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MSU MMG 301 - Lecture 14

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Lecture 141. Recall the different stages of binary fission a. Cell elongation (width stays the same)b. Septum formationc. Completion of septum; formation of walls; cell separationd. Generation time varies by species and environment2. Recall the different steps and enzymes involved in peptidoglycan synthesisa. Peptidoglycan are synthesized in the cytoplasm and transported through the cytoplasmic membraneb. G-M-pentapeptide precursor units are attached to bactoprenolc. Bactoprenol transports the precursor unit across the cytoplasmic membraned. Autolysins make holes in peptidoglycane. Transglycosylase connects the amino sugars of the precursor to the existing peptidoglycan strandsf. Transpeptidase connects the peptide portion of the precursors to form crosslinks in peptidoglycang. Peptidoglycan is a macromolecule unique to bacteria its synthesis is a target of antibioticsh. Bacitracin is an antibiotic that inhibits the function of bactoprenol. Works by not letting bactoprenol back into the cytoplasmic cell to load another precursori. Penicillin inhibits the transpeptidase enzyme. In the presence of penicillin some cells increase production of autolusin= cell lysisj. Transpeptidase is also known as penicillin-binding proteini. In the presence of penicillin, some growing cells increase production of autolysins, resulting in cell lysis3. Explain how microorganisms can have generation times shorter than the time needed for chromosome duplication a. By using multiple replication forks to increase the chromosome replication rate.b. Cells can contain more than 2 complete chromosomes at any one time4. Based on the number of bacterial cells in one large square of a Petrov- Hausser counting chamber, calculate the bacterial concentration in cells/mla. Whole grid has 25 squares, area 1mm^2, volume .02mm^3, and height is .02mmb. Number of bacteria per ml of sample: 12 bact x 25 squares x 50 x 10^3 = 1.5 * 10^7c. # bacteria x # of squares X 50 x 10^35. Explain how spectrophotometers measure bacterial concentration in cultures a. Light shines through a prism and creates incident light (I0), Light goesthrough a filter after a filter only a single wavelength.b. Shines light on cell suspension, detects light unscattered by cell suspension. It will be lower in intensity than I0. Intensity is because of bacterial density in the culture, light scattered = optical density (proportional to density of cells in suspension) 6. Explain why spectroscopic measurements of turbidity at high cell concentrations underestimate the bacteria population in a sample a. At high cell concentrations light is refractedb. Concentration appears to be higher than it actually is because light is refracted and some bacteria is hidden. If one bacteria is hiding behind another bacteria light wont hit it and wont be counted. 7. Recall how viable cell count by plating works a. Take 1mL of the sample and put into broth.b. Take 1 mL of broth and put into sterile broth, do this to dilute 5 times. (6 tubes)c. Then plate .1mL samples (spread plate method) 5 times. (6 plates)d. CFU per mL = # colonies X dilution factor X correction volume plated (159 X 10^3 X 10)8. Explain why viable cell counts should be calculated based on dilution plates containing between approximately 30 and 300 colonies a. 30-300 colonies is a statistically significant range9. Compare and contrast the methods of viable cell count by plating and by filtration a. Both- only count viable cells, time consumingb. Plating- 30 to 300 colonies, don’t count cells that are viable but non able toculturec. Filtration- can sense very low population density but can handle large sample size, less effort intensive. Selective and differential 10. Explain why the viable cell count by filtration method often focuses on counting coliform bacteria a. Filters are plated on selective medium for gram-negative bacteria and differential for lactose fermentationb. Is good because it measures bacteria with low population density and onlymeasure live cells11. Compare and contrast the different methods to measure bacterial populationsa. Direct microscope counti. Count bacteria in one square then multiple by 25 (the number of squares) and get the numberb. Turbidimetric (light scattering) methods i. Count number of cell not if dead or alive, fast ii. Need higher density of bacteria to workc. Differnece between the 2i. Not all cells are counted because they hide behind other cells and are not counted in turbid metric 12. Determine which method(s) for measurement of bacterial populations is (are) most appropriate in given conditions a. Direct microscope counti. Fastii. Drop if cultureiii. Just counting number of cells don’t need to know if dead or alive b. Turbidimetric (light scattering) methodsi. Fastii. In labs for microbial cultures that do not flocculate iii. High concentration of bacteria c. Viable cell count by plating i. Only viable cellsii. Long and take more material iii. 99% of bacterial are non- culturabled. Viable cell count by filtrationi. For very low bacterial population densities ii. Does not measure dead cells iii. Could have other bacteria in itiv. Not too much


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MSU MMG 301 - Lecture 14

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