1. Lectures prepared by Christine L. Case
Chapter 6
Microbial Growth
Lectures prepared by Christine L. Case
© 2013 Pearson Education, Inc. 1

3. Microbial Growth Increase in number of cells, not cell size
Populations
Colonies

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

5. Physical Requirements
Temperature
Minimum growth temperature
Optimum growth temperature
Maximum growth temperature

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

7. Applications of Microbiology 6
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
Cause food spoilage

9. Figure 6.2 Food preservation temperatures.
Temperatures in this range destroy most microbes,
although lower temperatures take more time.
Very slow bacterial growth.
Rapid growth of bacteria; some may produce toxins.
Danger zone
Many bacteria survive; some may grow.
Refrigerator temperatures; may allow slow growth
of spoilage bacteria, very few pathogens.
No significant growth below freezing.

10. 15 cm (6′′) deep 5 cm (2′′) deep Refrigerator air
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
Approximate temperature
range at which Bacillus cereus multiplies in rice
Refrigerator air

11. 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

12. Osmotic Pressure
Hypertonic environments, or an increase in salt or sugar, cause plasmolysis
Extreme or obligate halophiles require high osmotic pressure
Facultative halophiles tolerate high osmotic pressure

13. Cell in isotonic solution. Plasmolyzed cell in hypertonic solution.
Figure 6.4 Plasmolysis.
Plasma
membrane
Plasma
membrane
Cell wall
H2O
Cytoplasm
Cytoplasm
NaCl 0.85%
NaCl 10%
Cell in isotonic solution.
Plasmolyzed cell in hypertonic solution.

14. Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2

15. Chemical Requirements
Nitrogen
In amino acids and proteins
Most bacteria decompose proteins
Some bacteria use NH4+ or NO3–
A few bacteria use N2 in nitrogen fixation

16. Chemical Requirements
Sulfur
In amino acids, thiamine, and biotin
Most bacteria decompose proteins
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus

17. Chemical Requirements
Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors

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

19. Toxic Oxygen Singlet oxygen: 1O2− boosted to a higher-energy state
Superoxide free radicals: O2
Peroxide anion: O22–
Hydroxyl radical (OH•)
Superoxide dismutase
O2 + O H+
H2O2 + O2
Catalase
2 H2O2
2 H2O + O2
Peroxidase
H2O2 + 2 H+
2 H2O

20. Organic Growth Factors
Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines

21. Biofilms Microbial communities Form slime or hydrogels
Bacteria attracted by chemicals via quorum sensing

22. Clumps of bacteria adhering to surface Migrating clump of bacteria
Figure 6.5 Biofilms.
Clumps of bacteria adhering to surface
Migrating clump of bacteria
Surface
Water currents

23. Biofilms
Share nutrients
Sheltered from harmful factors

24. Applications of Microbiology 3.2 Pseudomonas aeruginosa biofilm.
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25. Biofilms
Patients with indwelling catheters received contaminated heparin
Bacterial numbers in contaminated heparin were too low to cause infection
84–421 days after exposure, patients developed infections

26. Culture Media Culture medium: nutrients prepared for microbial growth
Sterile: no living microbes
Inoculum: introduction of microbes into medium
Culture: microbes growing in/on culture medium

27. Agar Complex polysaccharide
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

28. Culture Media
Chemically defined media: exact chemical composition is known
Complex media: extracts and digests of yeasts, meat, or plants
Nutrient broth
Nutrient agar

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

30. Table 6.3 Defined Culture Medium for Leuconostoc mesenteroides

31. Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

32. Anaerobic Culture Methods
Reducing media
Contain chemicals (thioglycolate or oxyrase) that combine O2
Heated to drive off O2

33. Envelope containing sodium bicarbonate and sodium borohydride
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Clamp with clamp screw
Lid with
O-ring gasket
Envelope containing sodium bicarbonate and sodium borohydride
Palladium catalyst pellets
Anaerobic indicator (methylene blue)
Petri plates

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

35. Capnophiles Microbes that require high CO2 conditions CO2 packet
Candle jar

36. Biosafety Levels BSL-1: no special precautions
BSL-2: lab coat, gloves, eye protection
BSL-3: biosafety cabinets to prevent airborne transmission
BSL-4: sealed, negative pressure
Exhaust air is filtered twice

37. Figure 6.8 Technicians in a biosafety level 4 (BSL-4) laboratory.

38. Differential Media
Make it easy to distinguish colonies of different microbes

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

40. Selective Media
Suppress unwanted microbes and encourage desired microbes

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

42. Enrichment Culture Encourages growth of desired microbe
Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria
Inoculate phenol-containing culture medium with the soil, and incubate
Transfer 1 ml to another flask of the phenol medium, and incubate
Only phenol-metabolizing bacteria will be growing

43. Obtaining Pure Cultures
A pure culture contains only one species or strain
A colony is a population of cells arising from a single cell or spore or from a group of attached cells
A colony is often called a colony-forming unit (CFU)
The streak plate method is used to isolate pure cultures

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

45. Preserving Bacterial Cultures
Deep-freezing: –50° to –95°C
Lyophilization (freeze-drying): frozen (–54° to –72°C) and dehydrated in a vacuum

46. Reproduction in Prokaryotes
Binary fission
Budding
Conidiospores (actinomycetes)
Fragmentation of filaments
ANIMATION Bacterial Growth: Overview

47. (a) A diagram of the sequence of cell division
Figure 6.12a Binary fission in bacteria.
Cell wall
Cell elongates and DNA is replicated.
Plasma membrane
Cell wall and plasma membrane begin to constrict.
DNA (nucleoid)
Cross-wall forms, completely separating the two DNA copies.
Cells separate.
(a) A diagram of the sequence of cell division

48. Partially formed cross-wall
Figure 6.12b Binary fission in bacteria.
Partially formed cross-wall
DNA (nucleoid)
Cell wall
(b) A thin section of a cell of Bacillus licheniformis starting to divide
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49. Figure 6.13a Cell division.

50. Figure 6.13b Cell division.

51. Log number of cells (end) − Log number of cells (beginning)
Generation Time
If 100 cells growing for 5 hours produced 1,720,320 cells:
Number of generations =
Log number of cells (end) − Log number of cells (beginning)
0.301
Generation time =
60 min × hours
Number of generations
= 21 minutes/generation
ANIMATION Binary Fission

52. (1,048,576) Log10 = 6.02 Log10 = 4.52 Log10 of number of cells
Figure 6.14 A growth curve for an exponentially increasing population, plotted logarithmically (dashed line) and arithmetically (solid line).
(1,048,576)
Log10 = 6.02
Log10 = 4.52
(524,288)
Log10 = 3.01
Log10 of number of cells
Number of cells
(262,144)
Log10 = 1.51
(131,072)
(65,536)
(32,768)
(32)
(1024)
Generations

53. Figure 6.15 Understanding the Bacterial Growth Curve.
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).
Staphylococcus spp.

54. 1 ml 1 ml 1 ml 1 ml 1 ml Original inoculum Dilutions 1:10 1:100 1:1000
Figure 6.16 Serial dilutions and plate counts.
1 ml
1 ml
1 ml
1 ml
1 ml
Original inoculum
9 m broth
in each tube
Dilutions
1:10
1:100
1:1000
1:10,000
1:100,000
1 ml
1 ml
1 ml
1 ml
1 ml
Plating
1:10
1:100
1:1000
1:10,000
1:100,000
(10-1)
(10-2)
(10-3)
(10-4)
(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.)

55. Plate Counts
After incubation, count colonies on plates that have 25–250 colonies (CFUs)

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

57. Figure 6.18 Counting bacteria by filtration.

58. Most Probable Number
Multiple tube MPN test
Count positive tubes

59. Volume of Inoculum for Each Set of Five Tubes
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.

60. Most Probable Number
Compare with a statistical table

61. Figure 6.19b The most probable number (MPN) method.
(b) MPN table.

62. Grid with 25 large squares
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
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.
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).

63. Direct Microscopic Count
Number of bacteria/ml =
Number of cells counted
Volume of area counted 14
8 × 10−7
= 17,500,000

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

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