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Microbial Growth1Microbial GrowthIncrease in number of cells, not cell sizePopulationsColonies2The Requirements for GrowthPhysical requirementsTemperaturepHOsmotic pressureChemical requirementsCarbonNitrogen, sulfur, and phosphorousTrace elementsOxygenOrganic growth factor3

Typical Growth Rates and Temperature453 temperature adaptation groups

63 cardinal temperaturesMinimum temperature lowest temperature that permits a microbes growth and metabolismMaximum temperature highest temperature that permits a microbes growth and metabolismOptimum temperature promotes the fastest rate of growth and metabolism73 temperature adaptation groupsPsychrophiles optimum temperature below 15oC, capable of growth at 0oCMesophiles optimum temperature 20o-40oC, most human pathogensThermophiles optimum temperature greater than 45oCPsychrotrophsGrow between 0C and 2030CCause food spoilage8

Food Preservation Temperatures9pHMost bacteria grow between pH 6.5 and 7.5Molds and yeasts grow between pH 5 and 6Acidophiles grow in acidic environments10Osmotic PressureHypertonic environments, or an increase in salt or sugar, cause plasmolysisExtreme or obligate halophiles require high osmotic pressureFacultative halophiles tolerate high osmotic pressure

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Plasmolysis12Elements of Microbial Nutrition, Ecology and Growth14Microbial nutritionMacronutrients required in large quantities; play principal roles in cell structure & metabolism proteins, carbohydrates Micronutrients or trace elements required in small amounts; involved in enzyme function & maintenance of protein structuremanganese, zinc, nickel15NutrientsInorganic nutrients atom or molecule that contains a combination of atoms other than carbon and hydrogenmetals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate), gases (oxygen, carbon dioxide) and waterOrganic nutrients- contain carbon and hydrogen atoms and are usually the products of living thingsmethane (CH4), carbohydrates, lipids, proteins, and nucleic acids16Chemical composition of cytoplasm70% waterproteins96% of cell is composed of 6 elementsCarbonHydrogenOxygenPhosphorousSulfur

17Obtaining CarbonHeterotroph an organism that must obtain carbon in an organic form made by other living organisms such as proteins, carbohydrates, lipids and nucleic acids

Autotroph - an organism that uses CO2, an inorganic gas as its carbon sourcenot dependent on other living thingsChemical RequirementsCarbonStructural organic molecules, energy sourceChemoheterotrophs use organic carbon sourcesAutotrophs use CO21819Nitrogen Main reservoir is nitrogen gas (N2)79% of earths atmosphere is N2Nitrogen is part of the structure of proteins, DNA, RNA & ATP these are the primary source of N for heterotrophsSome bacteria & algae use inorganic N nutrients (NO3-, NO2-, or NH3)Some bacteria can fix N2Regardless of how N enters the cell, it must be converted to NH3, the only form that can be combined with carbon to synthesis amino acids, etc.Chemical RequirementsNitrogenIn amino acids and proteinsMost bacteria decompose proteinsSome bacteria use NH4+ or NO3A few bacteria use N2 in nitrogen fixation2021Oxygenmajor component of carbohydrates, lipids and proteinsplays an important role in structural & enzymatic functions of cellcomponent of inorganic salts (sulfates, phosphates, nitrates) & waterO2 makes up 20% of atmosphereessential to metabolism of many organisms22Hydrogenmajor element in all organic compounds & several inorganic ones (water, salts & gases)gases are produced & used by microbesroles of hydrogenmaintaining pHforming H bonds between moleculesserving as the source of free energy in oxidation-reduction reactions of respiration23Phosphorousmain inorganic source is phosphate (PO4-3) derived from phosphoric acid (H3PO4) found in rocks & oceanic mineral depositskey component of nucleic acids, essential to geneticsserves in energy transfers (ATP)

24Sulfurwidely distributed in environment, rocks, sediments contain sulfate, sulfides, hydrogen sulfide gas and sulfuressential component of some vitamins and the amino acids: methionine & cysteinecontributes to stability of proteins by forming disulfide bondsChemical RequirementsSulfurIn amino acids, thiamine, and biotinMost bacteria decompose proteinsSome bacteria use SO42 or H2SPhosphorus In DNA, RNA, ATP, and membranesPO43 is a source of phosphorus25Chemical RequirementsTrace elementsInorganic elements required in small amountsUsually as enzyme cofactors2627Important mineral ionsPotassiumSodiumCalciumMagnesiumIron

28Growth factorsorganic compounds that cannot be synthesized by an organism & must be provided as a nutrientessential amino acids, vitamins29Carbon sourceEnergy sourcephotoautotrophsCO2sunlightchemoautotrophsCO2Simple inorganic chemicalsphotoheterotrophsorganicsunlightchemoheterotrophsorganicMetabolizing organic cpds

The Effect of Oxygen (O2) on Growth30Singlet oxygen: O2 boosted to a higher-energy stateSuperoxide free radicals: O2

Peroxide anion: O22

Hydroxyl radical (OH)

Toxic Oxygen31

BiofilmsMicrobial communitiesForm slime or hydrogelsBacteria attracted by chemicals via quorum sensing

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BiofilmsShare nutrientsSheltered from harmful factors

33BiofilmsPatients with indwelling catheters received contaminated heparinBacterial numbers in contaminated heparin were too low to cause infection84421 days after exposure, patients developed infections

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BiofilmsPseudomonas fluorescens was cultured from the catheters

3537Binary Fission

38Population growth

39Growth curve

40Growth curveLag phase flat period of adjustment, enlargement; little growth

Exponential growth phase a period of maximum growth will continue as long as cells have adequate nutrients & a favorable environment

Stationary phase rate of cell growth equals rate of cell death cause by depleted nutrients & O2, excretion of organic acids & pollutants

Death phase as limiting factors intensify, cells die exponentially in their own wastesCulture MediaCulture medium: Nutrients prepared for microbial growthSterile: No living microbesInoculum: Introduction of microbes into mediumCulture: Microbes growing in/on culture medium42AgarComplex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deepsGenerally not metabolized by microbesLiquefies at 100CSolidifies at ~40C43Culture MediaChemically defined media: Exact chemical composition is knownComplex media: Extracts and digests of yeasts, meat, or plantsNutrient brothNutrient agar44

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46Anaerobic Culture MethodsReducing mediaContain chemicals (thioglycolate or oxyrase) that combine O2Heated to drive off O247

Anaerobic Jar48

An Anaerobic Chamber49CapnophilesMicrobes that require high CO2 conditionsCO2 packetCandle jar

501: No special precautions2: Lab coat, gloves, eye protection3: Biosafety cabinets to prevent airborne transmission4: Sealed, negative pressureExhaust air is filtered twiceBiosafety Levels51

Biosafety Level 4 (BSL-4) Laboratory52

Selective MediaSuppress unwanted microbes and encourage desired microbes53

Differential MediaMake it easy to distinguish colonies of different microbes.54Enrichment CultureEncourages growth of desired microbeAssume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteriaInoculate phenol-containing culture medium with the soil, and incubateTransfer 1 ml to another flask of the phenol medium, and incubateTransfer 1 ml to another flask of the phenol medium, and incubateOnly phenol-metabolizing bacteria will be growing55Obtaining Pure CulturesA 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)The streak plate method is used to isolate pure cultures

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The Streak Plate Method57Preserving Bacterial CulturesDeep-freezing: 50 to 95CLyophilization (freeze-drying): Frozen (54 to 72C) and dehydrated in a vacuum58Binary fissionBuddingConidiospores (actinomycetes)Fragmentation of filamentsReproduction in Prokaryotes59

Binary Fission60

Binary Fission61

Cell Division62If 100 cells growing for 5 hours produced 1,720,320 cells:

Generation Time63

Bacterial Growth Curve64

Phases of Growth65

Serial Dilutions66

Plate Counts67

Plate CountsAfter incubation, count colonies on plates that have 25250 colonies (CFUs)68

Counting Bacteria by Membrane Filtration69

Most Probable NumberMultiple tube MPN testCount positive tubes70

Most Probable NumberCompare with a statistical table.71

Direct Microscopic Count72

Direct Microscopic Count73Figure 6.21

Turbidity74

Turbidity75Measuring Microbial GrowthDirect MethodsPlate countsFiltrationMPNDirect microscopic countIndirect MethodsTurbidityMetabolic activityDry weight76Isolation

78The 5 Is of culturing microbesInoculation introduction of a sample into a container of mediaIncubation under conditions that allow growthIsolation separating one species from anotherInspectionIdentification79IsolationIf an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colonyA colony consists of one species80Isolation technique

81Media providing nutrients in the laboratoryMost commonly used:nutrient broth liquid medium containing beef extract & peptonenutrient agar solid media containing beef extract, peptone & agaragar is a complex polysaccharide isolated from red algaesolid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oCprovides framework to hold moisture & nutrientsnot digestible for most microbes

82Types of mediasynthetic contains pure organic & inorganic compounds in an exact chemical formulacomplex or nonsynthetic contains at least one ingredient that is not chemically definablegeneral purpose media- grows a broad range of microbes, usually nonsyntheticenriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes83Enriched media

84selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbesdifferential media allows growth of several types of microbes and displays visible differences among desired and undesired microbes85selective & differential media

86Selective media

87Differential media

88Miscellaneous mediareducing medium contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteriacarbohydrate fermentation medium- contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi89Carbohydrate fermentation media

Identification Identifying Bacteria

Identifying a Gram Negative, Oxidase Negative RodNumerical Identification

Design a rapid test for a Staphylococcus aureus.

The Western Blot

Staining

The Gram Stain MechanismCrystal violet-iodine crystals form in cellGram-positiveAlcohol dehydrates peptidoglycanCV-I crystals do not leaveGram-negativeAlcohol dissolves outer membrane and leaves holes in peptidoglycanCV-I washes out