Bacteriology

BacteriologyThe Study of BacteriaBacterial UltrastructuresBiofilmsBacterial MetabolismBacterial SporolationBacterial Reproduction and Growth KineticsBacterial CultivationEnvironmental Factors of GrowthDetection, Identification and CharacterizationTopics2BacteriologyBiofilmsA biofilm is any group of microorganisms in which cells stick to each other on a surface. These adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substance (EPS).Factorswhich may include cellular recognition of specific or non-specific attachment sites on a surfacenutritional cuesin some cases, by exposure of planktonic cells to sub-inhibitory concentrations of antibiotics.When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated.Definition

Biofilm FormationInitial attachment:Irreversible attachment:Maturation I:Maturation II:Dispersion:There are five stages of biofilm development (see illustration at right):Bacterial UltrastructuresBiofilmsBacterial MetabolismBacterial SporolationBacterial Reproduction and Growth KineticsBacterial CultivationEnvironmental Factors of GrowthDetection, Identification and CharacterizationTopics7Bacteriology Microbial GrowthIncrease in cell numberBinary FissionMicrobial GrowthThere are five main conditions for bacterial growthTemperatureMoistureTimePh levelOxygenConditions for bacterial growthBacteria grow best at 37C which is body temperature. They reproduce quickly between 3C and 63C which is the danger zone. Cook chilled food should be stored at 0C to 3C.To control temperature food should beCooled to below 3CHeated to above 63CTemperatureAbove 121C all bacteria and spores are killed72C temperature of reheated food63C Food should be heated above this temperature3-63C danger zone for bacterial growth-18C temperature of freezer-22 temperature to freeze food.Key temperaturesPsychrophilesMesophilesThermophilesTemperatureTrue Psychrophiles sensitive to temperature lower than 20COptimum temp at 15CPsychotrophsBetween 20 30CResponsible for most low temperature food spoilagePsychrophiles: Cold LovingMost pathogens and common spoilage organisms25 40COptimum Tem: 37CAdapted to live in the bodies of animalsMesophiles Middle Loving50 60CCannot grow below 45CAdapted to live in sunlit soil, compost piles, and hot springsExtreme Thermophiles: ArchaebacteriaThermophiles: Heat Loving

TemperatureMoistureBacteria like moist conditions. Many foods contain liquidControls for moistureDehydration-removing the water like in dried milkHigh sugar content- makes less water available like in jamSalt removes water by osmosis like in bacon.Freezing turns water into solids

TimeBacteria multiply rapidly. One bacterium can become one million in less than seven hours.To control bacteria multiplying you shouldEat food as soon after it is madeCool quickly and store in a fridge or freezer.

PH LevelBacteria grow best in a neutral PH between 6.6 and 7.5. They cannot survive below ph 4.5.Ph levels can be controlled by acidity regulators which keep food below ph4.5.Vinegar has a ph 3.5 and is used to preserve foods like onions

AcidophilesNeutrophilesAlkaliphilesPH Levels0.1 5.4LactobacillusAcidophiles5.4 8.5Includes most human pathogensNeutrophiles7 12 or higherVibrio cholerae and Alkaligenes faecalis pH 9Agrobacterium pH 12AlkaliphilesOxygenSome bacteria but not all need oxygen to reproduce.To prevent bacteria getting oxygen manufacturers use vacuum pack foods of a system known as MAP where the oxygen is removed and replaced with a less active gas.

Obligate Aerobes require oxygen to livePseudomonasFacultative AnaerobesCan use oxygen, but can grow in its absenceE. coli, Staphylococcus, yeasts, many intestinal bacteriaObligate AnaerobesCannot use oxygen and are harmed by the presence of OxygenClostridium

Oxygen Requirements

Growth in thioglycollate broth reveals oxygen preferencesObligate aerobesFaultative anaerobesObligate anaerobesAerotolerant anaerobesMicroaerophilesOxygen gradient Thyoglycollate binds molecular oxygen, reducing it and removing it: R-SH + O2 R-SO2

Resazurin dye is red in the presence of oxygen

Anaerobic and Low O2 Culture Methods

Brewer or anaerobic jarCO2 packetCandle jar80 90% waterHypertonic Solutions used to control spoilage and microbial growthHypotonic Solution microbe may lyse or burst if cell wall is weakOsmotic PressureHalophilesModerate to large salt concentrationsOcean Bacteria (3.5% salt)Extreme or Obligate Halophiles20 30%Bacteria in the Dead SeaFacultative HalophilesDo not require high salt concentrations for growthBut tolerate 2% or moreOsmotic PressureCarbonNitrogen, Sulfur, and PhosphorousChemical RequirementsMakes up 50% of dry weight of cellChemoheterotrops carbon from their energySource: lipids, proteins, and carbohydratesChemoautotrophs and PhotoautotropsCarbon from Carbon dioxideCarbonNitrogen, Sulfur, and Phosphorous14% of dry cell weightProteinAmmoniumNitrogen GasNitratesNitrogenUsed to form proteins and some vitaminsSourcesProteinHydrogen SulfideSulfatesSulfureSource: Mainly inorganic phosphate salts and buffersPhosphorousCalciumRequired for the synthesis in Gram positive bacteriaTrace ElementsUsed as enzyme cofactorsCommonly found in tap waterIronCopperMolybdenumZincOther elementsBacterial UltrastructuresBiofilmsBacterial MetabolismBacterial SporolationBacterial Reproduction and Growth KineticsBacterial CultivationEnvironmental Factors of GrowthDetection, Identification and CharacterizationTopics38BacteriologyBacterial ReproductionBacterial Reproduction

40IntroductionBacteria can reproduce in one of two ways:

AsexuallySexually

41Asexual ReproductionBinary Fission:one parent involvedoffspring are identical to parent & each other

Advantages:simple: only 1 parentoffspring are fully formed (no maturation needed)very fast (20 min. in ideal conditions) after 24 hrs: 1 bacterium 2x106 kg of cells (enough to cover the earth)

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Asexual ReproductionDisadvantages: no genetic varietyone unfavourable environmental condition can wipe out whole population

43Asexual ReproductionBinary Fission:

44Sexual Reproductiontwo parents involvedoffspring are genetically different to parents & to each otherAdvantages: genetic variety i.e. some are able to adapt to unfavourable conditions (ex: antibiotic resistance)

Disadvantages:more complex: slower because must find a compatible partner

no new individuals produced (i.e. no increase in population)

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Bacterial Conjugation

plasmid copies itself passes through pili (cytoplasmic bridge) into recipient cell

cells separate with both cells containing the plasmid

plasmid donor recipient cell (+) cell ( - ) pili

46Mechanisms That Produce VariationIf binary fission produces clonal offspring, why are bacteria so genetically diverse???Two factors contribute to genetic diversity among and within bacterial speciesMutationRecombination

MutationA mutation is a change in the DNA of a gene, ultimately leading to genetic diversity

Mutations can be spontaneous or caused by mutagensSpontaneous - Errors during DNA replicationMutagens - Chemical or physical factors that damage DNA

Spontaneous mutations are extremely rare, occurring on average only once in 10 million cell divisions, per gene

Because bacteria divide rapidly & exponentially, mutation is a relevant factor generating genetic diversityGenetic RecombinationThe combining of DNA from two sources

In sexually reproducing organisms this is the main way genetic variation is produced

In eukaryotes, it involves the sexual processes of meiosis and fertilization

In prokaryotes three other processes are used transformation, transduction, and conjugation (= bacterial sex)

Results in horizontal gene transfer the transfer of genetic material within a generation, instead of from one generation to the next a major force in the long-term evolution of bacteriaConjugationThe direct transfer of genetic material between two bacteria cells that are temporarily joined

DNA transfer is one-way, from male to female

The donor (male) uses an appendage called the sex pilus that forms a cytoplasmic mating bridge

DNA gets transferred via this bridge in the form of a plasmid

The plasmid encodes the ability to mate as well as other traits such as antibiotic resistance

TransformationThe alteration of a bacterial cells genotype and phenotype by the uptake of naked, foreign DNA from the surrounding environmentMany bacteria possess cell surface proteins that facilitate transformation in natural populationsE. coli is used in biotechnology applications of genetic recombination (genetic engineering)Cells are cultured in high CaCl2 to become competentCells are then transformed with human genes that code for proteins such as insulin or growth hormone that are needed in large amounts

TransductionPhages (viruses that infect bacteria) carry bacterial genes from one host cell to another as a result of mistakes in the phage reproductive cycleIn the process called generalized transduction, this transfer is random

Figure 18.16!

Bacterial Populations Evolve RapidlyNatural selection operates on genetic (heritable) variation, such as is generated readily by mutation in bacteria

A mutation that confers a reproductive advantage increases in frequency in subsequent generations, and eventually becomes fixed in the population

Bacteria reproduce quickly and therefore have a short generation time relative to most other organisms

The rapid evolution of antibiotic resistance in bacteria is a medically important example of natural selection at work

BacteriologyGrowth CurveGrowth KineticsGrowth patterns and kinetics in batch culture- growth phases In batch culture:- lag phase- logrithmic or exponential growth phase- deceleration phase- stationary phase- death phase

Typical growth curve for a bacterial populationLag phase

A period of adaptation for the cells to their new environment

New enzymes are synthesized.

A slight increase in cell mass and volume, but no increase in cell number

Prolonged by low inoculum volume, poor inoculum condition (high % of dead cells), age of inoculum, nutrient-poor medium

Multiple lag phases: (diauxic growth) medium contains more than one carbon sourceBatch Growth Kinetics

Typical growth curve for a bacterial populationDiauxic growth

Typical growth curve for a bacterial populationBatch Growth KineticsExponential growth phase

In this phase, the cells have adjusted to their new environment and multiply rapidly (exponentially)

Balanced growth all components of a cell grow at the same rate.

Growth rate is independent of nutrient concentration, as nutrients are in excess.

Typical growth curve for a bacterial population

Typical growth curve for a bacterial populationBatch Growth KineticsDeceleration growth phase

Very short phase, during which growth decelerates due to either:

Depletion of one or more essential nutrients

The accumulation of toxic by-products of growth (e.g. Ethanol in yeast fermentations)

Period of unbalanced growth: Cells undergo internal restructuring to increase their chances of survival

Typical growth curve for a bacterial populationBatch Growth KineticsStationary Phase:

With the exhaustion of nutrients (S0) and build-up of waste and secondary metabolic products

The growth rate equals the death rate.

There is no net growth in the organism population.

Cells may have active metabolism to produce secondary metabolites.Primary metabolites are growth-related: ethanol by S. cerevisae.Secondary metabolites are non-growth-related: antibiotics, pigments.Kinetic Pattern of Growth and Product Formation

Growth-associatedNon growth-associatedMixed-growth-associatedBatch Growth KineticsDeath Phase:The living organism population decreases with time, due to a lack of nutrients and toxic metabolic by-products.

The rate of death usually follows:

Figure 6.14

Growth Curve for Bacteria (Logarithmic Plot)Chapter 6Phases of GrowthLagAdapt to nutrientsLogActive growthStationaryDeath = Growth rateDeathNutrients consumedpH too low (why?)Optimize curves in production

BacteriologyCulture Media71 Culture mediumis the mixture of various nutrients that is suitable for the growth of microorganisms. Types of Culture Mediabased on the function and chemical componentsbased on the physical state72Based on the function and the chemical components:

Basic Medium --contains the basic nutrients for the most bacterial growth; --the base of other kind of media. --e.g. broth. Nutrient Medium/Enriched Medium Additional or special nutrients (e.g., serum, growth factors, trace elements) are added to support some fastidious bacterial growth. e.g. blood agar.

73Selective Medium the medium that can prevent the certain bacterial growth while permitting others. e.g. SS agar Differential Medium Some special substrates and indicators are added into the media in order to produce a visual differentiation when several bacteria grow on the same kind of medium. e.g. EMB agar (Eosin-methylene blue agar). 74E.coli on EMB

S.dysenteriae on EMB75Double sugar iron slant

Citrate slant76Anaerobic Medium a medium for the cultivation of certain anaerobes. The medium contains reducing agent, such as non-saturation fatty acid.77Based on the physical stateLiquid medium: Without agar.for the proliferation of bacteria.Solid medium: 1.5-2.5% agar.for the isolation and identification of bacteriae.g., slant, Petri dishes/plates.Semisolid medium: 0.3-0.5% agar.for the observation of bacterial motility and preservation of bacteria. 78Bacterial growth patterns In liquid medium: Superficial growth;Turbidity/diffuse;Precipitate growing;(sediment)

In solid medium: Confluent growth / Smear; Colony: a cluster of microorganisms growing on a solid medium. It is directly visible and arises from a single cell.

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In semi-solid medium: Only grow along the line of inoculationGrow diffusely 81

Goal: To chemically (or physically) suppress unwanted microbes and encourage desired microbes.Selective MediaFigure 6.9b, cMannitol salt agar : selective for halophiles with 7% salt (osmotic challenge) and differential for mannitol fermenters: good for skin bacterial cultures.

EMB Agar: kills gram positives with eosin and methylene blue, selective for gram negatives. Differential for lactose fermenters. Good for growing enterics.

McConkey Agar: supresses gram positives with crystal violet and bile salts; also differential for

MSAEMBMADistinguish between different species based on a metabolic ability.Differential MediaFigure 6.9a

Blood agar(sheeps blood) reveals if hemolytic

Mannitol salt agar contains the pH sensitive dye phenol red (yellow when acidic)

Se SaEncourages growth of desired microbe by providing special growth conditions or added growth factorsEnrichment Media

ThioglycollateAnaerobic or Brewer Jar

Glucose Salts Agar (enriches for microbes that can growth only on glucose and some inorganic nutrientsLysed red blood cells provide unique nutrients in blood/chocolate agar

A pure culture contains only one species or strainA colony is a population of cells arising from a single cell or spore or from a group of attached cellsA colony is often called a colony-forming unit (CFU)

Pure Cultures Used To Study Characteristics Of A Particular SpeciesBacteriologyBacterial EnumerationEstimating Bacterial Numbers by Indirect methodsDirect MeasuresPlate counts of viable bacterial forming coloniesCounting low viable bacterial numbers by filtrationCounting viable bacteria with Most Probable NumberCounting bacteria per ml in direct microscopyIndirect MeasuresTurbidity/Absorbance with a spectrophotometerMetabolic activity tracking conversion of colored moleculesDry weight by weighing a set volume and knowing weight of one cellAfter incubation, count colonies on plates that have 30-300 colonies (CFUs)Plate Assays: Spread Plate or Pour Plate MethodsFigure 6.15

The dilution in a particular tube = ml of fluid added to tube/total volume after addition; e.g. 1ml/(9ml + 1ml) = 1/10 = 10-2

Direct Measurements of Microbial GrowthFigure 6.19Filtration: Good for measuring very dilute samples of bacteriaDirect Measurements of Microbial GrowthFigure 6.17a, b

Multiple tube MPN testCount positive tubes and compare to statistical MPN tableProduces a range of concentrations Direct Measurements of Microbial GrowthFigure 6.18b

Estimating Bacterial Numbers by Indirect methodsDirect MeasuresPlate counts of viable bacterial forming coloniesCounting low viable bacterial numbers by filtrationCounting viable bacteria with Most Probable NumberCounting bacteria per ml in direct microscopyIndirect MeasuresTurbidity/Absorbance with a spectrophotometerMetabolic activity tracking conversion of colored molecules/enyzme assayDry weight by weighing a set volume and knowing weight of one cellTurbidityEstimating Bacterial Numbers by Indirect MethodsFigure 620

Metabolic Conversion/Enzyme Assay1 bacterium produces 4.6 x 1012 NADH/sec/cell under idea growth conditions.

In a 1 ml sample of growing cells, 5.2 x 1023 NADH/sec/ml are produced per second (as revealed by a color-based assay of NADH on the sample)

Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023 NADH/sec/cell) = 2.3 x 1024 cells/ml

Determining dry mass of a fixed volume

An E. coli cell has a dry mass of about 7.0 x 10-19 mg.

A 1 ml sample with a dry mass of 2 mg therefore has:

2 mg/ml x 1 cell/7 x 10-19 mg

= 2.8 x 1020 cells/mlMicrobial GrowthPhysical Requirements of MicrobesTemperature (optimal enzyme operation)Psychrophiles, mesophiles, thermophilespH (optimal enzyme operation)Using buffers in mediaMolds & yeasts versus bacteriaChemical RequirementsCarbon source in medium Nitrogen, sulfur, phosphorous, trace elementsOxygen requirementsObligate aerobes, anaerobes, facultative anaerobesFree radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobesCulture Media for MicrobesChemically defined vs. complex mediaAnaerobes: reducing media/Brewer jarOther: animals, eggs, tissue culture, CO2Media typesSelective, Differential, EnrichmentBacterial Population GrowthGrowth Curve: Lag, Log, Stationary, DeathQuantifying Growth

Bacterial UltrastructuresBiofilmsBacterial MetabolismBacterial SporolationBacterial Reproduction and Growth KineticsBacterial CultivationEnvironmental Factors of GrowthDetection, Identification and CharacterizationTopics97