Chapter 10: Classification of Microorganisms

1 Chapter 10: Classification of Microorganisms2. Methods of Identification1. The Taxonomic Hierarchy1. The Taxonomic HierarchyPhylogenetic Tree of the 3 Domains TaxonomicHierarchy DomainKingdomPhylumClassOrderFamilyGenus Species 8 successive taxaare used to classifyeach species:**species can also contain different strains**Scientific Nomenclature To avoid confusion, every type of organism must be referred to in a consistent current system of nomenclature (naming) has been in use since the 18thcentury: every type of organism is referred by its genusnamefollowed by its specific epithet( , species name)Homo sapiens(H. sapiens)Escherichia coli (E. coli) names are Latin (or Latinized Greek) with the genusbeing a noun and the specific epithet an adjective name should be in italicsand only the genus is capitalizedwhich can also be abbreviated**strain info can be listed after the specific epithet ( , E.)

2 Coli DH5 )**2. Methods of IdentificationBiochemical Testing In addition to morphological ( , appearance under the microscope)and differential staining characteristics, Microorganisms can also be identified by their biochemical signatures : the nutrient requirements and metabolic by-products ofof a particular microorganism different growth media can be used to test the physiological characteristics of a microorganism , medium with lactose only as energy source , medium that reveals H2S production**appearance on test medium reveals + or result!**Commercialdevices for rapid Identification Performmultiple testssimultaneouslyEnterotube IISuch devices involve the simultaneous inoculationof various test media: ~24 hrs later the panel of results reveals ID of organism!

3 Use of dichotomous keys Series of yes/no biochemical tests to ID organism. tests done in alogical order,each test resultindicates nexttest to be done collectiveresults of multiple testscreate a profileallowing ID ofmicroorganismSerology ( , antibodies) Specific antibodies can be used to ID bacteria: antibodies are produced by animals to anything foreign animals (rabbits, ) are routinely injected with biological material for which antibodies are needed antibodies present in theanimal serum can then beused in various ID , the agglutinationtestdifferences in antibody reactivity can reveal different bacterial strains orserovarsPhage (virus) Typing Bacteriophages (viruses that infect bacteria) have very specific hosts and can be use to ID bacteria.

4 Grow a lawn of bacteriato be tested on agar plate dot different test phagesamples on surface after ~24 hr, clear zonesappear where bacteriahave been infected & killed profile of phage sensitivitycan reveal ID of bacteriaDNA Base Composition Members of the same genera or species have nearly identical DNA sequences, and hence the same proportions of G/C base pairs & A/T base pairs: because they base pair, G = C and A = T G/C + A/T = 100% ( , if G/C = 40% then A/T = 60%)Determining the G/C content of the DNA from a testorganism and comparing to known values is a quick way to eliminate possible identities: if %G/C is different, cannot be a match! if %G/C is same, might be a match but additional testingis necessary to confirmThe Use of DNA Hybridization With enough heat, DNA strands will allows complementary strands to base pair.

5 This technique isused in a varietyof ways to see ifDNA from twodifferent sourcesare similar usually the DNAfrom one sourceis immobilized,the other is labeled to allowdetection FISH Fluorescent in situ hybridization:1) label DNA probe (fr. species of interest) w/fluorescent tag 2) chemically treat cells to allow DNA to enter, hybridize3) wash & view with fluorescence microscopy**cells w/DNA complementary to probe will fluoresce!**PCR selectively amplifies only desired DNA(if present) , DNA fromsuspectedpathogenPolymeraseChainReac tionPCR is a technique that involves manipulating DNA replication in of PCR Technique artificial primers specific for DNA of interest heat-stableDNA polymeraseEvery PCR reaction requires the following: DNA source to be tested (or amplified) free nucleotides (dNTP s)Plus an automated thermocyclerto facilitaterepeated cycles of:1) denaturationof DNA (separation of strands) @ ~95o C2) hybridizationof primers to template @ ~50-60o C3) DNA synthesis@ ~72o CRibosomal RNA (rRNA) ComparisonProkaryotic ribosomes contain 3 different rRNA mol.

6 : large subunit contains 23S (2900 nt) & 5S (120 nt) rRNA small subunit contains 16S (1500 nt)16S rRNA sequence is typically used for ribotyping: sequence is highly conserved (varies little) degree of difference reflects evolutionary distance **primary method for classifying prokaryotic species**Key Terms for Chapter 10 serology hybridization phage typing FISH dichotomous key PCR, thermocyclerRelevant Chapter Questions rvw: 4-10, 13, 14 MC: 2-8 ribotypingChapter 3:Microscopy1. Types of Microscopy2. Staining1. Types of MicroscopyaScale of Magnification Light microscopyElectron microscopy(TEM & SEM): limit of resolution ~ nm sufficient to see subcellulardetail, large molecular complexes limit of resolution* ~ m sufficient to see most organelles, bacteria*resolution= ability to distinguish objects close to each otherLight Microscopy Most common type isthe CompoundLightMicroscope:1) condenser lens focuseslight source on sample2) objective lens magnifiesthe image3) ocular lens furthermagnifies image123 Oil Immersion & Light Refraction Different media (air, water, glass, ) bend lightto different degrees.

7 , have differentrefractive indexes the oil immersionlens is too smallto capture all lightrefracted by air immersion oil hasrefraction indexsimilar to glass,allows more lightto enter the lensBright & Dark Field Microscopy Bright Field Microscopy standard or default type of light microscopyDark Field Microscopy barrier in condensereliminates all direct light only light reflected byspecimen enters theobjective lensPhase Contrast & DIC Microscopy Phase-Contrast Microscopy provides internal detail,contrast, w/o staining useful for livespecimensDifferential InterferenceContrast (DIC) Microscopy variation on phase-contrastwith a 2ndlight source greater detail, contrastFluorescence Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific UV light, dye fluoresces, onlylabeled cells or structures are MicroscopyOnly light from a given depth or plane is transmitted, out of focus light excludedElectron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen.

8 Electron beams have veryshort wavelengths allows far greater resolutionthan with light microscopyTransmission EM (TEM) thin sections of specimen,highest resolutionScanning EM (SEM) reveals surface features2. StainingWhy the Need for Stains?Because, no matter how high the magnification or resolution, you need contrastto be able to see contrast is not sufficient in the sample orthe microscopic method used, stainingcanprovide the necessary contrast: stains used for viewing bacteria via light microscopy aretypically positively charged chromophores(basicdyes) chromophore = color-bearing ion of a salt bacteria have a net negative charge ( , bind positive ions) General Types of StainsSimplestain dye that non-specifically stains all organisms, featuresDifferentialstain dye that binds various structures or organisms differently Negativestain dye that stains background, not specimenSpecialstain dye that specifically stains certain subcellular structures**a mordantis any chemical added to enhance a stain**Counterstain a 2nddye added that is a different color than original dyeGram Staining A very common stain to distinguish 2 bacterial types:Process.

9 1) primary stain2) add mordant3) decolorize*4) counter stain retain primarystainGram positive thick cell wall,NOouter membrane retain only thecounter stainGram negative thin cell wall, have outer membrane123*4* key stepAcid-Fast Staining Most bacteria are not acid-fast ( , don t retainstain afteracid wash). only stainsspecies in thegenera: non-acid-fast cells revealedby counterstainacid fastnon-acid fastMycobacteriumNocardiaOther Types of StainingNegative Staining , capsule stainSpore Staining specific for endosporesFlagella StainingKey Terms for Chapter 3 fluorescent, confocal microscopy refraction & oil immersion transmission vs scanning EM bright & dark field, phase contrast microscopy resolution simple, differential, counter, negative stains gram & acid-fast stains, mordant, chromophoreRelevant Chapter Questions rvw: 1-13 MC: 10