Bacterial Growth and Growth Curve

• Bacterial growth occurs through binary fission, where one cell divides into two.
• This process leads to exponential growth.
• A single bacterium can multiply to a massive number in a short time under optimal conditions.
• Generation or doubling time varies among species.
• Doubling time depends on environmental factors such as temperature, nutrient availability, and waste accumulation.
• Exponential growth would result in a bacterium becoming 4.6 x 10^21 in one day with unlimited resources and a 20 min doubling time.

What Happens in Reality

• Exponential growth doesn't continue indefinitely due to temperature, lack of nutrients, and waste accumulation.

Growth Curve in Broth Culture

• Microbial growth in a closed system involves distinct phases.
• The nutrients are rich at the start, but no new are added.
• Wastes are not removed in a closed system.
• There are 4 phases of growth:
• Lag phase
• Exponential (log) phase
• Stationary phase
• Death phase

Lag Phase

• This occurs when bacteria transition from a "resting" state to optimal growth conditions.
• New enzymes are synthesized in the lag phase.
• DNA replication starts during the lag phase.
• Nutrients are plentiful but not replaced.
• Wastes are not removed during the lag phase.

Exponential (Log) Phase

• Nutrients are abundant during this phase.
• Cell numbers increase exponentially, forming a straight line on a logarithmic scale.
• The cells are most vulnerable to antibiotics like penicillin.
• Lots of primary metabolism and cell component synthesis occurs.

Transition or Late Log Stage

• Conditions are no longer optimal for growth.
• Nutrient concentration becomes limited.
• Waste products accumulate.

Stationary Phase

• Growth is very limited with a balance between cell division and cell death.
• Living bacteria consume nutrients from dying bacteria.
• Cells stay alive, but conserve energy.

Die Off

• Starvation occurs during this phase.
• Some cells die and all metabolism stops.
• Hibernation stages may be initiated, if possible.
• Mutations that prolong survival may be selected.
• This phase can last for years or more.

Growth Stages

• During exponential stage, nutrients are plentiful and the focus is on growth.
• During stationary stage, there are some nutrients and the focus is on competition.
• Cutthroat competition occurs in the stationary phase.

Tough Conditions

• This involves transition from growth to survival.
• It includes secondary metabolites, like antibiotics.

Colony Growth on Agar

• The position of a single cell determines its environment.
• At the edge of the colony there is little competiotion for O2 or nutrients.
• The center of the colony has depleted O2 and nutrients and has accumulated toxic wastes.
• Cells at the edges of the colony grow exponentially, while those in the center are in the death phase.
• Overlapping colonies stop growing on the edge between them.

Continuous Culture

• This uses a chemostat in the lab or industry and provides an open system for sustained growth.
• Constant nutrient levels and addition rates give a constant growth rate and cell density.
• Harvest commercially valuable products.
• The human digestive tract is an example in nature.

Commercial Production

• Normal metabolites (ethanol) are best produced during the exponential phase.
• Secondary metabolites (penicillin) are best produced during the transitional or early stationary phase.

Environmental Factors

• Temperature, oxygen, pH, water availability, and nutritional factors affect cell growth.
• Life is found if water is sometimes liquid, and some energy source is available.

Temperature Ranges

• High temperatures can denature proteins and unbind DNA, but proteins can evolve to be heat-resistant.
• Endospores stabilize protein and DNA by reducing water content and suspending metabolism.
• Low temperatures slow reactions, but can preserve bacterial species.
• There needs to be a balance between the temperature and protein/membrane structure and flexibility.

Temperature Groups

• Optimal growth is usually close to the upper end of tolerance.
• Psychrophiles grow from -5°C to 15°C and live in Arctic and Antarctic regions.
• Psychrotrophs grow from 15°C to 30°C and cause refrigerated food spoilage.
• Mesophiles grow from 25°C to 45°C and include most infectious agents.
• Human pathogens grow optimally at 35°C to 40°C.
• Thermophiles grow from 45°C to 70°C and live in hot springs and compost heaps.
• Hyperthermophiles grow at 70°C or greater and are usually archaea found in hydrothermal vents.

Oxygen

• Original microorganisms did not originally use oxygen.
• Chemolithotrophs get energy from minerals.
• Cyanobacteria evolved to produce oxygen from sunlight.
• Superoxide and Peroxide are oxygen by-products that damage cells.
• Life evolved to hide from oxygen, tolerate it, or use it.

Oxygen Strategies

• Obligate aerobes require O2.
• Facultative aerobes use O2, but don't require it.
• Obligate anaerobes cannot use O2 and are usually killed it.
• Microaerophiles require small amounts of O2 only.
• Aerotolerant anaerobes are obligate fermenters.
• Aerotolerant anaerobes, also grow in O2 presence, but don't use it.
• The most efficient, fast-acting enzymes on the planet are Superoxide Dismutase and Catalase.
• Facultative anaerobes get 16x as much energy from a glucose molecule if using oxygen.
• In limited-oxygen environments, they scavenge any trace O2, to maintain an anaerobic environment.

pH

• Most bacterial interiors have a neutral pH, maintained through active transport.
• Neutrophiles thrive in pH 5-8 environments while acidophiles thrive in pH below 5.5.
• Picrophilus oshimae has optimum pH of less than 1.
• Alkaliphiles grow optimally at pH above 8.5

Water Availability

• Bacteria vary in their ability to grow in high salt conditions.
• Salting preserves food!
• Halotolerant bacteria tolerate up to 10% salt.
• Halophiles require high salt concentration.
• Extreme halophiles survive in salt saturated water (e.g., evaporating lakes)

Why We Care?

• Knowing a microorganism's growth needs helps prevent or promote its growth.
• It also aids in understanding disease symptoms.

H. Pylori

• Helicobacter pylori survives in the stomach by breaking down urea to create a non-acidic environment.

Energy Sources

• Phototrophs obtain energy from sunlight (plants, algae, photosynthetic bacteria).
• Chemotrophs extract energy from chemicals (mammalian cells, fungi, prokaryotes, sugars, amino acids, fatty acids).

Nutritional Factors

• Carbon sources include heterotrophs (organic carbon) and autotrophs (inorganic carbon).
• CHONPS and certain minerals are required for growth.

Growth Factors

• Some organisms (e.g., E. coli) make the needed factors from glucose and trace elements.
• Other organisms (including humans) need compounds provided by diet or symbiosis (e.g., gut bacteria!).
• E. coli can grow in diverse environments, its behavior is impacted.
• In nutrient broth at 37°C, E. coli divides every 20 minutes.
• In minimal essential media", it divides more like 40 minutes to an hour.
• In the large intestine (natural home!) it divides more like 12 hours.

Reality

• Pure cultures are important for lab study.
• The real world is more complex.
• Media is often undefined, and almost always a complex mixture of species of organisms.
• Changing and complex conditions in nature differ greatly from lab conditions.
• Changing and complex conditions in nature impact microbial growth and behavior.
• Cells adjust to changes in surroundings by sensing various chemicals.
• Cells produce materials appropriate for the situation

Biofilms

• Most attach to surfaces and live in polymer-encased communities termed biofilms.
• Examples of biofilms are slipperiness of rocks in stream beds, slimy gunk in sink drains, scum in toilet bowls, and dental plaque
• Biofilms start with free cells attaching to a surface and multiplying .
• Polymers are released, allowing unrelated cells to attach and grow.
• Nutrients and wastes pass through channels.

Cell Communication

• Mixed microbial cell interactions can be cooperative or competitive.
• They can foster growth for needy species.
• They can consume the O2 to allow strict anaerobes to grow.
• Metabolic waste may be uptaken by others.
• They can synthesize toxic compounds or use needle-like structures to eliminate the competition.

Medical Lab

• The lab is used to identify infection and any special characteristics to give insight.
• Start by using Streak-Plate onto Agar for colony isolation.

Selective and Differential Media

• Selective media inhibits the growth of unwanted speices.
• Differential media contains substances that can determine if growth is wanted or not.
• MacConkey agar is selective for Gram-negative rods.
• Crystal violet inhibits Gram-positive bacteria.
• Bile salts inhibit most non-intestinal bacteria.
• Blood agar is differential.
• Beta hemolysis produces clear zone on blood agar.
• Alpha hemolysis (not a true lysis) produces zone of greenish partial clearing on blood agar.
• Selective or differential media can be used to detect or isolate a species from a mixed population.

Measuring Microbial Growth

• Use membrane filtration where a known volume is passed through a sterile filter
• The membrane captures and concentrates microbes.
• The filter is incubated on appropriate agar medium.

Atmospheric Conditions

• Most obligate aerobes and facultative anaerobes can be incubated in standard air concentrations.
• Broth cultures can be shaken to provide maximum aeration.
• Grow medically important bacteria will increased CO2.
• Microaerophilic environments require low O2 and can be made with a candle or chemical packet.