Lecture #4 - Microbial Growth Flashcards
Growth:
measured as an increase in the number of cells
- get bigger –> increase in size of the organism
Binary fission:
cell division following enlargement of a cell to twice its minimum size
1 cell –> 2 cells
- process by which the growth occurs b/c:
1. cells are relatively constant in size
2. organism is unicellular
What happens at the SAME time for binary fission?
cell elongation, septum formation, completion of septum; formation of walls; cell separation
- v. fast & efficient
Generation time:
time required for microbial cells to DOUBLE in number
What are 2 imp. factors of generation time?
- NOT all organisms have the same generation time
2. Conditions affect this (pH, temp, salty, water avail, nutrients etc)
During cell division…
(process of binary fission) each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell
- 2 size identical & genetically identical daughter cells & have all the same comp’s needed for life
In bacteria and Archaea growth in cell size, chromosome replication and even septum formation typically occur…
SIMULTANEOUSLY (multitasking)
In bacteria and Archaea, growth in cell size, chromosome replication and even septum formation typically occur simultaneously
Contrary to Eukaryotic cells where…
growth, replication of DNA and separation via mitosis are separated into interphase and mitosis
- 1st with interphase: duplication of organelles (cell in prep for making 2)
- but then & only then do you progress into S phase - where there is replication of DNA
- then cell will change to make more of what it may not have had (D2 phase), then you go to divide by mitosis (ORDERED)
Mitosis does not occur in…
bacteria and Archaea
EUK ONLY
Most bacteria have ____ generation times than eukaryotic microbes
SHORTER
euk ~10hrs
Generation time is dependent on…
growth medium and incubation conditions: carbon source, pH, temperature, etc
- need to be met for organism to thrive, otherwise will be affected
Exponential growth
Growth of a microbial population in which cell numbers DOUBLE at a CONSTANT and SPECIFIC time interval
A relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth:
Nt =No x2n
- Nt is the final cell number
- N0 is the initial cell number
- n is the number of generations during the period of exponential growth (# of doubling)
Note: constant interval of time between doublings in this example
What is problematic with exponential growth?
it’s v. challenging to take a time point of 0 & 1 cell, & put it on a graph with a time point of 10 & have 1 million cells
- graph won’t have any meaning b/c on time scale its a small range of #’s, but on the cell # scale, you have 1 & a million & just a few # of data points & in the middle everything is really low compared to 1 million
- so, to create a meaningful, graphical representation of growth isn’t possible, so we show the log plot
Because cells increase exponentially in numbers, the…
increase in cell number is initially slow but increases at an ever faster rate following an exponential curve
• Only when plotting on a log scale can one appreciate that the cells are doubling at a constant rate
Diff. b/t graphical # of cells vs. logarithmic # of cells/reason we DON’T want to graph the # of cells…
b/c they exhibit exponential growth & as a result of exhibiting exponential growth, you see a FLAT section, where a lot is happening but we just can’t see it graphically b/c we’re trying to squeeze such expanded data points on the same y-axis
- b/t 0-100, there’s a lot going on, but its squeezed all together so we all get the jump
What is a way around using an exponential graph?
take log of the # of cells & graph that against time
- becomes clear to us that we have exponential growth (i.e. an increase in cell # over time)
- linear relationship
- can be fit to the equation of a line, y = mx+b –> allow opp. to use the x-value (time), to determine the log # of cells (y-value)
- imp. for counting (makes sense of how many cells will be present with a partic. GM)
When growth is UNLIMITED it is called _____ growth because it generates a curve whose slope ______ ________
UNLIMITED (nothing will get in the way of this growth - b/c as long as they are given nutrients & conditions that they favour; they’ll keep growing, provided that they won’t run out of their necessities)
EXPONENTIAL
INCREASES CONTINUOUSLY (constant b/c of the cells double with each gen. time that passes)
Why is exponential growth a key characteristic?
b/c they can complete binary fission
- b/c organisms can do this doubling (1 cell split in 2) every single time they generate
Growth rate (k) is…
the rate of increase in POPULATION NUMBER (ex: 10 cells initially, now we have 20 - opp. to increase cell # that can be used to gauge pop. size - # of cells present) or BIOMASS (the ORGANIC material that comprises the cell)
When cell doubles, so does the…
DNA, protein, RNA etc. (AKA the ORGANIC material increase as a conseq of doubling)
Since bacteria and archaea grow by binary fission, the growth rate is expressed as the…
number of DOUBLINGS per hour
- meaning 1 cell splits into 2
- ex: 2 doublings per hour etc., which will characterize that partic. species
Are any organisms that same in terms of growth rate?
NO 2 organisms are the same, b/c growth rate characterize a partic. species & its capabilities
Growth can also be looked as the time it takes for each cell to become 2 cells, this is called the…
generation time (g)
The specific growth rate (k) can be calculated using the formula:
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
If there’s 2 doubles per hour = k, what does that mean for the gen. time?
What does that mean about the time it takes for the doubling to occur?
k=2gen/hr
30 mins
k =
gen/hr
g =
of mins/gen
If we try the formula using this graph:
- N0 = 0.50 x 107 cells
- Nt = 1.00 x 107 cells
- Dt=4.2–3.5=0.7hr
What does the k value mean about the gen time?
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
Now that we know the growth rate (k) = 1.43 gen/hr, we can use it to calculate the generation time
(g):
g=1/k
g=1/1.43
= 0.70 hr/gen (less than a hr to complete a gen)
= 42.0 min/gen
For each organism there is a ________ that is the fastest growth rate in the best growth medium at optimal temperature
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)
Clostridium perfringens Details of Growth
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
Escherichia coli Details of Growth
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
Mycobacterium tuberculosis Details of Growth
CANNOT grow faster than one doubling every 24 h
- SLOWER than what it takes 1 of our cells to do mitosis
- LONG time
If you take a sputum sample from a person’s who’s potentially infected by TB. You spread it on a petri dish & you come back next day & see…
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
You’re worried that a patient has Mycobacterium TB. Are you gonna take a sputum sample & spread it on a petri dish & put it in an incubator to do a diagnosis?
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)
What is the treatment you use for Mycobacterium TB, in terms of the time, if you were told that antibiotics typically work against organisms that are actively growing. Is it an antibiotic therapy that’s 3 days, 5 days, or 10 days etc?
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
Batch culture:
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
Typical growth curve for population of cells grown in a closed system is characterized by four phases:
- Lag phase
- Exponential phase
- Stationary phase
- Death phase
Lag phase
• 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
LAG phase - if it was a SPORE
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
Exponential phase
• 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
Briefly describe continuous culture. What is different from the batch culture?
supports INDEFINITE GROWTH
diff.
- they live
- they come back & give more food & collect waste (clean envir. to thrive in)
Stationary phase
- 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
As a conseq. of growth & death being = to one another @ that partic. portion of the curve, what might be happening if an organism is capable of endospore formation?
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
Death phase
- 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
Continuous Culture:
an OPEN-system microbial culture of fixed volume
- feed you, remove waste & repeat (consistently come & go)
- can control
Chemostat
- 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
Dilution rate:
rate at which fresh medium is pumped in and spent medium is pumped out (in chemostat)
- therefore dilute organisms that’ll be present within
Real-life ex that can emulate dilution rate?
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
Concentration of a limiting nutrient controls…
the population size and the growth rate (IN CHEMOSTAT)
A [ ] of some limiting nutrient will serve to control the size of the pop. & what it’s able to reach…
- 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
Other than waste, what else is coming out/loosing the effluent (in chemostat)?
CELLS & SOME NUTRIENTS (some good stuff - sacrifice b/c you’re adding more fresh at top anyway & cells will double)
Microbial counts
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)
Microbial cells can be enumerated by direct microscopic observations using a Petroff-Hausser counting chamber
DESCRIBE STEPS
- Take volume of sample & place it on microscope slide & cover it with the cover slip
- 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
Limitations of microscopic counts
• 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
Flow Cytometry
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
Flow Cytometry can be used to enumerate:
- 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)
Viable cell counts:
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)
Two main ways to perform plate counts:
- Spread-plate method - spread sample around on top of plate
- 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
Issues with viable cell counts
• 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
What does, “Selective culture media and growth conditions target only particular species”, mean?
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
The great plate anomaly
• 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)
The great plate anomaly
• Direct microscopic counts of natural samples reveal far more organisms than those recoverable on plates
• 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)
Why is this?
- 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
Spectrophotometric Counts
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)
Spectrophotometric Counts are based on the fact that…
bacteria do behave like small particles and absorb and scatter light
Spectrophotometric Counts
Only a portion of the incident light makes it to the…
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
Spectrophotometric Counts
Caution:
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
If a lot of bacteria is present in sample, you’d expect the spectrophotometer to be…
v. turbid (cloudy)
If v. few bacteria is present in sample, you’d expect the spectrophotometer to be…
clear & relatively translucent
Spectrophotometric Counts process
- 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
What is the outcome of the Spectrophotometric Count?
amount of light making it through the photocell will reach spectrophotometer is gonna be characteristic of the # of cells in there
HIGHER # of cells, the OD =
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
How would Spectrophotometric Counts be diff. from a direct count?
direct count - have to count
whereas here, you put it in a cuvette, put in machine & reading comes up instantaneously - therefore is diff.
Turbid =
opaque (difficult to see through)
Turbidity measurements
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
The samples used in Spectrophotometric Counts have all kinds of origin
- 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)
To relate a direct cell count to a turbidity value (optical density), a…
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)
OD reading ___ with turbidy in the sample
INCREASES
- can fit to an equation –> unknown & measure OD value to plug in as “y” (solve for x)
How does the actual line differ from theoretical line for the Spectrophotometric Counts?
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
Higher cell density, more likely…
reflected?
Lower cell density…
either is reflected or not
Problems with optical density (spectrophotometric counts)
• 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
What is a problem if the sample diffuses to bottom?
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
OD of unknown sample is 1.4 (y-value). What would be the problem using this equation?
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
Other Counting Techniques
Total mass of cells (dry cell weight):
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
Other Counting Techniques
There are other spectrometric techniques to measure specific components of the cell:
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
