Chapter 11 Flashcards
What are the goals of a clinical microbiology laboratory?
Identify the microorganism in the patient involved in the disease
Provide antimicrobial susceptibility of the isolated microorganism
What is the identification process of a microorganism?
Identification of microorganisms by isolations and culture (presence or number of microorganisms, susceptibility)
Identification of a specific microbial gene or product (presence of a resistance gene)
Detection of specific antibodies to a pathogen for pathogens that are high risk or cannot be cultivated, retrospective diagnosis
Where are the two possible sites for specimen collection?
Sterile sites (blood, urine, CSF) and sites with commensal flora (skin, GI tract, urethra) Need to make sure collection from the sterile site isn't contaminated.
What are some of the downsides of culture-based methods for specimen growth?
Can take up to 2 days (>18 hours), organism must be culturable
What must be done specially for blood culture?
Low numbers of cells thus rely on detection of an antibody response to a pathogen,
What are the advantages of non-culture methods?
Fast, less labour intensive and suitable for organisms that cannot be cultured in the lab
What are some non-culture methods?
Microscopy (light and electron), immunodiagnostics and molecular diagnostics
What are the types of light microscopy? What are they used for?
Bright field used for stained preparations (gram stain, acid fast for mycobacteria) and wet preparations (for macromolecules like vesicles, granules).
Dark field for motility and thin cells
Fluorescent for naturally fluorescent or organisms and antibodies stained with fluorescent dyes
What colours show up in a gram stain?
Gram positive are purple
Gram negative are pink
How does electron microscopy work? What is it used for?
Uses a beam of electrons and magnets rather than light
Specimen must be cut into thin sections
Used for viruses (not usually used in a clinical lab)
How does a gram stain work?
Crystal violet, iodine (forms complex with crystal violet), alcohol (removes complex not trapped in gram + peptidoglycan) then pink dye
What could be the reason nothing show up under the microscope after a gram stain?
Mycobacterium or viral infection
What is the difference between a scanning (SEM) and transmission (TEM) electron microscope?
Scanning simply scans the surface of the cell
Transmission is used to see cell structures as it passes through
What organisms commonly cause bacterial meningitis?
Streptococcus pneumoniae, haemophilus influenzae, niesseria meningitis in CSF
How can immunodetection be done?
A specific antibody is coated onto the latex bead and when it binds to the organism there is visible clumping for bacterial meningitis
Much faster than culturing
What may cause a false positive in immunodetection?
If not enough of the patient’s specimen is tested. Cross reacting antigens
What is ELISA? How does it work?
Enzyme linked immunosorbent assay
Direct: Coat plate with antibodies, add patient sample (antigens bind) and then add fluorescent or chemically labelled antibodies will bind to patient antigen
Indirect: Coat plate with antigens then add patient sample (antibodies bind) then add a labelled anti-immunoglobulin
What is the difference between direct and indirect immunodetection?
Direct detects the antigen, indirect detects the antibody (more frequently used, can use same secondary antibody for multiple tests)
What can ELISA be used for?
HIV, HBV and herpes
What are monoclonal antibodies used for?
To distinguish between species and between strains of the same on the basis of antigenic differences Diagnostic tests (really specific)
How are monoclonal antibodies formed?
From hybridomas produced by fusion of immortal B cell tumours and normal antibody-producing cells
What is the probe-based method for specific gene detection? What is it used for?
Label a single-stranded nucleic acid fragment to hybridize with the target DNA.
A with T, G with C hydrogen bonding
Hepatitis C virus RNA
What are some problems with a probe-based method?
Must know the sequence of the target gene
Can’t make the strand too long or else it will bind to itself or if too small it will not be specific enough.
What is an amplification based method for specific gene detection?
Polymerase chain reaction (PCR) is the most common
Amplifies a target sequence from any source and copies it.
Design specific primers for the gene sequence using taq polymerase from 5’ (works at high temperature when DNA strands separate)
Doubling of DNA content for 30-40 cycles
Tells if gene is absent or present
How are culture-based methods done?
Use solid (isolation, differentiation) and liquid (turbidity shows presence) media Selective, differential, antimicrobial susceptibility
Why is agar used as a solid growth media?
Because it is not broken down by most bacteria
What is a selective growth media?
A media that allows the growth of some organisms while inhibiting the growth of others. Know the natural conditions the organism grows in.
Add bile salts for GI tract bacteria
What are some examples of differential growth media?
Lactose medium for salmonella and E. coli (produces acid, use pH indicator)
Blood medium to see which is capable of hemolysis (zone of clearing)
What are the different types of hemolysis? How do they show up on a blood medium?
Beta gives a clear zone (complete hemolysis)
Alpha gives green zone (heme) (incomplete hemolysis)
Gamma is no hemolysis
How can we culture viruses, chlamydia and rickettsia?
In cell or tissue culture (eukaryotic growth cultures)
Observe the presence by the cytopathic effect (changing of cell morphology due to cell killing)
What are some problems with eukaryotic growth cultures?
Labour intensive and slow, contamination because culture is so rich.
Usually use immunodetection or molecular methods instead
What are the steps taken when you want to identify a gram positive organism in culture?
Gram stain, shape and grouping, coagulase and hemolysis
(all for cocci)
Rods are aerobic or anaerobic
What are the steps taken when you want to identify a gram negative organism in culture?
Gram stain, shape, lactose fermentation, oxidase
(all for rods, aerobic)
Also cocci
What are fastidious organisms?
Hard to grow, must add additional growth factors. You have seen the cells but they aren’t growing on the plate
How are fungi and protozoa identified?
Fungi can be identified by their colonial characteristics and cell morpholoy
Protozoa is identified by direct examination
How are biochemical tests used to identify microbes in culture?
Phenotypic array
Can test acid production, using sugar as a food source, lower pH, degradation of vitamins, ability to resist antibiotics
