1Microbial GrowthBIO162Microbiology for Allied HealthBinary FissionMesosomeBacteria Growth in vitro• Binary fission – Splitting parent cell to form two similar-sized daughter cells to increase number of cells• Generation time– Duration of each division– Determined by type of bacteria – Example: E. coli (20 min)2The Requirements for Growth: Chemical Requirements• Nutrition– taking in chemicals, assimilating them and extracting energy– used in metabolism and growth• Water– Requirement for all living cells (70-90% water)– Bacterial endospores and protozoa cysts can survive in low moistureThe Requirements for Growth: Chemical RequirementsMacronutrients• required in large amounts• play role in cell structure and metabolism• Examples: Carbon, hydrogen, oxygen, nitrogen, phosphate, sulphurMicronutrients• required in trace amounts• involved in enzyme function and protein structure• Examples: zinc, copper, iron• Present in tap water and distilled waterThe Requirements for Growth: Chemical RequirementsGrowth factors– Organic compounds that cannot be synthesized by bacteria– must be provided as a nutrient; obtained from the environment– Some bacteria are “fastidious”– examples: amino acids, purines & pyrimidines(DNA components)3Culturing bacteria in vitro• in vitro – (latin, in glass) An in vitro biological study is one which is carried out in isolation from a living organism • Obligate intracellular pathogens need to be cultured in chick embryos, lab animals or animal/human cell cultures• Fungi require different culture medium than bacteria. Low pH and anti-bacterial agents usually added to prevent growth of bacteria in medium for fungal growth e.g. Sabouraud’s agarCulture media– Defined medium – exact ingredient is known– Complex medium – exact content unknown; digested extract of organs or cells; support growth of many fastidious microbes• Enriched medium – extra nutrients to promote growth of certain microbes. e.g. chocolate agar (haemoglobin) for N. gonorrhoeae– Selective medium – added inhibitors to discourage growth of certain microbes. e.g. mannitol salt (MSA) agar for salt-tolerant microbes (Staphylococcus carnosus)Use of selective medium4Culture media– Defined medium – exact ingredient is known– Complex medium – exact content unknown; digested extract of organs or cells; support growth of many fastidious microbes• Enriched medium – extra nutrients to promote growth of certain microbes. e.g. chocolate agar (haemoglobin) for N. gonorrhoeae– Selective medium – added inhibitors to discourage growth of certain microbes. e.g. mannitol salt (MSA) agar for salt-tolerant microbes (Staphylococcus carnosus)– Differential medium – allow differentiation of microbes in a microbial community. e.g. MacConkey agar differentiates lactose-fermenting from non-fermenting Gram-ve bacteria; thioglycollate broth differentiates microbes with different O2requirements.The use of blood agar as a differential mediumS. pyrogenesS. pneumoniaE. faecalisMacConkey agar can be used as both a selective and differential medium5Physical Requirements for Growth• Temperature– Each microbe finds its niche in the environment– Range of temperatures for microbial growth– Minimum, optimum and maximum temperatureTemperature requirement for growth– Psychrophile – optimum below 15oC– Psychrotrophs – optimum between 20oC-30oC– Mesophile – optimum between 25oC – 40oC– Thermophile – optimum higher than 45oC– Extreme thermophiles – optimum above 65oCTemperature6Preserving Bacteria Cultures:In the presence of a protective agent (cryoprotectant), freezing stops all microbial activity without killing the bacteria. They can be recovered after long period of time.• Deep-freezing: – 20% glycerol– -50°to -95°C• Lyophilization (freeze-drying): – 20% skim milk, 12% sucrose 10% serum– Frozen (-54°to -72°C) and water is removed in a va cuum (ice to vapor)– Lyophilized bacteria are stored under vacuum and <8ºCTemperatureThe Requirements for Growth: Physical Requirements• pH – acidity or alkalinity of a solution• Effects of pH– Acidophile – prefer below 7– Neutrophile – prefer 7– Alkalinophile – prefer above 7– Most bacteria grow between pH 6.5 and 7.5– Molds and yeasts grow between pH 5 and 6The Requirements for Growth: Physical Requirements• Physical effects of water:– Osmosis-diffusion of water molecules across a selectively permeable membrane (e.g. cell membrane of a bacteria) with an attempt to equalize the concentration on both sides – A salt solution contain NaCl dissolved in water– solute = NaCl– solvent = water– Water move from a low solute concentration to a high solute concentration– Osmotic pressure– force exerted on a membrane by the solutions on both sides7Osmosis• water flows from the solution with the lower solute concentration into the solution with higher solute concentration• Equilibrium is reached once sufficient water has moved to equalize the solute concentration on both sides of the membrane, and at that point, net flow of water ceases (water molecules still move between two sides of the membrane)Semipermeable membrane0.5M0.2MH2O movementLess solutes more solutesMore H2O less H2OMore dilute less diuteLess solutes more solutesLess H2O more H2OSame concentrationPhysical effects of water in the environments– Isotonic• External concentration of solutes is equal to cell’s internal environment • Diffusion of water equal in both directions• No net change in cell volume– Hypotonic• External concentration of solutes is lower than cell’s internal environment• Cells swell and burst – Hypertonic• Environment has higher solute concentration than cell’s internal environment• Cells shrivel (crenate)Osmoregulation in animal cells– Burst (lyse) in Hypotonic solutions because water rushes into the cell – Remain normal in Isotonic solutions– Shrink (shrivel) in Hypertonic solutions because water flows out8Osmoregulation in bacteriaHYPERTONICISOTONICWhat happen when you place bacteria in a hypotonic environment?The Requirements for Growth: Physical Requirements• Osmotic Pressure– Most microbes prefer isotonic solutions– Hypertonic environments (increase salt or sugar) usually cause plasmolysis– Extreme or obligate halophiles require high osmotic pressure