Week 8 Exam 3 Flashcards
Classification
Linnaeus - order & organization - predictions on structure & function - understand evolutionary connections organisms are grouped into taxa
Taxonomy:
Namenclature:
Classification:
Identification:
Taxonomy: science of classifying things (reflects phylogeny)
Namenclature: assigns scientific names to taxonomic categories & individuals
Classification: orderly arrangement of organisms into a hierarch(trees)
Identification: process of discovering & recording traits for taxonomic placement
- bacterial strain: population of cells that arose from a single cell
- species share at least 70% common DNA sequences
Carl Woese
- created 3 domain system : bacteria, eukaryote, and archaea
- created by: comparing rRNA sequences
- rRNA: ribosomal RNA, in all organisms, crucial for protein synthesis
Microbial Methods for Identification
- phenotypic: physical characteristics, biochemical tests, phage typing
- serological tests: immunologic(antibody/antigen binding)
- genotypic: analysis of nucleic acids
Phenotypic Methods
observation of traits/appearence/behavior
- types of enzymatic activities it can carry out
- physical conditions it thrives in
- antibiotics its susceptible to
- chemical composition of cell wall
Immunologic methods
- antibody response: patients sample is tested for presence of antibodies to a suspected pathogen (easier than testing for the microbe) ex. covid PCR, mono, group a strep
Genotypic Methods advantages
advantages: culturing is not necessary, rapid results, more precise than phenotypic methods, can be used for microbes not easily grown in the lab, can tell you the strain/species
Proper specimen collection:
- aseptic technique
- sterile sample containers & tools
only infected site should be sampled
Sputum collection techniques
- coughing into container
- catheter
(contamination with saliva should be avoided due to millions of bacteria per milliliter)
Urine collection samples
- aseptically: catheter
- clean catch: washing external urethra and collecting urine midstream (any contamination will be differentiated)
- mucus lining of urethra, vagina, or cervix with swab or applicator stick
sterile body fluids collection samples
- blood, cerebrospinal fluid and tissue fluid by sterile needle aspiration
antisepsis of puncture site in extremely important
other specimen sources:
- eye, ear canal, synovial fluid, nasal cavity: swab
- diseased tissue: surgically removed (biopsied)
Phenotypic methods: Immediate examination
-direct microscope observation of fresh or stained specimen
most rapid
stains often used: acid fast & gram
Phenotypic methods: 24 hour examination
growth of specimen on a media
- specialized media: grow pathogen over the amount of microbiota (increase in numbers of pathogen)
- selective media: used for non sterile specimens that contain a large amount of normal microbiota
- differential media: looks for distinctive characteristics and fermentation
Phenotypic methods: physical characteristics
after growth
- identified based on cellular morphology
- differential staining or structural staining
- distinct colony morphology of appearance
Phenotypic methods: biochemical tests
useful if microbes can grow under lab conditions provides evidence of enzymatic systems -fermentation of carbs -utilize specific substrates -produce waste products -fatty acid composition
Automated microscan system
- tool for rapid identification based on metabolic characteristics PHENOTYPIC
- multi-welled plate (98), use variety of chemical tests
- color change=presence of a particular metabolic reaction
dichotomous key
physical characteristics are based on D. key
-branching decision tree to help identify a microorganism
antimicrobial sesceptibility testing
useful in deterring which drugs will be used in treatment because of the rise of antimicrobial resistance
-phenotypic test
phage typing
helps identify bacterial strains
- phages: specific to host they infect and can be used to identify bacterial strains
- USE: bacteriophage (viruses that infect bacteria) to detect strain of bacteria
swab plate w/ bacteria you are wanting to identify, pipet small values of bacteriophage, a plaque(where the bacteriophage has infected the bacterium)
- bacteriophage infection observed using plaque assay
-phenotypic test
Drawbacks of Phenotypic methods:
- the microbe needs to be cultured: takes minimum of 18-24 hours
- many can be noonculturable, leaving the possibility that e culture normal microbiota
specificity
sensitivity
- specificity: focus on certain antigen and not react with unrelated antigen
- sensitivity: detection of small amount of antibodies
most effective immune testing has these
immunological methods: serology
- in vitro testing of serum, urine, cerebrospinal fluid, whole testing and saliva for presence of specific antibodies
- antibodies have an extreme specificity of antigens
- viewing interaction of antigens and antibodies microscopically or macroscopically provides a powerful tool for detecting, identifying, and quantifying them
- determines: immunologic status of patient, confirm a suspected diagnosis, and screen individuals for a disease
immunological tests: agglutination and perception reactions
- antigen is interlinked by many antibodies to form insoluble aggregates that settle in solution
- forms clumps
immunological tests: immunochromoatography
- lateral flow test (pregnancy and rapid strep tests)
- plastic cartridge contains porous material or polymer that directs fluid
- patient sample will encounter antibodies
- if patient sample contains correct antigen, it will bind the antibodies
- then a third molecule is impregnated on the paper in a strip form
immunological tests: immunofluorescence
- florescent antibodies (FAbs): monoclonal antibodies labeled by florescent dye
direct testing: - unknown antigen is fixed to a slide and exposed to a FAb solution
- if antigen-antibody complexes form, they will stay bonded to the sample and can be seen under a florescence microscope
- valuable for: locating microbial antigens on cell surfaces or tissues
indirect testing:
- FAbs recognize the Fc reign of antibodies in patient sera
- known antigen is added to the test serum
- binding of fluorescent antibody is seen through florescence microscope
- fluorescing aggregates or cells indicate the FAbs have complexed with microp-specific antibodies
Enzyme-linked immunosorbent assay (ELISA)
- uses an enzyme-linked indicator antibody to visualize Ag-Ab reactions
- relies on microtiter plate that will absorb the reactions
indirect:
- detects microbe specific antibodies in patient area
- known antigen is absorbed to the surface or a well and mixed with unknown antibody
- if ag-ab complex forms, in added indicator antibody will bind and produce a color change
direct:
- known antibody is absorbed to the bottom of the well and incubated with an unknown antigen
- if ag-ab complex forms, it will attract the indicator antibody and produce a color change
antibody titers
- concentration of antibodies in a sample
- determined by: serial dilution of patient serum
- the more the sample can be diluted, the greater the concentration of antibodies and its titer
- used to: diagnose autoimmune disorders and determine past exposure to diseases
sterotyping
- ag-ab technique for identifying, classifying, and subgrouping bacteria into categories
- employs antisera (antibodies) against cell antigens like capsule, flagellum and cell wall
- used for: identifying salmonella species and strain
- basis for differentiating pneumococcal and streptococcal sterotypes
genotypic methods: polymerase chain reaction PCR
- production of numerous identical copies of DNA or RNA molecules
- can amplify little amounts of nucleic acids present in a sample
-real-time PCR (qPCR) q=quantitative
uses fluorescent labeling during amplification
level of fluorescence is measured in real time as it is running
fully automated and faster, analysis of DNA not needed
-reverse-transcriptase PCR (RT-PCR)
creation of DNA out of RNA
used for identification of RNA viruses
genotypic methods: whole genome sequencing
- useful for rapid analysis of outbreaks and drug-resistant organisms
- will become so cheap and routine we will test from the patient to find microbes causing symptoms
genotypic methods: pulse-field gel electrophoresis: microbial fingerprints
- separation of DNA fragments
- slowly applying alternating voltage levels to the gel from 3 different directions
- allows similarly sized DNA fragments to separate
- used in: food borne outbreaks
genotypic methods: microbial fingerprints: pulsenet
- program established by CDC to assist in the investigation of disease food borne outbreaks
- sciencists from across the country can help and it will take hours
genotypic methods: nucleic acid composition
- prokaryotic G + C content varies 20%-80% between different species
- closer the percentage between the two bacteria, the more closely related they are
