Prokaryotic and Eukaryotic Classification & Identification

Prokaryotic and EukaryoticClassification & 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 fur feathers verses 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 Haeckel’s 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 A’s” 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 it’s 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.

Prokaryotic and Eukaryotic Classification & Identification