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1 MICROBIAL GROWTH I. Growth: Increase in cell # & cell mass Binary fission: Individual cell enlarges & divides 2 progeny of equal size Asexual reproduction: Does not involve genetic recombination II. The Growth Curve Used to study changes in total population number A. Lag phase: Cells don't divide Metabolically active adapting to new medium: Synthesizing: ATP Cofactors Ribosomes Enzymes B. Log phase: Cells grow and divide at their maximal rate Rate of growth is constant B362 2 Cell # increases exponentially Exponential growth: 1 1st generation 2 2nd generation 4 3rd generation 8 4th generation Generation time: Time required to double the # of cells in a population during the log phase of growth Depends on: Species Nutrients O2 pH Temperature Cell # increases by 2n where n = # of generations Large populations develop quickly: During log phase population uniform in: Chemical composition Physiological characteristics Log phase cultures used in: biochemical studies physiological studies C. D. Stationary phase: # of cells dying = # of cells dividing Cell # remains constant: Population reaches maximum density (109 cells/ml) Caused by: Accumulation of: acidic wastes toxic products Depletion of: O2 nutrients Death phase: Cells die faster than new cells are produced Caused by: Depletion of nutrients B362 3 Accumulation of wastes Decrease in pH Usually logarithmic: Constant # of cells dies every hour III. MEASUREMENT OF GROWTH A. Total cell count: Measures # of cells in a given volume of sample Place on slide & count: Petroff-Hausser counting chamber: Special slide with grids: holds given volume Advantages of total cell count: Quick Inexpensive Requires small volume of sample Gives information about: Cell size Cell morphology Disadvantages: Hard to count small cells Doesn't distinguish between living & dead cells B. Viable cell count more accurate Counts only living cells (cells able to divide & produce colonies) Colony - asexual progeny of a single cell growing on solid medium Use: Spread plates: Spread O.1 ml sample of bacteria on agar plate: Incubate until colonies appear. Every cell is a completely separate colony Pour plates: Sample mixed with liquid agar medium: Poured into sterile Petri dishes Cells stuck in agar are colonies Sample should be 25 - 250 colonies/plate: Thick suspensions must be diluted B362 4 Plate 1 ml sample of each dilution Incubate: Count colonies Total # of colonies equals the # of viable organisms in the diluted sample Multiply plate count by dilution factor: Reciprocal of dilution: If dilution is 10-6 dilution factor would be 106 Find # of cells present in 1 ml of the original undiluted sample Plate counts - spread plates & pour plates used to do viable cell counts Results expressed as colony forming units (CFU) since it is not absolutely certain that each colony arose from an individual cell Viable cell counts very sensitive: Any viable cell colony Allow: Identification of organisms Isolation of pure cultures Widely used to count bacteria in: Soil Food Water Cosmetics IV. The Influence of Environmental Factors on Growth: Growth greatly influenced by the chemical and physical nature of surroundings: A. Solutes and water activity: Microorganisms: Affected by changes in the osmotic concentration of their surroundings Rigid cell wall maintains: Shape Integrity B362 5 B. Compatible solutes: Solutes compatible with growth and metabolism at high concentration: Choline Betaine Proline Glutamic acid Other Amino acids Used to increase internal osmotic concentration of cell: Keeps cell membrane firmly pressed against cell wall If placed in hypotonic environment: Plasmolysis occurs H2O leaves cell Cell membrane pulls away from cell wall: Cell is dehydrated C. Water Activity: Quantitative expression of the degree of water available to a microorganism: 1% relative humidity of a solution: Equals the ratio of the solution's vapor pressure (Psoln) to that of pure H2O (Pwater): Aw = Psoln Pwater Most microorganisms only grow well at aw = 0.98 (seawater): Large quantities of : Sugar Salt are effective in preserving foods Osmotolerant organisms: Maintain high internal solute conc.: Retain H2O Grow over a wide range of water activity: Staphylococcus aureus Saccharomyces rouxii Dunaliella viridis B362 6 Halophiles: Extremely specialized: Found only in: Dead Sea Great Salt Lake Other highly salty habitats Require high levels of NaCl (2.8 -6.2 M) Have modified: Proteins Membranes Ribosomes D. Acidity or alkalinity: Measured on log scale Expressed in terms of pH: pH 7 is neutral: Number of H+ = Number of OHpH above 7 is alkaline: More OH- ions than H+ pH below 7 is acidic: More OH- ions than H+ Each species has definite pH growth range & pH growth optimum Most organisms: Grow best at pH of about 7: Will not grow if medium is: Too acid Too alkaline (basic) No matter what the optimum environmental for growth: The pH in cytoplasm of all organisms is maintained at @ 7.0: H+ & OH- ions actively pumped out of cell Acidophiles: Growth optima between pH 1.0 & 5.5: Important in production of fermented foods: Fermented milk products Pickles Vinegar Wine Beer B362 7 Examples of acidophiles: Thiobacillus Yeasts Molds Alkalophiles: Grow best between pH 8.5 & 11.5 Example of Alkalophiles: Vibrio cholerae NEUTROPHILES: Growth optima between 5.5 & 8.5 Note: In general pH of media should be at about 7: Add NaOH if too acid Add HCl if too basic During growth pH of medium tends to change: Becomes more acid as metabolic waste products accumulate: E. Buffer: Added to keep pH constant Can absorb large numbers of H+ & OH- ions Must be non-toxic to microorganisms Proteins & PO4 often used as buffers V. Temperature: A. Generalities Important component of organism's environment: Rate of chemical reactions depends on temperature: Increased temperature increases the rate of enzymatic reactions: Rate doubles for every 10o increase in temperature Affects: Rate of growth Metabolism Morphology B362 8 Pigment Production After certain temp growth slows: Enzymes & other proteins denature DNA denatures Microorganisms as a group grow over a wide temperature range: Growth range for a particular species spans about 30o: Stenothermal: Narrow temp range: Neisseria gonorrhoea B. Eurythermal: Wide temp range: Streptococcus faecalis Cardinal Temperatures: Temperatures at which an organism can grow Specific for each group: Each group has characteristic temp dependence Three cardinal temperatures for each group: 1. Maximum temperature: Temperature above which an organism will not grow C. 2. Minimum temperature: Temperature below which the organism will not grow 3. Optimum temperature: Temperature at which a particular organism grows fastest: Psychrophiles: Organisms with temperature optima at 15oC or lower: Maximum around 20oC: Grow well at 0oC: Enzyme systems: Function well at low temp. B362 9 Protein synthesizing mechanisms: Function well at low temp. Cell membranes have: High levels of unsaturated fatty acids: Function well at low temperatures: But begin to leak cellular components at 25 - 30oC Found in: Arctic & Antarctic habitats: Snow fields Glaciers Oceans (average temp = 5oC) Examples: Pseudomonas Flavobacterium Achromobacterium Alcaligenes D. Facultative Psychrophiles or Psychrotrophs: Growth optima between 20 & 30oC & maxima at about 35oC: Can grow at 0oC Major factors in the spoilage of refrigerated foods E. Mesophiles: Organisms with growth optima between 20-45oC: Maximum is 45oC or lower: Includes most organisms: Saprophytes Pathogens F. Most human pathogens have temperature optima of around 37oC (body temperature) Thermophiles: Grow at temperatures above 55oC: Grow minimum is about 45oC Optimum usually between 55 & 60oC Some maxima are above 100oC Heat stable: B362 10 Enzyme systems Function well at high temperatures Protein synthesizing mechanisms: Function well at high temperatures Cell membranes have high levels of saturated fatty acids: Have higher melting points then those of mesophiles: Remain intact at high temperatures Found in: Hot springs Areas of volcanic activity Compost Hay stacks Hot water lines Mostly bacteria but also includes a few algae VI. Oxygen Requirements: A. Obligate Aerobes: Require oxygen Cannot generate energy by fermentation Require O2 for the synthesis of unsaturated fatty acids & sterols B. Facultative Anaerobes: Grow best when O2 is present but are able to grow in its absence. C. Microaerophiles: Damaged by normal 20% O2 in the atmosphere but require between 2-10% O2 D. Obligate Anaerobes: Die when exposed to O2: Poisoned by O2 Examples: Clostridium Bacteroides B362 11 Special techniques are used protect obligate anaerobes from O2 Gaspak Thioglycolate medium E. F. Aerotolerant Anaerobes: Ignore O2: Grow equally well whether it is present or not: But do not use O2 as the terminal electron acceptor during electron transport Oxygen: Toxic in high conc. even for aerobic organisms: Toxic oxygen products produced during respiration and electron transport: H2O2 Superoxide radical (O2-) Hydroxyl radical (OH ) Obligate aerobes & facultative anaerobes have enzymes which destroy toxic oxygen products: Obligate aerobes: Catalase Superoxide dismutase Aerotolerant anaerobes: Catalase No superoxide dismutase Obligate anaerobes No catalase No superoxide dismutase B362