Chapter 6 Microbial Growth. Bacterial Cell Division New cells are formed by cell fission New cells...
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Transcript of Chapter 6 Microbial Growth. Bacterial Cell Division New cells are formed by cell fission New cells...
Bacterial Cell DivisionBacterial Cell Division
New cells are formed by cell New cells are formed by cell fissionfission
Cells do not grow – they double Cells do not grow – they double their cytoplasmic contents and their cytoplasmic contents and membranemembrane
They synthesize essential They synthesize essential molecules needed for their molecules needed for their metabolic processesmetabolic processes
PartitioningPartitioning
Prior to cell division, Prior to cell division, bacteria copy their bacteria copy their DNA( replicate their DNA)DNA( replicate their DNA)
They then partition the They then partition the DNA by constructing a cell DNA by constructing a cell wall between the two wall between the two molecules of DNAmolecules of DNA
This insures that the new This insures that the new cell receives a copy of the cell receives a copy of the chromosomechromosome
The division or partitioning The division or partitioning of chromosomes is more of chromosomes is more difficult in those organisms difficult in those organisms that have more than one that have more than one chromosomechromosome
Prokaryote vs. Prokaryote vs. EukaryoteEukaryote Prokaryote cells do not go Prokaryote cells do not go
through the cell cycle like through the cell cycle like eukaryote cellseukaryote cells
They divide by fissionThey divide by fission In some species there is some In some species there is some
linkage which forms tetrads, linkage which forms tetrads, sarcinae, and even staphylococcisarcinae, and even staphylococci
GrowthGrowth Increase in cellular constituents that Increase in cellular constituents that
may result in:may result in:– increase in cell numberincrease in cell number
when microorganisms reproduce by budding or when microorganisms reproduce by budding or binary fissionbinary fission
– increase in cell sizeincrease in cell size coenocyticcoenocytic microorganisms have nuclear divisions microorganisms have nuclear divisions
that are not accompanied by cell divisions. Fungi that are not accompanied by cell divisions. Fungi have a syncytium and their nuclei are not have a syncytium and their nuclei are not separated.separated.
Microbiologists usually study population Microbiologists usually study population growth rather than growth of individual growth rather than growth of individual cellscells
The Growth CurveThe Growth Curve
Observed when microorganisms Observed when microorganisms are cultivated in are cultivated in batch culturebatch culture– culture incubated in a closed culture incubated in a closed
vessel with a single batch of vessel with a single batch of mediummedium
Usually plotted as logarithm of Usually plotted as logarithm of cell number versus timecell number versus time
Usually has four distinct phasesUsually has four distinct phases
Figure 6.1
no increase
maximal rate of divisionand population growth
population growth ceases
decline inpopulationsize
Lag PhaseLag Phase
Cell synthesizing new Cell synthesizing new componentscomponents– to replenish spent materialsto replenish spent materials– to adapt to new medium or other to adapt to new medium or other
conditionsconditions varies in lengthvaries in length
– in some cases can be very short or in some cases can be very short or even absenteven absent
Exponential PhaseExponential Phase
Also called Also called log phaselog phase Rate of growth is constantRate of growth is constant Population is most uniform in Population is most uniform in
terms of chemical and physical terms of chemical and physical properties during this phaseproperties during this phase
Each individualcell divides at aslightly differenttime
Curve risessmoothly ratherthan as discretesteps
Balanced growthBalanced growth
During log phase, cells exhibit During log phase, cells exhibit balanced growthbalanced growth– cellular constituents manufactured cellular constituents manufactured
at constant rates relative to each at constant rates relative to each otherother
Unbalanced growthUnbalanced growth
Rates of synthesis of cell Rates of synthesis of cell components vary relative to each components vary relative to each otherother
Occurs under a variety of conditionsOccurs under a variety of conditions– change in nutrient levelschange in nutrient levels
shift-up (poor medium to rich medium)shift-up (poor medium to rich medium) shift-down (rich medium to poor medium)shift-down (rich medium to poor medium)
– change in environmental conditionschange in environmental conditions
Stationary PhaseStationary Phase
total number of viable cells total number of viable cells remains constantremains constant– may occur because metabolically may occur because metabolically
active cells stop reproducingactive cells stop reproducing– may occur because reproductive may occur because reproductive
rate is balanced by death raterate is balanced by death rate
Possible reasons for Possible reasons for entry into stationary entry into stationary phasephase
nutrient limitationnutrient limitation limited oxygen availabilitylimited oxygen availability toxic waste accumulationtoxic waste accumulation critical population density critical population density
reachedreached
Starvation responsesStarvation responses
morphological changesmorphological changes– endospore formationendospore formation
decrease in size, protoplast decrease in size, protoplast shrinkage, and nucleoid shrinkage, and nucleoid condensationcondensation
production of starvation proteinsproduction of starvation proteins long-term survivallong-term survival increased virulenceincreased virulence
Death PhaseDeath Phase
cells dying, usually at exponential cells dying, usually at exponential raterate
deathdeath– irreversible loss of ability to irreversible loss of ability to
reproducereproduce in some cases, death rate slows in some cases, death rate slows
due to accumulation of resistant due to accumulation of resistant cellscells
The Mathematics of The Mathematics of GrowthGrowth Generation (doubling) timeGeneration (doubling) time
– time required for the population to time required for the population to double in sizedouble in size
Mean growth rate constantMean growth rate constant– number of generations per unit timenumber of generations per unit time– usually expressed as generations usually expressed as generations
per hourper hour
The Generation TimeThe Generation Time
The generation time for most species The generation time for most species is between twenty minutes and 24 is between twenty minutes and 24 hours.hours.
Some organisms take a longer time to Some organisms take a longer time to go through the lag phasego through the lag phase
Some organisms due to their Some organisms due to their characteristics like characteristics like Mycobacterium Mycobacterium tuberculosistuberculosis grow slowly due to the grow slowly due to the cell wallcell wall
Synchronous GrowthSynchronous Growth
Cells doubling or dividing every Cells doubling or dividing every 20 minutes20 minutes
Measurement of Measurement of Microbial GrowthMicrobial Growth
Can measure changes in number Can measure changes in number of cells in a populationof cells in a population
Can measure changes in mass of Can measure changes in mass of populationpopulation
Measurement of Cell Measurement of Cell NumbersNumbers
Direct cell countsDirect cell counts– counting chamberscounting chambers– electronic counterselectronic counters– on membrane filterson membrane filters
Viable cell countsViable cell counts– plating methodsplating methods– membrane filtration methodsmembrane filtration methods
Counting chambersCounting chambers
easy, easy, inexpensive, inexpensive, and quickand quick
useful for useful for counting both counting both eucaryotes and eucaryotes and procaryotesprocaryotes
cannot cannot distinguish distinguish living from dead living from dead cellscells Figure 6.5
Electronic countersElectronic counters
microbial suspension forced microbial suspension forced through small orificethrough small orifice
movement of microbe through movement of microbe through orifice impacts electric current orifice impacts electric current that flows through orificethat flows through orifice
instances of disruption of current instances of disruption of current are countedare counted
Electronic counters…Electronic counters…
cannot distinguish living from cannot distinguish living from dead cellsdead cells
quick and easy to usequick and easy to use useful for large microorganisms useful for large microorganisms
and blood cells, but not and blood cells, but not procaryotesprocaryotes
Direct counts on Direct counts on membrane filtersmembrane filters cells filtered through special cells filtered through special
membrane that provides dark membrane that provides dark background for observing cellsbackground for observing cells
cells are stained with fluorescent cells are stained with fluorescent dyesdyes
useful for counting bacteriauseful for counting bacteria with certain dyes, can distinguish with certain dyes, can distinguish
living from dead cellsliving from dead cells
Plating methodsPlating methods
Measure Measure number of number of viable cellsviable cells
Population Population size is size is expressed expressed as as colony colony forming forming unitsunits (CFU)(CFU)
plate dilutions of plate dilutions of population on population on suitable solid suitable solid
mediummedium
count number of count number of
coloniescolonies
calculate number of calculate number of
cells in populationcells in population
Spread PlateSpread Plate
Samples are diluted by using 1 ml of Samples are diluted by using 1 ml of broth culture and 9 ml of sterile nutrient broth culture and 9 ml of sterile nutrient brothbroth
MixMix Then 1 ml of the 1:10 ( first dilution) is Then 1 ml of the 1:10 ( first dilution) is
added to another 9ml of fresh nutrient added to another 9ml of fresh nutrient brothbroth
MixMix Samples are diluted by using 1ml of broth Samples are diluted by using 1ml of broth
culture and 9 ml of sterile nutrient brothculture and 9 ml of sterile nutrient broth MixMix
Spread plateSpread plate
A ml of each dilution is pipetted A ml of each dilution is pipetted with a plastic transfer pipet to the with a plastic transfer pipet to the center of an agar platecenter of an agar plate
A spreader( looks like a hockey A spreader( looks like a hockey stick) is used to spread the cells stick) is used to spread the cells across the surfaceacross the surface
This is designed to produce an This is designed to produce an even distribution throughouteven distribution throughout
Colony CounterColony Counter
To make an exact count of the colonies you To make an exact count of the colonies you place the plate on a gridplace the plate on a grid
You then illuminate the plate.You then illuminate the plate. You count the colonies in the grid by going You count the colonies in the grid by going
across a horizontal row and then vertically to across a horizontal row and then vertically to the next row until you have covered the whole the next row until you have covered the whole plateplate
The final count is multiplied x the dilution The final count is multiplied x the dilution factor. This number is the number of bacteria factor. This number is the number of bacteria that were in 1 ml of culturethat were in 1 ml of culture
It is assumed that each colony is equal to 1 It is assumed that each colony is equal to 1 original cell in the broth cultureoriginal cell in the broth culture
Applications of this Applications of this technique commonly technique commonly used in the laboratoryused in the laboratory Determination of coliforms in the Determination of coliforms in the
environment( E. coli)environment( E. coli) Determination of cells Determination of cells
transformed by genetic transformed by genetic engineeringengineering
Determination of bacteria Determination of bacteria contaminating soil in the contaminating soil in the environmentenvironment
Problems with colony Problems with colony counts using platescounts using plates There is error in this methodThere is error in this method If the dilutions are homogeneous, If the dilutions are homogeneous,
there can be errorsthere can be errors This may not capture all This may not capture all
organisms in a broth because organisms in a broth because some may not be able to grow on some may not be able to grow on the chosen mediathe chosen media
Pour PlatesPour Plates
Add 1 ml of a serial Add 1 ml of a serial dilution to 9 ml of dilution to 9 ml of melted and slightly melted and slightly warm nutrient agarwarm nutrient agar
MixMix Pour into a Petri dish Pour into a Petri dish
and allow it to hardenand allow it to harden Colonies will develop Colonies will develop
both in the media and both in the media and on the mediaon the media
Cells may be damaged Cells may be damaged by the hot agar in this by the hot agar in this experimentexperiment
Plating methods…Plating methods…
simple and sensitivesimple and sensitive widely used for viable counts of widely used for viable counts of
microorganisms in food, water, microorganisms in food, water, and soiland soil
inaccurate results obtained if cells inaccurate results obtained if cells clump togetherclump together
Most Probable NumberMost Probable Number
Most probable number is used for Most probable number is used for environmental samplesenvironmental samples
Trying to determine the presence of an Trying to determine the presence of an organismorganism
Use dilution factors as previously describedUse dilution factors as previously described Use multiple tubes for dilutionsUse multiple tubes for dilutions Check broth for cloudiness or turbidity( signs Check broth for cloudiness or turbidity( signs
of bacterial growth)of bacterial growth) Use culture tubes containing sugars( lactose, Use culture tubes containing sugars( lactose,
sucrose, glucose) These can be checked for sucrose, glucose) These can be checked for the presence of gas with a small tube on the the presence of gas with a small tube on the interior called a Durham tube.interior called a Durham tube.
See chart on page 149 for clarificationSee chart on page 149 for clarification
Membrane filtration Membrane filtration methodsmethods
Figure 6.6especially useful for analyzing aquatic samples
Measurement of Cell Measurement of Cell MassMass
dry weightdry weight– time consuming and not very sensitivetime consuming and not very sensitive
quantity of a particular cell constituentquantity of a particular cell constituent– protein, DNA, ATP, or chlorophyllprotein, DNA, ATP, or chlorophyll– useful if amount of substance in each cell is useful if amount of substance in each cell is
constantconstant turbidometric measures (light turbidometric measures (light
scattering)scattering)– quick, easy, and sensitivequick, easy, and sensitive
The Continuous Culture The Continuous Culture of Microorganismsof Microorganisms
growth in an open systemgrowth in an open system– continual provision of nutrientscontinual provision of nutrients– continual removal of wastescontinual removal of wastes
maintains cells in log phase at a maintains cells in log phase at a constant biomass concentration constant biomass concentration for extended periodsfor extended periods
achieved using a achieved using a continuous continuous culture systemculture system
The ChemostatThe Chemostat
rate of incoming rate of incoming medium = rate medium = rate of removal of of removal of medium from medium from vesselvessel
an essential an essential nutrient is in nutrient is in limiting limiting quantitiesquantities
Figure 6.9
Dilution rate and Dilution rate and microbial growthmicrobial growth
Figure 6.10
dilution rate – rate atwhich medium flowsthrough vesselrelative to vessel size
note: cell densitymaintained at widerange of dilutionrates and chemostat operates best at low dilution rate
The TurbidostatThe Turbidostat
regulates the flow rate of media regulates the flow rate of media through vessel to maintain a through vessel to maintain a predetermined turbidity or cell predetermined turbidity or cell densitydensity
dilution rate variesdilution rate varies no limiting nutrientno limiting nutrient turbidostat operates best at high turbidostat operates best at high
dilution ratesdilution rates
Importance of Importance of continuous culture continuous culture methodsmethods
constant supply of cells in exponential constant supply of cells in exponential phase growing at a known ratephase growing at a known rate
study of microbial growth at very low study of microbial growth at very low nutrient concentrations, close to nutrient concentrations, close to those present in natural environmentthose present in natural environment
study of interactions of microbes study of interactions of microbes under conditions resembling those in under conditions resembling those in aquatic environmentsaquatic environments
food and industrial microbiologyfood and industrial microbiology
The Influence of The Influence of Environmental Factors Environmental Factors on Growthon Growth
most organisms grow in fairly most organisms grow in fairly moderate environmental moderate environmental conditionsconditions
extremophilesextremophiles– grow under harsh conditions that grow under harsh conditions that
would kill most other organismswould kill most other organisms
Solutes and Water Solutes and Water ActivityActivity water activity (awater activity (aww))
– amount of water available to amount of water available to organismsorganisms
– reduced by interaction with solute reduced by interaction with solute molecules (osmotic effect)molecules (osmotic effect)
higher [solute] higher [solute] lower a lower aww
– reduced by adsorption to surfaces reduced by adsorption to surfaces (matric effect)(matric effect)
Osmotolerant Osmotolerant organismsorganisms grow over wide ranges of water activitygrow over wide ranges of water activity
many use many use compatible solutescompatible solutes to to increase their internal osmotic increase their internal osmotic concentrationconcentration– solutes that are compatible with metabolism solutes that are compatible with metabolism
and growthand growth some have proteins and membranes some have proteins and membranes
that require high solute concentrations that require high solute concentrations for stability and activityfor stability and activity
Effects of NaCl on Effects of NaCl on microbial growthmicrobial growth halophileshalophiles
– grow optimally at grow optimally at >0.2 M>0.2 M
extreme extreme halophileshalophiles– require >2 Mrequire >2 M
Figure 6.11
pHpH
negative negative logarithm of the logarithm of the hydrogen ion hydrogen ion concentrationconcentration
pHpH
acidophilesacidophiles– growth optimum between pH 0 and pH 5.5growth optimum between pH 0 and pH 5.5
neutrophilesneutrophiles– growth optimum between pH 5.5 and pH 7growth optimum between pH 5.5 and pH 7
alkalophilesalkalophiles– growth optimum between pH8.5 and pH growth optimum between pH8.5 and pH
11.511.5
pHpH most acidophiles and alkalophiles most acidophiles and alkalophiles
maintain an internal pH near neutralitymaintain an internal pH near neutrality– some use proton/ion exchange mechanisms to some use proton/ion exchange mechanisms to
do sodo so some synthesize proteins that provide some synthesize proteins that provide
protectionprotection– e.g., acid-shock proteinse.g., acid-shock proteins
many microorganisms change pH of their many microorganisms change pH of their habitat by producing acidic or basic waste habitat by producing acidic or basic waste productsproducts– most media contain buffers to prevent growth most media contain buffers to prevent growth
inhibitioninhibition
TemperatureTemperature
organisms organisms exhibit exhibit distinct distinct cardinal cardinal growth growth temperaturestemperatures– minimalminimal– maximalmaximal– optimaloptimal Figure 6.13
Adaptations of Adaptations of thermophilesthermophiles
protein structure stabilized by a variety protein structure stabilized by a variety of means of means – more H bondsmore H bonds– more prolinemore proline– chaperoneschaperones
histone-like proteins stabilize DNAhistone-like proteins stabilize DNA membrane stabilized by variety of membrane stabilized by variety of
meansmeans– more saturated, more branched and higher more saturated, more branched and higher
molecular weight lipidsmolecular weight lipids– ether linkages (archaeal membranes)ether linkages (archaeal membranes)
Oxygen Oxygen ConcentrationConcentration
Figure 6.15
needoxygen
preferoxygen
ignoreoxygen
oxygen istoxic
< 2 – 10%oxygen
Basis of different Basis of different oxygen sensitivitiesoxygen sensitivities oxygen easily reduced to toxic oxygen easily reduced to toxic
productsproducts– superoxide radical superoxide radical – hydrogen peroxidehydrogen peroxide– hydroxyl radicalhydroxyl radical
aerobes produce protective enzymesaerobes produce protective enzymes– superoxide dismutase (SOD)superoxide dismutase (SOD)– catalasecatalase
PressurePressure
barotolerant organismsbarotolerant organisms– adversely affected by increased adversely affected by increased
pressure, but not as severely as pressure, but not as severely as nontolerant organismsnontolerant organisms
barophilic organismsbarophilic organisms– require or grow more rapidly in the require or grow more rapidly in the
presence of increased pressurepresence of increased pressure
Radiation damageRadiation damage
ionizing radiationionizing radiation– x rays and gamma raysx rays and gamma rays– mutations mutations death death– disrupts chemical structure of many disrupts chemical structure of many
molecules, including DNAmolecules, including DNA damage may be repaired by DNA repair damage may be repaired by DNA repair
mechanismsmechanisms
Radiation damage…Radiation damage…
ultraviolet (UV) radiationultraviolet (UV) radiation– mutations mutations death death– causes formation of thymine dimers in causes formation of thymine dimers in
DNADNA– DNA damage can be repaired by two DNA damage can be repaired by two
mechanismsmechanisms photoreactivationphotoreactivation – dimers split in presence of – dimers split in presence of
lightlight dark reactivationdark reactivation – dimers excised and replaced – dimers excised and replaced
in absence of lightin absence of light
Radiation damage…Radiation damage…
visible lightvisible light– at high intensities generates at high intensities generates singlet singlet
oxygenoxygen ( (11OO22)) powerful oxidizing agentpowerful oxidizing agent
– carotenoid pigmentscarotenoid pigments protect many light-exposed protect many light-exposed
microorganisms from photooxidationmicroorganisms from photooxidation
Microbial Growth in Microbial Growth in Natural EnvironmentsNatural Environments
microbial environments are microbial environments are complex, constantly changing, complex, constantly changing, and may expose a and may expose a microorganism to overlapping microorganism to overlapping gradients of nutrients and gradients of nutrients and environmental factorsenvironmental factors
Growth Limitation by Growth Limitation by Environmental FactorsEnvironmental Factors
Leibig’s law of the minimumLeibig’s law of the minimum– total biomass of organism determined total biomass of organism determined
by nutrient present at lowest by nutrient present at lowest concentrationconcentration
Shelford’s law of toleranceShelford’s law of tolerance– above or below certain environmental above or below certain environmental
limits, a microorganism will not grow, limits, a microorganism will not grow, regardless of the nutrient supplyregardless of the nutrient supply
Responses to low Responses to low nutrient levelsnutrient levels oligotrophic environmentsoligotrophic environments morphological changes to morphological changes to
increase surface area and ability increase surface area and ability to absorb nutrientsto absorb nutrients
mechanisms to sequester certain mechanisms to sequester certain nutrientsnutrients
Counting Viable but Counting Viable but Nonculturable Nonculturable Vegetative ProcaryotesVegetative Procaryotes
stressed microorganisms can stressed microorganisms can temporarily lose ability to grow using temporarily lose ability to grow using normal cultivation methodsnormal cultivation methods
microscopic and isotopic methods for microscopic and isotopic methods for counting viable but nonculturable cells counting viable but nonculturable cells have been developedhave been developed
Quorum Sensing and Quorum Sensing and Microbial PopulationsMicrobial Populations
quorum sensingquorum sensing– microbial microbial
communication communication and cooperationand cooperation
– involves involves secretion and secretion and detection of detection of chemical signalschemical signals
Figure 6.20
Processes sensitive to Processes sensitive to quorum sensing: gram-quorum sensing: gram-negative bacterianegative bacteria
bioluminescence (bioluminescence (Vibrio fischeriVibrio fischeri)) synthesis and release of virulence factors synthesis and release of virulence factors
((Pseudomonas aeruginosaPseudomonas aeruginosa)) conjugation (conjugation (Agrobacterium tumefaciensAgrobacterium tumefaciens)) antibiotic production (antibiotic production (Erwinia carotovora, Erwinia carotovora,
Pseudomonas aureofaciensPseudomonas aureofaciens)) biofilm production (biofilm production (P. aeruginosaP. aeruginosa))
Quorum sensing: gram-Quorum sensing: gram-positive bacteriapositive bacteria
often mediated by oligopeptide often mediated by oligopeptide pheromonepheromone
processes impacted by quorum sensing:processes impacted by quorum sensing:– mating (mating (Enterococcus faecalisEnterococcus faecalis))– transformation competence (transformation competence (Streptococcus Streptococcus
pneumoniaepneumoniae))– sporulation (sporulation (Bacillus subtilisBacillus subtilis))– production of virulence factors (production of virulence factors (Staphylococcus Staphylococcus
aureusaureus))– development of aerial mycelia (development of aerial mycelia (Streptomyces Streptomyces
griseusgriseus))– antibiotic production (antibiotic production (S. griseusS. griseus))
Requirements for NitrogenRequirements for Nitrogen
Nitrogen is required for the synthesis of amino acids that Nitrogen is required for the synthesis of amino acids that compose the structure of proteins, purines and pyrimidines the compose the structure of proteins, purines and pyrimidines the bases of both DNA and RNA, and for other derivative molecules bases of both DNA and RNA, and for other derivative molecules such as glucosamine.such as glucosamine.
Many microorganisms can use the nitrogen directly from amino Many microorganisms can use the nitrogen directly from amino acids. The amino group ( NH2) is derived from ammonia acids. The amino group ( NH2) is derived from ammonia through the action of enzymes such as glutamate through the action of enzymes such as glutamate dehydrogenase.dehydrogenase.
Most photoautotrophs and many nonphotosynthetic Most photoautotrophs and many nonphotosynthetic microorganisms reduce nitrate to ammonia and assimilate microorganisms reduce nitrate to ammonia and assimilate nitrogen through nitrate reduction. A variety of bacteria are nitrogen through nitrate reduction. A variety of bacteria are involved in the nitrogen cycle such as involved in the nitrogen cycle such as RhizobiumRhizobium which is able which is able to use atmospheric nitrogen and convert it to ammonia. ( Found to use atmospheric nitrogen and convert it to ammonia. ( Found on the roots of legumes like soy beans and clover) These on the roots of legumes like soy beans and clover) These compounds are vital for the Nitrogen cycle and the incorporation compounds are vital for the Nitrogen cycle and the incorporation of nitrogen into plants to make nitrogen comounds.of nitrogen into plants to make nitrogen comounds.
PhosphorousPhosphorous
Phosphorous is present in phospholipids( Phosphorous is present in phospholipids( membranes), Nucleic acids( DNA and membranes), Nucleic acids( DNA and RNA), coenzymes, ATP, some proteins, RNA), coenzymes, ATP, some proteins, and other key cellular components. and other key cellular components.
Inorganic phosphorous is derived from Inorganic phosphorous is derived from the environment in the form of the environment in the form of phosphates. Some microbes such as phosphates. Some microbes such as E. E. colicoli can use organophosphates such as can use organophosphates such as hexose – 6-phosphates . hexose – 6-phosphates .
MixotrophyMixotrophy
Chemical energy – source organicChemical energy – source organic Inorganic H/e- donorInorganic H/e- donor Organic carbon sourceOrganic carbon source
Requirements for Requirements for Nitrogen, Phosphorus, Nitrogen, Phosphorus, and Sulfurand Sulfur
Needed for synthesis of important Needed for synthesis of important molecules (e.g., amino acids, nucleic molecules (e.g., amino acids, nucleic acids)acids)
Nitrogen supplied in numerous waysNitrogen supplied in numerous ways Phosphorus usually supplied as Phosphorus usually supplied as
inorganic phosphateinorganic phosphate Sulfur usually supplied as sulfate via Sulfur usually supplied as sulfate via
assimilatory sulfate reductionassimilatory sulfate reduction
Sources of nitrogenSources of nitrogen
organic moleculesorganic molecules ammoniaammonia nitrate via assimilatory nitrate nitrate via assimilatory nitrate
reductionreduction nitrogen gas via nitrogen fixationnitrogen gas via nitrogen fixation
Growth FactorsGrowth Factors
organic compoundsorganic compounds essential cell components (or essential cell components (or
their precursors) that the cell their precursors) that the cell cannot synthesizecannot synthesize
must be supplied by environment must be supplied by environment if cell is to survive and reproduceif cell is to survive and reproduce
Classes of growth Classes of growth factorsfactors amino acidsamino acids
– needed for protein synthesisneeded for protein synthesis purines and pyrimidinespurines and pyrimidines
– needed for nucleic acid synthesisneeded for nucleic acid synthesis vitaminsvitamins
– function as enzyme cofactorsfunction as enzyme cofactors
Bases of nucleic acidsBases of nucleic acids Adenine and guanine Adenine and guanine
are purinesare purines
Cytosine, thymine, Cytosine, thymine, and uracil are and uracil are pyrimidinespyrimidines
Also found in energy Also found in energy triphosphates( ATP triphosphates( ATP and GTPand GTP))
Practical importance of Practical importance of growth factorsgrowth factors
development of quantitative development of quantitative growth-response assays for growth-response assays for measuring concentrations of measuring concentrations of growth factors in a preparationgrowth factors in a preparation
industrial production of growth industrial production of growth factors by microorganisms factors by microorganisms
Uptake of Nutrients Uptake of Nutrients by the Cellby the Cell Some nutrients enter by Some nutrients enter by passive passive
diffusiondiffusion Most nutrients enter by:Most nutrients enter by:
– facilitated diffusionfacilitated diffusion– active transportactive transport– group translocationgroup translocation
Passive DiffusionPassive Diffusion
molecules move from region of molecules move from region of higher concentration to one of higher concentration to one of lower concentration because of lower concentration because of random thermal agitationrandom thermal agitation
HH22O, OO, O22 and CO and CO22 often move often move across membranes this wayacross membranes this way
Active TransportActive Transport
energy-dependent processenergy-dependent process– ATP or proton motive force usedATP or proton motive force used
moves molecules against the moves molecules against the gradientgradient
concentrates molecules inside cellconcentrates molecules inside cell involves carrier proteins involves carrier proteins
(permeases)(permeases)– carrier saturation effect is observedcarrier saturation effect is observed
TransportersTransporters
“Molecular Properties of Bacterial Multidrug
Transporters” – Monique Putnam, Hendrik van Veen, and Wil Konings – PubMed Central. Full Text available .
Microbiol Mol Biol Review. 2000 December; 64 (4): 672–693
ABC transportersABC transporters
ATP-binding ATP-binding cassette cassette transporterstransporters
observed in observed in bacteria, bacteria, archaea, archaea, and and eucaryoteseucaryotes
Figure 5.3
Group TranslocationGroup Translocation molecules are molecules are
modified as modified as they are they are transported transported across the across the membranemembrane
energy-energy-dependent dependent processprocess
Figure 5.5
Fe uptake in Fe uptake in pathogenspathogens The ability of pathogens to obtain iron The ability of pathogens to obtain iron
from transferrins, ferritin, hemoglobin, and from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host other iron-containing proteins of their host is central to whether they live or dieis central to whether they live or die
Some invading bacteria respond by Some invading bacteria respond by producing specific iron chelators - producing specific iron chelators - siderophores that remove the iron from the siderophores that remove the iron from the host sources. Other bacteria rely on direct host sources. Other bacteria rely on direct contact with host iron proteins, either contact with host iron proteins, either abstracting the iron at their surface or, as abstracting the iron at their surface or, as with heme, taking it up into the cytoplasm with heme, taking it up into the cytoplasm
Iron and signallingIron and signalling
Iron is also used by pathogenic bacteria Iron is also used by pathogenic bacteria as a signal molecule for the regulation of as a signal molecule for the regulation of virulence gene expression. This sensory virulence gene expression. This sensory system is based on the marked system is based on the marked differences in free iron concentrations differences in free iron concentrations between the environment and intestinal between the environment and intestinal lumen (high) and host tissues (low)lumen (high) and host tissues (low)
ListeriaListeria Pathogenesis and Molecular Virulence Determinants Pathogenesis and Molecular Virulence Determinants
José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 José A. Vázquez-Boland,1,2* Michael Kuhn,3 Patrick Berche,4 Trinad Chakraborty,5 Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Gustavo Domínguez-Bernal,1 Werner Goebel,3 Bruno González-Zorn,1 Jürgen Wehland,6 and Jürgen Kreft3Wehland,6 and Jürgen Kreft3
Pathogens and Iron Pathogens and Iron uptakeuptake
Burkholderia cepaciaBurkholderia cepacia Campylobacter jejuniCampylobacter jejuni Pseudomonas aeruginosaPseudomonas aeruginosa E. coliE. coli Listeria monocytogenesListeria monocytogenes
Iron UptakeIron Uptake ferric iron is very ferric iron is very
insoluble so uptake insoluble so uptake is difficultis difficult
microorganisms use microorganisms use siderophoressiderophores to aid to aid uptakeuptake
siderophore siderophore complexes with ferric complexes with ferric ionion
complex is then complex is then transported into celltransported into cell
Figure 5.6
ListeriosisListeriosis
One involves the direct transport One involves the direct transport of ferric citrate to the bacterial cell of ferric citrate to the bacterial cell
Another system involves an Another system involves an extracellular ferric iron reductase, extracellular ferric iron reductase, which uses siderophores which uses siderophores
The third system may involve a The third system may involve a bacterial cell surface-located bacterial cell surface-located transferrin-binding proteintransferrin-binding protein
Iron bacteria in the Iron bacteria in the environmentenvironment There are several non-disease producing There are several non-disease producing
bacteria which grow and multiply in water bacteria which grow and multiply in water and use dissolved iron as part of their and use dissolved iron as part of their metabolism. They oxidize iron into its metabolism. They oxidize iron into its insoluble ferric state and deposit it in the insoluble ferric state and deposit it in the slimy gelatinous material which surrounds slimy gelatinous material which surrounds their cells. their cells.
These filamentous bacteria grow in stringy These filamentous bacteria grow in stringy clumps and are found in most iron-bearing clumps and are found in most iron-bearing surface waters. They have been known to surface waters. They have been known to proliferate in waters containing iron as low proliferate in waters containing iron as low as 0.1 mg/l. as 0.1 mg/l.
Culture MediaCulture Media preparations devised to support preparations devised to support
the growth (reproduction) of the growth (reproduction) of microorganismsmicroorganisms
can be liquid or solidcan be liquid or solid– solid media are usually solidified with solid media are usually solidified with
agaragar important to study of important to study of
microorganismsmicroorganisms
Synthetic or Defined Synthetic or Defined MediaMedia
all all components components and their and their concentrations concentrations are knownare known
Complex MediaComplex Media contain some contain some
ingredients of ingredients of unknown unknown composition composition and/or and/or concentrationconcentration
Some media Some media componentscomponents peptonespeptones
– protein hydrolysates prepared by partial protein hydrolysates prepared by partial digestion of various protein sourcesdigestion of various protein sources
extractsextracts– aqueous extracts, usually of beef or yeastaqueous extracts, usually of beef or yeast
agaragar– sulfated polysaccharide used to solidify sulfated polysaccharide used to solidify
liquid medialiquid media
Types of MediaTypes of Media
general purpose mediageneral purpose media– support the growth of many support the growth of many
microorganismsmicroorganisms– e.g., tryptic soy agare.g., tryptic soy agar
enriched mediaenriched media– general purpose media supplemented by general purpose media supplemented by
blood or other special nutrientsblood or other special nutrients– e.g., blood agare.g., blood agar
Types of media…Types of media…
Selective mediaSelective media– Favor the growth of some Favor the growth of some
microorganisms and inhibit growth microorganisms and inhibit growth of othersof others
– MacConkey agarMacConkey agar selects for gram-negative bacteriaselects for gram-negative bacteria Inhibits the growth of gram-positive Inhibits the growth of gram-positive
bacteriabacteria
Types of media…Types of media… Differential mediaDifferential media
– Distinguish between different groups Distinguish between different groups of microorganisms based on their of microorganisms based on their biological characteristicsbiological characteristics
– Blood agarBlood agar hemolytic versus nonhemolytic bacteriahemolytic versus nonhemolytic bacteria
– MacConkey agarMacConkey agar lactose fermenters versus nonfermenterslactose fermenters versus nonfermenters
Selective and differential media
Selects for Gram –
Differentiates between bacteria based upon fermentation of lactose( color change)
Organism
Salt Tolerance
Mannitol Fermentation
1. S. aureus
Positive - growth
Positive (yellow)
2. S. epidermidis
Positive*- growth
Negative( color does not change) – no fermentation of mannitol with production of acid
3. M. luteus
Negative
N/A**
http://www.austin.cc.tx.us/microbugz/20msa.html
Web References on Media
http://www.jlindquist.net/generalmicro/102diff.html - General Reference
http://medic.med.uth.tmc.edu/path/macconk.htm - MacConkey Agar
http://www.indstate.edu/thcme/micro/hemolys.html - Blood Agar
Figure 5.7
1. dispense cells ontomedium in petri dish
2. - 3. sterilize spreader
4. spread cellsacross surface
Spread-plate Spread-plate techniquetechnique
Isolation of Pure Isolation of Pure CulturesCultures Pure culturePure culture
– population of cells arising from a population of cells arising from a single cellsingle cell
Spread plateSpread plate, , streak platestreak plate, and , and pour platepour plate are techniques used to are techniques used to isolate pure culturesisolate pure cultures
Colony Morphology and Colony Morphology and GrowthGrowth
individual individual species form species form characteristic characteristic coloniescolonies
Figure 5.10b
Terms1. Colony shape and size: round, irregular, punctiform (tiny)2. Margin (edge): entire (smooth), undulate (wavy), lobate (lobed)3. Elevation: convex, umbonate, flat, raised4. Color: color or pigment, plus opaque, translucent, shiny or dull5. Texture: moist, mucoid, dry (or rough).
Colony growthColony growth
Most rapid at edge of colonyMost rapid at edge of colony– oxygen and nutrients are more oxygen and nutrients are more
available at edgeavailable at edge Slowest at center of colonySlowest at center of colony In nature, many microorganisms In nature, many microorganisms
form biofilms on surfacesform biofilms on surfaces