Exam 3 Flashcards
Explain the difference between culturability and viability
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Culturability: the organism is able to grow and reproduce under the laboratory conditions you provide
- <0.1% of microbes can be cultured in a lab
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Viability: viable counts are capable of growing and reproducing
- assumption - each viable cell will grow and divide to yield one colony which means colony numbers are a reflection of cell number
- Both are methods of measurement
Explain why viable counts have limited usefulness in determining bacterial population size in environmental samples such as seawater or soils
- Some species are not culturable
- Viability is theoretical
- viable only under right conditions
- organisms can shift; may be viable under conditions you provide initially until they start starving
Describe how the most abundant organism on earth was identified and isolated
Pelagibacter Ubique was first discovered in 2002 in the Sargasso Sea.
It was first identified by sequencing the 16s rRNA gene, which was labled the SAR II gene (this was the original name of the unknown organism).
Every sea sample they took contained the SAR II gene.
Describe the abundance, size, and growth rate of Pelagibacter ubique
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Abundance:
- over a quarter of ribosomal genes in ocean water
- dominates most systems
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Size:
- VERY small genome (1.3 x 106 bp) - this is as small as you can get without becoming an obligate endosymbiont
- smallest free living and replicating organism known
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Growth rate:
- µ = 0.4 - 0.58 per day; increases 50% d -1
- ultimate oligotroph - grows slowly no matter what the conditions are
Tests used to isolate Pelagibacter ubique
- Dilution-to-extinction
- FISH (Fluorescence In Situ Hybridization)
Describe dilution-to-extinction
Dilution-to-extinction -> MPN (most probable number calculation)
- Do dilution series in culture tubes (can’t count colonies; can’t grow on agar plates)
- Dilute to extinction
- Use microscope once diluted to extinction
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Spot test (pull a little bit out -> add color reagant -> turns pink) or turbidity (optical density) is used to determine growth
- Dark tubes are positive for growth
- Use statistical (probability) tables to estimate population size
- How many positive tubes are in each column?
- take a cluster of three columns (P1, P2, P3)
- once you find that number, multiply by the middle column (P2)
- i.e. if the P2 dilution is -6, multiple by 106 cells/mL
Describe FISH
Fluorescence In Situ Hybridization
- Staining technique
- ssDNA probe has the basic sequence of the gene they know their organism has (i.e. 16s rRNA gene for SAR II gene). The probe is fluorescently labeled, so when you shine a wavelength on it, it fluoresces and reemits as a different wavelength
- Add the probe to the sample, dilute everything else out, and the cell takes up the probe. When the probe comes in contact with the homologous region on the chromosome, the gene probe will align and become incorporated
- Rinse off the probe
- Fluorescence is determined - any cell that has that gene will fluoresce
- Example: FISH is used to identify spatial arrangement of ammonium-oxidizing (AOB) and nitrite-oxidizing bacteria (NOB) in biofilms
- Fluorescent probes are used for each
- AOB shows up red
- NOB shows up green; NOB lives off the waste of AOB, so they will both fluoresce
- The fluorescent probes can often be used to identify organisms that contain a nucleic acid sequence complementary to the probe
The different growth phases of bacteria in batch culture
- Lag phase
- Log phase
- Stationary phase
- Death phase
Describe the lag phase and why it occurs
- Period of induction, adjustment, and derepression (removal of repression, such as of an operon so that gene transcription occurs or is enhanced)
- Growth happens after this phase
- May be brief or extended depending on:
- history of cells used as inocula
- composition of growth medium
- growth conditions
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When this phase is absent:
- If bacteria is transferred during the exponential growth phase (log) then there is no lag phase
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When this phase is present:
- If the bacteria are transferred from a nutrient rich to a nutrient poor environment, or if the inoculum is taken from an old culture, then this phase is present because time is needed for biosynthesis of new enzymes
- during this phase, the bacteria is rapidly synthesizing proteins
- If the bacteria are transferred from a nutrient rich to a nutrient poor environment, or if the inoculum is taken from an old culture, then this phase is present because time is needed for biosynthesis of new enzymes
Describe the log phase and why it occurs
Exponential phase
- Exponential growth rate varies
- Depends on genetics of organism and growth conditions
- smaller organisms grow faster
- It’s hard to predict growth rate until the ideal conditions are met
- Changing slope depends on time and number of cells in solution
- Exponential phase can only happen for a certain period of time
- When the population doubles at regular intervals, this is the healthiest state and most desirable for laboratory studies
slope equation for exponential phase
dN/dt = (µmax)(N)
Describe stationary phase and why it occurs
- No population growth
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Cryptic growth (hidden growth)
- death = growth
- individual cells may be growing but there is no change in population size
- occurs halfway through stationary phase
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Cryptic growth (hidden growth)
- Resources depleted and waste products accumulated
- Secondary metabolites begin to be produced
- microbes change metabolism and change some enzyme systems to deal with starvation
- kill off competition
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maintenance energy requirements
- energy is needed to replace enzymes and keep a charged membrane
- break down storage granules
- break down own enzymes
- break down RNA
- break down DNA
- energy is needed to replace enzymes and keep a charged membrane
- Length of stationary phase will determine how you measure population size
- this phase can last months to years whereas others die quickly
- species variation (i.e. ocean organisms are in a state of constant starvation, so growth rate = zero)
- this phase can last months to years whereas others die quickly
What happens to cells during the batch culture phases?
- viable and culturable cells during the lag and log phase
- viable and nonculturable cells during stationary phase (starvation)
- nonviable but alive (don’t grow or reproduce)
- dead cells
- cell fragments (lyse)
Describe the death phase and what occurs (methods of measurements)
- See a decrease in population size, but no change in density
- Occurs as an exponential function, but the death rate is much slower than the exponential growth rate (takes months to years)
- optical density delines
- plate counts decline
- metabolic stain with direct microscopic counts decline
- total microscopic counts decline (only declines when cells start to lyse)
Define psychrophile
temperature <15ºC
Define mesophile
temperature 20-50ºC
Define thermophile
Temperature >45ºC
Define hyperthermophile (extreme)
temperature >80ºC
What special adaptations allow organisms to exist in extreme environments?
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Membrane
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For high temperatures, you want higher saturation, which makes the membrane more rigid = less fluid = more stable
- want this for thermophiles
- Psychrophiles want polyunsaturated fatty acids with kinks in them = membrane less rigid = more fluid
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For high temperatures, you want higher saturation, which makes the membrane more rigid = less fluid = more stable
- Length of fatty acid (influences hydrophobic interaction)
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Enzyme activity (ability to change shape)
- If it’s too stable, it won’t change shape at cold temperatures and won’t have any activity
- If it’s too lose, it will denature at high temperatures and will be unstable
What appears to be one of the primary restrictions limiting the distribution of living organisms (temperature)?
Organisms don’t have the energy, resources, or the genetics to synthesize and maintain all of the temperature adaptations, so they have to pick a lane and stay in it
What terms describe microorganisms capable of living at pH extremes?
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Alkaliphiles
- basic
- pH optima of 8 or higher (adjust to environment)
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Neutrophiles
- optimally at a pH value in the range termed circumneutral (pH 5.5 to 7.9)
- maintain cytoplasm at 7 regardless of the outer pH
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Acidophiles
- acidic
- pH optima of 5.5 or lower (adjust to environment)
- chemolithotrophs (pH around 2)
- sulfuric acid
- ferric acid
Describe three effects of environmental pH on microbial growth
- nutrient solubility
- elemental toxicity (too soluble)
- ???
Provide an equation that describes the effect of substrate concentration on the growth rate constant.
Be able to provide appropriate units for the Monod constants, µmax and Ks
Describe, using a graph, what the constants in the equation refer to
Describe how these constants would differ between copiotrophs and oligotrophs
Give an example of a “ubiquitous” oligotroph
- Monod Equation: microbial growth rate constant relating substrate concentration
µ = (µmax[s]) / (ks+[s])
ks : substrate concentration that produces 0.5µmax
µmax : plateau of the graph
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copiotroph
- adapted to high substrate concentrations
- faster growing
- higher growth rate
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oligotroph
- adapted to low substrate concentrations
- slow growing
- ubiquitous oligotroph: Pelagibacter ubique
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Aerobes
- obligate aerobe -> cannot grow without oxygen
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facultative anaerobes -> prefer oxygen, but can use something else
- grows better in presense of oxygen because it uses oxygen as a terminal electron acceptor
- use fermentation or anaerobic respiration
- microaerophiles -> too much oxygen is toxic (because of free radicals)
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Anaerobes
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obligate anaerobe -> requires absence of oxygen
- examples: Clostridium (botulinum, tetani, perfringens, septicum)
- these produce endospores
- examples: Clostridium (botulinum, tetani, perfringens, septicum)
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aerotolerant anaerobes -> can grow in presence of oxygen, but don’t use oxygen as terminal electron acceptor
- doesn’t grow any better in presence of oxygen
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obligate anaerobe -> requires absence of oxygen
Provide several terms that describe microorganisms based on their oxygen requirements
***Describe the free radical forms of oxygen that are toxic to some microorganisms and how particular enzymes deal with these free radicals
- Free radicals of oxygen are unstable because they oxidize anything around them
- cytochrome can only carry one electron -> only putting one electron onto oxygen
O2 + e- -> O2•- (super oxide)
- Superoxide is the most common; it is more reactive than H2O2 but not as reactive as the hydroxyl radical
O2•- + e- -> O2-2 (peroxide)
- Peroxide is the weakest oxidizing species and most stable; not as damaging
O2-2 + e- -> H2O + OH• (hydroxyl radical)
- Hydroxyl radical is the strongest oxidizing species and most dangerous because it is extremely reactive
ENZYMES
- catalase
- peroxidase
- superoxide dismutase
Describe two measures of water “availability” and know how the values change as water availability changes
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Water potential (MPa)
- Way of measuring negative pressure
- the amount of pressure/suction it takes to remove water from a water binding molecule
- the drier it gets, the more suction will be required to pull water away from the sample
- more commonly used
- water potential is zero at the highest to negative infinity
- units: megapascals
- higher water potential = less solute, more water, hypotonic
- lower water potential (more negative) = more solute, less water, hypertonic
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Water activity (aw)
- humidity
- goes from 1 (pure water) to 0
- unitless
- how it’s done: put a cap on it. if water is pure, humidity will form. if water has ions in it, no humidity above sample
- = partial pressure of water sample / partial pressure of oxygen
- used in food microbiology
Define the term B-lactam, be able to identify one based on its chemical structure, and give three examples.
What action do these chemicals have on bacteria, and what is the specific mechanism by which bacteria prevent this?
- B-lactam is a broad spectrum antibiotic that blocks the process of cell wall synthesis for bacteria, causing built up pressure which leads to membrane bursting
- binding of the beta-lactam ring to DD-transpeptidase inhibits its cross-linking activity and prevents new cell wall formation by interfering with important feature called transpeptidation
- bacteria prevent this by producing beta-lactamase which destroys or “cuts” the beta-lactam ring
- binding of the beta-lactam ring to DD-transpeptidase inhibits its cross-linking activity and prevents new cell wall formation by interfering with important feature called transpeptidation
- Examples:
- penicillin
- cephalosporins
- carbapenems
Enveloped Helical Nucleocapsid
i.e. Influenza virus
Naked Icosahedral virus
i.e. Norovirus, human papilloma virus
***Describe the variation in replication and synthesis shown by different viruses
- dsDNA
- injected into cell as circular loop
- just like normal replication
- ssDNA - rolling circle replication
- 1) injected into cell
- 2) creates circular dsDNA called the replicated form
- 3) a segment of mRNA transcribes proteins
- 4) a nick is created and a strand of DNA is displaced
- 5) DNA incorporates into the protein
Name and describe the different phases of a “growth curve” for a virulent bacterial virus and ***what technique is needed to measure the length of those phases
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adsorption
- viral particles in cell until lysis and release
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latent period
- includes the eclipse (virus went inside bacteria) and early maturation phases
- viral nucleic acid replicates and protein synthesis occurs
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maturation period
- virus nucleic acid and protein are assembled into mature virions and released at the end
- number of infectious virions inside host cell rises dramatically
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burst size
- number of virion release per cell
Discuss the problem encountered by a virus that uses RNA as it genetic material, and how +ssRNA and -ssRNA viruses solve these problems
- problem: dependent on DNA template to produce RNA
- solution:
- +ssRNA produces proteins and RDRP which can create the complementary strand and undergo rolling circle replication, giving us more +ssRNA
- -ssRNA must be injected with RDRP which is used as a template for mRNA to produce proteins and RDRP, which will undergo rolling circle replication to produce more
- -ssRNA to be incorporated into protein coats of virus with one copy of RDRP
Describe a unique characteristic of retroviruses, and name one type of retrovirus
- have +ssRNA using reverse transcriptase
- all retroviruses are animal viruses
- example: HIV
