โครงการหอ้ งเรยี นวิทยาศาสตร์ (วมว.) Microbial growthเอกสารประกอบการสอน วิชา ว 30245 ชวี วิทยาประยุกต์ ชดุ ที่ 3 อ. ธนสุ รา เหล่าเจรญิ สขุ 1
Microbial growth Refers to an increase in cell number, not in cell size. Bacteria grow and divide by binary fission, a rapid and relatively simple process. 2
Bacterial growth is the asexual reproduction, or cell division, of a bacterium into two daughter cells in a process called binary fission. 3
Culture Media Nutrient material prepared for microbial growth in the laboratory. 4
Culture Media : Requirements Must be sterile Contain appropriate nutrients Must be incubated at appropriate temperature 5
Culture Culture: Microbes that grow and multiply in or on a culture medium. 6
Culture Media Solid Media: Nutrient material that contains a solidifying agent (plates, slants, deeps). The most common solidifier is agar, first used by Robert Koch. 7
Agar plateAgar slant Agar deep 8
Growth of Bacterial Cultures Bacterial Division: Occurs mainly by binary fission. A few bacterial species reproduce by budding. 9
Binarry fission in Bacteria 10
Hyphomicrobium buding cell 11
Generation Time Time required for a cell to divide, and its population to double. 12
Generation time varies considerably E. coli divides every 20 minutes. Most bacteria divide every 1 to 3 hours. Some bacteria require over 24 hours to divide. 13
Bacterial Growth: Binary Fission 14
Growth of Bacterial Cultures Logarithmic Representation of Bacterial Growth : We can express the number of cells in a bacterial generation as 2n,where n is the number of doublings thathave occurred. 15
Growth of Bacterial Cultures 16
การศกึ ษา generation time ใชข้ อ้ มูลต่อไปน้ี1. จานวนแบคทีเรยี เรม่ิ ตน้2. จานวนแบคทเี รยี ในครง้ั สดุ ทา้ ย3. ระยะเวลาทงั้ หมดท่ีแบคทีเรยี ใชใ้ นการเจรญิ 17
การศกึ ษา generation time แบคทีเรยี เรมิ่ ตน้ 1 เซลล์ เวลาผ่านไป 1 generation time จะไดแ้ บคทีเรยี 2 เซลล์ เม่ือระยะเวลา 2 generation timeจะได้ แบคทเี รยี 4 เซลล์ 18
generation time แบคทีเรยี เรม่ิ ตน้ a เซลล์ มีการแบ่งตวั n generation time จะไดแ้ บคทีเรยี สดุ ทา้ ยเป็ น a x 2n เมื่อระยะเวลาผ่านไปในแต่ละ generation time จานวนแบคทีเรยี จะเพมิ่ เป็ น 2 เท่าเสมอ 19
Phases of Growth Bacterial Growth Curve : When bacteria are inoculated into a liquid growth medium, we can plot of the number of cells in the population over time. 20
Four phases of Bacterial Growth1.Lag phase2. Logarithmic phase (log phase)3. Stationary phase4. Death or decline phase 21
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1. Lag phase Period of adjustment to new conditions. Little or no cell division occurs, population size doesn’t increase. 23
1. Lag phase Phase of intense metabolic activity, in which individual organisms grow in size. May last from one hour to several days. 24
2. Logarithmic phase Cells begin to divide and generation time reaches a constant minimum. Period of most rapid growth. Number of cells produced > Number of cells dying 25
2. Logarithmic phase Cells are at highest metabolic activity. Cells are most susceptible to adverseenvironmental factors at this stage.• Radiation• Antibiotics 26
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3. Stationary phase Population size begins to stabilize. Number of cells produced = Number of cells dying Overall cell number does not increase. Cell division begins to slow down. 28
3. Stationary phase Factors that slow down microbial growth:• Accumulation of toxic waste materials• Acidic pH of media• Limited nutrients• Insufficient oxygen supply 29
4. Death or decline phase Population size begins to decrease. Number of cells dying > Number of cells produced Cell number decreases at a logarithmic rate. 30
4. Death or decline phase Cells lose their ability to divide. A few cells may remain alive for a long period of time. 31
32Bacterial Growth Phases
Measuring Microbial Growth Direct Methods of Measurement Indirect Methods of Measurement 33
Direct Methods of Measurement 1. Plate count 2. Filtration 3. Most Probable Number (MPN) 4. Direct Microscopic Count 34
1. Plate count Most frequently used method of measuring bacterial populations. Inoculate plate with a sample and count number of colonies. 35
1. Plate count Assumptions: • Each colony originates from a singlebacterial cell. • Original inoculum is homogeneous. • No cell aggregates are present. 36
1. Plate countAdvantages:• Measures viable cells 37
1. Plate countDisadvantages:• Takes 24 hours or more for visible colonies toappear.• Only counts between 30 and 300 colonies areaccurate.• Must perform serial dilutions to get appropriatenumbers/plate. 38
10 serial dilution 39
Serial dilutions are used with the plate count method to measure numbers of bacteria 40
1. Plate count 1.1 Pour plate 1.2 Spread plate 41
1.1 Pour plate Introduce a 1.0 or 0.1 ml inoculum into an empty Petri dish. Add liquid nutrient medium kept at 50oC. Gently mix, allow to solidify, and incubate. 42
1.1 Pour plate Disadvantages:• Not useful for heat sensitive organisms.• Colonies appear under agar surface. 43
1.2 Spread plate Introduce a 0.1 ml inoculum onto the surface of Petri dish. Spread with a sterile glass rod.Advantages:• Colonies will be on surface and notexposed to melted agar. 44
spread plate method 45
Pour plate versus Spread plate 46
Incubator 47
Bacterial colony on agar plate 48
Colony counter 49
Colony counter 50
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