Do different organisms require specific diets and environments?
Elements of Microbial Growth, Nutrition and Environment
Why do we care about growth?
• To Encourage the microbes we want
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•
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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
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.
• Pathogens
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
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
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Generation Time
Four phases of growth in a bacterial culture
• 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
1. Lag Phase
• Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites
• Not doubling at their maximum growth rate
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?
2. Exponential Growth Phase (Log)
• Maximum exponential growth rate of cell division
• Adequate nutrients
• Favorable environment
• Most sensitive to antibiotics. Why?
3. Stationary Phase
• Cell birth and cell death rates are equal
MRSA
• Survival mode –
depletion in nutrients, released waste can inhibit growth
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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
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
Direct: Standard Plate Counts • 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
Direct: Microscopic Count
• Advantages
– Easy and fast
• Disadvantages
– Uses special microscope counting slide
– Does not differentiate between live and dead bacteria
Direct: Membrane Filtration
Direct: Coulter Counter
Uses an electronic sensor to detect and count the number of cells.
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Indirect: Turbidity Using Spectrometer
Indirect: Metabolism Activity
• The metabolic output or input of a culture may be used to estimate viable count.
• Examples: The greater the turbidity, the larger the population size.
• 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
Which culture (left or right) has more bacteria?
Indirect: Dry Weight
Indirect: Genetic Methods
• To calculate the dry weight of cells
– cells must be separated from the medium – then dried – the resulting mass is then weighed
• Use real‐time PCR to “count” how many bacterial genes there are in a sample.
Which techniques distinguish between live and dead cells?
– Standard Plate counts – Direct Microscopic
– Filtration
– Coulter counter
– Turbidity
– Metabolic activity
– Dry weight
– Genetic Probing
Which techniques distinguish between live and dead cells?
– Standard Plate counts – Direct Microscopic
– Filtration
– Coulter counter
– Turbidity
– Metabolic activity
– Dry weight
– Genetic Probing
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Chemical Composition of an E. coli Cell
What are the requirements for microbial growth?
Microbial Nutrition
Cellular Transport
•Macronutrients:
- carbon, hydrogen, and oxygen
- required in relatively large quantities and play principal roles in cell structure and metabolism
• Passive Transport
– Molecules transported along concentration gradient
– Does not require energy
•Micronutrients:
- present in much smaller amounts - manganese, zinc, nickel
• Active Transport
•Inorganic nutrients: - Can have carbon OR hydrogen, but not both
– Molecules transported against concentration gradient
– Requires energy!
•Organic nutrients:
- Contain a carbon‐hydrogen bond
Passive Transport
• Simple Diffusion –
transport of small, neutral, hydrophobic molecules pass through membrane (H2O, CO2, O2)
• Facilitated Diffusion –
passive transport of large, charged, hydrophilic molecules
– Channel Proteins
– Carrier Proteins
Active Transport
• Carrier‐Mediated: molecules are pumped into and out of cell via protein pumps
• Group Translocation: Molecules are moved across membrane and converted into useful substance simultaneously
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Bulk Active Transport
• Transport of large molecules Microbial Nutrition
• All cells require the following for metabolism and growth:
– Exocytosis
– Endocytosis
– Carbon source
– Energy source
• Which direction do each go?
Phagocytosis‐
internalizing solid particles (ex. Bacteria)
Pinocytosis‐small particles and water are brought into the cell
Microbial Nutrition
• Growth factors (some bacteria are fastidious/picky and require extra supplements)
Microbial Nutrition
•Photo and chemo is telling us information about the energy source the organism uses:
•Hetero and auto is telling us information about whether the organism uses organic or inorganic sources for carbon: •Phototroph: microbes that photosynthesize
•Heterotroph: Organic carbon is carbon source
•Chemotroph: microbes that gain energy from ingesting and breaking down chemical compounds
•Autotroph: inorganic CO2 as its carbon source
- has the capacity to convert CO2 into organic compounds
- not nutritionally dependent on other living things
Which is most likely the category pathogens fall in?
Which is most likely the category pathogens fall in?
Microbial Nutrition: Autotrophs
• Photoautotrophs:
- Energy: Photosynthetic
- Carbon source: Produce organic molecules using CO2
- Ex: Cyanobacteria, algae
• Chemoautotrophs:
- Energy: Ingest organic or inorganic compounds for energy - Carbon source: CO2
- Ex: Archaea bacteria
Microbial Nutrition: Heterotrophs
• Photoheterotrohps:
- Energy: Photosynthetic
- Carbon source: Organic (uses carbohydrates, fatty acids and alcohols)
- Ex: Purple and green photosynthetic bacteria
• Chemoheterotrophs:
- Energy and Carbon source: organic compounds
- The vast majority of microbes causing human disease are chemoheterotrophs
- Ex: Most bacteria (that we know of), all protists, all fungi, and all animals
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Environmental (Physical) Factors Effecting Bacterial Growth
Metabolism: Building Blocks
Carbon Source
Energy Source
Photo‐
Energy from Sun
Chemo‐
Energy from chemicals
Auto‐
Carbon from inorganic molecules
Photoautotrophs
Chemoautotrophs
Hetero‐
Carbon from organic molecules
Photoheterotrophs
• 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
Chemoheterotrophs
Environmental Factors: Temperature
Temperature and Bacterial Growth
• 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
Five categories of microbes based on temperature ranges for growth
• Two gases that most influence microbial growth
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Minimum
Maximum
Psychrophile
Psychrotroph
Mesophile
Thermophile
Extreme thermophile
– Oxygen
Rate of Growth
Optimum
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Environmental Factors: Gases
• O2 has the greatest impact on microbial growth
• O2 is an important respiratory gas and a powerful oxidizing agent
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0
10
20
30
40
50
60
70
80
90
100
110
120
Temperature °C
Which category do human pathogens usually fall into? Why?
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– Carbon dioxide
• Waste for bacteria
• Carbon source for others (non‐pathogens)
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Oxygen Requirements
• As oxygen enters cellular reactions, it is transformed into several toxic products
– Oxygen is highly reactive and an excellent oxidizing agent (meaning it removes e‐ from molecules)
•Most cells have enzymes that scavenge and neutralize reactive oxygen byproducts
• Resulting oxidation causes irreparable damage to cells by attacking enzymes and proteins
•Two‐step process requires two enzymes:
Oxygen Requirements
If bacteria do not have superoxide dismutase or
catalase they can not tolerate oxygen.
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‐)
Catalase Test
Oxygen Requirements
• Obligate Aerobes
• Obligate Anaerobes
• Facultative anaerobes
• Aerotolerant anaerobes
• Microaerophiles
Microaerophiles
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
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 8
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
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?
Oxygen Requirements: Aerotolerant
• Can live with, but do not use oxygen
• Able to break down peroxides (not using catylase)
Oxygen Requirements: Microaerophiles
• Require small amounts of oxygen
• Ex. H. pylori
• Ex. Some lactobacilli and streptococci
Culturing Technique for Anaerobes
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 9
Example of the use of a selective medium for pH
Bacterial colonies
Fungal colonies
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
pH 7.3
pH 5.6
Environmental Factors: Water
• Microbes require water to dissolve enzymes and nutrients
• Water is an 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: Water pressure due to gravity (depth) 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!
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
Associations Between Organisms – Organisms live in association with different species
– Often involve nutritional interactions
• Antagonistic relationships
• Synergistic relationships
• Symbiotic relationships
Associations Between Organisms
Non symbiotic
Symbiotic
Organisms live in close
nutritional relationships;
required by one or both members.
Mutualism
Obligatory,
dependent;
both members
benefit.
Commensalism
The commensal
benefits; other
member not
harmed.
Parasitism
Parasite is
dependent
and benefits;
host harmed.
Organisms are free-living;
relationships not required
for survival.
Synergism
Members
cooperate
and share
nutrients.
Antagonism
Some members
are inhibited
or destroyed
by others.
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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?!
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
A biocontrol agent on the right (a bacteria) is making a material that is keeping the pathogen on the left (a fungus) from growing.
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
• Gum disease, dental caries, and some bloodstream infections involve mixed infections of bacteria interacting synergistically
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
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