Chapter 10:
Classification of Microorganisms

1. The Taxonomic Hierarchy 2. Methods of Identification

1. The Taxonomic Hierarchy

Phylogenetic Tree of the 3 Domains

Taxonomic
Hierarchy

• 8 successive taxa are used to classify each species:

Domain
Kingdom
Phylum
Class

Order
Family
Genus
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 18^th century:

• every type of organism is referred by its genus name followed by its specific epithet (i.e., species name)

Homo sapiens (H. sapiens) Escherichia coli (E. coli)

• name should be in italics and only the genus is capitalized which can also be abbreviated

• names are Latin (or “Latinized” Greek) with the genus being a noun and the specific epithet an adjective

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

2. Methods of Identification
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” of of 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!**

Commercial devices for rapid
Identification

Perform multiple tests simultaneously

Enterotube II

Such devices involve the simultaneous inoculation of 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 a logical order, each test result indicates next test to be done

• collective results of multiple tests create a profile allowing ID of microorganism

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 the animal serum can then be used 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 bacteria to be tested on agar plate

• “dot” different test phage samples on surface

• after ~24 hr, clear zones appear where bacteria have been infected & killed

• profile of phage sensitivity can 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 test organism 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 testing is necessary to confirm

The Use of DNA Hybridization

With enough heat, DNA strands will separate. Cooling allows complementary strands to base pair.

• this technique is used in a variety of ways to see if DNA from two different sources are similar

• usually the DNA from one source is immobilized, the other is labeled to allow detection

“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

Polymerase Chain Reaction

• selectively amplifies only desired DNA (if present)

• e.g., DNA from suspected pathogen

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

Overview of PCR Technique

Every PCR reaction requires the following:

• DNA source to be tested (or amplified) • artificial primers specific for DNA of interest • heat-stable DNA polymerase • free nucleotides (dNTP’s)

Plus an automated thermocycler to facilitate repeated cycles of:

1) denaturation of DNA (separation of strands) @ ~95^o C 2) hybridization of primers to template @ ~50^-60^o C 3) DNA synthesis @ ~72^o 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**

Key Terms for Chapter 10

• dichotomous key • serology • phage typing • hybridization • FISH • PCR, thermocycler • ribotyping

Relevant Chapter Questions

rvw: 4-10, 13, 14 MC: 2-8

Chapter 3: Microscopy

1. Types of Microscopy 2. Staining

1. Types of Microscopya
Scale of Magnification

Light microscopy

• limit of resolution* ~0.2 μm • sufficient to see most organelles, bacteria

Electron microscopy (TEM & SEM):

• limit of resolution ~2.5-20 nm • sufficient to see subcellular detail, large molecular complexes

*resolution = ability to distinguish objects close to each other

Light Microscopy

Most common type is the Compound Light Microscope:

3

1) condenser lens focuses light source on sample

2

2) objective lens magnifies the image

1

3) ocular lens further magnifies image

Oil Immersion & Light Refraction

Different media (air, water, glass, oil…) bend light to different degrees.

• i.e., have different refractive indexes

• the oil immersion lens is too small to capture all light refracted by air

• immersion oil has refraction index similar to glass, allows more light to enter the lens

Bright & Dark Field Microscopy

Bright Field Microscopy Dark Field Microscopy

• • standard or “default”
• • barrier in condenser

• type of light microscopy
• eliminates all direct light

• only light reflected by specimen enters the objective lens

Phase Contrast & DIC Microscopy

Phase-Contrast
Microscopy
Differential Interference
Contrast (DIC) Microscopy

• • provides internal detail,
• • variation on phase-contrast

contrast, w/o staining

with a 2^nd light source

• • useful for live specimens
• • greater detail, contrast

Fluorescence Microscopy

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

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

standard

confocal

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 very short wavelengths

• allows far greater resolution than with light microscopy

Transmission EM (TEM)

• thin sections of specimen, highest resolution

Scanning EM (SEM)

• reveals surface features

2. Staining