2. Microbial Growth  Increase in number of cells, not cell size  Colonies- groups of cells large enough to be seen without a microscope

3. The Requirements for Growth  Physical requirements  Temperature  pH  Osmotic pressure  Chemical requirements  Carbon  Nitrogen, sulfur, and phosphorous  Trace elements  Oxygen  Organic growth factors

4. Physical Requirements  Temperature  Minimum growth temperature- lowest temp. an organism will grow  Optimum growth temperature- organisms grow BEST  Maximum growth temperature- highest temp. an organism will grow

5. Physical Requirements  Temperature  Psychrophiles- cold loving microbes; 0- 25 C; deep oceans and ice  Mesophiles- moderate temperature loving microbes; 25-40 C; PATHOGENS  Thermophiles- heat loving microbes; 50- 60 C; hot springs and volcanoes  F to °C- Deduct 32, then multiply by 5, then divide by 9  °C to °F- Multiply by 9, then divide by 5, then add 32

6. Figure 6.1 Typical growth rates of different types of microorganisms in response to temperature. Psychrophiles Psychrotrophs Mesophiles Thermophiles Hyperthermophiles

7. 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.

8. Psychrotrophs  Grow between 0°C and 20–30°C (refrigerator)  Cause food spoilage  Mold, slime, change in colors and taste

9. pH  Most bacteria grow between pH 6.5 and 7.5  Molds and yeasts grow between pH 5 and 6  Acidophiles grow in acidic environments

10. Osmotic Pressure  Hypertonic environments, or an increase in salt or sugar, cause plasmolysis (shrinking)  Extreme or obligate halophiles require high osmotic pressure  Facultative halophiles tolerate high osmotic pressure

11. Figure 6.4 Plasmolysis. Plasma membrane Cell wall Cytoplasm H2OH2O NaCl 10% Cytoplasm Plasma membrane Cell in isotonic solution.Plasmolyzed cell in hypertonic solution. NaCl 0.85%

12. Chemical Requirements  CARBON  Structural organic molecules, energy source  Chemoheterotrophs- carbon from organic carbon sources  Autotrophs- carbon from CO 2

13. Chemical Requirements  NITROGEN  Makes amino acids and proteins  Most bacteria decompose proteins to get Nitrogen  Some bacteria use ammonium or nitrate  A few bacteria use nitrogen gas from the atmosphere in nitrogen fixation

14. Chemical Requirements  SULFUR  In amino acids, thiamine, and biotin  Most bacteria decompose proteins  Sulfate or Hydrogen Sulfide- source of sulfur  PHOSPHORUS  In DNA, RNA, ATP, and cell membranes  Phosphate-source of phosphorus

15. Chemical Requirements  TRACE ELEMENTS  Inorganic elements required in small amounts  Usually as enzyme cofactors

16. Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

17. Organic Growth Factors  Organic compounds obtained from the environment  Organism is unable to synthesize them on their own  Vitamins, amino acids, purines, and pyrimidines

18. Biofilms  Microbial communities  Form slime  Usually attached to surfaces  Quorum Sensing- bacteria are attracted to each other through chemical  Coordinate their activities  Benefit each other  Share nutrients  Sheltered from harmful factors- MORE RESISTANT

19. Figure 6.5 Biofilms. Clumps of bacteria adhering to surface SurfaceWater currents Migrating clump of bacteria

20. Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm. © 2013 Pearson Education, Inc.

21. Biofilms in Health  CDC- 70% of infections due to biofilms  Nosocomial infections  Indwelling catheters  Heart valves

22. Culture Media  Culture medium : media with nutrients prepared for microbial growth  Sterile: no living microbes; media must FIRST be this  Inoculum : introduction of microbes into medium  Culture : microbes growing in/on culture medium

23. Agar  Polysaccharide from algae  Used as solidifying agent for culture media in Petri plates, slants, and deeps  Generally not metabolized by microbes  Liquefies at 100°C  Solidifies at ~40°C

24. Culture Media  Must contain energy, chemicals and growth factors  Chemically defined media : exact chemical composition is known  Complex media : extracts and digests of yeasts, meat, or plants  Nutrient broth  Nutrient agar

25. Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

26. Anaerobic Culture Methods  Reducing media  Contain chemicals that combine with O 2  Heated before use to get rid of any O 2

27. Figure 6.7 An anaerobic chamber. Arm ports Air lock

28. Capnophiles  Microbes that require high CO 2 conditions  CO 2 packet  Candle jar

29. Selective Media  Suppress or inhibit unwanted microbes from growing while encouraging desired microbes  Ex: MacConkey Agar

30. Differential Media  Make it easy to distinguish colonies of different microbes by creating a VISUAL change  A type of SELECTIVE MEDIA  Ex: Mannitol Salt Agar, Blood Agar Plate

31. Figure 6.10 Differential medium. Uninoculated Staphylococcus epidermis Staphylococcus aureus

32. Figure 6.9 Blood agar, a differential medium containing red blood cells. Bacterial colonies Hemolysis

33. Enrichment Culture  Encourages growth of desired microbe (SELECTIVE)  Increase growth as well  Used for fecal or soil samples with many microorganisms

34. Obtaining Pure Cultures  Pure Culture- contains only one species  Colony (Colony Forming Unit CFU)- population of cells arising from a single cell or spore or from a group of attached cells  Streak plate method - used to isolate pure cultures  Sterile inoculating loop is used to streak plate in 4 quadrants

35. Figure 6.11 The streak plate method for isolating pure bacterial cultures. Colonies 1 2 3

36. Preserving Bacterial Cultures  Deep-freezing : –50° to –95°C  Lyophilization (freeze-drying) : frozen (–54° to –72°C) and dehydrated in a vacuum  Container is sealed by heat

37. Reproduction in Prokaryotes  Binary fission  Budding  Conidiospores ANIMATION Bacterial Growth: Overview

38. Figure 6.12b Binary fission in bacteria. (b) A thin section of a cell of Bacillus licheniformis starting to divide Cell wall DNA (nucleoid) Partially formed cross-wall © 2013 Pearson Education, Inc.

39. Figure 6.13a Cell division.

40. Generation Time  The time it take for a bacteria to DOUBLE it’s population  Varies from organism to organism

41. Phases of Growth  Lag Phase- cells are getting use to their new environment  Not much cellular division  Metabolism is occuring  1hr- several days  Log Phase- period of growth and celluar division  Most active

42. Phases of Growth  Stationary Phase- number of new cells being made EQUALS the number of cells dying  Conditions begin to deteriorate  Death Phase- number of deaths are greater than number of new cells being made  All or most of the cells die as conditions continue to deteriorate

43. Lag Phase Intense activity preparing for population growth, but no increase in population. Log Phase Logarithmic, or exponential, increase in population. Stationary Phase Period of equilibrium; microbial deaths balance production of new cells. Death Phase 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). Figure 6.15 Understanding the Bacterial Growth Curve. Staphylococcus spp.

44. ANIMATION Bacterial Growth Curve Phases of Growth

45. Plate Counts  Most frequently used  Sample is diluted several time in a process called SERIAL DILUTION before inoculated onto a plate.  Inoculation -  Pour Plate  Spread Plate  After incubation, count colonies on plates that have 25–250 colonies (CFUs)

46. Serial Dilutions  FIRST dilution-10,000 bacteria/1ml  SECOND dilution- add 1ml from 1 st dilution to 9ml(sterile) then you would have 10,000 bacteria/10ml which would be 1000/1ml.  THIRD dilution- add 1ml from 2 nd dilution to 9ml(sterile) then you would have 1000ml/10ml which would be 100/1ml.  This would be a COUNTABLE plate.

47. Figure 6.16 Serial dilutions and plate counts. Original inoculum- 10,000/1ml 1 ml 9 m broth in each tube 1000/1ml100/1ml10/1ml 1 ml Plating 1000 colonies100 colonies10 colonies

48. Plate Counts Pour Plate 1. 1ml or 0.1ml of sample 2. Add melted agar 3. Mix 4. Media solidifies with colonies on and in media  Only good for a count not identification. Colonies can be damaged. Spread Plate 1. Inoculate 1ml of sample onto solid agar. 2. Spread evenly. 3. Colonies only grow on the surface.  Better for identification of colonies.

49. Figure 6.17 Methods of preparing plates for plate counts. The pour plate method The spread plate method Inoculate empty plate. Add melted nutrient agar. Swirl to mix. Colonies grow on and in solidified medium. 1.0 or 0.1 ml0.1 ml Bacterial dilution Inoculate plate containing solid medium. Spread inoculum over surface evenly. Colonies grow only on surface of medium.

50. Filtration  Used in samples with small amounts of organisms. 1. Liquid is passed through a membrane with small pores. 2. Filter is transferred to a petri dish with nutrient broth and colonies grow on the filter.

51. Figure 6.18 Counting bacteria by filtration.

52. Direct Microscopic Count  A measured volume of bacterial suspension is placed within a defined area on a slide.  Dye is added to view bacteria.  Look within a large square (1ml). Count colonies.  Each colony= 1,250,000 bacteria  14 colonies x 1,250,000 = 17,500,000 cells/ml

53. 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 Cover glass Slide 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). Microscopic count: All cells in several large squares are counted, and the numbers are averaged. The large square shown here has 14 bacterial cells. 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. Location of squares

54. Turbidity  Cloudiness in a liquid which signifies growth.  Spectrophotometer- light is transmitted through the liquid.  Percentage is measured.  100% light transmitted- no turbidity

55. Figure 6.21 Turbidity estimation of bacterial numbers. Light source Light Blank Spectrophotometer Light-sensitive detector Scattered light that does not reach detector Bacterial suspension

56. Measuring Microbial Growth Direct Methods  Plate counts  Filtration  MPN  Direct microscopic count Indirect Methods  Turbidity  Metabolic activity  Dry weight