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Prokaryotic and Eukaryotic Classification & Identification

Kathy Huschle Northland Community and Technical College

All Species Inventory


in 2001 international project launched to identify and record every species on Earth in the next 25 years a very challenging undertaking considering that to date 1.5 million organisms have been named it is estimated that anywhere from 7 100 million living species exist science of organizing organisms into groups those with similar properties being grouped together similarities are due to relatedness phylogeny is the study of evolutionary history of organisms organization of organisms reflect phylogeny or evolutionary relationships

Taxonomy
three separate but interrelated disciplines are involved in taxonomy identification characterizing organisms classification arranging into similar groups nomenclature naming organisms

Taxonomy
organizing larger organisms based on morphology is often quite simple:

verses

feathers
verses

fur

fins legs with prokaryotes, it is not as simple

Prokaryote Classification
technologies used to characterize and ID prokaryotes microscopic examination culture characteristics biochemical testing nucleic acid analysis combination of the above is most accurate

Taxonomic Classification Categories


arranged in hierarchical order species is basic unit

Domain Kingdom Phylum or Division Class Order Family Genus Species

Classification Systems: a short history


in 1750, Carl Linnaeus devised the first classification system, placing plants and animals in separate systems Linnaeus placed all microorganism in one genus he named Chaos

Classification Systems: a short history


in 1866, Ernst Haeckel divided animals, plants, and microorganisms into 3 kingdoms Animalia Plantae Protista

3 Kingdoms

Haeckels Tree of Life

Classification Systems: a short history


in 1969, Robert Whittaker created a 5 Kingdom classification system, using obvious morphological differences Animalia Plantae Fungi Protista Monera

Protists include algae and protozoa

Classification Systems: a short history


in 1970, Carl Woese, by analyzing RNA, developed the 3 domain classification system archaebacteria bacteria eucarya

This system is currently favored by microbiologists.

Classification Systems
classification systems continue to evolve and will change as new information is discovered emerging technology increases the knowledge base of organisms

Click on the icon to enter a virtual lab. After connection, click bacterial identification.

Classification of Viruses
viruses are not included in the 3 Domain classification system viruses are not composed of cells the ecological niche of a virus is the host cell viruses may be more related to their host than to other viruses though not included in the 3 domain system, classification of animal viruses is currently based on genome structure single, double, DNA, RNA??? virus particle structure isometric, helical, pleomorphic??? presence or absence of viral envelope

Nomenclature
organisms must have a Latin suffix name is italicized or underlined all organisms have a binomial name 1st part of the binomial name is genus group of closely related species Canus 1st letter capitalized 2nd part or the binomial name is the species epithet written in all lower case latrans lupus familiaris

Nomenclature
species name include both genus and species epithet can abbreviate Canus letrans - coyote C. lupus - wolf C. familiaris - dog

C. lupus

Canus letrans

C. familiaris

Phenotypic Characteristics for Identifying Prokaryotes


often does not require sophisticated equipment can easily be done anywhere

Microscopic Phenotypic Exam


size and shape and arrangement information is retrieved quickly in some cases size and shape of a microorganisms is enough information for diagnosis of certain infections

yeast cells

Microscopic Phenotypic Exam


Gram positive

Gram stain distinguishes between Gram + and Gram bacteria narrows the possibilities quickly

Gram negative

Microscopic Phenotypic Exam


special stain allows for the distinction of microorganisms with unique characteristics capsule acid fast staining detects the waxy presence of Mycobacterium tuberculosis

Capsule staining

Acid fast staining of M. tuberculosis

Metabolic Phenotypic Exam


cultural approaches required for positive diagnosis of infection isolation and ID of pathogen accuracy, reliability, and speed specimen collection is important commonly used for positive identification of most prokaryotes methods used include culture characteristics biochemical reactions process

Metabolic Phenotypic Exam


culture characteristics organisms grown in a pure culture are easier to identify due to the high number of organisms obtained

E. coli

Metabolic Phenotypic Exam


culture characteristics use of selective or differential media can provide additional information selective media inhibits the growth of organisms other than the one being sought differential media contains substances that particular bacteria change in a recognizable way

Metabolic Phenotypic Exam


cultural characteristic examinations are speedy and accurate

MacConkey agar, differential for lactose fermentation and selective for Gram rods.

Urine sample swabbed on MacConkey agar results in the formation of pink colonies. E. coli, the most common causative agent for urinary tract infections, ferments lactose and is a Gram - rod

Metabolic Phenotypic Exam


cultural testing will again narrow the field of choices, but biochemical tests are generally used for conclusive ID biochemical testing utilizes pH indicators chemical reactions

Metabolic Phenotypic Exam


When identifying a suspected organism, a series of differential media is inoculated. After incubation, each medium is observed to see if specific end products of metabolism are present. This can be done by adding indicators to the medium that react specifically with the end product being tested, giving some form of visible reaction such as a color change. The results of these tests on the suspected microorganism are then compared to known results for that organism to confirm its identification.

Metabolic Phenotypic Exam


Some bacteria will produce the enzyme catalase. Catalase will break down hydrogen peroxide releasing oxygen, which is indicated by the bubbles that have formed.

Metabolic Phenotypic Exam


commercial tests have been developed using the qualities of the traditional methods of biochemical testing many tests can be incorporated in to one, saving labor

Enterotube, with each compartment containing a different type of selective or differential media. A metal rod touches the colony of bacteria and as it is withdrawn, it inoculates all of the media

Serological Testing Phenotypic Exam


serology uses the differences between the proteins and polysaccharides that make up the bacteria for identification distinguishing these differences relies on interactions between the antibody and antigen, which form insoluble aggregates of antibody-antigen complexes this formation is referred to as agglutination

Serological Testing Phenotypic Exam


serological testing uses ELISA testing fast and easy to use

Genotypic Characteristics for Identifying Prokaryotes


the use of genotypic testing has increased with the availability of technology genotypic testing is particularly useful in the case of organisms that are difficult to identify several techniques include gene probes PCR sequencing rRNA

Genotypic Characteristics for Identifying Prokaryotes


gene probes single stranded DNA that has been labeled with a identifiable tag, such as a fluorescent dye are complementary to target nucleotide sequences unique in DNA of pathogen

Microbe gene probed

Genotypic Characteristics for Identifying Prokaryotes


If there is a suspicion, based on symptoms or other environmental parameters that indicates that the organism to be identified may be organism A, a single strand of organism As DNA is introduced with a tag attached (such as fluorescent dye). If the introduced DNA binds to the unknown organism, then it is identified as organism A. If it does not bind to the unknown organism, then the unknown is not organism A.

Genotypic Characteristics for Identifying Prokaryotes


PCR: polymerase chain reaction used to detect small amounts of DNA present in a sample (blood, food, soil) the PCR chain reaction is used to amplify the amount of DNA present

Genotypic Characteristics for Identifying Prokaryotes


sequencing ribosomal RNA of particular use for identifying prokaryotes impossible to grow in a culture focus is place on the 16S molecules of the RNA because of its size approximately 1500 nucleotides once the 16S molecule is sequenced, it can then be compared to the sequences of known organisms
Machine used to pick colonies containing wanted DNA

Difficulties in Classifying Prokaryotes


historically prokaryotes have been grouped according to phenotypic attributes problems with this approach include mutation resulting in phenotypic changes just because they look alike, does not mean that they are even closely related according the prokaryotics new molecular approaches are providing better insight to the relatedness of microorganisms the more similar the nucleotide sequence, the more closely related

DNA extraction

Genotypic Characteristics used in Classifying Prokaryotes


comparison of nucleotide sequences differences in DNA sequence can assist in determination of divergence of evolutionary path for organisms DNA hybridization single strands of DNA anneal 16S ribonucleic acid comparing sequence of ribosomal RNA relatedness to other organisms can be determined using numerical taxonomy determined by the percentage of characteristics two organisms have in common The more you have in common phenotypically with another organism the closer related you are to that organism.