phosphorus and iron) are present in lower concentrations compared to demand.
• Trace elements, required in small amounts by all cells, are essential for enzymatic function.

Carbon and Energy Sources

• Heterotrophs use organic carbon for growth.
• Autotrophs utilize inorganic carbon (CO2) and are crucial for carbon fixation.
• Phototrophs derive energy from light.
• Chemotrophs obtain energy from chemical reactions.

Growth Factors

• Some bacteria lack the necessary enzymes to synthesize certain organic molecules and thus require external growth factors.
• Growth factor necessities vary among different microbes.
• Fewer enzymes for small molecule synthesis correlate with higher growth factor needs.

Cultivating Microorganisms in the Lab

• Media types:
  • Complex media (e.g., meat juices, peptones, beef extracts, nutrient broth, nutrient agar) – derived from complex substances.
  • Chemically defined media – utilize pure chemical components to define precise compositions.
• Selective media: inhibit the growth of unwanted organisms.
• Differential media: exhibit visible changes in the presence of specific bacteria (e.g., color changes, hemolysis).
• Examples include MacConkey Agar, Thayer-Martin Agar, Blood agar.

Differential Media

• Blood agar is a complex, differential media used to detect bacterial hemolysin production; not selective.
• Clear zones around colonies indicate beta-hemolysis (e.g., Streptococcus pyogenes).
• Greenish zones denote alpha-hemolysis (e.g., some Streptococcus species).

Selective and Differential Media

• MacConkey agar is both selective and differential, used to isolate Gram-negative intestinal bacteria.
• Selective due to the presence of crystal violet and bile salts.
• Differential due to lactose and pH indicator (resulting in pink colonies for lactose fermenters like E. coli).
• Treponema pallidum, the syphilis organism, cannot be cultivated in typical bacterial media.

Next Lecture

• Review material from chapters 10 and 12.

Test your knowledge on the principles of microbial growth, including bacterial reproduction methods and generation times. Discover how these concepts relate to health implications and various bacterial species. Perfect for students studying microbiology!