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Microbial Growth Chapter 6 Biology 1009-Summer 2003 Johnson Microbial Growth n Refers to the number of cells, not the size of the cells n 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 moderate temperature loving 20C-40C •Thermophile •Range heat loving 40C-100C 1 Physical Requirements (cont.) n pH – Most bacteria grow between pH 6.5-7.5 – Very few grow below pH 4 – Some foods (pickles & sauerkraut) preserved from spoilage by acids made by bacterial fermentation Physical Requirements (cont.) n 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 n Carbon-50% of bacteria cell dry wt – Microbes obtain from organic materials or CO2 n Iron, copper, and zinc (trace) – Used to make enzymes n Nitrogen, sulfur, and phosphorus-14% of bacterial cell dry wt – Needed for synthesis of cellular material 2 Other chemical requirements n Oxygen – Bacteria classified based on their oxygen requirements • 4 main classifications Obligate Aerobes n Require oxygen to live Obligate Anaerobes n No growth in the presence of oxygen 3 Facultative n Bacterial growth with or without oxygen Microaerophilic n Grow best in low concentrations of oxygen (as compared with the normal 21%) Oxygen Requirements 4 Toxic Forms of Oxygen n Singlet oxygen-normal molecular oxygen that has been boosted into high energy state n Superoxide free radicals-formed by all living organisms during respiration n Peroxide anion-found in hydrogen peroxide Superoxide dismutase SOD n O2 + O2 +2 H+ -----àH2O2 SOD-neutralizes oxygen free radicals *ok, but hydrogen peroxide also toxic… Catalase catalase n2 H2O2 ----------à 2 H2O + O2 – Obligate anaerobes lack catalase and superoxide dismutase-they die in the presence of oxygen 5 Culture media n 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 n 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 More special culturing procedures… n Microaerophilic Bacteria – Grow best at reduced oxygen levels and increased carbon dioxide – Utilizes a candle jar Normal oxygenCandle jar oxygenNormal carbon dioxideCandle jar carbon dioxide- Candle Jar-used to grow microaerophilic microbes Selective Media n 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 Differential Media n 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 n Increases the number of cells, not size ØBinary Fission-single cell divides into 2 identical cells ØMost bacteria:1 to 3 hr generation time ØE. coli-generation time of 20 minutes *20 generations (about 7 hours) • yields 1 million cells Binary Fission 8 Other bacterial division methods… n Budding-asexual form in which an outgrowth is formed, enlarges, and breaks free n Fragmentation-portion of cell breaks off and generates a new cell – Filamentous species only (Actinomycetes) Limiting Environmental Factors that Affect Bacterial Growth n Lack of food and water n Space n Accumulation of waste products n Lack of oxygen n Changes in pH n Temperature fluxuations Phases of Growth n Graph indicating the growth of a bacterial population over time n Four phases – Lag – Log – Stationary – Death 9 Lag Phase n Bacteria first introduced into environment or media n Bacteria get used to their surroundings n Bacteria very active metabolically n Little to no change in cell # n Lasts 1 hr to several days Log Phase n Rapid (exponential) cell growth population doubles every generation n Bacteria sensitive to adverse conditions n Bacterial Stationary Phase n n 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 Death Phase n Number of deaths exceeds the number of new cells formed n 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 (30-300 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 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