Bacterial Growth.pptx

Bacterial Growth  Pure culture techniques Pure culture, a population of cells arising from a single cell, to characterize an individual species Robert Koch - transformed microbiology Within about 20 years after the development of pure culture techniques most pathogens responsible f...

Bacterial Growth  Pure culture techniques Pure culture, a population of cells arising from a single cell, to characterize an individual species Robert Koch - transformed microbiology Within about 20 years after the development of pure culture techniques most pathogens responsible for the major human bacterial diseases had been isolated  The Spread Plate If a mixture of cells is spread out on an agar surface – colony - macroscopically visible growth or cluster of microorganisms The spread plate is an easy, direct way of achieving this result A small volume of dilute microbial mixture containing around 30 to 300 cells is transferred to the center of an agar plate and spread evenly over the surface with a sterile bent-glass rod  The dispersed cells develop into isolated colonies Because the number of colonies should equal the number of viable organisms in the sample, spread plates can be used to count the microbial population  The streak plate The microbial mixture is transferred to the edge of an agar plate with an inoculating loop or swab and then streaked out over the surface in one of several patterns After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector & so on….  this is essentially a dilution process In both spread-plate and streak plate techniques, successful isolation depends on spatial separation of single cells  The Pour Plate Extensively used with prokaryotes and fungi, a pour plate also can yield isolated colonies original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating  small volumes of several diluted samples are mixed with liquid agar that has been cooled to about 45°C, and the mixtures are poured immediately into sterile culture dishes After the agar has hardened, each cell is fixed in place and forms an  Culture Media Much of the study of microbiology depends on the ability to grow and maintain microorganisms in the laboratory A nutrient material prepared for the growth of microorganisms in a laboratory is called a culture medium  To be effective, the medium must contain all the nutrients the microorganism requires for growth all microorganisms need sources of energy, carbon, nitrogen, phosphorus, sulfur, and various minerals Precise composition of a satisfactory medium will depend on the species one is trying to cultivate Knoweledge of habitat often is useful  Culture media can be classified on the basis of several parameters:  Defined or synthetic medium all chemical components are known often used to culture photolithotrophic autotrophs such as cyanobacteria and photosynthetic protists Many chemoorganotrophic heterotrophs also can be grown in defined media with glucose as a carbon source and an ammonium salt as a nitrogen source  Defined media are used widely in research, as it is often desirable to know what the experimental microorganism is metabolizing Assay for vitamins – fastidious microbes such as lactobacilli are used in tests that determine the concentration of a particular vitamin  Complex Media Ingredients of unknown chemical composition Single complex medium may be sufficiently rich to completely meet the nutritional requirements of many different microorganisms Nutritional requirements of some microbes are unknown – defined medium cant be used  fastidious bacteria that have complex nutritional or cultural requirements; they may even require a medium containing blood or serum Complex media contain undefined components like peptones, meat extract, and yeast extract  Selective media Favor the growth of particular microorganisms Salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-  Endo agar, eosin methylene blue agar, and MacConkey agar – routinly for E. coli from water A medium containing only cellulose as a carbon and energy source is quite effective in the isolation of cellulose-digesting bacteria The possibilities for selection are endless, and there are dozens of special selective media in use  Enriched media it is designed to increase very small numbers of the desired type of organism to detectable levels Enrichment culture is usually liquid and provides nutrients and environmental conditions that favor the growth of a particular microbe Chocolate agar, an enriched medium used to grow fastidious organisms such as Neisseria gonorrhoeae Brown color is the result of heating red blood cells and lysing them before adding them to the medium  Differential /indicator media Distinguish among different groups of microbes and even permit tentative identification of microorganisms based on their biological characteristics Blood agar – hemolysis pattern (α, β, γ) [Staphylococci & Streptococci]  MacConkey agar – selective and differential Lactose fermenter (Pink)& non lactose fermenter (Pale) Neutral red indicator Mannitol salt agar – 7.5% NaCl – selective for staphylococci- phenol red indicator Mannitol fermenter – yellow Non fermenter - pink Macconkeys agar MSA  Assay Media - prescribed compositions are used for the assay of vitamins, amino acids, and antibiotics. Media of special composition are also available for testing disinfectants Media for Enumeration - Specific kinds of media are used for determining the bacterial content of such of Bacteria – Glucose yeast extract agar & Tryptic soy agar Maintenance Media - Satisfactory maintenance of the viability and physiological characteristics of a culture over time may require a medium different from that which is optimum for growth THE GROWTH CYCLE/CURVE  Binary fission and microbial culture other cell division processes bring about an increase in the number of cells in a population Population growth is studied by analyzing the growth curve of a  When microorganisms are cultivated in liquid medium, they usually are grown in a batch culture or closed system—that is, they are incubated in a closed culture vessel with a single batch of medium. Because no fresh medium is provided during incubation, nutrient concentrations decline and concentrations of wastes increase.  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  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 Although cell division does not take place right away and there is no net increase in mass, the cell is synthesizing new components  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 Whatever the causes, eventually the cells retool, replicate their DNA, begin to increase in mass, and finally divide  The lag phase varies considerably in length – 1. Condition of organism 2. Nature of medium Long- inoculum is refrigerated, chemically different medium Short or absent- young, vigorously growing exponential phase from same chemical medium  Exponential Phase During the exponential or log phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing Rate of growth is constant – cells are dividing and 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. The population is most uniform in terms of chemical and physiological properties - are usually used in biochemical and physiological studies. Exponential growth is balanced growth. That is, all cellular constituents are manufactured at constant rates relative to each other.  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 Primary metabolites  Stationary Phase Because this is a closed system, eventually population growth ceases and the growth curve becomes horizontal bacteria at a population level of around 109 cells per ml (final  In the stationary phase the total number of viable microorganisms remains constant balance between cell division and cell death (cryptic growth), or the population may simply cease to divide but remain metabolically active Reasons – 1. nutrient limitation; if an 2. accumulation of toxic waste products – anoxic conditions, acidity Bacteria in a batch culture may enter stationary phase in response to starvation This probably often occurs in nature because many environments have low nutrient levels Decrease somewhat in overall size, often accompanied by protoplast shrinkage and nucleoid condensation  Production of starvation proteins, which make the cell much more resistant to damage in a variety of ways increase peptidoglycan crosslinking and cell wall strength The Dps (DNA binding protein from starved cells) protein protects DNA Chaperone proteins prevent protein denaturation and repair damaged proteins  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 These changes are so effective that some bacteria can survive starvation for years Some other bacterial pathogens become more virulent when starved Clearly, these considerations are of great practical importance in medical and industrial microbiology  Senescence and Death Population will enter the death phase of the growth cycle, which occurs as an exponential function, however, the rate of cell death is much slower than the rate of exponential growth  For many years, the decline in viable cells following stationary cells was described simply as the “death phase” Nutrient derivation, toxic waste - irreparable harm resulting in loss of viability If transferred to new medium no growth observed - cells died but did not lyse  Mathematics of Growth Knowledge of microbial growth rates during the exponential phase is indispensable to microbiologists Growth rate studies contribute to basic physiological and ecological research and are applied in industry During exponential phase population will double in number during a specific length of time called the generation time or doubling time  Suppose that a culture tube is inoculated with one cell that divides every 20 minutes, the population will be 2 cells after 20 minutes, 4 cells after 40 minutes, and so forth Because the population is doubling every generation, the increase in population is always 2n where n is the number of generations The resulting population increase is exponential or logarithmic  The mean generation time (g) can be determined directly from a semilogarithmic plot of the growth data and the growth rate constant calculated from the g value The generation time also may be calculated directly from the previous equations  Synchronous culture Microbiological culture – all cells are in same stage of growth a synchronous culture can be treated as a single cell quantitative experimental results can simply be divided in the number of cells to obtain values that apply to a single cell extensively used to address questions regarding cell cycle and growth, and the effects of various factors on these  Continuous Culture It is possible to grow microorganisms in an open system, a system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes These conditions are met in the laboratory by a continuous culture system A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods in a continuous culture system  The Chemostat chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed The culture medium for a chemostat possesses an essential nutrient (e.g., an amino acid) in limiting quantities growth rate is determined by the rate at which new medium is fed into the growth chamber, and the final cell density depends  The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml) D = f/V  The Turbidostat Turbidostat has a photocell that measures the absorbance or turbidity of the culture in the growth vessel The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity or cell density Dilution rate varies best at higher rates Nutrients excess not limiting  Applications of countinuous culture Provide a constant supply of cells in exponential phase and growing at a known rate They make possible the study of microbial growth at very low nutrient levels, concentrations close to those present in natural environments These systems are essential for research in many areas—for example, in studies on interactions between microbial species under environmental conditions resembling those in a freshwater lake or pond Continuous systems also are used in food and industrial microbiology Measurement of growth  Direct microscopic count Breed’s count - a measured volume of a bacterial suspension is placed within a defined area on a microscope slide A 0.01-ml sample is spread over a marked square centimeter of slide stain is added, sample is viewed under the oil immersion  area of the viewing field of this objective can be determined. Once the number of bacteria has been counted in several different fields, the average number of bacteria per viewing field can be calculated. From these data, the number of bacteria in the square centimeter over which the sample was spread can also be calculated Petroff-Hausser cell counter  Petroff-Hausser cell counter – bacteria and sperm Haemocytometer – Neubar chamber – yeast and blood cells Spread plate and pour plate Estimating Bacterial Numbers by Indirect Methods  estimating turbidity is a practical way of monitoring bacterial growth. As bacteria multiply in a liquid medium, the medium becomes turbid, or cloudy with cells. In the spectrophotometer, a beam of light is transmitted through a bacterial suspension to a light sensitive detector  As bacterial numbers increase, less light will reach the detector. This change of light will register on the instrument’s scale as the percentage of transmission. Also printed on the instrument’s scale is a logarithmic expression called the absorbance (sometimes called optical density, or OD, which is calculated as Abs = 2 − log of % transmittance). The absorbance is used to plot bacterial growth.  More than a million cells per milliliter must be present for the first traces of turbidity to be visible. About 10 million to 100 million cells per milliliter are needed to make a suspension turbid enough to be read on a spectrophotometer. Therefore, turbidity is not a useful measure of contamination of liquids by relatively small numbers of bacteria.  Metabolic Activity Another indirect way to estimate bacterial numbers is to measure a population’s metabolic activity This method assumes that the amount of a certain metabolic product, such as acid or CO2, is in direct proportion to the number of bacteria present An example of a practical application of a metabolic test is the microbiological assay in which acid production is used to determine amounts of vitamins  Dry Weight For filamentous bacteria and molds, the usual measuring methods are less satisfactory A plate count would not measure this increase in filamentous mass. In plate counts of actinomycetes and molds, it is mostly the number of asexual spores that is counted instead. This is not a good measure of growth.  One of the better ways to measure the growth of filamentous organisms is by dry weight. In this procedure, the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator It is then weighed. For bacteria, the same basic procedure is followed.