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3/19/2009 TORTORA ⏐ FUNKE ⏐ CASE ninth edition MICROBIOLOGY an introduction Microbial Growth Microbial growth is the increase in number of cells, not cell size 6 Microbial Growth PowerPoint® Lecture Slide Presentation prepared by Christine L. Case Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Requirements for Growth: Physical Requirements Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Temperature Temperature Minimum growth temperature Optimum growth temperature Maximum g growth temperature p Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Psychrotrophs Psychrotrophs Figure 6.1 Grow between 0°C and 20-30°C Cause food spoilage Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.2 1 3/19/2009 The Requirements for Growth: Physical Requirements The Requirements for Growth: Physical Requirements pH Osmotic pressure Most bacteria grow between pH 6.5 and 7.5 Hypertonic environments, increase salt or sugar, Molds and yeasts grow between pH 5 and 6 cause plasmolysis Acidophiles p g grow in acidic environments Extreme or obligate g halophiles p require q high g osmotic pressure Facultative halophiles tolerate high osmotic pressure Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Requirements for Growth: Physical Requirements The Requirements for Growth: Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs p use CO2 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Requirements for Growth: Chemical Requirements Figure 6.4 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Requirements for Growth: Chemical Requirements Nitrogen In amino acids and proteins Trace elements Most bacteria decompose proteins Inorganic elements required in small amounts Some bacteria use NH4+ or NO3– Usually as enzyme cofactors A few bacteria use N2 in nitrogen fixation Sulfur S lf In amino acids, thiamine and biotin Most bacteria decompose proteins Some bacteria use SO42– or H2S Phosphorus In DNA, RNA, ATP, and membranes PO43– is a source of phosphorus Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 2 3/19/2009 The Requirements for Growth: Chemical Requirements Toxic Forms of Oxygen Oxygen (O2) Singlet oxygen: O2 boosted to a higher-energy state Superoxide free radicals: O2– Peroxide anion: O22– Hydroxyl radical (OH•) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Table 6.1 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Requirements for Growth: Chemical Requirements Culture Media Organic growth factors Culture medium: Nutrients prepared for microbial Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Agar Culture Media Complex polysaccharide Chemically defined media: Exact chemical composition Used as solidifying agent for culture media in Petri plates, slants, and deeps Generallyy not metabolized by y microbes is known Complex media: Extracts and digests of yeasts, meat, or p plants Liquefies at 100°C Nutrient broth Solidifies ~40°C Nutrient agar Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 3 3/19/2009 Culture Media Growth in Continuous Culture A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Tables 6.2, 6.4 Basic Chemostat System Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Our Chemostat System Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Anaerobic Culture Methods Anaerobic Culture Methods Reducing media Anaerobic Contain chemicals (thioglycollate or oxyrase) that jar combine O2 Heated to drive off O2 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.5 4 3/19/2009 Anaerobic Culture Methods Capnophiles Require High CO2 Anaerobic Candle jar chamber CO2-packet Figure 6.6 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.7 Selective Media Selective Media Suppress unwanted Inhibits the growth of some bacteria while selecting for microbes and the growth of others Example: encourage desired Brilliant Green Agar g microbes. dyes inhibit the growth of Gram (+) bacteria selects for Gram (-) bacteria Most G.I. Tract infections are caused by Gram (-) bacteria Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9b–c Selective Media EMB (Eosin Methylene Blue) dyes inhibit Gram (+) bacteria Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Differential Media Make it easy to distinguish colonies of different microbes. selects for Gram (-) bacteria G.I. Tract infections caused by y Gram (-) () bacteria Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.9a 5 3/19/2009 Some media are both selective and differential Selective and Differential Media Mannitol salt agar is both selective and differential Selective: Staphylococcus aereus can grow on mannitol salt agar that has a high concentration of salt; the growth of other organisms will be inhibited Differential: Staphylococcus aureus ferments mannitol and the medium will change color Other organisms that grow on high salt will grow on mannitol salt agar but may not ferment mannitol; the media will not change colors Mannitol Salt Agar used to identify Staphylococcus aureus Mannitol M i l Salt S l A Agar High salt conc. (7.5%) inhibits most bacteria sugar Mannitol pH Indicator (Turns Yellow when acid) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Selective and Differential Media Enrichment Media Encourages growth of desired microbe MacConkey’s Agar used to identify Salmonella MacConkey’s Agar Bile salts and crystal violet (inhibits Gram (+) bacteria) lactose pH Indicator Many Gram (-) enteric non-pathogenic bacteria can ferment lactose, Salmonella can not Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil and incubate Transfer 1 ml to another flask of the phenol medium and incubate Transfer 1 ml to another flask of the phenol medium and incubate Only phenol-metabolizing bacteria will be growing Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Streak Plate 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 colonyy is often called a colony-forming y g unit ((CFU). ) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.10a–b 6 3/19/2009 Preserving Bacteria Cultures Reproduction in Prokaryotes Deep-freezing: –50°to –95°C Binary fission Lyophilization (freeze-drying): Frozen (–54° to –72°C) Budding Conidiospores (actinomycetes) and dehydrated in a vacuum Fragmentation g of filaments Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Binary Fission Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.11 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.12b Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.13 If 100 cells growing for 5 hours produced 1,720,320 cells: PLAY Animation: Bacterial Growth Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 7 3/19/2009 1. Lag Phase Bacteria are first introduced into an environment or media Bacteria are “checking out” their surroundings cells are veryy active metabolicallyy # of cells changes very little 1 hour to several days Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.14 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 3. Stationary Phase 2. Log Phase Rapid cell growth (exponential growth) Death rate = rate of reproduction population doubles every generation cells begin to encounter environmental stress lack of nutrients microbes are sensitive to adverse conditions antibiotics lack of water anti-microbial agents not enough space metabolic wastes oxygen pH Endospores would form now Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 4. Death Phase Measuring Microbial Growth Death rate > rate of reproduction Direct methods Indirect methods Due to limiting factors in the environment Plate counts Turbidity Filtration Metabolic activity MPN Dryy weight g Direct microscopic count Dry weight Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 8 3/19/2009 Direct Measurements of Microbial Growth Plate Count Plate counts: Perform serial dilutions of a sample Inoculate Petri plates from serial dilutions Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15, step 1 Plate Count Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.16 Direct Measurements of Microbial Growth After incubation, count colonies on plates that have Filtration 25-250 colonies (CFUs) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.15 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.17 Direct Measurements of Microbial Growth Direct Measurements of Microbial Growth Multiple tube Direct microscopic count MPN test. Count positive tubes and compare to statistical MPN table. Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.18b Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 9 3/19/2009 Direct Measurements of Microbial Growth Estimating Bacterial Numbers by Indirect Methods Turbidity Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.19, steps 1, 3 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 6.20 10
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