Microbial Growth. Microbial Growth. Physical Requirements. Chapter 6 Biology 1009-Summer 2003 Johnson

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1 Microbial Growth Chapter 6 Biology 1009-Summer 2003 Johnson Microbial Growth Refers to the number of cells, not the size of the cells Requirements for growth *Two general types: Physical: temp, ph, osmotic pressure Chemical: elements, including C, N, S, O, and P Physical Requirements Temperature Psychrophile: cold loving Range 0C-20C Mesophile: Range Thermophile Range moderate temperature loving 20C-40C heat loving 40C-100C 1

2 Physical Requirements (cont.) ph Most bacteria grow between ph Very few grow below ph 4 Some foods (pickles & sauerkraut) preserved from spoilage by acids made by bacterial fermentation Physical Requirements (cont.) Osmotic Pressure Microbes obtain most of their nutrients in solution from surrounding water Many foods (honey, salted fish) preserved this way: high sugar or salt { } draws water out of bacterial cells, preventing their growth Chemical Requirements Carbon-50% of bacteria cell dry wt Microbes obtain from organic materials or CO2 Iron, copper, and zinc (trace) Used to make enzymes Nitrogen, sulfur, and phosphorus-14% of bacterial cell dry wt Needed for synthesis of cellular material 2

3 Other chemical requirements Oxygen Bacteria classified based on their oxygen requirements 4 main classifications Obligate Aerobes Require oxygen to live Obligate Anaerobes No growth in the presence of oxygen 3

4 Facultative Bacterial growth with or without oxygen Microaerophilic Grow best in low concentrations of oxygen (as compared with the normal 21%) Oxygen Requirements 4

5 Toxic Forms of Oxygen Singlet oxygen-normal molecular oxygen that has been boosted into high energy state Superoxide free radicals-formed by all living organisms during respiration Peroxide anion-found in hydrogen peroxide Superoxide dismutase SOD O2 + O2 + 2 H H2O2 SOD-neutralizes oxygen free radicals *ok, but hydrogen peroxide also toxic Catalase catalase 2 H2O H2O + O2 Obligate anaerobes lack catalase and superoxide dismutase-they die in the presence of oxygen 5

6 Culture media Nutrient material prepared for the growth of microbes Two main types Chemically defined: Exact chemical composition is known Used to grow very specific organisms Complex: Exact chemical composition unknown Most bacteria and fungi grown on this type Special Culturing Procedures Anaerobic microbes Require reducing media that removes free oxygen Example-fluid thioglycollate media Require anaerobic containers Example-gas pak jar Require agar stab inoculations Anaerobic Gas Pak 6

7 More special culturing procedures Microaerophilic Bacteria Grow best at reduced oxygen levels and increased carbon dioxide Utilizes a candle jar Normal oxygen- Candle jar oxygen- Normal carbon dioxide- Candle jar carbon dioxide- Candle Jar-used to grow microaerophilic microbes Selective Media Inhibits the growth of some bacteria while selecting for the growth of other organisms Examples: *Brilliant green: isolates Gram Salmonella *EMB: isolates Gram GI tract microbes 7

8 Differential Media Differentiate between different organisms growing on the same plate Example: Blood agar plate (TSA with 10% sheep blood) Used to differentiate types of Streptococci»Alpha, beta, and gamma hemolysis Bacterial Division Increases the number of cells, not size Binary Fission-single cell divides into 2 identical cells Most bacteria:1 to 3 hr generation time E. coli-generation time of 20 minutes *20 generations (about 7 hours) yields 1 million cells Binary Fission 8

9 Other bacterial division methods Budding-asexual form in which an outgrowth is formed, enlarges, and breaks free Fragmentation-portion of cell breaks off and generates a new cell Filamentous species only (Actinomycetes) Limiting Environmental Factors that Affect Bacterial Growth Lack of food and water Space Accumulation of waste products Lack of oxygen Changes in ph Temperature fluxuations Phases of Growth Graph indicating the growth of a bacterial population over time Four phases Lag Log Stationary Death 9

10 Lag Phase Bacteria first introduced into environment or media Bacteria get used to their surroundings Bacteria very active metabolically Little to no change in cell # Lasts 1 hr to several days Log Phase Rapid (exponential) cell growth Bacterial population doubles every generation Bacteria sensitive to adverse conditions Stationary Phase Death Rate=rate of reproduction Cells begin to encounter environmental stress Lack of water Lack of nutrients ph change High amounts of metabolic waste Lack of space Note: Endospores form at this time! 10

11 Death Phase Number of deaths exceeds the number of new cells formed Due to the limiting factors in the environment Bacterial Growth Curve Direct Measurement of Microbial Growth 1) Plate counts-most used method/require serial dilutions *example-pour plates ( colonies countable) 2) Filtration-passage of liquid through a grid containing small pores (<.45 um) 3) Most Probable Number-statistical method used with bacteria that won t grow on media 4) Direct Microscopic Count-known volume of bacterial suspension placed on special cell counting slide 11

12 Direct Microscopic Count Indirect Methods of Estimating Bacterial Numbers 1) Turbidity-cloudiness produced as media becomes filled with bacteria 2) Metabolic Activity-assumes certain amounts of metabolic wastes are in direct proportion to # of bacteria 3) Dry Weight-organisms removed from media, filtered, and weighed 12

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