上海交通大学：《微生物学 Microbiology》课程教学资源（双语课件）Lecture 5.2 Microbial Growth（Measuring Microbial Growth 微生物生长的测定（计数））

上泽充通大￥ Shanghai Jiao Tong University Lecture 5-2 Microbial Growth CHAPTER 5 in BROCK BIOLOGY OF MICROORGANISMS Zhao Liping,Chen Feng School of Life Science and Biotechnology Shanghai Jiao Tong University http://micro.sjtu.edu.cn

Lecture 5-2 Microbial Growth Zhao Liping, Chen Feng School of Life Science and Biotechnology Shanghai Jiao Tong University http://micro.sjtu.edu.cn CHAPTER 5 in BROCK BIOLOGY OF MICROORGANISMS

上帝充通大睾 Shanghai Jiao Tong University III.Measuring Microbial Growth 微生物生长的测定（计数）

III. Measuring Microbial Growth 微生物生长的测定（计数）

上降文通大学 Shanghai Jiao Tong University Growth measurements How to count cell numbers in a population Direct count of microbial growth: ·Total cell count,总菌数记数法 ·Counting chambers记数器 ·Viable count活菌记数法 ·Plate counting平板记数 Indirect count of microbial growth:Turbidimetric measurement浊度记数法 ·Photometer分光计 No.3 Shanghai Jiao Tong University

No.3 Shanghai Jiao Tong University Growth measurements How to count cell numbers in a population Direct count of microbial growth: • Total cell count总菌数记数法 • Counting chambers记数器 • Viable count活菌记数法 • Plate counting平板记数 Indirect count of microbial growth: Turbidimetric measurement浊度记数法 • Photometer分光计

上泽充通大学 Shanghai Jiao Tong University 5.7 Direct measurements of microbial growth:total and viable counts Total and viable counting methods each have their strengths and weaknesses.总菌计数与活菌计数法各有利弊。 No.4 Shanghai Jiao Tong University

No.4 Shanghai Jiao Tong University 5.7 Direct measurements of microbial growth: total and viable counts Total and viable counting methods each have their strengths and weaknesses.总菌计数与活菌计数法各有利弊

上泽充通大学 Shanghai Jiao Tong University 5.7.1 Direct Measurements 1:Total cell counts Direct microscopic count:The number of cells in a population can be measured by counting a sample under the microscope.直接显微计数法， With liquid samples,special counting chambers must be used.液体样品需使用计数室/血球计数板 No.5 Shanghai Jiao Tong University

No.5 Shanghai Jiao Tong University 5.7.1 Direct Measurements 1: Total cell counts Direct microscopic count :The number of cells in a population can be measured by counting a sample under the microscope.直接显微计数法, With liquid samples, special counting chambers must be used. 液体样品需使用计数室/血球计数板

Cover glass Chamber holding (a) bacteria (b) (c)

Figure 5.14 Ridges that support coverslip To calculate number Coverslip per milliliter of sample: 12 cells×25 large squares×50×103 Number/mm2(3×102) Sample added here.Care must be taken not to allow overflow;space Number/mm3(1.5×104) between coverslip and slide is 0.02 mm Microscopic observation;all mm.Whole grid has 25 large cells are counted in large squares,a total area of 1 mm2 and square(16 small squares): Number/cm3(ml)(1.5 x 107) a total volume of 0.02 mm3. 12 cells.(In practice,several large squares are counted and the numbers averaged.) Direct microscopic counting procedure using the Petroff-Hausser counting chamber

Figure 5.14 Coverslip Ridges that support coverslip To calculate number per milliliter of sample: 12 cells  25 large squares  50  103 Number/mm2 (3  102 ) Number/mm3 (1.5  104 ) Number/cm3 (ml) (1.5  107 ) Sample added here. Care must be taken not to allow overflow; space between coverslip and slide is 0.02 mm ( mm. Whole grid has 25 large squares, a total area of 1 mm2 and a total volume of 0.02 mm3 . Microscopic observation; all cells are counted in large square (16 small squares): 12 cells. (In practice, several large squares are counted and the numbers averaged.) 1 50 Direct microscopic counting procedure using the Petroff-Hausser counting chamber

上泽充通大学 Shanghai Jiao Tong University Limitations of microscopic count Dead cells and living cells are all counted Small cells are difficult to count Need phase contrast microscope Need high cell concentration,106 cells/ml (why?) Precision is difficult to achieve Motile cells must be immobilized before counting No.8 Shanghai Jiao Tong University

No.8 Shanghai Jiao Tong University Limitations of microscopic count Dead cells and living cells are all counted Small cells are difficult to count Need phase contrast microscope Need high cell concentration, 106 cells/ml (why?) Precision is difficult to achieve Motile cells must be immobilized before counting

上泽克通大学 Shanghai Jiao Tong University 5.7.2 Direct Measurements 2: Viable Cell Count活菌记数 No.9 Shanghai Jiao Tong University

No.9 Shanghai Jiao Tong University 5.7.2 Direct Measurements 2: Viable Cell Count 活菌记数

Figure 5.15 Two methods of a viable count(plate count/colony count):spread- plate method and pour-plate method Spread-plate method Surface colonies Incubation Sample is pipetted onto Sample is spread evenly Typical spread-plate surface of agar plate over surface of agar results (0.1 ml or less) using sterile glass spreader Pour-plate method Surface colonies Solidification Subsurface Bun可Bu and incubation colonies Sample is pipetted into Sterile medium is Typical pour-plate sterile plate added and mixed results well with inoculum 活菌记数的两种平板记数法：涂布法与混菌法

Figure 5.15 Sample is pipetted onto surface of agar plate (0.1 ml or less) Sample is spread evenly over surface of agar using sterile glass spreader Typical spread-plate results Surface colonies Surface colonies Subsurface colonies Typical pour-plate results Sample is pipetted into sterile plate Sterile medium is added and mixed well with inoculum Spread-plate method Pour-plate method Incubation Solidification and incubation Two methods of a viable count (plate count/colony count): spread￾plate method and pour-plate method 活菌记数的两种平板记数法: 涂布法与混菌法