Lecture #4 - Microbial Growth Flashcards

Question

The specific growth rate (k) can be calculated using the formula:

Answer 1

k=(LogNt –LogN0) 0.301 Dt k - determine how many gen's/hr this organism is characterized by Where: • N0 = number of cells at time1 • Nt = number of cells at time2 • Dt = time2 – time1

Answer 2

k=2gen/hr 30 mins

Answer 3

of mins/gen

Answer 4

k=(Log1x107 –Log0.5x107) (0.301)(0.7) k = 1.43 gen/hr - LESS than a hour - b/c your able to produce more than 1 gen in a 60 min period

Answer 5

(g): g=1/k g=1/1.43 = 0.70 hr/gen (less than a hr to complete a gen) = 42.0 min/gen

Answer 6

specific growth rate • Different for each organism - every organism has their best - working as hard as they can under the best of conditions - if you take them from their best conditions that support their best growth & move them away from that value, the values will change (get best k & g value - due to optimal nutrient avail, optimal pH, etc. that will characterize microbial growth)

Answer 7

can DOUBLE in numbers every 10 minutes under optimal growth conditions (e.g. nice warm stew on a warming plate) - DANGEROUS for food borne illness b/c it only takes 10 mins to double from for ex 50 cells to 100 - #'s increasing exponentially in v. short amounts of elapsed time

Answer 8

LESS than 30 min in a rich medium - can do a lot of DAMAGE too - hamburger disease (v. fast), become sick when eating b/c organism will be so plentiful - or could spread up urethra to bladder, feeds off N & other things & then enters into blood which is life threatening infection

Answer 9

CANNOT grow faster than one doubling every 24 h - SLOWER than what it takes 1 of our cells to do mitosis - LONG time

Answer 10

NOTHING, even if there were 50 or 100 cells that came out of that person's sputum, b/c it only doubles every 24 hrs, you're actually leaving that plate in an incubator for weeks - long time to see any indicator of growth

Answer 11

NO - you still do it, but have to be considerate of the long growth time - do an AFS & a genetic test (look for nucleic acid sequence of mycobacterium TB & also looks for resistance patterns to see what drugs might work against the organism)

Answer 12

the antibiotic therapy is 6 months long - has to take the pills everyday for 6 months b/c its growing so slowly, so that's what it takes to effectively eliminate all of them (compliance, education & supervision is a problem) - makes organism in a high priority position for developing resis. for what it is going on - if its an antibiotic resis. strain of TB that the person has, the therapy can be as long as 2 years, but its critical b/c untreated ~2/3 of people will die

Answer 13

a CLOSED-system microbial culture of fixed volume - give bacteria: nutrients, fresh growth media & put them in & lock door & walk away - these organisms will DIE - WANT that b/c this batch culture is used to access the properties of growth think: come into lecture theatre & prof gives food, drink & takes garbage & locks door & doesn't ever come back - eventually die - b/c starve to death & waste (toxic) start to build up b/c not one's removing them

Answer 14

1. Lag phase 2. Exponential phase 3. Stationary phase 4. Death phase

Answer 15

• Interval between inoculation of a culture and beginning of growth (growth & death are =) - SLOPE OF 0 -> means organisms AREN'T increasing their # - b/c organism have just been added to GM think: just checked into hotel, you take a look around & get used to surroundings & what the hotel offers - therefore, don't start growing b/c have to syn. enzymes for growth & prepare themselves

Answer 16

it's just coming out of the spore stage (if & only if there were spores to begin with) - temp is good & has everything it needs to become a vegetative cell think: just checked into hotel, you take a look around & get used to surroundings & what the hotel offers - therefore, don't start growing b/c have to syn. enzymes for growth & prepare themselves

Answer 17

• Cells in this phase are typically in the HEALTHIEST STATE - LOG PHASE - period of exponential growth - slope is steep & (+) b/c growth > death that's occuring - eventually nutrients wear out & wastes accumulate to point where you enter into stationary phase

Answer 18

supports INDEFINITE GROWTH diff. - they live - they come back & give more food & collect waste (clean envir. to thrive in)

Answer 19

* Cells METABOLICALLY ACTIVE, but growth rate of population is ZERO * Either an essential nutrient is used up, or waste product of the organism accumulates in the medium SLOPE = 0 again - b/c growth & death are = to one another - b/c organism is leveling out due to nutrient inavail & waste accumulation

Answer 20

notices he's running out of nutrients & will die soon so he will form a spore - but one's that can't form spores will inevitably have a (-) slope for next phase b/c death > growth

Answer 21

* If incubation continues after cells reach stationary phase, the cells will eventually DIE * NOT ALL bacteria die, some bacteria form spores/cysts or dormant stages that allow a significant proportion of cells to survive for a long time DEATH > GROWTH LOG PHASE

Answer 22

an OPEN-system microbial culture of fixed volume - feed you, remove waste & repeat (consistently come & go) - can control

Answer 23

* Most common type of CONTINUOUS culture device * Both growth rate and population density of culture can be controlled INdependently and SIMULTANEOUSLY * Dilution rate: rate at which fresh medium is pumped in and spent medium is pumped out * Concentration of a limiting nutrient controls the population size and the growth rate

Answer 24

rate at which fresh medium is pumped in and spent medium is pumped out (in chemostat) - therefore dilute organisms that'll be present within

Answer 25

YOUR COLON as a chemostat - lose bacteria as you deficate, but other nutrients get brought in, the organisms that are left over use them in order to grow & increase their number

Answer 26

the population size and the growth rate (IN CHEMOSTAT)

Answer 27

- limit amount of N --> therefore organisms will NOT have enough nutrients avail. for them - can choose to limit other nutrients to observe growth - to test reliance of what that nutrient might be for that organism THEREFORE, able to CONTROL the amount of fresh media that's coming to feed them or the effluent which is what you're letting out bottom

Answer 28

CELLS & SOME NUTRIENTS (some good stuff - sacrifice b/c you're adding more fresh at top anyway & cells will double)

Answer 29

Microbial cells can be ENUMERATED by DIRECT MICROSCOPIC OBSERVATIONS (looking through microscope & see cells that you count) using a Petroff-Hausser counting chamber • Each square corresponds to a calibrated volume • Results can be UNRELIABLE (riddled with error - just an estimate)

Answer 30

1. Take volume of sample & place it on microscope slide & cover it with the cover slip 2. Hemocytometer - use squares present on plate, which allows opp. to enumerate the # cells physically present - count how many are in the cells & then use the square volume that's present to work back to find how many cells are in big box (which is # of cells per ml or cm3) - allows enumeration of BC's, but in this case we use it to enumerate bacterial cells

Answer 31

• CANNOT DISTINGUISH between live and dead cells WITHOUT special stains - might count only dead cells - might not be an indication of what's infectious - stain; if alive they will take up cells differently then if their dead - similar to way you can change properties of cells in order to access whether or not their something going on (like cervical cancer vs. non cancer) • SMALL cells can be OVERLOOKED - count is TOO LOW • PRECISION is DIFFICULT to achieve (need a lot of counts) - human error with eyes • PHASE-CONTRAST microscope required IF a stain is not used • Cell suspensions of LOW DENSITY (<106 cells/ml) hard to count - b/c they aren't v. prevalent • Motile cells need to IMMOBILIZED - so you don't count more than once * DEBRIS in sample can be MISTAKEN for cells * Cells may move (Brownian motion - dancing on the spot; may count by accident more than once), some form clumps (like staphylococcus - hard to distinguish) Based on random distribution and dispersal of the cells BUT serves purpose of being a QUICK estimate

Answer 32

Flow Cytometry is an alternative method that can be used to count the total number of cells • Uses laser beams, fluorescent dyes, and electronics EFFECTIVE - cells flow through (that are either fluorescently stained or fluorescent on own) - laser - able to enumerate them b/c of how they engage with laser & add to tally

Answer 33

- diff types of leukocytes (WBC's) - not just BACTERIAL cells, but even EUK CELLS - & can sort them to show if it has a collection of B cells for ex (b/c it puts cells in piles as it pass through detection sorter)

Answer 34

Measure only LIVING cells • Cells capable of growing to form a POPULATION (genetically identical group of cells; all same species) (eleviate problems we had from counting dead cells - dead ones are there but not growing)

Answer 35

1. Spread-plate method - spread sample around on top of plate 2. Pour-plate method - add sample to sterile empty dish & then add slightly cool molten agar into that & swish it around both get colonies that you can count

Answer 36

• Requires LOTS of preparation (dilution tubes, agar plates), and incubation time (overnight or more) to get the measurements for a single culture - req's lots of time & effort • Plate counts can be HIGHLY UNRELIABLE when used to assess total cell numbers of natural samples (Ex: soil and water - lots of contaminants) - ex: when its highly [ ]'ed - lots of bacteria - when you come back they'll be organisms everywhere & you can't count anything b/c its an overgrown mass (organism grown into 1 another) • Selective culture media and growth conditions target ONLY PARTICULAR SPECIES (means NOT a large amount of growth - only have some present on dish b/c you select against some & only provide nutrients that some don't like) - A single medium will never grow every microbe - Can only count the types of bacteria that can grow in the medium you selected to use ex: CANNOT grow Chlamydia - NEVER be included apart of your count & DOESN'T mean person isn't affected with it

Answer 37

means NOT a large amount of growth - only have some present on dish b/c you select against some & only provide nutrients that some don't like • A single medium will never grow every microbe • Can only count the types of bacteria that can grow in the medium you selected to use ex: CANNOT grow Chlamydia - NEVER be included apart of your count & DOESN'T mean person isn't affected with it

Answer 38

• DIRECT MICROSCOPIC COUNTS of natural samples REVEAL FAR MORE organisms than those recoverable on plates (a lot of variation/diversity - reason is that the organism is incapable of growing, but doesn't mean it's not there - present with that envir, but just not sure what it takes to be able to grow it) • Modern genomic techniques suggest that only 1-10% of microbial diversity is culturable from most environmental samples (including the diversity of organisms in our own microbiomes) - can be displayed as a result of you growing these organisms on a dish (only shows a small fraction of the reality of organisms that are there - play a role in health b/c we assume nothing is there b/c nothing would grow)

Answer 39

* Microscopic methods count dead cells, whereas viable methods do not * Different organisms may have vastly different requirements for growth * We do not know the specific requirements for all organisms

Answer 40

Using spectrophotometry to count bacteria • Turbidity measurements are indirect, rapid, and useful counting methods • Most often turbidity is measured with a spectrophotometer, and measurement is referred to as optical density (OD) coming diff. b/t opaqisty (b/c of turbidity of high cell # & translucency of having low cell # - you can anticipate that the amount of light that'll pass through will be diff for both those options as well)

Answer 41

bacteria do behave like small particles and absorb and scatter light

Answer 42

photocell because particles (including cells) scatter light. The larger the number of particles, the greater the absorbance, the lower the light transmission to the photocell b/c bacteria that are present within your sample or any cell for that manner are behaving exact same as any small particle would, engaging with light passing through & shunting it to diff directions get an optical density reading that you can use, that you'll have to use a partic. wavelength

Answer 43

absorbance does NOT distinguish dead cells from living cells - similarly, artifacts like bubbles, fluff, etc. will impact light flow, which in turn will impact results you get - imp. for accurate measurements that you come up with a value that's clean b/c you've been taking care to prepare you're sample

Answer 44

v. turbid (cloudy)

Answer 45

clear & relatively translucent

Answer 46

- Prism: separates everything out - see light will engage with sample *more cells present within sample, the more that light will be diverted in diff. directions b/c of all that scattering - amount of light that's gonna be able to come through as a result of what has happened is gonna be the unscattered light & it makes it to photocell where the detections are & then it'll be determined & related to what you'll sample look like - cuvette - clear tube light can pass through - bacterial cells engage with light

Answer 47

amount of light making it through the photocell will reach spectrophotometer is gonna be characteristic of the # of cells in there

Answer 48

HIGHER = HIGHER absorbance light is absorbed by organisms rather than being transmitted to the machine - engaging with physical particles which bacterial cells constitute * - means that dead & living cells are both providing a signal - similar to direct count

Answer 49

direct count - have to count whereas here, you put it in a cuvette, put in machine & reading comes up instantaneously - therefore is diff.

Answer 50

opaque (difficult to see through)

Answer 51

indicated how dense or opaque the material is * Quick and easy to perform * Typically do not require destruction (NO DAMAGE TO CELL - NO STAIN, CHEMICAL or LIGHT) or significant disturbance of sample (some spectrophotometers are specifically designed to use growth tubes as cuvette) THEREFORE PRESERVE SAMPLE - if you didn't have enough sample that's a problem

Answer 52

- whatever it is in that vial is a timepoint of that infection (for ex) that you'll never be able to emulate again - therefore, if you waste sample that'll cause limitations down the line (b/c if you run out of it you lost opp.) - more to work with is better than less (perserved & there for reuse in an add. sample)

Answer 53

standard curve must first be established to another counting method • Viable cell counts OR • Weight of biomass produced (dehydrate it) - Measured as dry cell weight corresponding to a specific volume of cell culture - all cells will have protein, nucleic acid, PL's etc. collectively this means to you that if you had x # of grams of this material, it should give you a rough estimate of the # of cells (not fully accurate but provides advan. that a direct count won't give you)

Answer 54

INCREASES | - can fit to an equation --> unknown & measure OD value to plug in as "y" (solve for x)

Answer 55

deviates from linearity - not following eq of the line - meaning if you were to plug in an OD value from a sample that would lose its accuracy - relationship of linearity isn't true anymore b/c as it becomes v. dense with bacteria you would have expected normally that as light hit it it reflected away which will decrease transmission through to detection source

Answer 56

reflected?

Answer 57

either is reflected or not

Answer 58

• Has a FINITE LINEAR RANGE of measurement - once pop. density becomes too high, the linear range of measurement is lost • Only works if the cells are EVENLY DISTRIBUTED throughout the medium (no clumps or biofilms) • CUVETTE must NOT have SCRATCHES - scratches & fingerprints will create a skewed path for light & interfere with movement of light through • Culture may need to be DILUTED when the cells are at VERY HIGH DENSITY

Answer 59

it goes straight through middle - nothing is being scattered, therefore won't get OD value that's accurate * mix sample well (will disperse) don't mix aggressively b/c it will have gas bubbles & have consequences on beh of light & huge problems with OD value

Answer 60

wouldn't be a straight line (or maybe it is, but you wouldn't know - b/c you don't have data for that, don't have data point that would've been involved in fitting the eq of the line to the curve) - if you plug 1.4 into the eq, you're making an assumption (could be error) * so take sample with OD value = 1.4 & dilute - then mix & measure again - will fit y=mx+b - multiply x-value by dilution factor is imp. to correct dilution you did

Answer 61

a specific aliquot (volume) cells are concentrated, washed to remove media components, concentrated and dried - more wash = more pure - get measurement of dry weight, which is indicative of the total cell # in a rough estimate

Answer 62

protein, DNA etc. which are proportional to the whole mass of cells b/c all cells have a fair amount of protein - *problem is that their will be more protein in 1 cell that's more metabolically active but you assume it to be an error DNA - every cell will have a single circular chromosome & its characteristic of individuals, so when you get a certain amount of DNA is an indication of how many cells might be there & is always proportional to whole cell mass

Lecture #4 - Microbial Growth Flashcards

(87 cards)