Lecture 9 - Growth Measuring Growth Learning Objectives Be able to sketch and label a bacterial growth curve (x- and y-axis as well as phases). Explain what happens in each phase of a bacterial growth curve. Be able to use the growth equation to model bacterial growth. Explain the difference between primary and secondary metabolites, and give an example of each. Explain why it takes a very long time for 100% of the bacteria in a culture to die. Cite three differences between a continuous culture and a batch culture. Know HOW and WHEN to quantitatively measure bacterial growth by each of the following methods Direct counts Petroff-Hauser chamber Coulter counter Viable (Indirect) counts Dilution series + pour plate Dilution series + spread plate Membrane filtration method Most probable number method Biomass measurements Turbidity Total cell mass Know how bacterial growth can be quantified by measuring the accumulation of primary metabolites such as acid and gas, or by measuring ATP formation. Vocabulary lag phase exponential phase stationary phase death phase growth rate constant generation time primary metabolite secondary metabolite persister cells batch culture continuous culture chemostat direct cell count Petroff-Hauser chamber Coulter counter viable cell count pour plate spread plate membrane filter most probable number (MPN) turbidity (= optical density) biochip conductivity Durham tube luciferase The Bacterial Growth Curve Growth equation based on binary fission (same as the compound interest formula) Nt = N0emt N = number of cells m = growth rate constant ( / hr.) t = time (hr) How long will it take one cell dividing at a rate of 0.5 per hour to become a colony of 100,000 cells? 100,000 = (1) e0.5t t = 23 hrs What is the growth rate if it takes a cell 45 minutes to divide? Nt = N0emt 2 = (1) em(45/60) ln2 = 0.75m m = 0.92 / hr Production of metabolic products follows growth curve kinetics Decline phase can be VERY long, especially in mixed cultures especially due to persister cell survival Batch versus Continuous Culture • Batch is in a flask – growth stops when – wastes build up – nutrients run out – the cells become too crowded to get O2 • Continuous is in a chemostat – nutrients continually added at slow rates – bacteria perpetually in exponential phase – growth rate determined by nutrient resupply rate Counting Bacteria • Direct cell counts (all cells, live and dead ones) – Petroff-Hauser chamber – Coulter counter • Viable cell counts (only live cells) – Dilution and plating • pour plate • spread plate – filter plating – most probable number method • Biomass measurements – turbidity – weigh cells • Measure cell products – acid – gas – ATP Petroff-Hauser chamber allows microscopic counts of all cells within a grid square of known size Coulter counter measures interruption of electric current as cells pass through counter Dilution and plating schemes count all cells that can form colonies Spread plates put diluted culture ON TOP OF the agar Pour plates put diluted culture WITHIN the agar Membrane filtration method allows large volumes (100s of ml) to be counted MPN is a statistical method based on +/- growth in a large number of tubes at 3 different dilutions Turbidity is based on OD measurements with a spectrophotometer • more cells = more light scattered = higher OD • barely any scatter ~107 cells/ml • need to correlate OD with cell number for a given species Weigh cells directly • rules of thumb: – “typical” cell weighs ~1 pg wet – cell is ~70% water – cell is ~50% protein – You can harvest ~ 1g of cells (wet) from a liter of culture in a rich, complex medium Measurement of cell products depends on metabolism of cells being studied • acid from fermentation – easy to measure with pH electrode – micro-scale applications in food science Gases measured in Durham tube or by pH if gas is acidic ATP can be measured by luciferase reaction • sometimes done to see if any viable cells remain in a culture (no ATP = no metabolism = dead) • not quantitative since cells produce different amounts of ATP in different phases of growth