6.Microbial Growth CHAPTER 6 Microbial Growth Outline 6.1 The PH 123 e12 65 63 The Con 64
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 CHAPTER 6 Microbial Growth Membrane filters are used in counting microorganisms. This membrane has been used to obtain a total bacterial count using an indicator to color colonies for easy counting. 6.1 The Growth Curve 113 Lag Phase 113 Exponential Phase 114 Stationary Phase 114 Death Phase 115 The Mathematics of Growth 115 6.2 Measurement of Microbial Growth 117 Measurement of Cell Numbers 117 Measurement of Cell Mass 119 6.3 The Continuous Culture of Microorganisms 120 The Chemostat 120 The Turbidostat 121 6.4 The Influence of Environmental Factors on Growth 121 Solutes and Water Activity 121 pH 123 Temperature 125 Oxygen Concentration 127 Pressure 129 Radiation 130 6.5 Microbial Growth in Natural Environments 131 Growth Limitation by Environmental Factors 131 Counting Viable But Nonculturable Vegetative Procaryotes 132 Quorum Sensing and Microbial Populations 132 Outline
6.1 The Growth Curve 113 Concepts nary phas When m uch as on.In wide ostnam Time 6.1 The Growth Curve Population growth is studied by analyzing the growth curve of a medium.they usually at is,they are incu bated in a closed culture vessel with hapter 5 emphasizes that microorganisms need access to and the raw st have phospho tions of number of viable cels versus the incubation time.The resulting ses.the most important of which curve has four distinct phases (figure 6.1). n,act and group translocation Lag Phase call on microbial growth completes the chapter zing new components.A lag phase prio The cells may be old and depleted of TP ents.I ean sm was grov ng in pr eviously.Here new enzymes would be progeny of approximat to u bly the r gan causes,eventually the cells ret replicale their DNA.begin to inc lly div derably in length with the tigate the growth and reproduction of in tion of the organism and the nature of the n.Thi owth.mib ated Inocula ation of a culture into he total population number. The cell cycle (pp.87:285-86 chemically different medium also results in a longer lag phase
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 6.1 The Growth Curve Population growth is studied by analyzing the growth curve of a microbial culture. 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 (figure 6.1). 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. Although cell division does not take place right away and there is no net increase in mass, the cell is synthesizing new components. A lag phase prior to the start of cell division can be necessary for a variety of 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. 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 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. 6.1 The Growth Curve 113 Concepts 1. Growth is defined as an increase in cellular constituents and may result in an increase in a microorganism’s size, population number, or both. 2. When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation. In an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods. 3. A wide variety of techniques can be used to study microbial growth by following changes in the total cell number, the population of viable microorganisms, or the cell mass. 4. Water availability, pH, temperature, oxygen concentration, pressure, radiation, and a number of other environmental factors influence microbial growth. Yet many microorganisms, and particularly bacteria, have managed to adapt and flourish under environmental extremes that would destroy most higher organisms. 5. In the natural environment, growth is often severely limited by available nutrient supplies and many other environmental factors. 6. Bacteria can communicate with each other and behave cooperatively using population density–dependent signals. The paramount evolutionary accomplishment of bacteria as a group is rapid, efficient cell growth in many environments. —J. L. Ingraham, O. Maaløe, and F. C. Neidhardt Chapter 5 emphasizes that microorganisms need access to a source of energy and the raw materials essential for the construction of cellular components. All organisms must have carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, and a variety of minerals; many also require one or more special growth factors. The cell takes up these substances by membrane transport processes, the most important of which are facilitated diffusion, active transport, and group translocation. Eucaryotic cells also employ endocytosis. Chapter 6 concentrates more directly on the growth. The nature of growth and the ways in which it can be measured are described first, followed by consideration of continuous culture techniques. An account of the influence of environmental factors on microbial growth completes the chapter. 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 also results when cells simply become longer or larger. If the microorganism is coenocytic—that is, a multinucleate organism in which nuclear divisions are not accompanied by cell divisions— growth results in an increase in cell size but not cell number. It is usually not convenient to investigate the growth and reproduction of individual microorganisms because of their small size. Therefore, when studying growth, microbiologists normally follow changes in the total population number. The cell cycle (pp. 87; 285–86) Time Lag phase Exponential (log) phase Death phase Stationary phase Log number of viable cells Figure 6.1 Microbial Growth Curve in a Closed System. The four phases of the growth curve are identified on the curve and discussed in the text
6.Microbial Growth Chapter Micrbial Grwth position.the lag phase will be short or absent. envi nental conditions bial gr limited by the lo Exponential Phase or vit conditions during the xponential phase:that is.the microo The shape of the curve seems to reflect the rate of nutrient uptake not rise further with increasing nutrient concentration. ost un orm in terms of ogical assays (p.99): transport systems (pp.100-4) iological stud Stationary Phase d rowth.That is.all cellulat eases and the growth curve be ults This is growth during which the o not reach such high served in a shift-up experiment in which bacteria are transferred nutrient availability and other factors,as well as the type of mic cells firs ance between cell division and cell death.or the population may hesis.Finally.the expected nha Unbala a人品e网 ulswhen a bacterial population One opu ponents directly from the medium.When shifted to a nutrition very soluble and may be depleted so quickly that ony the surface plicationcontin after the way.Population growt (growing in the abence of ).Fo example. from sugar fermentation that their medium becomes acidic and
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 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. 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. Their rate of growth is constant during the exponential phase; that is, the microorganisms 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 (figure 6.1). The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures 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. If nutrient levels or other environmental conditions change, unbalanced growth results. This is growth during which the rates of synthesis of cell components vary relative to one another until a new balanced state is reached. This response is readily observed in a shift-up experiment in which bacteria are transferred from a nutritionally poor medium to a richer one. 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. Protein and DNA synthesis (sections 11.3 and 12.2) Unbalanced growth also results when a bacterial population is shifted down from a rich medium to a poor one. The organisms may previously have been able to obtain many cell components directly from the medium. When shifted to a nutritionally inadequate medium, they 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. Then balanced growth is resumed and the culture enters the exponential phase. Regulation of nucleic acid synthesis (pp. 275–83) These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions. When microbial growth is limited by the low concentration of a required nutrient, the final net growth or yield of cells increases with the initial amount of the limiting nutrient present (figure 6.2a). This is the basis of microbiological assays for vitamins and other growth factors. The rate of growth also increases with nutrient concentration (figure 6.2b), but in a hyperbolic manner much like that seen with many enzymes (see figure 8.17). The shape of the curve seems to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high nutrient levels the transport systems are saturated, and the growth rate does not rise further with increasing nutrient concentration. Microbiological assays (p. 99); Nutrient transport systems (pp. 100–4) Stationary Phase Eventually population growth ceases and the growth curve becomes horizontal (figure 6.1). This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml. Other microorganisms normally do not reach such high population densities, protozoan and algal cultures often having maximum concentrations of about 106 cells per ml. Of course final population size depends on nutrient availability and other factors, as well as the type of microorganism being cultured. 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 though remaining metabolically active. Microbial populations enter the stationary phase for several reasons. One obvious factor is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow. 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. Population growth also may cease due to the accumulation of toxic waste products. This factor seems to limit the growth of many anaerobic cultures (cultures growing in the absence of O2). For example, streptococci can produce so much lactic acid and other organic acids from sugar fermentation that their medium becomes acidic and 114 Chapter 6 Microbial Growth Growth rate (hr–1 ) Total growth (cells or mg/ml) Nutrient concentration Nutrient concentration Figure 6.2 Nutrient Concentration and Growth. (a) The effect of changes in limiting nutrient concentration on total microbial yield. At sufficiently high concentrations, total growth will plateau. (b) The effect on growth rate. (a) (b)
6.Microbial Grow 6.1 The Growth Curve 115 Table 6.1 An Example of Exponential Growth ary ph Time 29 esult from eral fa ors operating in 0= have see 2 well bco ironments hav be a p what in o rall size se linking and ce damaged protein As a result of the and many othe ls become harder to kill and more re osmotic dar edical and industrial microbiology.The e is even evidence tha m more hr pathogcns be Death Phase Detrimental environme ental changes like nutrient deprivation and crobial population,like its growth during the exponential pha otal cell number mains constant because the cels mply fail to no eTowandrprodcc.itisasmcdiocd lead.That is, eath is de Although most of microbial population lydies ina Minutes of incub n,the the po s.For this and other reasons, Figure 6.3 Exp ential Microbial Gr wth.The data from table 6.1 The Mathematics of Growth ity of the log plo y and nat a cu will be ninutes. cells after 40 minutes drngspecific length of mcaed the generation time or dou ting pop ulation increase is exponential or logarithmic (figure 6.3)
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 growth is inhibited. Streptococcal cultures also can enter the stationary phase due to depletion of their sugar supply. Finally, there is some evidence that growth may cease when a critical population level is reached. Thus entrance into the stationary phase may result from several factors operating in concert. As we have seen, bacteria in a batch culture may enter stationary phase in response to starvation. This probably often occurs in nature as well because many environments have quite low nutrient levels. Starvation can be a positive experience for bacteria. Many do not respond with obvious morphological changes such as endospore formation, but only decrease somewhat in overall size, often accompanied by protoplast shrinkage and nucleoid condensation. The more important changes are in gene expression and physiology. Starving bacteria frequently produce a variety of starvation proteins, which make the cell much more resistant to damage in a variety of ways. They increase peptidoglycan cross-linking and cell wall strength. The Dps (DNA-binding protein from starved cells) protein protects DNA. Chaperones prevent protein denaturation and renature damaged proteins. 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. These changes are so effective that some bacteria can survive starvation for years. Clearly, these considerations are of great practical importance in medical and industrial microbiology. There is even evidence that Salmonella typhimurium and some other bacterial pathogens become more virulent when starved. 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, like its growth during the exponential phase, is usually logarithmic (that is, a constant proportion of cells dies every hour). This pattern in viable cell count holds even when the total cell number remains constant because the cells simply fail to lyse after dying. 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. That is, death is defined to be the irreversible loss of the ability to reproduce. Although most of a microbial population usually dies in a logarithmic fashion, the death rate may decrease after the population has been drastically reduced. This is due to the extended survival of particularly resistant cells. For this and other reasons, the death phase curve may be complex. The 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 the solution of applied problems in industry. Therefore the quantitative aspects of exponential phase growth will be discussed. During the exponential phase each microorganism is dividing at constant intervals. Thus the population will double in number during a specific length of time called the generation time or doubling time. This situation can be illustrated with a simple example. Suppose that a culture tube is inoculated with one cell that divides every 20 minutes (table 6.1). 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 (figure 6.3). 6.1 The Growth Curve 115 Table 6.1 An Example of Exponential Growth Division Population Timea Number 2n (N0 2n ) log10Nt 0 020 1 1 0.000 20 1 21 2 2 0.301 40 2 22 4 4 0.602 60 3 23 8 8 0.903 80 4 24 16 16 1.204 100 5 25 32 32 1.505 120 6 26 64 64 1.806 a The hypothetical culture begins with one cell having a 20-minute generation time. 90 80 70 60 50 40 30 20 10 0 1.500 1.000 0.500 0.000 Log10 number of cells ( ) Number of cells ( ) 0 20 40 60 80 100 120 Minutes of incubation Figure 6.3 Exponential Microbial Growth. The data from table 6.1 for six generations of growth are plotted directly (•–•) and in the logarithmic form ( °–° ). The growth curve is exponential as shown by the linearity of the log plot.
6.Microbial Growth 6 Chapter 6 Microbial Growth These observations can be equations for the Let No=the initial population number Nthe population at time ho) n=the number of generations in time t Then inspection of the results in table 6.1 will show that N=×2” Solving for n,the number of generations.where all loga rithms are to the base 10. log N,=log No+nlog 2.and -s水-s The time it takes a populatic to double in size thatis the m N=2No Time (hours) k-s2g4.s2+g-s Figu re 6.4 Gen tion Time Determination.The tion tim ve.I re plotted wi h the of c The mean generation time is the reciprocal of the mean growth rate constant The mean generation time ()can be determined directly from semilogarithmic plot of the growth data (figure 6.4)and the times in nature are usually much longer than in culture. 2.Defne ubalanced gwthif-uxrmt alimiting utrient have on the yield 20 gen/r0.5 hr/gen.or 30 min/gen Generation times vary markedly with the species of mi croorganism and environmental conditions.They range from less
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 These observations can be expressed as equations for the generation time. Let N0 the initial population number Nt the population at time t n the number of generations in time t Then inspection of the results in table 6.1 will show that Nt N0 2n . Solving for n, the number of generations, where all logarithms are to the base 10, log Nt log N0 n · log 2, and log Nt log N0 log Nt log N0 n ______________ ______________ log 2 0.301 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. n log Nt log N0 k __ ______________ t 0.301t The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g), can now be calculated. If the population doubles (t g), then Nt 2 N0. Substitute 2N0 into the mean growth rate equation and solve for k. log (2N0) log N0 log 2 log N0 log N0 k ________________ _____________________ 0.301g 0.301g 1 k __ g The mean generation time is the reciprocal of the mean growth rate constant. 1 g __ k The mean generation time (g) can be determined directly from a semilogarithmic plot of the growth data (figure 6.4) and the growth rate constant calculated from the g value. The generation time also may be calculated directly from the previous equations. For example, suppose that a bacterial population increases from 103 cells to 109 cells in 10 hours. log 109 log 103 9 3 k _______________ ______ 2.0 generations/hr (0.301)(10 hr) 3.01 hr 1 g _________ 0.5 hr/gen. or 30 min/gen. 2.0 gen./hr Generation times vary markedly with the species of microorganism and environmental conditions. They range from less than 10 minutes (0.17 hours) for a few bacteria to several days with some eucaryotic microorganisms (table 6.2). Generation times in nature are usually much longer than in culture. 1. Define growth. Describe the four phases of the growth curve in a closed system and discuss the causes of each. 2. Define balanced growth, unbalanced growth, shift-up experiment, and shift-down experiment. 3. What effect does increasing a limiting nutrient have on the yield of cells and the growth rate? 4. What are the generation or doubling time and the mean growth rate constant? How can they be determined from growth data? 116 Chapter 6 Microbial Growth Time (hours) Number of cells (×107 ) 1 2 3 45 g 0 0.10 0.50 1.00 2.00 3.00 Lag phase Exponential (log) phase Figure 6.4 Generation Time Determination. The generation time can be determined from a microbial growth curve. The population data are plotted with the logarithmic axis used for the number of cells. The time to double the population number is then read directly from the plot. The log of the population number can also be plotted against time on regular axes.
6.Microbial Grow 117 Table 6.2 rglas mfor Selected 525050 4m0 6.2 Measurement of Microbial Growth e 6.5 The Petroff-Hausser Cou e.(e)An creases in both.The most commonly vt×400 dthe dvantage late the concer nofcd nental situation bacteria/mm(bacteria/square)(5). Measurement of Cell Numbers The most obvious way to determine microbial numbers is bacteria/cm'=(28 bacteria)(25 squares(501(10'=3.5 x 10? of microrPctrotl-H counting chambe can used for counting procaryotes:;he are stained,or when a phase-contrast ora fluorescence micro
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 6.2 Measurement of Microbial Growth 117 Table 6.2 Generation Times for Selected Microorganisms Temperature Generation Time Microorganism (°C) (Hours) Bacteria Beneckea natriegens 37 0.16 Escherichia coli 40 0.35 Bacillus subtilis 40 0.43 Staphylococcus aureus 37 0.47 Pseudomonas aeruginosa 37 0.58 Clostridium botulinum 37 0.58 Rhodospirillum rubrum 25 4.6–5.3 Anabaena cylindrica 25 10.6 Mycobacterium tuberculosis 37 12 Treponema pallidum 37 33 Algae Scenedesmus quadricauda 25 5.9 Chlorella pyrenoidosa 25 7.75 Asterionella formosa 20 9.6 Euglena gracilis 25 10.9 Ceratium tripos 20 82.8 Protozoa Tetrahymena geleii 24 2.2–4.2 Leishmania donovani 26 10–12 Paramecium caudatum 26 10.4 Acanthamoeba castellanii 30 11–12 Giardia lamblia 37 18 Fungi Saccharomyces cerevisiae 30 2 Monilinia fructicola 25 30 6.2 Measurement of Microbial Growth There are many ways to measure microbial growth to determine growth rates and generation times. Either population mass or number may be followed because growth leads to increases in both. The most commonly employed techniques for growth measurement are examined briefly and the advantages and disadvantages of each noted. No single technique is always best; the most appropriate approach will depend on the experimental situation. Measurement of Cell Numbers The most obvious way to determine microbial numbers is through direct counting. Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. Petroff-Hausser counting chambers can be used for counting procaryotes; hemocytometers can be used for both procaryotes and eucaryotes. Procaryotes are more easily counted in these chambers if they are stained, or when a phase-contrast or a fluorescence microCover glass Chamber holding (a) bacteria (b) (c) Figure 6.5 The Petroff-Hausser Counting Chamber. (a) Side view of the chamber showing the cover glass and the space beneath it that holds a bacterial suspension. (b) A top view of the chamber. The grid is located in the center of the slide. (c) An enlarged view of the grid. The bacteria in several of the central squares are counted, usually at 400 to 500 magnification. The average number of bacteria in these squares is used to calculate the concentration of cells in the original sample. Since there are 25 squares covering an area of 1 mm2 , the total number of bacteria in 1 mm2 of the chamber is (number/square)(25 squares). The chamber is 0.02 mm deep and therefore, bacteria/mm3 = (bacteria/square)(25 squares)(50). The number of bacteria per cm3 is 103 times this value. For example, suppose the average count per square is 28 bacteria: bacteria/cm3 = (28 bacteria) (25 squares)(50)(103 ) = 3.5 × 107 . scope is employed. These specially designed slides have chambers of known depth with an etched grid on the chamber bottom (figure 6.5). The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and any
w2 6.Microbial Grewth Chapter 6 Microbial Growtl ints.Low counts will result if clumps of cells are not bro ken upand cult to distinguish hetween living and dead cells in counting dividual cell.the results are often expressed in terms of colom chambers without special techniques unted with electronic coun sults.Of course the counts ill also be low if the agar medium em al Co he microbial su le m organisms pr h the cell is counted.The and other problems separate colony.A colony count gives the number of micro and gaims in the pcinTcdacanbcuscdt In mos counting proced res.ad ute used to count bac surface.Each micro ate memb acridine orange or DAPI and observed microscopically.Acridin in a sample. ely used for v o stain live and dead cells differe ntly are now microorganisms in a sample ()
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 sample dilutions required. There are some disadvantages to the technique. The microbial population must be fairly large for accuracy because such a small volume is sampled. It is also difficult to distinguish between living and dead cells in counting chambers without special techniques. Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted with electronic counters such as the Coulter Counter. The microbial suspension is forced through a small hole or orifice. An electrical current flows through the hole, and electrodes placed on both sides of the orifice measure its electrical resistance. Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted. The Coulter Counter gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems. Counting chambers and electronic counters yield counts of all cells, whether alive or dead. There are also several viable counting techniques, procedures specific for cells able to grow and reproduce. In most viable counting procedures, a diluted sample of bacteria or other microorganisms is dispersed over a solid agar surface. Each microorganism or group of microorganisms develops into a distinct colony. The original number of viable microorganisms in the sample can be calculated from the number of colonies formed and the sample dilution. For example, if 1.0 ml of a 1 106 dilution yielded 150 colonies, the original sample contained around 1.5 108 cells per ml. Usually the count is made more accurate by use of a special colony counter. In this way the spread-plate and pour-plate techniques may be used to find the number of microorganisms in a sample. Plating techniques are simple, sensitive, and widely used for viable counts of bacteria and other microorganisms in samples of food, water, and soil. Several problems, however, can lead to inaccurate counts. Low counts will result if clumps of cells are not broken up and the microorganisms well dispersed. Because it is not possible to be absolutely certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU) rather than the number of microorganisms. The samples should yield between 30 and 300 colonies for best results. Of course the counts will also be low if the agar medium employed cannot support growth of all the viable microorganisms present. The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates. Spread-plate and pour-plate techniques (pp. 106–8) Microbial numbers are frequently determined from counts of colonies growing on special membrane filters having pores small enough to trap bacteria. In the membrane filter technique, a sample is drawn through a special membrane filter (figure 6.6). The filter is then placed on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separate colony. A colony count gives the number of microorganisms in the filtered sample, and special media can be used to select for specific microorganisms (figure 6.7). This technique is especially useful in analyzing aquatic samples. Analysis of water purity (pp. 653–57) Membrane filters also are used to count bacteria directly. The sample is first filtered through a black polycarbonate membrane filter to provide a good background for observing fluorescent objects. The bacteria then are stained with a fluorescent dye such as acridine orange or DAPI and observed microscopically. Acridine orange–stained microorganisms glow orange or green and are easily counted with an epifluorescence microscope (see section 2.2). Usually the counts obtained with this approach are much higher than those from culture techniques because some of the bacteria are dead. Commercial kits that use fluorescent reagents to stain live and dead cells differently are now available. This makes it possible to directly count the number of live and dead microorganisms in a sample (see figure 2.13d). 118 Chapter 6 Microbial Growth Membrane filter removed and placed in plate containing the appropriate medium Water sample filtered through membrane filter (0.45 µm) Membrane filter on a filter support Incubation for 24 hours Typical colonies Figure 6.6 The Membrane Filtration Procedure. Membranes with different pore sizes are used to trap different microorganisms. Incubation times for membranes also vary with the medium and microorganism
6.Microbial Growh 119 Figure 6.7 Colonie variety of media Measurement of Cell Mass in a population are of roughly constant size.the amount of scat teria r about cells(1)per ml,the mediumap slightly clou centr d,dried in an oven,and weighed.This is an especially use so little,it may be necessary several hundred milli- growth can be e the puthe am crobial cells scatter light striking them.Because microbial cells tal quantity of that cell constituent is directly related to the total
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 Measurement of Cell Mass Increases in the total cell mass, as well as in cell numbers, accompany population growth. Therefore techniques for measuring changes in cell mass can be used in following growth. The most direct approach is the determination of microbial dry weight. Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity. More rapid, sensitive techniques depend on the fact that microbial cells scatter light striking them. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. When the concentration of bacteria reaches about 10 million cells (107 ) per ml, the medium appears slightly cloudy or turbid. Further increases in concentration result in greater turbidity and less light is transmitted through the medium. The extent of light scattering can be measured by a spectrophotometer and is almost linearly related to bacterial concentration at low absorbance levels (figure 6.8). Thus population growth can be easily measured spectrophotometrically as long as the population is high enough to give detectable turbidity. If the amount of a substance in each cell is constant, the total quantity of that cell constituent is directly related to the total 6.2 Measurement of Microbial Growth 119 Figure 6.7 Colonies on Membrane Filters. Membrane-filtered samples grown on a variety of media. (a) Standard nutrient media for a total bacterial count. An indicator colors colonies red for easy counting. (b) Fecal coliform medium for detecting fecal coliforms that form blue colonies. (c) m-Endo agar for detecting E. coli and other coliforms that produce colonies with a green sheen. (d) Wort agar for the culture of yeasts and molds. (a) (b) (c) (d) Spectrophotometer meter Photocell or detector Tube of bacterial suspension Lamp 10 9 8 7 4 5 6 3 2 1 0 0 .0 .1 .6 .5 .4 .3 .2 .8 .7 1.9 1. 10 9 8 7 4 5 6 3 2 1 0 0 .0 .1 .6 .5 .4 .3 .2 .8 .7 1.9 1. Figure 6.8 Turbidity and Microbial Mass Measurement. Determination of microbial mass by measurement of light absorption. As the population and turbidity increase, more light is scattered and the absorbance reading given by the spectrophotometer increases. The spectrophotometer meter has two scales. The bottom scale displays absorbance and the top scale, percent transmittance. Absorbance increases as percent transmittance decreases
6.Microbial Growth 120 Chapter 6 Microbial Growth Nutrient concentration Dilution rate t Dilution Rate and Microbial Growth.The of flow of continuous culture system. The Chemostat pltiomwilberceflcteod protein】 mined bythe rae at which wmediumis fedn Why are plate count resutofexpresed as cooy forming units? as the dilution rate (D)the rate at which medium flows through 6.3 The Continuous Culture of Microorganisms D- Forme iffismand Vis1ml,thediltionte is Up to this point the focus has been on systems called batch 0.30 h the
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 microbial cell mass. For example, a sample of washed cells collected from a known volume of medium can be analyzed for total protein or nitrogen. An increase in the microbial population will be reflected in higher total protein levels. Similarly, chlorophyll determinations can be used to measure algal populations, and the quantity of ATP can be used to estimate the amount of living microbial mass. 1. Briefly describe each technique by which microbial population numbers may be determined and give its advantages and disadvantages. 2. Why are plate count results often expressed as colony forming units? 6.3 The Continuous Culture of Microorganisms Up to this point the focus has been on closed systems called batch cultures in which nutrient supplies are not renewed nor wastes removed. Exponential growth lasts for only a few generations and soon the stationary phase is reached. However, 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 Two major types of continuous culture systems commonly are used: (1) chemostats and (2) turbidostats. A chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed (figure 6.9). The culture medium for a chemostat possesses an essential nutrient (e.g., an amino acid) in limiting quantities. Because of the presence of a limiting nutrient, the growth rate is determined by the rate at which new medium is fed into the growth chamber, and the final cell density depends on the concentration of the limiting nutrient. 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 For example, if f is 30 ml/hr and V is 100 ml, the dilution rate is 0.30 hr1 . Both the microbial population level and the generation time are related to the dilution rate (figure 6.10). The microbial population density remains unchanged over a wide range of dilution rates. The generation time decreases (i.e., the growth rate rises) as the dilution rate increases. The limiting nutrient will be almost 120 Chapter 6 Microbial Growth Fresh medium Control valve Air supply Air filter Culture vessel Receptacle Figure 6.9 A Continuous Culture System: The Chemostat. Schematic diagram of the system. The fresh medium contains a limiting amount of an essential nutrient. Growth rate is determined by the rate of flow of medium through the culture vessel. Measurement value Nutrient concentration Generation time Dilution rate Cell density or biomass Figure 6.10 Chemostat Dilution Rate and Microbial Growth. The effects of changing the dilution rate in a chemostat.
。 6.Microbial Grow factors on growth 121 ls inferus even seem able to live over 1.5 miles below the nt to use i rth's surface. in d causes a rise in cause of the effec called extrem ntal emphasis will be iven to solutes and water acti H.temp of their response to these factors Solutes and Water Activity The Turbidostat Because a selectively croorganisms from their environment,they can be affected by changes in the os sel is auto ed to maintain a predet ined tur ause it to burs atically from the chem h veryusefulbecause they pro Most bacteria.agac. and fungi have cell walls tha isms with rigid cell walls are placed ina hypertonic envir water leaves and the plasn ne:the cell usually become en microbial TCnlalconditionsre e and e ke in food and industrial microbioloy. their protopla se o 1.How does an open system differ fromaclosed culture systemo compatible with metabolism and growth when a sis or uptake o ctaine,proline,glu e and fune mploy su and polyols for exar Po e,arabitol,glycer 6.4 The Influence of Environmental solutes for this funeton be pt e Factors on Growth and functio A fo As we have seen(pp.114-15),microorganisms must be able to sium ions (se m ions also eles ed but no salt s fo surroundings see section20.3 protozoa do no have a cel wall.the water when ivin in hy ents of microorganisms. tective function of the cell wall (p.61)
Prescott−Harley−Klein: Microbiology, Fifth Edition II. Microbial Nutrition, Growth, and Control 6. Microbial Growth © The McGraw−Hill Companies, 2002 completely depleted under these balanced conditions. If the dilution rate rises too high, the microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate. The limiting nutrient concentration rises at higher dilution rates because fewer microorganisms are present to use it. At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate. This is because of the effect of nutrient concentration on the growth rate, sometimes called the Monod relationship (figure 6.2b). Only a limited supply of nutrient is available at low dilution rates. Much of the available energy must be used for cell maintenance, not for growth and reproduction. As the dilution rate increases, the amount of nutrients and the resulting cell density rise because energy is available for both maintenance and growth. The growth rate increases when the total available energy exceeds the maintenance energy. The Turbidostat The second type of continuous culture system, the 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. The turbidostat differs from the chemostat in several ways. The dilution rate in a turbidostat varies rather than remaining constant, and its culture medium lacks a limiting nutrient. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates. Continuous culture systems are very useful because they 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. 1. How does an open system differ from a closed culture system or batch culture? 2. Describe how the two different kinds of continuous culture systems, the chemostat and turbidostat, operate. 3. What is the dilution rate? What is maintenance energy? 6.4 The Influence of Environmental Factors on Growth As we have seen (pp. 114–15), microorganisms must be able to respond to variations in nutrient levels, and particularly to nutrient limitation. The growth of microorganisms also is greatly affected by the chemical and physical nature of their surroundings. An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms. The ability of some microorganisms to adapt to extreme and inhospitable environments is truly remarkable. Procaryotes are present anywhere life can exist. Many habitats in which procaryotes thrive would kill most other organisms. Procaryotes such as Bacillus infernus even seem able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C. Microorganisms that grow in such harsh conditions are often called extremophiles. In this section we shall briefly review how some of the most important environmental factors affect microbial growth. Major emphasis will be given to solutes and water activity, pH, temperature, oxygen level, pressure, and radiation. Table 6.3 summarizes the way in which microorganisms are categorized in terms of their response to these factors. Solutes and Water Activity Because a selectively permeable plasma membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration of their surroundings. If a microorganism is placed in a hypotonic solution (one with a lower osmotic concentration), water will enter the cell and cause it to burst unless something is done to prevent the influx. The osmotic concentration of the cytoplasm can be reduced by use of inclusion bodies (see pp. 49–52). Procaryotes also can contain pressure-sensitive channels that open to allow solute escape when the osmolarity of the environment becomes much lower than that of the cytoplasm. Most bacteria, algae, and fungi have rigid cell walls that maintain the shape and integrity of the cell. When microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the plasma membrane shrinks away from the wall, a process known as plasmolysis. This dehydrates the cell and may damage the plasma membrane; the cell usually becomes metabolically inactive and ceases to grow. Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes, so that the plasma membrane is always pressed firmly against their cell wall. Compatible solutes are solutes that are compatible with metabolism and growth when at high intracellular concentrations. Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions are also involved to some extent. Algae and fungi employ sucrose and polyols—for example, arabitol, glycerol, and mannitol—for the same purpose. Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme structure and function. A few procaryotes like Halobacterium salinarium raise their osmotic concentration with potassium ions (sodium ions are also elevated but not as much as potassium). Halobacterium’s enzymes have been altered so that they actually require high salt concentrations for normal activity (see section 20.3). Since protozoa do not have a cell wall, they must use contractile vacuoles (see figure 27.3) to eliminate excess water when living in hypotonic environments. Osmosis and the protective function of the cell wall (p. 61) 6.4 The Influence of Environmental Factors on Growth 121
