identification and classificatin of prokaryotes
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Prokaryotic and Eukaryotic
Classification & Identification
Kathy HuschleNorthland Community and Technical College
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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
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Taxonomy
three separate but interrelated disciplines are involved intaxonomy
– identification
characterizing organisms
– classification
arranging into similar groups
– nomenclature
naming organisms
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Taxonomyorganizing larger organisms based on morphology is
often quite simple:
verses
feathers
verses
legs
with prokaryotes, it is not as simple
fur
fins
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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
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Taxonomic Classification Categories
arranged in hierarchical order species is basic unit
Domain
KingdomPhylum or Division
Class
Order
FamilyGenus
Species
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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
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Classification Systems:
a short history
in 1866, Ernst Haeckel dividedanimals, plants, and
microorganisms into 3 kingdoms – Animalia
– Plantae
– Protista
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Haeckel’s Tree of
Life
3 Kingdoms
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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
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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.
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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.
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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
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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
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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
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Phenotypic Characteristics for
Identifying Prokaryotes
often does not require sophisticated equipment
can easily be done anywhere
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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
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Microscopic Phenotypic Exam
Gram stain
– distinguishes between
Gram + and Gram –
bacteria
– narrows the possibilities
quickly
Gram positive
Gram negative
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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
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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
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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
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Metabolic Phenotypic Exam
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
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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
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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
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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. Theresults of these tests on the suspected microorganism are
then compared to known results for that organism to confirm
its identification.
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Metabolic Phenotypic Exam
Some bacteria will
produce the enzyme
catalase. Catalasewill break down
hydrogen peroxide
releasing oxygen,
which is indicated by
the bubbles that have
formed.
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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 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
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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
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Serological Testing
Phenotypic Exam
serological testing uses ELISA
testing
– fast and easy to use
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Genotypic Characteristics for
Identifying Prokaryotes
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
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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
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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 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”.
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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
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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
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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
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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