L12: Classification of Microorganisms Flashcards
taxonomy definition
the science of classifying organisms by degree of similarities among them
taxonomy nomenclature
provides universal names for organisms
provides reference for identifying new organisms
systematics/phylogeny definition
study of the evolutionary history of organisms
true or false: bacteria and archaea, the 2 prokaryotes, share many similarities
false; bacteria and archaea differ in biochemistry, function, and etc.
true or false: archaea are more similar to eukaryotes than either are similar to bacteria
true; see tree of life diagram
endosymbiotic theory
eukaryotes came from prokaryotes that mutated and got ingested/infected by other prokaryotes, becoming endosymbionts; first began with bacteria evolving into mitochondria and chloroplasts
what is the evidence for endosymbiotic theory?
mitochondria and chloroplasts share similarities w/ bacteria:
- contain 70S ribosomes
- have tiny circles of DNA
- divide via binary fission
- share the first amino acid (Formylmethionine)
phylogenetics pt. 1
each species retains some characteristics of its ancestor
phylogenetics pt. 2
grouping organisms by common properties implies that a group of organisms from a common ancestor
phylogenetics are based on…
anatomy
fossils
rRNA seq similarity
scientific nomenclature
is binomial: genus + specific epithet
italicized OR underlined
genus is ALWAYS capitalized
specific epithet is ALWAYS lowercase
taxonomic hierarchy: phylum to species
phylum
class
order
family
genus
species
classification definition
placing organisms in groups of related species
identification definition
matching characteristics of an “unknown” organism to lists of known organisms
prokaryotic species definition
a population of cells with similar characteristics
culture
a population of bacterial cells grown in laboratory media
clone
population of cells derived from a single cell
strain
genetically different cells within a clone in which strains are differentiated based on the 16S ribosome
how do strains differ from species?
strains are closely related enough to where they are considered the same species, but have some genetic differences
strain example
harmless lab E. coli strains vs. E. coli O157:H7
do bacteria have kingdoms?
no; bacteria and archaea are domains
in the domain eukarya, what are the 4 kingdoms?
animalia
plantae
fungi
protista
animalia traits
multicellular
NO cell walls
chemoheterotrophic
plantae traits
multicellular
cellulose cell walls
photoautotrophic (mainly)
fungi traits
multi- OR uni-cellular
chitin cell walls
develop fr. spores, hyphal fragments
protista traits
wide variety
grouped into clades by rRNA
what is special about protists/protozoans?
protista is the “miscellanous” category for eukaryotes that do NOT fit the criteria for the other kingdoms
eukaryotic species definition
a group of closely-related organisms that breed among themselves
most are diploid or polyploid; most reproduce sexually
eukaryotes being diploid or polyploid, and able to reproduce sexually limits interbreeding to…
only eukaryotic organisms that are closely related
viral species definition
population of viruses w/ similar characteristics that occupy a particular ecological niche
what is special about viral species?
viral species are hard to classify
what are the 3 main identification methods?
analyzing morphological characteristics
differential staining
biochemical tests
morphological characteristics
useful for identifying eukaryotes
somewhat useful for bacteria and viruses
differential staining
includes Gram staining, acid-fast staining
useful for identifying bacteria
biochemical tests
determines presence of bacterial enzymes
what are the identification methods for bacteria?
- metabolic biochemical tests
- immunoassays
- flow cytometry
- protein profiling by mass spectrometry
- fatty acid profiling
- bacteriophage typing
- DNA methods
metabolic biochemical tests
require growing bacteria in a culture from a sample
immunoassays
the most common diagnostic test
uses antibodies
can identify antigens (bacteria) based on what antibodies are produced, etc.
flow cytometry
uses differences in electrical conductivity between different types of cells
in some cases, are part of immunoassays
how does flow cytometry identify microbes?
differential fluorescence in cells; can be produced by staining w/ fluorescent dye that stains some cells but not others
fluorescence = signifier of infection
how is flow cytometry incorporated into immunoassays?
cells can also be labeled w/ an antibody that selectively binds some cells; the antibody can be attached to a fluorescent dye
protein profiling by mass spectrometry
cellular proteins are profiled chemically and compared to known standards for particular bacterial stains
fatty acid profiling
AKA fatty acid methyl esters
compares differences in FA profiles to standards
bacteriophage typing
an older, now less-used method
set of phages (viruses) infects a bacterial strain to determine the strain identity
phage
viruses that attack bacteria
what is the mechanism of phage typing?
some strains of a bacterial species will be sensitive to a particular phage, and others will be resistant
how to use phage typing?
know which bacteria are sensitive and which are resistant to a set of well-characterized phage so you can use the phages to identify the bacteria in an unknown sample
clear spots signify that…
the strain is extremely sensitive to that phage
partially clear spots signify that…
the strain is partially resistant
opaque spots signify that…
the strain is completely resistant to that phage
DNA methods
- DNA fingerprinting methods
- rDNA sequencing
- hybridization
- whole genome sequencing
DNA methods: rDNA sequencing
identifies bacteria via the 16S rDNA gene (ribotyping); is amplified by polymerase chain reaction (PCR)
DNA methods: older methods such as ______ and ________ are being phased out
DNA fingerprinting
DNA base composition
DNA methods: DNA fingerprinting
electrophoresis of restriction enzyme digests
DNA methods: DNA base composition
measures % of G + % of C (GC%); bacteria have very low to very high ranges of GC%
DNA methods: DNA hybridization
requires a “DNA probe”
serological tests (immunoassays)
uses antibodies (AKA immunoglobulins)
what are the main types of immunoassays/serological tests?
- slide agglutination test
- ELISA (enzyme-linked immunosorbent assay)
slide agglutination test
latex beads w/ a known antibody attached are combined on a slide w/ a sample containing the unidentified organism
various subtypes of this test can be used for specific properties (enzymes, proteins, etc.)
slide agglutination test: if the test antibodies recognize “epitopes” in the sample…
large aggregates of latex + antibody bound to the recognized organism will be formed
a positive slide agglutination test appears…
slightly translucent and foamy; expanded
a negative slide agglutination test appears…
mostly white and opaque
ELISA (enzyme-linked immunosorbent assay)
uses 2 antibodies and an enzyme substrate in a dish to identify the antigen/organism
ELISA: general steps?
-known “primary” antibodies attached to dish
- a positive reaction results in the organism/antigen being captured by the antibody
ELISA: constant region definition
the Y-shaped structure where the antibodies “stick”
what are the 2 main types of ELISAs?
“sandwich”
direct
what is the target in each type of ELISA?
sandwich = antigen
direct = antibody
when an ELISA is used to determine if a patient has been exposed to and begun to respond to a pathogen, what type of ELISA is used?
antibody-capture, direct ELISA
when an ELISA is used to determine if a patient has an active infection of a pathogen, what type of ELISA is used?
antigen-capture, indirect/sandwich ELISA
when an ELISA is used to determine if a patient has been exposed to and begun to respond to a pathogen, the “bait” bound to the wells of the assay dish is…
antigen from the pathogen
when an ELISA is used to determine if a patient has an active infection of a pathogen, the “bait” bound to the wells of the assay dish is…
prepared (primary) antibody that recognizes the pathogen
when an ELISA is used to determine if a patient has an active infection of a pathogen, the secondary antibody used for detection in this type of assay must recognize and bind specifically to the…
human antibody constant region
when an ELISA is used to determine if a patient has an active infection of a pathogen, the secondary antibody used for detection in this type of assay must recognize and bind specifically to the…
antigen from the pathogen
ELISA Method A - Sandwich: step 1?
attach known “primary” antibody to the bottom of the dish
ELISA Method A - Sandwich: step 2?
add antigen; antigen will bind to the primary antibody
ELISA Method A - Sandwich: step 3?
add secondary antibody
ELISA Method A - Sandwich: step 4?
if the organism/antigen has bound to the primary antibody, the secondary antibody is captured
ELISA Method A - Sandwich: step 5?
secondary antibody covalently links to an enzyme
ELISA Method A - Sandwich: step 6?
substrate is added, and serves as a colorimetric indicator; goes through a color change once catalyzed by enzyme
ELISA Method B - direct: step 1?
attach antigen to the bottom of the dish
ELISA Method B - direct: step 2?
add antibodies (patient serum)
ELISA Method B - direct: step 3?
add secondary antibody that binds specifically to human antibodies
ELISA Method B - direct: step 4?
secondary antibody covalently links to an enzyme
ELISA Method B - direct: step 5?
add substrate; will connect to enzyme
enzyme substrate is a colorimetric indicator
what is the difference between the sandwich and direct ELISA?
sandwich: antigen capture (entire or part of the antigen)
- use antibody –> capture antigen
direct: antibody capture
- use antigen –> capture antibody
western blot
use of gel electrophoresis to transfer proteins to determine the presence of antigens
modern classification is heavily dependent on…
nucleic acid sequence (genome, rRNA)
nucleic acid hybridization
a DNA probe is hybridized to the denatured total DNA of an organism
the probe is tagged with a marker that allows it to be visualized
in nucleic acid hybridization, how is the probe generated?
PCR (polymerase chain reaction)
chemical synthesis
as new biotechnology develops and improves, older methods are…
being phased out and replaced by sequencing
