SPECIAL TOPICS 19 SPECIAL TOPICS Endospores bacec opinary y ospores are bodi s that do not stair and other environmental factors that may also provide antag spores show a well-defined multila d exosporium,an electron-dense ible i-15 Growth Growth of a cell is the culmination of an ordered interplay 1.Entrance of basic nutrients into the cell 2.Conversion of these compounds into energy and vital cell constituents 3.Replication of the chromosome 4.Increase in size and mass of the cell 5.Division of ecltdaugher cellseachconing acopy of thegenome
SPECIAL TOPICS 19 SPECIAL TOPICS Endospores A few bacteria such as Bacillus and Clostridium produce specialized structures called endospores. Endospores are bodies that do not stain with ordinary dyes and appear as unstained highly refractile areas when seen under the light microscope. They provide resistance to heat, desiccation, radiation, and other environmental factors that may threaten the existence of the organism. Endospores also provide a selective advantage for survival and dissemination of the species that produce them. Under the electron microscope, spores show a well-defined multilayered exosporium, an electron-dense outer coat observed as a much darker area, and a thick inner coat. In the spore interior, the darkly stained ribosomes and the nuclear material may also be visible (Fig. 1-15). Growth Growth of a cell is the culmination of an ordered interplay among all of the physiological activities of the cell. It is a complex process involving 1. Entrance of basic nutrients into the cell 2. Conversion of these compounds into energy and vital cell constituents 3. Replication of the chromosome 4. Increase in size and mass of the cell 5. Division of the cell into two daughter cells, each containing a copy of the genome and other vital components C CX N E R Fig. 1-15. Mature spore of Clostridium botulinum. Shown is a well-defined, multilayered exosporium (E), an electron-dense outer coat layer, a thick inner coat (C) and a less dense cortex (CX). The darkly stained ribosomes (R) and nucleoid areas (N) are clearly differentiated in the spore interior. Bar equals 0.2 µm. (Source: From Stevenson, K. E., R. H. Vaughn, and E. V. Crisan, 1972. J. Bacteriol. 109:1295.)
INTRODUCTON TO MICROBIAL PHYSIOLOGY A.study of the inr ase in population implies that each cell,as it is produced,is capable of producing new progeny. Growth Cycle.Under ideal circumstances in which cell division commences ered fashion for a period oftme ws a geometric progress 20→2→223→24-→25→26→27 →28→29→ete. b=1×2” b=ax2 where a is the number of organisms present in the original inoc um.Since the Plotting the numbe rof organisms pre sent as a function of time gener ates a curvilinea 01 This because the logarithm to the base 10ofa number isequal to3010 times the logarithm n assumed that the individual ration time (ie.the ells i n the population. shown in Figure I 16,the cells initially experience to the nev of the n me rec and there may be a drop in the number of viable adjust to the new exhausted).Since e plotting the cell number logarithm during this period results in inear functi ms phase of h is referred to as the logarithmic (log)phase or. organi eventually reach a maximum population density in stationary phase.Entry into this ult from sev events 上xhausto development of an the for the decline in the
20 INTRODUCTION TO MICROBIAL PHYSIOLOGY Microbiologists usually consider the phenomenon of growth from the viewpoint of population increase, since most current techniques do not allow the detailed study of individual cells. A study of the increase in population implies that each cell, as it is produced, is capable of producing new progeny. Growth Cycle. Under ideal circumstances in which cell division commences immediately and proceeds in unhampered fashion for a protracted period of time, prokaryotic cell division follows a geometric progression: 20 −−−→ 21 −−−→ 22 −−−→ 23 −−−→ 24 −−−→ 25 −−−→ 26 −−−→ 27 −−−→ 28 −−−→ 29 −−−→ etc. This progression may be expressed as a function of 2 as shown in the line above. The number of cells (b) present at a given time may be expressed as b = 1 × 2n The total number of cells (b) is dependent on the number of generations (n = number of divisions) occurring during a given time period. Starting with an inoculum containing more than one cell, the number of cells in the population can be expressed as b = a × 2n where a is the number of organisms present in the original inoculum. Since the number of organisms present in the population (b) is a function of the number 2, it becomes convenient to plot the logarithmic values rather than the actual numbers. Plotting the number of organisms present as a function of time generates a curvilinear function. Plotting the logarithm of the number, a linear function is obtained as shown in Figure 1-16. For convenience, logarithms to the base 10 are used. This is possible because the logarithm to the base 10 of a number is equal to 0.3010 times the logarithm to the base 2 of a number. Up to this point it has been assumed that the individual generation time (i.e., the time required for a single cell to divide) is the same for all cells in the population. However, in a given population, the generation times for individual cells vary, so the term doubling time encompasses the doubling time for the total population. As shown in Figure 1-16, the cells initially experience a period of adjustment to the new environment, and there is a lag in the time required for all of the cells to divide. Actually, some of the cells in the initial inoculum may not survive this lag phase and there may be a drop in the number of viable cells. The surviving cells eventually adjust to the new environment and begin to divide at a more rapid rate. This rate will remain constant until conditions in the medium begin to deteriorate (e.g., nutrients are exhausted). Since plotting the cell number logarithm during this period results in a linear function, this phase of growth is referred to as the logarithmic (log) phase or, more correctly, the exponential phase. All cultures of microorganisms eventually reach a maximum population density in the stationary phase. Entry into this phase can result from several events. Exhaustion of essential nutrients, accumulation of toxic waste products, depletion of oxygen, or development of an unfavorable pH are the factors responsible for the decline in the
SPECIAL TOPICS 2 10 9 Stationary Phase 8 7 6 se 4 3 Lag Phase 0 2 46 8101214161820 Time. Hours Fig.1-16.A typical growth curve for a bacterial culture ium between thenumber of cells able to divide and the number that are unable the death of in the lation results in a decline in the viable populati th p wil bute th the o sm under observat an h e加 such as during heat e cell numbers decline logarithmicall sing th cells ina population ()is equal to the number of cells in the initial inoculum ()x2 b=a×2" Then log2 b logz a+n log1ob logioa+n log1o 2 log1ob logo+(n x 0.3010 nlogb-logoa 0.3010
SPECIAL TOPICS 21 10 9 8 7 6 5 4 3 2 1 0 0 2 4 6 8 10 Time-Hours Lag Phase Exponential or "Log" Phase Stationary Phase Log10 Number Viable Organisms 12 14 16 18 20 Fig. 1-16. A typical growth curve for a bacterial culture. growth rate. Although cell division continues during the stationary phase, the number of cells that are able to divide (viable cells) are approximately equal to the number that are unable to divide (nonviable cells). Thus, the stationary phase represents an equilibrium between the number of cells able to divide and the number that are unable to divide. Eventually, the death of organisms in the population results in a decline in the viable population and the death phase ensues. The exact shape of the curve during the death phase will depend on the nature of the organism under observation and the many factors that contribute to cell death. The death phase may assume a linear function such as during heat-induced death where viable cell numbers decline logarithmically. Some additional considerations of the growth curve are important in assessing the effect of various internal as well as external factors on growth. Since the number of cells in a population (b) is equal to the number of cells in the initial inoculum (a) × 2n, b = a × 2n Then log2 b = log2 a + n log10 b = log10 a + n log10 2 log10 b = log10 a + (n × 0.3010) Solving the equation for n, the number of generations that occurred between the time of inoculation and the time of sampling is n = log10 b − log10 a 0.3010
INTRODUCTON TO MICROBIAL PHYSIOLOGY 1.=t/n Continuous Culture Usually bacteria are grown in"atch"culture in which a flask containing media nocul lowed to rates 2 g as the nuth the cell um will be too low for certain analyses.To grow bacteria at slow es and at hig stat is used.In thi us,fres mediur rate.The volume in the culture vessel is kep t constant by an overfl ow device that and c s at the same e as n m m is added.In a culture vessel.The faster the limiting nutrient is added.the faster the growth rate. FACTORS AFFECTING GROWTH Nutrition All living organism have widely in their nu tritional requirements.Twe main groups of organis are clas manner in w hey s nitroge 1.Lithotrophs utilize carbon dioxide as the sole source of carbon and gain energy compounds organic substrates as as ce of e hs utilize or compounds for growth. Alth al requirements are remarkably simple components and ide the e y for this activityth ough the oxidation of ino compo cha acteristic of stri tive chemolithotrophs can genous ources.Che such the e b isph (see Chapter 9)
22 INTRODUCTION TO MICROBIAL PHYSIOLOGY The generation time (tg) or doubling time may be determined by dividing the time elapsed (t) by the number of generations (n): tg = t/n Continuous Culture Usually bacteria are grown in “batch” culture in which a flask containing media is inoculated and growth is allowed to occur. This is a closed system where it is actually very difficult to manipulate growth rate. In batch cultures, growth rate is determined internally by properties of the bacteria themselves. A batch culture can be used to grow bacteria at different rates as long as the nutrient added is at a concentration that does not support maximal growth. But, to accomplish this, the cell density, and thus the cell number, will be too low for certain analyses. To grow bacteria at slow growth rates and at high cell density, a chemostat is used. In this apparatus, fresh medium containing a limiting nutrient is added from a reservoir to the culture vessel at a set rate. The volume in the culture vessel is kept constant by an overflow device that removes medium and cells at the same rate as fresh medium is added. In a chemostat, growth rate is determined externally by altering the rate-limiting nutrient added to the culture vessel. The faster the limiting nutrient is added, the faster the growth rate. FACTORS AFFECTING GROWTH Nutrition All living organisms have certain basic nutritional requirements: sources of carbon, nitrogen, energy, and essential growth factors (minerals and vitamins) are needed to support growth. Microorganisms vary widely in their nutritional requirements. Two main groups of organisms are classified on the basis of their ability to gain energy from certain sources and the manner in which they satisfy their carbon and nitrogen requirements for growth: 1. Lithotrophs utilize carbon dioxide as the sole source of carbon and gain energy through the oxidation of inorganic compounds (chemolithotrophs or “rock eaters”) or light (photolithotrophs). Inorganic nitrogen is utilized for the synthesis or organic compounds. 2. Organotrophs generally prefer organic substrates as a source of energy and carbon. Photoorganotrophs utilize light as a source of energy for the assimilation of carbon dioxide as well as organic compounds. Chemoorganotrophs utilize organic compounds for growth. Although their nutritional requirements are remarkably simple, chemolithotrophic bacteria must be metabolically complex since they synthesize all of their cellular components and provide the energy for this activity through the oxidation of inorganic compounds. One fundamental characteristic of strict chemolithotrophs is that they are unable to grow on or assimilate exogenous organic compounds. Facultative chemolithotrophs can utilize exogenous organic carbon sources. Chemolithotrophs possess unique mechanisms for carbon dioxide fixation such as the ribulose bisphosphate (Calvin-Benson) cycle and the reductive carboxylic acid (Campbell-Evans) cycle (see Chapter 9)
FACTORS AFFECTING GROWTH 23 esoecabonsouiceOgaoopmeyueioeaicnioeabunhd the groupg better when sup wit cocci als r one or more nitrogen sources as amino acids,purines. Fatty acic达ae rquired byom。aiay n the bc。the end rement by ion of the en sApaing2aeiomRpeofaeiviasecnepediananygovhfho nts. a vitamin cannot be regarded as a true growth requirement since it can be synthesized in the presence of the requisite vitamin. TABLE 1-2.Nutritional Requirementsof Some Organotrophs Basic Nutrients equired by all for maximum growth in defined mediur Additional Requireme None Tryptophan Nicotinic acid Nicotinic acid 10 amino acids minoacid Tween 80 ro Canie c
FACTORS AFFECTING GROWTH 23 Some organotrophic organisms utilize carbon dioxide as a source of carbon, but most prefer organic carbon sources and generally cannot subsist on carbon dioxide as the sole carbon source. Organotrophs may use inorganic nitrogen, but most members of the group grow better when supplied with organic nitrogen compounds. For example, E. coli, Enterobacter aerogenes, yeasts, and molds grow luxuriantly on glucose as the only organic nutrient. Other organotrophs such as streptococci and staphylococci also exhibit specific requirements for one or more nitrogen sources as amino acids, purines, or pyrimidines (see Table 1-2). Fatty acids are required by some organisms, particularly in the absence of certain B vitamins. Replacement of a growth factor requirement by the addition of the end product of a biosynthetic pathway in which the vitamin normally functions is referred to as a sparing action. This type of activity has been reported for many growth factors, including amino acids, purines, pyrimidines, and other organic constituents. If a vitamin can completely replace a particular organic nutrient in a defined medium, that nutrient cannot be regarded as a true growth requirement since it can be synthesized in the presence of the requisite vitamin. TABLE 1-2. Nutritional Requirements of Some Organotrophs Escherichia coli Salmonella typhi Staphylococcus aureusa Leuconostoc paramesenteroidesb Basic Nutrients Glucose NH4 + Mn2+ Mg2+ Fe2+ Required by all for maximum growth in defined medium K+ Cl− SO4 2− PO4 3− Additional Requirements None Tryptophan Nicotinic acid Nicotinic acid Thiamine Thiamine 10 amino acids Pantothenate Pyridoxal Riboflavin Cobalamin Biotin p-Aminobenzoate Folate Guanine Uracil 16 Amino acids Sodium acetate Tween 80 aFrom Gladstone, G. P. 1937. Br. J. Exp. Pathol. 18:322. bFrom Garvie, E. I. 1967. J. Gen. Microbiol. 48:429