Testing in the Diagnostic Microbiology Laboratory Flashcards
What is done during microbiology lab diagnostic testing?
Detection of pathogens Identification of pathogens Antimicrobial susceptibility testing
What is the emphasis of microbiological testing?
Quick diagnosis
What are the types of lab tests?
Microscopy Culture Detection of microbial components Serology
What are the types of microscopy?
Bright field Dark field Phase contrast Fluorescence Electron
What is bright field micrscopy?
Sample illumination is transmitted (i.e., illuminated from below and observed from above) white light, and contrast in the sample is caused by attenuation of the transmitted light in dense areas of the sample.
What kind of microscopy is used for gram stains?
bright field microscopy
What is dark field microscopy?
Using a dark background and allowing light to bend on the organism and into the eye piece making the organism show up on a black background
What is phase contrast microscopy?
Similar to bright field but 2 lights shine through with a phase difference of 1/4 wavelength
What is fluorescence microscopy?
Fluorescent light is shone onto labelled molecules to create a light image of the slide
How is the electron beam focused in electron microscopy?
Using magnets
How is the sample prepared on electron microscope?
It is prepared on a metal grid that is 3mm in diameter
What influences resolution in microscopy?
The wavelength
What is the purpose of culturing bacteria?
To determine if pathogens are present To provide enough of the organism to perform tests on To perform antibiotic susceptibility testing
How do lab staff determine which bacteria to culture for?
They use knowledge of the location that the specimen was taken from
How do lab staff determine which bacteria to culture for?
They use knowledge of the location that the specimen was taken from. (eg sputum: S.pneumoniae, h. influenzae) Doctors also make requests for ordering the tests. However, a particular set of conditions will not suit all pathogens. Instead particular culture conditions must be used.
What conditions must be accounted for when looking for pathogens using cultures?
Nutrition requirements Gaseous atmosphere Incubation time and temperature
What must be done when looking for an unusual pathogen?
Lab must be informed of special microscopy or stains as well as any special culture conditions. If you don’t know what you are looking for you must provide good clinical information on the request form. Specialists can also be called and asked about these situations.
How are bacteria identified in the lab?
Traditionally bacteria are identified using biochemical tests (based on enzymes possessed by bacteria) eg: catalase, coagulase, and oxidase tests
How are biochemical tests conducted?
Set of tests conducted (test kits) and the pattern of positives and negatives forms a number which correlates to a code and a diagnosis. Kit can also be added onto a vitek card and in turn a machine.
What is a MALDI-TOF?
It is a type of mass spectroscopy
How does MALDI-TOF work?
Bacterial suspension from colony is inoculated onto a steel plate and put into a machine and a laser beam shines on the plate from well to well and breaks up proteins in the bacteria and the speed they get to the ion detector depends on their mass and the mass achieved is compared to a database.
What microbial components can be used for diagnosis?
Antigens and nucleic acids
What is serology?
Detection of specific antibodies to a microorganism in the serum
Why is microscopy and culture not practical for some pathogens?
Can’t be visualized under microscope using routine stains (eg chlamydia and mycoplasma) Too small for routine microscopy (viruses) Grow too slowly Too difficult to grow in labs Microscopy and culture not sensitive enough
How can unique components of microorganisms be used to detect them?
Monoclonal antibodies against microbial antigens can be produced in lab animals and used in tests. Antibodies can be radioactively, enzymatically or fluorescently labelled.
How does nucleic acid amplification work?
Utilises the unique nature of genes. Genetic sequence must be known and a target within the sequence must first be chosen, Gene probes are used
When choosing a nucleic acid sequence to amplify what must target sequence have?
A unique sequence not shared. Ubiquity within the species (It must be present in all strains of the pathogen)
How many copies are made per cycle of PCR?
Copies are made 2^n per cycle where n is the number of cycles.
What is done with PCR samples for detection?
The resulting nucleic acids are then put into acrylamide gel with ethidium bromide for gel electrophoresis. Product can be put into conventional thermal cyclers. Real time PCR produces a light signal from each amplicon and as numbers increase the light intensity increases.
How does serological testing work?
It is designed to test whether or not a patient has produced an antibody to the pathogen.
How long does IgM usually persist in blood?
2 to 3 months
How long does IgG last in the blood?
Can last decades or for life
What does IgM in blood tell us?
Patient had infection within the last several months
True or False: IgM testing is prone to false positives.
True
True or False: Persistance of IgM will vary from pathogen to pathogen, from host to host.
True. eg, alpha-virus antibodies can persist for months to years.
What does specific IgG tell us about patient?
They had the infection at some time in the past. Could be a short time or a long time.
Can IgG be used to detect acute/recent infection?
Yes but only if these criteria are met: Seroconversion can be demonstrated between serum samples (acute to convalescent) Significant increase in IgG titre between acute and convalescent samples. (the samples are taken 10 - 14 days)
How different must both samples be in IgG titre before deciding that there is acute infection?
>4x increase (specimen 7 and 8)
How is a titre made?
A microtitre plate is used with 8 rows. Each row represents a sample from 1 patient. Each well contains 50 microliters of diluent and 50 microliters of the patient’s serum. (Diluted to 1:5) 50 microlitres is taken from the first well and put into second (1:20 dilution) This is done to the rest of the row. The titre is 1:1280 diluted by the 12th well. Each well is then tested for antibody and each well is tested either positive or negative.
Which serological test formats only give a detected/not detected result?
EIA
IFA
LPA
Tests for IgM
(these formats are generally not titred but they can be)
What serological formats give a titre?
Complement fixation
Haemagglutination inhibition
