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Microbial Growth (Ch 6) Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature. Thermophiles Hyperthermophiles Mesophiles Psychrotrophs Psychrophiles Applications of Microbiology 6.1 A white microbial biofilm is visible on this deep-sea hydrothermal vent. Water is being emitted through the ocean floor at temperatures above 100°C. Figure 6.2 Food preservation temperatures. Temperatures in this range destroy most microbes, although lower temperatures take more time. Very slow bacterial growth. Danger zone Rapid growth of bacteria; some may produce toxins. Many bacteria survive; some may grow. Refrigerator temperatures; may allow slow growth of spoilage bacteria, very few pathogens. No significant growth below freezing. Figure 6.3 The effect of the amount of food on its cooling rate in a refrigerator and its chance of spoilage. 15 cm (6′′) deep 5 cm (2′′) deep Refrigerator air Approximate temperature range at which Bacillus cereus multiplies in rice Figure 6.4 Plasmolysis. Plasma membrane Cell wall Plasma membrane H2O Cytoplasm NaCl 0.85% Cell in isotonic solution. Cytoplasm NaCl 10% Plasmolyzed cell in hypertonic solution. Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria www.facebook.com/RespectBacteria Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria Simmons’ Citrate Agar Tryptic Soy Broth Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates. Lid with O-ring gasket Clamp with clamp screw Envelope containing sodium bicarbonate and sodium borohydride Anaerobic indicator (methylene blue) Petri plates Palladium catalyst pellets Figure 6.9 Blood agar, a differential medium containing red blood cells. Bacterial colonies Hemolysis Figure 6.10 Differential medium. Uninoculated Staphylococcus epidermis Staphylococcus aureus Biosafety levels http://www.cdc.gov/training/quicklearns/biosafety/ Figure 6.7 An anaerobic chamber. Air lock Arm ports Biological Safety Cabinet. Arm ports labconco.com Biological Safety Cabinet. Arm ports genomica.uaslp.mx Biological Safety Cabinet. Arm ports usu.edu Biosafety level 3 (BSL-3) http://upload.wikimedia.org Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory. Figure 6.11 The streak plate method for isolating pure bacterial cultures. 1 2 3 Colonies Figure 6.12a Binary fission in bacteria. Cell elongates and DNA is replicated. Cell wall and plasma membrane begin to constrict. Cell wall Plasma membrane DNA (nucleoid) Cross-wall forms, completely separating the two DNA copies. Cells separate. (a) A diagram of the sequence of cell division Figure 6.12b Binary fission in bacteria. Partially formed cross-wall DNA (nucleoid) (b) A thin section of a cell of Bacillus licheniformis starting to divide © 2013 Pearson Education, Inc. Cell wall Figure 6.13a Cell division. Figure 6.13b Cell division. Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line). Log10 = 4.52 (524,288) Log10 = 3.01 Log10 = 1.51 (262,144) (131,072) (32) (65,536) (32,768) (1024) Generations Number of cells Log10 of number of cells (1,048,576) Log10 = 6.02 Figure 6.15 Understanding the Bacterial Growth Curve. Lag Phase Log Phase Stationary Phase Death Phase Intense activity preparing for population growth, but no increase in population. Logarithmic, or exponential, increase in population. Period of equilibrium; microbial deaths balance production of new cells. Population Is decreasing at a logarithmic rate. The logarithmic growth in the log phase is due to reproduction by binary fission (bacteria) or mitosis (yeast). Staphylococcus spp. Figure 6.16 Serial dilutions and plate counts. 1 ml Original inoculum 1 ml 1 ml 1 ml 1 ml 9 m broth in each tube Dilutions 1:10 1 ml 1:100 1 ml 1:1000 1:10,000 1 ml 1 ml 1:100,000 1 ml Plating 1:10 (10-1) 1:100 (10-2) 1:1000 (10-3) 1:10,000 (10-4) 1:100,000 (10-5) Calculation: Number of colonies on plate × reciprocal of dilution of sample = number of bacteria/ml (For example, if 54 colonies are on a plate of 1:1000 dilution, then the count is 54 × 1000 = 54,000 bacteria/ml in sample.) Figure 6.17 Methods of preparing plates for plate counts. The pour plate method The spread plate method 1.0 or 0.1 ml Inoculate empty plate. 0.1 ml Inoculate plate containing solid medium. Bacterial dilution Add melted nutrient agar. Spread inoculum over surface evenly. Swirl to mix. Colonies grow on and in solidified medium. Colonies grow only on surface of medium. Figure 6.18 Counting bacteria by filtration. Figure 6.19a The most probable number (MPN) method. Volume of Inoculum for Each Set of Five Tubes (a) Most probable number (MPN) dilution series. Figure 6.19b The most probable number (MPN) method. (b) MPN table. Figure 6.20 Direct microscopic count of bacteria with a Petroff-Hausser cell counter. Grid with 25 large squares Cover glass Slide Bacterial suspension is added here and fills the shallow volume over the squares by capillary action. Bacterial suspension Microscopic count: All cells in several large squares are counted, and the numbers are averaged. The large square shown here has 14 bacterial cells. Cover glass Slide Location of squares Cross section of a cell counter. The depth under the cover glass and the area of the squares are known, so the volume of the bacterial suspension over the squares can be calculated (depth × area). The volume of fluid over the large square is 1/1,250,000 of a milliliter. If it contains 14 cells, as shown here, then there are 14 × 1,250,000 = 17,500,000 cells in a milliliter. Figure 6.21 Turbidity estimation of bacterial numbers. Light source Spectrophotometer Light Scattered light that does not reach detector Blank Bacterial suspension Light-sensitive detector Figure 6.5 Biofilms. Clumps of bacteria adhering to surface Surface Water currents Migrating clump of bacteria Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm. © 2013 Pearson Education, Inc.