页数：94页文件大小：25.41 MB来源： 微生物学ppt课件
本课件为英文版微生物生长章节，介绍纯培养的分离与培养方法，讲解分批培养生长曲线各时期特点与数量测定技术，分析温度、pH、氧气等环境因素的影响，并给出消毒、灭菌与菌种保藏手段。
Chapter 7 Microbial GrowthHow to cultivate microorganisms?How microorganismsgrow?What factors affect the microbial growth? (Requirements for microbial growth)How to control the microbial growth?
7.1 Measurement of microbial growth7.1.1 Isolation of pure culturesIn natural habitats microorganisms usually grow in complex,mixed populations containing severals pieces. This presents a problem for microbiologist because a single type of microorganism cannot be studied.One needs a pure culture, a population of cells arising from a single cell,to characterize a individual species.
Isolation of puremicroorganisms:spread-plate method
Streak plate method
7.1.2 Culture Methods of MicroorganismsBe divided into two major categories depending on the requirement of oxygen：aerobic cultivationanaerobic cultivationBe divided into two major categories depending on the physical peculiarityof culture medium：liquid cultivationsolid cultivation
1) Solid aerobic cultivating methodsSolid cultivation in the test tubeand petri dish7.1.3 Aerobic cultivating methods
Solid culture of mushroom
2）Aerobicliquid cultivating methodsShaking incubator
Small-scale automatic fermentor for lab use
Measurement of cell numbers1)Petroff-Hausser counting chamber
2) Filtration
3）Viable count usingserial dilutions of thesample
Measureofcellmass1.Freshweight2.DryWeight3.Metabolic Activity4. Turbidity
Turbidity
7.2 GrowthMicrobial growth is at the level of both the individual and the populationIndividual growth = Increase in size and/or mass of an organism with timePopulation growth= increase in numbers of micro-organisms in microbial populationsthe knowledge of these two aspects can lead to the understanding of the rules of microbial growth
Bacterial growthRapidlygrowing-chromosomeisreplicatedbeforecelldivisionSlowlygrowing-chromosomeisreplicatedonebeforedivisionOriFigure 4-1
2、
Yeastcellgrowth1、Binary fissionFigure 4-3 Binary fission
2、buddingFigure 4-4 budding
ApicalgrowthofFilamentousfungiFigure 4-6Figure4-5Saprolegnia ferax0:001:00
Becausemicroorganismaresosmall,thegrowthofindividualmicroorganismsrarelystudied,instead,whenwediscussmicrobialgrowthweinevitablyrefertomicrobialpopulationsPopulation growth?
Growth of unicellular microbial populationsunicellularmicrobesincludeprocaryoticbacteriaandeucaryoticyeast.Thegrowthofthepopulationsistheincreaseinthenumberofcells.Therateofmicrobialpopulationsistheincreaseinnumberandincellmassperunittime.
1. A characteristic growth pattern–exponential phase
Logarithm of thenumber Of bacteria isrelated to timeNumberofbacteriaLogarithm of the number ofbacteriaTime
FomulaNt= N0×2nlgNt=lgN0+nlg2n=lgNt-lgN0=lgNt-lgN0lg2 0.301N Number of generationsNtFinal Concentration of CellsNoOriginal concentration of cells.301Conversion Factor to Convert Log2 to Log10Generation Time: The Time Required for a Cell to Divide
2、growth curvePhases of Microbial Growth
1）Lag PhaseDuring this period, the cell increases its sizeIt will synthesis new protoplasm, enzymes, coenzymes etcThe bacterium will adjust the physiological environmentThe cells are metabolically activeAt the end of lag phase, each cell will start reproduction
Factors culture ageinoculation size mediacomposition
2）exponential phase-All the cells are uniform chemical composition, metabolic activity and physiological characters-At this time, the generation time will be of constant -The cells will be sensitive to chemical inhibitors-This is the ideal stage to study the metabolic activity of the bacterium
3）Stationary Phase-death rate = rate of reproduction-A time of physiological change-Gram-positive bacteria may begin to become variable in the gram stain as the integrity of peptidoglycan comples deteriorates
Population size remains constant?-key nutrients are depleted-toxic metabolic byproducts accumulate-encounter environmental stress, such as oxygen, pH
Practical significance
4）Death or decline phase-Death rate > rate of reproduction-The cell lysis occur during this stage
ContinuouscultureBatchandcontinuousculturebatch culture，A suspension culture in which cells grow in a finite volume of liquid nutrient medium and follow a sigmoid pattern of growth. All cells are harvested at the same timeContinuous culture，A suspension culture continuously supplied with nutrients by the inflow of fresh medium. The culture volume is normally constant. a process in which cells or micro-organisms are maintained in culture in the exponential growth phase by the continuous addition of fresh medium that is exactly balanced by the removal of cell suspension from the bioreactor.
Continuous culturechemostatturbidostat
Synchronous growth and cultureSynchronous growth of bacteria : All the cells of a bacterial culture a same stage of their growth cycle is referred as synchronous growthsynchronous culture :A culture in which the microbial cell is synchronized for the majority of the cells present.
Synchronous non-Synchronous Growth
Microbial growth in natural enviromentsNatural enviroment ：-Complex-Constantly changingMicrobes’response ：-changes in the morphology-synthesis of special structures and enzymes
Growth limitation by environmental factors1、Liebig'sLawoftheMinimum：Thegrowthandreproductionofanmicroorganismisdependentonthenutrientsubstancethatisavailbleinminimumquantity2、Shelford’s law of tolerances：For an organism to succeed in a given environment, each of a complex set of conditions must remain within the tolerance range of that organism and if any one condition exceeds the minimum or maximum tolerance of that organism, it will fail to thrivethese two rules also can be applied to the cultured microorganisms
Some studies showed when bacteria have lost their ability to grow on conventional laboratory media using standard culture techniques, they still might be able to play a role in infectious diseases.Practical sense１, diversity２, new resource3, knowledge of new pathogen and its mechanisms
Quorum sensing and Microbial population More recently, it has become clear that many bacteria can communicate with one another and behave cooperatively through quorum sensing or autoinductionQuorum sensing –bacteria monitor their own population density through sensing the level of signal molecules, and some times called autoinducers because they can stimulate the cell that releases them. The concentration of these signal molecules increases along with the bacteria population until is rises to a specific threshold and signals the bacteria that the population density has reached a critical level or quorum. The bacteria then begin expressing sets of quorum-dependent genes
Important function of quorum sensingIf extracellular enzymes were released by only a few bacteria, they would diffuse away and be rendered ineffective because of dilution. With control by quorum sensing, the bacteria reach a high population density before they release enzymes, and as a consequence enzyme levels are concentrated enough to have significant effects.If a pathogen can reach high levels at a particular site before producing virulence factors and escaping into surrounding host tissues, it has a much better chance of counteracting hose defenses and successfully spreading throughout the host’s body.It is very important in many bacteria that establish symbiotic or parasitic relationship with hosts.
The influence ofenvironmental factors on growth
Temperature and growth
pH1. Acidophiles grow best between pH 0 and 5.52. Neutrophiles grow best between pH 5.5 and 8.03. Alkalophiles grow best between pH 8.5 and 11.54. Extreme alkalophiles grow best at pH 10.0 or higher5. Despite wide variations in habitat pH, the internal pH of most microorganisms is maintained near neutrality either by proton/ion exchange or by internal buffering
Accordingtotheeffectofthecontrollingsterilization:theprocessthatcandestroyorremoveallviablemicroorganisms,includingvirusesinagivenarea；disinfection:thewaysthatcankill,removeorreducethepathogenicorganismsfromanobject；antisepsis:chemicalsappliedtobodysurfacestodestroyorinhibitvegetativepathogens。
AccordingtotheprinciplesandmethodsofthecontrollingPhysicalmethodsforcontrollingChemicalmethodsforcontrolling7.4controlling microorganisms
Factors that influence the rate at which microbes are killed by antimicrobial agents：The total original microbialRelative resistanceLength of exposure to the agent Action of the agent
①the katogene of hot steam to cell component is stronger②the penetrating power of hot stream is stronger than hot air③latent hot of stream is strong, a great quantity of heat is released when gas changes into liquid ,therefore the temperature of sterilization is increased rapidly.
Boilingwater：exposuretothewaterwhichis1000Cformorethan15min。autoclave:appliedwidely.Puttheobjectsintosterilizingchamberwherethereisalittlewater.Assteamflowsfromthesteamjacketintothesterilizingchamber,thecoolairisforcedoutandaspecialvalveincreasesthepressuregraduallyandthetemperaturerisesto121andthesuper-heatedwatermoleculesrapidlyconductheatintothemicroorganisms.Thetimefordestructionofthemostresistantbacterialsporeisabout15minto20min.Thelowertemperature--1150Ccanalsobeutilizedbutshouldbekeptfor35min.Theautoclaveoccupiesanimportantpositioninthecontrolofmicroorganismsinhospitals,laboratoriesandfermentationplant.2、moist heatboiling water ,autoclave, intermittent sterilization, pasteurization
Note that it is not the pressure of the autoclave that kills the microorganisms, but the high temperature that can be achieved when steam is placed under pressure. As we shall know, increasing pressure can increase the boiling point of water, so that the temperature of steam can be increased.
（2）coldtreatmentTheprincipalbenefitofcoldtreatmentistoslowgrowthofculturesandmicrobesinfoodduringprocessingandstoragebyslowingtherateofenzymes’action.1．chillingKeepthefreshfoodat5℃(usuallythetemperatureofrefrigerator’ssafe)fromputrescence.However,itcanbekeptonlyfordaysbecausesomepsychrophilesgrowveryslowlyevenatlowtemperatures.2．freezingInfamiliesorfoodindustriesthefoodisfrozenat-10℃~-20℃atwhichthemostmicroorganismscannotgrow.Soitcanbekeptlongerbyfreezingthanbychilling.
(3)dryingandhypso-osmoticpressureSincemicroorganismsdonotgrowatlowwateractivities,microbialgrowthcanbecontrolledbydryingorcontrollingosmoticpressure.1.DrynessDryingistodehydratethematerials.Dryingcanseldomkillthemicroorganismsbutcaninhibitthegrowthofthem.Sensitivitytodrynessofdifferentmicroorganisms：MicrococcusgonococcussensitivetodrynessdieafterafewhoursBacillustuberculosisresistdrynessaliveat100℃for20minchaincoccusresistdrynessbepreservedforyearswithoutlosingpathogenicitycystosporestronglystranddrynessremainviableforlengthyperiodsinaridconditions2.osmoticpressureIncreasingosmoticpressureisanotherwaytoinhibitthegrowthofmicroorganismsbyloweringwateractivity.Foodspreservedbyadditionofsugararemainlyfruits.Saltedproductsareprimarilymeatsandfish.
（4）radiationFourtypesofradiation:UVradiation,ionizingradiation,strongvisibleradiationinagivencondition,microwave，whichcanbeusedascontrolthegrowthofmicroorganismsandpreservethefood.
1.UVradiation
2.ionizingradiation
3.StrongvisibleradiationThestrongvisibleradiationranginginwavelengthfrom400to700nmalsohasdirecteffectofsterilization.theycanoxidizetheintracellularphotaesthesia,suchaslactochromeandporphyrinring(thecompositionofoxidase).Thebacteriacultureinthelabshouldbeavoidedexposingtostrongvisibleradiation.CaesarredandMethyleneBluecanabsorbtheenergyofstrongvisibleradiationandoxidizetheproteinsandnucleicacids,thereforetheyareoftencombinedassterilizationtobacteriaandvirus.4.microwavesMicrowavesareinfraredrays.Microwavescannotkillthemicroorganismsdirectly,whiletheycausetheobjectstobecomehot,whichkillthemicroorganisms.However,italwayshappensthatmicroorganismsescapeheatingofmicrowavesbecauseofunevenmatter.
Chemical methods mainly use a serious of chemical agents to control the growth of microorganisms.antimicrobial chemical agentsdifferent physical condition：liquid, gasiform and soliddifferent effect and influence on human being or animals ：antiseptics, antiseptics, chemotherapeutic agent7.4.2 chemical methods for controlling microorganisms
7.4.2.1Disinfectantandantisepticsdisinfectantarechemicalsthatkillmicroorganismsbutalsodoharmtothehumansandareusedoninanimateobjectssuchasthesurfaceofobjects,equipmentsanddejectures.antisepticsarechemicalagentsthatkillorinhibitgrowthofmicroorganismsandthataresufficientlynontoxictobeappliedtolivingtissues.Theycanpreventthesurfaceofbodies,suchasskins,mucosaandwoundsetcfrominfecting.Theyarealsousedtoantisepticizefoods,drinkinganddrugproducts.Butnow,thedistinctionofdisinfectantandantisepticsisnotobviousstrictly.Forexample,highconcentrationalcoholphenyl（3%-5%）isusedtodisinfectthesurfaceofutensil，whilelowconcentration（0.5%）isusedtoantisepticizebiologicalproducts.Theidealchemicaldisinfectantandantisepticsshouldtakeaquickandgoodeffectbuthaslittletissuedamagewithstrongpenetrativityandlittlecorrosion.Theyshouldbemadeeasilyandbestablewithcheapnessandwithoutunpleasantsmell.
7.4.2.2 Chemical AgentsChemical Agents it must be able to kill certain microbes especially and selectivly, but it should also be nontoxic to animals or humans, it could be used to therapy disease caused by microbes.
1、Chemotherapeutic AgentsAgents which are similar to the necessary metabolite of cell in chemical structure , and can interfere normal metabolism activity.Sulfanilamidewasthefirstofagroupofchemotherapeuticagentsknownassulfonamides.Oneofthecomponentsoffolicacidispara-aminobenzoicacid（PABA）issimilartoSulfanilamideinchemicalstructure.Folicacidisacoenzyme,itplaysanimportantroleinthesynthesisofaminoacid,vitamin,nucleicacidandprotein.Certainbacteriamustsynthesizefolicacidforuseinnucleicacidproduction.
Why Sulfanilamide is nontoxic to human cells?Humans cannot synthesize their own folic acid because of the lack of enzymes --that can synthesize folic acid from PABA. therefore they cannot use PABA to synthesize folic acid, and must have it supplied to them, either in foods or in the pure form in vitamin capsules.
2、AntibioticsAntibiotics is composed by two Greece words,anti means “ antagonize ”，bios means “being”，join together，antibiotics means ability to kill beings,that is, antibiotics is a group of matters that can kill microorganisms.Antibiotics are chemical substances produced by certain microorganisms which are active against other microorganisms. Antibiotics constitute a special class of chemotherapeutic agents, distinguished most importantly by the fact that they are natural products (products of microbial activity) .They constitute one of the most important classes of substances produced by larger-scale microbial processes.
AntibioticswasfirstnoticedbyanEnglishscientistA.Fiemingin1928,andusedaschemotherapeuticagentsin1940’s.Penicilin inhibit the growth Micrococcus luteusAntimicrobial spectrum of antibiotics：The range of objects effected by antibiotics.Antibiotics which have effect on many classes of bacteria were called broad spectrum antibiotics, e.g. acheomycin and terramycin, have effect on both gram-positive and gram-negative bacteria;those which only have effect on a few bacteria were called narrow spectrum antibiotics，e.g. penicillin only have effect on gram-positive bacteria.
antibioticstiter:ameasuringunityofthequantityactivecomponentofmicroamountantibiotics.Someusetheweightofcorrespondbiologicalactivityasunit.e.g.1μg=1Unit(U)，phytomycinmuriaficuswasexpressedbythisway.Someusetheactualweightofbiologicalactivityofpureantibioticas1Unit,e.g.penicillinunitwasfirstmeasuredbyitsabilitytousetheleastquantitybuttotallypreventStaphylococcusaureusin50mlbouillonculture-mediumas1Unit.Afterthepurificationofpenicillin,1Unitwasdefinitedtobe0.5988μgbenzylpenicillinsodium，so0.5988μgbenzylpenicillinsodiumwasusedas1Unitpenicillin.
Antibiotic mechanism of action
Inherent resistance to antibiotic
Spread of resistance factors
ReferencesPrescottLM,HarleyJP,andKleinDA.,Microbiology(5thed.),HighereducationpressandMcGraw-HillCompanies,Inc.2002MichaelTM,JohnMM,JackP.Brock.,BiologyofMicoorganisms，Internationaledition,PearsonEducation,Inc.2003TalaroK.P.FoundationsinMicrobiology(FifthEdition),HighereducationpressandMcGraw-HillCompanies,Inc.2005ZhouDe-qing，Microbiologytextbook，secondedition，higheducationpublishinghouse，2002
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