3/7/2016 MICROBIAL GROWTH I

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1 MICROBIAL GROWTH I Dosen Pengampu: Prof. Dr. Ir. Sri Kumalaningsih, M.App.Sc Prof. Dr.Ir. Wignyanto, MS Dr.Ir. M. Hindun Pulungan, MS Dr.Ir. Nur Hidayat, MP IrniaNurika, STP, MP, PhD. Sakunda Anggarini, STP, MSc Cell Growth Cell Growth depends on : Metabolism a large number of chemical reactions Fueling some transformation of energy Biosynthesis synthesis of small molecules (building blocks, coenzymes, vitamins, etc) Polymerization reactions that make the macromolecules Most Important As these are synthesized and accumulated in the cell they are assembled into cell structure the cell grows in size and eventually divides Transferse/Binary Fission Most common means of unicellular microbial reproduction The term binary expresses the fact that two cells have arisen from one cell Usually involves an equal partitioning of material (DNA, proteins, etc) Cells elongate to approximately twice their length and then form a partition that eventually separates the cell into two cells Septum results from invagination of the cell membrane and cell wall Invagination is the inward growth of the cytoplasmic membrane and cell wall from opposing directions 1

2 FTS Proteins and Cell Division Fts (filamentous temperature sensitive) proteins are essential fol cell division, they are present in all Bacteria Mutations in genes encoding these proteins cause the cell to not divide normally Such as long filamentos cells that fail to divide Fts Proteins interact to form the divisome (a protein complex) Formation begins with attachment of molecules of FtsZ in a ring around the center of the cell ( proteins needed to polymerize) attracts other divisome proteins FtsZ ring defines the cell-division plane ZipA: anchor that connects FtsZ ring to the cytoplasmic membrane Fts A: protein that also helps connect the ring to the membrane Divisome Cell already elongating, but the DNA is replicated before divisome forms Divisome is responsible for reglating synthesis of new cell membrane and wall material Elongated cell divides, yielding two daughter cells 2

3 FtsZ Ring & Cell Division Divisome septum is produced at the center of the dividing cell Cell reaches 2x its length cell divides into 2 cells 1. FtsZ ring not formed 2. FtsZ ring appears as nucleoids start to segregate 3. Full FtsZ ring forms as cell elongates 4. Breakdown of FtsZ ring and cell division DNA Replication & Cell Division Minc and Mind proteins help dictate the center of the cell by inhibiting FtsZ ring formation outside the cell s center Prevents cell division at the poles by being the most abundant protein at the cell poles Mind oscilates and helps Minc find the cytoplasmic membrane MreB and Crescentin Cytoskeleton protein MreB is an actin analog that winds as a coil throgh the long axis of the rod shaped cells, making contact with cell membrane;if inactive cocci shapped cell In vibrio shaped bacteria, crescentin is found along with MreB, this protein is similar to keratin. 3

4 Peptidoglycan Synthesis Autolysins cut the preexisting peptidoglycan by breaking down the β-1,4-glycosidic bond There s insertion of peptidoglycan precursors simultaneously Cell wall is outside the cell membrane therefore the peptidoglycan precursors must be transported Bactoprenol is a C56 lipid (hydrophobic protein) that binds peptidoglycan precursors and transports them through the cell membrane Interacts with transglycosylase so precursors can be inserted Transglycolases make the glycosidic bond Transpeptidation Transpeptidation is the final step in cell wall/peptidoglycan synthesis Ftsl catalyzes the transpeptidase reaction in E. Coli and other cells Cells are penicillin sensitive becase penicillin binds to Ftsl and other penicillin binding proteins no cross-linking occurs with new material In absence of crosslinks and new cell wall synthesis, continued activity of autolysins weakens cell wall causing osmotic lysis (autolysis) Budding : Yeast Budding : A type of division which results in unequal distribution of cellular material between newly synthesized bud and the original cell Yeast also divide by binary fission: A type of division which results in equal distribution of cellular material 4

5 During growth both the total cell number and mass double Both can be quantitatively measured Usually equivalent because of non-synchronous growth NOTE: text indicates growth as increase in cell numbers, but we use either # or mass Usually expresses as : #cells/ml or mg cells/ml Cell mass is continuous whereas cell division is discontinuous Can be proved by synchronous cultures All cells are in the same stage in the cell cycle Cells increase in size without dividing until generation time is reached Exponential growth is the pattern population increase where there is a doubling in the nmber of cells Synchronous Cultures Measures made in this type of culture is equivalent to a single cell Mass increases continuously Cell number increases discontinuously Step - wise Bacterial Growth Curve The mathematics/kinetics of bacterial growth does not represent: The normal pattern of bacterial growth It reflects one selected portion of a normal growth curve, namely the Logaritmic/Exponential phase Cells divide at regular intervals known as generation time (g) Although exponential growth is characteristic of microbial populatios, it does not normally continue forlong periods of time During exponential growth cells divide according to g Applies to populations of cells, not a single cell 5

6 Phases Of Bacterial Growth Lag Phase : does not always occur in the growth of a bacterial population and when it does the duration can very considerably (k=0) Expnential or Log Phase: cannot continue indefinitely, limited (k=positive, reasches a max) Stationary Phase : no increase in cell number or cell mass (k=0) Death Phase : Cells die exponentially (k=negative) k = specific growth rate Lag Phase GROWTH CYCLE When a microbial population is inoculated into a fresh medium, growth usually does not take place immediately but only after a period of time (short or long) This time is called the lag phase The lag phase occurs because for growth to occur in a particlar culture medium the cells must have a complete complement of enzymes for the synthesis of the essential metabolits All phases are at maximal rate and cells multiply according to g Biphasic growth : two different carbon and energy sources Biphasic Growth Stationary culture cells are inoclated into a medium containing both glucose and lactose Growth first on most rapidly metabolized carbon source (glucose) Then lag before growth on lactose 2 extra enzymes needed Lactase permease to bring in the lactose and β-galactosidase to break lactose down to galactose and glucose If inoculate cells growing exponentially on glucose are placed into lactose, lag is seen 6

7 Exponential Phase The pattern of population increase where the number of cells doubles during each unit of time period is referred to as exponential growth Growth rate is maximal Cells are the healthhiest Prokaryotes grow faster than eukaryotes Smaller cells grow faster than larger cells The Mathematics of Exponential Growth Relationship between n and Nt Derivation of Equation n Nt n = generation number Nt= total final cell number Sample Problem 10 bacteria multiply into 100,000 bacteria in 8 hours. What is g?? 7

8 GROWTH CYCLE Stationary Phase In a tube or flask with limited nutrients the exponential growth cannot occur indefinitely E. Coli : grows for 48 hours with g = 20 minutes Stationary phase occurs because either : 1. An essential nutrient of the medium is used up 2. Waste by-product of the organism builds up to an inhibitory level In stationary phase there is no net increase or decrease in cell number, but many cell fnctions can continue. Cryptic growth some cells die while some cells are doubling ONE single E. Coli cell in 48 hours, without limits, could grow to 4,000x the weight of Earth! But thankfully this does not occur Sample Problem GROWTH CYCLE Death Phase GROWTH CYCLE When the cells are no longer able to find the nutrients needed, the cells can die The death phase is also exponential, but is lower than the growth exponential rate NOTE: the terms lag phase, exponential phase, stationary phase and death phase do not apply to individual cells, but only to populations of cells 8

9 Measurement of Growth Direct Microscopic CountsGrowth A special slide and cover slip which contains a known volume The number of bacteria per milliliter (ml) = number of cells conted X dilution (if used) X 50,000 Ex: 300 cells x 50,000 = 1.5 x 107 cells/ml Viable Plate Count Measurement of Growth Measurement of Growth 9

10 Measurement of Growth Indirect Counting Turbidity Spectrophotometer Measures light passing throgh the solution (unscattered, unabsorbed) Nepholometer Measures scattered light 10

11 Measurement of Growth TURBIDITY MEASUREMENTS OF MICROBIAL GROWTH Typical growth curve data for two organisms growing at different growth rates Relationship between cell number or dry weight and turbidity readings. Note that the one-to-one correspondence between these relationships breaks down at high turbidities Continuous Culture A continuous culture is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained Continuous Culture Schematic For A Chemostat In the chemostat, both growth rate and cell density of the culture can be controlled independently and simultaneously. Two factors govern growth rate and cell density respectively : the dilution rate, which is the rate at which fresh medium is pumped in and spent medium is removed the concentration of a limiting nutrient, such as a carbon or nitrogen source, present in the sterile medium entering the chemostat vessel. 11

12 Continuous Culture Experimental Uses of the Chemostat The chemostat has been used in microbial ecology as well as in microbial physiology the chemostat can easily mimic the low substrate concentrations that often prevail in nature Chemostats have also been used for enrichment and isolation of bacteria. From a natural sample, one can select a stable population under the nutrient and dilution-rate conditions chosen and then slowly increase the dilution rate until a single organism remains. MERCI BEAUCOUP 12