1. How many arrows do you see in the following shape?

2. Microbial Growth Microbiology 2314

3. Bacterial Growth Bacterial Growth is Bacterial Reproduction The Numbers of Bacteria are Increasing We see: 1. Observable Increases in Colonies Growing on Solid Media 2. Turbidity, Sediment, Scum or a Change in Color in Broth Cultures

5. Turbidity indicates bacterial numbers are increasing.

6. Binary Fission

8. Generation Time The Time Required for a Cell to Divide

9. Rapid Growth of Bacterial Population

11. Cockroaches Left Unchecked and Allowed to Multiply At Will

12. Generation Time N = (log 10 N f – log 10 N o ) /.301 N Number of generations N f Final Concentration of Cells N o Original concentration of cells.301Conversion Factor to Convert Log 2 to Log 10

13. What is a Logarithm? Logarithm is a function that gives the exponent in the equation b n = x. It is usually written as log b x = n. For example: 3 4 = 81 Therefore log 3 81 = 4

14. Example 1 Example 1 N = (log 10 N f – log 10 N o ) /.301 Measure Culture at 9:00 a.m.10,000 cells / ml Measure Culture at 3:00 p.m.100,000 cells / ml Calculate N N = (log 10 N f – log 10 N o ) /.301 N = (5-4)/0.301 N = 1/0.301 N = 3.33 Generations in 6 Hours We Know:6 Hours = 360 Minutes Therefore:Generation Time = 360 Minutes / 3.33 Generations N = 108 Minutes to Generate

15. Example 2 Example 2 N = (log 10 N f – log 10 N o ) /.301 Measure Culture at 9:00 a.m.10,000 cells / ml Measure Culture at Noon1,000,000 cells / ml Calculate N

16. Example 2 Example 2 N = (log 10 N f – log 10 N o ) /.301 Measure Culture at 9:00 a.m.10,000 cells / ml Measure Culture at Noon1,000,000 cells / ml Calculate N N = (log 10 N f – log 10 N o ) /.301 N = (6-4)/0.301 N = 2/0.301 N = 6.64 Generations in 3 Hours We Know:3 Hours = 180 Minutes Therefore:Generation Time = 180 Minutes / 6.64 Generations N = 27 Minutes to Generate

17. Example 3 Example 3 N = (log 10 N f – log 10 N o ) /.301 Measure Culture at 9:00 a.m.2000 cells / ml Measure Culture at 1:00 p.m.18,000 cells / ml Calculate N

18. Example 3 Example 3 N = (log 10 N f – log 10 N o ) /.301 Measure Culture at 9:00 a.m.2000 cells / ml Measure Culture at 1:00 p.m.18,000 cells / ml Calculate N N = (log 10 N f – log 10 N o ) /.301 N = (4.25-3.30)/0.301 N =.95/0.301 N = 3.16 Generations in 4 Hours We Know:4 Hours = 240 Minutes Therefore:Generation Time = 240 Minutes / 3.16 Generations N = 75.9 Minutes to Generate N = 1.27 Hours to Generate

19. Bacterial Growth Can Be Modeled With Four Different Phases

20. Phases of Microbial Growth

21. Typical bacterial exponential Growth Curve. In a rich culture medium bacteria, grown under aerobic conditions, achieve a final concentration of 2- 5 x 10 9 cells per ml in about 12-18 hours. Although plotted on a different time scale the human growth curve looks the same; the human population at similar points on the growth curve are shown in red.

22. Remember the Four Main Stages Lag Phase Initial Phase / Metabolic Activities Exponential Phase 2 nd Phase / Optimum Growth / Doubling Stationary Phase 3 rd Phase / Exhaustion of Nutrients / Accumulation of Wastes Death Phase Final Phase / Continued Accumulation 90% of Cells Die, then 90% of Remaining Cells Die, etc.

23. Quantification of Bacteria Cell Numbers Total Mass of the Population Population Per Media cells / ml or cells / gram Direct County Methods and Indirect Counting Methods

24. Direct Counting Methods Normally Viable Counts Remember that a Colony Starts Out as 1 Bacteria that Reproduces Colonies May Not All Be The Same Size

25. Types of Direct Measurements 1.Plate Count a. Spread (Streak) Plate b. Pour Plate 2. Direct Observation on Slides a. Petroff-Hausser Chamber Slide 3. Filtration 4. Most Probable Number

26. Direct Count Spread or Streak Plate

31. Advantages to a Streak Plate? Disadvantages?

32. Direct Count Pour Plate

34. Advantages to a Pour Plate? Disadvantages?

35. Petroff-Hausser Chamber Slide

36. What are You Counting on a Petroff-Hausser Slide?

37. Direct Method Filtration

39. Most Probable Number Statistical Procedure used to estimate the number of bacteria that will grow in liquid media. Gives a 95% probability that the bacterial numbers will fall within a certain range.

40. Indirect Measurements Turbidity a. No turbidity = < 10 7 cells/ml b. Slight = 10 7 – 10 8 cells/ml c. High = 10 8 – 10 9 cells/ml d. Very High = > 10 9 cells/ml Metabolic Activity Dry Weight

41. There Are More Accurate Methods to Determine Turbidity Levels

42. Chemical and Physical Requirements for Bacterial Growth

43. Physical Requirements

46. Why is Mexican Food Spicy?

47. Oklahoma is #1

48. Cardinal Temperatures Minimum Temperature Optimum Temperature Maximum Temperature

49. Classification of Bacteria by Temperature Requirements

50. Buffers Are Added to Media to Maintain Proper pH 1.Phosphates 2.Peptones 3.Amino Acids

51. Classification of Bacteria by pH Requirements Acidophiles1.0 to 5.5 Neutrophiles5.5 to 8 Alkalophiles8.5 to 11.5 Extreme Alkalophiles> = 10.0

52. Osmotic Pressure Effects

53. Bacterial response to osmotic effects is the reason we dry food.

54. Hams can be sugar cured or salt cured to preserve them. Are they cooked?

55. What About Jerky?

56. Chemical Requirements for Bacteria Water (80-90%) Carbon (Backbone of Hydrocarbons) Nitrogen (Amino Acids & Vitamins) Sulfur (Amino Acids &Vitamins) Phosphorus (Nucleic Acids, ATP) Minerals (Fe, Cu, Mg, etc. / as Cofactors) Oxygen (aerobes only)

57. What About Oxygen?

60. Special Culture Techniques Gas Pack Jar Is Used for Anaerobic Growth

61. Special Culture Techniques Candle Jar Candle Jar

63. Peritoneal Fluid Mixed Anaerobic Infection

64. Types of Media Media can be classified on three primary levels 1. Physical State 2. Chemical Composition 3. Functional Type

65. Physical States of Media Liquid Media Semisolid Solid (Can be converted into a liquid) Solid (Cannot be converted into a liquid)

66. Liquid Media Water-based solutions Do not solidify at temperatures above freezing / tend to be free flowing Includes broths, milks, and infusions Measure turbidity Example: Nutrient Broth, Methylene Blue Milk, Thioglycollate

67. Semi-Solid Media Exhibits a clot-like consistency at ordinary room temperature Determines motility Used to localize a reaction at a specific site. Example: SIM for hydrogen sulfide production and indole reaction

68. Solid Media Firm surface for discrete colony growth Advantageous for isolating and culturing Two Types 1. Liquefiable (Reversible) 2. Non-liquefiable Examples: Gelatin and Agar (Liquefiable) Rice Grains, Cooked Meat Media, Potato Slices (Non-liquefiable)

71. Chemical Composition of Culture Media 1.Synthetic Media Chemically defined Contain pure organic and inorganic compounds Exact formula (little variation) 2.Complex or Non-synthetic Media Contains at least one ingredient that is not chemically definable (extracts from plants and animals) No exact formula / tend to be general and grow a wide variety of organisms

72. Selective Media Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe. Example: Mannitol Salt Agar encourages the growth of S. aureus.

73. Differential Media Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates. Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies.

75. Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.

77. Enrichment Media Is used to encourage the growth of a particular microorganism in a mixed culture. Ex. Manitol Salt Agar for S. aureus

78. Microbes are Managed and Characterized by Implementing the Five I’s 1.Inoculation 2.Incubation 3.Isolation 4.Inspection 5.Identification

79. Inoculation Sample is placed on sterile medium providing microbes with the appropriate nutrients to sustain growth. Selection of the proper medium and sterility of all tools and media is important. Some microbes may require a live organism or living tissue as the inoculation medium.

80. Incubation An incubator can be used to adjust the proper growth conditions of a sample. Need to adjust for optimum temperature and gas content. Incubation produces a culture – the visible growth of the microbe on or in the media

81. Isolation The end result of inoculation and incubation is isolation. On solid media we may see separate colonies, and in broth growth may be indicated by turbidity. Sub-culturing for further isolation may be required.

82. Inspection Macroscopically observe cultures to note color, texture, size of colonies, etc. Microscopically observe stained slides of the culture to assess cell shape, size, and motility.

85. FYI

86. Identification Utilize biochemical tests to differentiate the microbe from similar species and to determine metabolic activities specific to the microbe. Utilize immunologic tests and genetic analysis. The Major Purpose of the 5 I’s is to Encourage Growth of a Microorganism so that the Lab Worker Can Determine the Type of Microbe, Usually to the Level of Species.