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Medical Microbiology I > General Principles of Laboratory Diagnosis > Flashcards

General Principles of Laboratory Diagnosis Flashcards

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Biology 101 flashcards USMLE® Step 1 flashcards
1
Q

General diagnostics methods

A
  • Microscopy
  • In Vitro Culture
  • Molecular Diagnosis
  • Serologic Diagnosis
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2
Q

Microscopic methods

A
  • Field largely defined by development & use of the microscope
  • Initial detection of microbes
  • Preliminary or definitive identification
  • 5 general methods
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3
Q

5 general methods of microscopy

A
  • Brightfield (light) (most used)
  • Darkfield
  • Phase-contrast
  • Fluorescent
  • Electron (not used in routine clinical microbiology)
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4
Q

Brightfield light microscope

A
  • Most commonly used method
  • Light source
  • Condenser
  • Ocular lens
  • Objective lenses
  • 10X, 40X, 100X (OI)
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5
Q

Microscopy direct smear examination

A
  • Clinical specimens/samples of growing culture

- Right on glass slide and examined

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6
Q

Gram stain

A
  • Best known & most widely used stain

- Basis for phenotypic classification of bacteria as Gram +/-

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7
Q

Degree to which the organism retains stain

A
  • Function of the organism
  • Culture conditions
  • Staining skills of microscopist
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8
Q

Gram stain differentiates between

A
  • Gram (+) = thick peptidoglycan cell walls
  • Gram (-) = thin peptidoglycan cell walls, outer membranes can be dissolved with alcohol or acetone
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9
Q

Gram positive clusters & chains

A
  • Staphylococcus aureus

- Streptococcus pyogenes

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10
Q

Gram positive rods (bacilli)

A
  • Bacillus cereus

- Clostridium perfringens

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11
Q

Darkfield microscopy setup

A
  • Same lenses as brightfield
  • Special condenser illuminates from oblique angle
  • Subject illuminated against a black background
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12
Q

Darkfield micrscopy usage

A
  • Detects organisms that are too thin to be observed by brightfield microscopy
  • Internal structures not visible
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13
Q

Phase-contrast microscopy setup

A
  • Illuminates objects with parallel beams of light that move out of phase relative to each other
  • Allows objects to appear as three-dimensional (3D) structures
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14
Q

Phase-contrast microscopy usage

A
  • Observing internal structures

- Identifying filamentous fungi

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15
Q

Fluorescent microscopy setup

A
  • High-pressure mercury, halogen, or xenon vapor lamps that emit a short wavelength of light to illuminate the object
  • A series of filters block heat and infrared light, and select a specific wavelength of light emitted by the object
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16
Q

Fluorescent microscopy usage

A
  • Organisms with stained with specific fluorescent dyes
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17
Q

Fluorescence

A
  • Observed as a brightly illuminate object against a dark background
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18
Q

Fluorescent antibody stains

A
  • Specific stains where antibodies are attached to a fluorochrome (such as fluorescein)
  • The antibody-antigen binding is detected by the fluorescence
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19
Q

Fluorescent antibody stain examples

A
  • Pneumocystis

- Varicella-Zoster virus

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20
Q

Calcofluor white stain

A
  • Used to detect yeasts and molds in clinical specimens

- This fluorescent dye binds to chitin in the fungal cell wall

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21
Q

False positive calcofluor white stains may occur if

A
  • Cotton fibers are present in the specimen because the dye will also bind to cellulose
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22
Q

Acid-fast stains

A
  • Ziehl-Neelsen
  • Kinyoun
    Fluorochrome
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23
Q

Ziehl-Neelsen stain

A
  • Original stain
  • Heat slide after basic fuchsin is added
  • Stain penetrates into the bacteria
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24
Q

Kinyoun stain

A
  • Cold acid-fast stain

- No heat, the concentrations of basic fuchsin and phenol are increased

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25
Q

Modified Kinyoun stain

A
  • Cold acid-fast stain

- Differs from the Kinyoun stain by using a weak acid solution in alcohol

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26
Q

Modified Kinyoun stain fuchsin retention

A
  • Nocardia, Rhodococcus, Gordonia, and Tsukamurella will retain some of the basic fuchsin stain (weak solution)
  • Higher concentration of acid prevents stain retention
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27
Q

Fluorochrome stain

A
  • Replaces basic fuchsin with two fluorescent dyes, auramine and rhodamine
  • Weak acid-fast stain so all acid-fast organisms will stain
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28
Q

Auramine rhodamine stain

A
  • Essentially the same as a Kinyoun stain, but with auramine and rhodamine
  • Examined under UV illumination using a fluorescent microscope
  • High contrast between the fluorescing bacilli and the black background, so more sensitive than the Kinyoun stain
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29
Q

Success of in vitro culture methods is determined by

A
  • Biology of the organism
  • Site of the infection
  • Patient’s immune response
  • Timing of specimen collection
  • Quality of the culture media
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30
Q

Types of in vitro culture media

A
  • Enriched nonselective
  • Selective
  • Differential
  • Specialized
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31
Q

Enriched nonselective media

A
  • Support the growth of most organisms without fastidious growth requirements
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32
Q

Selective media

A
  • Designed for the recovery of specific organisms that may be present in a mixture of other organisms
  • Supplemented with inhibitors that suppress the growth of unwanted organisms
33
Q

Differential media

A
  • Specific ingredients added that allow the identification of an organism in a mixture
  • Often combined with a selective media
34
Q

Specialized media

A
  • Created for the detection of specific organisms that may be fastidious, typically present in a large mixture of organisms, or require specific nutrients for cultivation
35
Q

Enriched nonselective media examples

A
  • Blood agar
  • Chocolate agar
  • Thioglycolate broth
  • Mueller-Hinton agar
36
Q

Blood agar

A
  • Many types
  • Contain two primary components:
  • Basal medium (tryptic soy, brain heart infusion)
  • Blood (sheep, horse, and rabbit)
37
Q

Chocolate agar

A
  • Modified blood agar medium
  • Blood/hemoglobin in heated basal media turns brown (resembling chocolate)
  • Supports the growth of most bacteria, including some that do not grow on blood agar
38
Q

Thioglycolate broth

A
  • Common enrichment broth
  • Recovers low numbers of aerobic and anaerobic bacteria
  • Various formulations used
  • More anaerobic recovery with hemin and vitamin K
39
Q

Mueller-Hinton agar

A
  • Susceptibility testing of bacteria
  • Well-defined composition of beef and casein extracts, salts, divalent cations, and soluble starch that is necessary for reproducible test results
40
Q

MacConkey agar

A
  • Selective agar for gram-negative bacteria

- Differential for differentiation of lactose-fermenting and lactose-nonfermenting bacteria

41
Q

MacConkey agar mechanisms

A
  • Bile salts and crystal violet inhibit gram-positive bacteria
  • Bacteria that ferment lactose produce acid (precipitates bile salts, turns red)
42
Q

MacConkey agar evaluation

A
  • Lactose fermenting colonies = pink or red
  • E. coli precipitates bile around the colonies
  • Lactose nonfermenters = colorless
43
Q

Hektoen enteric agar

A
  • Differentiation of lactose & sucrose fermenters

- Differentiation of nonfermenters and H2S producers and nonproducers

44
Q

Hektoen enteric agar mechanism

A
  • Bile salts and bromthymol blue inhibit gram-positive bacteria
  • Bacteria that ferment lactose or sucrose produce acid (yellow-orange color, ferric ammonium citrate for H2S detection)
45
Q

Hektoen enteric agar evaluation

A
  • Lactose and/or sucrose fermenting colonies appear yellow or orange
  • H2S producers - black colonies
  • Colorless and H2S producing colonies will be further identified from stool cultures
46
Q

Mannitol salt agar

A
  • Selective for isolation of Staphylococci

- Differential for S. aureus

47
Q

Mannitol salt agar mechanism

A
  • Digests of casein & animal tissue, beef extract, mannitol, salts, & phenol red
  • Staphylococci can grow in the presence of high salt concentration
  • S. aureus can ferment mannitol, producing yellow-colored colonies on this agar
48
Q

Mannitol salt agar evaluation

A
  • High salt (7.5%) inhibits most bacteria

- Differentiates mannitol fermentation with phenol red acid indicator

49
Q

Lowenstein-Jensen (LJ) medium

A
  • Isolation of mycobacteria
  • Contains glycerol, potato flour, salts, and coagulated whole eggs to solidify the medium
  • Malachite green is added to inhibit growth of gram-positive bacteria
50
Q

Middlebrook agar

A
  • Isolation of mycobacteria
  • Contains nutrients required for the growth of mycobacteria (i.e., salts, vitamins, oleic acid, albumin, catalase, glycerol, glucose) and malachite green for the inhibition of gram-positive bacteria
  • In contrast with LJ medium, it is solidified with agar
51
Q

Phenylethyl alcohol agar (PEA)

A
  • Selective for streptococci & staphylococci

- Inhibits most gram-negative organisms

52
Q

Thayer Martin agar

A
  • Selective for pathogenic Neisseria

- Vancomycin, colistin, nystatin, trimethoprim lactate

53
Q

CHROMAgar

A
  • Both selective and differential
  • Chloramphenicol to inhibit bacterial growth
  • Chromogenic substrates for yeasts
54
Q

CHROMAgar evaluation

A
  • Colonies of C. albicans appear light to medium green
  • C. tropicalis colonies appear dark blue to purple
  • C. krusei colonies appear as light pink, flat colonies with a whitish border
  • Other yeasts may appear light to dark mauve
55
Q

Bacterial detection and identification

A
  • Microscopy
  • Direct Antigen Detection
  • Nucleic Acid Detection
  • Culture
  • Serology (antibody response)
56
Q

Antigen detection

A
  • Often directly from specimen
57
Q

Culture

A
  • Many common organisms can be identified meeting 2 to 3 criteria by growth characteristics & rapid spot testing
  • Analysis of preformed enzymes, small scale fermentation to identify organism same day as growth
58
Q

Molecular diagnosis

A
  • DNA, RNA or proteins of an infectious agent in a clinical sample can be used to identify the agent
59
Q

Molecular diagnosis is used with

A
  • Slow growing organisms (mycobacteria)
  • Difficult to cultivate or fastidious organisms (Chlamydia / Neisseria gonorrhoeae)
  • Viruses (largest area of use in routine diagnosis and prognostic evaluation of treatment)
60
Q

Polymerase chain reaction (PCR)

A
  • Amplifies single copies of DNA millions of times by incubating the target DNA with 2 short DNA pieces (primers)
  • Primers are complementary to the ends of the genetic material of interest
61
Q

Key to PCR technological development

A
  • Discovery of a heat stable DNA polymerase enzyme (Taq) in 1985
  • Many variations of this assay later developed
62
Q

Serologic diagnosis

A
  • Many bacteria and viruses are detected by antigen or antibody detection as a rapid means for a diagnosis
  • Many immunoassays can be done in 1 to 2 hours to specifically identify presence of soluble antigen in a patient’s sample or for antibody detection in blood
63
Q

Antibodies can be used as

A
  • Sensitive and specific tools to detect, identify, and quantitate the antigens from a virus, bacterium, fungus, or parasite
64
Q

Specific antibodies may be obtained from

A
  • Convalescent patients or prepared in animals
65
Q

Polyclonal antibodies

A
  • Heterogeneous antibody preparations

- Can recognize many epitopes on a single antigen

66
Q

Monoclonal antibodies

A
  • Recognize individual epitopes on an antigen

- Commercially available for many antigens, especially for lymphocyte cell surface antigens as diagnostic reagents

67
Q

Advantages of monoclonal antibodies

A
  • Specificity can be confined to a single epitope on an antigen
  • Can be prepared in “industrial-sized” tissue culture preparations
68
Q

Major disadvantage of monoclonal antibodies

A
  • Often too specific
  • If specific for one epitope on a viral antigen of one strain, may not be able to detect different strains of the same virus
69
Q

Agglutination and flocculation assays

A
  • Some can be completed in 5 to 10 minutes
  • Syphilis testing (RPR)
  • Infectious mononucleosis
70
Q

Assays for antigen of antibody

A
  • Enzyme immunoassay (EIA)
  • Multiple steps in assay
  • Can be done as qualitative or quantitative assays
71
Q

Flow cytometer

A
  • Used to analyze the immunofluorescence of cells in suspension
  • Especially useful for identifying and quantitating lymphocytes (immunophenotyping)
72
Q

Flow cytometer mechanism

A
  • Laser is used in the flow cytometer to excite the fluorescent antibody attached to the cell
  • Determines the size of the cell by means of light-scattering measurements
  • Cells flow past the laser at rates of more than 5000 cells per second, and analysis is performed electronically
73
Q

Fluorescence-activated cell sorter (FACS)

A
  • Flow cytometer that can also isolate specific subpopulations of cells for tissue culture growth on the basis of their size and immunofluorescence
74
Q

Rapid EIA (serologic diagnosis)

A
  • Lateral flow self contained EIA cartridges can detect antigen or antibody in 20 minutes
  • Group A Streptococcus from throat swabs
  • Legionella antigen in urine
  • Rotavirus in stool
  • RSV, FLU (respiratory)
75
Q

Serology

A
  • Evaluation of humoral immune response
  • Often used to identify viruses and other agents that are difficult to isolate and cultivate in the lab
  • Used to evaluate the time course of an infection
76
Q

Antibody titer

A
  • The greatest dilution (lowest concentration) of patient serum that retains activity in the immunoassay
77
Q

Seroconversion

A
  • Occurs when antibody is produced in response to an infection
78
Q

Specific IgM antibody detected

A
  • Good indication of a recent primary infection

- Specific IgG evaluation along with IgM

Medical Microbiology I (8 decks)

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