Microbial Growth Chapter 9 PDF

Microbial Growth Chapter 9 Learning Objectives Define "generation time" for growth based on binary fission Describe phases of binary growth in a growth curve Compare & contrast methods that quantify growth Describe methods capable of measuring viable growth Understand stages of biofil...

Microbial Growth Chapter 9 Learning Objectives Define "generation time" for growth based on binary fission Describe phases of binary growth in a growth curve Compare & contrast methods that quantify growth Describe methods capable of measuring viable growth Understand stages of biofilm formation Explain use of quorum formation Identify and describe different categories of microbes with requirements for growth with or without oxygen Identify and describe the different categories of microbes with pH requirements for growth Identify and describe different categories of microbes with temperature requirements for growth Ch 9. 1 Binary Fission Most common form of bacterial reproduction 4 Basic Steps: 1. Growth of cell size and increase in cell components 2. Replication of DNA 3. Division of the cytoplasm (cytokinesis) 4. Septum formation and division of daughter cells Ch 9. 1 Binary Fission Cytokinesis w/ Chromosome FtsZ protein duplicates Cell size increases Separation of daughter cells Animation Ch 9. 1 Z ring Assembly Cytokinesis is directed by FtsZ protein FtsZ assembles Z ring to form divisome Divisome activates production of peptidoglycan and septum M. smegmatis with red labelled FtsZ Courtesy of Boutte Lab Ch 9. 1 Generation Time Generation Time (Doubling Time) – time takes to double population Varies greatly among species E. coli = 20 min. S. aureus = 30 min. B. subtilis = 120 min. M. tuberculosis = 15-20 hrs. Ch 9. 1 Calculating Population Size Growth is exponential (if resources are no concern) Population can be predicted from any starting size Nn= N02n n - number of generations N0 – initial number of cells Number of generation may need to be calculated Ex. Generation time of 30 min = 16 generations in 8 hours Ch 9. 1 Growth Curve Closed cultures have finite resources (i.e. nutrients) Predictable pattern occurs 1. Lag phase – cells adjust to culture medium; no change in population 2. Log (exponential) phase – binary fission occurs; cell replication > cell death 3. Stationary phase – resources become depleted; endospores can start forming cell replication = cell death 4. Death phase – endospores persist cell replication < cell death Ch 9. 1 Growth Curve Ch 9. 1 Growth Curve Open system cultures have infinite resources Nutrients & air are replenished Dead cells & waste are removed Beneficial for industrial microbiology Measuring Growth Quantifying populations size is important for determining infection, contamination of water or food supply, etc. Methods: Microscopic cell count Fluorescent staining for alive & dead cells Coulter count Viable cell count Optical Density Ch 9. 1 Measuring Growth Microscopic cell count – cells are counted under a microscope Known volume is transferred to a calibrated slide and cells are manually counted Cannot distinguish live vs. dead Ch 9. 1 Measuring Growth Coulter counter – detects electrical resistance change due to cell density Does not differentiate live/dead Ch 9. 1 Measuring Growth Optical Density (turbidity) Animation Measured w/ spectrophotometer Light is passed thru culture and measured on other side Population increase = turbidity increase Includes dead & live cells Ch 9. 1 OD Growth Curve Ch 9. 1 Measuring Growth Fluorescence Staining – cells are counted under a microscope or flow cytometer Red stain binds to damaged cells to indicate dead cells Ch 9. 1 Measuring Growth Viable cell count – samples are diluted and grown on solid media Results expressed in colony forming units per volume (CFU/ml) Limited only to easily cultured species Serial dilution is plated and counted via pour plate or spread plate technique Countable range is traditionally 30-300 CFU/ml (statistically most accurate) 300 - TNTC Ch 9. 1 Measuring Growth Counting viable CFU requires distinguishable colonies Achieved thru serial dilution to achieve the 30-300 CFU/ml range Often dilutions are on log scale Dilution “factor” is used to determine original CFU count Measuring Growth Ch 9. 1 Measuring Growth 10-1 10-2 10-3 10-4 10-5 TNTC TNTC Correct range for calculating CFU/ml = 50 x 10 * 0.1 ml = 5.0 x 10 4 6 viable count Measuring Growth 10-1 10-2 10-3 10-4 10-5 TNTC TNTC CFU/ml = 50 x 10^5 = 5.0 x 10^6 2 x 10^6 CFU/ml 389 x 10^4 = 3.89 x 10^6 CFU/ml Ch 9. 1 Measuring Growth Most probable number (MPN) – statistical method used when counts are very low (