lecture 6) clinical microbiology 2 Flashcards
why study the cause of infection?
prognosis
treatment
isolation
care
name 5 ways the cause of infection can be determined
microscopy molecular testing biochemical tests rapid test and immunoassays culture
what type of microscopy is used to identify infections of bacteria, fungi and protozoa?
light
what type of microscopy is used to identify viral infections?
electron
what molecular test can you do to identify infections of bacteria, fungi, protozoa and viruses?
nucleic acid amplification, sequencing, fingerprinting etc
what biochemical tests can be used to identify bacterial and fungal infections?
identification and sensitivity
what rapid tests can be used to identify bacterial and fungal infections?
biochemical identification
what immunoassay tests can be used to identify bacterial, fungal, protozoa and viral infections?
ELISA
flow cytometry
complement fixation
how would you culture bacterial and fungal infections?
purify and amplify
how would you culture viral infections?
cytopathology
what are the 4 steps in historical workflow of determining the cause of infection?
1) stain based methodologies
2) microbial cultures
3) biochemical/antigenic techniques
4) antimicrobial susceptibility testing
what is the aim of stain based methodologies and what do they support?
classification of microscopic morphology
supports early diagnostic and therapeutic decisions
what does microbial culture involve in historical workflow?
propagtion of organism on agar/liquid medium
what do biochemical/antigenic techniques involve and what does this lead to?
metabolic and phenotypic analysis
leads to identification of organism
what does antimicrobial susceptibility testing lead to?
confirming or tailoring therapeutic agents
give 3 examples of stains for microscopy
gram stain
acid fast stain
periodic acid schiff (PAS)
what is acid fast stain also known as?
zahl-neeson stain
how is acid fast stain done and what does it test?
performed directly on sputum (saliva + mucus coughed up from resp tract), no need for culture, specific
performed on mycobacteria
what is PAS used for?
stains glycoproteins in fungi
what type of background is needed for PAS?
high background
what is a disadvantage of PAS?
not very clear as it reacts with other cells that contain glycoproteins
name a selective media used for microbial culture
mannitol salt agar that is used for isolation of staphylococci
what does a selective media do?
supports the growth of one group of organisms out of a highly variable sample eg faeces
name a differential media used for microbial culture
MacConkey agar for the recovery of enterobacteriaceae
what property of mannitol salt agar plates prevents many bacterial strains from growing?
7.5% sodium chloride
what species of bacteria thrive in mannitol salt agar plates?
staphylococcus
ferment mannitol and produce an acidic product
what is the colour change for a positive result on mannitol salt agar?
red to yellow
what dye is used on mannitol salt agar plates?
phenyl red
what does differential media do?
supports the growth of many bacteria and then you can isolate certain ones
what type property of bacteria would cause a positive result on MacConkey agar plates?
lactose fermenters
what is the colour change for a positive result on MacConkey?
red to pink
what dye is used for MacConkey plates?
neutral red
why are tests that differentiate staphylococcus and streptococcus important?
because staph and strep are both gram positive bacteria that are found on the skin
name 3 biochemical techniques that can be used after gram staining?
coagulase
beta galactosidase
catalase
what does coagulase detect?
detects the presence of coagulase that causes plasma to clot
what bacteria does coagulase differentiate?
staphylococcus aureus and epidermis
what does beta galactosidase detect?
detects the presenc of an enzyme that cleaves lactose to form glucose and galactose
what does beta galactosidase differentiate?
separates the enterics and identifies pseudonomads
what does the biochemical test catalase detect?
catalse
converts hydrogen peroxide to water and oxygen
what dose catalse differentiate between?
staphylococcus and streptococcus
name 2 tests that differentiate between staphylococcus and streptococcus
catalase
mannitol salt agar plate
what test is the first form of information?
gram staining
what is used to carry out biochemical tests?
API strips (analytical profile index)
how many biochemical tests does API test for?
20
what does ELISA stand for?
enzyme linked immunoabsorbent assay
why would indirect ELISA be used?
if you want to find out if an individual has been infected with a certain pathogen
what are the steps involved in indirect ELISA?
take a serum sample from the individual
clone the antigen and stick it to the plastic wells
if they have antibodies for the antigens on the well they will stick to it and others wont
wash everything that doesnt stick away
indicator antibody linked to enzyme attached to any bound antibody
rinse well to get rid of anything bound to them
colourless substrate added
enzymes that are linked to the indicator antibody hydrolyse the substrate releasing a dye
what results are positive and negative from indirect ELISA?
coloured wells are positive for the antibody
colourless wells are negative for th antibody
what viruses can be detected using antibody sandwich ELISA?
orthohantavirus
measles virus
explain the steps in antibody sandwich ELISA
antibody absorbed to the well
test antigen added
enzyme linked antibody specific to test antigen binds to another antigen forming a sandwich
enzyme substrate added
if positive there will be a colour change
what type of infections do agglutination assays commonly test for?
viral infections
what do agglutination assays use?
uses the patients immune system to tell us what we have got
what beads are used in agglutination of red blood cells and what do they do/tell us?
latex beads coated with an antigen
latex beads coated with a protein belonging to the virus
if you are infected with that virus you probably have antibodies for the virus therefore the antibodies with bind to the antigens on the latex beads
agglutination of the antibodies to the antigens on the beads can be monitored in the blood sample
what happens to red blood cells that arent agglutinated?
the will pellet at the bottom of the well
explain how you would test for antimicrobial suscpetibility using an agar plate
put the antibiotic on the agar plate
dot the microbe around the agar plate
if there is mirobial growth = resistant to antibiotic
no microbial growth = susceptible to antibiotic
what is an Etest strip and what is it used for?
graded concentration strip
used for antibiotic susceptibiliy testing
name 5 techniques used in modern clinical microbiology
next generation sequencing of whole bacterial genomes
metagenomics and community profiling
multiplex PCR
mass spectrometry
which is the best technique used in modern microbiology?
mass spectrometry
what can also be measured in mass spectrometry?
PCR
how is mass spectrometry used?
detects mass/charge ratio
fragment will be in a certain pattern
describe NGS of bacterial genomes
currently culture dependent
species identification
becoming cheaper
gives a wealth of information
what are some of the advantages of using multiplex PCR?
differentiates strains based on the bands you have
no need for culture
cheap and sensitive
no comparison standard
what are some of the disadvantages of multiplex PCR?
need 1 set of primers specific for each organism
too laborious
prone to error in set up
what is used as an alternative to multiplex PCR?
NAAT
PCR
what is culturomics also known as?
metagenomics and community profiling
give some examples of gram positive bacteria
streptococcus staphylococcus mycobacteria listeria bacillus clostridium
give some examples of common negative bacteria
neisseria
enteronacteriacae
bacteriodes
campylobacter
mycobacteria are classed as being gram positives. what would make them qualify as being gram positive and gram negative?
1 phospholipid bilayer
1 peptiodglycan layer
no asymmetric membrane (gram positive)
mycolic acids serve as pseudophospholipid bilayer
retain gram stain
