Bacterial Growth Curve and its mathematics
- 1. Bacterial Growth Curve Nandhana Satheesan I MSc Microbiology DBT, CUSAT
- 2. Growth It is a complex process involving 1. Entrance of basic nutrients into the cell 2. Conversion of these compounds into energy and vital cell constituents 3. Replication of the chromosome 4. Increase in size and mass of the cell 5. Division of the cell into two daughter cells, each containing a copy of the genome and other vital components
- 3. The Growth Curve • The most common means of bacterial reproduction is binary fission; one cell divides, producing two cells. • Population growth is studied by analyzing the growth curve of a microbial culture. • Evident in batch culture or closed system where no nutrient medium is provided and there is depletion of waste materials . • The growth of microorganisms reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases.
- 5. Lag Phase • When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, so this period is called the lag phase. • No net increase in mass, the cell is synthesizing new components. Necessity of lag phase 1.The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. 2. The medium may be different from the one the microorganism was growing in previously. 3. Possibly the microorganisms have been injured and require time to recover.
- 6. The lag phase varies considerably in length with the condition of the microorganisms and the nature of the medium. • This phase may be quite long if the inoculum is from an old culture or one that has been refrigerated. • Inoculation of a culture into a chemically different medium also results in a longer lag phase. • When a young, vigorously growing exponential phase culture is transferred to fresh medium of the same composition, the lag phase will be short or absent.
- 7. Exponential Phase • microorganisms are growing and dividing at the maximal rate possible. • Their rate of growth is constant that is, the microorganisms are dividing and doubling in number at regular intervals ; the curve rises smoothly. • The population is most uniform in terms of chemical and physiological properties, exponential phase cultures are usually used in biochemical and physiological studies. • Exponential growth is balanced growth - all cellular constituents are manufactured at constant rates relative to each other.
- 8. • If nutrient levels or other environmental conditions change, unbalanced growth results where the synthesis of cell components vary relative to one another until a new balanced state is reached. • Unbalanced growth is readily observed in two types of experiments- shift up and shift down. shift-up experiment * culture is transferred from a nutritionally poor medium to a richer one • There is a lag while the cells first construct new ribosomes to enhance their capacity for protein synthesis. This is followed by increases in protein and DNA synthesis. * Finally, the expected rise in reproductive rate takes place.
- 9. shift-down experiment • A culture is transferred from a rich medium to a poor one. • there is a lag in growth because cells need time to make the enzymes required for the biosynthesis of unavailable nutrients. • Consequently cell division and DNA replication continue after the shift-down, but net protein and RNA synthesis slow. • The cells become smaller and reorganize themselves metabolically until they are able to grow again These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions.
- 10. Stationary phase • Population growth ceases and the growth curve becomes horizontal. • This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml. • In the stationary phase the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death, or the population may simply cease to divide but remain metabolically active. Microbial populations enter the stationary phase for several reasons • nutrient limitation • Population growth also may cease due to the accumulation of toxic waste products.
- 11. For example, streptococci can produce so much lactic acid and other organic acids from sugar fermentation that their medium becomes acidic and growth is inhibited. Bacteria in a batch culture may enter stationary phase in response to starvation. Prokaryotes have evolved a number of strategies to survive starvation. • Many do not respond with obvious morphological changes such as endospore formation, but only decrease in overall size • They increase peptidoglycan crosslinking and cell wall strength • . • The Dps (DNA binding protein from starved cells) protein protects DNA. • Chaperone proteins prevent protein denaturation and renature damaged proteins.
- 12. • As a result of these and many other mechanisms, the starved cells become harder to kill and more resistant to starvation itself, damaging temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine. There is even evidence that Salmonella enterica serovar Typhimurium (S. typhimurium), and some other bacterial pathogens become more virulent when starved.
- 13. Senescence and Death • It was assumed that detrimental environmental changes like nutrient deprivation and the buildup of toxic wastes caused irreparable harm resulting in loss of viability. • Loss of viability was often not accompanied by a loss in total cell number, it was assumed that cells died but did not lyse. • This view is currently under debate. There are two alternative hypotheses for this.
- 14. Hypothesis 1 : Programmed Cell Death Programmed cell death predicts that a fraction of the microbial population is genetically programmed to commit suicide. • In this case, nonculturable cells are dead and the nutrients that they leak enable the eventual growth of those cells in the population that did not initiate suicide. • The dying cells are thus altruistic—that is to say, they sacrifice themselves for the benefit of the larger population.
- 15. Hypothesis 2 : Viable But Non Culturable (VBNC) • Some microbiologists believe starving cells that show an exponential decline in density have not irreversibly lost their ability to reproduce. Rather, they suggest that microbes are temporarily unable to grow, at least under the laboratory conditions used. • This phenomenon, in which the cells are called viable but nonculturable (VBNC), is thought to be the result of a genetic response triggered in starving, stationary phase cells.
- 16. • Once the appropriate conditions are available VBNC microbes resume growth. • VBNC microorganisms could pose a public health threat. Phase of Prolonged Decline Exponential decline in viability is sometimes replaced by a gradual decline in the number of culturable cells. The bacterial population continually evolves so that actively reproducing cells are those best able to use the nutrients released by their dying siblings and best able to tolerate the accumulated toxins.
- 17. • This dynamic process is marked by successive waves of genetically distinct variants. Thus natural selection can be witnessed within a single culture vessel.
- 18. Mathematics of Growth • If we start with a single bacterium, the increase in population is by geometric progression: n = the number of generations The total population N at the end of a given time period would be expressed as However, under practical conditions, the number of bacteria N0 inoculated at time zero is not I but more likely several thousand, so the formula now becomes
- 19. Solving for n, The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate constant (k). This is the number of generations per unit time, often expressed as the generations per hour. the mean generation time or mean doubling time (g)—can now be calculated.
- 20. The mean generation time is the reciprocal of the mean growth rate constant. For example, suppose that a bacterial population increases from 103 cells to 109 cells in 10 hours.
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