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Copyright © 2010 Pearson Education, Inc. Lectures prepared by Christine L. Case Chapter 6 Microbial Growth
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Copyright © 2010 Pearson Education, Inc. Learning Objectives Upon completion of this chapter, you should be able to: Classify microbes into 5 groups based on their temperature preference Explain the importance of osmotic pressure in microbial growth Name a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth Explain how microbes are classified on the basis of oxygen requirements Identify ways in which aerobes avoid damage from toxic forms of oxygen Describe the formation of biofilms and their potential for causing infection Distinguish chemically defined and complex media Differentiate biosafety levels (1, 2, 3, and 4) Justify the use of the following: anaerobic culture techniques, living host cells, candle jars, selective and differential media, enrichment media Define colony Describe how a pure culture can be isolated using the streak plate method Define bacterial growth, including binary fission Compare the phases of microbial growth Explain 4 direct methods for measuring cell growth Explain 3 indirect methods of measuring cell growth
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Copyright © 2010 Pearson Education, Inc. Microbial Growth Increase in number of cells, not cell size Populations Colonies
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Copyright © 2010 Pearson Education, Inc. The Requirements for Growth Physical requirements Temperature pH Osmotic pressure Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor
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Copyright © 2010 Pearson Education, Inc. Physical Requirements Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature
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Copyright © 2010 Pearson Education, Inc. Figure 6.1 Typical Growth Rates and Temperature
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Copyright © 2010 Pearson Education, Inc. Psychrotrophs Grow between 0°C and 20–30°C Cause food spoilage
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Copyright © 2010 Pearson Education, Inc. Figure 6.2 Food Preservation Temperatures
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Copyright © 2010 Pearson Education, Inc. pH Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments
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Copyright © 2010 Pearson Education, Inc. Osmotic Pressure Hypertonic environments, or an increase in salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure
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Copyright © 2010 Pearson Education, Inc. Figure 6.4 Plasmolysis
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Copyright © 2010 Pearson Education, Inc. Chemical Requirements Carbon Structural organic molecules, energy source Chemoheterotrophs use organic carbon sources Autotrophs use CO 2
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Copyright © 2010 Pearson Education, Inc. Chemical Requirements Nitrogen In amino acids and proteins Most bacteria decompose proteins Some bacteria use NH 4 + or NO 3 – A few bacteria use N 2 in nitrogen fixation
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Copyright © 2010 Pearson Education, Inc. Chemical Requirements Sulfur In amino acids, thiamine, and biotin Most bacteria decompose proteins Some bacteria use SO 4 2– or H 2 S Phosphorus In DNA, RNA, ATP, and membranes PO 4 3– is a source of phosphorus
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Copyright © 2010 Pearson Education, Inc. Chemical Requirements Trace elements Inorganic elements required in small amounts Usually as enzyme cofactors
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Copyright © 2010 Pearson Education, Inc. Table 6.1 The Effect of Oxygen (O2) on Growth
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Copyright © 2010 Pearson Education, Inc. Singlet oxygen: O 2 boosted to a higher-energy state Superoxide free radicals: O 2 – Peroxide anion: O 2 2– Hydroxyl radical (OH) Toxic Oxygen
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Copyright © 2010 Pearson Education, Inc. Organic Growth Factors Organic compounds obtained from the environment Vitamins, amino acids, purines, and pyrimidines
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Copyright © 2010 Pearson Education, Inc. Figure 6.5 Biofilms Microbial communities Form slime or hydrogels Bacteria attracted by chemicals via quorum sensing
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Copyright © 2010 Pearson Education, Inc. Applications of Microbiology, p. 57 Biofilms Share nutrients Sheltered from harmful factors, such as antibiotics
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Copyright © 2010 Pearson Education, Inc. Biofilms Patients with indwelling catheters received contaminated heparin Bacterial numbers in contaminated heparin were too low to cause infection 84–421 days after exposure, patients developed infections
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Copyright © 2010 Pearson Education, Inc. Biofilms Mucoid form of Pseudomonas aeruginosa was cultured from the CF sputum. What causes the difference between the original strain and the CF strain?
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Copyright © 2010 Pearson Education, Inc. Culture Media Culture medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium
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Copyright © 2010 Pearson Education, Inc. 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 at ~40°C
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Copyright © 2010 Pearson Education, Inc. Culture Media Chemically defined media: Exact chemical composition is known Complex media: Extracts and digests of yeasts, meat, or plants Nutrient broth Nutrient agar
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Copyright © 2010 Pearson Education, Inc.
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Anaerobic Culture Methods Reducing media Contain chemicals (thioglycolate or oxyrase) that combine O 2 Heated to drive off O 2
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Copyright © 2010 Pearson Education, Inc. Figure 6.6 Anaerobic Jar
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Copyright © 2010 Pearson Education, Inc. Figure 6.7 An Anaerobic Chamber
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Copyright © 2010 Pearson Education, Inc. Capnophiles Microbes that require high CO2 conditions CO2 packet Candle jar
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Copyright © 2010 Pearson Education, Inc. 1: No special precautions 2: Lab coat, gloves, eye protection 3: Biosafety cabinets to prevent airborne transmission 4: Sealed, negative pressure Exhaust air is filtered twice Biosafety Levels
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Copyright © 2010 Pearson Education, Inc. Figure 6.8 Biosafety Level 4 (BSL-4) Laboratory
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Copyright © 2010 Pearson Education, Inc. Figure 6.10 Selective Media Suppress unwanted microbes and encourage desired microbes
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Copyright © 2010 Pearson Education, Inc. Figure 6.9 Differential Media Make it easy to distinguish colonies of different microbes.
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Copyright © 2010 Pearson Education, Inc. Enrichment Culture Encourages growth of desired microbe 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 Only phenol-metabolizing bacteria will be growing
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Copyright © 2010 Pearson Education, Inc. Obtaining Pure Cultures 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) The streak plate method is used to isolate pure cultures
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Copyright © 2010 Pearson Education, Inc. Figure 6.11 The Streak Plate Method
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Copyright © 2010 Pearson Education, Inc. Preserving Bacterial Cultures Deep-freezing: –50° to –95°C Lyophilization (freeze-drying): Frozen (–54° to – 72°C) and dehydrated in a vacuum
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Copyright © 2010 Pearson Education, Inc. Binary fission Budding Conidiospores (actinomycetes) Fragmentation of filaments ANIMATION Bacterial Growth: Overview Reproduction in Prokaryotes
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Copyright © 2010 Pearson Education, Inc. Figure 6.12a Binary Fission
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Copyright © 2010 Pearson Education, Inc. Figure 6.12b Binary Fission
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Copyright © 2010 Pearson Education, Inc. Figure 6.13b Cell Division
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Copyright © 2010 Pearson Education, Inc. Figure 6.14 Bacterial Growth Curve
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Copyright © 2010 Pearson Education, Inc. Figure 6.15 ANIMATION Bacterial Growth Curve Phases of Growth
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Copyright © 2010 Pearson Education, Inc. Figure 6.16 Serial Dilutions
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Copyright © 2010 Pearson Education, Inc. Figure 6.17 Plate Counts
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Copyright © 2010 Pearson Education, Inc. Figure 6.16 Plate Counts After incubation, count colonies on plates that have 25–250 colonies (CFUs)
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Copyright © 2010 Pearson Education, Inc. Figure 6.18 Counting Bacteria by Membrane Filtration
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Copyright © 2010 Pearson Education, Inc. Figure 6.19 Most Probable Number Multiple tube MPN test Count positive tubes
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Copyright © 2010 Pearson Education, Inc. Figure 6.19 Most Probable Number Compare with a statistical table.
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Copyright © 2010 Pearson Education, Inc. Figure 6.20 Direct Microscopic Count
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Copyright © 2010 Pearson Education, Inc. Direct Microscopic Count
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Copyright © 2010 Pearson Education, Inc. Figure 6.21 Turbidity
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Copyright © 2010 Pearson Education, Inc. Figure 6.21 Turbidity
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Copyright © 2010 Pearson Education, Inc. Measuring Microbial Growth Direct Methods Plate counts Filtration MPN Direct microscopic count Indirect Methods Turbidity Metabolic activity Dry weight
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