CLINICAL MICROBIOLOGY
COLLECTION:
Overall, the results obtained in the clinical laboratory are only as
good as the quality of the specimen collected for analysis. Specimens
may be collected by several methods using aseptic technique.
Aseptic technique refers to specific procedures used to prevent
unwanted microorganisms from contaminating the clinical
specimen. Each method is designed to ensure that only the proper
material will be sent to the clinical laboratory.
The most common method used to collect specimens from the
anterior nares or throat is the sterile swab. A sterile swab is a
rayon-, calcium alginate, or dacron-tipped polystyrene applicator.
Manufacturers of swabs have their own unique container design
and instructions for proper use. For example, many commercially
manufactured swabs contain a transport medium designed to preserve
a variety of microorganisms and to prevent multiplication of
rapidly growing members of the population . However,
with the exception of the nares or throat, the use of swabs for
the collection of specimens is of little value and should be discouraged
for two major reasons: swabs are associated with a
greater risk of contamination with surface and subsurface microorganisms,
and they have a limited volume capacity (0.1 ml).
Needle aspiration is used to collect specimens aseptically
(e.g., anaerobic bacteria) from cerebrospinal fluid, pus, and blood.
For both samples stringent antiseptic techniques are used to avoid
skin contamination. To prevent blood from clotting and entrapping
microorganisms, various anticoagulants (e.g., heparin, sodium citrate)
are included within the specimen bottle or tube.
Intubation [Latin in, into, and tuba, tube] is the inserting of
a tube into a body canal or hollow organ. For example, intubation
can be used to collect specimens from the stomach. In this procedure
a long sterile tube is attached to a syringe, and the tube is either
swallowed by the patient or passed through a nostril
into the patient’s stomach. Specimens are then withdrawn
periodically into the sterile syringe. The most common intubation tube is the Levin tube.
A catheter is a tubular instrument used for withdrawing or introducing
fluids from or into a body cavity. For example, urine specimens
may be collected with catheters to detect urinary tract infections
caused by bacteria and from newborns and neonates who
cannot give a voluntary urinary specimen. Three types are commonly
used for urine. The hard catheter is used when the urethra is
very narrow or has strictures. The French catheter is a soft tube used
to obtain a single specimen sample. The most common method used for the collection of urine is
the clean-catch method. After the patient has cleansed the urethral meatus (opening), a small container is used to collect the urine. The
optimal time to use the clean-catch method is early morning because
the urine contains more microorganisms as a result of being
in the bladder overnight. In the clean-catch midstream method, the
first urine voided is not collected because it will be contaminated
with those transient microorganisms normally occurring in the
lower portion of the urethra. Only the midstream portion is collected
since it most likely will contain those microorganisms found
in the urinary bladder. If warranted for some patients, needle aspirations
also are done directly into the urinary bladder.
Sputum is the most common specimen collected in suspected
cases of lower respiratory tract infections. Specifically, sputum
is the mucous secretion expectorated from the lungs, bronchi, and
trachea through the mouth, in contrast to saliva, which is the secretion
of the salivary glands. Sputum is collected in specially designed sputum cups.
Handling: Immediately after collection the specimen must be properly labeled
and handled. The person collecting the specimen is responsible
for ensuring that the name, hospital, registration number, location in the hospital, diagnosis, current antimicrobial therapy,
name of attending physician, admission date, and type of specimen
are correctly and legibly written or imprinted on the culture
request form. This information must correspond to that written or
imprinted on a label affixed to the specimen container. The type
or source of the sample and the choice of tests to be performed
also must be specified on the request form.
Transport:
Speed in transporting the specimen to the clinical laboratory after
it has been obtained from the patient is of prime importance.
Some laboratories refuse to accept specimens if they have been in
transit too long.
Microbiological specimens may be transported to the laboratory
by various means. For example, certain specimens
should be transported in a medium that preserves the microorganisms
and helps maintain the ratio of one organism to another. This is
especially important for specimens in which normal microorganisms
may be mixed with microorganisms foreign to the body location.
Special treatment is required for specimens when the microorganism
is thought to be anaerobic. The material is aspirated with a needle and syringe. Most of the time it is practical to remove
the needle, cap the syringe with its original seal, and bring
the specimen directly to the clinical laboratory. Transport of these
specimens should take no more than 10 minutes; otherwise, the
specimen must be injected immediately into an anaerobic transport
vial . Vials should contain a transport medium
with an indicator, such as resazurin, to show that the interior of
the vial is anaerobic at the time the specimen is introduced. Swabs
for anaerobic culture usually are less satisfactory than aspirates or
tissues, even if they are transported in an anaerobic vial.
Many clinical laboratories insist that stool specimens (the fecal
discharge from the bowels) for culture be transported in various
buffered preservatives. Preparation of these transport media
is described in various manuals (see Additional Reading).
Transport of urine specimens to the clinical laboratory must
be done as soon as possible. No more than 1 hour should elapse
between the time the specimen is obtained and the time it is examined.
If this time schedule cannot be followed, the urine sample
must be refrigerated immediately.
Cerebrospinal fluid (CSF) from patients suspected of having
meningitis should be examined immediately by skilled personnel
in the clinical microbiology laboratory. CSF is obtained by lumbar
puncture under conditions of strict asepsis, and the sample is
transported to the laboratory within 15 minutes. Specimens for
the isolation of viruses are iced before transport, and can be kept
at 4°C for up to 72 hours; if the sample will be stored longer than
72 hours, it should be frozen at 72°C.
Identification of Microorganisms From Specimens:
The clinical microbiology laboratory can provide preliminary or
definitive identification of microorganisms based on (1) microscopic
examination of specimens, (2) study of the growth and
biochemical characteristics of isolated microorganisms (pure cultures),
(3) immunologic tests that detect antibodies or microbial
antigens, (4) bacteriophage typing (restricted to research settings
and the CDC), and (5) molecular methods.
Microscopy: Wet-mount, heat-fixed, or chemically fixed specimens can be examined
with an ordinary bright-field microscope. These preparations
can be enhanced with either phase-contrast or dark-field microscopy.
The latter is the procedure of choice for the detection of
spirochetes in skin lesions associated with early syphilis or in
blood specimens of people with early leptospirosis. The fluorescence
microscope can be used to identify certain acid-fast microorganisms
(Mycobacterium tuberculosis) after they are
stained with fluorochromes such as auramine-rhodamine.
Growth and Biochemical Characteristics:
Typically microorganisms have been identified by their particular
growth patterns and biochemical characteristics. These characteristics
vary depending on whether the clinical microbiologist is
dealing with viruses, rickettsias, chlamydiae, mycoplasmas, grampositive
or gram-negative bacteria, fungi (yeasts, molds), or parasites
(protozoa, helminths).
Viruses:
Viruses are identified by isolation in conventional cell (tissue) culture,
by immuno diagnosis (fluorescent antibody, enzyme immuno-assay radioimmunoassay, latex agglutination, and immuno peroxidase)
tests, and by molecular detection methods such as nucleic acid probes
and amplification assays. Several types of systems are available for
virus cultivation: cell cultures, embryonated hen’s eggs, and experimental animals.
Cell cultures are divided into three general classes:
1) Primary cultures consist of cells derived directly from
tissues such as monkey kidney and mink lung cells that
have undergone one or two passages since harvesting.
2) Semi-continuous cell cultures or low-passage cell lines are
obtained from subcultures of a primary culture and usually
consist of diploid fibroblasts that undergo a finite number
of divisions.
3) Continuous cell cultures, such as HEp-2 cells, are derived
from transformed cells that are generally epithelial in
origin. These cultures grow rapidly, are heteroploid (having
a chromosome number that is not a simple multiple of the
haploid number), and can be subcultured indefinitely.
Each type of cell culture favors the growth of a different array of
viruses, just as bacterial culture media have differing selective
and restrictive properties for growth of bacteria.
Viral replication in cell cultures is detected in two ways:
(1) by observing the presence or absence of cytopathic effects
(CPEs), and (2) by hemadsorption.
A cytopathic effect is an observable morphological change
that occurs in cells because of viral replication .
Examples include ballooning, binding together, clustering, or
even death of the culture cells. During the incubation
period of a cell culture, red blood cells can be added. Several
viruses alter the plasma membrane of infected culture cells
so that red blood cells adhere firmly to them. This phenomenon is called hemadsorption.
Embryonated hen’s eggs can be used for virus isolation. There
are three main routes of egg inoculation for virus isolation: (1) the
allantoic cavity, (2) the amniotic cavity, and (3) the chorioallantoic
membrane . Virus replication is recognized by the
development of pocks on the chorioallantoic membrane, by the
development of hemagglutinins in the allantoic
and amniotic fluid, and by death of the embryo.
Laboratory animals, especially suckling mice, are used for
virus isolation. Inoculated animals are observed for specific signs
of disease or death.
Several new serological tests for viral identification make
use of monoclonal antibody-based immuno fluorescence. These
tests detect viruses such as the cytomegalovirus and
herpes simplex virus in tissue-vial cultures.
Cited By Kamal Singh Khadka
MSc Microbiology, TU.
Fig: Needle Aspiration
Overall, the results obtained in the clinical laboratory are only as
good as the quality of the specimen collected for analysis. Specimens
may be collected by several methods using aseptic technique.
Aseptic technique refers to specific procedures used to prevent
unwanted microorganisms from contaminating the clinical
specimen. Each method is designed to ensure that only the proper
material will be sent to the clinical laboratory.
The most common method used to collect specimens from the
anterior nares or throat is the sterile swab. A sterile swab is a
rayon-, calcium alginate, or dacron-tipped polystyrene applicator.
Manufacturers of swabs have their own unique container design
and instructions for proper use. For example, many commercially
manufactured swabs contain a transport medium designed to preserve
a variety of microorganisms and to prevent multiplication of
rapidly growing members of the population . However,
with the exception of the nares or throat, the use of swabs for
the collection of specimens is of little value and should be discouraged
for two major reasons: swabs are associated with a
greater risk of contamination with surface and subsurface microorganisms,
and they have a limited volume capacity (0.1 ml).
Needle aspiration is used to collect specimens aseptically
(e.g., anaerobic bacteria) from cerebrospinal fluid, pus, and blood.
For both samples stringent antiseptic techniques are used to avoid
skin contamination. To prevent blood from clotting and entrapping
microorganisms, various anticoagulants (e.g., heparin, sodium citrate)
are included within the specimen bottle or tube.
Intubation [Latin in, into, and tuba, tube] is the inserting of
a tube into a body canal or hollow organ. For example, intubation
can be used to collect specimens from the stomach. In this procedure
a long sterile tube is attached to a syringe, and the tube is either
swallowed by the patient or passed through a nostril
into the patient’s stomach. Specimens are then withdrawn
periodically into the sterile syringe. The most common intubation tube is the Levin tube.
A catheter is a tubular instrument used for withdrawing or introducing
fluids from or into a body cavity. For example, urine specimens
may be collected with catheters to detect urinary tract infections
caused by bacteria and from newborns and neonates who
cannot give a voluntary urinary specimen. Three types are commonly
used for urine. The hard catheter is used when the urethra is
very narrow or has strictures. The French catheter is a soft tube used
to obtain a single specimen sample. The most common method used for the collection of urine is
the clean-catch method. After the patient has cleansed the urethral meatus (opening), a small container is used to collect the urine. The
optimal time to use the clean-catch method is early morning because
the urine contains more microorganisms as a result of being
in the bladder overnight. In the clean-catch midstream method, the
first urine voided is not collected because it will be contaminated
with those transient microorganisms normally occurring in the
lower portion of the urethra. Only the midstream portion is collected
since it most likely will contain those microorganisms found
in the urinary bladder. If warranted for some patients, needle aspirations
also are done directly into the urinary bladder.
Sputum is the most common specimen collected in suspected
cases of lower respiratory tract infections. Specifically, sputum
is the mucous secretion expectorated from the lungs, bronchi, and
trachea through the mouth, in contrast to saliva, which is the secretion
of the salivary glands. Sputum is collected in specially designed sputum cups.
Handling: Immediately after collection the specimen must be properly labeled
and handled. The person collecting the specimen is responsible
for ensuring that the name, hospital, registration number, location in the hospital, diagnosis, current antimicrobial therapy,
name of attending physician, admission date, and type of specimen
are correctly and legibly written or imprinted on the culture
request form. This information must correspond to that written or
imprinted on a label affixed to the specimen container. The type
or source of the sample and the choice of tests to be performed
also must be specified on the request form.
Transport:
Speed in transporting the specimen to the clinical laboratory after
it has been obtained from the patient is of prime importance.
Some laboratories refuse to accept specimens if they have been in
transit too long.
Microbiological specimens may be transported to the laboratory
by various means. For example, certain specimens
should be transported in a medium that preserves the microorganisms
and helps maintain the ratio of one organism to another. This is
especially important for specimens in which normal microorganisms
may be mixed with microorganisms foreign to the body location.
Special treatment is required for specimens when the microorganism
is thought to be anaerobic. The material is aspirated with a needle and syringe. Most of the time it is practical to remove
the needle, cap the syringe with its original seal, and bring
the specimen directly to the clinical laboratory. Transport of these
specimens should take no more than 10 minutes; otherwise, the
specimen must be injected immediately into an anaerobic transport
vial . Vials should contain a transport medium
with an indicator, such as resazurin, to show that the interior of
the vial is anaerobic at the time the specimen is introduced. Swabs
for anaerobic culture usually are less satisfactory than aspirates or
tissues, even if they are transported in an anaerobic vial.
Many clinical laboratories insist that stool specimens (the fecal
discharge from the bowels) for culture be transported in various
buffered preservatives. Preparation of these transport media
is described in various manuals (see Additional Reading).
Transport of urine specimens to the clinical laboratory must
be done as soon as possible. No more than 1 hour should elapse
between the time the specimen is obtained and the time it is examined.
If this time schedule cannot be followed, the urine sample
must be refrigerated immediately.
Cerebrospinal fluid (CSF) from patients suspected of having
meningitis should be examined immediately by skilled personnel
in the clinical microbiology laboratory. CSF is obtained by lumbar
puncture under conditions of strict asepsis, and the sample is
transported to the laboratory within 15 minutes. Specimens for
the isolation of viruses are iced before transport, and can be kept
at 4°C for up to 72 hours; if the sample will be stored longer than
72 hours, it should be frozen at 72°C.
Identification of Microorganisms From Specimens:
The clinical microbiology laboratory can provide preliminary or
definitive identification of microorganisms based on (1) microscopic
examination of specimens, (2) study of the growth and
biochemical characteristics of isolated microorganisms (pure cultures),
(3) immunologic tests that detect antibodies or microbial
antigens, (4) bacteriophage typing (restricted to research settings
and the CDC), and (5) molecular methods.
Microscopy: Wet-mount, heat-fixed, or chemically fixed specimens can be examined
with an ordinary bright-field microscope. These preparations
can be enhanced with either phase-contrast or dark-field microscopy.
The latter is the procedure of choice for the detection of
spirochetes in skin lesions associated with early syphilis or in
blood specimens of people with early leptospirosis. The fluorescence
microscope can be used to identify certain acid-fast microorganisms
(Mycobacterium tuberculosis) after they are
stained with fluorochromes such as auramine-rhodamine.
Growth and Biochemical Characteristics:
Typically microorganisms have been identified by their particular
growth patterns and biochemical characteristics. These characteristics
vary depending on whether the clinical microbiologist is
dealing with viruses, rickettsias, chlamydiae, mycoplasmas, grampositive
or gram-negative bacteria, fungi (yeasts, molds), or parasites
(protozoa, helminths).
Viruses:
Viruses are identified by isolation in conventional cell (tissue) culture,
by immuno diagnosis (fluorescent antibody, enzyme immuno-assay radioimmunoassay, latex agglutination, and immuno peroxidase)
tests, and by molecular detection methods such as nucleic acid probes
and amplification assays. Several types of systems are available for
virus cultivation: cell cultures, embryonated hen’s eggs, and experimental animals.
Cell cultures are divided into three general classes:
1) Primary cultures consist of cells derived directly from
tissues such as monkey kidney and mink lung cells that
have undergone one or two passages since harvesting.
2) Semi-continuous cell cultures or low-passage cell lines are
obtained from subcultures of a primary culture and usually
consist of diploid fibroblasts that undergo a finite number
of divisions.
3) Continuous cell cultures, such as HEp-2 cells, are derived
from transformed cells that are generally epithelial in
origin. These cultures grow rapidly, are heteroploid (having
a chromosome number that is not a simple multiple of the
haploid number), and can be subcultured indefinitely.
Each type of cell culture favors the growth of a different array of
viruses, just as bacterial culture media have differing selective
and restrictive properties for growth of bacteria.
Viral replication in cell cultures is detected in two ways:
(1) by observing the presence or absence of cytopathic effects
(CPEs), and (2) by hemadsorption.
A cytopathic effect is an observable morphological change
that occurs in cells because of viral replication .
Examples include ballooning, binding together, clustering, or
even death of the culture cells. During the incubation
period of a cell culture, red blood cells can be added. Several
viruses alter the plasma membrane of infected culture cells
so that red blood cells adhere firmly to them. This phenomenon is called hemadsorption.
Embryonated hen’s eggs can be used for virus isolation. There
are three main routes of egg inoculation for virus isolation: (1) the
allantoic cavity, (2) the amniotic cavity, and (3) the chorioallantoic
membrane . Virus replication is recognized by the
development of pocks on the chorioallantoic membrane, by the
development of hemagglutinins in the allantoic
and amniotic fluid, and by death of the embryo.
Laboratory animals, especially suckling mice, are used for
virus isolation. Inoculated animals are observed for specific signs
of disease or death.
Several new serological tests for viral identification make
use of monoclonal antibody-based immuno fluorescence. These
tests detect viruses such as the cytomegalovirus and
herpes simplex virus in tissue-vial cultures.
Cited By Kamal Singh Khadka
MSc Microbiology, TU.
Fig: Needle Aspiration
Fig: Catheter
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