Chapter 4 Microbial Growth Measurement BIOL 230 PDF

Chapter 4 Microbial Growth Measurement General Microbiology Course Code: BIOL 230 Growth in bacteria: increase in number or population Binary fission: most of bacteria, exponential growth Budding: few bacterial species  Generation time The time required for a cell...

Chapter 4 Microbial Growth Measurement General Microbiology Course Code: BIOL 230 Growth in bacteria: increase in number or population Binary fission: most of bacteria, exponential growth Budding: few bacterial species  Generation time The time required for a cell to divide ( population to double) Varies among species and with environmental conditions ( temperature) Can be as short as 20 minutes ( E. coli) to > 24h (Mycobacterium tuberculosis) Most bacteria have a generation time of 1 to 3 hours Populations grow fast Eg, consider a 20 minute generation time 1 cell becomes 1 million in 20 generations (6.7 hrs) 1 billion in 30 generations (10 hrs)  Bacterial populations increase in number rapidly The number of bacteria doubles in each generation. The superscript indicates the generation: 25= 5 generations Bacterial Growth Curve: Arithmetic vs. Logarithmic Representation It is difficult to graph population change of such big arithmetic numbers ( solid line) Growth curve Population changes cannot plotted using be shown in the early stages logarithmic of growth scale Hence we use log scales to graph bacteria growth Growth curve plotted using arithmetic scale Phases of bacterial growth:  Bacterial Growth Curve Lag Phase Period of little or no cell division / period of initial adjustment The physiological adaptation of the cell to the culture conditions. Horizontal line of the graph Last for several hours or several days Cells are not dormant Undergo intense metabolic activity (synthesis of enzymes and various molecules )  Bacterial Growth Curve Log/ exponential phase characterized by a period of the exponential growth Cells begin to divide and enter period of growth or logarithmic increase Most active cellular reproduction phase Cells are most active metabolically and is prefrrerd for industrial purposes During exponential growth, the number of cells increases in the geometric progression 20 , 21 , 22 , 23 , 24 until, after n divisions, the number of cells is 2n. If the initial cell number is X0, the number of cells after n doublings is X0 2n  Bacterial Growth Curve The stationary phase state of no net growth ( population stabilized) Although there is no net growth in the stationary phase, cells still grow and divide. Growth simply balanced by an equal number of cells dying (reproduction rate equal to the equivalent death rate) Growth rates slow down : number of microbial death balances the number of new cells. Exhaustion of nutrients, accumulation of waste products , production of secondary metabolites, change in pH Bacterial Growth Curve Death / decline phase The final phase of the growth curve which is characterized by a net loss of culturable cells – number of deaths exceed number of new cell cells Direct Measurements of Microbial Growth Plate counts Grow microbial sample on agar plate Count resulting colonies Advantages – only viable (live) cells counted Disadvantage – takes time for colonies to form  CFU- Colony forming units Only limited colonies should grow ( 25-300 colonies) Serial dilutions ensure this range Plate count : Pour plate or spread plate Pour plate: not suitable for heat sensitive microbes ( damaged by melted agar) Most Probable Number (MPN) Statistical estimation technique based on principle : greater the number of bacteria the more dilution is required to a point where no bacteria are left to grow Used when bacteria cannot grow on solid media But, numbers are an approximation ~95% accurate Additional Direct Measurements method of choice for low coun Direct Measurements of Microbial Growth Direct microscopic count: Counting chambers (slides- Hemocytometer/ Petroff-Hauser chambers) for microscope Number of microbes counted in microscope No incubation time required Most accurate, but … Motile cells difficult to count Dead cells look like live cells Need high cell numbers to count accurately Estimating Bacterial Numbers by Indirect Methods Turbidity Cloudiness, or density, of a liquid culture Detected using a spectrophotometer at specific wavelgth of light Higher cell number = increased cloudiness/ turbidity Fast, convenient and easy method of obtaining number Nondestructive sampling No additional incubation but … Require large cell numbers to measure turbidity accurately Cannot distinguish between live and dead cells , clumping can cause inaccurate results Estimating Bacterial Numbers by Indirect Methods Estimating Bacterial Numbers by Indirect Methods Metabolic activity Assumes higher number of bacteria produces higher amount of metabolic product Eg, measure CO2 build-up Can be used when cells can’t be cultured Dry Weight :measuring dry weight of the microbial cells Removal of microbes from growth medium, dried, and weighed Useful for filamentous bacteria, molds Cannot disntinguish between live and dead ones Requires high concentration.