1. Elements of Microbial Growth, Nutrition and Environment

2. Do different organisms require specific diets and environments?

3. Why do we care about growth?
To Encourage the microbes we want
Brewery, winery, food production
Vaccine and drug production
Microbial fuel cells
Bioremediation, Sewage treatment plant, oil spill clean up
Resident microbiota-probiotics to aid microbial antagonism and perform other functions
To Discourage the microbes we don’t want
Pathogens

4. What is Growth?
In microbiology, we define growth in relation to the number of cells, not the size of cells.
Concentrate on population growth
Bacterial cells divide via binary fission, not mitosis.

5. Binary fission The division of a bacterial cell
Parental cell enlarges and duplicates its DNA
Septum formation divides the cell into two separate chambers
Complete division results in two identical cells

6. Generation Time
The time required for a complete division cycle (doubling)
Length of the generation time is a measure of the growth rate
Growth is exponential not arithmetic
Dependent on chemical and physical conditions

7. Generation Time Average generation time is 30 – 60 minutes
shortest generation times can be 10 – 12 minutes
E. coli GT=20 min.
Mycobacterium leprae has a generation time of 10 – 30 days
11 million cells (20 generations) in 7 hours
most pathogens have relatively short generation times

8. Which is bacterial growth curve?

10. Four phases of growth in a bacterial culture
(Log)

11. 1. Lag Phase
Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites
Not doubling at their maximum growth rate

12. 2. Exponential Growth Phase
Maximum exponential growth rate of cell division
Adequate nutrients
Favorable environment
Most sensitive to antibiotics. Why?

13. Exponential Growth Phase
A person actively shedding bacteria in the early and middle stages of infection is more likely to spread it than a person in the later stages. Why?
MRSA

14. 3. Stationary Phase Cell birth and cell death rates are equal
Survival mode – depletion in nutrients, released waste can inhibit growth

15. 4. Death Phase
A majority of cells begin to die exponentially due to lack of nutrients or build up of waste
Slower than the exponential growth phase

16. How do we measure microbial growth?
Direct measurement
Standard Plate counts
most common, need to DILUTE to get individual, countable colonies
Microscopic Count
count with microscope
Filtration
when # microbes small,
water run thru filter and filter applied to TSA plate and incubated
Coulter Counter
Automated cell counter
Indirect (Estimation)
Turbidity
more bacteria, more cloudiness
can measure w/ spectrophotometer or eye
Metabolic Activity
assumes amount of metabolic product is proportional to #
Dry Weight
used for filamentous organisms, like molds
Genetic Probing
Real-time PCR

17. Direct: Standard Plate Counts

18. Direct: Microscopic Count
Advantages
Easy and fast
Disadvantages
Uses special microscope counting slide
Does not differentiate between live and dead bacteria

19. Direct: Membrane Filtration

20. Direct: Coulter Counter
Uses an electronic sensor to detect and count the number of cells.

21. The greater the turbidity, the larger the population size.
Indirect: Turbidity Using Spectrometer
The greater the turbidity, the larger the population size.
Which culture (left or right) has more bacteria?

22. Indirect: Metabolism Activity
The metabolic output or input of a culture may be used to estimate viable count.
Examples:
Measure how fast gases and/or acids are formed in a culture
Or the rate a substrate such as glucose or oxygen is used up

23. Indirect: Dry Weight To calculate the dry weight of cells
cells must be separated from the medium
then dried
the resulting mass is then weighed

24. Indirect: Genetic Methods
Use real-time PCR to “count” how many bacterial genes there are in a sample.

25. Which techniques distinguish between live and dead cells?
Standard Plate counts
Direct Microscopic
Filtration
Coulter counter
Turbidity
Metabolic activity
Dry weight
Genetic Probing

26. Which techniques distinguish between live and dead cells?
Standard Plate counts
Direct Microscopic
Filtration
Coulter counter
Turbidity
Metabolic activity
Dry weight
Genetic Probing

27. What are the requirements for microbial growth?

28. Chemical Composition of an Escherichia coli Cell

29. Microbial Nutrition Macronutrients: carbon, hydrogen, and oxygen
required in relatively large quantities and play principal roles in cell structure and metabolism
Micronutrients:
present in much smaller amounts
manganese, zinc, nickel
Inorganic nutrients:
Can have carbon OR hydrogen, but not both
Organic nutrients:
Contain carbon and hydrogen

30. Microbial Nutrition
All cells require the following for metabolism and growth:
Carbon source
Energy source
Growth factors (some bacteria are fastidious/picky and require extra supplements)

31. Microbial Nutrition Heterotroph: Organic carbon is carbon source
Autotroph: inorganic CO2 as its carbon source
has the capacity to convert CO2 into organic compounds
not nutritionally dependent on other living things
Phototroph: microbes that photosynthesize
Chemotroph: microbes that gain energy from injesting chemical compounds

32. Microbial Nutrition: Autotrophs
Photoautotrophs:
Photosynthetic
Produce organic molecules using CO2
Ex: Cyanobacteria, algea
Chemoautotrophs:
Ingest organic or inorganic compounds for energy
Carbon source is CO2

33. Microbial Nutrition: Heterotrophs
Chemoheterotrophs:
organic compounds for both carbon and energy source
derive both carbon and energy from processing these molecules through respiration or fermentation
The vast majority of microbes causing human disease are chemoheterotrophs
Ex: Most bacteria, all, protists, all fungi, and all animals

34. Diffusion: Review
Transport of necessary nutrients occurs across the cell membrane, even in organisms with cell walls
Diffusion:
Atoms or molecules move in a gradient from an area of higher concentration to lower concentration
Diffusion of molecules across the cell membrane is largely determined by the concentration gradient and permeability of the substance

35. Osmosis: Review Isotonic: Equal solutes in cell and in environment
Osmosis: the diffusion of water through a selectively permeable membrane
Isotonic: Equal solutes in cell and in environment
parasites living in host tissues are most likely to be living in isotonic habitats
Hypotonic: More solutes in cell than in environment
A slightly hypotonic environment can be favorable to bacteria cells
Hypertonic: Less solutes in cell than in environment
hypertonic solutions such as concentrated salt and sugar solutions act as preservatives for food (salted ham is an example)

36. Osmosis: Review

37. Chemoautotrophs
chemoorganic autotrophs: use organic compounds for energy and inorganic compounds as a carbon source
lithoautotrophs: rely totally on inorganic minerals and require neither sunlight nor organic nutrients

38. Environmental (Physical) Factors Effecting Bacterial Growth
Temperature
Gas pH
Osmotic pressure
Other factors
Microbial association
Survival in a changing environment is largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat

39. Environmental Factors: Temperature
Effect of temperature on proteins:
Too high, proteins unfold and denature
Too low, do not work efficiently
Effect of temperature on membranes of cells and organelles:
Too low, membranes become rigid and fragile
Too high, membranes become too fluid 39

40. Temperature and Bacterial Growth

41. Five categories of microbes based on temperature ranges for growth
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Minimum
Psychrophile
Maximum
Optimum
Psychrotroph
Mesophile
Thermophile
Extreme thermophile
Rate of Growth
-20
-10 10 20 30 40 50 60 70 80 90
100
110
120
130
Temperature °C
Which category do human pathogens usually fall into? Why?

42. Environmental Factors: Gases
Two gases that most influence microbial growth
Oxygen
O2 has the greatest impact on microbial growth
O2 is an important respiratory gas and a powerful oxidizing agent
Carbon dioxide
Waste for bacteria
Carbon source for others (non-pathogens)

43. Oxygen Requirements
As oxygen enters cellular reactions, it is transformed into several toxic products
highly reactive and excellent oxidizing agents
Resulting oxidation causes irreparable damage to cells by attacking enzymes and proteins 43

44. Oxygen Requirements
As oxygen enters cellular reactions, it is transformed into several toxic products:
singlet oxygen (O)
superoxide ion (O2-)
hydrogen peroxide (H2O2)
hydroxyl radicals (OH-)
Most cells have enzymes that scavenge and neutralize reactive oxygen byproducts
Two-step process requires two enzymes:

45. Oxygen Requirements
If bacteria do not have superoxide dismutase or catalase they can not tolerate oxygen.
Catalase Test

46. Oxygen Requirements Aerobes Anaerobes Facultative anaerobes
Aerotolerant anaerobes
Microaerophiles 46

47. Determining Oxygen Requirements
Thioglycollate broth to demonstrate oxygen requirements.
Oxygen levels throughout the media are reduced via reaction with sodium thioglycolate.
Producing a range of oxygen levels in the media that decreases with increasing distance from the surface

48. Oxygen Requirements: Obligate Aerobe
Requires oxygen for metabolism
Have enzymes that neutralize toxic oxygen metabolites
Ex. Most fungi, protozoa, and bacteria, such as Bacillus species and Mycobacterium tuberculosis

49. Oxygen Requirements: Facultative Anaerobe
Does not require oxygen, but can grow in its presence
During oxygen free states, anaerobic respiration or fermentation occurs
Possess superoxide dismutase and catalase
Ex. Many Gram-negative pathogens
Prefer oxygenated environments because more energy is produced during aerobic respiration compared to anaerobic respiration or fermentation
Why is this the “best” for pathogens?

50. Oxygen Requirements: Obligate Anaerobes
Cannot use oxygen for metabolism
Do not possess superoxide dismutase and catalase
The presence of oxygen is toxic to the cell and will kill it
Ex. Many oral bacteria, intestinal bacteria

51. Oxygen Requirements: Aerotolerant
Can live with, but do not use oxygen
Able to break down peroxides (not using catylase)
Ex. Some lactobacilli and streptococci

52. Oxygen Requirements: Aerotolerant
Can live with, but do not use oxygen
Able to break down peroxides (not using catylase)
Ex. Some lactobacilli and streptococci

53. Oxygen Requirements: Microaerophiles
Require small amounts of oxygen
Ex. H. pylori

54. Culturing Technique for Anaerobes

55. Environmental Factors: pH
Most cells grow best between pH 6-8
strong acids and bases can be damaging to enzymes and other cellular substances
Pathogens like our neutral pH
Yeast & Molds like acidic conditions

56. Environmental Factors: pH
Acidophiles
thrive in acidic environments.
Ex. Helicobacter pylori
Alkalinophiles
thrive in alkaline conditions
Ex. Proteus can create alkaline conditions to neutralize urine and colonize and infect the urinary system

57. Example of the use of a selective medium for pH
Bacterial colonies
Fungal colonies
pH 7.3
pH 5.6

58. Environmental Factors: Water
Microbes require water to dissolve enzymes and nutrients
Water is important reactant in many metabolic reactions
Most cells die in absence of water
Some have cell walls that retain water
Endospores cease most metabolic activity
Two physical effects of water
Osmotic pressure
Hydrostatic pressure 58

59. Environmental Factors: Water
Osmotic pressure:
Halophiles (Salt lovers)
Requires high salt concentrations
Withstands hypertonic conditions
Ex. Halobacterium
Facultative halophiles
Can survive high salt conditions but is not required
Ex. Staphylococcus aureus

60. Other Physical Factors Influencing Microbial Growth
Radiation- UV, infrared
Barophiles – withstand high pressures
Spores and cysts- can survive dry habitats
Microbes require different nutrients and different environments specific to survive. They are very specialized!

61. Associations Between Organisms
Organisms live in association with different species
Often involve nutritional interactions
Antagonistic relationships
Synergistic relationships
Symbiotic relationships
Associations Between Organisms
Symbiotic
Non symbiotic
Organisms live in close
nutritional relationships;
required by one or both members.
Organisms are free-living;
relationships not required
for survival.
Mutualism
Obligatory,
dependent;
both members
benefit.
Commensalism
The commensal
benefits; other
member not
harmed.
Parasitism
Parasite is
dependent
and benefits;
host harmed.
Synergism
Members
cooperate
and share
nutrients.
Antagonism
Some members
are inhibited
or destroyed
by others. 61

62. Associations Between Organisms
Antagonism: free-living species compete
Antibiosis: the production of inhibitory compounds such as antibiotics
The first microbe has a competitive advantage by increasing the space and nutrients available to the competitor
Remember importance of microflora?!
A biocontrol agent on the right (a bacteria) is making a material that is keeping the pathogen on the left (a fungus) from growing.

63. Associations Between Organisms
Synergism: free-living species benefits together but is not necessary for survival
Together the participants cooperate to produce a result that none of them could do alone
Gum disease, dental caries, and some bloodstream infections involve mixed infections of bacteria interacting synergistically

64. Associations and Biofilms
Complex relationships among numerous species of microorganisms
Many microorganisms more harmful as part of a biofilm
Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by
Plaque (biofilm) on a human tooth 64

65. Associations and Biofilms
Benefits of biofilm
large, complex communities form with different physical and biological characteristics
the bottom may have very different pH and oxygen conditions than the surface
partnership among multiple microbial species
cannot be eradicated by traditional methods

66. Now that you know more about the nutritional needs of bacteria let’s look at using this information to ID bacteria! 66

67. Survey of Microbial Diseases
How to identify bacteria in patient specimens or
in samples from nature? Or the MM project;)
phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry
immunologic: serological analysis
genotypic: genetic techniques
increasingly being used as a sole resource
for identifying bacteria
Data from these methods can provide a unique
profile for any bacterium

68. Survey of Microbial Diseases:
Phenotypic Methods
Physiological/Biochemical Characteristics
Traditional mainstay of bacterial identification
Enzyme production and other biochemical properties are reliable ways to ID microbes
Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities:
fermentation of sugars
capacity to digest complex polymers
production of gas
sensitivity to antibiotics
nutrient sources

69. Blood agar as a differential medium
Beta-hemolysis
Blood agar as a differential medium
Alpha-hemolysis
No hemolysis (gamma-hemolysis)

70. Tests for fermentation and gas production
Survey of Microbial Diseases:
Phenotypic Methods
Tests for fermentation and gas production
Durham tube (inverted tube to trap gas)
No fermentation
Acid fermentation with gas

71. Phenotypic Methods: Direct Examination of Specimen
Direct observation of fresh or stained specimen
Stains most often used
Gram stain
acid-fast stain

72. Survey of Microbial Diseases:
Phenotypic Methods
Isolation Media and Morphological Testing
Selective media: encourage the growth of only the suspected pathogen
Differential media: used to identify definitive characteristics and fermentation patterns

73. MacConkey Agar: Selective and Differential
Selects for Gram-negative and tells you if the bacterium ferments lactose

74. Phenotypic Methods: Biochemical Testing
Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for cytochrome c oxidase what does that tell you?

75. Phenotypic Methods: Biochemical Testing
Unknown
microbe +
different
substrates
DNPG
ADH
LDC
ODC |
CIT |
H2S
URE
TDA
IND | VP | |
GEL |
GLU
MAN
INO
SOR
RHA
SAC
MEL
AMY
ARA
Results (+/–) – – + + – – – – + – – + – – – – – – – –
Enzyme-mediated metabolic reactions often
visualized by a color change
microbe is cultured in a medium with a special substrate, then tested for a particular end product
microbial expression of the enzyme is made visible by a colored dye

76. Flowchart: We will use this to ID our MM!
Cocci
Gram (+)
Gram (–)
Catalase (+),
irregular clusters,
tetrads
Catalase (–),
pairs, chain
arrangement
Aerobic,
oxidase (+),
catalase (+)
Anaerobic,
oxidase (–),
catalase (–)
Streptococcus
Strictly
aerobic
Facultative
anaerobic
Neisseria
Branhamella
Moraxella
Veillonella
Micrococcus
Staphylococcus
Planococcus

77. Phenotypic Methods: Phage Typing
Testing for sensitivity to various phage groups
a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block
cleared areas corresponding to lysed cells indicate sensitivity to that phage
Ex. S. aureus Phage Group I vs. Group II

78. Determining Clinical Significance of Cultures
Important to rapidly determine if an isolate from a specimen is clinically important or if it is merely a contaminant or normal biota
a few colonies of E. coli in a urine sample can indicate normal biota, but several hundred can mean an active infection
a single colony of a true pathogen such as Mycobacterium tuberculosis in a sputum culture, or an opportunist in a sterile site, is highly suggestive of disease
repeated isolation of a relatively pure culture of any microorganism can mean it is an agent of disease