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Question 1

What is Sensitivity?

Answer 1

the ability of a test to single out those with disease as positiveIf high sensitivity- a negative result rules out disease

Question 2

What is Specificity?

Answer 2

the ability of the test to classify people who do nothave illness as negativeIf high specificity- a positive test result rules in disease

Question 3

What are the three main methods of specimen collecting?

Answer 3

• Direct Specimen - when the pathogen is located in an otherwise sterile site, e.g. deep abscess• Collect surgically• Needle aspiration• Indirect Sample – when the pathogen is located in an otherwise sterile site, but must pass through a site containing normal flora• Expectorated sputum• Voided urine• Sample from site with normal flora – sample collected is a mixture, then the normal flora are inhibited under growth conditions for analysis• Throat swab• Stool sample

Question 4

The acid-fast stain is aka?

Answer 4

Ziehl-Neelsen

Question 5

What is a negative stain sued for?

Answer 5

india ink is excluded from capsules to expose it

Question 6

What is one of the main reasons for culturing?

Answer 6

to obtain purified colonies in case confirmative testing is needed

Question 7

Are BAP differential or selective?

Answer 7

differential only

Question 8

What are MacConkey agar plates selective for? differential?

Answer 8

selective- dyes that are selective for gram negative rodsdifferential- for lactose+ (bright color) vs. lactose- (same red color) like EMB

Question 9

MacConkey plates that contain sorbitol instead of lactose are used for what?

Answer 9

selective for GNR and differential for E. Coli O157 (will be white while all other E. coli colonies will be red)

Question 10

What are Thayer-Martin agar plates used for?

Answer 10

Chocolate agar + AGX (brownish color). Blood has been heated and lysed that release iron and other things bacteria can grow on, The addition of certain antibiotics make it SELECTIVE for Neisseria species because they kill any other bacteria there.

Question 11

When are Thayer-Martin plates used?

Answer 11

Used in the isolation of N gonorrheae from genital secretions but only chocolate agar (without antibiotics) is used for isolation of N. meningitidis from CSF because the CSF is normally sterile so we don’t have to worry about other contaminants that might compete with the Neisseria

Question 12

How should blood cultures be collected?

Answer 12

• Obtain at least three 10-mL samples in a 24-hr period to increase chance of getting good sample and for replicability• Cleanse the site with 2% iodine before puncture to cut back on contamination (draw from different spots to get your samples)• Add to rich growth medium (brain-heart infusion broth) to capture anything thats on there • Need to consider anaerobic incubation in addition to aerobic• Check for turbidity or CO2 production daily for up to 7 days

Question 13

What kinds of things would make you want to do a blood culture?

Answer 13

Sepsis (Staph aureus)Endocarditis (Strep pneumo)Osteomyelitis (E. coli)Meningitis (K. pneumoniae)Pneumoni (Ps. aeruginosa)

Question 14

How/When would you perform a throat culture?

Answer 14

• 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)• Note: Gram stain is of little use because of viridans Strep present

Question 15

What kinds of bugs would you be looking for on a throat swap?

Answer 15

DiphtheriaGonococcal pharyngitis Thrush (Candida)

Question 16

How would you do a sputum culture?

Answer 16

Make sure sample is sputum, not saliva• >25 leukocytes,

Question 17

What kinds of bugs would you be looking for on a sputum culture?

Answer 17

Strep pneumoniae Staph aureusK pneumoniaPs aeruginosaM tuberculosis thinking Pneumonia or TB

Question 18

How would you do a spinal fluid culture?

Answer 18

Lumbar puncture• Consider this a medical emergency, send to lab immediately (bacterial/viral meningitis)• Centrifuged samples are cultured but observed microscopically for the presence of bacteria• Rich medium to capture anything- Blood agar and chocolate agar

Question 19

What kinds of bugs would you be looking for on a spinal fluid culture?

Answer 19

MeningitisS pneumoniae N meningitidis H influenzae

Question 20

How would you do a stool culture?

Answer 20

• Direct examination of stool sample• Methylene blue staining of leukocytes would be seen in inflammatory diarrhea (opposite is watery diarrhea without leukocytes)• Culture on selective, differential media such as EMB or MacConkey because theres tons of normal flora there • Grossly bloody diarrhea should be grown on MacConkey Sorbitol to look for EHEC O157• Anaerobic cultures are NOT needed because of abundance of normal flora

Question 21

What kinds of bugs would you be looking for on a stool culture?

Answer 21

EnterocolitisSalmonella Campylobacter ShigellaE coli O157

Question 22

How would you do a urine culture?

Answer 22

• Urine is sterile (because bladder is), but will become contaminated as it is released • Capture first morning, clean-catch midstream because bacteria are concentrated then • Start cultures within 1 hr or refrigerate to avoid growth • 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• Count colonies to determine whether >100,000/mL (count on late should be over 100 for diagnosis)

Question 23

When should a urine culture be performed?

Answer 23

Pyelonephritis or CystitisE coli (gram-)Enterobacter (gram-)Proteus (gram-)Enterococcus (gram positive)Staph saprophyticus (gram positive)

Question 24

What are some things to consider on wound and abscess cultures?

Answer 24

Frequently involve aerobic and anaerobic mixed infections• Culture aerobically and anaerobically on a variety of media

Question 25

Infection of a brain, lung, or abdominal abscess would probably be caused by which bacteria

Answer 25

bacteriodes, Staph aureus, Strep pyogenes (GAS)

Question 26

Infection of a surgical wound would probably be caused by which bacteria?

Answer 26

Staph aureus

Question 27

Infection of a traumatic open wound would probably be caused by which bacteria?

Answer 27

C. perfringens

Question 28

Infection of a cat or dog bite would probably be caused by which bacteria?

Answer 28

Pasteurella multocida

Question 29

How is viral culturing performed?

Answer 29

Start with a specimen in a transport medium, then do a low speed centrifugation to pellet large things (our cells, bacterial cells, fungal cells) to the bottom. Small viruses will remain in the supernatant. Then add antibiotics to the supernatant and then inoculate a cell monolayer grown under culture. The virus will cause cytopathic effect and the appearance of the monolayer changes

Question 30

What is the gold standard for bacterial infection diagnosis?

Answer 30

Four-fold rise in specific convalescent vs acute titer

Question 31

How is syphillis testing performed?

Answer 31

• Non-specific testing• Rapid plasma reagin (RPR)• Venereal Disease Research Lab (VDRL)• Beef heart cardiolipin (diphosphatidylglycerol) is the antigen, conjugated to carbon particles, which clumps in the presence of antibody to T. pallidumif positive, follow up with:• Specific testing• Fluorescent treponemal antibody absorption (FTA-ABS)• Patient serum is absorbed with non-Tp treponemes, then reacted with fixed T pallidum on a slide• Fluorescent anti-human IgG is added to visualize the bound bacteria

Question 32

Why is the FTA-ABS expensive?

Answer 32

T. palladium cannot be grown in culture (just rabbit testicles)

Question 33

What is the advantage of antigen detection over antibody detection when possible?

Answer 33

Antigen may be present at higher levels earlier in an infection than antibodies while the immune response is being formed

Question 34

How are viral neutralization assays performed?

Answer 34

detecting antigenhave source with virus in it and add it to a series of tubes with commercial neutralizing antibodies against suspected causative antigens and then look on a monolayer plate for inhibition of the virus. Need a control to make sure its working properly.

Question 35

How are Rapid Antigen Detection tested (RADT) performed?

Answer 35

often used for strep throat and for flu infectionsCalled lateral flow immunoassays and can typically read in about 15 minutes and they are accurateTake collected samples (throat swab) and put it in a medium that breaks up the bacteria to release the antigens and then we add a droplet of sample onto a well on a wickable paper so that the sample will flow. It will encounter antibody to antigens present in that organism to make sure the organism is present. Once they bind they are concentrated and trapped to a line via another antibody fixed to the slide. The second fixed line binds the antibody to make sure its present as a control. Doesn’t tell you which antigen you are binding, you assume it is the right antigen

Question 36

How is HIV testing done?

Answer 36

• ELISA (antibody testing)• Murex Single Use Diagnostic System (SUDS)-Assays antibodies and presence of p24 • OraQuick (home testing)-Saliva antibodies • Western blot (confirmatory test)-Positive: p24, gp41 and/or gp120/160-Indeterminate: some bands, but not those above-Negative: no bands

Question 37

How is direct probing done?

Answer 37

denature nucleic acid of target organism and then bring primers to amplify the DNA via PCR

Question 38

When are Nucleic Acid Amplification Tests (NAT or NAAT used)?

Answer 38

• Particularly useful when organism is difficult to routinely grow and isolate• 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• Can be used to determine pathogen load• Uses (there are plenty others)• HIV• HBV, HCV• Chlamydia, GC

Question 39

Why is probe hybridization preferred over an NAAT in BV?

Answer 39

PCR could be too specific and would give false positives if other flora were introduced

Question 40

Metabolite Analysis

Answer 40

useful in identification of gram- rodsuses enterotubes with agar ‘cubes’ that can change color depending on whats in it. Use an inoculating rod with organism for each rod and can see each the change in each cube. Uses a scoring system to identify

Question 41

Urine Dipstick

Answer 41

common UTi-causing bacteria can reduce nitrate to nitrite. Can be assayed using the dipstick format

Question 42

Mass Spec

Answer 42

Matrix-Assisted Laser Desorption Ionization Time-of-Flight add bacteria in a sample and use a laser to ionize the proteins and then line them up through an electrostatic field. They are then sprayed and they “fly” through an analyzer and the small proteins fly faster than the heavier ones. Read on computer.