Growth of bacteria in culture Culture systems based on having: a pure culture suitable nutrient medium satisfactory gro

1. Growth of bacteria in culture Culture systems based on having: a pure culture suitable nutrient medium satisfactory growth conditions sterile technique to maintain pure culture

2. Heterotrophic E.coli Different micro-organisms have different nutrient requirements • Heterotroph- organism that uses complex organic carbon compounds as a source of carbon and energy • Photoautotroph- uses light as source of energy and CO2 as carbon source • Chemoautotroph- uses oxidation of simple inorganic compounds for energy and fixes CO2 as a source of carbon Chemoautotrophic bacteria appear purple, Phototrophic cyanobacteria

3. Bacteria can be grown on solid media or liquid media: • agar in Petri plates • agar in glass bottles or deep tubes (less dehydrating and anaerobic conditions) • flat sided bottles allowing growth as a monolayer • Liquid culture in a culture flask

4. Dynamics of growth usually studied in liquid culture • Many cells can be grown as a homogeneous single celled suspension e.g. many bacteria, yeast, some animal cells…. Need to be able to monitor the growth of organisms with time as 1. Want to make sure growth is occurring 2. Want to monitor for contamination 3. Want to follow stage of growth as some products produced during growth, others at the end of growth Batch culture • Cells inoculated into a sterile vessel containing a fixed amount of nutrients. Growth in the vessel normally follows the predicable “growth curve” • No nutrients are added or removed (a closed system) • Cells grow by binary fission, as cell divides population increases exponentially

6. A. Lag- • No increase in cell number, cells getting ready to grow and divide • length related to how the cells used as inoculum were previously treated • B. Log/exponential- • Cells growing at maximum rate for these conditions. • Growth is balanced i.e. nothing is limiting • C. Stationary- • Cell growth begins to be limited by lack of particular nutrients or accumulation of inhibitory compounds • No. Cells dividing are equal to cells dying, a plateau is reached. • D. Death Phase- • cells dying faster than dividing

7. Measuring growth 1. Direct counts e.g. using a haemocytometer 2. electronic 3. culture based e.g. dilution and spread plating 4. using a spectrophotometer

8. Direct counts using a Hemocytometer

9. Sample is viewed on special slide with fixed volume • Sample may be stained first to increase visability or to discern whether cells are viable • A count of number of cells in a primary square is made • This is repeated for several other squares • An average cells per primary square is calculated • As volume counted is known cells per ml can be calculated Direct counts using a Hemocytometer

10. Electronic counters e.g. Coulter counter The device counts because whenever a particle goes through the hole, the electronic system detects a sudden and momentary increase in resistance (a partial interruption of current flow) and a green vertical line appears on the screen, as shown at the top.

11. Issues with coulter counter • Does not distinguish between live and dead • Clumping is a problem • Interference is an issue • expense Other electronic devises include Flow cytometers that incorporate fluorescence and lasers to distinguish and count cells. These are very expensive and require dedicated technologists

12. Culture based methods 1. Pour plate- cells are mixed with molten agar and poured into petri plate, allowed to grow and colonies counted 2. Spread plate- cells are spread on the surface of an agar plate allowed to grow and colonies counted Both methods assume one cell gives rise to one colony

17. Colorimetric/spectrophotometric methods: Turbidity is a measure of the # of microbes: • measured by light scattering or absorbance in a spec • Cells must be single cells in suspension • Clumping makes the relationship non linear • Can make a standard curve for specific organism of cell number versus absorbance so can correlate absorbance with number of cells