Tools and Techniques Flashcards by Cal Schiller

rapid, sensitive but possibly less specific, less expensive

Screening/Presumptive

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Less rapid, more specific/expensive

Defininitve/confirmatory

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Immunodiagnostic tests

Antibody in patient’s serum

Antigen in patient’s specimen

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moderately enriched medium on which colonies can be differentiated by hemolytic pattern – grows almost all bacteria, except Neisseria gonorrhoeae, Haemophilus influenzae, and a few others which require SCA. Permits visual observation of blood cell hemolysis

Blood Agar Plate (BAP) (usually sheep blood)

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highly enriched media, grows most medically significant bacteria, but without visual display of certain colony characteristics

Supplemented Chocolate Agar (SCA)

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Selective & differential media for gram-negative bacilli – Contain inhibitors for gram-positive cocci/bacilli and gram-negative cocci

MacConkey Agar (MAC)

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Thioglycollate

Trypticase Soy

Broth media (liquid)

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Bacterial colony morphology

General Appearance
Size
Surface texture
color
Hemolysis (only on blood agar - alpha, beta, gamma)

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Alpha (hemolysis)

green zone around colony

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Beta (hemolysis)

clear zone around colony

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Gamma (hydrolysis)

non change in RBCs around colony

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Bact. Colony Morphology timeline?

24ish hours for observable traits

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Yeast cultivation

similar agar/media to bacteria (2-7 days)

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Mold cultivation

grow on media with antibiotics (e.g. Sabouraud with antibiotics) which inhibit the growth of bacteria.

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Mycelial Masses visible in

1-4 weeks

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Yeast ID

presumptive by microscopy; biochemical tests; antigen tests

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Mold ID

direct microscopic exam of specimen; microscopic exam of culture; few biochemical tests; few antigen detection tests

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Virus Cultivation

grow in living tissue; 2-21 days

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Virus ID

antibody detection, antigen detection, gene probes

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Antigen-Antibody Reactions requirements:

(1) Epitopes must be detectable – sufficient quantity needed
(2) Epitopes must be highly specific in order to produce accurate test result.
(3) Possibility of Cross-reactive – some Ab Fab sites will bind with very similar, but incorrect antigens

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

Sensitivity

all true-positives are detected, even if that means a low percentage of false-positives are included.

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

Specificity

Distinguish cross-reactive and false-positive results from true positives

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Overall degree of accuracy of the immunological test depends on quantity of ____, exposure of antigen, and any cross-reactivity between non-matching antigen and antibody

antigen or antibody

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if __ is unknown, then Ag must be known

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Usually test ____

serum

The term ____ refers to some means of removing or blocking the unbound reactants so that unintended binding will not occur

“wash off”

captures Ag or Ab, and detector reaction makes it visible

Capture molecule LFI (Lateral Flow Immunoassay):

visible clumping without label

Agglutination

Fluorescent molecule produces light of detectible wavelength when excited by UV light

Immunofluorescence

Color-producing enzyme reacts with substrate

ELISA

Refer to pg 5 for LFI graphic

Neat

Direct agglutination tests -- results in .... | timeline

2-20 mins Relatively low sensitivity (i.e. requires greater quantity of Ag or Ab in the unknown sample)

Very sensitive and specific; especially useful for certain types of agents

Immunofluorescence

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

Enzyme Immunoassay (EIA) or Enzyme Linked Immunosorbent Assay (ELISA) Method Can also display an amount (as opposed just to a pos or neg)

test kits used to detect the presence of significant gene sequences

Gene probes

increase the number of copies of significant microbial gene sequences so they can be detected, e.g. polymerase chain reaction (PCR)

gene amplification

Gene detection is NOT

the same as immunoassay

Most common type of gene amplification

Polymer Chain Reaction (PCR)

Gene amplification will also pick up genes from non-living organisms (i.e., not infectious/active), so be sure to corroborate with ____

clinical SSx

Major components of a microscope?

``` Base Arm Substage Stage Body ```

Commonly used objectives (microscope)

Low power: 10x High (dry): 40-45x Oil immersion high: 90-100x

Eyepiece (lens) usual magnification?

10x

the power (magnification) of the objective lens times the power of the ocular lense?

Total magnification e.g., high dry power = 450x total (45 * 10)

Abbe condenser

align & condense light rays

Matching of lens and lighting

low power - low intensity high dry power - low-medium light oil immersion high power lens - high light intensity

Matchup of lens/magnification for microbes...

Gram stain for bacteria - oil immersion high power KOH prep for molds - low power Other wet mounts - low or high dry (not oil immersion)

Staining enables bacterial cells to be seen using brightfield microscopy (unstained cells are invisible)

Gram stain

Enables bacterial differentiation based on morphologic and staining differences (different cell wall composition)

Gram stain

Bacteria stain either gram-positive or gram-negative on the basis of differences in their

cell wall composition

Fix the smear to the slide by flooding the dry smear with _____ for about 1 minute (gram stain)

methanol

Staining steps

Primary Stain (Crystal Violet) Mordant (gram's iodine) Decolorizer (Acetone/Alcohol) Counterstain (safranin)

cells appear blue-violet

gram pos

cells appear pink-red

gram neg

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

Wet Mount Microscopic Examination

Yeast cells with capsules in cerebrospinal fluid (CSF) are revealed by the addition of ___ to the CSF

India Ink

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

Potassium Hydroxide Wet Preparation (KOH)

_____ preparation of vaginal exudate reveals yeast cells and motile Trichomonas organisms

Saline wet

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

Antimicrobic Susceptibility Testing (AST)

Decisio to perform AST based on clinical significane of specimen:

- non-pathogens - empiric therapy - likelihood of resistant stains

Each antimicrobic has a maximum concentration (dosage) that can be administered safely. This dosage leads to a specific concentration in the blood (or urine or tissue).

Antimicrobic Susceptibility Testing (AST)

Bacteria can tolerate (resist) only a certain (low) concentration of the drug. Each antimicrobic can be safely administered only up to its maximum safe concentration (dosage).

Antimicrobic Susceptibility Testing (AST)

Each antimicrobic has a maximum concentration (dosage) that can be administered ___. This dosage leads to a specific concentration in the blood (or urine or tissue).

safely.

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.

resistance

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.

Susceptible

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.

Intermediate (susceptible dose dependent)

Each drug has a concentration range at which it is effective and safe. ____ vary among antimicrobics. Test determines if the antimicrobic is effective within its safe dosage.

Concentrations (i.e., a lower dose of one drug can be as effective as a higher dose of another drug)

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

Disk diffusion testing method | very rigid procedures and quality control

Disk diffusion is only useful for ____

rapidly growing aerobic and facultative bacteria

(Disk diffusion) A ____ will occur where the antimicrobic concentration is sufficiently high. A large zone of inhibited growth indicates that greater concentration of antibiotic has migrated farther away from the disk AND/OR the bacteria is killed at a low concentration

zone of inhibition (no growth)

(Disk Diffusion) Measurement of size of zone of inhibition of growth is interpreted using a detailed chart that converts zone size (millimeters) into _____

“Susceptible”, “Intermediate” or “Resistant”.

(Disk Diffusion) Paper disks containing known, standard concentrations of antimicrobics (each type of disk contains a particular antimicrobic) are applied to an inoculated _____ using strict standardized procedures

agar plate

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

Minimum Inhibitory Concentration

The bacterium is tested against various concentrations of each antimicrobic

Minimum Inhibitory Concentration

The achievable concentration of antimicrobic at the site of infection varies with the site/tissue and the route of administration. Example: Most antimicrobics can be administered in higher doses via intravenous vs oral route.

Interpration/Clinical Application of Minimum Inhibitory Concentration

Select the most effective “Susceptible” drug based on site of infection, toxicity, route of administration, etc. (a) Concentration in certain tissues/organs will be lower (b) Kidneys increase concentration of certain antibiotics

Minimum Inhibitory Concentration

"Susceptible" interpretation means the antibiotic concentration of at least ____ times the MIC can be achieved at the site of infection when administered by an appropriate route.

2 Minimum Inhibitory Concentration

____– antibiotic concentration of 2X the MIC cannot be achieved even with the maximum safe dosage.

“Resistant” and “Intermediate” Minimum Inhibitory Concentration

Tools and Techniques Flashcards

(77 cards)