Topic 6 - Culture and Control Flashcards
metabolism - catabolism =?
releases energy
metabolism - anabolism =?
consumes energy
catabolism and anabolism
CATABOLISM
from: chemicals, light (energy source)
towards:
- energy for biosynthesis
- energy for motility, transport of nutrients, etc
- waste products
ANABOLISM
from: nutrients for biosynthesis
towards:
(biosynthesis) -> macromolecules and other cell components
nutritional requirements!
macronutrients
- required by ALL cells to build macromolecules always
- C, N, P, S, O
micronutrients
- required by some cells (like for enzyme parts)
- incl. Fe, Cu, Na, Mg, Mn, etc
metabolism naming steps/parts (3)
- energy source
- electrons
- carbon source
1) photo/chemo
2) litho/organo
3) hetero/auto
troph
- energy source
- for oxidation, providing electrons for ETC
PHOTO (light energy, photosynthetic: organic or inorganic e-)
or
CHEMO (chemical energy: organic or inorganic e-)
- electrons
ORGANO (organic) (e.g., glucose, acetate, things with C)
or
LITHO (inorganic) (e.g., H2, NH3, H2O)
- carbon source
- for cell maintenance and division
HETEROTROPH (fixed, organic, C-C bonds)
or
AUTOTROPH (gaseous, inorganic, CO2)
nutrition: humans are?
chemoorganoheterotrophs
nutrition: cyanobacteria are?
photolithoautotroph
energy sources (chemoorganotrophs, chemolithotrophs, phototrophs)
chemoorganotrophs
- energy and electrons from oxidation of organic compounds
- i.e. glucose + O2 -> CO2 + H2O
chemolithotrophs
- energy and electrons from oxidation of inorganic compounds
- found only in prokaryotes
- i.e. H2 + O2 -> H2O
phototrophs
- energy from light captured by pigments
- may be oxygenic or anoxygenic (don’t make oxygen, e.g., oxidize Fe)
carbon sources (autotrophs/heterotrophs)
carbon needed for energy storage/manipulation, structure
autotrophs
- not all phototrophs! chemoautotrophs exist
- “primary producers”
- fix C directly from CO2
heterotrophs
- use organic molecules produced by autotrophs
acquisition of N: assimilation of ammonia into ____ is most common? with what enzyme?
assimilation of ammonia into glutamate/glutamine is most common!
glutamine synthetase
what is N needed for?
- AAs
- nucleic acids
key nutrient available in ___ amount will limit growth
lowest
obligate aerobes
require O2
microaerophiles
grow best in low lvl of O2
aerotolerant anaerobes
not harmed by O2 but don’t use it
obligate anaerobes
can’t grow when O2 is present
facultative anaerobes
can grow in absence of O2, grows better with O2
why are obligate anaerobes affected by O2?
its defenses!
- oxidizing agents: oxidizes things, is toxic
- some lipids can be repaired but some electrons are permanently taken
catalase test
- some microorganisms produce catalase
- pos test = bubbles
- neg test = nothing
- H2O2 + H2O2 -> 2H2O + O2
effects of pH
- affects macromolecule structures and transmembrane electrochemical gradients
- each microbe has optimal pH range
- regardless of pH preference, INTRACELLULAR pH stays ~NEUTRAL (some lil low/high in pH extremes)
acidophiles =?
love acid, low pH
neutrophiles =?
neutral pH
alkalophiles
love alkaline, high pH
water activity
aw
- interactions with solutes can decrease aw values
- pure water (1.0) has higher aw than seawater
- most bacteria need aw > 0.9
water activity formula
aw = vp of air in equilibrium with substance or solution / vp of air with pure water
- vp of air with pure water would be 100%, max value
- measure of available water
gram-positive bacteria are bit better at ____ aw survival
lower
fungi tend to be ____ (dry, low aw)
xerophiles
cytoplasm typically a ___ solute conc than the external env
higher!
water tends to move into cell
positive water balance
water loss prevented by ____ internal solute conc
+ how (2)?
increasing
- pumping in inorganic ions from env
- synthesis/concentration of organic solutes
what can temp affect? (3)
- macromolecular structure, membrane fluidity, enzyme func
why are eukaryotes limited to mostly being mesophiles or psychrophiles?
- eukarya have mitochondria which aren’t thermophiles, so limited to med/low temp range
thermophile / mesophile / psychrophile, how is it determined?
thermophile - hot
mesophile - med
psychrophile - cold
determined through optimal temp
effects on cell over optimal temp curve
min - membrane gelling; transport processes so SLOW that growth cannot occur
middle - enzymatic rxn occurring faster at higher temp
optimum - enzymatic rxn occurring at max rate
STEEP drop into max - protein denaturation; collapse of cytoplasmic membrane; thermal lysis
psychrophiles temp range
min <0 deg C
optimum <15 deg C
max ~20 deg C
ex. ocean water
- often v sensitive to moderate temp (enzymes denature)
- higher proportion of unsaturated fatty acids in membrane phospholipids than mesophiles
(kinks discourage gelling in membrane)
Psychrotolerant temp range (how are they diff from psychrophiles?)
- can grow ~0-4 deg C
- optimum 20-40 deg C (why they are diff from psychrophiles)
- mesophiles capable of low-temp growth
- found in temperate climates, many soil microorganisms
hyperthermophiles
- ex. supervolcano
- ~50-100 deg C, some go up to 350
features that provide thermal stability
- critical AA substitutions in key locations produce heat-tolerant folds
- increases in ionic bonds between acidic and basic residues: resists unfolding in aqueous cytoplasm
- certain solutes help stabilize proteins (Di-inositol phosphate, diglycerol phosphate)
heat-related modifications in cytoplasmic membrane
- saturated fatty acids in bacteria (decrease fluidity) when hot
- phospholipid monolayer in archaea more thermotolerant than bilayers
agar media
- polysaccharide derived from algae
- gel at certain temp
- not degraded by most microbes
- solidifying agent in micro media
what components immediately makes a medium complex?
yeast extract
peptone/tryptone
media types (2)
complex media
defined media
specialized media (selective/differential/enriched)
selective
- allows for isolation of microbes w/ specific properties
differential
- allows certain microbes to be recognized based on visual reactions in medium
enriched
- used to increase a population of microbes with a specific property
Selective AND differential media (3)
might be FYI
brilliant green
eosin methylene blue (EMB)
MacConkey
3 methods for separating cells on a plate (for isolated colony)
- streak plate method
- spread plate method
- pour plate method
spread plate vs pour plate
- spread plate uses less bacterial suspension and its on top
- pour plate uses more bacterial suspension and has colonies inside and on top of the agar
3 ways of quantifying microbe
direct counts
viable cell counts
turbidity measurements
direct count
- known volume is loaded onto slide with a grid
- cells counted under light microscope
- cheap, fast, easy
- can’t differentiate between dead/viable cells
viable cell count
serial dilutions and CFUs
- culture diluted in a series of tubes then plated
- after incubation colonies are counted
CFUs
colony-forming units
- CFUs per mL of initial culture calc through multiplying CFUs by inverse of dilution factor
- 25-250 units on a plate for counting is good
what if cells are too diluted?
- a filter apparatus can concentrate the cells
turbidity
spectrophotometer sends light through a culture
- light absorbance can give rough measure of cell density in tube
microbial growth curve (what scale? measured with? 4 stages?)
- measured w spectrophotometer
- logarithmic scale
4 basic phases
- lag phase (microbes preparing for steady growth)
- exponential phase (replicating at a constant and steady exponential rate)
- stationary phase (replication has halted increasing or it’s equal to death rate)
- death phase (nutrients depleted, waste levels high, cells dying at steady exponential rate)
what is generation time?
time to double the population in exponential phase
what is growth rate?
number of generations/unit of time (inverse of generation time)
what is growth yield?
maximum population density and/or amount of cellular material produced by culture
controls of microbial growth (4)
filtration
temp
radiation
chemical control
typical filters (2) & size
nylon/teflon filters with pore size of 0.2 or 0.45 um
How can viruses be filtered out? what’s the filter size? what does it require?
ultrafiltration
- reducing pore size10-100 nm
- requires high pressure
0.2 um pore size common; removes particles from ___ and ___
ideal for?
liquids and gasses!
ideal when material is heat- or radiation-sensitive
3 types of filters
- depth filter
- conventional membrane filter
- nucleopore filter
depth filter
- fibrous sheet or mat
– randomly overlapping fibers of diff substances (e.g., paper, glass, like filter paper) - used as a pre-filter
- BIG pore size, NOT for sterilization
“trapping action”
conventional membrane filter
- used for ROUTINE STERILIZATION
- polymer filter (0.45 or 0.22 um)
– cellulose acetate or cellulose nitrate - pore diameter variable
“sieve-like action)
nucleopore filters
- not default for sterilization
- for capturing cells for IMAGING
- thin polycarbonate film (~10 um thick)
– made w radiation, cracks enlarged by chemical “etching”
– consistent pore size - used for microscopy (filtered material on a single surface plant (1 depth))
what is used filter small volumes vs big volumes
small vol. = syringe filters
big vol. = peristaltic pump
___ heat is more efficient than ____ heat
moist (e.g., steam)
dry
how does heat sterilize things? how about autoclave? potential problems?
- denatures proteins and nucleic acids
- 100 deg C kills most microbes quickly
- an autoclave adds pressure, keeping fluids from evaporating during high temp
problems: hyperthermophiles, endospores, some material can’t be heated
autoclave (conditions, efficiency affected by)
- steam under pressure (pressure cooker)
- 121 deg C, 15 psi above atm
- efficiency determined by:
– destruction of endospores
– vegetative cells
T/F pressure helps to kill microbes along with heat in an autoclave
False! Just heat
how long is sterilization time? does it change?
always 15 mins
what would happen if an autoclave’s pressure was relieved too quickly?
liquids will boil
3 stages of autoclave cycle
- autoclave time
- sterilization
- exhaust
T/F pasteurization sterilizes things
False, only reduces microbial load!
Pasteurization (purpose, 3 processes)
reduces microbial load
- destroys pathogens; 90-99% other microbes killed
- increases shelf life but does not sterilize
common process: high-temp short time (HTST)
- 72 deg C for 15sec
other processes:
- UHT (ultra high temp): 135 deg C for <1 sec
- ESL (extended shelf life): filtration, then lower temp treatment
what’s the purpose of low heat and freezing in pasteurization?
lower heat - reduces microbe numbers
freezing - can damage cells by forming ice crystals, can stop biochemical rxn
- good for long-term preservation
electromagnetic radiation
for “sanitization” not really sterilization
- UV radiation of 260-280 nm wavelengths
- can dmg DNA by forming thymine dimers (can kill cell)
- exploited to control microbial growth on non-living surfaces and in water
ionizing radiation
sterilization! dependent on dose
- protein dmg
- DNA dmg
– double strand breaks
– stray e-
– hydroxide ions
– hydride radicals
- higher energy (measured in Grays Gy), better penetration
- limited to large industrial operations
(ex. medical supplies, grafting tissue)
chemicals (Antiseptics, disinfectants, antibiotics)
antimicrobial action
- agents target diff groups
– microbicidal, bacteriocidal, fungicidal, algicidal, viricidal
- agents vary wrt selective toxicity
– non-selective
– selective
selective agents useful for treating disease
non-selective agents are NOT for internal use
definitions: bacteriostatic/bacteriocidal/bacteriolytic
bacteriostatic - growth inhibitory (same viable cell count, same total cell count)
bacteriocidal - kills cells (no viable cell count, same total cell count)
bacteriolytic - causes cell lysis (no viable cell count, no total cell count)
*can be conc-dependent, not just on chemical identity
disinfectants vs antiseptics
disinfectants: used on NON-LIVING surfaces to kill potentially infectious microbes
antiseptics: used on LIVING tissue to kill potentially infectious microbes (topical only usu)
what makes a chemical a good microbe killer?
- kills wide range of microbes
- not corrosive or too toxic
- doesn’t leave residue or emit fumes
- cheap
- temp stable
what are antibiotics?
- antimicrobial agents produced by microbes
- cidal, static, or lytic (conc-dependent)
- most work by binding to proteins or cellular organelles and disrupt essential functions necessary for growth and survival of microorganisms
details about tetracycline, polymyxin B, penicillin!
broad spectrum: tetracycline
narrow spectrum: polymyxin B, penicillin
- pencillin: inhibit cell wall synthesis (gram pos)
- tetracycline: disrupt protein synthesis
- polymyxin b: disrupt cell outer membrane (gram neg)
how measure effectiveness of killing targe organism
decimal reduction time (D value)
- time require to kill 90% of target organism
(reducing pop size by a decimal)
