Transcript General Medical Microbiology Introduction Specimen Collection  The failure to isolate the causative agent of an infectious disease is frequently the result of faulty.

General Medical Microbiology
Introduction
Specimen Collection
 The failure to isolate the causative agent
of an infectious disease is frequently the
result of faulty collecting or transport
techniques. Therefore, when collecting a
specimen for microbiological examination,
the primary general considerations that
need to be addressed are:
General Considerations
 The specimen obtained must be
representative of the disease process. For
example, a throat swab can’t substitute for
sputum in cases of pneumonia. WHY?
 Sufficient material must be collected to ensure
a complete and accurate exam.
 The specimen must be obtained in a manner
that avoids contamination by the patient’s
own normal flora
General Considerations
 If at all possible, the specimen should be
obtained before initiating antibiotic therapy.
 It is best to obtain the specimen at the acute
phase of the disease when the causative
organism is most likely to be found in large
numbers.
 The specimen should be delivered promptly to
the laboratory. WHY?
General Considerations
 The laboratory should be provided with
sufficient clinical information to guide the
microbiologist in the selection of the suitable
media and incubation conditions. (Some
organisms require special media for growth,
while other organisms require special
incubation conditions).
 The following specimens should normally be
sterile (i.e., have no normal flora):
General Considerations
 Blood
 Cerebral spinal fluid (CSF)
 Tissue
 Serous fluids
 Specimens from the lower respiratory tract
 Urine taken directly from the bladder or kidney
 The following specimens are likely to be
contaminated with normal flora:
 Specimens from the upper respiratory tract,
including the mouth and nose
General Considerations
 Sputum
 Feces
 Genital tract specimens
 Skin
 The following specimens may be
contaminated with normal flora, but the
numbers of contaminating organisms are
likely to be quite low:
 Conjunctiva of the eyes
 External ears
Circumventing Normal Flora
 There are several different ways that one
may use to circumvent problems of
contamination with indigenous normal
flora:
 Antisepsis – antiseptics such as tincture of
iodine should be applied to the skin prior to
aspiration of abscesses and various normally
sterile body fluids such as blood and CSF
Circumventing Normal Flora
 Decontamination – procedures that selectively
inhibit or destroy microorganisms other than
those of specific interest may be used. For
example sputum may be treated with NaOH
prior to culturing for mycobacteria
 Use of selective media – use media that
selectively inhibits growth of normal flora and
allows for growth of pathogens, i.e. MSA
(more on this later)
Circumventing Normal Flora
 Quantification – the classic example of
quantification procedures is the procedure
used for urine specimens. A calibrated loop
that delivers 1 ul of urine (1/1000 of a ml) is
used and if the colony count is > 100
colonies, the result is considered to be
significant and indicative of infection (How
many organisms would this be/ml?)
Urine colony counts
Circumventing Normal Flora
 Microscopy –
 A cytological exam should be done to look for the
presence of squamous epithelial cells in urine,
sputum, or wound specimens which, when
present, indicate likely contamination with skin or
mucosal flora.
 A new specimen should be requested when
numerous squamous epithelial cells are present in
the specimen.
Inappropriate sputum specimen
Good sputum specimen
Circumventing Normal Flora
 Invasive procedures that allow the physician
to avoid normal flora in collecting the
specimen including:
 Transtrachael aspirate
 Suprapubic aspirate
 Bronchoscopy
 Needle biopsies
Transtrachael aspirate
Suprapubic aspirate
General instructions for
collection of specimens
 General instructions for specimen collection:
 Identification – the specimen should be labeled with
the following:
Patient’s name and identification number
Patient’s location
Patient’s physician
Site/source of specimen
Type of exam requested (bacterial, fungal, viral, parasites)
Tentative diagnosis (Why is this important?)
Date and time of specimen collection (Why is this
important?)
 If antibiotics have been administered the type, dosage and
time given should be provided
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General instructions for
collection of specimens
 Transport containers, devices, media:
 Swabs are convenient and economical, but:
 They are often inadequate in terms of the amount of
specimen required for multiple types of culturing.
 The recovery of bacteria is usually< 10% of the original
inoculum.
 Swabs are often used for throat cultures and for cervical,
vaginal and urethral secretions.
 They should not be used when pus or exudate is
available, for surgical specimens, or for cultures for
anaerobes or mycobacteria.
General instructions for
collection of specimens
 Syringes – these are good for aspirates. The tip of the
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needles should be plugged with a sterile stopper or the
needle should be removed and the syringe capped.
Tubes, bottles, and jars should be sterile and leak proof.
Fecal transport systems – polyvinyl alcohol fixative should be
used for preservation of fecal parasites
For sexually transmitted diseases it is best to inoculate media
directly at the bedside of the patient or to use a
swab/transport media system specifically designed for
optimal recovery of sexually transmitted pathogens.
For specimens for viral examination or for anaerobic
culturing, appropriate transport media should be used.
General instructions for
collection of specimens
 The specimen must be promptly transported to the
laboratory to preserve the viability of fastidious
organisms and to prevent overgrowth of fastidious
organisms by more rapidly growing bacteria which
may be insignificant.
 Sometimes refrigeration of the specimen is warranted (i.e.
urine specimens),
 Sometimes refrigeration will kill fastidious organisms (i. e.
Streptococcus pneumoniae or Neisseria gonorrhoeae from
sputum or genital tract specimens, respectively)
Specific guidelines
 Specific guidelines for specimen collection:
 Septicemia means that organisms, or their toxins are
present, and growing in the blood.
 Bacteremia simply means the presence of organisms
in the blood without causing infection (Can you think
of examples where you might find this?)
 For septicemia, 2-3 cultures should be collected by
venipuncture in a 24 hour period
 20-30 mls should be collected for each sample.
 The samples should be inoculated into culture media directly
at the bedside of the patient.
Venipuncture
Specific guidelines
 Infections of other normally sterile body fluids
 Meningitis and encephalitis – collect CSF via a lumber
puncture
 Pleural, pericardial and synovial fluids – aspirate and collect
sufficient quantities for a thorough exam.
 Wounds
 The best specimens are aspirates of pus or exudate.
 A swab is usually not a good way to collect specimens from
wounds.
Lumbar puncture
Specific guidelines
 Upper respiratory tract infections – a swab is
sufficient for collecting URT specimens
 Lower respiratory tract infections – collect
sputum. Alternatively, may use:
 Transtrachael aspiration,
 Bronchial wash, or
 Lung puncture for collecting the specimen.
 Urinary tract infection
 Use a clean voided midstream specimen
Clean voided midstream
Specific guidelines
 Use catherization
 Use suprapubic aspiration of bladder or kidney
 Gastroenteritis – collect a stool sample in a sterile
container.
 For intestinal parasites, three separate specimens should be
collected because some organisms are only present
intermittently.
 Genital tract infections – a swab or aspirate of
exudate plus direct inoculation on media is best
 Ocular infections – a swab is sufficient
 Tissue specimens – that should be obvious!
Direct exam
 Direct examination of specimens
 Gross examination of the specimen may yield
important information:
 If CSF is cloudy this probably indicates infection
 Sputum – the color, consistency and odor may give clues as
to the causative agent of infection
 If the stool contains mucous and blood, this is typical of
dysentery
 If the odor is foul, the specimen may contain anaerobes
 Visible granules (which are aggregates of actinomycetes) are
found in actinomycete infections
Initial microscopic examination
 Initial microscopic examination
 Differential stains
 Gram stain –
 Allows one to determine if organisms are Gram positive or
Gram negative (Why would this be important for a physician?)
 To determine the shape of bacteria it is usually necessary to
use oil. On low power it is possible to see fungi, some parasites
and WBCs. (The hallmark of an acute bacterial infection is
numerous PMNs).
 Positive and negative controls should always be done (Why?)
 A Gram stain of a direct smear can provide important
information for some specimens, but is useless for others (Give
examples of each)
 It is important to not over interpret Gram stain results
Gram stain of Bacillus species
Gram stain of Staphylococcus aureus
Gram stain of Neisseria species
Gram stain of Haemophilus species
Initial microscopic examination
 Acid fast stain –
 Divides organisms into acid-fast and non acid-fast
organisms
 Acid fast staining results are clinically very important for
diagnosing tuberculosis. Mycobacteria tuberculosis
grows so slowly that it may be 6-8 weeks before a
culture report is signed out. It is important that the
physician know if acid-fast bacilli are seen in the direct
smear so that appropriate antimicrobic therapy can be
initiated as soon as possible.
Acid fast stain
Initial microscopic examination
 Special stains
 Spore stain The position of the spore may be
diagnostically important
Initial microscopic examination
 Capsule stain – usually the background and the
organism are stained while the capsule is left
unstained.
Initial microscopic examination
 Trichrome stain – for permanent stained smears of
intestinal parasites:
Initial microscopic examination
 Iron-hematoxylin stain – another way to make
make permanent stained smears of intestinal
parasites
Initial microscopic examination
 Wrights and Giemsa stains of blood – parasites
and bacteria in the blood may be seen
Initial microscopic examination
 Wet mounts
 India ink mount for Cryptococcus neoformans
Initial microscopic examination
 Lactophenol cotton blue – to observe fungi
Initial microscopic examination
 10% KOH – to observe fungi from skin scrapings.
The KOH destroys the epithelial cells without
harming the fungal elements.
Initial microscopic examination
 Iodine stain – used for stool examination for
parasites