Block 2 - Tools & Tech

Question 1

Within screening and presumptive tests, rapid/sensitive but possibly less specific/less expensive can be categorized as?

Answer 1

screening / presumptive

Question 2

With regard to screening tests, this category is less rapid/more specific, and more expensive.

Answer 2

definitive / confirmatory

Question 3

There are two types of immunodiagnostic tests.

These two tests come from what?

Answer 3

antibody in patient’s serum

antigen in patients specimen

Question 4

Within culture media, what are the four types?

Answer 4

nutrient

enriched

selective

differential

Question 5

On blood agar, what three types can it differentiate?

Answer 5

Alpha / Beta / Gamma

Question 6

With regard to cultivation and identification,

define the type of microbe:

a. Cultivation – grows on blood agar plate and other special media similar to bacteria, 2-7 days
b. Identification – presumptive by microscopy; biochemical tests; antigen tests

Answer 6

Yeast

Question 7

With regard to cultivation and identification,

define the type of microbe:

a. Cultivation – grow on media with antibiotics (e.g. Sabouraud with antibiotics) which inhibit the growth of bacteria. Mycelial masses (colonies) will be visible in 1 to 4 weeks
b. Identification – direct microscopic exam of specimen; microscopic exam of culture; few biochemical tests; few antigen detection tests

Answer 7

Molds

(remember that molds will grow on media with antibiotics to inhibit bacterial growth!)

Question 8

With regard to cultivation and identification,

define the type of microbe:

a. Cultivation – grow in living tissue culture cells – 2 to 21 days to grow
b. Identification – antibody detection; antigen detection; gene probes

Answer 8

Viruses and some atypical bacteria

Question 9

What are the three types of “binding” in antigen-antibody reactions?

Answer 9

good binding

cross-reactive

non-reactive

Question 10

What is the difference between the specificity and sensitivity of a test?

Answer 10

Specificity – the ability of the test to accurately detect only the correct antigen or antibody while not reacting with incorrect antigen or antibody.

Sensitivity – the ability of the test to detect very low levels of antigen (or antibody) when it is present.

Question 11

Sensitivity in tests are expresssed as what two things?

Answer 11

limit of detection

percent accuracy

Question 12

True or False:

Tests with high sensitivity may be used as initial screening or presumptive tests.

Answer 12

True

Question 13

Specificity in tests distinguishes _____-_______ and _______-_______ results from true positives.

Answer 13

cross-reactive

false-positive

Question 14

In the summary of immunoassay reaction procedures, what are the 3 general steps.

Answer 14

1. antigens bind to specific antibodies
2. remove or wash off unbound antibody
3. detect antigen-antibody binding

Question 15

In a lateral flow immunoassay, what are the 3 basic steps?

Answer 15

a. Known molecule and detection molecules are embedded in (attached to) a membrane
b. Specimen is deposited on one end of the membrane and a wicking pad draws the fluid containing the specimen and reagents through the membrane; thus bringing them into contact.
c. Antigen-Antibody binding (reaction) is visible by deposit of the bound molecules at the “test line”.

Question 16

The reaction of a particle-sized antigen (e.g. bacteria or red blood cell or antigen on a latex particle) with its corresponding antibody resulting in a macroscopic (visible) clumping.

Answer 16

Agglutination Method

Question 17

a. Antibody is labeled (tagged) with a fluorescent substance which gives off a particular color of light at a specified wavelength of ultraviolet light
b. Labeled antibody is reacted with unknown antigen and then unbound antibody is washed off
c. The reaction of the bound, labeled antibody with its corresponding antigen is made visible by exposure to ultraviolet light
d. Emitted light is detected by an instrument or by using a ultraviolet light microscope. NOTE: Some fluorescence tests are not antibody-related; rather, the chemical reaction is fluorescent.
e. Results available from “real-time” (as the reaction occurs) to 1 hour to 2 days after specimen is collected, depending on the nature of the specimen, the antigen, and test procedure.
f. Very sensitive and specific; especially useful for certain types of agents

Answer 17

Immunofluorescence Method

Question 18

a. Antibody (known) is labeled with a color-producing enzyme
b. Labeled antibody is reacted with unknown antigen and unbound antibody is washed off (NOTE: this may use a known antigen & unknown antibody, and create a sandwich layering effect)
c. The reacted antigens and antibodies are exposed to a color-producing

substrate to make the antigen-antibody reaction visible.

d. This is a very sensitive and accurate test, and is usually read by an instrument; however, some products are membrane-bound (lateral flow) and read by the naked eye.
e. Results available from a few minutes to 2 days after specimen is collected, depending on the nature of the specimen and the test; typical is 1 to 4 hours.

Answer 18

Enzyme Immunoassay (EIA) or Enzyme Linked Immunosorbent Assay (ELISA) Method

Question 19

Define this microscopic examination:

Purpose

(1) Staining enables bacterial cells to be seen using brightfield microscopy (unstained cells are invisible)
(2) Enables bacterial differentiation based on morphologic and staining differences (different cell wall composition)

Answer 19

Gram Stain

Question 20

Describe the staining steps for the Gram stain:

4 Steps

Answer 20

Staining steps

(a) Primary stain (Crystal Violet): 1 minute, then rinse with tap water
(b) Mordant (Gram’s Iodine): 1 minute, then rinse with water
(c) Decolorizer (Acetone & Alcohol): 2 to 5 seconds, then rinse with water
(d) Counterstain (Safranin): 30 to 60 seconds, then rinse with water and allow to dry

Question 21

Describe smear preparation for Gram Stains:

3 steps

Answer 21

(a) Smear the specimen onto a clean glass microscope slide
(b) Allow the smear to dry in the air without heating
(c) Fix the smear to the slide by flooding the dry smear with methanol for about 1 minute (Less favorable alternate: Heat the dried smear slightly to fix the bacteria to the slide)

Question 22

What has replaced the JBAIDS (Joint Biological Agent Identification & Diagnostic System)?

Answer 22

BioFire FilmArray

(NGDS-Next Generation Diagnostic System)

Question 23

Explain the Gram Stain examination:

3 steps

Answer 23

(a) Allow the slide to dry without heating (careful blotting without wiping is acceptable)
(b) Focus on objects using the low power lens and low light intensity; use coarse focus adjustment
(c) Change to oil immersion lens to observe details of bacteria; add a drop of oil to the slide; increase the light intensity; focus by using the fine focus knob

Question 24

What PCR Technology in DoD is moving to Role 1/2

Answer 24

NGDS-2 Far Forward PCR & Chemical Agent Exposure

Question 25

What type of examination is described below:

a. Principle:
Direct examination of certain unfixed (i.e. “wet”) specimens to permit examination of cells in their “natural” state

b. General procedure

(1) Place a portion of the specimen and any reagents required on a clean slide and cover with a thin coverglass
(2) Observe using low and/or high-dry power objectives and using reduced light intensity

Answer 25

Wet Mount Microscopic Examination

Question 26

What are three examples of “Wet Mount Microscopic Examinations”?

Answer 26

(1) Potassium Hydroxide Wet Preparation (KOH) – 10% KOH dissolves (clears) epithelial cells permitting visualization of fungal mycelial fragments in skin scrapings; performed by the clinician without about 10 minutes after specimen is collected
(2) Yeast cells with capsules in cerebrospinal fluid (CSF) are revealed by the addition of India Ink to the CSF
(3) Saline wet preparation of vaginal exudate reveals yeast cells and motile Trichomonas organisms

Question 27

What is the purpose to use Antimicrobial Susceptibility Testing?

Answer 27

To determine if the pathogen is Susceptible or Resistant to a particular set of antimicrobics

Question 28

The microorganism is not inhibited (can tolerate) by the maximum safe dose/concentration of the antimicrobic. Therefore, this drug should not be selected for therapy of this infection.

Answer 28

Resistant

Question 29

The microorganism is inhibited (or killed) by the maximum safe dose/concentration of the antimicrobic. This antimicrobic is a candidate for use in treating this infection.

Answer 29

Susceptible

Question 30

(Susceptible dose dependent) – the microbe may be inhibited by a high dosage that very near the resistant point. This is typically interpreted as if it is “resistant”. It may be possible to use this antibiotic for treatment under very carefully defined situations and controlled conditions.

Answer 30

intermediate

Question 31

Determine which antimicrobics are effective against a particular bacterium at the ‘breakpoint’ concentration of each antimicrobic.

Answer 31

Disk Diffusion Testing Method

Question 32

What technique is specifically useful only for rapidly gorwing aerobic & facultative bacteria?

Answer 32

Disk Diffusion Testing Method

Question 33

Determines the minimum concentration of each antimicrobic agent that is effective against the bacterial pathogen. Because this information permits more precise selection of antimicrobic and dosage, this is especially useful when using one of the more toxic antimicrobics, when treating the more resistant bacteria, or the patient has complications.

Answer 33

Minimum Inhibitory Concentration

Question 34

(1) The bacterium is tested against various concentrations of each antimicrobic
(2) For each antimicrobic tested, the minimum concentration required to inhibit the bacteria (no growth in the test), the MIC value (μg per mL), is reported. Additionally, the susceptible, intermediate, or resistant interpretation is included.

Answer 34

Minimum Inhibitory Concentration