Transcript Organic nutrients

Elements of Microbial Growth, Nutrition
and Environment
Do different organisms require specific
diets and environments?
Why do we care about growth?
• To Encourage the microbes we want
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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
• Pathogens
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.
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
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
Which is
bacterial
growth curve?
Four phases of growth in a bacterial culture
1. Lag Phase
• Cells are adjusting, enlarging, and synthesizing
critical proteins and metabolites
• Not doubling at their maximum growth rate
2. Exponential
Growth Phase
• Maximum
exponential growth
rate of cell division
• Adequate nutrients
• Favorable
environment
• Most sensitive to
antibiotics. Why?
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
3. Stationary Phase
• Cell birth and cell
death rates are equal
• Survival mode –
depletion in nutrients,
released waste can
inhibit growth
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
• 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: Standard Plate Counts
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.
Indirect: Turbidity Using Spectrometer
The greater the turbidity, the larger the population size.
Which culture (left or right) has more bacteria?
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
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
Indirect: Genetic Methods
• 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
What are the requirements for
microbial growth?
Chemical Composition of an Escherichia coli Cell
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
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)
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
Microbial Nutrition: Autotrophs
• Photoautotrophs:
- Photosynthetic
- Produce organic molecules using CO2
- Ex: Cyanobacteria, algea
• Chemoautotrophs:
- Ingest organic compounds for energy
- Produce organic
molecules using CO2
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
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
Osmosis: Review
•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)
Osmosis: Review
37
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
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
Temperature and Bacterial Growth
Five categories of microbes based on
temperature ranges for growth
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Minimum
Maximum
Psychrophile
Psychrotroph
Mesophile
Thermophile
Extreme thermophile
Rate of Growth
Optimum
-20
-10
0
10
20
30
40
50
60
70
80
90
100
110
120
Temperature °C
Which category do human pathogens usually fall
into? Why?
130
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
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
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 developed enzymes that scavenge and
neutralize reactive oxygen byproducts
•Two-step process requires two enzymes:
Oxygen Requirements
If bacteria do
not have
superoxide
dismutase or
catalase they
can not
tolerate
oxygen.
Catalase Test
Oxygen Requirements
• Aerobes
• Anaerobes
• Facultative anaerobes
• Aerotolerant anaerobes
• Microaerophiles
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
Oxygen Requirements: Facultative Anaerobe
• Does not require oxygen, but can grow in its
presence
• During minus oxygen 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
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
Thioglycollate broth enables the identification of aerobes, facultative
anaerobes, and obligate anaerobes.
Use of thioglycollate
broth to demonstrate
oxygen requirements.
Culturing Technique for Anaerobes
Anaerobes must grow in an oxygen minus environment, because toxic
oxygen metabolites cannot be neutralized.
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
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
Example of the use of a selective medium for pH
Bacterial colonies
pH 7.3
Fungal colonies
pH 5.6
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 and cysts cease most metabolic
activity
• Two physical effects of water
–Osmotic pressure
–Hydrostatic pressure
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
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 have
specialized over the years!
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.
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 it
-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.
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
Associations and Biofilms
– Biofilms
• Complex relationships among
numerous species of
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
Plaque
(biofilm) on a
human tooth
Biofilms: Quorum Sensing
•Quorum sensing: used by bacteria to interact with members of
the same species as well as members of other species that are
close by
•Structure of the 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 inhabitants
-cannot be eradicated by traditional methods
Now that you know more about the
nutritional needs of bacteria let’s look at
using this information to ID bacteria!
Survey of Microbial Diseases
•How to identify bacteria in patient specimens or
in samples from nature?
-
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
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
Beta-hemolysis
Blood agar as a differential
medium
Alpha-hemolysis
No hemolysis
(gamma-hemolysis)
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
Phenotypic Methods: Direct Examination
of Specimen
•Direct observation of
fresh or stained specimen
•Stains most often used
- Gram stain
- acid-fast stain
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
MacConkey Agar: Selective and Differential
Selects for Gram-negative and tells you if the bacterium
ferments lactose
Phenotypic Methods: Biochemical Testing
• Physiological reactions:
indirect evidence of
enzymes present in a
species. If bacteria tests +
for superoxide dismutase
(an oxidase) what does
that tell you?
Phenotypic Methods: Biochemical Testing
Unknown
microbe +
different
substrates
Results (+/–)
DNPG ADH LDC
–
–
+
ODC | CIT | H2S
+
–
–
URE TDA IND
–
–
+
| VP| | GEL | GLU
–
–
+
MAN INO SOR
–
–
–
RHA SAC MEL
–
–
–
AMY ARA
–
• 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
–
Flowchart: We will use this to ID our MM!
Cocci
Gram (+)
Gram (–)
Catalase (–),
pairs, chain
arrangement
Catalase (+),
irregular clusters,
tetrads
Aerobic,
oxidase (+),
catalase (+)
Anaerobic,
oxidase (–),
catalase (–)
Streptococcus
Strictly
aerobic
Micrococcus
Facultative
anaerobic
Staphylococcus
Planococcus
Neisseria
Branhamella
Moraxella
Veillonella
Phenotypic Methods: Phage Typing
-
used when morphological and biochemical tests are
insufficient. Ex. S. aureus Phage Group I vs. Group II
-
bacteriophage infect bacteria in a
species-specific and strain-specific
way, which is useful in identifying
some bacteria
-
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
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