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

2. Binary Fission in Prokaryotes

3. Dividing vegetative cells But what happens when stressed or starved?

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

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

6. Spore Germination: activation; germination; outgrowth

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

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

9. Exponential Growth Phase N t = N o + 2 n Number of generations (n) = (log N t – log N o ) / 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).

10. Time (hours) k = (log N t – log N o ) / 0.301 t Generation time (g) = doubling time

11. 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.

12. 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!