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Growth a

  1. 1. MICROBIAL GROWTH LECTURE
  2. 2. INTRODUCTION <ul><li>Growth = cell reproduction </li></ul><ul><li>Binary Fission = doubling viable cell number in popln </li></ul><ul><li>Growth is exponential - rate of increase dependant on doubling time </li></ul><ul><li>Growth curve (adaptation, reproduction, no net growth and decline) </li></ul><ul><li>Factors influencing bacterial growth rates - temp, salinity, pH, O 2 , other physical & chemical factors </li></ul><ul><li>Bacteria exhibit ranges of tolerance </li></ul>
  3. 3. <ul><li>GROWTH: steady increase in all chemical components of an organism, usually results in increase in size of cell and frequently results in cell division </li></ul><ul><li>Bacterial Cell Cycle: SIMPLE </li></ul><ul><li>Time of division of mother cell into 2 daughter cells then WHEN 1 daughter cell then divides into 2 more daughter cells </li></ul><ul><li>Characterized: continuous macromolecular synthesis </li></ul><ul><li>Cell elongation occurs along with genome replicated </li></ul>
  4. 4. <ul><li>Eukaryotic Cell Cycle: COMPLEX </li></ul><ul><li>Involves separate phases for cell enlargement, replication of the genome, separation of replicated genomes by mitosis and cell division (cytokenesis) </li></ul>
  5. 5. <ul><li>Cells grow by increasing cellular constituents and then dividing into 2 cells ( ASEXUAL PROCESS ) </li></ul><ul><li>Known as BINARY FISSION </li></ul><ul><li>Division is GEOMETRICAL (Population Doubles) </li></ul><ul><li>1 cell divides into 2 new cells </li></ul>
  6. 6. BACTERIAL GROWTH
  7. 8. BINARY FISSION <ul><li>Involves 3 processes : </li></ul><ul><li>Increase in cell size (cell elongation) </li></ul><ul><li>DNA replication </li></ul><ul><li>Cell division </li></ul><ul><li>NOTE: NOT ALL BACTERIA </li></ul><ul><li>Yeast-like budding </li></ul>
  8. 9. <ul><li>GROWTH RATE: time for cell to reproduce </li></ul><ul><li>KINETICS OF BACTERIAL REPRODUCTION </li></ul><ul><li>Binary fission results in doubling viable cell no’s </li></ul><ul><li>GENERATION TIME: Time required for a complete </li></ul><ul><li> fission cycle </li></ul><ul><li>i.e., time for 1 parent cell to form 2 new daughter cells </li></ul><ul><li>1st Generation = 2 cells </li></ul><ul><li>2nd = 4 cells </li></ul><ul><li>3rd = 8 cells </li></ul><ul><li>4th = 16 cells </li></ul><ul><li>5th = 32 cells </li></ul><ul><li>AND SO ON……. </li></ul>
  9. 10. <ul><li>GENERATION TIME </li></ul><ul><li>Formation of each new bacterial cell, its growth and eventual division into 2 cells </li></ul><ul><li>EXAMPLES: (Optimal conditions) </li></ul><ul><li>Bacillus stearothermophilus 11 mins </li></ul><ul><li>E.coli generation time 20 mins </li></ul><ul><li>Staphylococcus aureus 28 mins </li></ul><ul><li>Lactobacillus acidophilus 60-80 mins </li></ul><ul><li>Mycobacterium tuberculosis 360 mins </li></ul><ul><li>Treponema pallidum 1980 mins </li></ul>
  10. 11. <ul><li>QUANTITATIVE ASSEMENT </li></ul><ul><li>(1) Expressed as 2 1 , 2 2 , 2 3 , 2 4 …..2 n </li></ul><ul><li>The power value increases by 1 each generation </li></ul><ul><li>(the number of the generation) </li></ul><ul><li>Termed EXPONENTIAL </li></ul>
  11. 12. <ul><li>Logrithmic graphs are preferred - more accurate cell numbers during early growth </li></ul>
  12. 13. <ul><li>(2) SO starting with 1 cell, the total popln N after n generations </li></ul><ul><li>N = 1 x 2 n </li></ul><ul><li>(3) IF original popln was hundreds/thousands cells express as: </li></ul><ul><li>N = N o x 2 n </li></ul><ul><li>N o represents original popln size at time zero </li></ul>
  13. 14. <ul><li>(4) Expression to determine no’ of generations (n) </li></ul><ul><li>(USE THE LOGARITHM) </li></ul><ul><li>HENCE Log N = Log N o + n Log 2 </li></ul><ul><li>(5) Rearrange to solve for (n) </li></ul><ul><li>n = Log N- Log N o </li></ul><ul><li> Log 2 </li></ul><ul><li>(6) Simplify by substituting Log of 2 = 0.301 </li></ul><ul><li>n = Log N- Log N o </li></ul><ul><li> 0.301 </li></ul><ul><li>(7) THUS , knowing No and N (initial popln and total popln respect) can calculate no’ of generations ( n ) occurred over elapsed time ( t ) </li></ul>
  14. 15. <ul><li>Generation time g = time elapsed </li></ul><ul><li>no’ of generations </li></ul><ul><li> g = t </li></ul><ul><li> n </li></ul><ul><li>(8) OR Using (6) </li></ul><ul><li>g = 0.301 x t </li></ul><ul><li> Log N - Log N o </li></ul><ul><li>(9) Growth rate of culture (K) is no’ of generations per unit time i.e., reciprocal of g </li></ul><ul><li>K = Log N - Log N o </li></ul><ul><li>0.301 x t </li></ul>
  15. 16. <ul><li>EGG SANDWICH EXAMPLE </li></ul><ul><li>Calculate the number of Staphylococcus aureus cells present your egg sandwich which you made at 8am and has been sitting on the back seat of you car on True Blue campus for the past 4 hrs </li></ul><ul><li>Length of Time (t) = 4 hours (240 minutes) </li></ul><ul><li>Inoculum no’ (N o ) = 10 </li></ul><ul><li>Generation time (g) = 20 minutes </li></ul><ul><li>We know: N = N o x 2 n </li></ul><ul><li>No of generations (n) = t = 240 = 12 </li></ul><ul><li> g 20 </li></ul>
  16. 17. <ul><li>Therefore: N = N o x 2 n </li></ul><ul><li> = 10 x 2 12 </li></ul><ul><li> = 10 x 4096 </li></ul><ul><li> = 40960 cells present in sandwich after 4 hrs </li></ul>
  17. 18. PHYSIOLOGICAL EFFECT OF GROWTH <ul><li>Various consequences </li></ul><ul><li>Growth rate increases = increased cell mass </li></ul><ul><li>i.e., at faster growth rates they become larger </li></ul><ul><li>Contain increased cell components: DNA, RNA & protein (Increases at exponential rate) </li></ul><ul><li>RNA/PROTEIN: Ribosome No’s  </li></ul><ul><li>(Biosynthesis proteins, polymerize Aa’s) </li></ul><ul><li>DNA: fast growing cells initiate DNA replication prior to cell division </li></ul>
  18. 19. NORMAL GROWTH <ul><li>IN REALITY </li></ul><ul><li>Popln does not maintain its potential growth rate, </li></ul><ul><li>i.e., does not double endlessly </li></ul>
  19. 20. <ul><li>GROWTH CURVE </li></ul><ul><li>In a closed system: nutrients and space finite </li></ul><ul><li> no removal of waste products </li></ul>
  20. 21. <ul><li>(A) LAG PHASE </li></ul><ul><li>1. Newly inoculated cells, adjust to new environment </li></ul><ul><li>2. Cells not multiplying at maximum rate </li></ul><ul><li>3. Popln is sparse or dilute </li></ul><ul><li>(B) EXPONENTIAL (LOG) PHASE </li></ul><ul><li>Growth occurs at an exponential rate </li></ul><ul><li>Cells reach maximum rate of cell division </li></ul><ul><li>(Continues as long as nutrients and environment is favorable) </li></ul><ul><li>FACTORS: TEMPERATURE </li></ul><ul><li>E. coli @ 30 o C gtime = 1hr, @ 37 o C gtime = 30mins </li></ul>
  21. 22. <ul><li>(C) STATIONARY PHASE </li></ul><ul><li>Popln reaches maximum numbers </li></ul><ul><li>Rate of cell inhibition (death) = Rate of multiplication </li></ul><ul><li>FACTORS: pH changes, accumulation of waste, reduced O 2 </li></ul><ul><li>(D) DEATH PHASE </li></ul><ul><li>Decline in growth rate </li></ul><ul><li>Caused by depletion of nutrients , O 2 </li></ul><ul><li> excretion of toxic waste products </li></ul><ul><li> increased density of cells (limited space) </li></ul><ul><li>FACTORS: Same as STATIONARY PHASE </li></ul>
  22. 23. ENUMERATION OF BACTERIA <ul><li>Assess rate of microbial reproduction </li></ul><ul><li>Determine no’s of microbes present </li></ul><ul><li>Various Methods </li></ul><ul><li>Live bacteria (reproducing on media) </li></ul><ul><li>Dead & Live bacteria </li></ul>
  23. 24. SERIAL DILUTIONS
  24. 25. VIABLE COUNT PROCEDURES <ul><li>Viable Plate Count: living bacteria </li></ul><ul><li>STEP 1: Serial dilutions of suspension of bacteria </li></ul><ul><li>(addition of aliquot of specimen to sterile water) </li></ul><ul><li>If 1ml of sample added to 99ml of SW </li></ul><ul><li>Dilution = 1:100 (10 -2 ) </li></ul><ul><li>(Same 0.1ml sample to 9.9ml SW) </li></ul><ul><li>Greater dilutions: 1ml first dilution (10 -2 ) to 9ml SW = 10 -3 </li></ul><ul><li>1ml 10 -3 dilution to 9ml SW = 10 -4 </li></ul><ul><li>AND SO ON </li></ul>
  25. 26. <ul><li>STEP 2: Plate onto growth media (Spread or Pour) </li></ul><ul><li>SPREAD: drop of suspension (known volume) placed onto center agar plate & spread over surface (sterile glass rod) </li></ul>
  26. 27. <ul><li>POUR: suspension (known volume) added to tubes molten agar (42 o - 45 o C) & poured into petri dish </li></ul><ul><li>Alternatively: known volume added to center of plate and molten agar poured into plate </li></ul><ul><li>Colonies form throughout agar </li></ul>
  27. 28. <ul><li>Reproduction on medium  visible colonies (16-24hr) </li></ul><ul><li>(Assumption 1 colony arises from1bacterial cell) </li></ul><ul><li>STEP 3: Count colonies (CFU’s - Colony Forming Units) </li></ul><ul><li>Concentration of bacteria in original sample determined </li></ul><ul><li>(number bacteria/mL) ( MUST account for dilution factors) </li></ul>
  28. 29. VIABLE COUNT BY SAMPLE DILUTION <ul><li>POUR PLATE METHOD </li></ul>
  29. 30. SURFACE DROP (MILES + MISRA METHOD) <ul><li>Record Results: </li></ul><ul><li>10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 All show confluent growth TNTC (Too Numerous To Count) </li></ul><ul><li>10 -6 , 10 -7 Look for colonies  30- 300 colonies </li></ul><ul><li>10 -8 Has only 10 colonies TFTC (Too Few To Count) </li></ul><ul><li>10 -7 Has countable no’ of colonies used in calculation </li></ul>
  30. 31. <ul><li>CALCULATION </li></ul><ul><li>IF No’ of colonies = 40 </li></ul><ul><li>Dilution counted = 10 -3 </li></ul><ul><li>Volume of drop = 1 </li></ul><ul><li>THEREFORE No’ of bacteria present in original sample </li></ul><ul><li>= 40 x 1 x 10 3 = 4 x 10 4 /cm -3 </li></ul><ul><li>ASSUMING EACH COLONY ARISES FROM </li></ul><ul><li>1 VIABLE CELL </li></ul>
  31. 33. <ul><li>CELL COUNT: Microscopic </li></ul><ul><li>NOT Distinguish DEAD & LIVE CELLS </li></ul><ul><li>Calibrated slide Petroff-Hauser Counter </li></ul><ul><li>(Hemocytometer (RBC’s count) </li></ul><ul><li>Aliquot of culture under cover slip </li></ul><ul><li>Depth known </li></ul><ul><li>Caculate: No’ orgs/unit volume </li></ul>
  32. 34. <ul><li>CELL DENSITY: Spectrophotometer </li></ul><ul><li>Record optical density (OD) or Absorbance (A) units </li></ul><ul><li>As popln increases, turbidity (density) increases </li></ul>
  33. 35. <ul><li>QUESTION </li></ul><ul><li>A research student obtained a set of data at set time intervals (every 30 minutes) from a culture growing in a fermenter. The number of viable cells in each sample was then counted using the pour plate method </li></ul><ul><li>TIME VIABLE COUNT RESULTS </li></ul><ul><li>0 0.446 x 10 8 </li></ul><ul><li>30 0.512 x 10 8 </li></ul><ul><li>60 1.202 x 10 8 </li></ul><ul><li>90 3.36 x 10 8 </li></ul><ul><li>120 1.99 x 10 9 </li></ul><ul><li>180 2.29 x 10 9 </li></ul><ul><li>210 2.51 x 10 9 </li></ul><ul><li>240 2.51 x 10 9 </li></ul><ul><li>270 2.29 x 10 9 </li></ul><ul><li>300 1.99 x 10 9 </li></ul>
  34. 36. <ul><li>1. Plot data in most suitable form </li></ul><ul><li>( THINK FIRST ) </li></ul><ul><li>2. Label each phase of growth on the graph </li></ul><ul><li>Calculate the mean generation time </li></ul><ul><li>Take 2 popln values from each end of the LOG growth phase. Use these to calculate the MGT, using the following equation: </li></ul><ul><li>t x Log 2 </li></ul><ul><li> Log b - Log a </li></ul><ul><li>Where: </li></ul><ul><li>t = time interval (mins) </li></ul><ul><li>Log b = Log cell numbers at end of Log Growth Phase </li></ul><ul><li>Log a = Log cell numbers at beginning of Log Growth Phase </li></ul>
  35. 37. PHYSICAL & CHEMICAL FACTORS AFFECTING GROWTH
  36. 38. <ul><li>ENVIRONMENTAL FACTORS </li></ul><ul><li>Physical factors affecting MICROBIAL GROWTH </li></ul><ul><li>TEMPERATURE </li></ul><ul><li>3 Cardinal Temperatures </li></ul><ul><li>MINIMUM </li></ul><ul><li>Lowest temp, permits microbial growth + metabolism </li></ul><ul><li>MAXIMUM </li></ul><ul><li>Highest temp permits growth + metabolism </li></ul><ul><li>OPTIMUM </li></ul><ul><li>Small range of temp’s, (promotes fastest growth & metabolism rates) </li></ul><ul><li>Extremes of MIN & MAX beyond which growth is inhibited </li></ul>
  37. 39. <ul><li>TEMPERATURE RANGES (OPTIMA GROWTH) OF SOME BACTERIA </li></ul><ul><li>Bacterium Growth Temperature o C </li></ul><ul><li>Min Max Optimum </li></ul><ul><li>1. Pseudomonas fluorescens 2-4 36-38 25-30 </li></ul><ul><li>2. Pseu. aeruginosa 10-15 41-44 c. 37 </li></ul><ul><li>3 . Escherichia coli 15-20 45 37 </li></ul><ul><li>4 . Bacillus polymyxa 5-10 35-45 30-32 </li></ul><ul><li>5 . B. stearothermophilus 30-45 65-75 c. 55 </li></ul><ul><li>6 . Thermus sp. 40 79 70-72 </li></ul>
  38. 40. TEMPERATURE ADAPTATIONS <ul><li>PSYCHROPHILE: Psychrophilic </li></ul><ul><li>Opt temp below 15 o C </li></ul><ul><li>Capable of growth at 0 o C </li></ul><ul><li>Cannot grow above 20 o C </li></ul><ul><li>Found: SNOW FIELDS, POLAR ICE, DEEP OCEAN </li></ul><ul><li>EXAMPLES: Pseudomonas , Flavobacterium , Alcaligenes & Achromobacter sp. </li></ul><ul><li>FACULTATIVE PSYCHROPHILE: </li></ul><ul><li>Grow slowly in cold conditions </li></ul><ul><li>BUT have opt temp above 20 o C </li></ul><ul><li>EXAMPLES: Staphylococcus aureus , L. monocytogenes </li></ul><ul><li>CONCERN: Contaminants of food/dairy products </li></ul>
  39. 41. <ul><li>MESOPHILE: </li></ul><ul><li>Opt temp 20-40 o C </li></ul><ul><li>Capable of growth 10-50 o C </li></ul><ul><li>Group containing HUMAN PATHOGENS (30-37 o C) </li></ul><ul><li>EXAMPLE: E. coli </li></ul><ul><li>THERMOPHILE: </li></ul><ul><li>Opt temp >45 o C </li></ul><ul><li>Capable of growth 45-85 o C </li></ul><ul><li>Incapable of growth at usual body temp </li></ul><ul><li>(NOT INVOLVED in HUMAN INFECTIONS) </li></ul><ul><li>Found: VOLCANO, DIRECT EXPOSURE TO SUN </li></ul><ul><li>EXAMPLE: Bacillus stearothermophilus </li></ul><ul><li>( EXTREME THERMOPHILES: opt temp >80 o C) </li></ul>
  40. 42. TEMPERATURE RANGES FOR MICROBIAL GROWTH
  41. 43. GAS REQUIREMENTS <ul><li>Oxygen plays important role in MICROBIAL GROWTH </li></ul><ul><li>- terminal electron acceptor in respiration </li></ul><ul><li>Oxygen - limited solubility in water </li></ul><ul><li>Therefore can be limiting factor </li></ul><ul><li>Enzymes are required </li></ul><ul><li>Reduce Oxygen to water and toxic products </li></ul><ul><li>(hydrogen peroxide + superoxide) </li></ul>
  42. 44. <ul><li>Microbes convert toxic products to mole c Oxygen by: </li></ul><ul><li>1. CATALASE </li></ul><ul><li>H 2 O 2 H 2 O + O 2 </li></ul><ul><li>2. PEROXIDASE </li></ul><ul><li>H 2 O 2 + NADH + H + 2H 2 O + NAD + </li></ul><ul><li>3. SUPEROXIDE DISMUTASE </li></ul><ul><li> 2O 2 - + 2 H + H 2 O 2 + O 2 </li></ul><ul><li>Peroxide is metabolized by Catalase (as above) </li></ul>
  43. 45. <ul><li>BASED ON OXYGEN REQUIREMENTS </li></ul><ul><li>microbes divided into 4 groups </li></ul><ul><li>OBLIGATE AEROBES </li></ul><ul><li>Totally dependant on O 2 for growth </li></ul><ul><li>Requirement of 1 atmosphere (20%) </li></ul><ul><li>Produce H 2 O 2 and O 2 - but possess catalase and superoxide dismutase - can tolerate high [O 2 ] </li></ul><ul><li>MICROAEROPHILES </li></ul><ul><li>Grow in presence of O 2 BUT tolerate only 4% </li></ul><ul><li>Possess enzymes BUT if toxic products  , enzyme systems overload INHIBITING GROWTH </li></ul>
  44. 46. <ul><li>FACULTATIVE ANAEROBES </li></ul><ul><li>Grow in presence or absence of O 2 </li></ul><ul><li>Presence use Aerobic respiration </li></ul><ul><li>Absence fermentation for energy prodn </li></ul><ul><li>Grow best under AEROBIC CONDITIONS </li></ul><ul><li>e.g. Enterobacteriacea </li></ul>
  45. 47. <ul><li>ANAEROBES </li></ul><ul><li>Grow ONLY in ABSENCE of O 2 </li></ul><ul><li>Effect of presence variable - LETHAL or TOLERANCE </li></ul><ul><li>LETHAL - org’s lack enzymes to remove toxic products </li></ul><ul><li>TOLERANT - org’s lack enzymes to reduce O 2 to water or toxic products </li></ul><ul><li>e.g. LOWER GI ORG’S Clostridium sp , Bacteroides sp </li></ul><ul><li>OBLIGATE ANAEROBES: </li></ul><ul><li>Fermentative metabolism </li></ul><ul><li>EXAMPLE: Desulfovibrio , Archaebacteria & Protozoa </li></ul><ul><li>STRICT ANAEROBES: </li></ul><ul><li>Sensitive to O 2 </li></ul><ul><li>Brief exposure will KILL </li></ul>
  46. 48. Bacterial Enzymes that Protect the Cell Against Toxic Forms of Oxygen
  47. 49. OXYGEN & BACTERIAL GROWTH 1 2 3 4 5 1: Obligate Aerobe 2: Facultative Anaerobe 3: Aero-tolerant Anaerobe 4: Strict Anaerobe 5: Microaerophilic
  48. 50. EFFECTS OF pH <ul><li>pH - degree of acidity or alkalinity of a soln related to the [H + ] </li></ul><ul><li>pH = -log H+ (1/log H+) </li></ul><ul><li>Neutral Solutions (pH 7) </li></ul><ul><li>Alkaline (Basic) Soln (pH >7) </li></ul><ul><li>Acidic Soln (pH <7) </li></ul><ul><li>GROWTH RATES INFLUENCED BY pH VALUES </li></ul><ul><li>(NATURE OF PROTEIN) </li></ul>
  49. 51. <ul><li>INFLUENCE ON GROWTH & SURVIVAL </li></ul><ul><li>Most live between pH 6-8 ( Neutrophiles 6.5-7.5) </li></ul><ul><li>Fungi between pH 5-6 (acidic)generally wider range (5-9) </li></ul><ul><li>pH EXTREMES: </li></ul><ul><li>few microbes in stomach pH2 </li></ul><ul><li>( Lactobacillus acidophilus , Helicobacter pylori ) </li></ul><ul><li>ACIDOPHILE: growth at low pH Thiobacillus sp (pH2) </li></ul><ul><li>OBLIGATE ACIDOPHILE - Euglena mutabilis </li></ul><ul><li> (acid pools 0-1) </li></ul><ul><li>ALKALINOPHILES - high levels of minerals (salt) pH 9-11 </li></ul>
  50. 52. WATER ACTIVITY (Aw) <ul><li>ALL BACTERIA require water (growth & reproduction) </li></ul><ul><li>Essential solvent, biochemical reactions </li></ul><ul><li>Water activity = index amount of water free to react </li></ul><ul><li>Equivalent to atmospheric measure (Relative Humidity) </li></ul><ul><li>Absorption & Solution factors reduce availability (  Aw) </li></ul><ul><li>Pure distilled water (Aw =1) </li></ul><ul><li>E.g., Saturated soln NaCl (Aw = 0.8) </li></ul><ul><li>Seawater [NaCl]  3% (Aw = 0.98) </li></ul><ul><li>RH = 100 Aw Therefore, 90% RH = 0.90 Aw </li></ul>
  51. 53. <ul><li>Most bacteria Aw >0.9 (active metabolism) </li></ul><ul><li>Most microbes - grow opt Aw = 1.0 </li></ul><ul><li> Aw = slow growth rate </li></ul><ul><li>Below Aw 0.9 Bacteria unable to grow </li></ul><ul><li>EXCEPTIONS: </li></ul><ul><li>XEROTOLERANT: lower Aw </li></ul><ul><li>Fungi able to grow Aw 0.60 </li></ul><ul><li>Yeasts (conc sugar soln’s Aw = 0.60) </li></ul><ul><li>Salt-tolerant Bacteria - Halophiles (High [Solute], low Aw) </li></ul>
  52. 54. Effect of Aw on growth of Staphylococcus aureus in medium containing hydrolysate
  53. 55. The Interrelationships of Aw of various foods & susceptibility to microbial spoilage
  54. 56. OSMOTIC PRESSURE <ul><li>Results from: water diffusing across cell membrane in response to [solute] </li></ul><ul><li>Association with [salt] = SALINITY </li></ul><ul><li>OSMOTOLERANT: withstand high osmotic pressure </li></ul><ul><li>high [solute] </li></ul><ul><li>OSMOPHILES: require high [solute] for growth </li></ul><ul><li>E.g., Xeromyces (opt Aw =0.9) </li></ul>
  55. 57. <ul><li>SALINITY: </li></ul><ul><li>Most microbes - HYPOTONIC or ISOTONIC conditions </li></ul><ul><li>HALOPHILES (exceptions) - Require High [NaCl] </li></ul><ul><li>Moderate Halophiles: Marine bacteria 3% [salt] </li></ul><ul><li>Require 1.5% NaCl (maintain membrane integrity) </li></ul><ul><li>Extreme Halophiles: saturated brine soln’s </li></ul><ul><li>OBLIGATE HALOPHILES Halobacterium , Halococcus sp </li></ul><ul><li>Both 25% NaCl </li></ul><ul><li>Drying, Salting, Jamming (achieve low Aw) </li></ul><ul><li>EFFECTIVE methods of preservation </li></ul>
  56. 58. <ul><li>HYDROSTATIC PRESSURE </li></ul><ul><li>Normal pressure: 1 atmosphere (atm) </li></ul><ul><li>Ocean Depth (1000m +) HP = 600-1100 atm (2-3 o C) </li></ul><ul><li>Bacteria survive & adapt BAROPHILES </li></ul><ul><li>BAROTOLERANT:  pressure not adversely affected </li></ul>
  57. 59. NUTRIENT CONCENTRATION <ul><li>Must utilize various nutrients </li></ul><ul><li>Required for: energy production & macromolecular biosynthesis </li></ul><ul><li>Hence: </li></ul><ul><li>growth limited by concentration of required nutrient </li></ul><ul><li>Final net growth (cell yield) increases with initial amount of limiting nutrient </li></ul>
  58. 60. a) Effect of changes in limiting concentration on total microbial yield b) Effect on growth rate Total growth (cells or mg/ml) Nutrient concentration Nutrient concentration Growth rate (hr -1 )
  59. 61. <ul><li>Hyperbolic Curve: rate of nutrient uptake by microbial transport systems </li></ul><ul><li>High Nutrient Level: transport system saturated </li></ul><ul><li>NO FURTHER GROWTH (with increase in nutrients) </li></ul><ul><li>GROWTH YIELD: microbial mass produced from a nutrient </li></ul><ul><li>Y = mass of microorganisms formed </li></ul><ul><li> mass of substrate consumed </li></ul><ul><li>Expressed: grams of cells formed/ gram substrate used </li></ul><ul><li>(MOLAR GROWTH: grams cells/mole nutrient consumed) </li></ul>
  60. 62. MICROBIAL ASSOCIATIONS <ul><li>Influence on growth </li></ul><ul><li>SYMBIOTIC: org’s iving in close nutritional relationship </li></ul><ul><li>3 Types: MUTUALISM </li></ul><ul><li>COMMENSALISM </li></ul><ul><li>PARASITISM </li></ul><ul><li>MUTUALISM: orgs live in an obligatory BUT mutually beneficial relationship </li></ul><ul><li>EXAMPLE: </li></ul><ul><li>Lactobacillus arabinosus requires phenylalanine </li></ul><ul><li>Streptococcus faecalis requires folic acid </li></ul>
  61. 63. <ul><li>EXAMPLE: MUTALISM (MICROBES & ANIMALS) </li></ul><ul><li>Mammals unable digest cellulose </li></ul><ul><li>Microbes & Ruminants: ability to digest cellulose </li></ul><ul><li>Cellulose degradation in RUMEN </li></ul><ul><li>10 10 - 10 11 bacteria, 10 5 - 10 6 protozoa/gram contents </li></ul><ul><li>EXAMPLE: MUTALISM (MICROBES & PLANTS) </li></ul><ul><li>Rhizobium sp. & Legumes (clover, soybeans, alfalfa, peas) </li></ul><ul><li>Bacterium: Nitrogen fixation </li></ul><ul><li>Plant provides environment required </li></ul>
  62. 64. <ul><li>COMMENSALISM: commensal benefits, other org’s neither harmed nor benefit </li></ul><ul><li>Unidirectional relationship between populations </li></ul><ul><li>1. 1 microbe may destroy/neutralize an antimicrobial factor (enables 2nd org to grow) </li></ul><ul><li>2. One popln makes metabolic by-product serves as source (carbon, energy or growth factor) for another microbe </li></ul>
  63. 65. <ul><li>EXAMPLE </li></ul><ul><li>GI TRACT millions of bacteria live on the waste products within Large Intestine Bacteroides , Fusobacterium etc. </li></ul><ul><li>ORAL CAVITY anaerobes survive due to removal of O 2 by facultative anaerobes present </li></ul><ul><li>SKIN Staph. epidermidis reside on outer dead layer of skin in conjunction with Matassezia furfur </li></ul>
  64. 66. <ul><li>PARASITISM: host org provides parasitic microbe with nutrients and habitat. Multiplication of parasite HARMS HOST </li></ul><ul><li>EXAMPLE </li></ul><ul><li>Rickettsiae - obligate parasitic bacteria of humans. </li></ul><ul><li>Rocky Mountain Spotted Fever - children, young </li></ul><ul><li>adults. Org carried in tick, lice etc DOG TICK </li></ul>
  65. 67. <ul><li>NON SYMBIOTIC : org’s are free-living, relationship not required for survival </li></ul><ul><li>2 Types: SYNERGISM (PROTO-COOPERATION) </li></ul><ul><li>ANTAGONISM </li></ul><ul><li>SYNERGISM: relationship where org’s cooperate and share nutrients that are beneficial BUT not necessary for survival </li></ul>
  66. 68. <ul><li>SYNTROPHISM: form of synergism </li></ul><ul><li>Result of cross-feeding </li></ul><ul><li>2 popln’s supply each others nutritional needs </li></ul><ul><li>(Completion of metabolic pathway) </li></ul><ul><li>Compound A B C D </li></ul><ul><li>species 1 2 3 </li></ul>
  67. 69. <ul><li>ANTAGONISM: 1 popln has harmful effect on growth of another popln </li></ul><ul><li>Competition between free-living species where some members are inhibited or destroyed by others </li></ul><ul><li>EXAMPLE </li></ul><ul><li>Lactobacillus sp in vagina produces an acidic environment which prevents against infection by other microbes </li></ul>
  68. 70. <ul><li>QUESTION </li></ul><ul><li>A research student obtained a set of data at set time intervals (every 30 minutes) from a culture growing in a fermenter. The number of viable cells in each sample was then counted using the pour plate method </li></ul><ul><li>TIME VIABLE COUNT RESULTS </li></ul><ul><li>0 0.446 x 10 8 </li></ul><ul><li>30 0.512 x 10 8 </li></ul><ul><li>60 1.202 x 10 8 </li></ul><ul><li>90 3.36 x 10 8 </li></ul><ul><li>120 1.99 x 10 9 </li></ul><ul><li>180 2.29 x 10 9 </li></ul><ul><li>210 2.51 x 10 9 </li></ul><ul><li>240 2.51 x 10 9 </li></ul><ul><li>270 2.29 x 10 9 </li></ul><ul><li>300 1.99 x 10 9 </li></ul>
  69. 72. <ul><li>t x Log 2 </li></ul><ul><li> Log b - Log a </li></ul><ul><li>t = 120 - 60 = 60 mins </li></ul><ul><li>Log b = 9.30 </li></ul><ul><li>Log a = 8.08 </li></ul><ul><li>Therefore 60 x 0.301 </li></ul><ul><li> 9.30 - 8.08 </li></ul><ul><li>= 18.06 </li></ul><ul><li> 1.22 </li></ul><ul><li>MGT = 14.80 minutes </li></ul>

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