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Chapter 10:Classification of Microorganisms

2. Methods of Identification

1. The Taxonomic Hierarchy

1. The Taxonomic Hierarchy

Phylogenetic Tree of the 3 Domains

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TaxonomicHierarchy

Domain

Kingdom

PhylumClass

Order

Family

Genus

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 way.The current system of nomenclature (naming) has been in use since the 18th century:

• every type of organism is referred by its genus namefollowed by its specific epithet (i.e., 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 italics and only the genus is capitalizedwhich can also be abbreviated

**strain info can be listed after the specific epithet (e.g., E. coli DH5α)**

2. Methods of Identification

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Biochemical Testing In addition to morphological (i.e., 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

• e.g., medium with lactose only as energy source

• e.g., medium that reveals H2S production

**appearance on test medium reveals + or – result!**

Commercialdevices for rapid

Identification Performmultiple testssimultaneously

Enterotube II

Such devices involve the simultaneous inoculationof various test media:

• ~24 hrs later the panel of results reveals ID of organism!

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 ofmicroorganism

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Serology (i.e., antibodies) Specific antibodies can be used to ID bacteria:

• antibodies are produced by animals to anything “foreign”

• animals (rabbits, goats…) are routinely injected with biological material for which antibodies are needed

• antibodies present in theanimal serum can then beused in various ID tests:

e.g., the agglutination test

differences in antibody reactivity can reveal different bacterial strains or serovars

Phage (virus) Typing Bacteriophages (viruses that infect bacteria) have very specific hosts and can be use to ID bacteria:

• 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 bacteria

DNA 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% (e.g., 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 confirm

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The Use of DNA Hybridization With enough heat, DNA strands will separate.Cooling allows complementary strands to base pair.

• 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, hybridize

3) wash & view with fluorescence microscopy**cells w/DNA complementary to probe will fluoresce!**

PCR

• selectively amplifies only desired DNA(if present)

• e.g., DNA fromsuspectedpathogen

PolymeraseChainReaction

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PCR is a technique that involves manipulating DNA replication in vitro…

Overview of PCR Technique

• artificial primers specific for DNA of interest• heat-stable DNA polymerase

Every PCR reaction requires the following:• DNA source to be tested (or amplified)

• free nucleotides (dNTP’s)

Plus an automated thermocycler to facilitaterepeated cycles of:

1) denaturation of DNA (separation of strands) @ ~95o C

2) hybridization of primers to template @ ~50-60o C

3) DNA synthesis @ ~72o C

Ribosomal RNA (rRNA) Comparison

Prokaryotic ribosomes contain 3 different rRNA mol.:• 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**

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Key Terms for Chapter 10

• serology

• hybridization

• phage typing

• FISH

• dichotomous key

• PCR, thermocycler

Relevant Chapter Questions rvw: 4-10, 13, 14 MC: 2-8

• ribotyping

Chapter 3:Microscopy

1. Types of Microscopy

2. Staining

1. Types of Microscopya

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Scale of Magnification Light microscopy

Electron microscopy(TEM & SEM):

• limit of resolution ~2.5-20 nm

• sufficient to see subcellulardetail, large molecular complexes

• limit of resolution* ~0.2 μm

• sufficient to see most organelles, bacteria

*resolution = ability to distinguish objects close to each other

Light Microscopy Most common type isthe Compound LightMicroscope:

1) condenser lens focuseslight source on sample

2) objective lens magnifiesthe image

3) ocular lens furthermagnifies image

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2

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Oil Immersion & Light Refraction Different media (air, water, glass, oil…) bend light

to different degrees. • i.e., have different

refractive indexes

• the oil immersionlens is too smallto capture all lightrefracted by air

• immersion oil hasrefraction indexsimilar to glass,allows more lightto enter the lens

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Bright & Dark Field Microscopy

Bright Field Microscopy• standard or “default”

type of light microscopy

Dark Field Microscopy• barrier in condenser

eliminates all direct light

• only light reflected byspecimen enters theobjective lens

Phase Contrast & DIC Microscopy

Phase-Contrast Microscopy

• provides internal detail,contrast, w/o staining

• useful for live specimens

Differential InterferenceContrast (DIC) Microscopy

• variation on phase-contrastwith a 2nd light source

• greater detail, contrast

Fluorescence Microscopy Fluorescent dyes or antibodies with a fluorescent tag stick to specific targets.

Under UV light, dye fluoresces, onlylabeled cells or structures are seen.

confocalstandard

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Confocal Microscopy

Only light from a given depth or plane is transmitted, “out of focus” light excluded

Electron Microscopy Electromagnetic lenses focus electron beam onto metal-stained specimen.

• electron beams have veryshort wavelengths

• allows far greater resolutionthan with light microscopy

Transmission EM (TEM)• thin sections of specimen,

highest resolution

Scanning EM (SEM)• reveals surface features

2. Staining

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Why the Need for Stains?Because, no matter how high the magnification or resolution, you need contrast to be able to see anything.

If contrast is not sufficient in the sample orthe microscopic method used, staining canprovide the necessary contrast:

• stains used for viewing bacteria via light microscopy aretypically positively charged chromophores (basic dyes)

• chromophore = “color-bearing” ion of a salt

• bacteria have a net negative charge (i.e., bind positive ions)

General Types of StainsSimple stain

• dye that non-specifically stains all organisms, features

Differential stain• dye that binds various structures or organisms differently

Negative stain• dye that stains background, not specimen

Special stain• dye that specifically stains certain subcellular structures

**a mordant is any chemical added to enhance a stain**

Counter stain• a 2nd dye added that is a different color than original dye

Gram Staining A very common stain to distinguish 2 bacterial types:

Process:1) primary stain

2) add mordant

3) decolorize*

4) counter stain

• retain primarystain

Gram positive

• thick cell wall,NO outer membrane

• retain only thecounter stain

Gram negative

• thin cell wall, have outer membrane

1 2 3* 4

* key step

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Acid-Fast Staining Most bacteria are not “acid-fast” (i.e., don’t retain

stain afteracid wash).

• only stainsspecies in thegenera:

• “non-acid-fast”cells revealedby counterstainacid fast non-acid fast

MycobacteriumNocardia

Other Types of Staining

Negative Staining• e.g., capsule stain

Spore Staining• specific for endospores

Flagella Staining

Key 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, chromophore

Relevant Chapter Questions rvw: 1-13 MC: 10