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Microbial growth (Part-1) 140 MBIO panel (semester 2; 1437-1438H)
140MIC: Microbiology Lecture-11 Microbial growth (Part-1) 140 MBIO panel (semester 2; H) د. فاطمة العتيبي Dr. Fatmah Alotaibi د. كاكاشان بروين Dr. Kahkashan Perveen د. حميراء رضوان Dr. Humaira Rizwana أعدت العروض التقديمية منسقة المقرر: د. أسماء الصالح رقم المكتب 5T201 الموقع: إيميل
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Content Bacterial Cell Division Population growth
Cell Growth and Binary Fission Determinant Proteins for division and morphology (Fts, MreB). Population growth The Concept of Exponential Growth The Microbial Growth Cycle Measuring Microbial Growth Environmental Factors Affecting Growth Temperature Acidity and Alkalinity Osmotic Effects on Microbial Growth Oxygen and Microorganisms Toxic Forms of Oxygen © 2012 Pearson Education, Inc.
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Cell Growth and Binary Fission
Growth: increase in the number of cells Binary fission: cell division following enlargement of a cell to twice its minimum size Generation time: time required for microbial cells to double in number During cell division, each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell © 2012 Pearson Education, Inc.
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Determinant Proteins for division and morphology (Fts, MreB).
Fts (filamentous temperature-sensitive) Proteins Essential for cell division in all prokaryotes Interact to form the divisome (cell division apparatus) FtsZ: forms ring around center of cell; related to tubulin ZipA: anchor that connects FtsZ ring to cytoplasmic membrane FtsA: helps connect FtsZ ring to membrane and also recruits other divisome proteins DNA replicates before the FtsZ ring forms Location of FtsZ ring is facilitated by Min proteins : MinC, MinD, MinE FtsK protein mediates separation of chromosomes to daughter cells © 2012 Pearson Education, Inc.
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Minutes 20 40 60 80 Min CD Cell wall Cytoplasmic membrane Nucleoid
Cytoplasmic membrane Nucleoid 20 MinE 40 Divisome complex Figure 5.3 DNA replication and cell-division events. 60 FtsZ ring Septum 80 Nucleoid MinE © 2012 Pearson Education, Inc. 5
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Determinant Proteins for division and morphology (Fts, MreB).
Prokaryotes contain a cell cytoskeleton that is dynamic and multifaceted MreB: major shape-determining factor in prokaryotes Forms simple cytoskeleton in Bacteria and probably Archaea Forms spiral-shaped bands around the inside of the cell, underneath the cytoplasmic membrane Not found in coccus-shaped bacteria © 2012 Pearson Education, Inc.
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The Concept of Exponential Growth
Most bacteria have shorter generation times than eukaryotic microbes Generation time is dependent on growth medium and incubation conditions Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval During exponential growth, the increase in cell number is initially slow but increases at a faster rate © 2012 Pearson Education, Inc.
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The Concept of Exponential Growth
Batch culture: a closed-system microbial culture of fixed volume Typical growth curve for population of cells grown in a closed system is characterized by four phases: Lag phase: Interval between when a culture is inoculated and when growth begins Exponential phase: Cells in this phase are typically in the healthiest state Stationary phase: Growth rate of population is zero, either an essential nutrient is used up or waste product of the organism accumulates in the medium Death phase: If incubation continues after cells reach stationary phase, the cells will eventually die
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Growth phases Lag Exponential Stationary Death 1.0 10 0.75
9 Turbidity (optical density) Optical density (OD) Log10 viable organisms/ml 0.50 Viable count 8 0.25 7 Figure 5.10 Typical growth curve for a bacterial population. 6 0.1 Time © 2012 Pearson Education, Inc.
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Continuous Culture: The Chemostat
Continuous culture: an open-system microbial culture of fixed volume Chemostat: most common type of continuous culture device Both growth rate and population density of culture can be controlled independently and simultaneously Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out Concentration of a limiting nutrient In a chemostat The growth rate is controlled by dilution rate The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient In a batch culture, growth conditions are constantly changing; it is impossible to independently control both growth parameters. © 2012 Pearson Education, Inc.
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Fresh medium from reservoir Flow-rate regulator
Figure 5.11 Fresh medium from reservoir Flow-rate regulator Sterile air or other gas Gaseous headspace Culture vessel Culture Figure 5.11 Schematic for a continuous culture device (chemostat). Overflow Effluent containing microbial cells © 2012 Pearson Education, Inc.
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Microbial growth (Part-2) 140 MBIO panel (semester 2; 1437-1438H)
140MIC: Microbiology Lecture-12 Microbial growth (Part-2) 140 MBIO panel (semester 2; H) د. فاطمة العتيبي Dr. Fatmah Alotaibi د. كاكاشان بروين Dr. Kahkashan Perveen د. حميراء رضوان Dr. Humaira Rizwana أعدت العروض التقديمية منسقة المقرر: د. أسماء الصالح رقم المكتب 5T201 الموقع: إيميل
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Content Bacterial Cell Division Population growth
Cell Growth and Binary Fission Determinant Proteins for division and morphology (Fts, MreB). Population growth The Concept of Exponential Growth The Microbial Growth Cycle Measuring Microbial Growth Environmental Factors Affecting Growth Temperature Acidity and Alkalinity Osmotic Effects on Microbial Growth Oxygen and Microorganisms Toxic Forms of Oxygen © 2012 Pearson Education, Inc.
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Measuring Microbial Growth
Microscopic Counts Viable Counts Turbidimetric Methods © 2012 Pearson Education, Inc.
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Measuring Microbial Growth
Microscopic Counts Viable Counts Turbidimetric Methods Microbial cells are enumerated by microscopic observations Results can be unreliable Limitations of microscopic counts Cannot distinguish between live and dead cells without special stains Small cells can be overlooked Precision is difficult to achieve Phase-contrast microscope required if a stain is not used Cell suspensions of low density (<106 cells/ml) hard to count Motile cells need to immobilized Debris in sample can be mistaken for cells © 2012 Pearson Education, Inc.
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Figure 5.14 To calculate number per milliliter of sample: 12 cells 25 large squares 50 103 Ridges that support coverslip Coverslip Number/mm2 (3 102) Sample added here. Care must be taken not to allow overflow; space between coverslip and slide is 0.02 mm ( mm. Whole grid has 25 large squares, a total area of 1 mm2 and a total volume of 0.02 mm3. Number/mm3 (1.5 104) Microscopic observation; all cells are counted in large square (16 small squares): 12 cells. (In practice, several large squares are counted and the numbers averaged.) 1 50 Number/cm3 (ml) (1.5 107) Figure 5.14 Direct microscopic counting procedure using the Petroff–Hausser counting chamber. © 2012 Pearson Education, Inc.
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Measuring Microbial Growth
Microscopic Counts Viable Counts Turbidimetric Methods Viable cell counts (plate counts): measurement of living, reproducing population Two main ways to perform plate counts: Spread-plate method (Figure 5.15) Pour-plate method To obtain the appropriate colony number, the sample to be counted should always be diluted. © 2012 Pearson Education, Inc.
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Spread-plate method Pour-plate method Figure 5.15 Surface colonies
Incubation Sample is pipetted onto surface of agar plate (0.1 ml or less) Sample is spread evenly over surface of agar using sterile glass spreader Typical spread-plate results Pour-plate method Surface colonies Figure 5.15 Two methods for the viable count. Solidification and incubation Subsurface colonies Sample is pipetted into sterile plate Sterile medium is added and mixed well with inoculum Typical pour-plate results © 2012 Pearson Education, Inc.
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Too many colonies to count
Figure 5.16 Sample to be counted 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 9-ml broth Total dilution 1/10 (101) 1/100 (102) 1/103 (103) 1/104 (104) 1/105 (105) 1/106 (106) Plate 1-ml samples Figure 5.16 Procedure for viable counting using serial dilutions of the sample and the pour-plate method. 159 colonies 17 colonies 2 colonies 0 colonies Too many colonies to count 159 103 105 Plate count Dilution factor Cells (colony-forming units) per milliliter of original sample © 2012 Pearson Education, Inc.
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Measuring Microbial Growth
Microscopic Counts Viable Counts Turbidimetric Methods Turbidity measurements are an indirect, rapid, and useful method of measuring microbial growth Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.) To relate a direct cell count to a turbidity value, a standard curve must first be established Quick and easy to perform Typically do not require destruction or significant disturbance of sample Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium) © 2012 Pearson Education, Inc.
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Environmental Factors Affecting Growth
Temperature Acidity and Alkalinity Osmotic Effects on Microbial Growth Oxygen and Microorganisms Toxic Forms of Oxygen
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Effect of Temperature on Growth
Temperature is a major environmental factor controlling microbial growth Cardinal temperatures: the minimum, optimum, and maximum temperatures at which an organism grows. Microorganisms can be classified into groups by their growth temperature optima Psychrophile: low temperature Mesophile: midrange temperature Thermophile: high temperature Hyperthermophile: very high temperature © 2012 Pearson Education, Inc.
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Figure 5.20 Figure 5.20 Antarctic microbial habitats and microorganisms. © 2012 Pearson Education, Inc.
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Figure 5.22 Figure 5.22 Growth of hyperthermophiles in boiling water.
© 2012 Pearson Education, Inc.
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Figure 5.23 Figure 5.23 Growth of thermophilic cyanobacteria in a hot spring in Yellowstone National Park. © 2012 Pearson Education, Inc.
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Acidity and Alkalinity
The pH of an environment greatly affects microbial growth (Figure 5.24) Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles) Acidophiles: organisms that grow best at low pH (<6) Some are obligate acidophiles; membranes destroyed at neutral pH Stability of cytoplasmic membrane critical Alkaliphiles: organisms that grow best at high pH (>9) Some have sodium motive force rather than proton motive force The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic Microbial culture media typically contain buffers to maintain constant pH © 2012 Pearson Education, Inc.
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Increasing alkalinity
Figure 5.24 Moles per liter of: pH Example H OH 1 1014 Volcanic soils, waters Gastric fluids Lemon juice 101 1013 102 1012 Acid mine drainage Vinegar Increasing acidity Rhubarb Peaches 103 1011 Acidophiles Acid soil Tomatoes 104 1010 American cheese Cabbage 105 109 Peas Corn, salmon, shrimp 106 108 Neutrality 107 107 Pure water Seawater 108 106 Very alkaline natural soil 109 105 Figure 5.24 The pH scale. Alkaline lakes 1010 104 Increasing alkalinity Soap solutions Alkaliphiles Household ammonia Extremely alkaline soda lakes 1011 103 1012 102 Lime (saturated solution) 1013 101 1014 1 © 2012 Pearson Education, Inc.
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Osmotic Effects on Microbial Growth
Typically, the cytoplasm has a higher solute concentration than the surrounding environment, thus the tendency is for water to move into the cell (positive water balance) When a cell is in an environment with a higher external solute concentration, water will flow out unless the cell has a mechanism to prevent this Halophiles: organisms that grow best at reduced water potential; have a specific requirement for NaCl. Extreme halophiles: organisms that require high levels (15–30%) of NaCl for growth Halotolerant: organisms that can tolerate some reduction in water activity of environment but generally grow best in the absence of the added solute © 2012 Pearson Education, Inc.
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Halotolerant Halophile Extreme halophile Nonhalophile
Figure 5.25 Halotolerant Halophile Extreme halophile Example: Staphylococcus aureus Example: Aliivibrio fischeri Example: Halobacterium salinarum Growth rate Figure 5.25 Effect of sodium chloride (NaCl) concentration on growth of microorganisms of different salt tolerances or requirements. Nonhalophile Example: Escherichia coli 5 10 15 20 NaCl (%) © 2012 Pearson Education, Inc.
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Oxygen and Microorganisms
Aerobes: require oxygen to live Anaerobes: do not require oxygen and may even be killed by exposure Facultative organisms: can live with or without oxygen Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cannot use it Microaerophiles: can use oxygen only when it is present at levels reduced from that in air © 2012 Pearson Education, Inc.
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Oxygen and Microorganisms
Thioglycolate broth Complex medium that separates microbes based on oxygen requirements Reacts with oxygen so oxygen can only penetrate the top of the tube © 2012 Pearson Education, Inc.
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