Chapter 6 Part A – Microbial Growth

Definition & Scope of Microbial Growth

• “Growth” in microbiology = increase in the NUMBER of cells, not an enlargement of individual cell size.
• Central for food safety, clinical infection control, biotechnology, and ecological cycles.

Physical Requirements for Growth

Temperature

• Each species possesses a characteristic minimum, optimum, and maximum growth temperature.
• Cardinal-temperature model often resembles an asymmetric bell-shaped curve (Fig. 6.1 concept).

Major Temperature Classes

• Psychrophiles
  • True cold-lovers; optimum ≈ -5\,\text{to}\,15\,^{\circ}\text{C}.
  • Found in polar seas, alpine soils; seldom important in food spoilage because they grow slowly at refrigeration.
• Psychrotrophs
  • Grow between 0^{\circ}\text{C} and 20–30^{\circ}\text{C} (optimum near room temp).
  • Primary agents of refrigerated food spoilage; still capable of slow growth at 4^{\circ}\text{C}.
• Mesophiles
  • Optimum 25–40^{\circ}\text{C}; include most pathogens & normal microbiota whose optimum ≈ body temperature 37^{\circ}\text{C}.
• Thermophiles
  • Optimum 50–60^{\circ}\text{C}; important in composting, dairy pasteurization spoiling.
• Hyperthermophiles (Extreme thermophiles)
  • Grow 80–110^{\circ}\text{C}; archaeal domain; hot springs, deep-sea vents.
  • Enzymes stable at high T ⇒ biotechnological thermostable DNA polymerases.

Food-Safety Temperature Map (Fig. 6.2 logic)

• “Danger zone” \approx 20^{\circ}–50^{\circ}\text{C} where rapid bacterial multiplication & possible toxin production occur.
• Cooking temperatures > 60^{\circ}\text{C} destroy most microbes; the higher the T the shorter the required holding time.
• Refrigeration (0–4^{\circ}\text{C}) = very slow growth of spoilage microbes, inhibition of most pathogens.
• Freezing <0^{\circ}\text{C} = no significant growth though many cells survive.

pH

• Most bacteria prefer near-neutral pH 6.5–7.5.
• Molds/yeasts tolerate and often prefer acidic pH 5–6 ⇒ exploited in food preservation (cheese, pickles).
• Acidophiles thrive at extremely low pH (e.g., pH <2), important in industrial acid leaching & stomach pathogen Helicobacter pylori adaptation.
• Buffering (e.g., phosphate buffers) stabilizes pH in culture media.

Osmotic Pressure

• Hypertonic environments (high salt/sugar) draw water from cells ⇒ plasmolysis (cytoplasmic shrinkage) ⇒ growth inhibition ⇒ principle behind jams, jerky, salted fish.
• Obligate (Extreme) halophiles need high NaCl (up to 30\%) to maintain turgor; archaeal halophiles in Dead Sea.
• Facultative halophiles (e.g., Staphylococcus aureus) tolerate \le 10\% salt ⇒ concern for salted foods.
• Water follows osmotic gradients across plasma membrane; Figure 6.4 depicts normal 0.85 % NaCl vs. 10 % NaCl.

Chemical Requirements for Growth

Macronutrients

• Carbon (~50\% dry weight)
  • Backbone of all organic molecules.
  • Chemoheterotrophs use organic C sources; autotrophs fix CO_2.
• Nitrogen (~14\%)
  • Constituent of amino acids & nucleotides.
  • Sources: protein catabolism, NH4^+, NO3^-, or atmospheric N_2 (nitrogen fixation, e.g., Rhizobium).
• Sulfur & Phosphorus (~4\% Sulfur together with P)
  • S in amino acids cysteine & methionine and vitamins (thiamine, biotin). Sources: protein debris, SO4^{2-}, H2S.
  • P in DNA, RNA, ATP, phospholipids; supplied as PO_4^{3-}.

Trace Elements

• Fe, Cu, Zn, Mn, Mo required in minute (µM–nM) amounts; usually present as contaminants of water or glassware.
• Serve predominantly as enzyme cofactors, e.g., Fe in cytochromes.

Oxygen Relationships

• Presence/absence of O _2 dictates metabolic strategy & enzyme complement.

Toxic Forms of Oxygen

• Singlet O_2 (excited state).
• Superoxide radicals O2^- produced in respiration; detoxified by SOD: 2O2^- + 2H^+ \to O2 + H2O_2.
• Peroxide anion O2^{2-} in H2O2 neutralized by catalase: 2H2O2 \to 2H2O + O2 or peroxidase: H2O2 + 2H^+ \to 2H2O.

Organic Growth Factors

• Cell can’t synthesize; must be taken up: vitamins (e.g., B vitamins as coenzymes), amino acids, purines, pyrimidines.

Culture Media & Cultivation Technology

Definitions

• Culture medium: nutrient solution/gel designed for growth.
• Sterile: free of viable organisms ⇒ autoclaving, filtration.
• Inoculum: microbes introduced into medium.
• Culture: the resulting microbial growth.

Agar

• Complex polysaccharide (red-algal cell wall extract).
• Not digested by most microbes; melts at 100^{\circ}\text{C}, solidifies at \approx 40^{\circ}\text{C} ⇒ ideal for plate, slant, deep formulations.

Types of Media

• Chemically defined media: exact composition known; vital for fastidious autotrophs or metabolic studies.
• Complex media: extracts/digests of yeast, meat, or plants (nutrient broth/agar); rich but undefined.
• Reducing media: contain thioglycollate or oxyrase to chemically remove O_2; boiled/heated prior to inoculation.
• Selective media: suppress unwanted organisms, encourage target.
  • Example: EMB agar selects Gram-negatives.
• Differential media: visually distinguishes colonies.
  • Example: Blood agar (hemolysis patterns).
• Enrichment media: increases low-abundance microbes without necessarily inhibiting others.
  • Phenol-enrichment protocol isolates phenol-degraders from soil.

Anaerobic Culture Methods

• Anaerobic jar with GasPak: NaHCO3 + NaBH4 → CO2 + H2; palladium catalyzes H2 + O2 \to H_2O.
• Anaerobic glove box/chamber: continuous inert gas atmosphere.
• Candle jar / CO 2 packet: microaerophilic, CO2-enriched but not strictly anaerobic; supports capnophiles.

Isolation of Pure Cultures

• Pure culture = single species/strain.
• Colony originates (theoretically) from a single cell ⇒ colony-forming unit (CFU).
• Streak-plate method dilutes cells across agar surface to obtain isolated colonies (Fig. 6.10).

Preservation Techniques

• Deep-freezing -50^{\circ}\text{ to }-95^{\circ}\text{C} in glycerol/cryoprotectant.
• Lyophilization (freeze-drying): frozen at -54^{\circ}–-72^{\circ}\text{C} then vacuum-sublimated; long-term storage.

Microbial Reproduction & Population Dynamics

Asexual Reproduction Modes

• Binary fission (most common): cell elongates, DNA replicates, septum forms, daughter cells separate (Fig. 6.11).
• Budding (e.g., Caulobacter), conidiospores (filamentous actinomycetes), fragmentation of filaments.

Mathematical Description of Growth

• Cell number after n generations: N = N_0 2^{n}.
• Generation time (g): g = \dfrac{t}{n} where t = elapsed time.
• Example provided: 100 → 1,720,320 cells in 5 h;
  Solve n = \log_2 \left(\dfrac{1,720,320}{100}\right) \approx 14.1;
  g \approx \dfrac{5\,\text{h}}{14.1} \approx 21\,\text{min}.
• Semi-log plots (Fig. 6.13) linearize exponential phase; useful for generation-time estimation.

Bacterial Growth Curve (Batch Culture)

1. Lag phase: metabolic adjustment, enzyme synthesis, no net division.
2. Log (Exponential) phase: constant, maximal division rate; cells most susceptible to antibiotics/radiation.
3. Stationary phase: nutrient depletion & waste accumulation; growth rate = death rate; secondary metabolite (antibiotic) production peaks.
4. Death (Log decline) phase: viable cells decline exponentially; some populations enter long-term stationary or form spores.

Quantifying Microbial Growth

Direct Methods

• Plate count
  • Perform serial dilutions (10^{-1},10^{-2},…) ➔ spread/pour plates.
  • Choose plate with 25–250 CFUs; calculate:
  \text{CFU/ml} = \dfrac{\text{colonies} \times \text{dilution factor}}{\text{volume plated}}.
• Filtration
  • Vacuum pass large volume through membrane; filter placed on agar; useful for low-density aquatic samples.
• Most Probable Number (MPN)
  • Multiple-tube fermentation; statistical estimate by pattern of positive tubes compared to MPN table.
• Direct microscopic count (Petroff-Hausser cell counter)
  • Known chamber volume;
  \text{cells/ml} = \dfrac{\text{cells counted}}{\text{volume of squares counted}}; example: \dfrac{14}{8\times10^{-7}} = 1.75\times10^{7}.
  • Rapid, counts dead + live, requires ≥10⁷ cells/ml for accuracy.

Indirect Methods

• Turbidity (Spectrophotometry)
  • Optical density (OD) at \lambda=600\,\text{nm} proportional to biomass; requires standard curve.
• Metabolic activity
  • Measure rate of product formation/ substrate utilization (e.g., CO_2 evolution, acid production).
• Dry weight
  • Filter, oven-dry, weigh; common for fungi, filamentous bacteria where plating is unreliable.

Real-World & Ethical Connections

• Understanding growth parameters guides sterilization, pasteurization, and refrigeration standards (public health impact).
• Selective/differential media critical in clinical diagnostics to quickly identify pathogens (e.g., Streptococcus hemolysis).
• Enrichment & isolation fuel bioremediation (phenol degraders) and industrial strain development.
• Manipulating growth conditions can overproduce antibiotics, enzymes (biotechnology benefit) but also raises dual-use biosecurity considerations.
• Proper culture preservation prevents loss of genetic resources and maintains reproducibility—ethical duty of researchers.

Links to Foundational Principles & Previous Lectures

• Nutrient cycling (C, N, S) ties to microbial metabolism & ecological roles discussed earlier.
• Enzyme-catalyzed detoxification of oxygen radicals relates to prior coverage of oxidative phosphorylation & reactive oxygen species.
• Physical/chemical control strategies (heat, osmotic pressure) foreshadow upcoming lectures on antimicrobial treatments.