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Assisting in Microbiology
and Immunology
Chapter 55
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
1
Learning Objectives
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Define, spell, and pronounce the terms listed in
the vocabulary.
Apply critical thinking skills in performing patient
assessment and care.
Cite the protocols for the collection, transport,
and processing of specimens.
Identify the elements needed for microbial
growth.
Compare bacteria with viruses.
Describe the characteristics of common viral
diseases.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Learning Objectives
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Describe the bacterial structures used in
identification.
Compare bacteria with fungi, parasites, and
protozoa.
Describe various bacterial morphologies.
Explain the characteristics of common diseases
caused by bacteria.
Describe the unusual characteristics of chlamydia,
rickettsia, and mycoplasma.
Identify the characteristics of common diseases
caused by fungi as well as protozoan and parasitic
diseases.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Learning Objectives
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Perform patient education on the collection of a
stool specimen for ova and parasite testing.
Describe the equipment needed in a
microbiology laboratory.
List the different growth media used for
culturing.
Describe the preparation of a bacterial smear.
Perform the procedure for the inoculation of a
blood agar plate.
Perform a urine culture.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Learning Objectives
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Perform a screening urine culture test.
Prepare a direct smear or culture smear for
staining.
Compare and contrast the throat culture for
Streptococcus pyogenes with the rapid strep test.
Perform a rapid strep test.
Describe three microbiology tests that use a rapid
identification technique.
Describe the method used for antimicrobial
susceptibility testing.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Learning Objectives
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Explain how pinworm testing is done and when
it must be performed.
Perform a cellulose tape collection for
pinworms.
Discuss the purpose of immunology testing.
Describe three rapid immunology tests that
could be done in the POL.
Perform the Mono-test for mononucleosis.
Discuss legal and ethical issues in laboratory
testing.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Microbiology
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The medical assistant needs to understand the
role of microorganisms.
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Responsibilities include preventing nosocomial
infections and assisting with infection control
The main objective of microbiology procedures
is to identify the organisms responsible for
illness so that the physician can properly treat
the patient.
Microbiology procedures may be performed in
the POL or in the microbiology department of a
medical referral laboratory.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Specimen Collection and Transport
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Specimen collection and handling are among the most
critical considerations in patient care because any
results the laboratory generates are directly dependent
on the quality of the specimen and its condition on
arrival in the laboratory.
When collecting specimens for microbiological analysis,
the medical assistant should ask two questions:
“In what ways can I prevent extraneous microorganisms
from contaminating this sample?”
“What can I do to prevent myself from becoming
infected while I collect this sample?”
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Specimen Collection and Transport:
Table 55-1
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Microorganisms are living organisms and must be
provided with conditions that permit their survival but
do not permit their multiplication.
If microorganisms are allowed to multiply after their
collection, the culture results will not reflect the true
disease state.
Transport media
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Stuart medium or Amies medium
The medical assistant must strictly follow the
directions.
Transport the specimen directly after collection or by
courier.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Containers for Transport
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Collection Devices
From De la Maza
LM, Pezzlo MT,
Baron EJ: Color
atlas of diagnostic
microbiology, St
Louis, 1997,
Mosby.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Critical Thinking Application
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Aaron Mitchell, age 9 years, was brought into
the clinic this morning at 9 o’clock with
scabbing sores on his upper lip. Dr. Chowdry
suspects impetigo and orders a wound
culture. How will Anna collect this culture?
What device might she use? How should she
store this specimen until the courier, who
does not come until 3:00 pm, arrives? Anna
knows that impetigo is highly contagious.
How can she protect herself from becoming
infected?
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Classification of Microorganisms
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Bacteria, fungi, protozoa
Parasitic worms
Viruses
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Once inside a host cell they use the host cell’s
structures to multiply; obligate intracellular parasites
Table 55-2
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Naming of Microorganisms
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Binomial system of nomenclature
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Assigns two names: the first is the genus and the
second is the species
 genus name begins with a capital letter, species
name with a lowercase letter
 Neisseria gonorrhoeae
 The genus name of the organism may be
represented by a single letter after the organism’s full
genus and species name is written once in a report.
For example, it is common to refer to Escherichia coli
as E. coli
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Critical Thinking Application
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While preparing to collect the specimen from Aaron, Anna
receives a telephone call from BioStatLab, the referral
laboratory the clinic uses. The results from
Ms. Tina Walker’s urine culture and from
Mr. Robert Livore’s abscess culture are complete.
Anna listens carefully to the technician from the referral
laboratory regarding Mr. Livore’s abscess culture and jots
down “staphylococcus” on the reporting form. She pauses
when the technician stops and asks what species of
“staph.” Why is this important?
The technician also reports Ms. Walker’s test results,
indicating that the organism causing her urinary tract
infection was identified as Escherichia coli. How could E.
coli have infected the urinary tract?
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Typical Pathogenic Bacteria
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Bacteria are single-celled prokaryotic organisms
that reproduce by binary fission.
Often classified by their shape, staining
characteristics, and the environmental
conditions in which they thrive.
Three types of cell wall structures based on
reactions in specialized stains: gram positive,
gram negative, acid fast.
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Bacterial Shapes
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Critical Thinking Application
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Anna knows that impetigo is caused by
Staphylococcus aureus. Without using a
microscope, she knows what the organism
will look like. How does she know?
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Bacterial Oxygen Requirements
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Aerobes – require oxygen to live
Anaerobes – die in the presence of oxygen
Facultative anaerobes – flexible concerning
oxygen requirements and, although they are
anaerobes, can survive in the presence of
oxygen
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Bacterial Physical Structures
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Flagella – thin, long structures that aid in
propulsion
Capsules – thick gelatinous coats surrounding the
cell wall
Endospores – intracellular structures that allow
the cell to remain viable when environmental
conditions are not favorable
Tables 55-3 to 55-5 list some important infectious
diseases caused by typical pathogenic bacteria
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
20
Unusual Pathogenic Bacteria
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Viruses range between 10 and 100 nanometers (nm);
typical pathogenic bacteria are between 1000 and
5000 nm; these are tiny bacteria that fall between the
size range of viruses and typical pathogenic bacteria
Chlamydia – tiny bacteria; require host cells for growth
Mycoplasma – no PG in cell wall but are not obligate
parasites like rickettsia and chlamydia
Rickettsiae – tiny gram-negative bacteria that are
transmitted by blood-sucking arthropods; cannot
multiply outside of a host cell
Table 55-6
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Fungi: Table 55-7
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Mycology – study of fungi and diseases they cause
Fungi are eukaryotes, larger than bacteria
Present in soil, air, and water
Few cause disease
Transmission – direct contact with infected persons,
prolonged exposure to a moist environment,
inhalation of contaminated dust or soil
Treatment – resistant to antibiotics used for
bacterial infections; treated with drugs active
against their unusual cell walls
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
22
Parasites
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Parasitology – study of all parasitic organisms that live on
or in the human body
Transmitted by ingestion during the infective stage, direct
penetration of the skin, inoculation by a vector
Identified in urine, sputum, or tissue fluid or through tissue
biopsy
Helminths – worms; live on or in living organisms and
nourish themselves at the expense of the host organism
Protozoa – single-celled parasitic eukaryotes ranging in
size from microscopic to macroscopic; present in moist
environments and bodies of water; transmitted through
contaminated feces, food, and drink
Table 55-8
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
23
Parasites: Roundworms
and Whipworms
From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
24
The Microbiology Laboratory
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Most laboratories have:
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Refrigerator, autoclave, safety cabinet, microscope,
and incubator
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Inoculating Equipment
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Inoculating loops and needles
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Incineration Equipment
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Fastest way to sterilize reusable equipment
Used to heat-fix smears
Some laboratories use a Bunsen burner
connected to a natural gas supply but most use
an electric incinerator because of the reduced
fire hazard
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Incineration Equipment (cont’d)
From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Culture Media
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All-purpose or nutritive – supports growth of a wide
variety of bacteria; will not support fastidious bacteria
Selective – supports growth of one type of organism
while inhibiting growth of others
Differential – contains chemicals or dyes that alter the
appearance of certain bacterial types; used in
biochemical testing
Enriched – contains complex organic materials that
certain fastidious species must have in order to multiply;
blood agar, needed for the growth of Streptococcus
pyogenes, is made by adding sterile sheep blood to an
all-purpose medium; used to cultivate pathogens
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Culture Media
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Solid, liquid, or semisolid
From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
30
Inoculation of Media
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Quadrant streak – done with a loop to spread sample
thinly over agar medium in several directions;
separates bacteria so they grow in individual colonies
Lawn or spread streak – used when assessing
antibiotic effectiveness or counting colonies; spread
sample with a swab or loop continuously over the
entire plate
Plates are incubated in an inverted position so
condensation that accumulates on underside of lid
does not fall down onto the growth; temperature and
conditions of incubation depend on specimen source
and suspected pathogens
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
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Identification of Pathogens in the
Microbiology Laboratory
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Culture is incubated 18–24 hours; examined for
evidence of pathogens
Suspicious colonies are subcultured onto
appropriate medium to isolate them in pure culture
Throat culture – Streptococcus pyogenes, also known as
group A beta-hemolytic Streptococcus, causes septic sore
throat; grown on sheep blood agar plate; lysis indicates
beta-hemolytic “strep”
 Urine culture – count number of bacteria colonies present
in a sample; each colony that grows on the plate
represents 1000 colony-forming units per milliliter
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Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
32
Beta Hemolysis Around Strep Colonies
From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
33
Critical Thinking Application
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The technician from the referral laboratory
indicated that Ms. Walker had a urinary tract
infection. Anna recorded the results
“>100,000 cfu/ml” on the patient's record.
What does this number mean?
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
34
Differential Staining
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Staining
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Gram’s stain – Gram stain differentiates bacteria
into two categories according to cell wall
thickness; sequence of primary dyes applied to
slide; dyes are taken up differently according to
the chemical composition of the cell walls
• Gram-positive – stain purple
• Gram-negative – stain pink or red
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Acid-fast stain – differentiates bacteria into two
categories based on presence or absence of a
waxy lipid in the cell wall; is used in identification
of Mycobacterium species
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Gram-Positive and Gram-Negative
Cocci
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Gram stain
From De la Maza LM, Pezzlo MT, Baron EJ: Color atlas of diagnostic microbiology, St Louis, 1997, Mosby.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
36
Identification of Pathogens in the
Microbiology Laboratory
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Biochemical testing
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Performed to identify the genus and species
Rapid identification methods
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Designed to give the physician a positive indication
of the problem
Rapid strep testing
Influenza A and B testing
RSV testing
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
37
Rapid Identification Tests
From Stepp CA, Woods MA: Laboratory procedures for medical office personnel, Philadelphia, 1998, Saunders.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
38
Antimicrobial Susceptibility Testing
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“Culture and sensitivity” test – to determine the
appropriate antibiotic
Culture the organisms from a patient sample and
test to determine the organism’s susceptibility to
certain antibiotics
The physician’s decision for antimicrobial agents is
based on numerous factors.
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Test results, physical examination, and the knowledge of
the patient
Asepsis must be strictly observed to ensure safety
and good results; the organism must be isolated in
pure culture before the test
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39
Kirby-Bauer Antimicrobial
Susceptibility Test
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Inoculate sterile water with pure bacterial culture
Spread suspension in lawn pattern on agar medium
Disks containing an antimicrobic agent are placed on agar
After incubation, the zone of inhibition (area of no growth)
around each disk is measured in millimeters and
compared with values provided by the manufacturer of the
disks
Three determinations:
 S – susceptible; antibiotic effective against organism
 R – organism is resistant to antibiotic
 I – intermediate – additional testing must be performed to
determine the dosage of antimicrobial needed for
therapeutic treatment
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
40
Antimicrobial Susceptibility Testing
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
41
Critical Thinking Application
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Anna has recorded the results of
Ms. Walker’s urine culture and notes that
10 antimicrobial agents had been tested, but
the Escherichia coli was susceptible to only
five of them. How will Dr. Ling determine
which of these five antibiotics would be best
for Ms. Walker?
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
42
Pinworm Testing: Procedure 55-7
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Testing for pinworms
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Specimens best collected at night or early before
bowel movement, urination, or bathing.
Petroleum jelly paraffin swabs used to collect eggs.
Parent may also be instructed to bring child to office.
Copyright © 2011, 2007, 2003, 1999 by Saunders, an imprint of Elsevier Inc. All rights reserved.
43
Immunology Testing
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Provides information about past or present infections with
bacteria or viruses and certain cancers
Demonstrates the reaction between antigen and antibody
Amount of antibody at any given time can be measured
with serological testing and is referred to as the titer
Agglutination and precipitation – antibody/antigen
molecules clump together (agglutinate); they precipitate
to the bottom of a test tube
Immunochromatographic assay – when antigen-antibody
reaction occurs a color change is visible
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44
Immunology Testing
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The medical assistant may perform CLIA-waived
tests for infectious mononucleosis, H. pylori, HIV,
and Lyme disease.
Mononucleosis testing – CBC and serologic tests
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CBC shows increased number of lymphocytes that
appear atypical on the differential examination
 Antibodies react with surface antigens of horse
erythrocytes, causing agglutination
 Procedure 55-8
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45
Critical Thinking Application
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Tiffany Warhola, a seventh grade student,
visits Dr. Chowdry complaining of extreme
fatigue and sore throat. Dr. Chowdry orders a
rapid strep test and a mononucleosis test.
What sample will Anna need for the rapid
strep test? What sample will she need for the
mononucleosis test?
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46
Patient Education
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List of teaching topics to help educate patients
An explanation of patient’s type of infection
 How infection spreads
 Normal barriers to infection
 Risk factor
 Patient preparations, collection
 Hand washing, proper storage and cleaning
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47
Reportable Diseases
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Certain infectious diseases must be reported
to the CDC or local board of health.
Each state legislature determines how data
are reported as well as what diseases must
be reported.
National data published weekly by the CDC in
the MMWR – the Morbidity and Mortality
Weekly Report.
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48