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.
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
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