《微生物学》课件 Microbiology Chapter 4

1、Chapter 4Microbial GrowthGrowth is defined as an increase in the number, size or mass of microbial cells. Because a single cell of microorganism is very small, to measure the growth of a single microbial cell is difficult. So microbial growth is generally referred as population growth.Soil Microorga

2、nismsType of organismSize, mmNr of speciesFresh weight, kg/hectare (100 x100m)MicroorganismsViruses0.00002 - 0.0003?Bacteria0.0002 - 0.00530 000700Fungi0.01 - 0.02 (-0.4)1 500 0001000Protozoa0.002 - 0.1100 00030 - 200Algae0.001 - 0.160 000?Soil faunaNematodes0.1 (width)500 0002-50Earthworms250300010

3、 - 1000The numbers and diversity of organisms in soil is enormousHanksworth and Mound 19914.1 Microbial pure cultureIn natural environment, a single strain of microorganism usually occurs as only one component of a large and mixed population containing many other microbial strains. If we study or us

4、e a certain kind, that strain must be separated from all the others. A culture containing only one strain of microorganism, which is usually derived from a single cell or spore is called pure culture. Gram Stain of Staphylococcus aureus金黄色葡萄球菌Gram Stain of Escherichia coliSingle Rod (Bacillus)There

5、are several methods of isolating pure cultures.4.1.1 The pour plate and spread plate methodA sample to be tested usually contains billions of microbial cells. It is not possible to get well-separated colonies in a plate if the sample is not diluted. 4.1.1 The pour plate and spread plate methodThe fi

6、rst step of isolating pure culture is to make serial dilutions. The dilutions are made in a way that 1 ml of sample is added to 9 ml of sterile distilled water or saline solution.The pour plate and spread plate methods differ in the way the diluted sample is added to the plate. In the pour plate met

7、hod, the diluted sample is added to a petridish (plate), then melted agar medium is poured and mixed. In the spread plate method, the diluted sample is placed onto the surface of an already solidified agar medium plate and spread evenly over the surface by means of a sterile, bent glass rod. The pla

8、tes are incubated at a optimal temperature until individual colonies appear.The procedures of pour plate method for isolating pure cultureRhizobium nodules on a pea rootHow to get the rhizobia in the root nodules ?4.1.2 The streak plate methodBy means of a sterile transfer loop, a loop of sample is

9、diluted by moving the loop back and forth on the surface of solidified agar medium plate. As the loop is streaked back and forth, fewer and fewer microbial cells are deposited on the surface.Repeating this process several times can sufficiently dilute the microbial population on the agar surface so

10、that single colonies could be obtained after incubation. C2:C3:C4:C1: first set of streaks4.1.3 The micromanipulator methodThis technique is useful for isolating pure culture of fungi. To make a sufficient dilution is also the first important step. A device called the micromanipulator can be used to

11、 pick up a single spore (or cell) from a diluted sample under a microscope. 4.1.3 The micromanipulator methodThis method permits the operator to control the movement of a micropipette (a fine needle). So that a single spore (or cell) can be isolated. This technique requires a skilled operator.Low sp

12、eed centrifuge with large tubes (100 ml)Sieves of different sizesKnifeForcepsPipetteMicroscopic slides, cover glasses, mountant liquidsFor extracting AMF spores, only some simple tools are needed. Compared with other fungal spores, AMF spores are very big, 0.05 0.5 mmThe significance of pure culture

13、 In research: one gets wrong results In practice 4.2 Maintenance and preservation of pure culture The objectives: To keep the strains alive To preserve the characteristics of the strains 4.2 Maintenance and preservation of pure culture The methods: Preservation at 4 4.2 Maintenance and preservation

14、of pure culture The methods: Storage at -70 80 4.2 Maintenance and preservation of pure culture The methods: Freeze-Drying method 4.2 Maintenance and preservation of pure cultureInternational famous culture collection: ATCC美国模式典型物收集中心 USDA美国农业部 ACCC中国农业微生物菌种保藏管理中心 CCBAUCulture Collection, Beijing Ag

15、ricultural University 4.3 Quantitative measurement of microbial growthDirect cell counts measure the number of cells in a population under microscope. Viable counts measure only the living cells. Measurements of cell mass and cell activity are indirect.4.3.1 Total cell countDirect microscopic count

16、This method is a quick way of estimating microbial total cell number. A special counting chamber called Petroff-Hausser counter is needed. 4.3.1 Total cell countIn such a counting chamber, a grid is marked on the surface of the glass slide, with squares of known small area and known size. The number

17、 of cells per unit area of grid can be counted under the microscope. Direct Measurements of Microbial GrowthThis method has two limitations: dead cells can not be distinguished from living cells. small cells are difficult to see under the microscope.Turbidity The principle of this method is that mic

18、roorganisms in a suspension absorb and scatter the light passing through them. So that a sample containing more microbial cells appears more turbid. The turbidity can be measured by using a spectrophotometer（分光光度计）. TurbidityEstimating Bacterial Numbers by Indirect MethodsFigure 620However, this met

19、hod may not be possible to measure cultures grown in deeply colored media or cultures that contain suspended material other than microbial cells. One should realize that both living and dead cells contribute to turbidity. 4.3.2 Viable cell countThe Plate count method. Viable count is to determine th

20、e number of cells in the sample capable of forming colonies on a suitable agar medium plate. 4.3.2 Viable cell countThe plate count is well established and widely used viable count method. The assumption of this counting procedure is that each viable cell will yield one colony. The results of viable

21、 counts in the sample tested are usually expressed in colony forming units (CFU).The procedure of plate count is almost the same as the pour plate and spread plate method described for isolating of pure culture . However, the plate count needs more accuracy in each step. Otherwise, precision cannot

22、be achieved.Plate Counts: Perform serial dilutions of a sampleInoculate Petriplates from serial dilutionsPlate CountAfter incubation, count colonies on plates that have 25250 colonies (CFUs)Plate Count159153 103 =1.53 105 CFU/mlMembrane-filter count. This technique is particularly useful in determin

23、ing the number of microorganisms in a large sample (H2O, air) that has a small number of viable cells. The first procedure is trapping the viable cells by use of membrane filters which have a uniform porosity of predetermined size sufficiently small that can not let microorganisms pass through. The

24、membrane is then placed onto the surface of the medium plate. After incubation, colonies appear on the membrane surface.Filtration4.3.3 Cell massThe mass of the microbial cells can be determined directly by dry weight. Meanwhile, nitrogen and DNA content, the major constituents of cell material, can

25、 also be measured. 4.3.4 Metabolic activityMicrobial growth is closely related to the metabolic activities in a cell. 4.3.4 Metabolic activityThere are several metabolic properties can be used for indirect measurement of microbial growth, for example, respiration, oxygen consumption and enzyme activ

26、ities. High metabolic activity means more microbial cells present in a sample. 4.4 Bacterial population growth4.4.1 Growth curve Batch culture is a closed-system microbial culture of fixed volume. By means of batch culture method, a given number of bacterial cells are inoculated into a flask of liqu

27、id medium which is subsequently incubated. 4.4 Bacterial population growth4.4.1 Growth curve During the incubation period, we determine the viable cell number intermittently, and plot the logarithms of the number of cells versus time. We obtain a curve that is called bacterial growth curve. From the

28、 curve, it can be seen that there is： an initial period of that appears no growth (lag phase), followed by rapid growth (exponential-or logarithmic phase), then a leveling off (stationary phase), finally a decline in the viable population (death or decline phase).Bacterial population growthBacterial

29、 Growth CurveThe lag phase： The addition of inoculum to a new medium is not followed immediately by a doubling of the population. The population remains temporarily unchanged. There is a lag in cell division. But this does not mean that the cells are dormant . Bacterial Growth CurveOn the contrary,

30、during this stage the individual cells are very active, and synthesizing enzymesand molecules required for cell division. Time for adjustment in the new environment is needed. Bacterial Growth CurveHowever, since not all cells complete the lag phase simultaneously, there is gradual increase in the p

31、opulation until the end of this period.Bacterial Growth CurveThe logarithmic or exponential phase： During this period the cells divide steadily at a constant rate, and the log of the number of cells plotted against time results in a straight line. Bacterial Growth CurveThe increase in population is

32、by geometric progression. Moreover, the population is most nearly uniform in terms of chemical composition of cells, metabolic activity, and their physiological characteristics. So the exponential phase cultures are commonly used for studies of microbial metabolism.Generation time(G): the time requi

33、red for the population to double. The generation time can be calculated by the following formula: n：the number of generations;t: time;N0: the viable number of bacterial cells at time 0;Nt: the viable number of bacterial cells at log phase and at time t. Bacterial Growth CurveThe stationary phase The

34、 viable number of bacterial cells remains constant for a time. The reproduction rate is balanced by equivalent death rate. Bacterial Growth CurveThis can be attributed to a variety of circumstances, particularly the exhaustion of some nutrients, and the production of toxic products during growth.Bac

35、terial Growth CurveThe decline or death phase：Following the stationary phase the bacteria die faster than new cells are produced. The number of viable cells decreases exponentially. Bacterial Growth CurveA variety of conditions contribute to bacterial death, but the most important factors are the de

36、pletion of essential nutrients， and the accumulation of inhibitory products, such as acids.Continuous cultureIn both experimental research and in industrial processes, it is often desirable to maintain a bacterial population growing at a particular rate in the exponential phase. This condition is kn

37、own as steady-state growth. Continuous cultureThe culture volume and the cell concentration are both kept constant by allowing fresh sterile medium to enter the culture vessel at the same rate that “spent” medium containing cells is removed from the growing culture. Continuous cultureUnder these con

38、ditions, the rate at which new cells are produced in the culture vessel is exactly balanced by the rate at which cells are being lost through the overflow from the culture vessel. This culturing method is called continuous culture. 4.5 Factors affecting microbial growthMicrobial growth is deeply inf

39、luenced by the physical, chemical and biological factors in the environment. This is important to understand the microbial diversity, explore microbial resources and control undesirable microorganisms.4.5.1 physical factors4.5.1.1 TemperatureTemperature is one of the most important environmental fac

40、tors affecting growth and survival of microorganisms. Every species of microorganism grows over a range of temperature. (1) minimum temperature A minimum temperature is that below which growth no longer occurs(2) optimum temperature an optimum temperature is that at which growth is most rapid(3) max

41、imum temperature A maximum temperature means above which growth is not possibleMicroorganisms are divided into four major groups based on the range of temperature over which they can grow: psychrophiles嗜冷生物mesophiles嗜温生物thermophiles嗜热生物 hyperthermophiles嗜高温生物生长速率嗜冷生物如：黄杆菌属Flavobacteriumspecies嗜温生物如：

42、大肠埃希氏杆菌Escherichia coli嗜热生物如：嗜热脂肪芽孢杆菌BacillusStearotherm ophilus嗜高温生物如：速生热球菌Thermococcusceler嗜高温生物如：布氏热网菌Pyrodictiumbrockii0 10 20 30 40 50 60 70 80 90 100 110 12013396088105温度（）4.5.1.2 pHEach microorganism grows over a range of pH and has a well-defined pH optimum. Natural environments generally ha

43、ve pH values between 5 and 9, and microorganisms with optimum in this range are most common. Only a few species can grow at pH values of less than 2 or greater than 10. (1) Acidophiles can grow well in environments of extremely low pH. (2) Alkaliphiles can grow well in environments of extremely high

44、 pH. Growth pH of some microorganisms 微 生 物PH最 低最 适最 高褐球固氮菌4.57.47.69.0大豆根瘤菌4.26.87.011.0亚硝酸细菌7.07.88.69.4氧化硫杆菌1.02.02.84.06.0嗜酸乳酸杆菌4.04.65.86.66.8放 线 菌5.07.08.010.0酵 母 菌3.05.06.08.0黑 曲 霉1.55.06.09.0Oxygen Microorganisms can be divided into three major classes based on their need for oxygen. 4.5.1.3

45、 Oxygen(1) Aerobes are species capable of growth at the presence of oxygen. (2) Anaerobes can not use oxygen as a terminal electron acceptor. There are two kinds of anaerobes: Aerotolerant anaerobes can tolerate oxygen and can grow at the presence of oxygen. Obligate anaerobes are killed by oxygen.

46、(3) facultative Many microbes are facultative, they can grow under either aerobic or anaerobic conditionsFor example: Yeast, lactic acid bacteriaobligate aerobesFacultative anaerobesObligate anaerobesAerotolerant anaerobesMicroaerophilesAnaerobic jarAnaerobic Culture MethodsFigure 6.5碳酸氢钠硼氢化钠甲基蓝钯Ana

47、erobic chamberAnaerobic Culture MethodsCandle jarCO2-packetCapnophiles require high CO2Water activity water is the important component of microbial cells. Water availability is an important factor affecting microbial growth. 4.5.1.4 Water activityWater availability is generally expressed as water ac

48、tivity (aw). Microorganisms grow over a range of aw values between 0.66 and 0.99. Every species has its optimal value of aw .The optimal aw values of some microorganisms 微 生 物一般细菌一般酵母菌一般霉菌嗜盐细菌嗜盐真菌嗜高渗酵母0.910.880.800.760.650.60 Visible light and radiation phototropic bacteria need visible light as ene

49、rgy source. 4.5.1.5 Visible light and radiation Some edible fungi can form their fruiting body only when there is certain amount of sun light. Strong light and radiation can kill microorganisms.4.5.2 Chemical agentsMany chemical agents are poisonous to microorganisms. They can kill or inhibit microb

50、ial growth. These chemicals are useful for controlling the growth of undesirable microorganisms.4.5.3 Biological factorsMicroorganisms have close relationship with the living forms in the environment. Plant root extrudes some organic substances as nutrients for microbial growth. This is why that the

51、 number of microorganisms in the rhizosphere is much higher than that in the non-rhizosphere. Some microorganisms live in plant cells to form symbiotic structures, to benefit each other, such as root nodules, mycorrhiza. 4.5.3 Biological factorsAnimals are habitats of microorganisms. Some are living

52、 inside the animal organs, and some are living on the skin. Animals provide nutrients for microbial growth. 4.5.3 Biological factorsDifferent species of microorganisms in an environment affect the growth of each other.4.5.4 Controlling the harmful microorganismsThere are many different species of mi

53、croorganisms in environments. Some of them are harmful to human, animal and plant. They contaminate food and biochemical agents. Methods must be applied to kill or inhibit the growth of undesirable microorganisms. How to eliminate undesirable microbes in those products ?In practice, there are many w

54、ays to achieve these aims:4.5.5 Controlling the harmful microorganismsSterilization: it is a method that eliminate all the viable microorganisms by applying strong physical agents, e.g., heat and radiation. Methods of Sterilization4.5.4 .1 Heat most important and widely used. For sterilization alway

55、s consider type of heat, time of application and temperature to ensure destruction of all microorganisms. Endospores of bacteria are considered the most thermoduric of all cells so their destruction guarantees sterility. 4.5.4 Controlling the harmful microorganisms(1) Incineration: burns organisms a

56、nd physically destroys them. Used for needles , inoculating wires, glassware, etc. and objects not destroyed in the incineration process. (2) Boiling100 for 30 minutes. Kills everything except some endospores To kill endospores, very long or intermittent boiling is required. (3) Autoclaving (steam under pressure or pressure cooker): 121 for 15 minutes. Good for sterilizing almost anything, but heat-labile substances wi