1. Microbial Nutrition and Growth
Chapter 6
Microbial Nutrition and Growth

2. Metabolism Results in Reproduction Reproduction results in Growth
Microbial Growth
Metabolism Results in Reproduction
Reproduction results in Growth
What is microbial growth?
an increase in a population of microbes (rather than an increase in size of an individual)
Result of microbial growth?
a discrete colony – an aggregation of cells arising from single parent cell
Animations: Bacterial Growth Overview

3. Factors affecting growth: Nutritional Factors
Nutrients – chemicals taken in and used by organisms for energy, metabolism and growth
Water (Hydrogen and Oxygen)
Carbon
Nitrogen
Sulfur
Phosphorus
Trace Elements
Growth Factors

4. Factors affecting growth: Nutritional Factors
Macronutrients - Required in large amounts
Carbon
Needed for synthesis of cellular material and energy source
Nitrogen
Needed for protein synthesis, nucleic acids, ATP
Sulfur
Needed to synthesize amino acids and vitamins (thiamine, biotin)
Phosphorus
Needed to synthesize nucleic acids, ATP, phospholipids

5. Factors affecting growth: Nutritional Factors
Trace Elements
required in trace amounts
involved in enzyme function and protein structure
Examples: Zn, Cu, Fe
Present in tap water and distilled
Growth factors
Organic compounds that cannot be synthesized by bacteria
bacteria are “fastidious”
examples: amino acids, purines, pyrimidines, vitamins
Fastidious organisms require relatively large amounts of growth factors in the media. Can be used to test samples for presence of growth factors EXP. Euglena granulata reguires Vitamin B12 to grow and the amount of Vitamin B12 affects amount of growth.

6. Growth Requirements Nutrients: Chemical and Energy Requirements
Sources of carbon, energy, and electrons
Two groups of organisms based on source of carbon
Autotrophs
Heterotrophs
Two groups of organisms based on source of energy
Chemotrophs
Phototrophs

7. Sources of Carbon, Energy, and Electrons
Carbon sources
Organisms are categorized into two groups:
Autotrophs
Those using an inorganic carbon source (carbon dioxide)
Heterotrophs
Those catabolizing organic molecules (proteins, carbohydrates, amino acids, and fatty acids)

8. Sources of Carbon, Energy, and Electrons
Energy sources
Organisms are categorized into two groups:
Chemotrophs
Acquire energy from redox reactions involving inorganic and organic chemicals
Phototrophs
use light as their energy source

9. Groups of organisms based on carbon and energy source
Figure 6.1

10. Oxygen sources Oxygen Requirements
Found as gaseous O2 or covalently bound in compounds
Essential for aerobic respiration
Oxygen is the final electron acceptor
Deadly for some types of bacteria (anaerobes)
Toxic forms of oxygen are highly reactive
are excellent oxidizing agents
Results in irreparable damage to cells by oxidizing compounds such as proteins and lipids

11. Toxic Forms of Oxygen Singlet oxygen: 1O2
Oxygen boosted to a higher-energy state; extremely reactive
Superoxide free radicals: O2 O2 + 2H H2O2 +O2
Superoxide Dismutase
Peroxide anion: O22
2H2O H2O + O2
Catalase
H2O2 + NADH+H H2O
Peroxidase
Hydroxyl radical (OH)
Result of ionizing radiation & incomplete reduction of hydrogen peroxide;
extremely reactive but danger averted in aerobes because of catalase & peroxidase

12. Oxygen and Carbon Dioxide Requirements
Aerobes – must use oxygen and can detoxify it
Anaerobes- can not use oxygen nor detoxify it
Facultative anaerobes- do not require oxygen but can use and detoxify it
Aerotolerant anaerobes – can not use aerobic metabolism but have some enzymes to detoxify oxygen’s poisonous forms
Microaerophile – requires a small amount of oxygen for growth
Capnophile – requires higher CO2 tension (3-10%) than normally found in the atmosphere

13. Classification of Organisms Based on
Oxygen Requirements
Microbial Growth is affected by Oxygen Concentration
Obligate aerobes
Facultative anaerobes
Obligate anaerobes
Aerotolerant anaerobes
Microaerophiles

14. Nitrogen Requirements
Nitrogen Sources
Acquired from organic and inorganic nutrients
Recycled from amino acids and nucleotides to make other proteins and nucleotides
Nitrogen fixation-
Nitrogen gas reduced to ammonia
Essential to life on Earth because nitrogen is made available in a usable form

15. Factors that Affect Microbial Growth
Temperature –
Affects proteins and lipid membranes
If too low, membranes become rigid and fragile
If too high, membranes become too fluid
Categories based on Optimum Temperature
Psychrophile – optimum below 15oC
Mesophile – optimum between 20oC – 40oC
Thermophile – optimum higher than 45oC
Hyperthermophiles – optimum above 80oC

16. Effects of temperature on microbial growth
Figure 6.4

17. Catagories of Microbes Based on Temperature Range
Figure 6.5

18. Temperature

19. Use of Temperature to Preserve Microbes
Preserving Bacteria Cultures:
Refrigeration:
Storage for short periods of time
Deep-freezing:
-50° to -95°C
Preserves cultures for years
Lyophilization (freeze-drying):
Frozen (-54° to -72°C) and dehydrated in a vacuum
Can last decades

20. Classification of Microbes based on pH
Effects of pH
Classification of Microbes based on pH
Organisms sensitive to changes in acidity
H+ and OH– interfere with H bonding
Acidophiles – prefer below 7
Neutrophiles – prefer 7
Alkalinophiles – prefer above 7
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6

21. Physical Effects of Water
Microbes require water
to dissolve enzymes and nutrients required in metabolism; to react in many metabolic reactions
Some microbes have cell walls that retain water
Endospores and cysts stop most metabolic activity to survive in a dry environment for years
Two physical effects of water
Osmotic pressure
Hydrostatic pressure

22. Osmotic Pressure
Osmotic pressure The pressure exerted on the semipermeable membrane by a solution containing solutes, which cannot move across the membrane. Osmosis Diffusion of water across a semipermeable membrane driven by unequal concentration of solutes across the membrane.

23. Osmotic Variations in the Environment
Isotonic
External concentration of solutes is equal to cell’s internal environment
Diffusion of water equal in both directions
No net change in cell volume
Hypotonic
External concentration of solutes is lower than cell’s internal environment
Cells swell and burst
Hypertonic
Environment has higher solute concentration than cell’s internal environment
Cells shrivel (crenate)
Halophiles tolerate higher salt concentrations

24. Osmotic Pressure
ISOTONIC
Physiologic Saline
HYPERTONIC

25. Hydrostatic Pressure Water exerts pressure in proportion to its depth
For every addition of depth, water pressure increases 1 atm
Organisms that live under extreme pressure are barophiles
Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape

26. Microbial Growth Binary fission
Splitting parent cell to form two similar-sized daughter cells to increase number of cells
Generation time
Duration of each division
Determined by type of bacteria
Example: E. coli (20 min)

27. Exponential Growth by Binary Fission
DNA replication
Cell elongation
Septum formation
Septum completion leads to separation or further division
Process repeats
Figure 6.17a

28. Bacterial Growth Curve
Animation: Bacterial Growth Curve
Figure 6.20

29. Bacterial Growth Curve
Graph of a closed bacterial population over time
Lag phase
Acquire cell mass, no reproduction
Log (Exponential growth) phase
Cells dividing
Stationary phase
Cells stop growing, cells dividing and dying at same rate
Death phase
Cells dying due to lack of nutrients and increased waste products

30. Methods of Culturing Microbes
Specimen Collection
Taking a sample of infected material
Sterile (aseptic) technique required to avoid introducing unwanted microbes into the sample

31. Clinical Sampling
Table 6.3

32. Culturing Microorganisms
Inoculate
Implant microbes onto medium (broth or solid)
Inoculum
Sample of microbes from specimen
Culture
act of cultivating microorganisms
or the microorganisms that are cultivated

33. Streak Plate Method Pure Culture Most commonly used.
Figure Overview

34. Mixed Culture Can you see 12 different bacterial colonies?
Count colonies. A colony is defined as a population of similar cells that arose from one viable cell/ or one endospore.
Can you see 12 different bacterial colonies?
Figure Overview

35. Culture Media Used as solidifying agent for culture media
Nutrient preparation for microbial growth
Must provide all chemical requirements
Physical state depends on amount of AGAR
Used as solidifying agent for culture media
Composed of complex polysaccharides
Advantages of agar vs gelatin:
Generally not metabolized by microbes
Liquefies at 100°C
Solidifies ~40°C
Fanny Hesse used agar from seaweed in her jams and jellies, which she learned from a neighbor who had lived in Java (Indonesia).

36. Chemically Defined vs Complex Media
Comparison of Media

37. Types of Media Used in the Clinical Lab
Basic Nutrient
Designed to grow broad-spectrum microbes
Enriched
Add enrichment to encourage growth of microbes
Blood, growth factors, serum
Selective
Suppress unwanted microbes and encourage desired microbes to grow
Salt, dyes, alcohol

38. An example of the use of a selective medium
Figure 6.12

39. Types of Media Used in the Clinical Lab
Differential
To distinguish colonies of different microbes from one another
Dyes, pH indicators
Reduced (anaerobic) media
Contain chemicals (thioglycollate) that combine O2
Used for anaerobic cultures
Transport
Maintain and preserve microbes
Include atmospheric buffers
Prevent drying

40. MacConkey agar as a selective and differential medium
Figure 6.15

41. Anaerobic Culture Methods
Gas Pak Jar
Glove Box
Figure Overview

42. Capnophiles require high CO2
Candle jar (3-10% CO2)
CO2-packet

43. Environment to Culture Microbes
Incubation
Temperature
35-37oC – body temperature
25-30oC – room temperature
4-8oC – refrigerator temperature
Atmosphere
Aerobic - free oxygen present
Microaerophilic – free O2 present; increased CO2
Anaerobic – NO free O2 present
Time
18 – 24 hours
Longer for slow-growing microbes

44. Planktonic vs Sessile Bacteria
All lab tests use “pure cultures” of suspended cells called planktonic bacteria since they float around in liquid.
In fact, pure cultures are virtually absent in nature.
Most microbes exist as sessile
bacteria– attached to a surface –
and they live in communities called biofilms. 
Robert Koch
Courtesy of the
National Library of Medicine

45. Biofilms Biofilms Complex relationships among numerous microorganisms
Develop an extracellular matrix
Adheres cells to one another
Allows attachment to a substrate
Sequesters nutrients
May protect individuals in the biofilm
Form on surfaces often as a result of quorum sensing
Many microorganisms more harmful as part of a biofilm

46. Ingredients needed for a biofilm :
What is a biofilm ?
An organized, layered system of microbes
attached to a surface
Biofilms form when microbes adhere to a surface that is moist and contains organic matter
Ingredients needed for a biofilm :
Surface
Bacteria
Aqueous environment
Nutrients

47. How does a biofilm develop?
1. Planktonic cells attach to surface
2. Cells multiply ;Produce glycocalyx
3. Slime layer entraps nutrients, cells, microbes
4. Dynamic pillar-like layers form

48. How do biofilms communicate?
Cell to cell communication
- send and receive chemical signaling molecules
Quorum sensing
- accumulation of signaling molecules
- enables a cell to sense the cell density
Center for Biofilm Engineering Montana State University–Bozeman

49. Biofilm Behavior
Biofilm bacteria “turn on” a different set of genes than planktonic bacteria
Examples:
Turn off flagellar protein and turn on pili genes
Turns on genes for antibiotic resistance
Form a "division of labor" by nutrient cycling
Some cells turn on metabolic pathways that degrade particulate matter, while other adjacent cells of the same population use the degradation products to produce new cells that are dispersed in the environment

50. Where are Biofilms Found?

51. Biofilms Found in Health Care
Dental caries
Contact lenses
Lungs of Cystic Fibrosis patients
Indwelling medical devices
Endotracheal tube
Mechanical heart valves
Pacemakers
Urinary catheters
IV connectors
Prosthetic joints
Biofilm on a contact lens
Staphylococcus biofilm
on inner surface of
IV connector
Rodney M. Donlan, CDC

52. Where are Biofilms Found?
Biofilm on soft, daily-wear, contact lens
Biofilm on urinary catheter

53. Biofilms and Chronic Wounds
60 % of chronic wounds
had biofilms
Only 6% of acute wounds
Found normal flora and pathogens produced different chemicals for their communication.

54. Medical Importance of Biofilms
Are 1000X more resistant to antimicrobial agents than planktonic cells
Easily transfer genes to express new and sometimes more virulent phenotypes
Are more resistant to host defense mechanisms
80% of nosocomial infections are biofilm associated (NIH)
20% of patients with biofilm-related septicemia die