Growth of microbes in batch culture The Phases of Growth Growth in the bacterial context is normally described as an increase in cell number. Microorganisms, depending upon the specific species, increase their numbers by binary fission, budding or by filamentous growth. Binary fission is the separation of the initial cell, the mother cell, into two daughter cells of approximately equal size. This is a very common method of multiplication and most of the organisms we will investigate divide in this manner. Budding division Budding division involves the
asymmetric creation of a growing bud, on the mother cell. The bud increases in size and eventually is severed from the parental cell. After division is complete, the mother cell reinitiates the process by growing another bud. Yeast and some bacteria (Caulobacter is one example) use this form of division. Filamentous growth Filamentous growth is characterized by the formation of long, branching, nondivided filaments, containing multiple chromosomes. Sometimes the filaments will have cross walls separating chromosomes. As growth proceeds, the
filaments increase in length and number. Under nutrient limiting conditions, some filamentous microorganisms will go through developmental changes, with a fraction of the filaments differentiating to form spores. The structures and mechanisms used to form these spores can be spectacular. Streptomyces species and many molds grow in this manner. Binary fission The best characterized type of growth is binary fission and this is what we will focus on in this experiment. When grown in liquid medium, bacterial cultures progress through several distinguishable phases, which can be characterized by plotting the log
of the cell number vs. time. A typical growth curve has phases of growth, lag phase, exponential growth phase (also termed balanced growth), stationary phase and death phase. Binary fission requires: cell mass to increase chromosome to replicate cell wall to be synthesized cell to divide into two cells Exponential growth Exponential growth is a function of binary fission since at each division there are two new cells. The time between divisions is called generation time or the doubling time since this is the time for the population to double. These can range from minutes to days depending on the species of bacteria.
Growth rate is the change in cell number or mass per unit time. What do we mean by exponential growth? A population doubles each generation is exponential growth. Graphically on arithmetic coordinates the graph takes the shape of a J - a curve with ever increasing slope - growth rate. Plotted on semilogarithmic paper, where the Y axis is logarithmic (base 10) and the X axis is arithmetic (either generation or time), you get a straight line. Exponential growth Generation times - N = No2n where No is the original number of cells and n is the number of generations. g, generation time, equals t/n, time divided by generation. How do you calculate n? N = No2n log N = log No + nlog2
log N - log No = n log2 n = [log N - log No] / log2 n = [log N - log No] / 0.301 We also know that the slope of the semi-log line equals 0.301 divided by the generation time. A bacterial growth curve The four phases of growth (lag (1), exponential (2), stationary (3) and death phase(4)) are labeled. When an organism is inoculated into a fresh medium, it needs to adapt to the new nutrients available, synthesize RNA, protein and finally replicate its DNA before starting division. These processes take time and there is no net increase in cell numbers, thus a lag phase is observed. Once the appropriate enzymes for growth in a particular medium have been expressed cells begin to multiply. This period
of maximal division can last for several hours or days, depending upon the organism, and is called the log or exponential growth phase . Eventually the increase in cell number ceases, either because cells stop dividing or the rate of division equals the rate of cell death, resulting in a stationary phase. This is usually caused by limitation of a nutrient or the accumulation of a toxic waste product. Depending on the bacterium, stationary phase can last for several hours to many days. The final chapter of a growth curve is the death phase . An exponential decrease in the number of organisms due to cell death occurs during this phase. Some microorganisms never experience a death phase or it is greatly delayed due to their ability to survive for long periods without nutrients. Measurement of Bacterial Growth
How do we measure growth? Direct microscopic counts - use the microscope and a slide with a grid engraved on it. A coverslip and placed over the grid which captures a known volume of liquid. Problems : dead cells are difficult to distinguish, small cells are difficult to see method not suitable for dilute samples Viable counts - count only cells that are able to divide and form offspring. Referred to as plate counts or colony counts. Assumption each viable cell gives rise to a colony. Dilutions - to cover a cell density that ranges from 30 - 300 colony forming units per plate. Problems: not all species of bacteria will form colonies on any particular medium, small colonies are not counted. Turbidity - cell suspensions look cloudy because each cell scatters light as it passes through a suspension of cells. Take advantage of the light scattering properties of a suspension using a spectrophotometer which measures unscattered light as it passes through. The scatter is proportional to cell number (density of cells) up to high density cultures because cells begin to cause rescatter the light back into the path of unscattered
light. Therefore the optical density is not linear at high density suspensions. Need to develop a standard curve between OD and cell numbers (viable counts). Measurement of Bacterial Growth Growth of microorganisms can be measured by following the increase in cell number or the increase in a cellular macromolecule such as DNA or protein. In most cases, the increase in cell number is determined. Cell numbers can be measured in a variety of ways. For this experiment we will use the viable plate count, which you are familiar with, and turbidometric measurement. So which wavelength of light do we use to measure cell numbers? Typically the shorter the wavelength the greater the sensitivity of the measurement. Some wavelengths cannot be used, however, because cellular constituents absorb light, not scatter them. For example proteins absorb light at 280 nm. Likewise the color of the medium affects absorbance. For this experiment we will be measuring the growth of E. coli in either a yellow or a clear medium, 600 nm works well. Beware, however, that the choice of wavelength must be considered for each experimental condition.
The sample is placed in a sample chamber. The chamber will contain a holder, an entrance for the selected wavelength, and an exit, leading to the detector. A critical piece of equipment used in the sample chamber is the cuvette. A cuvette is a special test tube that holds the sample. Cuvettes must be clean and free of aberrations, both of which could scatter light, resulting in inaccurate readings. Most good cuvettes are expensive and must be treated with care! Measurement of Bacterial Growth The actual operation of a spectrophotometer is much simpler than understanding its parts. There are a few general, rules of thumb, when using any spectrophotometer. 1. Before you begin, be sure the wavelength selector is set for the wavelength of light you are going to use. This involves setting the correct wavelength on the selector, making sure the correct lamp is on for the wavelength you have selected and that you are using the correct cuvette. 2. Make sure to zero the spectrophotometer. When this is performed you are adjusting the machine to 100% transmittance. This is done by using a sample that contains all components of a mixture except the component to be measured. For example, if you are measuring the growth of B. cereus in nutrient broth, the machine would
be blanked with sterile, uninoculated nutrient broth. Realize that the machine is being blanked to read 100% T (or 0 absorbance) with a specific solution in a specific cuvette, with the cuvette in a specific orientation. This last consideration, nullifies any aberrations in the cuvette. 3. With proper technique, nothing should be spilled into the spectrophotometer. If it is, clean it up and immediately notify an instructor. This type of spillage usually happens if cuvettes are filled over a spectrophotometer, a very bad habit to form! 4. Clean the cuvette after each use by rinsing with distilled water and allowing to dry upside down in a test tube rack. For more stubborn stains use a dilute solution containing a mild soap (like Ivory). In rare cases, difficult blemishes can be removed with dilute acid (1-5%) or with ethanol. Never let a sample dry in a cuvette! Protein and nucleic acids form a strong bond with glass and can be impossible to remove. 5. When measuring the turbidity  of a cell suspension, absorbance values in the range of 0.1--0.8 are acceptable. Readings below 0.1 push the limits of the spectrophotometer, since it is not sensitive enough in that range. Absorbance values above 0.8 result in microorganisms casting shadows onto one another and not being seen by the spectrophotometer. If a reading is above this range, the culture must be diluted with sterile medium. If a reading is below this range, concentrate your culture using a centrifuge. 6. When graphing growth versus time, you must plot absorbance on a log scale. Even though absorbance is a
logarithmic unit, absorbance readings are proportional to cell mass which is increasing exponentially during growth. Generating a growth curve In this experiment, the classic bacterial growth curve will be demonstrated. A culture of Escherichia coli will be sampled at hourly or halfhourly intervals from the time of inoculation of the culture (0-time) through a 7 to 9-hour incubation period. The periodic samplings will be plated to determine viable counts (as colonyforming units per ml of culture) over the incubation period such that a growth curve may be plotted. From the graph, we may note the stages in the growth of the culture as it grows into the stationary phase. Additionally we will be able to determine the growth rate and generation time of E. coli under our
experimental conditions from two points in the exponential phase of the graph. Graphing of bacterial growth on a linear scale Generating a growth curve By definition, bacterial growth is cell replication i.e., growth of the culture. Most species of bacteria replicate by binary fission, where one cell divides into 2 cells, the 2 cells into 4, the 4 into 8, etc. If this cell division occurs at a steady rate - such as when the cells have adequate nutrients and compatible growing conditions - we can plot numbers of cells vs. time such as on the graph at right. Before too long, we will need to extend the paper vertically as the population
continues to double. For a culture where cells divide every 20 minutes, one cell can result in 16,777,216 (i.e., 224) cells after just 8 hours barring nutrient depletion or other growthaltering conditions. Graphing of bacterial growth with cell number on a log scale. Generating a growth curve If we were to convert our vertical axis to a logarithmic scale - as in the previous slide - we will not need as many sheets of graph paper, and we will find that a steady rate of growth is reflected as a straight line. (On the vertical axis, the same distance on the paper is covered with each doubling.) This type of graph paper is called semilogarithmic graph paper on which we will be plotting our class results.
The numbers we plot will fall on the graph at the same place the logarithms of these numbers would fall when plotted on conventional graph paper. In the next slide you will see a logarithmic paper. Generating a growth curve The example on the right shows the type of graph we may obtain from our class data. We can plot both colony-forming units (CFUs) per ml and absorbance on the same graph, remembering that the absorbance units should also be on a logarithmic scale. Rather than "connecting the dots," we draw the best straight line among our CFU/ml plots to represent the phases of growth - lag, exponential, and the start of the maximum stationary phase. Example data showing a plot of cell number by VPC and by turbidity.
For the growth rate formula we are about to use, we need to choose two points on the straight line drawn through the exponential phase, also making note of the time interval between them. As we will be converting our numbers to logarithms for the formula, why not choose two points for which the logs are easy to obtain? (For example, the log of 1X1010 is simply 10.) Higher CFU/ml = Xt = 1X1010 (at 5.75 hours) Lower CFU/ml = X0 = 1X108 (at 2.75 hours) Time interval (in hours) between the 2 points = t = 3 We find the growth rate which is the number of generations (doublings) per hour: Generating a growth curve With the second formula, we find the generation time which is the time it takes for the population to double:
When we graph the CFUs/ml and absorbance on the same graph, we would hope to see an upward trend for both. Sometimes the absorbance continues to rise after the CFUs/ml level off into the maximum stationary phase. What would be the cause of that? With a clear graph, one should be able to determine the generation time without the use of formulas. Just look for a doubling of the population and the time it takes for that to happen. For example - in the previous graph - the time it takes to go from 3 X 109 to 6 X 109 appears to be approximately 30 minutes, which is close to the generation time determined above. Environmental factors Temperature - as temperature increases, the growth rate increases until a point at which the growth rate declines.
Minimum temperature - below growth does not occur may be due to the stiffening of the cytoplasmic membrane. optimum temperature where the growth rate is maximum Maximum temperature- above which growth does not occur which reflects when proteins may be denatured, nucleic acids and other cellular components are irreversibly damaged. Environmental factors Classification of bacteria based on temperature optimum psychrophiles - low temperature optima <15 C - may even be killed by brief warming or thawing. mesophiles - midrange temperature optima 25 - 40 C
thermophiles - high temperature optima 40 - 80 C hyperthermophiles - very high temperature optima >80 C Psychrophiles open ocean water is between 1 and 3 C. Artic and Antartic regions are cold. Adaptation membranes rich in unsaturated fatty acids Thermophiles hot springs all over the world fermenting compost Adaption thermostable proteins with usually a few changes in the amino acid sequence when compared to a mesophile's protein
saturated fatty acids in their membranes Environmental factors Acidity and alkalinity Most environments are between 5 and 9 and optima are between these values, around neutral pH of 7. Acidophiles live at low pH Obligate acidophiles such as Thiobacillus. cytoplasmic membrane actually dissolves and the cell lysis at more neutral pH. Alkaliphiles live at high pH such as soda lakes and carbonate soils. Important to biotechnology since they
have hydolytic proteases that function at alkaline pH and are used in household cleaners. Environmental factors Water availability - bacteria need water as a solvent. Water availability is expressed as water activity - how much water is available. Solutes and surfaces affect water activity both decrease it. Water moves from high water activity values to lower values in the process of osmosis. Different bacteria have different tolerances towards low water activities. In fact, preservation process takes advantage of lower water activity which causes plasmolysis or pulling away of the membrane from the cell wall. This inhibits cell growth. Halophiles require 1-6% for mild halophiles and 7-15% salt for moderate halophiles. Extreme halophiles require 15-30% salt.
Environmental factors Oxygen 1. Aerobes require oxygen up to 21% as in air. 2. Microaerophilic bacteria require reduced levels of oxygen 3. strict or obligate anaerobes require the absence of oxygen. 4. Facultative anaerobes can grow aerobically if oxygen is present and switch to fermentation or anaerobic respiration if oxygen is absent. E. coli is a facultative anaerobe that grows aerobically and using anaerobic respiration when necessary. 5. Aerotolerant anaerobes don't use oxygen for growth but tolerate its presence. Can grow on the surface of solid medium with out the special anaerobic conditions required for the strict anaerobes. Anaerobic culture conditions - add reducing reagents such as thioglycolate, bubble nitrogen gas through your solutions to remove oxygen after autoclaving, add a dye such as resazurin to indicate when oxygen is penetrating, use an anaerobic jar with an atmosphere containing hydrogen gas and carbon dioxide. Why go to such great troubles for the strict anaerobes? Because they contain lots of flavins which react with oxygen to produce toxic oxygen species that are very reactive. Oxygen species - singlet oxygen which the valence electrons become highly reactive and oxidize organic matter readily. Superoxide anion, hydrogen peroxide, and hydroxyl radical which are inadvertant byproducts during respiration.
These can all damage cell macromolecules by oxidation processes. Measures to counter these toxic oxygens - catalase degrade hydrogen peroxide to oxygen and water. peroxidases - destroys hydrogen peroxides too but requires NADH. no oxygen evolved. super oxide dismutase produces hydrogen peroxide from super oxides. Aerobes and facultative aerobes generally contain catalase and super oxide dismutase. Environmental factors Big thanks to http://inst.bact.wisc.edu/inst/index.php?modul e=book&type=user&func=displaychapter&chap _id=43&theme=Printer AND http://microbiology.okstate.edu/faculty/demed 2/Notes/Microbial%20growthdoc.html
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