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Microbial Growth The Cell Cycle Microbial Growth and Cell Division Increase in mass Increase in cell numbers Mitosis in most eukaryotes Budding in yeasts Fragmentation in filamentous fungi Binary fission in bacteria Steps in Binary Fission Chromosome replication Chromosome attachment to cell membrane. Chromosomal segregation Septum formation Inward movement of cell wall and cell membrane dividing daughter cells Wall Elongation Binary Fission Bacterial Chromosome Replication Semiconservative Results in one new and one parental strand In Escherichia coli Bidirectionally from single origin Initiates replication Controls frequency of initiation events Segregates replicated chromosomes to daughter cells The Requirements for Growth: Physical Requirements Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature Temperature Ranges Figure 6.2 The Requirements for Growth: Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO2 The Requirements for Growth: Chemical Requirements Nitrogen Sulfur In amino acids, proteins Most bacteria decompose proteins Some bacteria use NH4+ or NO3 A few bacteria use N2 in nitrogen fixation In amino acids, thiamine, biotin Most bacteria decompose proteins Some bacteria use SO42 or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43 is a source of phosphorus The Requirements for Growth: Chemical Requirements Trace Elements Inorganic elements required in small amounts Usually as enzyme cofactors The Requirements for Growth: Chemical Requirements Oxygen (O2) obligate aerobes Faultative anaerobes Obligate anaerobes Aerotolerant Microaerophiles anaerobes Toxic Forms of Oxygen Singlet oxygen: O2 boosted to a higherenergy state Superoxide free radicals: O2 Peroxide anion: O22 Hydroxyl radical (OH) The Requirements for Growth: Chemical Requirements Organic Growth Factors Organic compounds obtained from the environment Vitamins, amino acids, purines, pyrimidines Logistic Growth Exponential growth is optimal, real growth follows a logistic pattern Lag phase Log phase Exponential growth Stationary phase A period when cells are metabolizing but not dividing yet Equal numbers of cells are dividing and dying Death phase Culture dies exponentially Bacterial Growth Curve Requirements for Culturing Bacteria Culture Medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium Isolation and Pure Culture A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU) Isolation Methods 1. 2. Streak Plate Pour plate Streak Plate Figure 6.10a, b General Types of Culture Media Culture Media Chemically Defined Media: Exact chemical composition is known Complex Media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar Culture Media Table 6.2 & 6.4 Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C Incubation Techniques Temperature Oxygen Content Reducing Media Anaaerobic jar Anaerobic glove box Candle jar Anaerobic Culture Methods Reducing media Contain chemicals (thioglycollate or oxyrase) that combine O2 Heated to drive off O2 Anaerobic Culture Methods Anaerobic jar Figure 6.5 Anaerobic Culture Methods Anaerobic chamber Figure 6.6 Capnophiles require high CO2 Candle jar CO2-packet Figure 6.7 Special Types of Media Selective Differential Selective/ Differential Enrichment Selective Media Suppress unwanted microbes and encourage desired microbes. Figure 6.9b, c Differential Media Make it easy to distinguish colonies of different microbes. Figure 6.9a Direct Measurements of Microbial Growth Plate Counts: Perform serial dilutions of a sample Figure 6.15, top portion Plate Count Inoculate Petri plates from serial dilutions Figure 6.16 Plate Count After incubation, count colonies on plates that have 25250 colonies (CFUs) Figure 6.15 Direct Measurements of Microbial Growth Filtration Figure 6.17a, b Direct Measurements of Microbial Growth Multiple tube MPN test Count positive tubes and compare to statistical MPN table. Figure 6.18b Direct Measurements of Microbial Growth Direct Microscopic Count Direct Measurements of Microbial Growth Figure 6.19 Estimating Bacterial Numbers by Indirect Methods Turbidity Figure 620 Estimating Bacterial Numbers by Indirect methods Metabolic activity Measure a metabolic product CO2 Dry weight Preserving Bacteria Cultures Deep-freezing: -50°to -95°C Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum