Microbial growth = increase in number of cells, not cell sizeChapter 6Microbial GrowthTwo categories of requirements for growth:1. PhysicalTemperature, pH, Osmotic Pressure2. ChemicalSources of: carbon, nitrogen, sulfur, phosphorus,trace elements, oxygen, and organic factorsWith a focus on Bacteria• Temperature– Minimum growth temperature– Optimum growth temperature– Maximum growth temperatureUsually within a 30-40 degree rangeFour general groups of bacteria based on preferred temperature:1. Psychrophilesmin -10°Cmax 30°Coptimal 15°C2. Mesophilesmin 10°Cmax 50°Coptimal 37°C3. Thermophilesmin 40°Cmax 70°Coptimal 60°C4. Hyperthermophilesmin 70°C max 110°Coptimal 95°C• pH– Most bacteria growbetween pH 6.5 and 7.5– Molds and yeasts growbetween pH 5 and 6– Acidophiles grow only inacidic environmentsAmy Warenda Czura, Ph.D.1SCCC BIO244 Microbial Growth Lab 2 (Text Chapter 6)• Osmotic Pressure– Hypertonic environments, (increased salt or sugar), causeplasmolysis– Cell wall protects bacteria from hypotonic environments– Extreme or obligate halophiles require high osmoticpressure– Facultative halophiles tolerate high osmotic pressureNormalbacterial cell is80-90% waterPlasmolysis in ahypertonic highsalt solution• Carbon– Structural organic molecules, energy source– Heterotrophs use organic carbon sources– Autotrophs use CO2The Requirements for Growth: Chemical Requirements• Nitrogen– In amino acids, proteins– Most bacteria decompose proteins– Some bacteria use NH4+ or NO3!– A few bacteria use N2 in nitrogen fixation• Sulfur– 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• Trace Elements– Inorganic elements required in small amounts (Potassium, Magnesium, Calcium, Iron, Copper, Zinc)– Usually as enzyme cofactors• Oxygen (O2)• Organic Growth Factors– Organic compounds obtained from the environment– Vitamins, amino acids, purines, pyrimidinesCulture Media• Culture Medium: Nutrients prepared formicrobial growth in the lab• Sterile: No living microbes• Inoculum: Introduction of microbes into medium• Culture: Microbes growing in/on culture mediumAmy Warenda Czura, Ph.D.2SCCC BIO244 Microbial Growth Lab 2 (Text Chapter 6)• 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°CAgarCulture Medium: liquid form = brothsolid gel form using agar = plates, slants, deeps• Chemically Defined Media: Exact chemicalcomposition is known• Complex Media: Extracts and digests of yeasts,meat, or plants– Nutrient broth, BHI broth (liquid)– Nutrient agar, BHIA (solid gel)Culture MediaFastidious organisms require many growth factors:must be grown in complex media• Suppress unwanted microbes and encouragedesired microbes.Selective Media• Make it easy to distinguish colonies of differentmicrobes.Differential Media• A pure culture contains only one species or strain• A colony is a population of cells arising from asingle cell or spore or from a group of attachedcells• A colony is often called a colony-forming unit(CFU)Streak Plate method usedto isolate a pure cultureAmy Warenda Czura, Ph.D.3SCCC BIO244 Microbial Growth Lab 2 (Text Chapter 6)Reproduction in ProkaryotesBinary FissionGeneration time - the time required for a cell todivide, to undergo one round of binary fissionCommon bacterial generation times range 1-3hrsE. coli has a generation time of 20 min: one cell in 20generations will become ~1 million cells (~7 hrs)Exponential growth is graphed on a logarithmic scale:A logarithum of a number X to base 10 is thepower/exponent to which 10 must be raised to give thatnumber XExponential growthLog scale of exponential growthBacterial Growth CurveAmy Warenda Czura, Ph.D.4SCCC BIO244 Microbial Growth Lab 2 (Text Chapter 6)Phases of Bacterial Growth in a New Culture-Lag phase: initial period of little to no cell division as bacteria acclimate to new media-Log phase: period of exponential growth with a constant generationtime-Stationary phase: cell growth is equal to cell death-Death phase: cell death exceeds cell growthQuantifying Microbial GrowthDirect MeasurementsIndirect EstimationsPlate CountsFiltrationMost Probable Number (MPN)Direct Microscopic CountTurbidityMetabolic ActivityDry Weight• Plate Counts: Perform serial dilutions of asample, plate, and count resulting coloniesDirect Measurements of Microbial Growth• FiltrationAmy Warenda Czura, Ph.D.5SCCC BIO244 Microbial Growth Lab 2 (Text Chapter 6)Count positive tubes and compare to statistical MPN table.• Multiple tube MPN (most probable number) test• Direct Microscopic CountPetroff-Hausser Cell Counter• TurbidityEstimating Bacterial Numbers byIndirect Methods• Metabolic activity• Dry weightAmy Warenda Czura, Ph.D.6SCCC BIO244 Microbial Growth Lab 2 (Text Chapter