Micro - Testing In Diagnostic Micro Lab Flashcards
what are the roles of testing
- detection of pathogens (in specimens collected or indirectly by detecting antibodies in patient)
- identification of pathogens
what are the 4 broad categories of tests
- microscopy
- culture and subsequent identification
- detection of microbial components
- serology
what are the 5 types of microscopy and their uses
A. Bright field - e.g. used for wet preparation of vaginal epithelial cells, gram stain, histopathological stain
B. dark field - e.g. used for the diagnosis of T. palladium from chancre fluid, bright morning spirochetes against dark background
C. phase contrast - 2 beams of light of phase by 1/2 wavelength - gives you a 3D image, e.g. for decal specimens, looking for parasite ova or other internal structures within a cell
D. fluorescence - non specific fluorphore, specific labelled monoclonal antibodies e.g. to identify Legionella, Ricketssiae in cell culture
E. electron - high resolution e.g. used for Herpes simplex, Adenoviruses, Influenza, Pox virus
explain advantages and disadvantages of bacterial culture
advantages - determines if pathogen is present, provides enough of organism to perform identification tests, can do antibiotic susceptibility testing
- once cultured biochemical tests can identify pathogen (based on enzymes produced by the bacteria) e.g catalase, coagulase, oxidase
disadvantages - particular culture conditions won’t suit all pathogens. need appropriate: nutrition, gaseous atmosphere, incubation temp/time.
- if looking for something unusual must let lab know because need special stains or microscopy, need special culture conditions
- sometimes many biochemical tests are needed for identification
explain the range of id tests used, from simple to high tech
- individual tests: eg fermentation of sugars, citrate test
- incorporation into primary agars so that pathogenic colonies take on a particular appearance
- biochemical kit tests - AP1 - positive or negative reactions give numerical biochemical reactions in miniature vital card, card inoculated with bacterial suspension, incubated, analysed by machine
- newer techniques - MALDI-TOF - bacterial suspension from colony inoculated onto steel plate, put in machine. laser creates protein fragments which acquire a change and fly down mass analyser, unique spectrum compared to database by software to produce genus and species id.
- 16s rRNA gene analysis: gene that has highly conserved regions and highly variable regions. conserved regions = universal PCR primers can be designed to enable amplification of hyper variable regions. when amplified - variable region sequenced and compared against database to provide genus and species id. usually reserved for difficult to id organisms.
how are fungi identified from a culture and why does this process take time
fungal culture - identification dependent on morphology
- most fungi grow slowly (weeks before results)
- grow as yeasts/moulds on agar
yeasts - often identified from biochemical reactions such as API kit
moulds - identification is visual i.e. colony appearance, colour, microscope of mycelia and canidia. features can take weeks to appear therefore slow ID
describe antigen detection and how it provides rapid detection/ID.
what are examples of different labels that can be used on the antibodies
monoclonal antibodies against microbial antigens can be produced in lab animals, used in detection tests in the lab. antibodies are labelled with fluourophores, enzymes or radioisotopes
describe the principles of PCR and its advantages.
what is gel based PCR
what is qPCR
nucleic acid amplification - must know part of sequence of pathogen been sought. need a sequence that is not shared by other microorganisms and is present in all strains of the pathogen.
PCR = number of copies of target = 2^n where n= number of cycles
1st cycle - melt, anneal, extend. need: parent strands, forward/reverse primers, NT’s
gel based PCR - amplicons detected by electrophoresis in polyacrylamide gel with ethidium bromide
qPCR - each amplicon produces light signal, as amplicon number increases, light intensity increases
describe the antibody response to infection and how this is applied to diagnostic testing
what are the criteria needed for diagnosis of a pathogen by this method
serology testing - detection of specific antibodies in a patients serum specific to a particular pathogen.
- indirect evidence that patient has been infected with a pathogen
upon first exposure to pathogen:
IgM specific to pathogen appears in several days (around 1 week), Igm persists for several months
IgG - appears in first or second week, persists for decades/life (at much higher concentrations that IgM)
upon second exposure
IgM response absent/small
IgG - brisk IgG response may be enough to prevent symptomatic infection.
if IgM is detected in serum, patient must have had infection with specific pathogen within last several months
if IgG detected patient has had infection at some point in the past (weeks - years). measurement only useful in diagnosing acute/recent infection if these criteria are met:
- seroconversion can be demonstrated between 2 samples (acute and convalescent i.e. negative and positive)
- significant increase in IgG titre between acute and convalescent samples (usually taken 10-14 days apart)
eg seroconversion - if acute sample negative (
explain the meaning of an antibody titre
- row of 8 wells of plastic mircolitre plate
- used to test serum of a patient
- add 50ul of diluent to each well
titre- series of dilution of patient serum
ie 50ul of patients serum (pre diluted 1:5) to first well, mix, patients serum now 1:10 dilution - take 50l from first well and put into second well, mix, serum now 1:20 dilution etc
wells now contain doubling dilutions of contents of first well.
next test each well for presence of antibodies. each well will be either positive or negative
