Microbiology Exam 2: Clinical Microbiology Labs

The clinical lab

samples of tissues or fluids are collected for microbiological, immunological, and molecular biological analyses. samples may include blood, urine, feces, sputum, cerebrospinal fluid, pus, or others.

specimen should be obtained from site of infection aseptically; sample size must be large enough; metabolic requirements for the organism must be maintained during sampling, storage, and transport.

taking the swab from the correct location may be criticall

bacteremia and septicemia

the presence of bacteria in the blood; blood is normally sterile.

blood infection.

draw 10 to 20 ml of blood and culture; one sample is cultured aerobically, while the other is cultured without oxygen (anaerobically).

urinary tract infections are common, especially in women. disease-causing agents are often normal flora.

general-purpose media (blood agar); selective media (MacConkey agar, eosin-methylene blue agar); additional differential media may be used to differentiate bacteria.

skin infections associated with injuries are sampled to determine the relevant and irrelevant microbes in the area; pus is drawn from abscesses and cultured.

Staphylococcus aureus may be resistant and is a common skin or wound infection.

a common selective and differential medium
causes MRSA colonies to appear pink, other bacterial colonies appear blue.

Neisseria gonorrhoeae is a common cause of purulent discharge.

Thayer-Martin media (MTM) is used to eliminate the normal microbiota during culture; colonies appear dark.

some pathogens are obligate anaerobic bacteria: the isolation, growth, and identification of obligate anaerobes can be complicated by specimen contamination; maintaining anoxic conditions during collection, transport, and culture is challenging; for anoxic conditions during collection, transport, and culture is challenging; for anoxic incubation, agar plates are placed in a sealed jar, which is made anoxic.

sabouraud dextrose plates

viruses are obligate intracellular pathogens; must be cultured in cells, not an agar plates; viruses cause cytopathic effect in cell culture.

supports growth of most aerobic and facultatively aerobic organisms (blood agar plate)

contain growth factors that enhance growth of certain fastidious pathogens; E. coli (lactose fermenter) and Pseudomonas (non-lactose fermenter) on EMB agar plate.

microscopy of microbes; culture and isolation of microbes; detection of microbial antigens; detection of microbial nucleic acids; serology - detection of antibodies against microbes in patient serum.

some pathogens can be readily identified by microscopic examination of tissue samples.

viruses are too small to be seen with a light microscope; microscopic identification of viruses requires an electron microscope.

specific identification of pathogens requires a combination of microbiological, immunological, and molecular biological techniques.

immunofluorescence assay - to detect microbial antigens in a patient specimen or from growth in culture. antibodies are chemically modified with fluorescent dyes to help detect antigens on microbes or infected cells; a rapid (within hours) and specific technique to confirm a pathogen.

patient specimen with microbial antigens placed on microscope slide; add specific antibody to microbe with fluorescent label; detect antigen with fluorescent microscope.

hybridization methods identify specific pathogens in patient samples by using unique nucleic acid probes to detect pathogen DNA.

requires a labeled nucleic acid probe consisting of DNA sequences from the specific microbe of interest.

fluorescent labeled staph DNA probe

useful method to detect microbial DNA or RNA in a patient specimen; extremely sensitive - may detect as little as one viral DNA molecule in a specimen and amplify over 1000 fold; requires specific DNA primers for the pathogen.

reaction buffer; target DNA servers as template for reaction; nucleotriphosphate bases dATP, dCTP, dGTP, dTTP; two 20-30 base oligonucleotide primers - usually 100 to 1000 bases from each other (must know DNA sequence of target DNA); thermostable DNA polymerase (tag) - not denatured at 95C, derived from a thermostable bacteria.

double-stranded DNA target into single-stranded DNA - 95C for 30 seconds.

two specific oligonunucleotide DNA primers (20 to 30) bases to ss DNA target - 55C for 2 minutes.

thermostable Taq polymerase extends primers by DNA synthesis - 72C for 3 minutes.

at 95C for 30 seconds to release DNA primers.

25 to 25 times to amplify DNA.

run amplified DNA on this and determine DNA sequence.

rapid (within hours) and specific approach to confirm a pathogen; the techniques require skilled technicians for accurate results.

serological assays

true

may take 7 to 10 days to generate detectable antibodies levels.

a very sensitive serological test used to detect antibodies in diseases such as AIDS

-electrophoresis of proteins, followed by transfer to a membrane and detection by addition of specific antibodies
-immunoblot methods detect antibodies to specific antigens or the antigens themselves; •HIV antigens on filter
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•add patient serum
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•add labeled secondary antibody against Fc component of IgG
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•add indicator