Chapter 4 Growth Of Microbial Population

Chapter 4: Growth of Microbial Population Introduction 1. As long as the environment is favorable, most prokaryotes reproduce continuously but some do form spore in response to environment, but seldom it is a result of differentiation. 2. Some bacteria have development cycles that are obligatory like Cauloabacter, which differentiates into two different kinds of cells at every cell division, where most just divides into equal progency cells and resembles that previous generation. 3. “Growth” indicates an increase in population as well as the size of an individual, where some growth are slow and some are fast in environments where they have to compete for nutrients and being able to utilize nutrients imparts a string selective advantage. 4. Fast growth is not the only available survival strategy, i.e. adhering to surface allows the long rage survival of organisms that do not compete well for food but are able to stay put in a particular environmental niche. 5. Photoautotropic obtain their energy from light and chemautotrophic obtain their energy from the oxidation of inorganic compounds. 6. Behavior of cultures of organism in mediums helps determine their identity and the use of differential medium is used to do this, where a selective medium are designed to let some organism grow and other inhibit others. (Shape and color of colony help identify the organism) 7. Nutrient broth the precise chemical composition is not know, but a define synthetic medium contains inorganic salts with glucose as the sole carbon source and addition of know amounts of nutrients. 8. Every medium is sterilize in a autoclave, which get rids of most bacteria except extreme thermophiles, but these bacteria usually don’t grow at 37C or liquid is passed through a membrane filter that retains cellular microbes, but not viruses, which is good for media that contain heat liabile components. How to Measure Growth of a Bacterial Culture 1. Following the rates of increase in number of cells, disappearance of nutrients, or accumulation of metabolites, but most widely used measurement is that of turbidity, which depends on the ability of bacterial cells scatter light and determined using a colorimeter or spectrophotometer. (not good when medium is cloudly) 2. Counting living cells determines on the ability of single bacterium to produce a colony, and to carry out a viable count, dilutions are made and spread on agar, incubate, and count the number of colonies that develop. 3. Total particle count can be carried out using a microscope where cells in a sample can be counted with a counted chamber, which is a glass slide with a central depression of known depth,, the bottom of which is ruled into squares of known area. (hemocytometer is used for animal cells) 4. Electronic counting is based on the principle that a bacterial cell conducts less electricity than a saline salt solution where two chamber are filled with saline solution connected by a small hole and the bacterial sample to one chamber and every time a bacterial cell pass through the hole it decreases electrical conductivity. When Should the Growth Rate be Determined? 1. Stationary phase of growth is when the culture grows over a period of time but then stops growing due nutrients being exhausted or waste products reached an inhibitory concentration. 2. Lag phase is the delayed period of time is takes a organism to start growing in a new medium, which depends on the organism, particular medium, and the length of time the culture has been in the stationary phase. (length process of spore germination in Bacillus subtilis is a example) 3. Growth that resumes at a steady rate is the exponential phase and this is when growth rate should be determined. 4. Culture in exponential phase of growth at a constant rate as long as the concentration of substrate being consumed does not fall below a working level, if at zero growth rate decreases. 5. The relationship of growth rate and substrate concentration approximates first order kinetics. The Laws of Growth 1. 2^n describes the increase in cell numbers, where n is the number of generation.

2. N=2^n multiple No, where N is number of cell after n generation starting with No cell, where log if the equation can be rearrange to give n= (logN-logNo)/.301 to determine the number of generations if number of beginning cells and number of ending cells are known. 3. If time interval over which the cell increase is known then the time it takes for cells to double can be calculated where generation time = time/ number of generation (g=t/n). 4. Calculating the rate of growth for any time interval requires a mathematical digression, dN/dt =kN where dN is the change in the number, N, of cells per unit volume over (t), and k is a proportionality constant called the specific growth rate (hours^-1). 5. k=2.303 (logN-logN0)/t, where specific growth rate, k, of the culture describes how fast a particular bacterium grows in a particular environment and we can also determine the relationship between k and g. 6. Exponential growth/ logarithmic growth mimics an autocatalytic or first order, chemical reaction. Balance Growth 1. As long as a culture is in the exponential phase, all cell constituents increase by the same proportion over the same interval of time, which is known as balance growth, but does not persist for long in environment because most bacteria alternate between periods of growth and nongrowth. ( going in and out of stationary phase) 2. Cultures in balance growth is the only phase of growth of a culture that is readily reproducible and can be replicated on different occasions and in different laboratories. 3. Balance growth suggests that the mean cell size remains constant, a condition that might at first glance appear paradoxical because as they grow, individual cells increase in size and eventually divides, so it really refers to the average behavior of cells in population, not to that of individual cells. 4. Some cell properties change early during the growth of a culture, long before the increase in mass slows down, which means balance growth conditions can be approximated only at low cell densities. 5. Unless growth is monitored throughout a physiological experiment. The results may not be reproducible. Continuous Growth 1. A culture can be maintained in balance growth by diluting it at set intervals with fresh medium, which will make the cell grow in an unrestricted manner, which can be done with an apparatus called a continuous-culture device or chemostat. 2. For a chemostat to function properly the bacterial density should not exceed that which allows balanced growth in a batch culture, which is achieved by making an essential nutrient limiting. 3. Important properties of a chemostat are as follows: the rate of addition of fresh medium (per volume) determines the growth rate in the culture vessel and density of bacteria in the culture vessel is constant and is determined by the concentration of limiting nutrient. (useful in studying mutagenesis and evolution) 4. Increase the rate of fresh medium, outflow will increase, cell will be lost at greater rate than they are formed, density of cells in vessel decrease using nutrients at lower rate so nutrients increases, growth rate will then increase to match the rate of the loss cells. How is the Physiology of the Cell Affected by the Growth Rate? 1. Many bacteria are particularly well adapted to exploit their nutritional environment and to convert it into their own special form of selective advantage, a high growth rate where both the macromolecule composition and cell size change with growth rate. 2. The growth rate that a particular medium supports, not its specific composition, determines the physiological state (cell size and macromolecular composition) of cells growing in it, which means that changes in physiological state are nutrition mediated but not nutrition specific. 3. Cell mass, protein, RNA and DNA change with growth rate: as the cell grows faster, it becomes bigger and contains more of each component, but each component changes concentration to different degrees to growth rate. ( RNA is faster than protein, DNA, which is efficient) 4. In slow growing culture, cells make ribosomes that are not used, which seem inefficient, but is there so cell that are in poor medium that move to a rich medium can protein synthesize fast as possible. Effects of Temperature, Hydrostatic Pressure, Osmotic Strength and pH

1. Microbes are in intimate contact with an environment that can change abruptly and become threatening, so they have evolve an array of mechanism to cope with environmental stresses and changes. 2. Members of extremophiles have volved not only tolerate but also thrive in environment at startling exteemes of temperature, pH, hydrostatic pressure or concentration of salt. I. Temperature 1. Microbes can grow wherever there is liquid water, regardless of the ambient temperature where boiling points and freezing point can extend well beyond there limits at sea level, where liquid is kept below freezing point by solutes and above boiling point by high pressure. 2. Broadly; bacteria (40C) and fungi dominate the lowest temperature and archaea dominate the highest, but all microbe specialize in one particular range of temperature. A. Effects on Growth Rate 1. Over the normal range, the temperature affects the growth rate the same way it does the rate of a chemical reaction, which obeys Arrhenius equation, which describes the logarithm of a chemical reaction rate as a linear function of the reciprocal of absolute temperature. 2. Ar he high end of temperature, the decline of growth rate passes through maximum (optimum growth temperature), and then fall sharply to zero (maximum growth tempertuare), at the low end, the decline is slower, but eventually the slope curve becomes vertical at the minimum growth temperature. B. Classifying the Temperature Response of Microbe 1. One way to classify temperature responses is by the microbe’s optimum temperature for growth: a microbe is psychrophile if it is low (Psychromonas ingrahamii -12c), a mesophile if it is very moderate, a thermophile if it is high, and a hyperthermophile if it is very high. C. Growth Limits at Temperature Extremes 1. The reason for stopping growth that high temperature is some vital cellular component, most likely protein becomes thermally inactivated and many mutation (heat-sensitive mutation) diminishes the thermal stability of essential protein, DNA melts at 80C becomes two strands). 2. In ordinary organisms, proteins, nucleic acids, and lipids are unstable at temperatures that are far lower than those required for the growth of extremophilic bacteria and archaea. 3. Heat resistance is genetically encoded and there are evidence that their thermostability depends on their tertiary structure, which means they have a large portion of charged amino acids. 4. Hyperthermophiles manufacture heat shock proteins, which proteins other proteins from heat denaturation, where some of these proteins are chaperone which facilitate the folding of other proteins or assembly of multiprotein complexes. 5. Thermopiles have both a thermoprotective DNA-binding protein and relatively high magnesium concentration that stabilize the molecules by neutralizing their phosphate, also have a reverse gyrase, which introduces positive supercoil into DNA. 6. Thermophiles, membrane are not composed of phospholipids bilayers but rather are isoprenoids linked to glycerol by highly stable ether bonds forming a monolayer, which lead the belief that Archaea first arise in high-temperature environment and then evolved to grow at lower temperatures. 7. Chemical reactions proceed more slowly as the temperature decrease, but they do not stop, in terms of microbial growth hydrophobic interactions are weaken by lower temperature by causing conformational changes in proteins. D. Lethal Effects 1. Temperatures just a few degrees higher than those that stop a microbe’s growth may kill it, but no clear answer of how high a temperature must be or how long at that temperature is required to sterilize, only probability.

2. Instead of a normally distributed bell shaped curved range of sensitivities, a microbes’s chance of dying in any period of time—immediately or after prolonged exposure—is constant, where the logarithm of the number of survivors is a linear function of time of exposure. (Single-hit kinetics) 3. To determined how long a treatment is required to eliminate a microbial population, need to know the decimal reduction time (D value), which is the time necessary to decrease the viable population by 1 log, the size of population and assurance that all cell will be killed. 4. Cold shock is sudden shift in temperature, where freezing kills microbes, but that is not a consequence of low temperature, it is the suspending medium being freeze exposing a high osmotic strength. II. Hydrostatic Pressure 1, Most microbes are protected from being crush to death by high hydrostatic pressure because their outer cell barrier, including cell membrane, is freely permeable to water so the pressure within the microbial cell rapidly balance the external pressure, but they are affected molecularly by favoring deceases in molecular volume. 2. Barophiles, grow faster at increased pressure, where some are obligate where they grow at pressure higher than 1 atmospheres. 3. If the volume of the molecule in the activated state is larger, the reaction will be inhibited by increase pressure; if the volume is smaller, the rate will be enhanced by or possibly even be dependent on high pressure. III. Osmotic Pressure 1. High concentration of salt completely stop the growth of microbe likely to be present in food, but some other microbe can tolerate such environment and the range can be as high as 5.2M of NaCl. (Halobacterium & Halomonas). 2. Environment with high osmotic pressure presents a microbe with two challenges, it decreases the activity of water, drying the cell out and it diminishes the cell’s turgor pressure by decreasing the difference in osmotic pressure between the inside and the outside. 3. When exposed to a high osmotic environment, cell maintain turgor by increasing the solute concentration of their cytoplasm , either by pumping solutes in or by synthesizing more, which inactivate it own proteins, but certain compatible solutes are less damaging and may even be protective. 4. Trahalose protects the membrane from drying and salt. IV. pH 1. Acidophiles grow at pH 1 and alkaliphiles grow at pH 11.5, but in general bacteria prefer a slightly alkaline pH and fungi prefer a acid pH. 2. Prokaryotes can grow over a wider range of pH than their proteins can tolerate, but they resist the pH by pumping protons in it out of their cells, instead of adapting to it.

Schaechter, M., Ingtaham, J., & Neidhardt, F. C. (2006). Microbe. Washington, D.C.: ASM Press.