Bacterial Cultivation Growth Culturing Microorganisms Binary fission sporulation

Bacterial Cultivation & Growth • Culturing Microorganisms – Binary fission & sporulation – What determines growth? – Media Types – Batch vs Continuous Culture Growth

Binary Fission in Prokaryotes

Dividing vegetative cells But what happens when stressed or starved?

Endospores: • Resting stage during “lean or stressful times”. • Resistant protein coat! • Develop in different locations of vegetative cell: • Schaeffer – Fulton Stain: Young (24 h) free; sub-terminal; central; terminal Old (96 h)

Endospores & Sporulation (cortex) Spore coat proteins resist toxic chemicals. exosporium Dipicolinic Acid & Calcium protects DNA from heat.

Spore Germination: activation; germination; outgrowth

Culturing Microorganisms • Tolerance to All Environmental Factors (Shelford’s Law of Tolerance) • • • Temperature Solute Concentration / Water Activity p. H (acidity versus alkalinity) Oxygen Concentration Barometric Pressure Electromagnetic Radiation • Growth Limiting Resource (Liebig’s Law of the Minimum): Nutrient in least supply relative to bacterial needs will cap growth yield.

Closed (“batch”) Culture Systems “growth curve” Unbalanced Growth Balanced Growth: Rates of RNA = Protein = DNA = binary fission

Exponential Growth Phase Nt = N o + 2 n Number of generations (n) = (log Nt – log No) / log 2 Growth Rate Constant (k) = n/t It is expressed in units of generations per hours (h-1) Generation time (g) = 1/k; it is expressed in units of hours (h).

k = (log Nt – log No) / 0. 301 t Generation time (g) = doubling time Time (hours)

Nutrient Concentration Effects in Batch Cultures: • Total growth will increase until limiting nutrients are exhausted (included oxygen for aerobes) or metabolic byproducts accumulate that change environmental conditions to inhibit growth (toxicity). • Growth rate will also increase with increasing nutrient concentration up to a some maximum value, beyond which there is no effect (transporters are saturated with there substrate.

Open (“continuous”) Culture Systems Chemostat: growth rate = dilution rate (D = f/V); constant dilution rate with nutrient limiting growth. Turbidostat: dilution rates varies to maintain constant turbidity (cell density); no limiting nutrient. Flow (f) Volume (V) Wash out!