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Clinical Microbiology and Immunology
CHAPTER OVERVIEW
This chapter describes the field of clinical microbiology, which is concerned with the detection and
identification of pathogens that are the etiological agents of infectious disease. Identification may be based on
the results of some combination of morphological, physiological, biochemical, and immunological procedures.
Time may be critical in life-threatening situations. Therefore, rapid identification systems and computers can
be used to greatly speed up the process. The chapter closes with a discussion of in vitro antigen-antibody
interactions. These are particularly useful in diagnostic procedures.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
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describe the functions and/or services performed by clinical microbiology laboratories
discuss the types of procedures used to identify microorganisms in specimens
describe molecular genetic techniques useful for identifying microorganisms in a clinical setting
describe the various in vitro antigen-antibody interactions, and give examples of diagnostic tests based on
them
CHAPTER OUTLINE
I.
II.
Overview of the Clinical Microbiology Laboratory
A. Clinical microbiologists isolate and identify pathogens from clinical specimens to provide
information to physicians
B. Universal precautions for handling specimens have been established by the Centers of Disease
Control and Prevention (CDC)
1. Specimen—human material that is tested, examined, or studied to determine the presence or
absence of specific microorganisms
a. Because of safety concerns, specimens must be handled carefully; universal safety
precautions have been recommended by the CDC to address safety issues in specimen
handling
b. Specimens should be:
1) Representative of the diseased area
2) Adequate in quantity for a variety of diagnostic tests
3) Devoid of contamination, particularly by microorganisms indigenous to the skin and
mucous membranes
4) Forwarded promptly to the clinical laboratory
5) Obtained prior to the administration of any antimicrobials
Identification of Microorganisms from Specimens
A. Microscopy
1. Direct examination of specimen, or examination of specimen after various staining procedures
that are specific for types of bacteria, fungi, viruses, or protists
2. Bacteria
a. Wet mounts for bright-field, phase-contrast, or dark-field microscopy; fluorescent
microscopy with nucleic acid stains or labeled antibodies
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b.
B.
C.
D.
E.
Hybridomas produce monoclonal antibodies specific for a single epitope; these can be
fluorescently labeled and used for rapid, accurate culture confirmation
c. Immunofluorescence stains specimens with fluorescent dyes that emit visible colored
light when excited
1) Direct immunofluorescence—after fixation to a slide, the specimen is stained with a
fluorescently labeled antibody directed at cell surface antigens
2) Indirect immunofluorescence—after a known antigen is attached to a slide, a
patient's serum (antiserum) is applied; if antibodies specific for the antigen are
present, these can be detected with a fluorescently labeled secondary antibody
3. Fungi—light microscopy to observe hyphae and spores are diagnostic for fungi; lactophenol
aniline blue stain often used
4. Parasites—direct microscopy to observe ova, trophozoites, and cysts; D'Antonio's iodine and
Giemsa stain often used
5. Viruses—can be observed with electron microscopy
Growth and biochemical characteristics
1. Bacteria
a. Isolation and growth of bacteria are required before many diagnostic tests can be used
b. Initial identity may be suggested by source of specimen; microscopic appearance and
Gram reaction; pattern of growth on selective, differential, and other media; and by
hemolytic, metabolic, and fermentative properties
c. After pure cultures are obtained, specific biochemical tests can be done and a
dichotomous key used for identification
d. Rickettsias—identified by immunoassays or by isolation in specialized laboratories
2. Fungi—examination of cultures on selective media, often supplemented with antibacterial
drugs
3. Parasites—generally not cultured
4. Viruses—identified by isolation in cell (tissue) culture, by immunodiagnosis, and by molecular
detection
a. Viral cultivation
1) Cell cultures—viruses are detected by cytopathic effects (observable morphological
changes in host cells) or by hemadsorption (binding of red blood cells to infected
cells)
2) Embryonated eggs—virus can be inoculated into allantoic cavity, amniotic cavity,
or the chorioallantoic cavity; virus is detected by development of pocks on the
chorioallantoic membrane, by development of hemagglutinins in the allantoic and
amniotic fluid, and by death of the embryo
3) Laboratory animals (e.g., suckling mice)—observed for signs of disease or death
Rapid methods of identification
1. Manual biochemical systems such as the API 20E system for enterobacteria
a. Consists of 20 microtube inoculation tests
b. Results are converted to a seven- or nine-digit profile number
c. The number is compared to the API Profile Index to determine the name of the bacterium
2. Automated systems (Phoenix™, VersaTREK®, MicroScan® WalkAway) test pure cultures in
miniaturized chambers for specific capabilities and antibiotic sensitivity
3. Monoclonal antibodies are available for the detection of many microorganisms; the antibodies
can be specific to the species or strain level
4. Biosensors can be based on microfluidic antigen sensors, rapid PCR, sensitive spectroscopy
systems, and liquid crystal amplification of immune complexes
Bacteriophage typing—based on the fact that host range specificities of bacteriophages are
dependent upon surface receptors on the bacteria
Molecular genetic methods
1. Group of accurate measures including protein comparisons, enzyme characterizations, nucleic
acid-base composition, nucleic acid hybridization, and nucleic acid sequencing
2. Nucleic acid-based diagnostic methods
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a.
DNA molecules that have been cloned from organism or prepared by PCR technology
can be used in hybridization procedures
b. Real-time PCR can monitor amplification of specific microbial genes; rRNA genes can
be sequenced to identify bacterial strains (ribotyping)
c. Genomic fingerprinting examines patterns produced from PCR amplification of repetitive
sequences; plasmid fingerprinting—separation and detection of the number, molecular
weight, and restriction patterns of different plasmids, that are often consistently present in
a strain of bacteria
III. Clinical Immunology
A. Detection of antigens and antibodies may be valuable diagnostically; interpretation of immunologic
test results can be difficult
B. Serotyping—antigen-antibody specificity is used to differentiate among various strains (serovars) of
an organism found in serum samples
C. Agglutination—visible clumps or aggregates of cells or of coated latex microspheres; if red blood
cells are agglutinated, the reaction is called hemagglutination
1. Widal Test—direct agglutination test for diagnosing typhoid fever
2. Latex agglutination tests are used in pregnancy tests and to diagnose mycotic, helminthic, and
bacterial infections
3. Mycoplasmas are typically identified by hemagglutinin reactions, PCR, and antigen-antibody
reactions
4. Viral hemagglutination inhibition tests are used to diagnose influenza and other viral infections
5. Agglutination tests can be used to measure antibody titer (the reciprocal of the greatest dilution
showing agglutination reaction)
D. Complement fixation—used to detect the presence of serum antibodies to a pathogen; currently used
to diagnose certain viral, fungal, rickettsial, chlamydial, and protozoan diseases
E. Enzyme-linked immunosorbent assay (ELISA)—involves linking enzymes to an antibody
1. Indirect immunosorbent assay—detects serum antibody
a. Well of a microtiter plate is coated with antigen specific to the antibody of interest
b. Test serum is added; if antibodies are present, they will bind antigen and will be retained
after washing
c. An antibody against the test immunoglobulin is added; the second antibody is conjugated
to an enzyme and will only be retained in the well after washing if the test antibody is
present in the well
d. Substrate of the enzyme is added; reaction only occurs if conjugated antibody (and
therefore test antibody) are present in the well; the colored product of the reaction can be
detected spectrophotometrically
2. Double antibody sandwich assay—detects antigens in a sample
a. Wells of a microtiter plate are coated with antibody specific to the antigen of interest
b. Test sample is placed in well; if it contains the antigen of interest, the antigen will be
retained in the well after washing
c. Second antibody is added; it is conjugated to an enzyme and is specific to the antigen; the
second antibody will be retained in the well after washing if the antigen was retained in
the previous step
d. Substrate of enzyme is added; reaction only occurs if conjugated enzyme (and therefore
antigen) is present in the well; produces a colored product that can be detected
F. Immunoblotting (Western Blot)—proteins are separated by electrophoresis, blotted into
nitrocellulose sheets, then treated with solution containing enzyme-tagged antibodies
G. Immunoprecipitation—soluble antigens form insoluble immune complexes that can be detected
H. Immunodiffusion—involves the precipitation of immune complexes in an agar gel
1. Single radial immunodiffusion (RID) assay is quantitative
2. Double diffusion assay (Öuchterlony technique)—lines of precipitation form where antibodies
and antigens have diffused and met; determines whether antigens share identical determinants
I.
Immunoelectrophoresis—antigens are first separated by electrophoresis according to charge, and
are then visualized by the precipitation reaction; greater resolution than diffusion assay
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J.
K.
Flow cytometry
1. Detects single or multiple microorganisms on the basis of a cytometric parameter or by means
of fluorochromes
2. Flow cytometer forces cells through a laser beam and measures light scatter or fluorescence as
the cells pass through the beam; cells can be tagged with fluorescent antibody directed against
specific surface antigen
Radioimmunoassay (RIA)—purified antigen labeled with a radioisotope competes with unlabeled
antigen sample for antibody binding
TERMS AND DEFINITIONS
Place the letter of each term in the space next to the definition or description that best matches it.
____
____
____
____
1.
2.
3.
4.
____ 5.
____ 6.
____ 7.
____ 8.
____ 9.
____ 10.
____ 11.
____ 12.
____ 13.
The insertion of a tube into a body canal or hollow organ
A tubular instrument used for withdrawing or introducing fluids from or into a body cavity
An observable change that occurs in cells as a result of viral infection
Phenomenon that occurs because of an alteration of the membrane of a virus-infected cell so that red
blood cells will adhere to it
A method of identifying bacteria based on the bacteriophage that infect them
A method of strain typing that involves preparing a Southern Blot of chromosomal DNA cleaved
with restriction endonucleases, and then probing the blot with specific rRNA probes
Visible aggregates or clumps formed by agglutination reactions
An agglutination reaction-based test that is used to diagnose typhoid fever
The agglutination of red blood cells by viruses
A colorless substrate that is acted on by an enzyme to produce a colored end product
A process in which a suspension of cells is forced through a laser beam; it can be used to detect,
count, separate, and characterize cells in the suspension
A process wherein a fluorescent dye is attached to an antibody and used to detect the antigen
specific for that antibody
The use of in vitro antibody-antigen reactions to differentiate strains of microorganisms
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
agglutinates
bacteriophage typing
catheter
chromogen
cytopathic effect
flow cytometry
hemadsorption
immunofluorescence
intubation
ribotyping
serotyping
viral hemagglutination
Widal test
FILL IN THE BLANK
1.
2.
The major concern of the __________ __________ is to rapidly isolate and identify microorganisms
from clinical specimens.
Viral replication in cell cultures is detected in two ways: by the observation of __________ __________
(observable changes in cell morphology) and by ____________ (alterations in the plasma membrane that
enable red blood cells to adhere firmly to virus-infected cells).
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
3.
4.
5.
6.
One of the most widely used serological tests is the ____________. It involves the linkage of an antibody
molecule to an enzyme whose activity can be detected by the formation of a colored product.
When a precipitation reaction occurs in an agar gel, it is called
. One assay based on this
phenomenon is the
assay. This assay is based on the diffusion of
antigen out of a well into agar containing an antibody. In another assay, the __________ __________
assay, also known as the
, antigens are placed in a set of wells in
agar and antibodies are placed in another well. Both antibodies and antigens diffuse
into the agar.
Serological procedures used to differentiate different strains of microorganisms are called
.
An important example of this is the __________ __________, which is used to classify streptococci
based on the antigenic nature their cell walls. One way of detecting these differences is the __________
__________, in which mixing antiserum with a solution of streptococci causes capsular swelling if the
antiserum is specific for that particular serovar.
Some mixtures of antigens are very complex, making it difficult to detect a particular antigen. In such
cases, __________ is useful. In this process, antigens are first separated based on their electrical charge
and then are visualized by precipitation reaction.
MULTIPLE CHOICE
For each of the questions below select the one best answer.
1.
2.
3.
4.
5.
Which of the following is NOT normally
used in the identification of microorganisms?
a. microscopic examination
b. growth or biochemical characteristics
c. immunological techniques that detect
antibodies or microbial antigens
d. All of the above are used in the
identification of microorganisms.
Which of the following is NOT normally
used to culture viruses?
a. growth on artificial media
b. growth in cell cultures
c. growth in embryonated hen’s eggs
d. growth in whole animals
Which of the following is normally used to
detect spirochetes in skin lesions in early
syphilis?
a. bright-field microscopy
b. phase-contrast microscopy
c. dark-field microscopy
d. immunofluorescence microscopy
Which of the following is NOT likely to be
useful in the identification of bacteria?
a. source of the culture specimen
b. growth patterns on selective and
differential media
c. hemolytic, metabolic, and fermentative
properties
d. All of the above are useful in the
identification of bacteria.
What is the basic principle underlying
plasmid fingerprinting?
a.
6.
7.
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Microbial isolates of the same strain
contain the same number of plasmids
with the same molecular weights.
b. Microbial isolates of different strains
have different plasmids (either in
number, molecular weight, or both).
c. Both (a) and (b) are correct.
d. Neither (a) nor (b) is correct.
Which of the following is NOT a reason that
immunological tests for antibodies against a
particular infectious organism might yield
negative results?
a. The person might not be infected with
the particular organism.
b. The organism might be poorly
immunogenic and may not stimulate
sufficient antibody production to be
detectable.
c. There might not have been sufficient
time since the onset of infection for an
antibody response to develop.
d. All of the above might yield negative
results.
Which of the following is NOT true about the
use of DNA:rRNA hybrids for identification
as compared to the use of DNA:DNA
hybrids?
a. DNA:rRNA hybrids are more sensitive,
and therefore, fewer microorganisms
are required.
b. DNA:rRNA hybrids are more specific,
and they show less cross-hybridization
to other species.
c.
8.
9.
DNA:rRNA hybrids are formed more
rapidly; test requires two hours or less
for results.
d. All of the above are true about the use
of DNA:rRNA hybrids as compared to
the use of DNA:DNA hybrids.
Which type of cell culture for cultivating
viruses makes use of transformed cells,
generally epithelial in origin?
a. primary cultures
b. secondary cultures
c. semicontinuous cell cultures
d. continuous cell cultures
When complement binds to an antibodyantigen complex, it becomes used up and is
no longer available to lyse sensitized red
blood cells. This can be used diagnostically
in an assay. What is this assay called?
a. complement utilization assay
b. complement titration assay
c. complement fixation assay
d. complement complexation assay
10. In one immunological assay, antigens are
separated by electrophoresis through a
polyacrylamide gel and then are transferred
to a sheet of nitrocellulose. This is then
probed with enzyme-tagged antibodies. What
is this called?
a. immunoprecipitation
b. immunodiffusion
c. ELISA
d. immunoblotting
11. Immunological assays can be used to
differentiate strains of microorganisms. What
are these assays called?
a. serotyping
b. immunotyping
c. isotyping
d. antigen typing
TRUE/FALSE
____ 1.
The Gram stain is used for bacteria that have cell walls, while the acid-fast stain is used primarily
for wall-less bacteria.
____ 2. Rickettsias are routinely isolated and identified by culture methods because these are relatively
inexpensive and safe to use.
____ 3. Bacteria can usually be identified by morphological examination, and biochemical tests are only
needed for confirmation of identification.
____ 4. Detection of an elevated antibody titer is used to indicate an active, ongoing infection.
____ 5. Strains of bacteria that are infected by different phage isolates are referred to as phagovars.
____ 6. Rapid ID systems such as the API 20E system identify bacteria based on substrate utilization
characteristics.
____ 7. Ribotyping involves probing Southern Blots of endonuclease digested chromosomal DNA with
rRNA genes probes.
____ 8. Immunoprecipitation reactions will occur as long as the antibody and antigen are present in nearly
equal amounts.
____ 9. Radioimmunoassays do not need specific antibodies.
____ 10. Flow cytometry can use lasers to detect specific cells in a stream of suspended cells.
CRITICAL THINKING
1.
Nucleic acid-based detection methods have a great deal of power; however, microscopy and biochemical
tests are still widely used. Why? Contrast these two approaches and compare them to current
immunoassays for detection of pathogens.
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2.
There are a variety of rapid methods for microbial identification. Discuss several of these methods and
how they might be applied. In recent years, the need for rapid tests has increased. Why? What kind of
new technologies are being developed?
ANSWER KEY
Terms and Definitions
1. i, 2. c, 3. e, 4. g, 5. b, 6. j, 7. a, 8. m, 9. l, 10. d, 11. f, 12. h, 13. k
Fill in the Blank
1. clinical microbiologist 2. cytopathic effect; hemadsorption 3. ELISA 4. immunodiffusion; single radial
immunodiffusion (RID); double diffusion agar; Ouchterlony technique; 5. serotyping; Lancefield system;
Quellung reaction 6. immunoelectrophoresis
Multiple Choice
1. d, 2. a, 3. c, 4. d, 5. c, 6. d, 7. b, 8. d, 9. c, 10. d, 11. a
True/False
1. F, 2. F, 3. F, 4. F, 5. T, 6. T, 7. T, 8. T, 9. F, 10. T
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