Prokaryotic Growth Kathy Huschle Northland Community & Technical College

ProkaryoticProkaryoticGrowthGrowth

Kathy HuschleKathy HuschleNorthland Community & Technical CollegeNorthland Community & Technical College

Pure CulturesPure Cultures

pure culture: population pure culture: population of organisms descended of organisms descended from one organismfrom one organism– only approximately 1% only approximately 1%

of all bacteria can be of all bacteria can be cultured successfully cultured successfully in the labin the lab

Vibro chlorae

Pure CulturePure Culture

colony, clonecolony, clone– begins with a single bacterial cell placed on a solid begins with a single bacterial cell placed on a solid

medium such as agarmedium such as agar

agaragar– provides specific nutrition for bacteria and a provides specific nutrition for bacteria and a

medium to grow onmedium to grow on

Nutritional AgarColonies on agar

Binary FissionBinary Fission

method of bacterial method of bacterial reproductionreproduction

cell divides exactly in halfcell divides exactly in half– single cell divisionsingle cell division– reproduction of the reproduction of the

entire organismentire organism

Binary FissionBinary Fission

asexualasexual

no genetic recombinationno genetic recombination– the DNA molecule the DNA molecule

replicates itself when replicates itself when bacterial reproduction bacterial reproduction takes placetakes place

E. coli undergoing cell division

Bacterial GrowthBacterial Growth

bacterial growth = bacterial cell reproductionbacterial growth = bacterial cell reproduction

the process of binary fission doubles the population each the process of binary fission doubles the population each time binary fission takes placetime binary fission takes place– this doubling time demonstrates exponential growththis doubling time demonstrates exponential growth

each generation results in a doubling of the population each generation results in a doubling of the population generation time is = to doubling timegeneration time is = to doubling time– measure of microbial growth ratemeasure of microbial growth rate

Bacterial Growth Curve:Bacterial Growth Curve:laboratory conditionslaboratory conditions

bacterial growth bacterial growth generally follows a generally follows a characteristic patterncharacteristic pattern

– 5 phases5 phases– normal growth curve, normal growth curve,

with optimum with optimum environmental and environmental and nutritional conditionsnutritional conditions


lag phaselag phase– no increase in cell no increase in cell

numbersnumbers– cells are adapting to cells are adapting to

the environment the environment – cells are preparing for cells are preparing for

reproductionreproduction

synthesizing new synthesizing new DNA, etc.DNA, etc.


log phaselog phase– exponential phaseexponential phase– maximal rate for maximal rate for

reproductionreproduction

this happens with a this happens with a specific set of growth specific set of growth conditionsconditions

those resources for those resources for growth are abundantly growth are abundantly available available


stationary growth phasestationary growth phase– maximum population for maximum population for

the resources availablethe resources availablerequired nutrients required nutrients become depletedbecome depletedinhibitory end inhibitory end products from cell products from cell metabolism metabolism accumulateaccumulate

– cell growth = cell deathcell growth = cell death


death phasedeath phase– cell death > new cell cell death > new cell

formationformation


phase of prolonged phase of prolonged declinedecline– can last from months can last from months

to yearsto years– ““survival of the fittest”survival of the fittest”

Solid MediaSolid Media

on solid mediaon solid media– cells do not disperse readilycells do not disperse readily– nutrients become limited in centernutrients become limited in center– death phase occurs in the center with death phase occurs in the center with

exponential phase at periphery of the exponential phase at periphery of the bacterial colonybacterial colony


most lab organisms are grown in a most lab organisms are grown in a batch culturebatch culture– closed systemclosed system

new materials are not addednew materials are not added

waste products are not removedwaste products are not removed– under these conditions bacteria populations follow under these conditions bacteria populations follow

distinct patterns of growthdistinct patterns of growth

Algae batch cultures


continuous culture maintainedcontinuous culture maintained– nutrients must be continually nutrients must be continually

suppliedsupplied– end products must be end products must be

removed removed – exponential growth phase exponential growth phase

maintainedmaintained

Continuous culture in lab

Natural ChemostatNatural Chemostat

chemostatchemostat– continuous continuous

culture culture devicedevice

A cow, with it’s four stomachs, is natures perfect chemostat; constantly grazing to add nutrients and continually belching and other such mechanics to remove bacterial metabolic end products

Environmental Parameters:Environmental Parameters:influencing bacterial growthinfluencing bacterial growth

not all bacteria favor the same environmental conditionsnot all bacteria favor the same environmental conditions– the effects of varying conditions are seen as the effects of varying conditions are seen as

differences in reproduction (bacterial growth)differences in reproduction (bacterial growth)

some environmental conditions that can affect bacterial some environmental conditions that can affect bacterial growth includegrowth include– temperaturetemperature– oxygenoxygen– salinitysalinity– pHpH

Environmental Influencing Factors:Environmental Influencing Factors:temperaturetemperature

temperaturetemperature– ideal temperature for growth varies ideal temperature for growth varies

between organismsbetween organisms– specified by the bacterial genomespecified by the bacterial genome


– temperature growth rangetemperature growth range

minimum to maximum temperatures for bacterial minimum to maximum temperatures for bacterial growthgrowth

– optimal growth temperatureoptimal growth temperature

temperature at which the highest rate of temperature at which the highest rate of reproduction occursreproduction occurs


5 divisions of 5 divisions of prokaryotes, based on prokaryotes, based on optimal growth optimal growth temperaturetemperature– psychrophilespsychrophiles– psychrotrophspsychrotrophs– mesophilesmesophiles– thermophilesthermophiles– hyperthemophileshyperthemophiles

Psychrophile:Desulfofaba gelida

Thermophile:Pyrococcus sp.

Hyperthermophile:Thermococcus barophilus


psychrophilespsychrophiles– optimum growth temperature: -optimum growth temperature: -

5500C – 15C – 1500CC– found in the Arctic and Antarctic found in the Arctic and Antarctic

regions of the worldregions of the world

Bacteria found in meltfrom a Russian outposton Lake Vostok

Desulfofrigus oceanense


psychotrophspsychotrophs– optimum growth optimum growth

temperature: 20temperature: 2000C – C – 303000CC

will grow at lower will grow at lower temperaturestemperatures

– most commonly found most commonly found in refrigerated food in refrigerated food spoilagespoilage

Stemphlium sarcinaeforme


mesophilesmesophiles– optimum growth temperature: optimum growth temperature:

252500C – 45C – 4500CC

most human pathogens are most human pathogens are mesophilesmesophiles

– adapted well to growth in adapted well to growth in the human body, whose the human body, whose normal temperature is normal temperature is around 37around 3700CC

Salmonella


thermophilesthermophiles– optimum temperature: 45optimum temperature: 4500C – 70C – 7000CC– commonly found in compost heaps commonly found in compost heaps

and hot springs, water heatersand hot springs, water heaters

Sulfur pots in Yellowstone

Sulfolobus

Thermophile in a hot spring


hyperthermophileshyperthermophiles– optimum growth optimum growth

temperature: 70temperature: 7000C – 110C – 11000CC– usually member of the usually member of the

ArchaeArchae domain domain– found in hydrothermal vents found in hydrothermal vents

in the depths of the oceanin the depths of the ocean

Deep Sea Vent

Temperature RangesTemperature Ranges

psychrophilespsychrophiles– -5-500 C to 15 C to 1500 C C

psychotrophspsychotrophs– 202000 C to 30 C to 3000 C C

mesophilesmesophiles– 252500 C to 45 C to 4500 C C

thermophilethermophile– 454500 C to 70 C to 7000 C C

hyperthermophileshyperthermophiles– 707000 C to 110 C to 11000 C C

Temperature ConsiderationsTemperature Considerations

food preservationfood preservation– refrigerationrefrigeration

inhibits fast growing inhibits fast growing mesophilesmesophiles

– psychrophiles can still grow in psychrophiles can still grow in refrigeration, but at a refrigeration, but at a diminished ratediminished rate

– freezing destroys freezing destroys microorganisms that require microorganisms that require water to growwater to grow

Temperature ConsiderationsTemperature Considerations

diseasedisease– body temperature varies: extremities are usually body temperature varies: extremities are usually

cooler than 37cooler than 3700C C – some microorganisms can cause disease in certain some microorganisms can cause disease in certain

body parts but not in others due to variations in body body parts but not in others due to variations in body temperaturestemperatures

Environmental Influencing Factors: Environmental Influencing Factors: oxygenoxygen

oxygen levels vary between environments and within the oxygen levels vary between environments and within the same environmentsame environment

based on Obased on O22 requirements, prokaryotes are separated into requirements, prokaryotes are separated into

the following groupsthe following groups– obligate aerobesobligate aerobes– obligate anaerobesobligate anaerobes– facultative anaerobesfacultative anaerobes– microaerophilesmicroaerophiles– aerotolerant anaerobesaerotolerant anaerobes


obligate aerobesobligate aerobes– need oxygen present need oxygen present

to multiplyto multiply

Giardia


obligate anaerobesobligate anaerobes– cannot multiply in the presence of oxygencannot multiply in the presence of oxygen– often killed by traces of oxygen in their environmentoften killed by traces of oxygen in their environment

C. perfringens


facultative anaerobesfacultative anaerobes– grow best with oxygen, but can grow best with oxygen, but can

grow without oxygengrow without oxygen– respiration if oxygen is availablerespiration if oxygen is available– fermentation if no oxygen is fermentation if no oxygen is

presentpresent– growth is greater in the presence growth is greater in the presence

of oxygen due to the production of oxygen due to the production of more ATP (energy source of of more ATP (energy source of the cell)the cell)

Aeromonas hydrophiliaon intestinal cells


microaerophilesmicroaerophiles– require oxygen but have maximal require oxygen but have maximal

growth at reduced oxygen growth at reduced oxygen concentrationconcentration

– high concentration of oxygen inhibit high concentration of oxygen inhibit growthgrowth

Helicobacter sp.

Helicobacter sp.


aerotolerant anaerobesaerotolerant anaerobes– indifferent to oxygenindifferent to oxygen

S. mutans

Environmental Influencing Factors: pHEnvironmental Influencing Factors: pH

based on pH of the based on pH of the environment, microorganisms environment, microorganisms are separated into the are separated into the following groupsfollowing groups– neutrophilesneutrophiles– acidophilesacidophiles– alkalophilesalkalophiles

Environmental Influencing Factors: pHEnvironmental Influencing Factors: pH

neutrophilesneutrophiles– optimum pH of 7 (neutral)optimum pH of 7 (neutral)– most microorganisms grow best between pH of 5 most microorganisms grow best between pH of 5

(acidic) and pH of 8 (alkaline)(acidic) and pH of 8 (alkaline)

acidophilesacidophiles– optimal growth, pH of less than 5.5optimal growth, pH of less than 5.5

alkalophilesalkalophiles– optimum pH of 8.5 or greateroptimum pH of 8.5 or greater

Copper

Copper tolerant acidophile

Urinary bacterial infectioncaused by alkaline urine

Environmental Influencing Factors: Environmental Influencing Factors: salinitysalinity

HH22O is required by all microorganisms for growthO is required by all microorganisms for growth

in some places Hin some places H22O is hard to come by such as in salt O is hard to come by such as in salt concentrationsconcentrations– if a cell is in an environment that has a greater solute if a cell is in an environment that has a greater solute

concentration than the interior of the cell, then by concentration than the interior of the cell, then by osmosis the water will leave the cell causing osmosis the water will leave the cell causing plasmolysis (shrinking of the cell)plasmolysis (shrinking of the cell)

Environmental Influencing Factors: Environmental Influencing Factors: salinitysalinity

halophiles are microorganisms that have adapted to halophiles are microorganisms that have adapted to this kind of environmentthis kind of environment– halophileshalophiles

require high levels of sodium chloriderequire high levels of sodium chloride

moderate halophilesmoderate halophiles– 3% salt concentration3% salt concentration

extreme halophiles: extreme halophiles: ArchaeaArchaea– require at least 9% salt solutionrequire at least 9% salt solution– found in the Dead Sea found in the Dead Sea

Dunaliella salina cell, near a salt crystal. 40X

Dead Sea

Nutritional Influencing FactorsNutritional Influencing Factors

major elementsmajor elements– C, O, H, N, S, P, K, MG, Ca FeC, O, H, N, S, P, K, MG, Ca Fe

essential components of protein, carbohydrates, essential components of protein, carbohydrates, lipids and nucleic acidlipids and nucleic acid

– needed to synthesize cell componentsneeded to synthesize cell components

Nutritional/Energy Influencing FactorsNutritional/Energy Influencing Factors

heterotrophsheterotrophs– utilize organic carbonutilize organic carbon

autotrophautotroph– utilize inorganic carbonutilize inorganic carbon

phototrophsphototrophs– harvest the energy of harvest the energy of

sunlightsunlight

chemotrophchemotroph– obtain energy by obtain energy by

metabolizing chemical metabolizing chemical compoundscompounds

Dinoflagellates

Myxobacteria

Purple Sulfur Bacteria: a chemotroph

Nutritional DiversityNutritional Diversity

prokaryotes are able to use diverse sources of carbon (an prokaryotes are able to use diverse sources of carbon (an essential element) and energyessential element) and energy– this ability allows them to thrive in virtually and this ability allows them to thrive in virtually and

environmentenvironment

Forms of Carbon

Nutritional DiversityNutritional Diversityphotoautotrophsphotoautotrophs– utilize the energy of sunlightutilize the energy of sunlight– obtain carbon from COobtain carbon from CO22

– primary producers of the microbial primary producers of the microbial worldworld

6CO6CO2 2 + 12H+ 12H22O CO C66HH1212OO6 6 + 6H+ 6H22O + O + 6O6O22

photoheterotrophsphotoheterotrophs– utilize the energy of sunlightutilize the energy of sunlight– obtain carbon from organic obtain carbon from organic

compoundscompounds

Cyanobacteria

Rhodobacter sphaeroides


chemolithoautotrophs chemolithoautotrophs – AKA asAKA as

chemoautotrophs or chemoautotrophs or chemolithotrophschemolithotrophs

– energy from inorganic energy from inorganic compounds such as hydrogen compounds such as hydrogen sulfidesulfide

– carbon from COcarbon from CO22

Thiobacillus denitrificans


chemoorganoheterotrophschemoorganoheterotrophs– AKAAKA

chemoheterotrophs or chemoorganotrophschemoheterotrophs or chemoorganotrophs– utilize organic compounds for energy and as a utilize organic compounds for energy and as a

carbon sourcecarbon source– most common group of microorganisms associated most common group of microorganisms associated

with humans and animalswith humans and animals– important organic degradersimportant organic degraders

B. vietnamiensisBrachionus calyciflorus

Prokaryotes in the LabProkaryotes in the Lab

studying microorganisms in their environment, studying microorganisms in their environment, enhances our ability to grow them in the labenhances our ability to grow them in the lab

lab growth is important for the study of the lab growth is important for the study of the microbial world and its effect on human lifemicrobial world and its effect on human life

Lab Cultivation of MicrobesLab Cultivation of Microbes

complex mediacomplex media– used for routine purposesused for routine purposes– variety of ingredients needed by the microorganism variety of ingredients needed by the microorganism

are included in the mediaare included in the media

nutrient agar, blood agar, PEA agar, Mannitol Salt nutrient agar, blood agar, PEA agar, Mannitol Salt agar are some examplesagar are some examples

S. aureus on blood agar


selective mediaselective media– formulated with

ingredients that inhibit the growth of some bacteria, such as an antibiotic, but enhance growth of the target organism

– ie: MacConkey agar ie: MacConkey agar can be used to isolate can be used to isolate Gram-negative rodsGram-negative rods


differential mediadifferential media– includes ingredients, such as chemical indicators,

that produce observable differences between species of bacteria

– ie: ph indicator may be incorporated with the agar ie: ph indicator may be incorporated with the agar medium allowing for the detection of acid producing medium allowing for the detection of acid producing microorganismsmicroorganisms

mannitol salt agar: pH indicator turns the agar yellow in the presence of a salt tolerant organism

Creating Appropriate Environmental Creating Appropriate Environmental ConditionsConditions

to enhance microbial to enhance microbial growth in a lab, certain growth in a lab, certain environmental conditions environmental conditions need to be createdneed to be created– atmospheric pressureatmospheric pressure– temperaturetemperature– oxygen availabilityoxygen availability


atmosphereatmosphere

– increase COincrease CO2 2 for some species of microbesfor some species of microbes

Candle jar used in lab to increase COCO22 concentration


anaerobic microorganisms require anaerobic microorganisms require anaerobic conditions required growthanaerobic conditions required growth– these are some of the most difficult these are some of the most difficult

types of microorganisms to culture types of microorganisms to culture in the lab, due to the fact that even in the lab, due to the fact that even a brief exposure to oxygen a brief exposure to oxygen generally results in the death of the generally results in the death of the organismorganism

Anaerobic jars used in labs


temperaturetemperature– controlled with the use controlled with the use

of an incubator of an incubator – allows for setting the allows for setting the

optimum temperature optimum temperature for individual for individual microorganismsmicroorganisms

Bacteria EnumerationBacteria Enumeration

lab techniques that monitor bacterial growthlab techniques that monitor bacterial growth– viable plate countviable plate count– direct countdirect count– most probable numbermost probable number– membrane filtrationmembrane filtration– measuring biomassmeasuring biomass

turbidityturbidity

total weighttotal weight

chemical constituentschemical constituents


viable plate countviable plate count– measure the number of cells in a sample based on measure the number of cells in a sample based on

the fact that one cell gives rise to one colonythe fact that one cell gives rise to one colony– utilizes a series of dilutions in order to calculate the utilizes a series of dilutions in order to calculate the

number of viable bacteria in the original samplenumber of viable bacteria in the original sample


direct countdirect count– using special equipment capable of making:using special equipment capable of making:

a direct microscopic counta direct microscopic count

a count of cells suspended in a a count of cells suspended in a suspensionsuspension

a count by analyzing the scattering of a count by analyzing the scattering of light as cells pass by a laserlight as cells pass by a laser


most probable numbermost probable number– a statistical analysis a statistical analysis

of cell numbers of cell numbers based on the theory based on the theory of probabilityof probability


membrane filtrationmembrane filtration– used when cell numbers are lowused when cell numbers are low– allows for a concentration of the microbes by filtering allows for a concentration of the microbes by filtering

before platingbefore plating

Membrane filtration equipment

Membrane filtration on mEnterococcus agar. The plate at the bottom is uninoculated. The red colonies typical of the Enterococci are clearly visible on the white membrane filters.


measuring biomassmeasuring biomass– turbidityturbidity– total weighttotal weight– chemical constituentschemical constituents

turbidity


turbidityturbidity– cloudiness, which indicates the presence of microbial cloudiness, which indicates the presence of microbial

growthgrowth– cell numbers can be measured with a cell numbers can be measured with a

spectrophotometerspectrophotometer


total weighttotal weight– tedious worktedious work

measure the wet weight, measure the wet weight, centrifuge and then measure dry centrifuge and then measure dry weightweight


chemical constituentschemical constituents– analyzing the quantity of chemical (metabolic analyzing the quantity of chemical (metabolic

byproduct) in a bacterial sample and using that byproduct) in a bacterial sample and using that information to calculate biomassinformation to calculate biomass

Spectroanalysis

Bacterial Growth in NatureBacterial Growth in Nature

similar to a continuous culturesimilar to a continuous culture– nutrients are continually added and byproducts are nutrients are continually added and byproducts are

removedremoved

generally multiply more slowly than under lab generally multiply more slowly than under lab conditionsconditions

often the waste of one microorganism is the nutrient often the waste of one microorganism is the nutrient of anotherof another

Microbial mat in Yellowstone


biofilmsbiofilms– polysaccharide-encased polysaccharide-encased

communitycommunity– slippery rocks, gunk in drains, slippery rocks, gunk in drains,

plaque on teeth, IV’s are all plaque on teeth, IV’s are all examples of biofilmsexamples of biofilms

– begins with adherence of a begins with adherence of a bacterium to a surfacebacterium to a surface

bacteria multipliesbacteria multipliessynthesizes a loose glycocalyx synthesizes a loose glycocalyx allowing unrelated cell to attach allowing unrelated cell to attach and growand grow

Methanogen biofilm


biofilmsbiofilms– medical problemsmedical problems

resist antibioticsresist antibiotics

65% of human infections 65% of human infections involve biofilmsinvolve biofilms

often times 100X more often times 100X more resistant to disinfectantsresistant to disinfectants

Biofilm on endotrachial tube


bioremediationbioremediation– bacteria used to degrade bacteria used to degrade

chemicals are enhanced by chemicals are enhanced by organisms present in biofilmorganisms present in biofilm

Acid from an abandoned mine. Microorganisms are introduced to this environment and are successfully able to clean up the “problem”.

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Prokaryotic Growth Kathy Huschle Northland Community & Technical College