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Chapter 3. Microbial Growth. Microbial Growth means. increase in number of cells, not cell size. It is very confusing with reproduction ( 繁殖 ) in other forms of life (e. g. animals). Growth requirements. Physical. 1 Temperature. 2 pH. 3 Osmotic pressure (water). 4 Light. Chemical.

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    1. Chapter 3 Microbial Growth

    2. Microbial Growth means increase in number of cells, not cell size. It is very confusing with reproduction (繁殖) in other forms of life (e. g. animals).

    3. Growth requirements Physical 1 Temperature 2 pH 3 Osmotic pressure (water) 4 Light Chemical 1 Carbon 5 Inorganic element 2 Nitrogen 6 Oxygen 3 Sulfur 7 growth factors 4 Phosphorus

    4. Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature Physical Requirements for Growth: 1 temperature

    5. 专性嗜冷 兼性嗜冷 中温型 嗜热 极端嗜热 Arctic/antarctic mammal/soils Spring/sea volcano compost Sea/refrige

    6. Question: Is it always safe if foods are stored in refrigerator for long time?

    7. Figure 6.2

    8. Physical Requirements for Growth: 2 pH • pH • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow between pH 5 and 6 • Acidophiles grow in acidic environments

    9. Physical Requirements for Growth: 3 osmotic pressure • Osmotic Pressure (=water availability) • Hypertonic (高渗) environments, increase salt or sugar, cause plasmolysis(质壁分离) • Extreme or obligate (专性) halophiles require high osmotic pressure • Facultative (兼性) halophiles tolerate high osmotic pressure

    10. Plasmolysis Cell in normal osmotic pressure environment Cell in hypertonic environment Figure 6.4

    11. Physical Requirements for Growth: 4 Light • Light (=radiation) • Necessary for phototrophic bacteria (using light as energy source) • Radiation in different wavelength has different effects (ultraviolet, x-ray, and gamma-ray kills bacteria while green light induces development of life cycle, e. g. mushrooms)

    12. Carbon Structural organic molecules, energy source (recall the chemical constituents of cells) Chemoheterotrophs (化能异养型) use organic carbon sources Autotrophs (自养型) use CO2 Chemical Requirements for Growth: 1 carbon

    13. Chemical Requirements for Growth: • Nitrogen • In amino acids, proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3 • A few bacteria use N2 in nitrogen fixation • Sulfur • In amino acids, thiamine (硫胺素=Vb 1), biotin (生物素 VH) • Most bacteria decompose proteins • Some bacteria use SO42 or H2S

    14. Phosphorus A. In DNA, RNA, ATP, and membranes B. PO43is a source of phosphorus Trace elements A. Inorganic elements required in small amounts B. Usually as enzyme cofactors Chemical Requirements for Growth

    15. Obligate aerobes 专性需氧菌 Obligate anaerobes 专性厌氧菌 Aerotolerant anaerobes 微耐氧菌 Micro-aerophiles 微需氧菌 Facultative anaerobes 兼性厌氧菌 O2 is not necessary but tolerable O2 is necessary Grow better if O2 is present O2 is toxic O2 is needed at low partial pressure Chemical Requirements for Growth: • Oxygen (O2)

    16. Singlet oxygen: O2 boosted to a higher-energy state Superoxide free radicals (超氧化物自由基): O2 Peroxide anion (过氧化物阴离子): O22 Hydroxyl radical 羟基(OH) Why O2 is toxic to some bacteria Some bacteria do not have these enzymes

    17. Chemical Requirements for Growth: • Organic Growth Factors • Organic compounds obtained from the environment • Vitamins, amino acids, purines (嘌呤), pyrimidines (嘧啶)

    18. Culture Media • Culture Medium: Nutrients prepared for microbial growth • Sterile: No living microbes • Inoculum: Introduction of microbes into medium • Culture: Microbes growing in/on culture medium

    19. Complex polysaccharide Used as solidifying agent for culture media in Petri plates (平板), slants (斜面), and deeps (深层培养) Generally not metabolized by microbes Liquefies at 100°C Solidifies ~40°C Agar

    20. Chemically defined media: exact chemical composition is known Complex media: Extracts and digests of yeasts, meat, or plants, e. g. Nutrient broth (营养肉汤) Nutrient agar (营养琼脂) Culture Media

    21. Examples of Culture Media

    22. Reduced media Contain chemicals (thioglycollate 巯基乙酸盐or cystine (胱氨酸) or ascorbate (抗坏血酸) to remove O2 Heated to drive off O2 Anaerobic Culture Methods

    23. Anaerobic Culture Methods • Anaerobic jar

    24. Anaerobic Culture Methods • Anaerobic chamber

    25. Some bacteria require high CO2 • Candle jar O2-packet

    26. Selective Media Differential Media • Enhance the growth of certain wanted organisms but suppress unwanted microbes. • Make it easy to distinguish colonies of different microbes. Figure 6.9b, c

    27. Enrichment Media • Encourages growth of desired microbe • Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria • Inoculate phenol-containing culture medium with the soil and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Transfer 1 ml to another flask of the phenol medium and incubate • Only phenol-metabolizing bacteria will be growing

    28. All-purposed (rich) medium • An all-purposed (rich) medium is rich in a wide variety of nutrients (including many growth factors) and will, therefore, support the growth of a wide range of bacteria.

    29. Minimal Medium • A Minimal medium supplies only the minimal nutritional requirements of a particular organism.

    30. Summary of Commonly-Used Constituents of Media • Substance Function Composition Source • AGAR Solidifying agent Impure polysaccharide marine algae • PEPTONES nutrient Animal/Plant proteins cow,soy • EXTRACTS nutrient Animal/Bacteria paste cow,yeast • BODY FLUIDS hormones Blood animals • BUFFERS pH K2PO4;NaHPO3;CaCO3 - • REDUCTANTS e- source thioglycolate - • SELECTIVES bacteriostat Antibiotics, sodium azide varies • INDICATORS pH bromothymol blue,phenol red - • WATER hydration H2O (DI & tap) -

    31. A few words • A pure culture contains only one species or strain • A colony is a population of cells arising from a single cell or spore or from a group of attached cells • A colony is often called a colony-forming unit (CFU)

    32. Streak Plate (平板划线)

    33. Medium sterilization

    34. Sterilization: all living cells, viable spores, viruses are killed or removed from object or habitat though: Irradiation: destroys/distorts nucleic acids X-rays & microwaves. UV commonly used on object surfaces Filtration: physical removal from liquid or gas. Sterilize soln’s that are denatured by heat, i.e., antibiotics, injectable drugs, amino acids, vitamins, etc Gas: formaldehyde, glutaraldehyde, ethylene oxide. Toxic chemicals (require gas chamber) used for large items Heat: important, widely used. Endospores (theromoduric) destruction guarantees sterility

    35. Treatment Temp Effectiveness Vaporizes organic material on non-flammable surfaces but may destroy many substances in the process Incineration >500o 30 minutes boiling kills microbial pathogens & vegetative forms of bacteria BUT may not kill endospores Boiling 100o 100o Intermittent Boiling 3x30 min intervals, followed by periods of cooling kills endospores

    36. Treatment Temp Effectiveness Autoclave/pressure cooker (steam under pressure) 121o/15mins @ 15lbs pressure Kills all forms of life including endospores. Sterilization requires maintenance at effective temp for full time period Dry heat (hot air oven) 160o/2hrs Materials that must remain dry Dry Heat (hot air oven) 170o/1hr Same as above NB: Increasing temp by 10o shortens sterilizing time 50%

    37. Treatment Temp Effectiveness Pasteurization (batch method) 63o/30mins Kills most vegetative bacterial cells including pathogens, i.e., streptococci, staphylococci & Mycobacterium tuberculosis Pasteurization (flash method) 72o/15secs Similar to batch method For milk conducive to industry fewer undesirable effects on quality & taste

    38. Some more words Sterilants: Chemical agent used in chemical sterilization Disinfectants: agents (chemical) used in disinfection only on inanimate objects Disinfection: killing, inhibition or removal of microbes that may cause disease Sanitization: related to disinfection. Microbial popln reduced to levels considered safe by public health standards Antisepsis: prevention of infection or sepsis Antiseptics: chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth (not toxic) Germicide: kills pathogens & non pathogens (not endospores) Bactericide, Fungicide, Algicide or Viricide: disinfectant/antiseptic effective against specific microbial group

    39. Autoclave to sterilize medium

    40. Filtration apparatus to sterilize medium

    41. How Bacterial Cells Divide Binary fission(二分裂殖): 1 cell divides into 2 new cells Growth rate: rate of cell to reproduction Generation(世代时): Time required for a complete fission cycle i.e., 1 parent cell = 2 new daughter cells 1st Generation = 2 cells 2nd = 4 cells 3rd = 8 cells

    42. N time t = 0 Log(N) 2 x N DT = Doubling Time time t = 0 “Exponential growth” “Log growth”

    43. Bacterium DT max Bacillus stearothermophilus 8 min E. coli 23 min Caulobacter crescentus 90 min Mycobacterium tuberculosis 6 hours

    44. Assume that cells are at the exponential growth stage. Then we can calculate cell numbers at ant time of the stage. N0 is the number of cells at time zero. Nt is the number of cells at time t. n is the generations G is the time needed to complete one generation.

    45. Growth stage in a batch culture Log(N) inoculate time stationary decline log lag

    46. LAG PHASE 1. Newly inoculated cells, adjustment (can be diauxic) 2. NO cell division taking place 3. Population is sparse or dilute LOG PHASE 1. Population growth at geometric/logarithmic rate 2. Cells reach maximum rate of cell division (while nutrients and environment are favorable) STATIONARY PHASE 1. Population reaches maximum numbers, rate of cell inhibition (death) = Rate of multiplication DEATH PHASE 1. Decline in growth rate (reverse Log phase) 2. Death in geometric fashion

    47. Media flow culture Out flow Continuous cultures: Cells in continuous culture vassal can be kept at log phase.

    48. The elemental composition of biomass The elemental composition of biomass is surprisingly constant across the variety of commercially utilised strains of bacteria and fungi. For Example: C/N mole ratio Escherichia coli CH1.77O0.49N0.24? Saccharomyces cerevisiae CH1.83O0.56N0.17? Pseudomonas C12B CH2.00O0.52N0.23 ? Average CH1.79O0.50N0.20? This average elemental composition can be utilised if no other value is available. However, values for many other organisms are available in literature.

    49. The end