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Which nutrients only need to undergo slight modification before incorporation into cellular material during metabolism?
Most nutrients, primarily N, S and P
Which nutrients undergo many transformations before incorporation into cellular material?
Sources of carbon and energy
What is the difference between macronutrients and micronutrients?
- Macro - needed in large amounts
- Micro - required in trace amounts
What are some examples of macronutrients?
- Carbon - CO2, organic compounds
- Nitrogen - NH3, NO3-, N2, organic nitrogen compounds (amino acids etc)
- Hydrogen - H2O, organic compounds
- Oxygen - H2O, O2, organic compounds
- Phosphate - PO4 3-
- Sulfur - H2S, SO4 2-, organic S compounds, metal sulfide (FeS, CuS, ZnS etc)
- Potassium - K+ in solution or various K salts
- Magnesium - Mg 2+ in solution or various Mg salts
- Calcium Ca 2+ in solution or various Ca salts
- Sodium - Na+ in solution or various Na salts
What are some examples of micronutrients?
- Iron - Fe 2+, Fe 3+
- Trace metals
What are growth factors?
- Vitamins, amino acids, purines, pyrimidines, or other orgnic molecules that the microorganism needs for growth but cannot synthsize itself
- Some growth factors for one organism are the by-products or waste of another microorganism
What is a key component of many enzymes involved in respiration and photosynthesis?
What is a limiting nutrient?
- All microorganisms compete for it
- Iron is an example
How is iron imported in anoxic conditions?
- Iron is in the ferrous, soluble form (Fe2+)
- Can be imported by the usual transporter
How is iron imported in oxic conditions?
- Iron is in the ferric, insoluble form (Fe3+)
- Special transport systems are needed: siderophore
What is a siderophore?
- The special transport mechanism used for iron under oxic conditions
- May also be used to acquire iron at very low iron concentration or to steal it from iron-binding proteins used by another organism to acquire or store iron
How is growth of the microorganism population defined?
Increas in number of cells or biomass
How do most prokaryotes multiply?
- Binary fission - cell grows in size until forms a partition (septum) that constricts the cells into 2 daughter cells
- Each daughter cell receives one copy of the chromosome and sufficient ribosomes, and other macromolecules, monomers and other molecules to exist as an independent cell
Division in prokaryotes requires a cell division apparatus called what?
How is the divisome formed?
Formation of the divisome with the formation of a ring, composed of FtsZ, precisely at the center of the bacteria
What is the purpose of the FtsZ ring?
- It is the scaffold for the orchestration of the function needed for division
- -synthesis of new membrane material
- -synthesis of peptidoglycan (or other cell wall molecules)
Where is the divisome complex located?
What blocks the formation of the FtsZ ring?
The nucleoid, so once the nucleoids start to segregat, the FtsZ ring can be assembled
What is the overall role of MinCD and MinE proteins?
To ensure that the FtsZ ring is assembled at the center of the cells
What does MinCD specifically do?
Block formation of the ring and polymerize preferentially at the pole (determined by proteins involved in segregation of the chromosome)
What does MinE specifically do?
- Forms a ring and oscillates from pole to pole, sweeping MinCD as it moves along
- The higher concentration of MinCD is found at the pole
- On average the center of the cells has the lower concentration of MinCD
Cell division requires ____ and ____ of wall material
- Synthesis of new wall material
- Destruction of existing wall material
What role do autolysins play in cell division?
- At the FtsZ ring, autolysins create some gaps in the peptidoglycan
- This allows rearrangement of the peptidoglycan and synthesis of new cell wall material
What are wall bands?
The scar between old and new peptidoglycan
How do gram positive bacteria grow on MacConkey selective medium?
- They do not - bile salts inhibit growth of gram + bacteria
- Lactose negative
How to gram negative bacteria grow on MacConkey selective medium?
- Gram -, enteric pathogens, are able to grow
- Lactose positive
How can you tell if E. coli is present on a MacConkey plate?
E. coli forms dark pink colonies with bile precipitate
What is Mannitol-Salt agar selective towards?
- High NaCl concentration inhibits most gram - and many gram + bacteria
- Used for selection of staphylococcus (mannitol positive)
How can you tell if a Mannitol-Salt agar is posituve or negative?
- positive - yellow
- negative - pink
What percent of all microbial species have never been grown as a pure culture in the lab?
What are some factors affecting growth?
- Radiation (visible light, UV)
What are extremophiles?
Microorganisms that grow preferentially under extreme conditions
What is generation time?
Time needed for the population to double
What is generation time dependent on?
Growth medium and conditions
When conditions are right, how to microorganisms grow?
Exponentially - population will double at a constant rate
How do you calculate generation time?
- G = t/n
- g - generation time
- t - time
- n - number of generations
What is the lag phase?
Time needed by the bacteria to adjust to the new conditions
What is the exponential phase?
Doubling of the population at a constant rate
What is the stationary phase?
- Limiting nutrients are depleted or accumulation of a waste product that inhibits growth
- Growth is stopped
- No net increase in cell number
- Cells still metabolically active
- Production of "survival" systems
What is the death phase?
- Cells start to die
- Metabolism is stopped
- In some cases cell death is accompanied by cell lysis
- Death phase is also an exponential function
What are batch cultures continually being affected by?
- Metabolic activites of growing microorganisms
- Depletion of nutrients
- Generation of toxic waste
What does it mean that most natural environments are open systems?
- Constant supply of nutrients and diffusion of waste
- Competition with other microorganisms
- Changing environmental conditions
What is equilibirum in environmental systems?
- Division rate equals death rate
- Over time this is reached
What is the main factor in some cases for establishing equilibrium in environmental systems?
Concentration of a limiting growth factor
How can a constant growth rate over a long period of time be attained in the lab?
Through the use of a chemostat
What is a chemostat?
- Fresh medium, supply of limiting nutrients, is constantly supplied at a regulated flow rate into the cultute within the culture vessel, along with aeration by sterile air or other gas
- Overflow, the death of old microorganisms containted in the effluent containing microbial cells, is drained out the bottom of the culture vessel
What are the limiting nutrients in a chemostat?
What does the dilution rate depend on in a chemostat?
- Addition of fresh medium
What happens once equilibrium is reached within a chemostat?
- Number of cells is constant
- Growth rate equals death rate (washout)
When is measuring microbial growth important?
- Evaluating contamination
- In processes requiring microorganisms (beer, yogurt)
- Evaluating antimicrobial agents
- Studying population within an ecosystem
- Measuring mutation of genes
How is a microscope count done?
- Sample added to slide, on a grid with 25 large squares
- All cells counted in large square, numbers averaged with other large squares
Why are microscopic counts beneficial?
- Count all cells dead or alive and these cannot be grown in lab, viability staining can be used to differentiate between dead/alive
- Fast, no need to wait until bacteria has grown
Why are microscopic counts not always the best option?
- Small cells can be missed
- Motile cells hard to count and must be immobilized
What are the benefits of flow cytometry?
- Better at counting big cells (protozoan, yeast, mammalian, etc)
- Detection of fluorescent dyes allow labeling of specific cell types or species
- Can be used to sort cells according to size, shape, labeling, etc.
How is flow cytometry done?
- Cells put in sample stream and pushed through nozzle
- Laser shines through stream
- Stream goes through light scatter and fluorescence detector
- Induces charge on selected droplets
- Deflection plates bounce samples in desired direction
- Samples are sorted in this way into receptacle
How are viable counts done?
2 methods: spread-plate method and pour-plate method
What is the spread-plate method?
- Viable count
- Sample pipetted onto agar surface
- Sample spread evenly on agar surface using sterile glass spreader
- Count surface colonies
What is the pour-plate method?
- Viable count
- Sample pipetted into sterile plate
- Sterile medium is added and mixed well with inoculum
- Count surface and subsurface colonies
Why are serial dilutions performed?
- Bacterial culture can reach high number of cells (billions)
- To get a viable count of such cultures, must be diluted
- Results and reproducibility strongly affected by skills of technician
How is serial dilution done?
- Sample pipetted from one sample into tube with known broth
- Sample taken out of previous tube and added to next tube of broth
- Total dilution depends on how many times it is done
What is the turbidimetric method?
- Light shone on prism
- Incident light goes through filter onto sample containing cells
- Unscattered light goes onto photocell, measuring the amount of unscattered light
- Spectrophotometer reads the optical density
What does the turbidimetric method measure?
The contribution of alive an dead cells to turbidity
What is the turbidimetric method affected by?
Properties of the cells (i.e. if they clump together, if they attach to the surface, size, shape, composition, cell inclusion)
What do the results of a turbidimetric method measurement look like?
- A standard curve must be made and relationship between OD and cell number must be established empirically
- Some mutation may affect this relationship
How does temperature affect microbial growth?
- At minimum: membrane gelling, transport processes too slow for growth
- Slight warming: enzymatic reactions occuring at increasingly rapid rates
- Almost to maximum: enzymatic reactions occuring at maximal possible rate
- At maximum: protein denaturation, collapse of cytoplasmic membrans, thermal lysis
What is the order, from least to most tolerant of temperature, of names of types of microbes?
Psychrophile < Mesophile < Thermophile < Hyperthermophile
What is the temperature range for most microorganisms?
Typically 25-40°C around optimal temperature
Organisms that can grow at 0°C but have optima around 20-40°C are called what?
How do microbes adapt to cold temperatures?
- Change protein structure and sequende so enzymes are active in cold
- Transport across membrane functions optimally at low temp
- Requires modification of the cytoplasmic membrane so it stays fluid at low T
- Cold-shock proteins which help keep protein active
What are cryoprotectants?
- Antifreeze proteins, glycerol
- Help prevent the formation of ice crystals that can puncture the cytoplasmic membrane
What kills microorganisms at cold temperatures?
Not the temp itself, but ice crystals
How can microbial cultures be preserved at -80°-196°C?
In liquid nitrogen
What does barophilic mean?
Grow best at high pressure
How do microbes adapt to high temperatures?
- Change in protein sequence so the enzymes are not denatured by the high T and can stay active (heat stable enzymes)
- Transport across membrane function optimally at high T
- Requires modification of the cytoplasmic membrane so it remains stable at high T
- (Example: lipid monolayer of Archaea)
- Heat-shock proteins which help keep protein active
- Protection mechanism to ensure stability of DNA
What part of the microorganism helps resist high temperature?
How to microorganisms adapt to low pH?
- Change of cytoplasmic membrane to resist high proton concentration
- Usually the membrane requires high proton concentration for stability
- Bacteria lyse at higher pH because membrane becomes unstable
How to microorganisms adapt to high pH?
- Change of cytoplasmic membrane to resist low concentrations of protons
- Use of Na+ gradient for transport and motility
- Keep H+ pump out by the elctron chain close to the ATPase
What is the internal pH of the bacteria like at extreme pH?
- Ideally close to the pH of the environment, but:
- DNA is acid-labile
- RNA is alkaline-labile
- Internal pH limits: 4.6-9.5 (with protection system)
Why are buffers usually used in growth medium?
To keep pH steady because bacterial waste tends to affect pH
What are halophiles?
- Microorganisms that can grow at high salt concentrations
- Usually require NaCl for growth
Obligate and facultative aerobes are usually grown in liquid with what?
Constant shaking of the culture to ensure sufficient O2 concentration in medium
What does thioglycolate do?
Reduces O2 to H2O which creates a gradient of oxygen concentration
What does resazurin do?
- It is a redox indicator used in tests to differentiate oxic and anoxic zones
- It turns pink when oxidized
What are toxic forms of oxygen?
- During aerobic respiration and oxygenic photosynthesis O2 is reduced to H2O
- During this process, toxic forms of oxygen are produced
- Flavoproteins, quinone and iron-sulfur proteins, present in virtually all cells, can also reduce O2 to O2-
- Toxic forms of oxygen oxidize cell components, stopping key metabolic pathways and destroying key structures
How are aerobes and facultative aerobes resistant to toxic forms of oxygen?
Usually contain catalase and superoxide dismutase
Why can't anaerobes that contain catalase and superoxide dismutase survive toxic forms of oxygen?
If they contain these enzymes, the activity is clearly not sufficient for the cell to survive