Transcript Growth C
I. Laboratory Culture of Microorganisms • 3.1 Cell Chemistry and Nutrition • 3.2 Culture Media • 3.3 Laboratory Culture Microbial Nutrition Categories Energy source (Electron Donor) ED Macronutrients Micronutrients Trace Elements Terminal Electron Acceptor (TEA) Energy Source Organotroph- Organic Molecules Lithotroph- Inorganic Molecules Phototrophs- light S + TEA------------- P (R/O) + Cell Mass + E Macronutrients C-Source CO@-CO2-Autotroph Organic-Heterotroph N-Source Organic-NH2 Inorganic NH3 or N03 P-Source Organic-RNA Inorganic H2P04 S-Source Organic-Amino Acid(Cysteine) Inorganic-SO4, S-2 3.1 Cell Chemistry and Nutrition • Carbon – Required by ALL cells – Typical bacterial cell is ~50% carbon (by dry weight) – Major element in ALL classes of macromolecules – Heterotrophs use organic carbon – Autotrophs use carbon dioxide (CO2) 3.1 Cell Chemistry and Nutrition • Nitrogen – Typical bacterial cell is ~13% nitrogen (by dry weight) – Key element in proteins, nucleic acids, and many more cell constituents 3.1 Cell Chemistry and Nutrition • Other macronutrients – Phosphorus (P) • Synthesis of nucleic acids and phospholipids – Sulfur (S) • Sulfur-containing amino acids (cysteine and methionine) • Vitamins (e.g., thiamine, biotin, lipoic acid) and coenzyme A – Potassium (K) • Required by enzymes for activity 3.1 Cell Chemistry and Nutrition • Other macronutrients (cont'd) – Magnesium (Mg) • Stabilizes ribosomes, membranes, and nucleic acids • Also required for many enzymes – Calcium (Ca) • Helps stabilize cell walls in microbes • Plays key role in heat stability of endospores – Sodium (Na) • Required by some microbes (e.g., marine microbes) 3.1 • Iron Cell Chemistry and Nutrition – Key component of cytochromes and FeS proteins involved in electron transport • Growth factors – Organic compounds required in small amounts by certain organisms • Examples: vitamins, amino acids, purines, pyrimidines – Vitamins • Most commonly required growth factors Micronutrients Cations- monovalent K+, Na+Di-valent Mg++. Ca++, Fe+2 cofactors/Enzymes Anions- Cl- Trace Elements Co, Zn, Mo, Mn, Ni,Se Vitamins, Yeast Extract Culture Media Difco, BBL Liquid Media- Cell Numbers Solid MediaAgar- Complex polysaccharide Sea Weed 1.5% Melts 80-90 Solidifies 40-42 C Inert Gel 3.2 Media and Laboratory Culture • For successful cultivation of a microbe, it is important to know the nutritional requirements and supply them in proper form and proportions in a culture medium Types of Culture Media 1. Synthetic or Defined Media Chemically Defined 2. Complex Media/General Purpose Chemical Composition not known Biological or plant Extract 3. Selective Media Selects one M.O. Over another Bile salts Selects GM- over GM + 4. Differential Media Distinguishes between a given group of Bacteria GP Media Difco Nutrient Agar Beef Extract 3g Peptone 5g Agar Reagent Water ml 1.5g 1000 3.2 Media and Laboratory Culture • Pure culture: culture containing only a single kind of microbe • Contaminants: unwanted organisms in a culture • Cells can be grown in liquid or solid culture media – Solid media are prepared by addition of a gelling agent (agar or gelatin) – When grown on solid media, cells form isolated Enrichment Media Environmental Tolerances Oxygen pH Osmotic Temperature Pressure Substrate organic inorganic Km (Affinity) Growth Rate Effect of Oxygen Obligate aerobes- Grow only in presence of Oxygen Facultative Anaerobes- With or with out Aerotolerant anaerobes- Ignore Oxygen Obilgate Anaerobes- Die in Oxygen Microaerophilis- 1-10% Oxygen Toxic By Products Superoxide (02- ): 02 + e- 02Hydrogen Peroxide: 02- + e- + H+ H2O2 Hydroxyl radical: H2O2 + e- + H+ -- H2O + 0H. Chlorophyll + hv------ Chlorophyll* Chl* + 02------ ‘02 Growth Binary Fission/Asexual Growth Clones 1 cell=2 Cells Cell Cycle= G G D C=Chromosome Replication G= distribution of cell synthesis D=division I. Bacterial Cell Division • • • • 5.1 5.2 5.3 5.4 Binary Fission Fts Proteins and Cell Division MreB and Cell Morphology Peptidoglycan Biosynthesis 5.2 Fts Proteins and Cell Division • Fts (filamentous temperature-sensitive) proteins (Figure 5.2) – Essential for cell division in all prokaryotes – Interact to form the divisome (cell division apparatus) • FtsZ: forms ring around center of cell; related to tubulin • ZipA: anchor that connects FtsZ ring to cytoplasmic membrane Outer membrane FtsI ZipA Peptidoglycan FtsA ATP FtsZ ring GTP GDP + Pi FtsK ADP + Pi Cytoplasmic membrane Divisome complex FtsZ ring Cytoplasmic membrane Figure 5.2 5.2 Fts Proteins and Cell Division • DNA replicates before the FtsZ ring forms (Figure 5.3) • Location of FtsZ ring is facilitated by Min proteins – MinC, MinD, MinE • FtsK protein mediates separation of chromosomes to daughter cells MinCD Minutes Cell wall Cytoplasmic membrane Nucleoid 0 MinE 20 40 Divisome complex 60 FtsZ ring Septum Nucleoid 80 MinE Figure 5.3 FtsZ Cell wall Cytoplasmic membrane MreB Sites of cell wall synthesis Figure 5.5a II. Population Growth • 5.5 Quantitative Aspects of Microbial Growth • 5.6 The Growth Cycle • 5.7 Continuous Culture 5.5 Quantitative Aspects of Microbial Growth • Most bacteria have shorter generation times than eukaryotic microbes • Generation time is dependent on growth medium and incubation conditions Growth =Population Doubles/Unit Time Generation Time=Time to double Population Complex Series of Events >2000 chemical reactions 1800 different Proteins 1 Complete Genomic DNA Replicated 1 Cytoplasmic membrane Synthesized Short Time= E. coli= 20 minutes 5.6 The Growth Cycle • Batch culture: a closed-system microbial culture of fixed volume • Typical growth curve for population of cells grown in a closed system is characterized by four phases (Figure 5.11): – Lag phase – Exponential phase – Stationary phase BATCH Culture: Growth curve Batch Culture Closed System Nutrients Limited Growth Conditions Optimize pH aeration substrate Incubate Enumerate cells#/Unit Time Exponential Growth Calculations Log Phase #/ T = Rise/Run= Kd Geometric Progression of the # 2 2°21222324----2n 1 2 4 8 16----n # generations Nt=No2n Scientific Notation LOG Transformation Scientific Notation: 1000000000= 1.0 X 109 (Coefficient/Base 10/Exponent) 0.000000001= 1.0 X 10-9 LOG10= Coefficient=1.0 Then LOG10=Exponent LOG10 2= 0.301(X 2.303=0.693) e=ln=Natural Logs=ln 2 =0.693 (/2.303=0.301) Multiplication/Division using LOGs To Multiply Numbers Written in Exponential Notation ADD the Exponents: (3X 104) X (2 X 103)= (3X2) X 104 + 3= 6 X 107 To Divide, Divide the Coefficients and Subtract the Exponents 3 X 104 = 3/2 X 104= 1.5 X 101 2 X 103 Logarithm Power a base number is raised to produce a given number Log10 0.00001= log10(1 X 105 = -5 Log101000000= log10(1 X 106) = 6.0000 Exponential Growth Calculations Log Phase #/ T = Rise/Run= Kd Geometric Progression of the # 2 2°21222324----2n 1 2 4 8 16----n # generations Nt=No2n Calculation of Growth Kinetics Nt=No2n gt= T/n gt= Time for population to double (Generation Time hr-1/division) T= Time n= # generations gt= T/n; n = t/gt; t = n X gt Log transformation of formula Log10 NT= log10 No + (log10 2)(n) Log10 NT/ log10 No = (log10 2)(n) n = Log10 NT- log10 No = # (log10 2) 0.301 Example of Growth rate Calculation Nt=No2n gt = 20 min N0 = 1 Staphyloccus aureus/gram potato salad Nt? After 4 hr? gt= 0.333 hr-1/division 2n = ? 212 Log transformation (12)(log 2) (12)(.301)= 3.612 Antilog 3.612 = 4092 cells Alternate way to calculate Growth Kinetics #/ T = Rise/Run= Kd Nt=Noet = logNt-Log No Page 143 (log 2) (t) = 8.0-7.69 (Nt = 1 X 108; No = 5 X 107) (.301)(t=2hr) = 0.5 div/hr-1 gt? = 1/ = 1/0.5 = 2 hr gt = t/n = 2hr/n=1 = 2 hr (k), gt, t, n One can calculate different organisms growing under different conditions What do we do with this Information? 1. Optimize culture conditions! 2. Test for “+” or “-” effects of an experimental Treatment! Anaerobic jars Anaerobic glove box Methods of Enumeration ? (k), gt, t, n Direct Methods Direct Microscopic Methods D Counting chambers i fixed Film r e Particle Sizer c t Indirect Methods (Viable Cell Counts) Plate Counting Techniques Pour Plate Spread Plate Most Probable Number (MPN) Membrane Filtration (MF) Turbidity (absorbance) Ridges that support coverslip To calculate number per milliliter of sample: 12 cells X 25 large squares X 50 X 103 Coverslip Number/mm2 (3 X 102) Sample added here. Care must be taken not to allow overflow; space between coverslip and slide is 0.02 mm 1 mm). Whole grid has 25 large ( 50 squares, a total area of 1 mm2 and a total volume of 0.02 mm3. Microscopic observation; all cells are counted in large square (16 small squares): 12 cells. (In practice, several large squares are counted and the numbers averaged.) Number/mm3 (1.5 X 104) Number/cm3 (ml) (1.5 × 107) Figure 5.15 5.8 Microscopic Counts • Limitations of microscopic counts – – – – – – – Cannot distinguish between live and dead cells without special stains Small cells can be overlooked Precision is difficult to achieve Phase-contrast microscope required if a stain is not used Cell suspensions of low density (<106 cells/ml) hard to count Motile cells need to immobilized Debris in sample can be mistaken for cells 5.9 Viable Counts • Viable cell counts (plate counts): measurement of living, reproducing population – Two main ways to perform plate counts: • Spread-plate method (Figure 5.16) • Pour-plate method • To obtain the appropriate colony number, the sample to be counted should always be diluted (Figure 5.17) Counting the number of viable cells by serial dilution and plate count Figure 6.5 (Part 1) Concentration of cells by membrane filtration Concentration of cells by membrane filtration Concentration of cells by membrane filtration Relationship between light absorbency and cell mass Relationship between nutrient concentration and total cell mass Continuous culture in a chemostat Steady-state relationship between substrate concentration and output of bacterial mass Response of bacterial growth to oxygen availability