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Shixue Yin (Prof Dr) CESE, Yangzhou University Chapter 3 Microbial Growth Shixue Yin (Prof Dr) CESE, Yangzhou University 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). Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Physical Requirements for Growth: 1 temperature • Temperature –Minimum growth temperature –Optimum growth temperature –Maximum growth temperature Shixue Yin (Prof Dr) CESE, Yangzhou University 专性嗜冷 兼性嗜冷 中温型 Arctic/antarctic mammal/soils compost Sea/refrige 嗜热 极端嗜热 Spring/sea volcano Shixue Yin (Prof Dr) CESE, Yangzhou University Question: Is it always safe if foods are stored in refrigerator for long time? Shixue Yin (Prof Dr) CESE, Yangzhou University Figure 6.2 Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Plasmolysis Cell in normal osmotic pressure environment Cell in hypertonic environment Figure 6.4 Shixue Yin (Prof Dr) CESE, Yangzhou University 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) Shixue Yin (Prof Dr) CESE, Yangzhou University Chemical Requirements for Growth: 1 carbon • 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Chemical Requirements for Growth: 1. Nitrogen A. B. C. D. In amino acids, proteins Most bacteria decompose proteins Some bacteria use NH4+ or NO3 A few bacteria use N2 in nitrogen fixation 2. Sulfur A. In amino acids, thiamine (硫胺素=Vb 1), biotin (生物素 VH) B. Most bacteria decompose proteins C. Some bacteria use SO42 or H2S Shixue Yin (Prof Dr) CESE, Yangzhou University Chemical Requirements for Growth 3. Phosphorus A. In DNA, RNA, ATP, and membranes B. PO43 is a source of phosphorus 4. Trace elements A. Inorganic elements required in small amounts B. Usually as enzyme cofactors Shixue Yin (Prof Dr) CESE, Yangzhou University Chemical Requirements for Growth: 5. Oxygen (O2) Obligate Facultative Obligate aerobes anaerobes anaerobes 专性需氧菌 兼性厌氧菌 专性厌氧菌 O2 is necessary Grow better if O2 O2 is toxic Aerotolerant anaerobes 微耐氧菌 O2 is not necessary but tolerable Microaerophiles 微需氧菌 O2 is needed at low partial Shixue Yin (Prof Dr) CESE, Yangzhou University Why O2 is toxic to some bacteria • 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) Some bacteria do not have these enzymes Shixue Yin (Prof Dr) CESE, Yangzhou University Chemical Requirements for Growth: 6. Organic Growth Factors A. Organic compounds obtained from the environment B. Vitamins, amino acids, purines (嘌呤), pyrimidines (嘧啶) Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Agar • 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Culture Media • 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) Shixue Yin (Prof Dr) CESE, Yangzhou University Examples of Culture Media Shixue Yin (Prof Dr) CESE, Yangzhou University Shixue Yin (Prof Dr) CESE, Yangzhou University Anaerobic Culture Methods • 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Anaerobic Culture Methods • Anaerobic jar 钯 Shixue Yin (Prof Dr) CESE, Yangzhou University Anaerobic Culture Methods • Anaerobic chamber Shixue Yin (Prof Dr) CESE, Yangzhou University Some bacteria require high CO2 • Candle jar O2-packet Shixue Yin (Prof Dr) CESE, Yangzhou University Selective Media • Enhance the growth of certain wanted organisms but suppress unwanted microbes. Differential Media • Make it easy to distinguish colonies of different microbes. Figure 6.9b, c Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University 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. Shixue Yin (Prof Dr) CESE, Yangzhou University Minimal Medium •A Minimal medium supplies only the minimal nutritional requirements of a particular organism. Shixue Yin (Prof Dr) CESE, Yangzhou University Summary of Commonly-Used Constituents of Media •Substance •AGAR Function Composition Solidifying agent Impure polysaccharide Source marine algae •PEPTONES nutrient Animal/Plant proteins cow,soy •EXTRACTS nutrient Animal/Bacteria paste cow,yeast •BODY FLUIDS hormones •BUFFERS pH animals K2PO4;NaHPO3;CaCO3 •REDUCTANTS e- source •SELECTIVES bacteriostat •INDICATORS pH •WATERhydration Blood thioglycolate - Antibiotics, sodium azide varies bromothymol blue,phenol red - H2O (DI & tap) - Shixue Yin (Prof Dr) CESE, Yangzhou University 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 colonyforming unit (CFU) Shixue Yin (Prof Dr) CESE, Yangzhou University Streak Plate (平板划线) Shixue Yin (Prof Dr) CESE, Yangzhou University Medium sterilization Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University Treatment Incineration Temp >500o Effectiveness Vaporizes organic material on non-flammable surfaces but may destroy many substances in the process Boiling 100o 30 minutes boiling kills microbial pathogens & vegetative forms of bacteria BUT may not kill endospores Intermittent Boiling 100o 3x30 min intervals, followed by periods of cooling kills endospores Shixue Yin (Prof Dr) CESE, Yangzhou University Treatment Temp Effectiveness Autoclave/pr 121o/15mins essure cooker @ 15lbs (steam under pressure 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% Shixue Yin (Prof Dr) CESE, Yangzhou University Treatment Pasteurization (batch method) Temp 63o/30mins Effectiveness Kills most vegetative bacterial cells including pathogens, i.e., streptococci, staphylococci & Mycobacterium tuberculosis Pasteurization 72o/15secs (flash method) Similar to batch method For milk conducive to industry fewer undesirable effects on quality & taste Shixue Yin (Prof Dr) CESE, Yangzhou University 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: effective against specific microbial group disinfectant/antiseptic Shixue Yin (Prof Dr) CESE, Yangzhou University to sterilize medium Autoclave Shixue Yin (Prof Dr) CESE, Yangzhou University Filtration apparatus to sterilize medium Shixue Yin (Prof Dr) CESE, Yangzhou University 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 Shixue Yin (Prof Dr) CESE, Yangzhou University N “Exponential growth” time t=0 Log(N) “Log growth” 2xN DT = Doubling Time time t=0 Shixue Yin (Prof Dr) CESE, Yangzhou University Bacterium DT max Bacillus stearothermophilus 8 min E. coli 23 min Caulobacter crescentus 90 min Mycobacterium tuberculosis 6 hours Shixue Yin (Prof Dr) CESE, Yangzhou University Assume that cells are at the exponential growth stage. Then we can calculate cell numbers at ant time of the stage. Nt N0 2 n 0.301t G lg N t lg N 0 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. Shixue Yin (Prof Dr) CESE, Yangzhou University Growth stage in a batch culture Log(N) stationary inoculate decline log lag time Shixue Yin (Prof Dr) CESE, Yangzhou University 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. 2. Decline in growth rate (reverse Log phase) Death in geometric fashion Shixue Yin (Prof Dr) CESE, Yangzhou University Continuous cultures: Media flow culture Out flow Cells in continuous culture vassal can be kept at log phase. Shixue Yin (Prof Dr) CESE, Yangzhou University 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. Shixue Yin (Prof Dr) CESE, Yangzhou University The end