General Principles of Laboratory Diagnosis Flashcards
General diagnostics methods
- Microscopy
- In Vitro Culture
- Molecular Diagnosis
- Serologic Diagnosis
Microscopic methods
- Field largely defined by development & use of the microscope
- Initial detection of microbes
- Preliminary or definitive identification
- 5 general methods
5 general methods of microscopy
- Brightfield (light) (most used)
- Darkfield
- Phase-contrast
- Fluorescent
- Electron (not used in routine clinical microbiology)
Brightfield light microscope
- Most commonly used method
- Light source
- Condenser
- Ocular lens
- Objective lenses
- 10X, 40X, 100X (OI)
Microscopy direct smear examination
- Clinical specimens/samples of growing culture
- Right on glass slide and examined
Gram stain
- Best known & most widely used stain
- Basis for phenotypic classification of bacteria as Gram +/-
Degree to which the organism retains stain
- Function of the organism
- Culture conditions
- Staining skills of microscopist
Gram stain differentiates between
- Gram (+) = thick peptidoglycan cell walls
- Gram (-) = thin peptidoglycan cell walls, outer membranes can be dissolved with alcohol or acetone
Gram positive clusters & chains
- Staphylococcus aureus
- Streptococcus pyogenes
Gram positive rods (bacilli)
- Bacillus cereus
- Clostridium perfringens
Darkfield microscopy setup
- Same lenses as brightfield
- Special condenser illuminates from oblique angle
- Subject illuminated against a black background
Darkfield micrscopy usage
- Detects organisms that are too thin to be observed by brightfield microscopy
- Internal structures not visible
Phase-contrast microscopy setup
- 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
Phase-contrast microscopy usage
- Observing internal structures
- Identifying filamentous fungi
Fluorescent microscopy setup
- 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
Fluorescent microscopy usage
- Organisms with stained with specific fluorescent dyes
Fluorescence
- Observed as a brightly illuminate object against a dark background
Fluorescent antibody stains
- Specific stains where antibodies are attached to a fluorochrome (such as fluorescein)
- The antibody-antigen binding is detected by the fluorescence
Fluorescent antibody stain examples
- Pneumocystis
- Varicella-Zoster virus
Calcofluor white stain
- Used to detect yeasts and molds in clinical specimens
- This fluorescent dye binds to chitin in the fungal cell wall
False positive calcofluor white stains may occur if
- Cotton fibers are present in the specimen because the dye will also bind to cellulose
Acid-fast stains
- Ziehl-Neelsen
- Kinyoun
Fluorochrome
Ziehl-Neelsen stain
- Original stain
- Heat slide after basic fuchsin is added
- Stain penetrates into the bacteria
Kinyoun stain
- Cold acid-fast stain
- No heat, the concentrations of basic fuchsin and phenol are increased
Modified Kinyoun stain
- Cold acid-fast stain
- Differs from the Kinyoun stain by using a weak acid solution in alcohol
Modified Kinyoun stain fuchsin retention
- Nocardia, Rhodococcus, Gordonia, and Tsukamurella will retain some of the basic fuchsin stain (weak solution)
- Higher concentration of acid prevents stain retention
Fluorochrome stain
- Replaces basic fuchsin with two fluorescent dyes, auramine and rhodamine
- Weak acid-fast stain so all acid-fast organisms will stain
Auramine rhodamine stain
- 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
Success of in vitro culture methods is determined by
- Biology of the organism
- Site of the infection
- Patient’s immune response
- Timing of specimen collection
- Quality of the culture media
Types of in vitro culture media
- Enriched nonselective
- Selective
- Differential
- Specialized
Enriched nonselective media
- Support the growth of most organisms without fastidious growth requirements
Selective media
- 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
Differential media
- Specific ingredients added that allow the identification of an organism in a mixture
- Often combined with a selective media
Specialized media
- 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
Enriched nonselective media examples
- Blood agar
- Chocolate agar
- Thioglycolate broth
- Mueller-Hinton agar
Blood agar
- Many types
- Contain two primary components:
- Basal medium (tryptic soy, brain heart infusion)
- Blood (sheep, horse, and rabbit)
Chocolate agar
- 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
Thioglycolate broth
- Common enrichment broth
- Recovers low numbers of aerobic and anaerobic bacteria
- Various formulations used
- More anaerobic recovery with hemin and vitamin K
Mueller-Hinton agar
- 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
MacConkey agar
- Selective agar for gram-negative bacteria
- Differential for differentiation of lactose-fermenting and lactose-nonfermenting bacteria
MacConkey agar mechanisms
- Bile salts and crystal violet inhibit gram-positive bacteria
- Bacteria that ferment lactose produce acid (precipitates bile salts, turns red)
MacConkey agar evaluation
- Lactose fermenting colonies = pink or red
- E. coli precipitates bile around the colonies
- Lactose nonfermenters = colorless
Hektoen enteric agar
- Differentiation of lactose & sucrose fermenters
- Differentiation of nonfermenters and H2S producers and nonproducers
Hektoen enteric agar mechanism
- 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)
Hektoen enteric agar evaluation
- 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
Mannitol salt agar
- Selective for isolation of Staphylococci
- Differential for S. aureus
Mannitol salt agar mechanism
- 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
Mannitol salt agar evaluation
- High salt (7.5%) inhibits most bacteria
- Differentiates mannitol fermentation with phenol red acid indicator
Lowenstein-Jensen (LJ) medium
- 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
Middlebrook agar
- 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
Phenylethyl alcohol agar (PEA)
- Selective for streptococci & staphylococci
- Inhibits most gram-negative organisms
Thayer Martin agar
- Selective for pathogenic Neisseria
- Vancomycin, colistin, nystatin, trimethoprim lactate
CHROMAgar
- Both selective and differential
- Chloramphenicol to inhibit bacterial growth
- Chromogenic substrates for yeasts
CHROMAgar evaluation
- 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
Bacterial detection and identification
- Microscopy
- Direct Antigen Detection
- Nucleic Acid Detection
- Culture
- Serology (antibody response)
Antigen detection
- Often directly from specimen
Culture
- 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
Molecular diagnosis
- DNA, RNA or proteins of an infectious agent in a clinical sample can be used to identify the agent
Molecular diagnosis is used with
- 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)
Polymerase chain reaction (PCR)
- 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
Key to PCR technological development
- Discovery of a heat stable DNA polymerase enzyme (Taq) in 1985
- Many variations of this assay later developed
Serologic diagnosis
- 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
Antibodies can be used as
- Sensitive and specific tools to detect, identify, and quantitate the antigens from a virus, bacterium, fungus, or parasite
Specific antibodies may be obtained from
- Convalescent patients or prepared in animals
Polyclonal antibodies
- Heterogeneous antibody preparations
- Can recognize many epitopes on a single antigen
Monoclonal antibodies
- Recognize individual epitopes on an antigen
- Commercially available for many antigens, especially for lymphocyte cell surface antigens as diagnostic reagents
Advantages of monoclonal antibodies
- Specificity can be confined to a single epitope on an antigen
- Can be prepared in “industrial-sized” tissue culture preparations
Major disadvantage of monoclonal antibodies
- 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
Agglutination and flocculation assays
- Some can be completed in 5 to 10 minutes
- Syphilis testing (RPR)
- Infectious mononucleosis
Assays for antigen of antibody
- Enzyme immunoassay (EIA)
- Multiple steps in assay
- Can be done as qualitative or quantitative assays
Flow cytometer
- Used to analyze the immunofluorescence of cells in suspension
- Especially useful for identifying and quantitating lymphocytes (immunophenotyping)
Flow cytometer mechanism
- 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
Fluorescence-activated cell sorter (FACS)
- Flow cytometer that can also isolate specific subpopulations of cells for tissue culture growth on the basis of their size and immunofluorescence
Rapid EIA (serologic diagnosis)
- 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)
Serology
- 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
Antibody titer
- The greatest dilution (lowest concentration) of patient serum that retains activity in the immunoassay
Seroconversion
- Occurs when antibody is produced in response to an infection
Specific IgM antibody detected
- Good indication of a recent primary infection
- Specific IgG evaluation along with IgM
