4. Microbial growth Flashcards
what are the essential elements needed for microorgs?
- which are relatively simple; slight modification needed before incorporation into cellular material?
- vs sources of (2) usually undergo many transformations
- CHNOPS + Se
- N, S, P
- carbon and energy
sources of nutrients: required in large amounts
MACRONUTRIENTS:
- where to get potassium, magnesium, calcium, sodium?
MICRONUTRIENTS: required in trace amounds
- which one is super important?
what is the 3rd category?
- from salts!
- iron! essential bc all enzymes that play a role in electron accepting/donor need Fe as cofactor! BUT a few pathogens don’t need Fe
- growth factors! ie vitamins, aa, purines, pyrimidines, other organic molecules –> microorganism needs for growth but cannot synthesize by itself! (ie fastidious bacteria)
- some growth factors are byproduct or waste of another microorg
why is it important to measure microbial presence/growth? (6)
- Identification of specific microbes in samples (ie diagnostic of infection)
- Evaluate contamination of food, water, etc.
- Ensure enough microorganisms are inoculated during process requiring them: beer, wine, yogurt, cheese, etc. (to make good bread)
- Evaluate the efficiency of antimicrobial agents.
- Study microbial populations from different ecosystems.
- Measure effect of mutation of genes involved in metabolic pathway, survival, protection, virulence, etc.
what is a selective medium?
vs differential medium
give 2 examples
- medium that restricts growth of certain microorganisms –> allows for microorgs that we want to grow better!
- differential: through production of acid ish or not –> will produce different colors on the plate (ie lactose fermenters (pink) vs lactose non-fermenters (colorless)
- MacConkey
- mannitol salt
describe selective medium: MacConkey:
- what is special –> allows growth for what?
- differentiate btw (2) –> explain
- which bacteria forms dark pink colonies with bile precipitate?
- bile salts! inhibit growth of gram pos BUT permissive for gram neg, enteric pathogens
- btw lactose fermenters (pink) and lactose non-fermenters (colorless):
*lactose –> glucose + galactose
*glucose –> glycolytic pathway –> fermentation –> lactate (lactic acid) –> reduce pH –> pink!
E.coli –> cause precipitation
selective medium: mannitol-salt:
- what is special -> allows growth for what?
- used for isolation of waht?
- differentiate btw what? (2)
- high NaCl! inhibits more gram neg and many gram pos
- of staphylococcus! (part of normal skin bacteria)
- mannitol fermenters = mannitol + (yellow) (ie staphylococcus aureus –> pathogen!) VS mannitol non-fermenters = mannitol - (pink) (ie staphylococcus epidermidis)
name the 5 different ways used to count/identify bacteria
- viable counting (spread plate method or pour plate method)
- serial dilution + plating
- microscopic counts
- flow cytometry
- turbidimetric method
what are the 2 methods to do viable counts?
- explain
- need what type of medium?
- results reliable when?
- colonies grow where?
- best for what species?
- POUR PLATE METHOD
- mix bacteria with warm agar –> pour onto sterile plate –> swirl and mix –> incubate plate until bacterial colonies grow
- need permissive growth medium
- reliable when btw 30 and 300 colonies
- grow IN agar and ON surface
- that dont require O2 (bc not much O2 in agar) - SPREAD PLATE METHOD:
- sample poured onto solid medium –> spread sample evenly on surface –> place incubated until bacterial colonies grow on surface
- need permissive growth medium
- reliable when btw 30 and 300 colonies
- grow on SURFACE of agar
- can have strict aerobes
SERIAL DILUTION
- explain method (how many mL) + dilution factor
- why do dilutions? needed for what?
- reliable number of colonies is btw what and what?
- results and reproducibility strongly affected by WHAT
- formula to calculate! (IMPORTANT)
- 1 mL of test sample + 9 ml Of broth = 1:10 dilution –> take 1mL of that 10 mL and put on plate to grow
- take 1mL of 1:10 + 9mL broth –> 1:100 dilution, and so on
- after 5 dilutions (took 1mL, 5 times) –> 1:100 000
- bacterial cultures can reach high number of cells (billions)! to get a viable count of such cultures, serial dilutions have to be made!
- btw 30 and 300
- skills of the technician!
colony forming unit (CFU) = number of colonies / (dilution_plated x volume_plated)
ie: 50 colonies/ 1:10 000 x 0.1
= 50 / 10^-4 x 10^-1 = 50 x 10^5 = 5 milllion
MICROSCOPIC COUNTS:
- using what equipment?
- usually for what types of cells?
- space btw slide and cover slip?
- 25 small squares = ____ mm^2
- how many bacteria in 1mL if you count 10 in 1 small square?
- Petroff-Hauser chamber (very fancy microscopic slid: calibrated)
- for larger cells (ie yeast, macrophages, eukaryotes)
- 0.2mm = height
- 25 small squares = 1 mm^2 –> 1 mm^2 x 0.2 mm = 0.2 mm^3
*0.2 mm^3 * 5 x 10^3= 1 cm^3 = 1mL - 10 x 25 x 5 x 10^3 = 1.25 x 10^6 cells
microscopic counts:
- what kind of cells are counted?
- what can be used to mitigate for that
- pro?
- con? (2)
- dead, alive and cells that cannot be grown in lab
- viability staining can be used to differentiate dead (red) and live (green) cells –> ie so you only count green cells
PRO: fast, no need to wait until bacteria have grown
CON: small cells can be missed + motile cells are hard to count and must be immobilized
FLOW CYTOMETRY
- explain principle
- better at counting which types of cells?
- detection of WHAT allows labeling of specific cell types or species
- can be used to sort cells according to (3)
- cells in small tube/en file indienne –> laser beam on them –> side scatter and secondary scatter detectors –> picked up by computer
- big cells! protozoan, yeast, mammalian cells, etc.
- fluorescent dyes! –> ie do a dead control, a live control, and then both at same time to see which are more green (alive) and which are more red (dead)
- size, shape, labeling
TURBIDIMETRIC METHOD:
- measure the contribution of (2) cells to __________
- describe
*affect by what?
- what (2) affects the behaviour of cells
- alive and dead cells to TURBIDITY (opaqueness)
- as bacteria grow a lot, more substances in the medium = more cloudy –> put in spectophotometer –> can measure the optical density (OD) (amount of light that is blocked)–> compare with standard curve of that specific bacteria
*OD is affected by properties of cell: size, shape, composition, cell inclusion
*a standard curve must be made and relationship btw OD and cell number (which depends on species) must be established empirically –> mutations may affect relationship! - clumping and attachment to surfaces
MICROBIAL GROWTH:
- growth of population = increase in (2) = increase in ____________
- most prokaryotes multiply by _________ _________ –> explain (2 points ish)
- increase in number of cells OR in size –> increase in biomass!
- BINARY FISSION! cells grow in size until it forms a partition (septum) that constricts the cell into 2 daughter cells
- each daughter cell receives 1 copy of the chromosome, sufficient ribosomes, macromolecules, monomers and other molecules to exist as an independent cell
- what is needed when bacteria are growing? ie cell division requires (2)
- what allows ________ subunit to be exported across cytoplasmic membrane?
- constantly breaking and remodeling peptidoglycan!!!! involves destruction of cell wall using autolysins and synthesis of new cell wall material using transpeptidation
- bactoprenol allows peptidoglycan subunit to be exported across cytoplasmic membrane
- what allows rearrangement of peptidoglycan and synthesis of new cell wall?
- similar mechanism in archaea?
at division ring (FtsZ ring), autolysins create some gaps in the peptidoglycan! (ie FtsZ ring is at middle of cell and shows where to make the cut to separate the 2 daughter cells!)
- similar mechanism in archaea with a cell wall!
- define generation time
- depends on (2)
- if I have 3 cells initially, how many cells will I have after 4 generations (use formula!)
- when the conditions are right, microorganisms can grow __________, population ________ at a constant rate
- how is population growth usually shown on a graph?
- time needed for population to double!
- growth medium and conditions
N = N_0 * 2^n
N = 3 * 2^4 = 3 * 16 = 48 cells! - exponentially –> population doubles!
- using a log scale! (so that its a straight line)
how to calculate generation time?
generation time = time/number of generation
(basically take 2 points on the line (of cells/mL (y) vs time (x) –> and calculate the slope = g
- what does batch culture mean?
- describe the 4 steps of growth cycle in batch culture
IMPORTANT for exam!
- batch culture = in a closed tube!
- LAG PHASE:
- no increase in number of living bacteria cells, slow growth
- adapting to the new environment –> ie synthesize new enzymes
- flat line - LOG PHASE:
- exponential increase in number of living bacterial cells (when conditions are perfect)
- doubling of pop at constant rate!
- straight line up - STATIONARY PHASE:
- plateau in # of living bacterial cells: rate of cell division and death roughly equal
- net growth stops, nutrients are depleted, lots of waste –> induce “survival” systems (ie if bacteria can make spores they will!)
- cells are still metabolically active!
- longer than log phase! can last for days, weeks, months, depending on species - DEATH OR DECLINE PHASE:
- exponential decrease in number of living bacterial cells
- cells start to die, metabolism has stopped
- straight line down!
most natural environments are in closed/open systems –> explain (4)
- over time, most environmental systems can reach an _______________
- in some cases, main factor is the concentration of WHAT?
OPEN systems! dynamic
- constant supply of nutrients and diffusion of waste
- competition with other microorgs
- predation (protozoans, worms)
- changing environmental conditions
- equilibrium! division rate = death rate
- concentration of a limiting growth factor
- in a lab, what can be used to keep microorgs in a constant growth rate over a long period of time?
describe - do microbes have a max growth rate?
- what controls growth rate?
CHEMOSTATS!
- fresh medium is continuously added = supply of limiting nutrients
- volume is also continuously removed –> reduces density of the culture (number of microorgs present)
*overflow = death of microorganism
- all microbes have a maximum growth rate (limit)
- concentration of limiting nutrients controls growth rate
- can most microbial species grow as pure cultures in laboratory?
- factors affecting growth (7)
- what are extremophiles?
- 99% of all microbial species have NEVER been grown as pure culture in laboratory –> cannot! bc don’t know how to do it/what they need
- nutrients, temp, pH, osmolarity, oxygen, pressure, radiation (visible light, UV light)
*most bacteria build partnerships with other microbes (ie one’s waste is used by the other) - microorgs that grow preferentially under extreme conditions (ie in exotic habitats that are over 37°C and under 20°C ish
explain the curve of how temperature can affect microbial growth rate
- at a minimum temperature: membrane gels (stops being fluid) –> transport processes so slow that growth cannot occur
- then, a steady increase in growth rate –> enzymatic reactions occurring at increasingly rapid rates! (the colder it is, the less efficient rxns are = less growth rate)
- growth rate increases until it reaches optimum temperature –> enzymatic rxns occur at maximal possible rate
- then, a VERY rapid decrease in growth rate –> reaches maximum temp (very close to optimum temp) –> protein denaturation, collapse of cytoplasmic membrane; thermal lysis
describe
PSYCHROPHILES
MESOPHILES
THERMOPHILES
HYPERTHERMOPHILES
PSYCHROTOLERANT
CRYOPHILES
- most microorgs have WHAT temp range as their optimal temperatures?
PSYCHROPHILES: -5 to 20°C ish
MESOPHILES: 15 to 45°C ish –> 37°C (“normal” bc human pathogens can grow at this temp)
THERMOPHILES: 42-80°C –> 60°C
HYPERTHERMOPHILES: 68-105°C –> 90°C
PSYCHROTOLERANT: can grow at 0°, but have optima around 20-40°C
*problem bc they can grow at fridge temp and inside your body!
CRYOPHILES: can grow at less than 0°C
- 25-40°C
- how do microorgs adapt to the cold temperatures? (5)
- what kills microorganisms in cold temp?
- microbial cultures can be preserved at ______ or _______ in liquid ____________ –> how to they not die?
- changes in protein structure and sequence so the enzymes are active at low temp
- transport across membrane functions optimally at low temp
- required mod of cytoplasmic membrane so it stays fluid at low temps
- cold-shock proteins which help keep proteins active (ie blankets)
- cryoprotectants are produced: antifreeze proteins or glycerol –> help prevent formation of ice crystals that can puncture cytoplasmic membrane
- cold temp doesn’t kill microorgs, ice crystals do!
- -80°C or -196°C in liquid nitrogen –> with anti-freeze so they can just stay there and not die
usually animals and plants live at what temperature? can survive til what?
- vs microorgs (eukaryotic)
vs prokaryote: bacteria vs archaea
- animals and plants: 37°C –> up to 50°C max
- eukaryotes: 56, 60, 62°C
- bacteria: 73, 95!
- archaea: 122°C!
*microbes will be found where they are the best at growing
what are barophilic microbes? what is another name for them?
- do microbes live in the compost?
- piezophilic: grow best at high pressure! ie bottom of ocean: hydrothermal vent: high temp and high pressure! (so you can have liquid water at 122°C)
- yes! can reach 70°C!
what are 5 microbial adaptations to high temperatures?
- Changes in protein sequence so the enzymes are not denatured by the high temp and can stay active. These enzymes are heat-stable.
- Transport across membrane functions optimally at high temperature.
- Requires modification of the cytoplasmic membrane so it remains stable at high temps (ie lipid monolayer of Archaea)
- Heat-shock proteins help keep proteins in the active conformation (chaperones help prots keep 3D shape)
- Protection mechanisms to ensure stability of DNA (GC rich). (triple bond vs AT = double bond)
does hot temperatures kill microorganisms?
- can endospores resist high temps? BUT?
yes! by thermal lysis (vs cold temps don’t kill microbes, ice crystals do)
yes! but not metabolically active
describe
- acidophile, neutrophile, alkaliphile
ACIDOPHILES:
- pH btw 1 and 5.5, average 3 ish
- volcanic soils/water, vinegar, tomatoes/fruits&veg
- many microbes that spoil f&v are acidophiles
NEUTROPHILES:
- pH btw 5.5 and 8.5 ish –> avg 7
ALKALIPHILES:
- pH btw 7.5 and 11.5 –> avg 9.5 ish
- alkaline lakes
- soap
- low protons!
- what are microbial adaptations to high pH (3) vs low pH (1)
HIGH pH:
- Changes of cytoplasmic membrane to resist low [protons] (stabilized by high pH)
- Use of Na+ gradient for transport and motility (low [protons] outside, pmf is hard to maintain)
- keep the electron transport chain close to the ATPase, so protons that are pumped out dont diffuse away
LOW pH:
- changes of cytoplasmic membrane to resist high concentration of protons. Usually, the membrane requires high concentration of protons for stability (phospholipids adapt). Bacteria lyse at higher pH, because the membrane becomes unstable.
what are the internal pH limits of a cell? why?
- what are used in growth medium to keep pH steady?
- internal pH: ideally close to the pH of the environment BUT
- DNA = acid-labile
- RNA = alkali-labile
- internal pH limits: 4.6-9.5 (with protection system, not fully understood yet)
- buffers! bc bacterial waste tends to affect pH (ie macConkey agar)
OSMOTIC EFFECT
- microorgs that can grow at HIGH salt concentrations are called ___________ –> what are 3 other categories?
- do microbes usually require NaCl for growth?
- how much NaCl % is sea water?
- HALOPHILES! btw 0 and 12% NaCl (ie Aliivibrio fischeri)
- NONHALOPHILES: 0 to 1% salt (ie E.coli)
- HALOTOLERANT: 0 to 10% (ie staphylococcus aureus)
- EXTREME HALOPHILE: 10 to >20% (ie halobacterium salinarum)
*great salt lake in Utah –> purple color is caused by high density of alga dunaliella and archaean halobacterium spp
Yes. Requires NaCl
3%: halotolerant and halophiles
factors affecting microbial growth: OXYGEN
- name the 5 different categories + relationship with O2 + type of metabolism
AEROBES:
a) OBLIGATE: require O2
- only do aerobic respiration
- ie nitrifying bacteria, sulfur bacteria, micrococcus luteus
b) FACULTATIVE: don’t require O2 but grow better with O2
- aerobic respiration + anaerobic respiration and/or fermentation
- ie E.coli
c) MICROAEROPHILIC: require O2 but at levels lower than atmospheric (ie 10% O2 saturation)
- aerobic respiration
- ie spirillum volutans and campylobacter jejuni
ANAEROBES:
a) AEROTOLERANT: don’t require O2 and grow no better with O2, but O2 not toxic for them
- fermentation
- ie streptococcus pyogenes (gives you pneumonia)
b) OBLIGATE: O2 = harmful/lethal –> lives in places where theres no O2
- fermentation or anaerobic respiration
- ie methanobacterium formicicum (sultur reducers, methanotrophs)
- which types of bacteria are usually growth in liquid with constant shaking of culture to ensure sufficient O2 concentration in medium?
- what is added to medium to create gradients of O2 concentration? what is the reaction?
- what indicator is used to differentiate which 2 zones?
- obligate and facultative aerobes!
*shaking = mix with air! - Thioglycolate! reduces O2 to H2O
- redox indicator reazurin –> pink when oxidized (oxic zone) vs normal color (anoxic zone)
- how are toxic forms of O2 produced?
- which (3) can further oxidize/reduce O2 to what?
- what do toxic forms of O2 do? (3)
what are examples of toxic forms of O2?
- during aerobic respiration, O2 is reduced to H2O –> during oxygenic photosynthesis H2O is oxidized to O2 –> during these processes, toxic forms of O2 are produced
- flavoproteins, quinone and iron-sulfur proteins, present in virtually all cells, can also reduce O2 to O2^-
- oxidize cell components –> stops key metabolic pathways + destroy key structures
- O2 + e- –> O2^- (superoxide)
- O2^- + e- + 2H+ –> H2O2 (hydrogen peroxide)
- H2O2 + e- + H+ –>H2O + OH° (hydroxyl radical)
- OH° + e- + H+ –> H2O
Reactive oxygen species!
what do aerobes and facultative aerobes usually have to resist toxic forms of O2?
- do anaerobes also have that?
- catalase, peroxidase, superoxide dismutase! –> detoxify ROS
- anaerobes may or may not contain such enzymes –> if they do, activity is clearly not sufficient to allow org to grow under oxic conditions –> may be sufficient for cell to survive
