Lecture 13 Basic Microbiology PDF

Lecture – 13 Farzana Hossain, Ph.D. Assistant Professor Dept. of Biochemistry and Microbiology * Growth of Bacterial Cultures  * Growth: *Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms repro...

Lecture – 13 Farzana Hossain, Ph.D. Assistant Professor Dept. of Biochemistry and Microbiology * Growth of Bacterial Cultures  * Growth: *Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission. *In the latter, individual cells enlarge and divide to yield two progeny of approximately equal size.  * Growth in Unicellular Microorganisms *Growth in unicellular microorganisms such as bacteria, yeasts and protozoans is defined in terms of an increase in the number of a given population. Growth in Multicellular Microorganisms *growth results in an increase in cell size but not cell number. Ex. Some algae (multicellular) * *  Bacterial growth refers to an increase in bacterial numbers, not an increase in the size of the individual cells. 1. Bacteria normally reproduce by binary fission. 2. A few bacterial species reproduce by budding. (they form a small initial outgrowth (a bud) that enlarges until its size approaches that of the parent cell, and then it separates). 3. Some filamentous bacteria (certain actinomycetes) reproduce by producing chains of conidiospores carried externally at the tips of the filaments. 4. A few filamentous species simply fragment, and the fragments initiate the growth of new cells.  * Definition of Binary Fission  Simple mode of asexual reproduction that results in two daughter cells which are exact copies of the parent cell.  The division of a bacterial cell occurs mainly through binary, or transverse, fission;  binary means that one cell becomes two,  transverse refers to the division plane forming across the width of the cell.  * *During binary fission, the parent cell enlarges, duplicates its chromosome, and forms a central transverse septum that divides the cell into two daughter cells. *This process is repeated at intervals by each new daughter cell in turn, and with each successive round of division, the population increases. * Step # 1: Replication of DNA Replication of DNA initiate binary fission process. The step of replicating nucleic acid begins from a replication origin. A replication bubble is formed, which separates the DNA strands. Each of the strands then serves as a template for synthesis of the complementary strand. Then, the DNA or genetic material is duplicated.  * Step # 2: Growth of Cell *As a preparatory step, it grows considerably and increases its size. *At the same time, the two circular DNA strands migrate and attach themselves to plasma membrane in different sites.  * Step # 3: Segregation of DNA Cell elongates and pulls apart in opposite poles. A division septum is created transversely in the cell. In short, the cell membrane extends and pinches inward of the cell. During this process, separation of the two chromosomes takes place. Step # 4: Splitting of Cells *The final binary fission step is splitting of the parental cell into two daughter cells, each having a nuclear material (chromosome) of its own.  * *The time required for a complete fission cycle— from parent cell to two new daughter cells—is called the generation, or doubling, time. *In bacteria, each new fission cycle or generation increases the population by a factor of 2, or doubles it. *Thus, the initial parent stage consists of 1 cell, the first generation consists of 2 cells, the second 4, the third 8, then 16, 32, 64, and so on. *For many common bacteria, the generation time is quite short, 20-60 minutes under optimum conditions. For most common pathogens in the body, the generation time is probably closer to 5-10 hours. *  * *One cell's division produces two cells; two cells' divisions produce four cells, and so on. *The number of cells in each generation is expressed as a power of 2 and the exponent tells the number of doublings (generations) that have occurred.  Logarithmic Representation of Bacterial Populations If binary fission continues, an enormous number of cells will be produced.  If a doubling occurred every 20 minutes- which is the case for E. coli under favorable conditions- after 20 generations a single initial cell would increase to over I million cells. This would require a little less than 7 hours.  In 30 generations or 10 hours, the population would be I billion. -difficult to graph population changes of such enormous magnitude by using arithmetic numbers. -logarithmic scales are generally used to graph bacterial Logarithmic Representation of Bacterial Populations Figure: A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line)  * *The method is used to observe the population growth pattern in a viable count technique, in which the total number of live cells is counted over a given time period. *In brief, this method entails; (1) placing a tiny number of cells into a sterile liquid medium (2) incubating this culture over a period of several hours (3) sampling the broth at regular intervals during incubation (4) plating each sample onto solid media (5) counting the number of colonies present after incubation.  * *There are four basic phases of growth: (1)lag phase (2) log phase (3) stationary phase (4) death phase.  * Lag Phase: *When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase. *there is no net increase in cell mass but the cell is synthesizing new components.  * Reasons: *The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. *The medium may be different from the one the microorganism was growing in previously. Here new enzymes would be needed to use different nutrients. *Possibly the microorganisms have been injured and require time to recover.  * Length of Lag Phase *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. *On the other hand, 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.  * Log Phase or Exponential Phase *During the exponential or log phase, microorganisms are growing and dividing at the maximal rate. *Their rate of growth is constant during the exponential phase (doubling in number at regular intervals). *Because each individual divides at a slightly different moment, the growth curve rises smoothly rather than in discrete jumps. * Practical Implication of Log Phase *The log phase is the time when cells are most active metabolically and is preferred for industrial purposes where, for example, a product needs to be produced efficiently. *However, bacteria are most sensitive (vulnerable) during exponential growth phase because the bacteria are most active during the log phase of growth. *Many antibiotics such as penicillin are only effective when cells are actively dividing, since they depend on disrupting new cell wall synthesis.  * Stationary Phase * The growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes. This period of equilibrium is called the stationary phase. * The growth curve becomes horizontal. * This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml. * Inthe stationary phase the total number of viable microorganisms remains constant. Reasons for Microbial Populations to Enter the Stationary Phase: 1. One obvious factor is nutrient limitation (starvation); if an essential nutrient is severely depleted, population growth will slow. 2. Aerobic organisms often are limited by O2 availability. Oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated in another way.  * 3. Population growth also may cease due to the accumulation of toxic waste products. 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. 4. Finally, there is some evidence that growth may cease when a critical population level is reached.  * Death Phase *Detrimental environmental changes like nutrient deprivation and the buildup of toxic wastes lead to the decline in the number of viable cells characteristic of the death phase. *The death of a microbial population is usually logarithmic (that is, a constant proportion of cells dies every hour). *Often the only way of deciding whether a bacterial cell is viable is by incubating it in fresh medium; if it does not grow and reproduce, it is assumed to be dead.