1. Chapter 5 Microbial Nutrition and Culture cont’d
Siti Sarah Jumali (ext 2123)
Room 3/14

2. Microbial Growth Requirements

3. The Requirements for Growth
CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS)
PHYSICAL REQUIREMENTS
Temperature pH
Oxygen
Hydrostatic Pressure
Osmotic pressure
Carbon
Nitrogen, sulfur, and phosphorous
Trace elements
Oxygen
Organic growth factor

4. Physical Factors Required for Bacterial Growth
Optimum pH: the pH at which the microorganism grows best (e.g. pH 7)
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
According to their tolerance for acidity/alkalinity, bacteria are classified as:
Acidophiles (acid-loving): grow best at pH
Neutrophiles: grow best at pH 5.4 to 8.0
Alkaliphiles (base-loving): grow best at pH

5. 2) Temperature
According to their growth temperature range, bacteria can be classified as:
Psychrophiles : grow best at 15-20oC
Psychrotrophs : grow between 0°C and 20–30°C
Mesophiles : grow best at 25-40oC
Thermophiles : grow best at 50-60oC
Typical Growth Rates and Temperature
Minimum growth temperature: lowest temp which species can grow
Optimum growth temperature: temp at which the species grow best
Maximum growth temperature: highest temp at which grow is possible

7. Food Preservation Temperatures

8. 3) Oxygen Aerobes: require oxygen to grow
Obligate aerobes: must have free oxygen for aerobic respiration (e.g. Pseudomonas)
Anaerobes: do not require oxygen to grow
Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
Microaerophiles: grow best in presence of small amount of free oxygen
Capnophiles: carbon-dioxide loving organisms that thrive under conditions of low oxygen
Facultative anaerobes: carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent
Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism
Obligate: organism must have specified environmental condition
Facultative: organism is able to adjust to and tolerate environmental condition, but can also live in other conditions

9. Patterns of Oxygen Use

10. 4) Hydrostatic Pressure
Water in oceans and lakes exerts pressure exerted by standing water, in proportion to its depth
Pressure doubles with every 10 meter increase in depth
Barophiles: bacteria that live at high pressures, but die if left in laboratory at standard atmospheric pressure

11. 5) Osmotic Pressure
Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells
Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell)
Hypoosmotic environment: cells gain water and swell and burst

12. Plasmolysis

13. Halophiles
Salt-loving organisms which require moderate to large quantities of salt (sodium chloride)
Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside
Why do halophiles require sodium?
1) Cells need sodium to maintain a high intracellular
potassium concentration for enzymatic function
2) Cells need sodium to maintain the integrity of their
cell walls

14. Responses to Salt

15. The Great Salt Lake in Utah

16. Chemical Requirement: Nutritional Factors
Carbon sources
Nitrogen sources
Sulfur and phosphorus
Trace elements (e.g. copper, iron, zinc, and cobalt)
Vitamins (e.g. folic acid, vitamin B-12, vitamin K)

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

18. 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

19. 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

20. Chemical Requirements
Trace elements
Inorganic elements (mineral) required in small amounts
Usually as enzyme cofactors
Ex: iron, molybdenum, zinc
Buffer
To neutralize acids and maintain proper pH
Peptones and amino acids or phosphate salts may act as buffers

21. Organic Growth Factors
Organic compounds obtained directly from the environment
Ex: Vitamins, amino acids, purines, and pyrimidines

22. Preparation of 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

23. 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

24. Types of Culture Media
Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter
Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition
Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

25. Types of Culture Media Type of Media Purpose Chemically Defined
Growth of chemoheterotrophs and photoautotrophs: microbiological assays
Complex
Growth of most chemoheterotrophic organisms
Reducing
Growth of obligate anO2
Selective
Suppresion of unwanted microbes; encouraging desired microbes
Differential
Differentiation of colonies of desired microbes from others
Enrichment
Similar to selective media but designed to increase numbers of desired microbes to detectable levels.

26. 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. Selective, Differential, and Enrichment Media
Selective medium: encourages growth of some organisms but suppresses growth of others
(e.g. antibiotics)
Differential medium: contains a constituent that causes an observable change (e.g. MacConkey agar)
Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified

30. Selective Media Differential Media
Suppress unwanted microbes and encourage desired microbes
Ex: Sabouraud’s Dextrose Agar: used to isolate fungi, has a pH of 5.6, outgrow most of bacteria
Differential Media
Make it easy to distinguish colonies of different microbes.
Ex: Blood Agar: bacteria that can lysed blood cells causing a clear areas around the colonies.

31. Three species of Candida can be differentiated in mixed culture when grown on CHROMagar Candida plates

32. Identification of urinary tract pathogens with
differential media (CHROMagar)

33. Enrichment Media 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

34. Culturing Bacteria
Culturing of bacteria in the laboratory presents two problems:
1. A pure culture of a single species is needed
to study an organism’s characteristics
2. A medium must be found that will support
growth of the desired organism

35. Obtaining Pure Cultures
Pure culture: a culture that contains only a single species or strain of organism
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

36. The Streak Plate Method uses agar plates to prepare pure cultures

37. The Streak Plate Method
Figure 6.11

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

39. Anaerobic Jar
Figure 6.6

40. To culture obligate anaerobes, all molecular oxygen must be removed and kept out of medium. Agar plates are incubated in sealed jars containing chemical substances that remove oxygen and generate carbon dioxide or water

41. Anaerobic Transfer

42. An Anaerobic Chamber
Figure 6.7

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

44. Preserved Cultures
To avoid risk of contamination and to reduce mutation rate, stock culture organisms should be kept in a preserved culture, a culture in which organisms are maintained in a dormant state
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum
Deep Freezing: –50° to –95°C
Refrigeration
Reference culture (type culture): a preserved culture that maintains the organisms with characteristics as originally defined

45. CHAPTER 6: MICROBIAL GROWTH

46. Growth and Cell Division
Microbial growth is defined as the increase in the number of cells, which occurs by cell division
Binary fission (equal cell division): A cell duplicates its components and divides into two cells
Septum: A partition that grows between two daughter cells and they separate at this location
Budding (unequal cell division): A small, new cell develops from surface of exisiting cell and subsequently separates from parent cell

47. Binary Fission

48. Binary Fission

49. Thin section of the bacterium Staphylococcus, undergoing binary fission

50. Budding in Yeast

51. Phases of Growth
Consider a population of organisms introduced into a fresh, nutrient medium
Such organisms display four major phases of growth in batch culture:
The lag phase
The logarithmic phase
The stationary phase
The death phase

53. The Lag Phase Organisms do not increase significantly in number
They are metabolically active
Grow in size, synthesize enzymes, and incorporate molecules from medium
Produce large quantities of energy in the form of ATP

54. The Log Phase Organisms have adapted to a growth medium
Growth occurs at an exponential (log) rate
The organisms divide at their most rapid rate
a regular, genetically determined interval (generation time)

55. Synchronous growth: A hypothetical situation in which the number of cells in a culture would increase in a stair-step pattern, dividing together at the same rate
Nonsynchronous growth: A natural situation in which an actual culture has cell dividing at one rate and other cells dividing at a slightly slower rate

56. Stationary Phase Decline (Death) Phase
1) Cell division decreases to a point that new cells
are produced at same rate as old cell die.
2) The number of live cells stays constant.
Decline (Death) Phase
1) Condition in the medium become less and less
supportive of cell division
2) Cell lose their ability to divide and thus die
3) Number of live cells decreases at a logarithmic
rate

57. Microbes growing continuously in a chemostat

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

59. - Pour plate or Spread Plate Method
Direct Methods
Plate Count
Must perform
- Serial Dilutions
- Pour plate or Spread Plate Method
often reported as colony-forming units (CFU)
Serial Dilution

60. Plate Counts
Figure 6.17

61. Plate Counts
After incubation, count colonies on plates that have 25–250 colonies (CFUs)
Figure 6.16

62. 2) Counting Bacteria by Membrane Filtration

63. 3) The Most Probable Number (MPN) Method
Method to estimate number of cells
Multiple tube MPN test
Count positive tubes
Compare with a statistical table.

64. 4) Direct Microscopic Counts
Another way to measure bacterial growth by:
1) Petroff-Hausser counting chamber
2) Colony counting chamber
In Petroff-Hausser counting chamber, bacterial suspension is introduced onto chamber with a calibrated pipette
Microorganisms are counted in specific calibrated areas
Number per unit volume is calculated using an appropriate formula

65. The Petroff-Hausser Counting Chamber

66. Direct Microscopic Count

67. Counting colonies using a bacterial colony counter

68. Bacterial colonies viewed through the magnifying glass against a colony-counting grid

69. Which of these plates would be the correct one to count? Why?
Countable number of colonies
(30 to 300 per plate)
Which of these plates would be the correct one to count? Why?

70. Indirect Methods 1) Turbidity
Practical way of monitoring bacterial growth.
Measure turbidity using spectrophotometer
A Spectrophotometer: This instrument can be used to measure bacterial growth by measuring the amount of light that passes through a suspension of cells

71. Turbidity

72. Turbidity
The less light transmitted, the more bacteria in sample.

73. 2) Metabolic Activity
Assuming the amount of a certain metabolic product, ex acids, CO2 produced = direct propotion of no of bacteria present
3) Dry Weight
For filamentous bacteria and molds
Apply filtration of amount of broth culture on filter paper and dried in a dessicator
Weight of dried culture = direct propotion of no of bacteria present

74. Biosafety Levels 1: No special precautions
2: Lab coat, gloves, eye protection
3: Biosafety cabinets to prevent airborne transmission
4: Sealed, negative pressure
Exhaust air is filtered twice

75. Biosafety Level 4 (BSL-4) Laboratory

76. Questions?