Optimum Temperature Optimum Temperature RequirementsRequirements

TemperatureTemperature Most human pathogens are ???Most human pathogens are ???

Physical RequirementsPhysical Requirements 65 -130 ˚C- most 65 -130 ˚C- most

bacteria destroyed by bacteria destroyed by enzyme denaturation.enzyme denaturation.

50-65 ˚C slow growth50-65 ˚C slow growth 15-50 ˚C Danger of 15-50 ˚C Danger of

food spoilagefood spoilage 5 -15 ˚C survival, and 5 -15 ˚C survival, and

slow growthslow growth 0-5 ˚C slow or no 0-5 ˚C slow or no

growthgrowth <0 ˚C no growth, but <0 ˚C no growth, but

may survive.may survive.

Physical RequirementsPhysical Requirements

pHpH <6.0 acidophiles<6.0 acidophiles 6 - 8 neutrophiles6 - 8 neutrophiles >8.0 basophiles>8.0 basophiles

Most pathogens are Most pathogens are neutrophiles.neutrophiles.

Physical RequirementsPhysical Requirements

Osmotic PressureOsmotic Pressure Isotonic - 0.85% NaCl - 300 mOsIsotonic - 0.85% NaCl - 300 mOs Hypotonic- low salt or pure waterHypotonic- low salt or pure water Hypertonic - high salt - halophilesHypertonic - high salt - halophiles

Nutritional ClassificationNutritional Classification

Photoautotroph – Energy = sunlight, Carbon Photoautotroph – Energy = sunlight, Carbon source = source = COCO22 Photosynthesis Photosynthesis

Photoheterotroph - Energy source = sunlight, Photoheterotroph - Energy source = sunlight, Carbon source = Carbon source = organicorganic compounds. compounds.

Chemoautotrophs - Energy source = Chemoautotrophs - Energy source = Oxidation of Oxidation of inorganicinorganic compounds Nitrates, compounds Nitrates, Iron, Sulfur. Carbon source = COIron, Sulfur. Carbon source = CO2 2

Chemoheterotroph Both Energy and Carbon Chemoheterotroph Both Energy and Carbon sources are sources are organicorganic compounds. compounds.

Chemical RequirementsChemical Requirements

AerotoleranceAerotolerance Obligate Aerobe – Uses oxygen as final Obligate Aerobe – Uses oxygen as final

Hydrogen acceptor. Has Superoxide Hydrogen acceptor. Has Superoxide dismutase and catalase. dismutase and catalase. Pseudomonas Pseudomonas aeruginosaaeruginosa

Facultative Anaerobe – Can use OFacultative Anaerobe – Can use O22, but other , but other hydrogen acceptors i.e., nitrates. They are hydrogen acceptors i.e., nitrates. They are also capable of fermentation. Efficiency also capable of fermentation. Efficiency decreases without oxygen. Has Superoxide decreases without oxygen. Has Superoxide dismutase and catalase. dismutase and catalase. E. coli, E. coli, Staphylococcus sp.Staphylococcus sp.

Obligate Anaerobe – unable to use oxygen for Obligate Anaerobe – unable to use oxygen for energy. Most are harmed by it. No enzymes energy. Most are harmed by it. No enzymes to detoxify oxygen forms. to detoxify oxygen forms. Clostridium spClostridium sp..

Chemical RequirementsChemical Requirements

Anaerobe jarAnaerobe jar Chemicals make COChemicals make CO22

and Hand H22

HH22 reacts with O reacts with O22 + + paladium --> Hpaladium --> H22OO

OO2 2 consumed.consumed.

Typical anaerobes:Typical anaerobes: Clostridium, Clostridium,

BacteroidesBacteroides

Aerotolerance continuedAerotolerance continued Aerotolerant Anaerobe – Cannot use oxygen Aerotolerant Anaerobe – Cannot use oxygen

i.e., i.e., FermentativeFermentative, but tolerate oxygen – , but tolerate oxygen – possess Superoxide dismutase. Lactic acid possess Superoxide dismutase. Lactic acid producers.producers.

Microaerophiles- Require oxygen but too much Microaerophiles- Require oxygen but too much causes the production of toxic oxygen causes the production of toxic oxygen compounds. No enzymes. Lower levels of compounds. No enzymes. Lower levels of oxygen required for grow. oxygen required for grow.

Capnophile – Some aerobic organisms require Capnophile – Some aerobic organisms require lower levels of oxygen and lower levels of oxygen and higher levels of higher levels of COCO22..Neisseria meningiditis or gonorrheaNeisseria meningiditis or gonorrhea

Special TechniquesSpecial Techniques

Anaerobic growthAnaerobic growth Extra COExtra CO22 - -

capnophilic -capnophilic -NeisseriaNeisseria Selective media- Selective media-

selects for specific selects for specific group.group.

Differential mediaDifferential media--allows you to tell one allows you to tell one group from another.group from another.

Toxic Forms of OxygenToxic Forms of Oxygen Normal Atmospheric Molecular Oxygen ONormal Atmospheric Molecular Oxygen O22

Most stable form of oxygen, but quite Most stable form of oxygen, but quite reactive.reactive.

Singlet oxygen – O single oxygen atom, Singlet oxygen – O single oxygen atom, higher energy state, much more reactive higher energy state, much more reactive than Othan O22. Found in phagocytes. Found in phagocytes

Superoxide Free Radical OSuperoxide Free Radical O22-- Produced Produced

during aerobic respiration must be detoxified during aerobic respiration must be detoxified by by superoxide dismutasesuperoxide dismutase..

Peroxide anion OPeroxide anion O222-2- very reactive and toxic. very reactive and toxic.

Removed by Removed by catalasecatalase. .

Chemical RequirementsChemical Requirements Carbon -Carbon -

Photosynthesis - Basis for all organic Photosynthesis - Basis for all organic compoundscompounds

NitrogenNitrogen From nitrates, ammoinum salts, amino acids, From nitrates, ammoinum salts, amino acids,

and nitrogen fixationand nitrogen fixation Needed for protein and nucleic acid synthesisNeeded for protein and nucleic acid synthesis

PhosphorousPhosphorous From phosphate salts.From phosphate salts. For DNA, RNA, ATP, etc For DNA, RNA, ATP, etc

SulfurSulfur From sulfate ions, hydrogen sulfide and sulfur From sulfate ions, hydrogen sulfide and sulfur

containing amino acidscontaining amino acids For protein synthesisFor protein synthesis

Trace ElementsTrace Elements Mg, Fe, Mn, Zn, Cu etc for metabolism.Mg, Fe, Mn, Zn, Cu etc for metabolism.

Culture MediumCulture Medium

MediumMedium Sterile - no living Sterile - no living

organisms present.organisms present. Simple - definedSimple - defined Inoculum- microbes Inoculum- microbes

that start the culturethat start the culture Culture = medium + Culture = medium +

microbesmicrobes Agar – polysaccharide Agar – polysaccharide

derived from seaweedderived from seaweed

Culture MediumCulture Medium MediumMedium

Agar - polysaccharide Agar - polysaccharide derived from seaweedderived from seaweed

Chemically definedChemically defined Complex - serum, beef Complex - serum, beef

or yeast extract, or yeast extract, peptones etcpeptones etc

Fastidious organisms Fastidious organisms have many nutritional have many nutritional requirements. Organic requirements. Organic growth factorsgrowth factors

Non-Selective MediaNon-Selective Media NutrientNutrient Agar Agar Blood AgarBlood Agar LB agar (no antibiotics)LB agar (no antibiotics) TTryptic ryptic SSoy oy AAgar –TSA gar –TSA

in petri dishesin petri dishes Nutrient BrothNutrient Broth Sugar tubesSugar tubes TSA Agar (tube)TSA Agar (tube) TSA brothTSA broth Brain Heart InfusionBrain Heart Infusion

Selective MediaSelective Media

E. coliE. coli on EMB on EMB InhibitorInhibitor - -

Methylene blueMethylene blue IndicatorIndicator- Eosin- Eosin Gives a dark purple Gives a dark purple

colony with green colony with green metallic sheenmetallic sheen

Mannitol Salt AgarMannitol Salt Agar Phenylethyl AlcoholPhenylethyl Alcohol MacConkey AgarMacConkey Agar

Enrichment MediaEnrichment Media Media to increase the numbers Media to increase the numbers

of desired species, but doesn’t of desired species, but doesn’t purify them.purify them.

Used in soil and fecal samples.Used in soil and fecal samples. SalmonellaSalmonella- Selenite broth.- Selenite broth. Bacto-Dubos - Bacto-Dubos - M. tuberculosisM. tuberculosis Grow overnight and then plate Grow overnight and then plate

on elective medium.on elective medium. Increases chances of isolation Increases chances of isolation

when numbers are very low or when numbers are very low or contaminants very high.contaminants very high.

Obtaining Pure CulturesObtaining Pure Cultures

Streak plate.Streak plate. Each new direction Each new direction

diltues organisms diltues organisms until you get until you get individual ones.individual ones.

CFUCFU Serial dilution.Serial dilution.

Dilute in broth until Dilute in broth until you get 1 organism. you get 1 organism. 1:10 --> 1:100 --> 1:10 --> 1:100 --> 1:0001:000

Obtaining Pure CulturesObtaining Pure Cultures

Preserving CulturesPreserving Cultures Large stock tubes - Large stock tubes -

4 to 6 months @ 4 to 6 months @ 4˚C4˚C

Freeze in medium with Freeze in medium with glycerol -20˚C- 1 year glycerol -20˚C- 1 year

Store -70˚C - 5 to 10 Store -70˚C - 5 to 10 yearsyears

Store liquid nitrogen - Store liquid nitrogen - indefinitely.indefinitely.

Lyophilization- freeze Lyophilization- freeze drying - indefinitely. drying - indefinitely.

Frozen Culture TrickFrozen Culture Trick Can’t thaw and freeze very Can’t thaw and freeze very

many times- kills organisms.many times- kills organisms. Aseptically add 0.2-0.3 mls Aseptically add 0.2-0.3 mls

of warm medium to top of of warm medium to top of the frozen vial.the frozen vial.

Swish with pipette tip and Swish with pipette tip and remove liquid.remove liquid.

This thaws only a small top This thaws only a small top portion keeping the portion keeping the remainder frozen.remainder frozen.

Can do this 10 times per Can do this 10 times per vial. vial.

Bacterial GrowthBacterial Growth

Binary FissionBinary Fissionone cell makes twoone cell makes two

Cell first grows in size.Cell first grows in size. Replicates its DNA (genome)Replicates its DNA (genome) Then divides.Then divides. Time it takes between Time it takes between

divisions is the divisions is the Generation Generation Time.Time.

Dynamics of Bacterial Dynamics of Bacterial GrowthGrowth

Generation Times GGeneration Times GTT.. E. coliE. coli - 20 minutes - 20 minutes Bacillus subtilisBacillus subtilis - 1-2 hours - 1-2 hours Borrelia burgdorferiBorrelia burgdorferi - 8-10 hours - 8-10 hours Mycobacterium tuberculosisMycobacterium tuberculosis- 1 day- 1 day Treponema pallidumTreponema pallidum - 35 hours ( in - 35 hours ( in

rabbits)rabbits) Mycobacterium leprae Mycobacterium leprae - > 30 days- > 30 days

Phases of GrowthPhases of Growth

LagLag - no growth. - no growth. Exponential Exponential

GrowthGrowth- rapid - rapid growth- Ggrowth- GTT..

Stationary PhaseStationary Phase- - equal numbers dying equal numbers dying and dividing.and dividing.

Logarithmic Death Logarithmic Death - - rapid death due to :rapid death due to : Lack of nutrientsLack of nutrients Hostile media Hostile media

conditions (acid, conditions (acid, toxic by-products)toxic by-products)

Methods to Measure Methods to Measure GrowthGrowth

DirectDirect Plate CountsPlate Counts Direct CountingDirect Counting

Indirect (estimations)Indirect (estimations) TurbidityTurbidity Metabolic activityMetabolic activity Dry WeightDry Weight

Plate CountsPlate Counts

Perform serial dilutionsPerform serial dilutions Plate out samples from Plate out samples from

dilutionsdilutions

Plate CountsPlate Counts

Can do spread Can do spread plates for plates for aerobes.aerobes.

Can do pour Can do pour plates for plates for facultative facultative anaerobes or anaerobes or microaerophiles.microaerophiles.

Direct CountsDirect Counts

Petroff-Hausser Petroff-Hausser chamberchamber Dilute sample.Dilute sample. Add volume of Add volume of

diluted medium to diluted medium to chamber.chamber.

Count bacteriaCount bacteria Calculate number Calculate number

in original sample.in original sample.

Indirect MethodsIndirect Methods

Metabolic ActivityMetabolic Activity By product - COBy product - CO22

More product / min More product / min = higher = higher raterate = =

more bacteriamore bacteria

Dry WeightDry Weight Collect by Collect by

centrifugationcentrifugation Decant liquidDecant liquid DryDry WeighWeigh

TurbidityTurbidity

Use spectrophotometer.Use spectrophotometer. Measure transmission in sterile medium.Measure transmission in sterile medium. Measure transmission in culture.Measure transmission in culture.