六微生物的生长及其控制.pptx

会计学1六微生物的生长及其控制六微生物的生长及其控制Section ADetermination of growth and reproduction（测定生长繁殖的方法）A.Determination of growth quantum（quantity）,suitable for all microbes.(测定生长量测定生长量)B.Counting reproduction number,only suitable for unicelled bacteria and yeast,not for the filamentous moulds and actinomyces.(计繁殖数计繁殖数)第1页/共107页A.Measuring of growth quantity（测生长量）（测生长量）（测生长量）（测生长量）n nDirect measuring(Direct measuring(直接法直接法直接法直接法)1.volumetry 1.volumetry 2.dry weight weighing 2.dry weight weighing n nIndirect measuring(Indirect measuring(间接法间接法间接法间接法)1.physiological index 1.physiological index 生理指标法生理指标法 nitrogen contentnitrogen content indexes carbon content indexes carbon content P,DNA,RNA,ATP,DAP,chitin etal.P,DNA,RNA,ATP,DAP,chitin etal.2.turbidimetry(nephelometry)2.turbidimetry(nephelometry)比浊法比浊法第2页/共107页turbidimetry第3页/共107页 B.Offspring counting 1.Direct ways(直接法直接法)Using blood counting plate under the light microscope,the result is the the total number including the dead cells.living cells and dead cells will be distinguished if staining with special dye before counting,thus both living cell number and the total number can be obtained respectively through this way.第4页/共107页Direct counting under light microscpe Structure of the counting area第5页/共107页 Colorless(living)Yeast(methylene blue)Blue(dead)Bacteria stain with acridine orange(吖啶橙吖啶橙)and observe under UV microscope Orange fluorescence(living cell)Green fluorescence(dead cell)第6页/共107页The growth of filamentous microbe第7页/共107页2.Indirect ways(living cell counting)The methods are designed on the basis that living microbe can increase the turbidity while growing in liquid medium and can form colony on the solid medium.The most common used method is colony-counting.第8页/共107页2.1 plate colony-counting methods(平板菌落计数平板菌落计数法法)The method is suitable for aerobic and anearobic microbes,every living cell will form a colony forming unit(菌落形成单位菌落形成单位cfu),the total number of living cell in the sample can be calculated from cfu dilution factor(稀释倍稀释倍数数).pour plate (浇注平板浇注平板)spread plate(涂布平板涂布平板)第9页/共107页225ml无菌无菌水加水加25g样品样品1ml1ml1ml试管中原始试管中原始装装9ml无菌水无菌水10-210-310-41ml第10页/共107页2.2 anaerobic colony-counting method Hungate roll-tube technique(亨盖特滚管技术亨盖特滚管技术)is designed by famous American microbiologist R.E.Hungate in 1950.its a epoch-making invention in the development history of microbiology which made the isolation and study for the obligate anearobic possible.The method above needs complicated equipment and the skillful technique.Zhou deqing invented semi-solid media deep agar method(半固体深层琼脂法半固体深层琼脂法)which is easily manipulated.第11页/共107页Section B Rules of microbial growthPart A cell growth and synchronous growthPart B typical growth curve of unicelled microbePart C continuous culturePart D high cell-density culture第12页/共107页Part A cell growth and synchronous growth Extremely complicated biochemical changes and cytological changes are going on in the tiny cells periodicallyalong with the cell growth,the observation of these changes are fairly difficult.The methods used present are:1.Ultra-thin section EM (电镜观察超薄切片电镜观察超薄切片)2.Synchronous culture (同步培养技术同步培养技术)第13页/共107页Synchronous culture trying to keep a group of cells synchronous in cell growth and at the same stages of cell cycle,thus the individual biochemical changes can be indirectly studied through the group.Ways to keep synchronous growth:1.Induced by the environmental condition(环境条件诱导法)2.Mechanically screening(机械筛选法)第14页/共107页 The sequence of events extending from the formation of a new cell to the next division is called the cell cycle.(细胞周期是指从一个细胞分裂结束到第二次新的细胞分裂结束所经历的顺序事件)In this cycle,a growing E.coli will double in length then divide into two cells of equal size,with each new cell containing at least one copy of the bacterial DNA.Consequently,during this time,a copy of the chromosome must be synthesized and the two chromosomes segregated into the two daughter cells.Cell cycle 第15页/共107页 DNA replication occurs during the C(chromosome replication)phase and chromosome segregation（分离）occurs in the G(gap)phase,which may be of variable length.Segregation of the chromosomes is achieved by attachment of the replicated DNA onto two adjacent sites on the membrane.Membrane growth between the sites pushes the chromosomes towards the poles of the cells.Finally,a cross wall(septum)is laid down between the two chromosomes and the cell divides into two(D phase).DNA replication第16页/共107页 Cell division and DNA replication have to be coordinated.（协调）Initiation of DNA replication at the origin(oriC),a short adenine and thymine rich sequence,is dependent on the cell reaching a critical mass(initiation mass)（DNA复制取决于细胞中起始物质所达到的临界量（准备DAN 复制相关的酶和蛋白）and requires a number of protein initiation factors.DNA segregation and division,however,are controlled by the length of the cell which must reach a particular threshold length（阈值）before the chromosomes are partitioned and cell division initiated.Coordimated replication and division第17页/共107页 Fig.Stages in the cell cycle of prokayotes.第18页/共107页Rapid growth When conditions for growth are favorable,E.coli can grow with a doubling time（生长倍增时间）of approximately 20 min.However,the time it takes to synthesize a complete copy of the E.coli chromosome is 40 min,under optimum conditions and segregation of the DNA and division takes another 20 min.Thus,the shortest cell cycle and,therefore,doubling time for E.coli should be 60 min.This is obviously not the case.For cells to divide faster than every 60.min,DNA replication must begin in one cycle and finish in another.Contradiction 第19页/共107页 When cells are growing fast(doubling time 60 min),initiation of replication occurs,as normal,producing two replication forks which move bidirectionally round the chromosome to the termination point.However,the origins on these new strands then initiate further rounds of replication before the previous round of DNA replication has finished(Fig.).Thus,when cell division occurs the DNA in the daughter cells is already replicating.The faster the cell growth rate,the more replication forks are formed such that the DNA in new cells may have multiple replication forks.Why?第20页/共107页 That repeated DNA replication can occur without cell division indicates that the control of the two processes is not linked.This is in contrast to eukaryotic cells where the two processes are directly connected.Indications 第21页/共107页Fig.2.Coordination of DNA synthesis and cell division in slow and fast growing bacteria.Slow growth Fast growth originsReplication forksInitiation of DNA synthesisDNA synthesis already initiated in new cells 第22页/共107页Part B Typical growth curve of unicelled microbe第23页/共107页 The best way of producing great numbers of microbes or their natural products is to culture in the liquid medium.Normally,the technique we use is called batch culture（分批（分批培养）培养）in which the bacteria are inoculated into flasks(三角瓶)of a suitable medium and grown at an appropriate temperature and degree of aeration（通气条件）.Batch culture第24页/共107页 Bacteria grown in this way show a particular pattern of growth which is referred to as the bacterial growth curve.The number of viable bacterial cells is measured over time（整个时间）and is plotted as a graph of the log10 viable cell numbers（活细胞数）against time,which is called a semi-logarithmic plot（半对数关系曲线）.The bacterial growth curve reveals four phases of growth.Growth curve第25页/共107页 A logarithmic scale（对数尺度）is used to plot bacterial growth owing to the large numbers of cells produced and to reveal the exponential nature of bacterial growth.If an arithmetic scale（算术尺度）is used to plot the increase in the number of cells,a curve of increasing gradient would be seen which is converted into a straight line when a logarithmic scale is used.（增加的剃度曲线变成直线）The doubling time for the bacteria can be read directly from the graph.Why use a logarithmic scale rather than arithmetic scale？第26页/共107页 When bacteria are first inoculated into a medium there is a period in which no growth occurs.During this phase the bacteria are adapting to the new environment,synthesizing new enzymes as required and increasing cell size ready for cell division.The length of this time depends on the nature of the inoculum(接种物，接种物，pl.inocula).If this comes from a fresh culture in the same medium,the lag phase will be short,but if the inoculum is old or the medium has been changed(especially moving bacteria from a rich medium to a poor one)the lag phase will be longer.1.Lag phase（延迟期）（延迟期）第27页/共107页Factors that affect the time of lag phase:(1)The nature of the inoculum,fresh or old.(接种龄)(2)Seed volume or pitching rate.(接种量)(3)The ingredients of the medium.(培养基成分)第28页/共107页 Once the bacteria start to divide,the numbers increase at a constant rate（恒速）（恒速）which reflects the doubling time of the bacteria.This is seen as a straight line part of the graph.2.Exponential(logarithmic)phase （指数期或对数期）（指数期或对数期）第29页/共107页This stage has the following features:(1)Maximum R(生长速率常数),the shortest generation time(代时)for cell fission or the shortest doubling time(倍增时间)for the doubling of the cytoplasm.(2)Balanced growth of the cell,so the ingredient of the cell is evenly distributed(细胞进行平衡生长,故菌体各部分成分均匀)(3)Active enzyme complex,blooming metabolism.(酶系活跃,代谢旺盛)第30页/共107页 Based on the advantages we discussed above,the microbes of the whole group have relatively consistent physiological attributes,therefore is the better material for the metabolism and physiological researches,and is the optimum host cell to multiply the bacteriophage,the suitable seed for the fermentation industry as well.usages第31页/共107页Factors that affect the period of the exponential phase:(1)Bacterial species(2)Ingredients of the medium(3)Concentration of the nutrients(4)Culture temperatureEscherichia coli 12.517min.Bacillus subtulis 2632Lactobacillus acidphilus 6687Streptococcus lactis 2648examples第32页/共107页 The concentration of the nutrient can affect both the growth rate(R)and the total amount of growth.The growth rate only can be affected while the nutrients concentration is rather low.When the nutrients concentration is gradually increased,only the final cell yield is affected and the growth rate isnt affected.If the concentration is further increased,both the growth rate and the growth amount wouldnt be affected any more.第33页/共107页Growth-limited factor (生长限制因子生长限制因子)The growth-limited factor is a kind of nutrient that can affect the growth rate and the growth amount in a small range of concentration.(凡处于较低浓度范围内可影响生长速率和菌体产量的某营养物,就称生长限制因子)第34页/共107页 As the bacteria increase in numbers they use up all the available nutrients and produce toxic waste products.Eventually a point is reached where there is no net increase in cell numbers,seen as a flattening off（变平的部分）of the growth curve.During this state of equilibriumcells are still functioning（在稳定阶段，细胞仍有功能）.There is some cell death which is balanced by some small amounts of controlled cell division.3.Stationary phase（稳定期或平衡期）（稳定期或平衡期）第35页/共107页 Once the cell growth enters the stationary phase,the inclusion bodies(细胞内含物)such as glucogen,metachromatic granules and fat become to accumulate,and the spore become to form in the bacillus,some microbes begin to synthesize the useful secondary metabolites(次生代谢物 eg.antibiotics)via complex secondary metabolism pathway from the precursorprimary metabolites(初生代谢物),thus the secondary metabolites also is called idiolites(稳定期产物).Special product of the stationary phase第36页/共107页Why the stationary phase comes?The reasons are:(1)The exhaustion of the nutrients especially the growth-limited factor.(2)The ratio disorder of the nutrients particularly C/N ratio.(3)The accumulation of the harmful metabolites for instance the acid,alcohol toxin or H2O2(4)the physiochemical conditions becomes more and more unsuitable(pH,redox potential).第37页/共107页The growth pattern of the stationary plays the instructive role in the practice:(1)its the optimum harvesting time for the fermentative production designed for the bacteria or the bacteria-growth paralleled metabolites.(产物的最佳收获期)(2)Its the best bioassay time for the amino acid,nitrogenous bases and amino acids.（最佳生物测定时期）(3)The study on the reason about the coming of the stationary phase accelerate the putting forward for the principles of continuous culture and the development of technology and techniques.(通过对稳定期到来原因的研究，还促进了连续培养原理的提出和工艺、技术的创建)第38页/共107页After a while the rate of cell death becomes greater than cell division and the number of viable cells drops.The whole group is in a negative-increasing state.The increasing proteinase activity resulted in the autolysis of some microbes and the culture becomes less turbid.some beneficial secondary metabolites(antibiotics)are further synthesized or released by some species in this stage,and for the bacillus,spore is also released during this time.4.Death phase（衰亡期）（衰亡期）第39页/共107页A typical bacterial growth curve.time第40页/共107页Part C microbial continuous culture Continuous culture also named open culture,it is put forward on the basis of batch culture or closed culture in which the unicelled microbes shows the typical growth curve.When continuous culture is used in practice,it is called continuous fermentation(连续发酵连续发酵).第41页/共107页 The problem with batch culture is that eventually cell growth stops due to the exhaustion of nutrients or the accumulation of toxic products.If these problems can be prevented by replacing the spent medium with fresh,then bacterial growth can be maintained indefinitely.Continuous culture systems such as the chemostat do just that.A major advantage of these systems is in the provision of constant conditions for physiological studies.This is impossible in batch culture as the growth of the bacteria changes the environmental conditions such as pH and nutrient concentration of the medium.Batch culture versus continuous culture第42页/共107页Batch cultureContinuous culturechemostatturbidostatContinuous medium flow inThe relationship between batch culture and continuous cultureTime：late stage of exponential phase第43页/共107页 They are flow systems in which fresh medium is constantly fed into a culture vessel which is kept at a constant volume by an overflow(溢流)system that removes the excess of liquid so the bacteria are kept in the exponential phase of growth.Consequently,once the system has reached equilibrium,cell numbers are kept constant and a steady-state growth rate is reached.This growth rate can be manipulated by altering conditions such as medium composition and flow rate.chemostat第44页/共107页cflmCulture vesselOverflow pipe to collecting vessel培养基流速控制搅拌器通入空气培养瓶至收容器的流出管Fig.4 a simplified diagram of a continuous culture system第45页/共107页Medium storing vessel filterMedium inflow pipe pumpchemostatCollecting vesselfilterSampling outletStirring mechanismAir filter第46页/共107页Classification of Continuous culture apparatusB one-step continuous fermenter Steps Multi-step continuous fermenterA turbidostat(恒浊器)controlling style chemostat(恒