Identification and Classificatin of Prokaryotes

7/30/2019 Identification and Classificatin of Prokaryotes

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Prokaryotic and Eukaryotic

Classification & Identification

Kathy HuschleNorthland 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 todate 1.5 million organisms have been named

– it is estimated that anywhere from 7 – 100 millionliving 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 intaxonomy

– identification

characterizing organisms

– classification

arranging into similar groups

– nomenclature

naming organisms



Taxonomyorganizing larger organisms based on morphology is

often quite simple:

verses

feathers

verses

legs

with prokaryotes, it is not as simple

fur

fins



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

KingdomPhylum or Division

Class

Order

FamilyGenus

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 microorganismin one genus he named Chaos




a short history

in 1866, Ernst Haeckel dividedanimals, plants, and

microorganisms into 3 kingdoms – Animalia

– Plantae

– Protista



Haeckel’s Tree of

Life

3 Kingdoms




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




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.

http://www.hhmi.org/biointeractive/vlabs/index.html



Classification of Viruses

viruses are not included in the 3 Domain classificationsystem

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 toother 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



Nomenclaturespecies name

– include both genus and species epithet – can abbreviate

Canus letrans - coyote

C. lupus - wolf

C. familiaris - dog C. lupus

C. familiaris

Canus letrans



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




Gram stain

– distinguishes between

Gram + and Gram –

bacteria

– narrows the possibilities

quickly

Gram positive

Gram negative




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




culture characteristics

– organisms grown in a pure culture are easier to

identify due to the high number of organisms

obtained

E. coli




culture characteristics

– use of selective or differential media can provideadditional information

selective media inhibits the growth of organisms

other than the one being soughtdifferential media contains substances thatparticular bacteria change in a recognizable way




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




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




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. Theresults of these tests on the suspected microorganism are

then compared to known results for that organism to confirm

its identification.




Some bacteria will

produce the enzyme

catalase. Catalasewill break down

hydrogen peroxide

releasing oxygen,

which is indicated by

the bubbles that have

formed.




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 compartmentcontaining 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 andpolysaccharides that make up the bacteria for identification

– distinguishing these differences relies on interactions

between the antibody and antigen, which forminsoluble 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


the use of genotypic testing has increased with theavailability 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





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





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 tagattached (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”.





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





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 relatedDNA

extraction

G t i Ch t i ti d i



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 usingnumerical taxonomy

– determined by the percentage of characteristics twoorganisms have in common

The more you have in common phenotypically with another

i th l l t d t th t i

Identification and Classificatin of Prokaryotes

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