MICROBIOLOGY & PARASITOLOGY
Chapter 13
Diagnosing Infectious Diseases
Introduction
The proper diagnosis of an infectious disease requires:
1. Taking a complete patient history
2. Conducting a thorough physical examination of
the patient
3. Carefully evaluating the patient’s signs and
symptoms
4. Implementing the proper selection, collection,
transport, and processing of appropriate clinical
specimens
Clinical Specimens
 Specimens collected from patients such as blood,
urine, feces, and cerebrospinal fluid (CSF), are
known as clinical specimens
 Specimen commonly submitted to the hospital’s
Clinical Microbiology Laboratory (CML) include:
blood, bone marrow, bronchial washings, sputum,
CSF, cervical and vaginal swabs, feces, hair and nail
clippings, pus, skin scrapings, synovial fluid, throat
swabs, tissue specimens, urethral discharge
material, urine, and urogenital secretions
 All specimens should be of the highest possible
quality
 The Role of Healthcare Professionals in the
Submission of Clinical Specimens:
o There should be a close working relationship
among the members of the healthcare team to
ensure a proper diagnosis of infectious diseases
o Healthcare professionals who collect and
transport specimens should exercise extreme
caution during the collection and transport of
specimens
o In the laboratory, all specimens are handled
carefully, exercising Standard Precautions
o Although laboratory professionals do not
themselves make diagnosis, they make
laboratory observations and generate test
results that assist clinicians to correctly diagnose
infectious diseases and initiate appropriate
therapy.
Importance of High-Quality Clinical Specimens
 High-quality clinical specimens are required to
achieve accurate, clinically relevant laboratory
results.
 The 3 components of specimen quality are:
Proper specimen selection
Proper specimen collection
Proper transport of the specimen to the
laboratory
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 The laboratory must provide written guidelines
(“Laboratory Policies and procedures Manual”)
 The person who collects the specimen is ultimately
responsible for its quality.
Proper Selection, Collection, and Transport of Clinical
Specimens
• Specimens must be properly selected.
• Specimens must be collected properly.
• Material (i.e., specimens) should be collected
from a site where the suspected pathogen is most
likely to be found.
• Specimens should be obtained before
antimicrobial therapy, if possible.
• The acute stage of the disease is the most
appropriate time to collect a specimen.
• Specimen collection should be performed with
care and tact to avoid harming the patient.
• A sufficient quantity of the specimen must be
obtained to provide enough material for all required
diagnostic tests.
• All specimens should be placed or collected into
a sterile container to prevent contamination.
• Specimens should be protected from heat and
cold and promptly delivered to the laboratory.
• Hazardous specimens must be handled with
even greater care to avoid contamination of
couriers, patients, and healthcare professionals.
• Whenever possible, a sterile, disposable
specimen container should be used.
• The specimen container must be properly
labeled and accompanied by an appropriate request
slip with adequate instructions.
• Specimens should be collected and delivered to
the lab as early in the day as possible to allow
sufficient processing time.
Types of Clinical Specimens Usually Required to
Diagnose Infectious Diseases
• Blood
– Usually sterile
– The presence of bacteria in the
bloodstream is known as bacteremia.
– Septicemia is a serious disease involving
chills, fever, prostration, and the presence of
bacteria or their toxins in the bloodstream.
– To prevent contamination of a blood
specimen with indigenous skin flora, extreme
care must be taken to use aseptic technique.
• Urine
– Normally sterile in the bladder;
becomes contaminated by indigenous microflora
of the distal urethra during voiding.
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– Contamination is reduced by collecting
a clean-catch, midstream urine.
– Urine culture involves 3 parts:
• A colony count (using a
calibrated loop)
• Isolation and identification of
the pathogen
• Antimicrobial susceptibility
testing
Urine Colony Count
• The colony count is a way of estimating the
number of viable bacteria that are present in a
urine specimen.
• A calibrated loop, either 0.01 mL or 0.001 mL, is
used to inoculate the entire surface of a blood
agar plate.
• After incubation at 37oC overnight, the colonies
are counted and the number is multiplied by the
dilution factor (either 100 for the 0.01 mL loop,
or 1000 for the 0.001 mL loop) to determine the
number of colony-forming units (CFUs).
– # Colonies x dilution factor = # CFUs/mL
• Cerebrospinal Fluid (CSF)
– Meningitis is inflammation or infection
of the membranes (meninges) that surround the
brain and spinal column.
– Encephalitis is inflammation or infection
of the brain.
– Meningoencephalitis is inflammation or
infection of both the brain and meninges.
– CSF is collected by lumbar puncture into
a sterile tube; this is a surgically aseptic
procedure performed by a physician.
– CSF is considered a STAT (emergency)
specimen in the lab!
• Sputum
– Sputum is pus that accumulates deep
within the lungs of a patient with pneumonia,
tuberculosis, or other lower respiratory
infection.
– Often, specimens labeled “sputum” are
actually just saliva; saliva specimens don’t
provide clinically relevant information.
– If TB is suspected, extreme care should
be taken!
– Better specimens can be obtain by
bronchial aspiration or transtracheal aspiration.
• Throat Swabs
– Routine throat swabs are used to
determine whether a patient has strep throat
– Specific cultures may be necessary
when Neisseria gonorrhoeae or
Corynebacterium diphtheriae are suspected
• Wound Specimens
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– Whenever possible, a wound specimen
should be an aspirate (i.e., pus collected by
needle and syringe), rather than a swab
– Specimens collected by swab are
frequently contaminated with indigenous
microflora
• GC Cultures (for Neisseria gonorrhoeae)
– N. gonorrhoeae is a fastidious,
microaerophilic, and capnophilic bacterium.
– Only Dacron, calcium alginate, or
nontoxic cotton swabs should be used to collect
GC specimens.
– Specimens (e.g., vaginal, cervical,
urethral, throat, and rectal swabs) are cultured
on special medium (e.g., Thayer-Martin medium)
and incubated in a CO2 incubator.
– Special transport media are available
and GC swabs should never be refrigerated.
• Fecal Specimens
– Ideally, fecal (stool) specimens should
be collected at the laboratory and processed
immediately to prevent a decrease in
temperature, which would allow the pH to drop
and cause the death of many Shigella and
Salmonella species.
– Bacteria in fecal flora are obligate-,
aerotolerant-, and facultative anaerobes.
– A combination of direct microscopic
examination, culture, biochemical tests, and
immunologic tests may be performed to identify
Gram-negative and Gram-positive bacteria,
fungi, intestinal protozoa, and intestinal
helminths isolated from fecal specimens.
The Pathology Department (“The Lab”)
• Clinical specimens are submitted to the Clinical
Microbiology Laboratory (CML), which is a part of
the Pathology Department.
• The Pathology Department (often referred to as
“the Lab”) is under the direction of a pathologist (a
physician who has specialized training in pathology).
• The pathology department is divided into 2
major divisions:
– Anatomical Pathology
– Clinical Pathology
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The CML may be under the direction of a

pathologist, a microbiologist, or a senior clinical
laboratory scientist.
• Responsibilities
– Primary mission of the CML is to assist
clinicians in the diagnosis and treatment of
infectious diseases.
– The 4 major day-to-day responsibilities
are to:
• Process various clinical
specimens that are submitted to the CML
• Isolate pathogens from those
specimens
• Identify (speciate) the
pathogens
• Perform antimicrobial
susceptibility testing, when appropriate to do
so

Isolation and Identification (Speciation) of Pathogens

• Anatomical Pathology
• Bacteriology Section
– Diseased organs, stained tissue
– Bacterial pathogens are isolated from
sections, and cytology specimens are examined
specimens, tests are performed to identify the
here.
bacterial pathogens, and antimicrobial
– Cytogenetic technologists,
susceptibility testing is performed whenever
cytotechnologists, histologic technicians,
appropriate to do so.
histotechnologists, and pathologist’s assistants
– CML professionals are very much like
are employed in this division.
detectives and crime scene investigators, in that
– In addition, autopsies are performed in
they gather clues about a pathogen until they
the morgue and some Pathology Departments
are able to identify it.
have an Electron Microscopy Laboratory.
– Numerous phenotypic characteristics
• Clinical Pathology
are used to identify the bacteria (e.g., Gram
– Consists of several laboratories in
reaction, cell shape, motility, presence and
addition to the Clinical Microbiology Laboratory:
location of spores, presence or absence of
Clinical Chemistry, Urinalysis,
various enzymes, etc.)
Hematology/Coagulation, Blood Bank, and
• Mycology Section
Immunology.
– Responsibility is to assist clinicians in
– Personnel include pathologists,
the diagnosis of fungal infections (mycoses)
chemists and microbiologists, clinical laboratory
– The specimens processed here are the
scientists (also known as medical technologists -
same as those that are processed in the
MTs), and clinical laboratory technicians (also
Bacteriology Section, with the addition of hair
known as medical laboratory technicians -
and nail clippings and skin scrapings.
MLTs).
– A variety of procedures are used to
The Clinical Microbiology Laboratory identify fungal pathogens including special
media, KOH preps, tease mounts, biochemical
tests (for yeasts), and a combination of
microscopic and macroscopic observations (for
moulds).
• Parasitology Section
– Assists clinicians in the diagnosis of
parasitic diseases
– Parasites are identified by observing
and recognizing various parasite life cycle stages
(e.g., trophozoites, cysts, microfilariae, eggs,

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larvae, adult worms) in specimens – identified

primarily by their physical appearance (e.g., size,
shape, internal details)
• Virology Section
– Assists clinicians in the diagnosis of viral
diseases
– Techniques used in the identification of
viruses include immuno-diagnostic tests,
cytologic or histologic examination, electron
microscopy, molecular techniques, virus
isolation by cell cultures, and cytopathic effect
(CPE) CML professionals are very much like detectives and
• Mycobacteriology Section (also called the TB crime scene investigators -- gathering clues about a
Lab) pathogen until they have enough information to
– Assists clinicians in the diagnosis of identify it.
tuberculosis (TB) Minisystems Used to Identify Bacteria
– Various types of specimens are
submitted, but sputum is the most common type
– Mycobacterium spp. are identified by
the acid-fast staining procedure and by using a
combination of growth characteristics (e.g.,
growth rate, colony pigmentation,
photoreactivity, and morphology) and a variety
of biochemical tests

Proper Method of Preparing the Venipuncture Site

when Obtaining Blood for Culture

API-20E for identification of Enterobacteriaceae

Obtaining a Urine Colony Count

Technique of Lumbar Puncture

Enterotube II for identification of Enterobacteriaceae

Diagram Illustrating the 3 Types of Hemolysis that can

be Observed on a Blood Agar Plate

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 MICROBIOLOGY & PARASITOLOGY

A Colony (Mycelium) of the Mould Aspergillus

fumigatus, a Common Cause of Pulmonary Infections
in Immunosuppressed Patients

Colonies (Mycelia) of a Penicillium Species

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