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Block 2 Microbiology > Laboratory Diagnosis > Flashcards

Laboratory Diagnosis Flashcards

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1
Q

What is a direct specimen?

A
  • Pathogen located in otherwise sterile site, e.g. deep abscess
    1. Collect surgically
    2. Needle aspiration
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2
Q

What is an indirect sample?

A
  • Pathogen located in otherwise sterile site, but must pass through a site containing normal flora
  • Expectorated sputum
  • Voided urine
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3
Q

What are two examples of samples from sites with normal flora?

A
  • Throat swab
  • Stool sample
  • Sample collected is a mixture, then the normal flora are inhibited under growth conditions for analysis
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4
Q

What is an acid-fast stain? What might you use it to detect?

A
  • Mycolic acid on surface resistant to gram staining
  • Mycobacterium
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5
Q

What is a negative stain used for?

A
  • Used to show capsules
  • Stains everything but the bacteria
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6
Q

What is this staining technique? What is it for?

A
  • KOH
  • Fungi
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7
Q

What is this staining technique? What might you use it for?

A
  • Dark field (specialized technique)
  • Used to test for T pallidum (syphilis) because even though it will gram stain, you can’t see it with that technique b/c it is too thin
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8
Q

What is this staining technique? Selective? Differential? Explain.

A
  • Blood agar
  • Non-selective
  • Differential -> hemolysis (alpha - green, beta - clear and yellow, gamma - no change)
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9
Q

What is this staining technique? Selective? Differential? Explain.

A
  • Mannitol salts agar
  • Selective (only Staph)
  • Differential for Staph: yellow aureus, no change epididermis
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10
Q

What is this staining technique? Selective? Differential? Explain.

A
  • Eosin-methylene blue agar
  • Selective: Gram- rods
  • Differential: lac/suc+ black (i.e., E. coli), lac/suc- no color (i.e., Shigella, Salmonella)
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11
Q

What is this staining technique? Selective? Differential? Explain.

A
  • MacConkey agar: used to distinguish among enterics, i.e., in diarrheal disease
  • Selective: Gram- rods
  • Differential: lac+ pink, lac- grey
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12
Q

What is this staining technique? Selective? Differential? Explain.

A
  • MacConkey Sorbitol
  • Selective: Gram- rods
  • Differential: E coli O157
  • E. coli O157 always sorbitol (-), and show up white, rather than pink on plate
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13
Q

What is this staining technique? Selective? Differential? Explain.

A
  • Thayer-Martin Agar: chocolate agar + ABs (T-M); blood heated up to inhibit certain enzymes
  • Selective: Neisseria
  • NOTE: Thayer-Martin used in isolation of N gonorrhea from genital secretions, but only chocolate agar used for isolation of N meningitides from CSF because no normal flora in CSF
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14
Q

Why does campylobacter need to be cultured in a special media?

A

Has to grow at a higher temperature (42 degrees C)

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15
Q

How should you obtain a blood culture?

A
  • At least three 10-mL samples in 24-hr period because:
    1. Presence in blood varies w/time
    2. Reduces likelihood of contamination
  • Cleanse site with 2% iodine before puncture
  • Add to rich growth medium (brain-heart infusion broth)
  • May need to consider anaerobic incubation in addition to aerobic
  • Check for turbidity or CO2 production daily for up to 7 days
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16
Q

What factors are important to consider in sputum cultures? What might you be testing for?

A
  • Make sure sample is sputum, not saliva
  1. >25 leukocytes, <10 epithelial cells per 100X field
  2. If necessary, use transtracheal aspirate or bronchial lavage
  • Gram or acid-fast stain may reveal organisms; routine culture is on blood agar, but special media may be required
  • Anaerobic culture may be needed if aspiration (because may be a pneumonia that came up from GI)
  • Bugs: Strep pneumoniae, S. aureus, K pneumonia, Ps aeruginosa, M tuberculosis
17
Q

Describe the basics of obtaining a throat culture.

A
  • Swab posterior pharynx and tonsils
  • Routine culture is on blood agar, but special media may be required
  • Confirm b-hemolytic colonies are GAS by sensitivity to Taxo A (bacitracin)
  • Mostly testing fro Strep, but also diphtheria, thrush, gonococcal pharyngitis, etc.
  • NOTE: Gram stain of little use because of viridans Strep present
18
Q

What might you be looking for in a spinal fluid culture?

A
  • S pneumoniae, N meningitidis, H influenzae
  • Goal is to rule in bacterial meningitis so you can treat it quickly -> medical emergency, send to lab immediately
  • Lumbar puncture
  • Centrifuged sample cultured & observed microscopically for presence of bacteria
  • Blood agar and chocolate agar
19
Q

What are the basics for acquiring a stool sample? What are you looking for?

A
  • Direct examination of stool sample
  • Methylene blue staining of leukocytes in inflammatory diarrhea
  • Culture on selective, differential media such as EMB or MacConkey
  • Grossly bloody diarrhea grown on MacConkey Sorbitol to look for EHEC O157
  • Anaerobic cultures are not needed because of normal flora (not diagnostic in this case)
  • Testing for enterocolitis: Salmonella, Campylobacter, Shigella, E coli O157
20
Q

What is the process for obtaining a urine sample? What are you looking for?

A
  • Urine sterile, but contaminated as it is released, so try to capture first thing in the AM, and with a clean catch, midstream
  • Start cultures w/in 1 hr or refrigerate to avoid growth (no good to you after 24 hours)
  • Culture must be quantitative to confirm infection
  • Use a 0.001mL calibrated loop to streak a BAP and EMB plate -> if no colonies on EMB plate, not a Gram negative infection; if colonies on both plates, likely Gram negative (i.e., E. coli)
  • Count colonies to determine whether >100,000 mL
  • Testing for: pyelonephritis, cystits - Enterobacter, E. coli, Proteus, Enterococcus, Staph saprophyticus
21
Q

What is important to consider when doing wound and abscess cultures? What might you be looking for?

A
  • Freq involve aerobic and anaerobic mixed infections
  • Culture aerobically and anaerobically on variety of media
  • May be testing for:
    1. Brain, lung, abdomen abscesses: Bacteroides, S. aureus, GAS
    2. Traumatic open wound: C. perfringens
    3. Surgical wound: S. aureus
22
Q

What is the difference between non-specific and specific Syphilis testing?

A
  • Non-specific testing: Rapid plasma reagin (RPR)
    1. Beef heart cardiolipin (diphosphatidylglycerol) is the antigen, conjugated to carbon particles, which clumps in the presence of antibody to T. pallidum
    2. 1=nonreactive; 2=weakly reactive; 3=strongly reactive -> run this test first b/c cheaper and quick. If (+), need to do specific test to confirm
  • Specific testing: fluorescent treponemal Ab absorption
  1. Pt serum absorbed with non-Tp treponemes, then reacted with fixed T pallidum on a slide
  2. Fluorescent anti-human IgG is added to visualize the bound bacteria (+ test shown in other notecard)
  3. Fluorescent microscopy
  4. Expensive test because you have to infect testicles and grow T pallidum for these assays
23
Q

What kind of rise in IgG would you expect to see in convalescent vs. acute titer?

A

4x

24
Q

What is the advantage of an Ag titer compared to an Ab titer?

A

Can occur before Ab titer sufficient

25
Q

How is viral neutralization used in Ag testing?

A
  • See image for process if you need to: this is a (+) test for Virus B because it has been tagged by Ab, and therefore does not lyse the cells when cultured (unlike A, which was not bound by Ab, and the control, which had no Ab added)
  • Virus-specific Ab can also be used in a fluorescent Ab format to identify virus in tissue samples or infected cultured cells
26
Q

What is a Rapid Ag Detection Test?

A
  • RADT
  • Lateral flow immunoassays that typically produce results in 15 minutes
  • Used for many different type of infections, i.e., strep throat and flu (also used for pregnancy)
  • Gold balls (attached to Abs) make pink color reaction – concentration at test line, and need confirmation at control line, all washing down to end result (i.e., one line negative, two lines positive, and zero lines means failed test)
27
Q

Briefly describe the 4 types of HIV testing.

A
  • ELISA
  • Murex Single Use Diagnostic System (SUDS): assays Abs and presence of p24 (p24 will catch earlier than Ab assays, but Ab assay stronger signal) -> rapid enzyme immunoassay
  • OraQuick (home testing): saliva antibodies
  • Western blot: GOLD STANDARD
    1. Positive: p24, gp41 and/or gp120/160; Indeterminate: some bands, but not those above; Negative: no bands
    3. Proteins separate by size; know exactly which proteins the Ab is seeing
28
Q

What is the nucleic acid amplification test?

A
  • NAAT: particularly useful when organism difficult to routinely grow and isolate (based on PCR; very sensitive assays that do not require much sample) -> used to detect virus or bacterium
  • Developed to shorten window period b/t infection and (+) Ab test -> includes any test that directly detects genetic material of the infecting organism or virus.
  • There are multiple methods that fall in this group, incl: 1) PCR (use a primer to rapidly make copies of the genetic material), 2) reverse transcriptase PCR (RT-PCR) for HIV and other RNA viruses, 3) branched DNA (quantiplex bDNA) tests
  • Often becoming the “gold standard” of diagnosis
  • Fairly rapid, and because of amplification, very sensitive
  • Can be used to detect pathogen before significant immune response, and can be used to determine pathogen load
  • Uses (there are plenty others): HIV, HBV, HCV, Chlamydia, GC
29
Q

What is the time to result for the tests discussed in this lecture (8)?

A
  1. RADT 5-20 min
  2. qPCR 1-2 hr; Std PCR 3-5 hr
  3. Probe hybridization 6-12 hr
  4. Metabolite analysis 1-2 days
  5. Bacterial growth 1-2 days typical, but can be weeks to months
  6. Viral CPE: several days to weeks
  7. Seroconversion (ELISA) 2-4 weeks
30
Q

Following identification of a bacteria, what additional test might you want to do?

A

Antibiotic sensitivity testing

31
Q

Briefly describe the 3 metabolite analysis tests?

A
  • Particularly important in some bacterial infections
  • Enterotube: stab all the way through to simultaneously inoculate all of the different indicators (saving you a single plate for each test -> creates a bio-code, ID-code for different infections)
  • Nitrate to nitrite test: common UTI-causing bacteria can reduce nitrate to nitrite; assayed in a urine dipstick format
  • Leukocyte esterase test: monocytes, neutrophils, and eosinophils produce an esterase that can be used as an indication of infection (urine dipstick format for UTIs)

Block 2 Microbiology (4 decks)

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