Microbial Growth

How Microbes Grow

Bacterial cell cycle: formation of new cells via DNA replication and partitioning.
Prokaryotic reproduction: asexual.
Most bacteria: single circular chromosome.

Binary Fission

Most common replication mechanism.
Cell growth: increases cellular components before division.
DNA replication: starts at the origin of replication and continues bidirectionally until terminus.

Binary Fission Key points

Offspring receive complete parental genome copy.
Cytokinesis: division of cytoplasm.
FtsZ protein: directs cytokinesis by forming a Z ring.
Divisome: structure formed by additional proteins on the Z ring, produces peptidoglycan cell wall, and builds a septum.

Generation Time

Eukaryotes: time between life cycle points in successive generations.
Prokaryotes: doubling time (time for population to double).
Doubling times vary: E. coli (20 min), M. tuberculosis (15-20 hours), M. leprae (14 days).

Calculating cell number

Exponential increase: $2^n$ (n = generations).
Formula: $N<em>n = N</em>02^n$ ( $N<em>n$ = cells at generation n, $N</em>0$ = initial cells).

Growth Curve

Models cell number in culture over time.
Closed culture: reproducible pattern, no nutrients added, waste not removed.
Culture density: cells per unit volume.

Growth curve phases

Lag: cells prepare for growth, increase size, synthesize proteins, repair damage; duration varies.
Log (exponential): active division by binary fission, number increases exponentially.
Stationary: total live cells plateau, waste accumulates, nutrients deplete.
Death (decline): dying cells exceed dividing cells, exponential decrease.

Sustaining microbial growth

Chemostat: maintains continuous culture in log phase with steady nutrient supply.

Measurement of Bacterial Growth

Bacterial counts: estimate cell number.
- Direct methods: counting cells.
- Indirect methods: measure presence/activity without counting.

Direct Cell Count Methods

Direct microscopic cell count: Uses calibrated slide (Petroff-Hausser chamber).
Electronic cell counting (Coulter counter): detects electrical resistance changes, doesn't differentiate live/dead cells.

Plate Count

Viable plate count: live cells form visible colonies, expressed as CFU/mL.
Methods: pour plate, spread plate, both begin with serial dilution.

Indirect Cell Counts

Turbidity: measures cloudiness using a spectrophotometer.
Spectrophotometer: measures light transmission, converted to % transmission or absorbance.
Dry weight: measures culture density, useful for filamentous microorganisms.

Alternative Cell Division Patterns

Asymmetrical division (budding).
Fragmentation.

Biofilms

Complex ecosystems on surfaces.
Structured communities influencing structure with environmental conditions such as nutrient availability.
Streamers (filamentous biofilms): form in flowing water.
Mushroom shape: forms in still water.

Biofilms and Human Health

Intestinal/respiratory microbiota: ward off pathogens.
Dental plaque: contributes to disease.
Wounds/medical devices: cause infections.

Oxygen Requirements

Reactive Oxygen Species (ROS) are unstable ions and molecules derived from partial reduction of oxygen and examples include:
- Singlet oxygen ( $O_2$ ●)
- Superoxide ( $O_2$ –)
- Peroxides ( $H<em>2O</em>2$ )
- Hydroxyl radical (OH●)
- Hypochlorite ion (OCl–)

Thioglycolate tube cultures

Used to observe oxygen requirements of microorganisms.

Types of Microorganisms Based on Oxygen Requirements

Obligate aerobes: require abundant oxygen.
Facultative anaerobes: thrive in presence/absence of oxygen (fermentation/anaerobic respiration).
Aerotolerant anaerobes: don't use oxygen, not harmed by it (fermentative metabolism).
Microaerophiles: require minimal oxygen.
Obligate anaerobes: killed by oxygen.

Clostridium

Gram-positive, rod-shaped obligate anaerobe.
Forms endospores for survival.
C. difficile: health-acquired infections.
C. tetani: tetanus.
C. perfringens: gas gangrene.

Studying Obligate Anaerobes

Anaerobic jar: removes oxygen, releases $CO_2$ .
Anaerobic chamber: enclosed box with oxygen removed.

Oxygen Concentration

Optimum: ideal concentration for growth.
Minimum permissive: lowest concentration allowing growth.
Maximum permissive: highest tolerated concentration.

Detoxification of Reactive Oxygen Species

Enzymes: superoxide dismutase, peroxidase, catalase.
Reaction 1: peroxidases
Reaction 2: superoxide dismutase (SOD) breaks down superoxide anions: $O<em>2^- + O</em>2^- + 2H^+ \rightarrow H<em>2O</em>2 + O_2$
Reaction 3: catalase converts hydrogen peroxide to water and oxygen: $2H<em>2O</em>2 \rightarrow 2H<em>2O + O</em>2$ .

Catalase Test

Distinguishes streptococci (aerotolerant, no catalase) from staphylococci (facultative anaerobes).
Positive result: bubbles released when mixed with 3% hydrogen peroxide.

Enzymes Present in Microorganisms Based on Oxygen Requirements

Obligate anaerobes: usually lack all 3 enzymes.
Aerotolerant anaerobes: have SOD but no catalase.

Capnophiles

Grow in higher $CO_2$ , lower oxygen than atmosphere.
Candle jar: consumes oxygen, releases $CO_2$ .

Effects of pH on Microbial Growth

pH < 7.0: acidic.
pH > 7.0: basic.

pH Growth Values

Optimum growth pH: most favorable pH.
Minimum growth pH: lowest tolerated pH.
Maximum growth pH: highest tolerated pH.

Microorganisms Based on pH

Neutrophiles: optimum pH near 7 (E. coli, staphylococci, Salmonella).
Acidophiles: optimum pH < 5.55 (Sulfolobus, Ferroplasma, Lactobacillus).
Alkaliphiles: optimum pH 8.0-10.5 (Vibrio cholerae, Bacillus firmus, Natronobacterium).

Temperature and Microbial Growth

Microbes classified by growth temperature range.
Highest growth rates: at optimum growth temperature.
Minimum growth temperature: lowest survival/replication temperature.
Maximum growth temperature: highest growth temperature.

Temperature Classifications

Mesophiles: 20-45°C (human microbiota/pathogens).
Psychrotrophs: high of 25°C to refrigeration of about 4°C (spoilage of refrigerated food).
Psychrophiles: grow at 0°C and below, optimum close to 15°C, usually do not survive above 20°C (found in permanently cold environments).
Thermophiles: optimum 50-80°C (Thermus aquaticus, Geobacillus).
Hyperthermophiles: 80-110°C (Pyrobolus, Pyrodictium).

Temperature Effects

Low temperatures: membrane fluidity loss, ice crystal damage, slow reactions, protein rigidity.
High temperatures: protein/nucleic acid denaturation.

Thermophiles/Hyperthermophiles adaptations

Increased saturated/polyunsaturated lipid ratio.
Higher guanine-cytosine nitrogenous bases in DNA.
Thermoenzymes: Taq polymerase from T. aquaticus (PCR), degradation enzymes in hot-water detergents.

Other Factors Affecting Growth

Salinity, barometric pressure, humidity, light.

Osmotic and Barometric Pressure

Halophiles: require high salt concentrations (3.5%).
Halotolerant: survive/divide in high salt.
Barophiles: require high atmospheric pressure.

Light

Photoautotrophs and photoheterotrophs need sufficient light intensity.
Photosynthetically Active Radiation (PAR): 400-700 nm.
Accessory pigments widen the range of wavelengths.

Nutritional Requirements

General all-purpose media: support growth of many organisms (Tryptic Soy Broth - TSB).
Enriched media: contains growth factors, vitamins, essential nutrients for fastidious organisms.
Chemically defined media: known chemical composition.
Complex media: extracts/digests of yeasts, meat, plant (Nutrient broth, TSB, brain heart infusion).