Sample collection, transport

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Sample collection, transport processingpg 76
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Importance of good samples

• Think like a microbe. Be aware that the microbes
  are everywhere. Always use aseptic technique as
  much as is possible and practical.
• Good microbiology cannot be performed on a bad
  specimen. A poor specimen can result in recovery
  of contaminants, failure to recover causative
  microorganisms, and or inaccurate diagnosis and
  treatment.

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A few ways of avoiding contamination

• Surface cleansing with alcohol and iodine for
  deep abscess fluid or blood cultures
• Rinsing oral cavity with water before collecting
  sputum
• Touching only inflamed area of the throat
  (usually tonsillar fossae) and avoiding gums,
  tongue, lips, etc.
• Cleansing the external genitalia before having
  the patient void the urine for a midstream
  collection for urine culture
• Use your head think like a microbe and it will
  be common sense

4
Importance of good samples

• Example
• Klebsiella pneumoniae causes acute bacterial
  pneumonia BUT it also colonizes the oral cavity
  of many hospitalized patients after a day or 2,
  so
• Collecting saliva instead of sputum can lead to a
  misdiagnosis of K. pneumoniae pneumonia.

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Importance of good samples

• Specimens must be from the actual infection site
• Sputum (deep cough) versus saliva (superficial)
• Drainage from deeper sites versus scabs on top
  of a skin lesion
• Aspiration of pus after skin decontamination is
  ideal

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Timing of sample collection

• Collect the specimen prior to administration of
  antimicrobial therapy
• Acute symptomatic phase is the most productive
• Example
• S. typhi (typhoid fever) can be cultured from
  blood during the first week of infection
• S. typhi can be cultured from feces and urine in
  second and third weeks
• S. typhi usually cannot be cultured after the
  acute symptomatic phase
• Serology is method of choice for diagnosing
  typhoid after first three weeks of infection

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General guidelines

• The number of specimen should be appropriate
• More than one routine throat, sputum, feces,
  urine, or wound culture within a 24h period is
  unnecessary
• However, a single blood culture is not adequate

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General guidelines

• The number of specimens should be appropriate
• A single blood culture is never adequate. Two to
  three blood cultures within 24hour period are
  needed per septic episode because
• multiple methods (routine diagnostic, serology,
  etc.) may be used making more sample necessary
• bacteremia is sometimes transient growth may
  show up in one sample and not in another
• unless multiple samples are collected, it is
  difficult to determine if the isolate is a
  contaminant

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General guidelines

• Sufficient quantity of specimen is required
• The single most important criterion that
  correlates with positive blood cultures is the
  volume of blood drawn
• Swabs, due to the small volume collected and the
  tendency to dry out, are generally inferior to
  collecting liquid specimens in a needle and
  syringe

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General guidelines

• Prompt processing is important
• The quicker the specimen is processed the better
• Pathogens may die in transit thus leading to
  false negative results (ideally specimens for
  culture should arrive in the lab within 30
  minutes of collection)
• If transport is delayed, a transport medium
  should be used

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Transport Media

• Stuarts (typical formula) dont memorize
• Sodium chloride
• Potassium chloride
• Disodium phosphate
• Monopotassium phosphate
• Calcium chloride
• Magnesium chloride
• Agar
• Distilled water
• pH 7.3, buffered

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Transport Media

• Contain no growth supporting nutrients
  (carbohydrates, peptones or other) the
  objective is to maintain viability without
  supporting growth commensal organisms would
  likely outcompete and ultimately kill pathogens
• Special purpose additives
• a reducing agent such as thioglycolate can be
  added to preserve anaerobes but allows aerobes to
  survive
• CO2 can be included to support viability of
  certain pathogens (e.g. Neisseria gonorrhoeae and
  Streptococcus pneumoniae)
• Charcoal, gelatin, or corn starch can be included
  to absorb toxic metabolic products of the host or
  the hosts normal microbes this is most critical
  for particularly fastidious pathogens (ex.
  Legionella)

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Transport Media

• Special purpose transport media
• Buffered glycerol is sometimes used to transport
  fecal samples to be shipped via the mail (but not
  adequate for shigellosis)
• Boric acid will preserve urine specimens for
  culture at room temperature when refrigeration is
  not possible
• Refrigerated urine for culture can be kept up to
  24h without specimen deterioration due to
  microbial growth

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General guidelines

• Some microbes do not survive well even in
  transport media and should be brought immediately
  to the lab or, better still, planted on media at
  the bedside, examples
• Shigella die quickly in feces
• Neisseria gonorrhoeae lose viability genital
  specimens due to low temperature
• Bordetella pertussis in oral secretions

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General guidelines

• Some swabs are better than others
• If swabs must be used the following guidelines
  are important
• Cotton may contain toxins to fastidious pathogens
• Calcium alginate inhibits Chlamydia and other
  pathogens
• Dacron (polyester) is the best all-around
  material for culture swabs

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Upper Respiratory Infections

• The upper respiratory tract (URT) includes the
  oral cavity, uvula, oropharynx, nasopharynx,
  epiglottis, eustachian tube, middle ear, and
  nasal cavity (sinuses)
• The oral cavity, oropharynx nasopharynx, are
  normally inhabited by a multitude of indigenous
  microbiota
• The eustachian tube, middle ear, and the nasal
  sinuses generally are free of indigenous
  microbiota but may occasionally have a few
  transients

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Upper respiratory tract (URT) sample collection

• The principle of collecting a throat specimen is
  the same as for collecting specimens from any
  site harboring indigenous microbes limit sample
  to the infected areas and avoid the indigenous
  microbiota to the extent possible
• A tongue blade should be used to press down on
  the tongue in order to get it out of the way

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URT samples

• The throat should be visualized using good
  lighting and the patient is asked to say aaah
  to retract the uvula
• Areas in the posterior pharynx that appear
  inflamed (swollen, red, suppurative) should be
  sampled

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URT samples

• The tonsilar fossae and the extreme posterior
  pharynx beyond the uvula are the sites most often
  inflamed
• The swab should not touch the tongue, lips,
  teeth, gums, and uvula (unless it is specifically
  implicated)
• Two or more swabs are collected if serology
  testing or other additional procedures are
  requested

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(No Transcript)
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URT samples

• It is always best to transport swabs and other
  samples to the lab ASAP to prevent drying,
  possibility of contamination, etc. If transport
  is delayed, most organisms maintain viability
  better if stored in an appropriate transport
  medium such as Stuarts.
• An exception to this rule, throat swabs for S.
  pyogenes culture (and antigen testing) do not
  have to remain moist by placing them in a
  transport medium. Studies have shown that dry
  swabs sent through the mail yielded more positive
  cultures of S. pyogenes than similar ones sent in
  a transport medium

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URT samples

• A properly collected throat swab is
  roll-inoculated onto one quadrant of SBA,
  streaked for isolated colonies, and stabbed
  with the loop in order to place organisms below
  the surface into a relatively low oxygen
  environment
• If Haemophilus infection is suspected, a
  chocolate agar plate should be similarly
  inoculated.

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URT infections

• Since the indigenous microbes are almost always
  present, the problem for the microbiologist is to
  be able to differentiate between the normal
  and the pathogenic microbes in URT cultures
• Also, what is normal in the oropharynx may be
  pathogenic in the sites that communicate with it
  (e.g. middle ear and sinuses and the lower
  respiratory tract)

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URT Infections

• Each URT site can be infected with a variety of
  bacteria, fungi, and viruses
• The site most commonly infected is the oropharynx
  (infected throat or pharyngitis)
• Pharyngitis is most frequently caused by viruses
  (70 to 80) and not by bacteria
• Viral phayngitis cultures are not rountinely
  conducted in the med lab as they require special
  cell culture techniques not available
• The exception to this rule is RSV which can be
  quickly diagnosed using a rapid serological
  method such as ELISA, yet this may not be
  conducted in the med lab either

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URT Infections

• Routine cultures of the oropharynx in the U.S.A
  is almost exclusively for S. pyogenes (group A
  beta hemolytic streptococcus or GAS) - the most
  common bacterial infection of the oropharynx in
  the U.S.A. Although the remainder of these URT
  throat slides are generic, the focus is actually
  on S. pyogenes for this reason.
• S. pyogenes pharyngitis is frequently associated
  with pain, swelling, fever and a purulent exudate
  from the affected area
• S. pyogenes can readily be detected by culturing
  throat swabs on sheeps blood agar (SBA), looking
  for characteristic colony morphology, confirming
  beta hemolysis, and serotyping to Lancefield
  group A, or running an ELISA rapid strep test.

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URT Infections

• Throat cultures for S. pyogenes
• Some studies have shown that the use of a
  selective blood agar medium is superior to using
  a non-selective blood agar medium for isolating
  GAS
• SBA containing antibiotics (e.g trimethoprim
  sulfomethoxizole) is an example of such a
  selective medium
• This is known as SXT blood agar

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URT samples - nasopharygeal

• A moistened swab on a thin flexible wire is used
  to sample the nasopharynx
• It is inserted through one of the nares and slid
  along the floor a nasal passage
• When the swab reaches the nasopharyngeal chamber
  it is held there for a few seconds and is then
  retrieved
• The procedure is repeated using the same swab in
  the other nasal passage
• Ideally the specimen should be placed on
  appropriate media by the bedside, especially if
  sampling for B. pertussis (frequently found in
  nasal fluids) as it dies quickly outside the body
• If C. diptheriae (diphtheria) or Bordetella
  pertussis (pertussis) are suspected, some of the
  nasopharyngeal sample should be used to conduct a
  specific direct fluorescent antibody procedure.

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URT samples - nasopharygeal

• Corynebacterium diptheriae (diphtheria) and
  Bordetella pertussis (pertussis or whooping
  cough) are rare in most industrialized countries
  due to very successful vaccination programs.
  Infants are given the DPT vaccine to prevent
  diphtheria(D), pertussis(P), and tetanus(T).
• However, some individuals are not immune. Either
  of these species can be detected using throat
  cultures but nasopharyngeal swabs yield positive
  results more often. Both organisms require
  special media for culturing.

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URT samples - nasopharygeal

• Blood or chocolate agar supplemented with
  tellurite as well as Tinsdales agar (which also
  contains tellurite) are selective and
  differential media for detecting C.diphtheriae.
  Organisms capable of starch utilization reduce
  tellurite which results in gray-black colonies.
• Loefflers solidified serum medium is
  non-selective but C. diphtheriae produces cells
  that have a characteristic morphology on it.
• B. pertussis will not grow on media containing
  peptone (such as TSA). Bordet-Gengou works well
  as it is a peptone-free agar medium containing
  sheeps blood, potato, and glycerol usually with
  added antibiotics.
• Regan Lowe is an antibiotic containing
  peptone-based selective medium that contains
  toxin-neutralizing charcoal to counteract the
  inhibition of B. pertussis.
• Dont learn this stuff for me at this point. We
  may cover this stuff later.

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URT samples sinus, inner ear

• Reliable cultures of the sinuses or inner ear
  involves aspirating fluid from the cavities using
  a needle and syringe. The needle is inserted
  through the eardrum (tympanocentesis) or through
  the roof of the mouth (sinuses)
• Since few pathogens are likely to cause sinusitis
  or otitis media, and since invasive sampling is
  traumatic, antibiotic therapy is usually
  under-taken rather than culture
• If empirical antibiotic therapy is not
  successful, the invasive procedures can be
  performed later

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LRT samples

• The lower respiratory tract (LRT) includes the
  larynx, trachea, bronchi, bronchioles, and
  alveoli
• Microbes that reach the LRT are usually quickly
  and efficiently removed by the ciliary elevator
  or destroyed by immune defenses such as alveolar
  macrophages. The LRT should be essentially
  microbe free.
• Direct LRT sampling requires invasive procedure
  which is contraindicated unless absolutely
  necessary as in the case of inner ear
• Non-invasive sputum collection is more common

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LRT samples - sputum

• Sputum is a mucous secretion from the LRT
• Saliva is not sputum saliva is secreted from
  the URT care should be taken to prevent their
  mixing
• The patient may produce a sufficient volume of
  sputum via coughing. If not, it can be induced
  by breathing aerosolized hypertonic salt or other
  solutions or by postural drainage . Respiratory
  therapist perform these procedures

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LRT samples - sputum

• Expectorated or induced sputum are often used but
  are not ideal for diagnosing LRT infections as
  they are invariably contaminated by indigenous
  microbiota of the URT
• Nursing personnel will instruct hospitalized
  patients about proper sputum collection
• It is usually the responsibility of the lab
  technician to instruct outpatients about specimen
  collection so which is it?
• If the patient is properly instructed and
  supervised, the quality of a sputum specimen can
  be improved significantly, otherwise patients
  often produce only saliva which is not acceptable

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LRT samples - sputum

• The patient should be made to understand that
  sputum comes from deep down in the chest
• Also they should wash their mouth thoroughly with
  water just prior to collection
• In spite all the precautions, sputum is still
  not the very best specimen for diagnosing LRT
  infections
• Novel approaches such as testing the urine for
  antigens derived from LRT pathogenic microbes
  (ex S. pneumoniae) are being evaluated as an
  alternateive to sputum cultures

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LRT samples - sputum

• Smears of expectorated sputum should be made,
  Gram stained, and examined microscopically
• The smear is scanned using low power
  magnification for immune cells, especially PMNs,
  and for contaminating URT cells, specifically
  squamous epithelial cells (SECs).
• Quantitative scoring systems are used to
  determine acceptability of sputum samples. One
  such standard states that a low power field (LPF)
  should contain gt PMNs and lt 10 SECs.
• Many variations of this systems are used.

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LRT samples

• Transtracheal Aspiration (TTA) Fig 3-2 pg 129
• TTA is performed (by a physician) for the
  following reasons
• If sputum specimen cannot be expectorated
• If routine sputum samples have failed to recover
  the causative organism in spite of clinical
  bacterial pneumonia
• If an anaerobic infection is suspected
• Procedure
• the skin over the larynx is anesthetized and
  disinfected
• A large bore needle is inserted into the larynx
  through the skin
• A polyethylene catheter is passed through the
  needle and into the lower trachea
• Secretions are aspirated with a syringe

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LRT samples - bronchoscopy

• Bronchoscopy is an alternative to transtracheal
  aspiration
• A fiberoptic bronchoscope is inserted through he
  upper respiratory tract down into the bronchi and
  lungs
• This allows the attending physician to visualize
  a lesion and directly sample it
• A protected bronchial brush can be inserted
  through the lumen of the bronchoscope without
  being heavily contaminated with URT microbes
• The brush can be uncovered temporally and be used
  to collect a sample of the lesion, an acceptable
  method for collecting anaerobic organisms as well
  as aerobes
• The cover is then put back over the brush and
  retracted

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LRT samples - bronchoscopy

• Fluid can be injected into the bronchi and lungs
  through the bronchoscope cannula and aspirated
  with a syringe, a method termed a bronchial
  washing
• Unlike bronchial brushes, bronchial washings are
  not suitable for anaerobic cultures as O2 is
  invariably introduced in the washing fluid
• A bronchoscope can be threaded deep into the
  alveoli, injected with fluid and then retrieved.
  This method is called bronchoalveolar lavage
  (BAL)
• BAL is considered by many to be the method of
  choice for diagnosing Pneumocystis carinii
  pneumonia in immunocompromised patients. Why?

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LRT Infections

• The recovery of Streptococcus pneumoniae,
  Klebsiella pneumoniae, Haemophilus influenzae,
  and Moraxella catarrhalis as predominating
  organisms from a respiratory culture supports
  their role as the cause of acute pneumonia
• If it is a hospitalized patient, Pseudomonas
  aeruginosa, Staphylococcus aureus, and enteric
  Gram negative rods can be added to the list
• Non-bacterial organisms such as opportunistic
  fungi, especially Aspergillus sp., and
  Pneumocystis carinii are found in
  immunocompromised host
• Viruses are not an infrequent cause of LRTs

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LRT Infectionsfastidious organisms - serology

• Mycoplasma pneumoniae is the cause of primary
  atypical pneumonia (PAP), mostly in
  non-hospitalized young adults (a.k.a walking
  pneumonia). This organism is normally diagnosed
  using serological methods.
• Legionellosis (caused by Legionella pneumophila)
  can be diagnosed by culture on special media such
  as charcoal yeast extract agar (CYE), but is most
  often diagnosed by serological methods or direct
  microscopic examination.

41
continued

• Mycobacteria, especially M. tuberculosis in the
  general population,and M. avian-intracellulare,
  in AIDS patients, are causes of LRT infections
  that require special culture techniques and
  special media (Covered in another Power Point
  series)

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LRT Infectionsroutine culture

• Sheeps blood agar (SBA) is used for routine
  culture because of its ability to grow most
  pathogens of the LRT
• S. pneumoniae, which is by far the most common
  cause of typical acute bacterial pneumonia,
  causes a unique and characteristic type of
  hemolysis (alpha) on blood and has a
  characteristic colony morphology on SBA.

43
continued

• Chocolate agar is included because H. influenzae
  is a possible cause of LRT infections and it will
  not grow on SBA
• MacConkey agar is included to isolate enteric
  gram-negative rods and nonfermenting
  gram-negative rods (especially P. aeruginosa and
  Burkholderia cepacia) which are occasional LRT
  pathogens

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LRT Infectionsdirect microscopic examination

• After assessing the quality of the sputum the
  smear is examined microscopically.
• If there appears to be is a predominating
  organism, its morphology should be noted and
  reported
• An experienced microbiologist often can make
  presumptive identifications of classical
  pathogens based on morphological criteria

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Blood Cultures

• Definitions
• Septicemia bacteria actively growing in the
  blood stream or along the endothelial lining of
  the blood stream, including the heart valves
• Bacteremia bacteria being transported via the
  blood stream from a focus of infection to other
  body sites. Bacteria may not be present in every
  sample sloughing is transient.
• Morbidity mortality occur in both cases, but
  septicemia is generally worse. Bacteremia can
  follow daily activities like brushing teeth.
  Bacteremia can be severe in cases of pre-existing
  conditions, such as heart valve damage.
• See table 3-8, pg 154

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Blood Cultures

• Definitions
• Occult bacteremia bacteremia with persistent
  fever but no other signs of sepsis. Seen mostly
  in children, is a sign of potential septicemia,
  but without identifiable focus of infection. In
  healthy children this frequently resolves
  spontaneously. Historically linked to bacterial
  pneumonia, but other conditions apply.
• Intermittent bacteremia occurs when microbes are
  intermittently released into the bloodstream from
  an established focus of infection - symptoms
  occur upon release of cells and disappear when
  destroyed.
• Persistent bacteremia patient is constantly
  symptomatic, risk of morbidity / mortality is
  greater likely preempts septicemia infection
  is most likely intravascular.
• Toxemia septicemia with persistent systemic
  toxins

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Blood Cultures

• Blood sample contamination with skin microbes is
  a serious issue, therefore strict disinfection
  procedures must be followed per your text
• Apply 12 iodine antiseptic, allow to dry 1-2
  minutes
• Remove iodine with 70 alcohol
• Tom
• Press firmly at and make an inside-out
  concentric pattern from

48
Blood Cultures - timing

• Taking multiple blood samples increases the
  likelihood of detecting infection, however,
  statistics indicate that taking more than 3
  samples in 24hrs is unnecessary
• Ideally a blood culture should be taken just as
  the patients fever spikes during early-accute
  phase
• If this is not possible (not all patients with
  bacteremia will have a fever) the blood should be
  drawn before antibiotics are given
• If antibiotics need to be given immediately, two
  to three blood cultures should be drawn
  one-after-another, using separate puncture sites
  if possible
• If antibiotic therapy is not immediately
  anticipated, two to three blood cultures should
  be performed at 30 minute intervals or up to one
  hour intervals

49
Blood Cultures - volume

• The volume of blood collected is the most
  important criterion for obtaining a positive
  blood culture. Too little blood may not allow
  detection of mild or early onset of bacteremia.
  This is more true of adults who generally have
  lower microbe concentrations than children. As
  little as 1 2mL will often suffice for
  detection of microbes in childrens blood.
• The minimum volume of blood for a single adult
  sample is 10 mL
• 20 to 30 mL per sample is advocated by many
  clinicians, with 30mL being an absolute maximum
• For each 1mL increase of blood/culture, there
  will be a 2-3 increase in the number of positive
  blood cultures

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Blood Cultures - media

• Blood contains many natural antimicrobial
  substances (e.g. phagocytic cells, lysozyme,
  complement, etc,).
• Additionally, when the blood is collected
  antibiotics may have already been administered.
• In order to negate these antimicrobials blood
  specimens need to be adequately diluted in a
  culture medium
• Common blood culture media are liquid and contain
  trypic soy (TSB) with peptone, BHI and other
  additives
• The ideal blood to media ratio (see above) is 1
  part blood with 9 parts media for a final
  dilution factor of 110.
• Many commercially automated blood culture systems
  use a 1 to 5 blood to medium ratio which
  manufacturers claim to be adequate for their
  systems.

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Blood Cultures - media

• Use of anticoagulants is essential to prevent
  microbes from becoming trapped in a fibrin clot.
  Trapped microbes may not grow in the liquid
  portion of the culture media giving false
  negative results
• Anticoagulants used must not inhibit the growth
  of important pathogenic microbes. Many
  anticoagulants have this undesirable property.
• Sodium polyanetholsulfonate (SPS) is an
  anticoagulant formulated for blood cultures. It
  is commonly used at a concentration of 0.024 to
  0.05 V/V (these values will not be on a test).

52
continued

• Other beneficial effects of SPS
• In addition to anticoagulant properties, SPS
  also inactivates phagocytes and neutralizes some
  antibiotics such as aminoglycosides and
  polymyxins.
• SPS interferes with the antibacterial action of
  complement.
• Undesirable effects of SPS
• SPS may inhibit the growth of certain pathogenic
  bacteria (P. anaerobius, N.gonorroheae, N.
  meningitidis and G. vaginalis), though this
  inhibitory effect of SPS can be overcome by
  adding gelatin to the blood medium (final
  concentration 1).

53
Conventional Blood Cultures

• A blood sample is distributed between two (or
  more) bottles of media. Bottles of media are
  under vaccum, with high CO2 and low O2
  conditions.
• One bottle of media, best suited for growing
  obligate aerobes and most facultative organisms,
  is vented to allow CO2 to escape and O2 to enter.
  
• The other bottle is not vented (?CO2 ?O2) and
  is better suited for growing obligate anaerobes,
  most facultative organisms, and aerotollerant
  anaerobes.
• Conventional blood cultures are examined
  manually twice a day (both macroscopically
  microscopically) for the first two days and once
  a day thereafter. They are incubated for 7 days
  before being reported as negative.
• Macroscopic examination bottles are observed for
  evidence of a)turbidity, b)hemolysis, c)gas
  bubbles, and d) microcolonies on the surface of
  the sedimented red blood cells or suspended in
  the broth

54
continued

• An aliquot of a well-mixed broth and blood is
  taken for microscopic exam and for subculture
  from bottles showing any evidence of growth.
• Blind subculture of aerobic bottles showing no
  evidence of growth (for the 1st 2 days) is
  required because viable microbes may actually be
  present a day or two after collection and display
  no evidence of growth. Pathogens can then die
  and never show evidence of their presence if
  subcultures are not done.
• Chocolate agar is the best medium for routine
  blind subculture. Chocolate agar contains X
  and V factors (NAD hematin) which are
  required by Haemophilus influenzae, a very
  important fastidious pathogen that is often
  isolated in blood cultures.
• Chocolate agar subcultures are incubated under
  elevated CO2 levels which favors growth of some
  fastidious pathogens, as well as anaerobes no
  need to subculture anaerobic bottles.

55
continued

• Advantages of a conventional broth-based-non-autom
  ated blood culture system
• savings automation is expensive
• Eyes machines dont have em
• Disadvantages of this type of system
• Continuous monitoring is not possible
• The whole process is labor intensive because
  subculturing is a monotonous repetitive task
• Results of positive conventional blood cultures
  are often delayed compared to automated systems
  (mainly due the inability to continuously
  monitor)
• Chance of human error

56
Automated Blood Culture - broth

• Blood samples are processed as usual, inoculated
  into the same broth media under the same
  conditions, but following inoculation there are a
  lot of differences, and advantages?...
• Once placed in the incubator/monitor/computer
  unit no action is taken until a positive is
  detected by the machine or the incubation time is
  completed
• Monitoring is continuous (usually once every ten
  minutes or so) during the entire incubation
  period
• Time course data - growth curve - kinetics
• Visual and/or audible results signals are given
• Routine blind subcultures are not necessary
• Incubation times are usually shorter than for
  conventional systems allowing negative reports to
  be rendered in five days or less versus seven
  days.

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Automated Blood Culture - broth

• Detection methods
• Optical density meter to detect turbidity
• CO2 evolution (or consumption?) indicating
  substrate catabolism, via pH colorimetry, pCO2
• Consumption of other gasses (O2, H2, etc)
• Other?
• Summary / considerations
• Cost of machine vs cost of labor
• Rapidly process large number of samples
• Continuous monitoring allows earlier detection of
  positive samples quicker diagnosis treatment

58
Automated blood culture methods not exclusively
broth-based

• Lysis centrifugation system
• lyses all formed elements in sample (RBC WBC)
• Centrifuges solids for collection
• Sediment inoculated onto plates of various agar
  media and incubated under various conditions
• Allows examination of colony morphology and
  generates pure cultures for further study AB
  sensitivity
• Allows detection of intracellular parasites such
  as Listeria and Brucella
• The system has its problems including necessity
  of extensive manual labor and an inherently high
  contamination rate that occurs during sample
  manipulation

59
continued

• Biphasic blood cultures
• Bottles of media containing broth and an agar
  slant are inoculated allowing culturing and
  examination of both - best of both worlds
• broth culture recovery / turbidity
• colony morphology on agar
• The unique closed-system design
• allows sub-culturing from broth to
• agar that is much easier, quicker
• and has much less manipulation
• which greatly decreases chances
• of contamination

60
Blood Cultures wrap-up

• Microscopic examination (e.g. gram reaction
  cellular morphology) is an absolute necessity,
  and is a first step, no matter which system is
  used i.e. automation does not replace this.
• Since septicemia and complications thereof has
  such a high mortality rate, a positive blood
  culture is a medical emergency and should be
  treated as such. Promptness and accuracy
• The results must be communicated to the physician
  or designee immediately by phone and followed up
  by a written report
• Lab results on cerebrospinal fluid are also
  treated as critical values

61
Fecal Specimens

• Several bacterial species (and other pathogens)
  cause gastrointestinal (GI) conditions (diarrhea,
  dysentery, colitis, etc) associated either with
  consumption of food or water contaminated with
  feces (ie. fecal-oral), or with food poisoning.
  Food poisoning primarily involves the ingestion
  of toxins present in food from previous growth of
  bacteria therein, although some bacterial growth
  and toxin production can occur in the patient.
  The lower GI tract is the primary focus for all
  of the above.
• Diarrhea can be limited to cramping loose
  stool. It can be mild or severe depending on the
  potency of the enterotoxins involved example
  S. aureus vs V. cholera.
• Enterotoxins attach to the intestinal mucosal
  cells and stimulates them to secrete water and
  electrolytes without physically damaging the cell
  - it is non-inflammatory.
• Other exotoxins (see conditions on next slide)
  can be inflamatory, causing varying degrees of
  cell destruction ex enterohemorrhagic E. coli
  verotoxin is deadly.

62
continued

• Colitis involves diarrhea with additional
  pathology resulting in inflammation of the colon,
  and most likely pain.
• Enterocolitis is similar but also involves the
  small intestine.
• Dysentery includes the above symptoms along with
  fever, and severe diarrhea, but most
  characteristic is the passage of blood (with
  PMNs) and mucus due to the enteroinvasive nature
  of the pathogens and toxins involved. Bacterial
  dysentery-like illness is most commonly
  associated with Shigella, Salmonella,
  enterohemorrhagic E. coli, Campylobacter jejuni,
  and Yersinia enterocolitica.

63
continued

• These and other GI conditions are much less
  common in industrialized countries due to
  infrastructural advantages hygienic practices,
  municipal sanitation, water systems, etc

64
Fecal Specimens

• Salmonella, Shigella, Campylobacter,
  enterohemorrhagic Escherchia coli, and
  Clostridium difficile are routinely tested for in
  most microbiology labs in the USA
• Culture and direct wet preparations for fecal
  leukocytes are standard methods for all of the
  above except C. difficile
• For years the only intestinal pathogenic
  bacteria routinely cultured for was non-typhoidal
  Salmonella and Shigella
• Through further study and improved methods it was
  over time determined that many other organisms
  were significant causers of GI pathology.
• Enteric pathogens that emerged include Salmonella
  typhi, Vibrio cholera, Vibrio parahaemolyticus,
  Yersinia entercolitica, Campylobacter jejuni,
  Bacillus cereus, S. aureus, several toxin
  producing and enteroinvasive strains of E. coli,
  and some viruses rotavirus, norwalk virus, HVA,
  etc.

65
Fecal specimens

• Collection and processing
• Collection of fecal samples is relatively
  uncomplicated.
• Feces (stool) is the specimen of choice for
  diagnosing fecal-orally contracted diarrhea,
  dysentery, or food poisoning caused by bacteria,
  protozoans viruses.
• Stool should be collected in a wide-mouth
  container with a tight fitting lid. The
  container need not be sterile, but should be free
  of preservatives, detergents and metal ions, and
  not previously contaminated with feces or urine.
• If the patient cannot produce a stool specimen
  (such as newborns debilitated adults) a rectal
  swab will suffice.
• Swab samples are generally processed more rapidly
  in the lab, and are therefore superior for
  recovery of those pathogens (Shigella and
  Clostridium difficile) that are especially
  susceptible to cooling, drying and progressive
  acidification that can result in a sample.
• It is always best to transport all samples
  directly to the lab within hoe hour of collection.

66
Fecal Specimens

• Collection and Processing
• Stool that cannot be processed within one hour
  should be placed in a transport medium as
  discussed previously.
• If intestinal parasites are suspected, samples
  should be placed in preservatives such as PVA and
  10 formalin.

67
Fecal direct examination

• The majority of GI infections are caused by
  morphologically similar Gram(-) rod-shaped
  bacteria. Most of these are members of the family
  Enterobacteriaceae (E. coli and his cousins
  Shigella, Salmonella, etc, - ie. the enterics).
• Therefore a Gram stain for bacterial cellular
  morphology is not generally clinically useful
• Fecal Gram stains for leukocytes (PMNs) are
  helpful for differentiating inflammatory from
  non-inflammatory infections.
• A Methylene blue wet prep is an easy alternative
  to the Gram stain for detecting fecal leukocytes

68
Fecal direct exam exceptions

• The presence of Gram () cocci in clusters
  presumptively suggests the involvement of S.
  aureus
• A preponderance of budding cells (with or without
  pseudohyphae) is suggestive of yeast infection.
  Candida albicans is an infrequent cause of
  diarrhea.
• Campylobacter is a tiny curved or loosely
  spiraled Gram (-) bacterium that can be seen
  on Gram stains in heavy infections.
  Characteristic darting motility may also be
  revealed in a wet mount of Campylobacter.

69
Fecal specimen culture methods

• Fecal specimens are heavily contaminated with
  indigenous microbiota and must be cultured on
  selective media
• Fecal microbes, in general, are resistant to
  bile, are facultative or obligately anaerobic,
  and can ferment a variety of carbohydrates
  (including lactose in some cases) producing acid
  and sometimes gas. There are exceptions to these
  rules.
• This allows application of various selective and
  differential media for characterization of fecal
  bacteria.
• Selective media for the isolation of Salmonella,
  Shigella, Campylobacter jejuni, and
  enterohemorrhagic E. coli should always be
  included.

70
continued

• Selective/differential media routinely used for
  Salmonella and Shigella usually contain bile
  salts and/or dyes with bile-like properties as
  inhibitors.
• The differential agents in these media commonly
  include lactose along with a pH indicator to
  indicate production of acid fermentation via
  color change of the media and or colonies.
  Salmonella and Shigella do not ferment lactose
  therefore their colonies appear clear and
  colorless. Many other fecal bacteria such as E.
  coli do ferment lactose, and their colonies have
  color on these media. Such media include
  MacConkeys and SS agar.
• Colony color in the case of some media (EMB) is
  due to accumulation of dye from the agar rather
  than pH change.

71
? MacConkey EMB? SS
agar ?
72
continued

• There is a commercially available selective
  medium for Y. entercolitica but, since this
  organism is almost exclusively found in cooler
  climates (Canada, N-C. Europe) it is not commonly
  tested for in the USA any further south than New
  England.
• Vibrio cholera and V. parahaemolyticus are not
  prevalent in the USA except near the Gulf of
  Mexico. Special selective media for these
  bacteria are not commonly used elsewhere in the
  USA.
• Aeromonas and Pleisiomonas are also diarrhea
  producing bacteria with a very low incidence in
  the USA.

73
Fecal cultures - enrichment broth media

• To aid in selection of the causative pathogens
  from the numerous commensals, fecal samples are
  inoculated into highly selective enrichment
  broth media. These media contain chemicals
  preventing the normal gut microbes from quickly
  entering the logarithmic growth phase while
  enteric pathogens are allowed to grow rapidly.
• Gram Negative (GN) and Selenite broths are two
  such types of enrichment broth media.
• It takes the normal gut bacteria six hours in GN
  broth to reach log phase, and 12 hours to reach
  log phase in selenite broth.
• Salmonella in selenite, and Salmonella and
  Shigella in GN reach the log phase of growth
  within an hour or two.
• After several hours of incubation, the enrichment
  broth is subcultured onto the appropriate
  selective agar.

74
Culturing Campylobacter

• Campylobacter jejuni and C. coli are thermophilic
  (they grow best at 42oC) and also microaerophilic
  (require sub-ambient O2 and elevated carbon
  dioxide conditions). This atmosphere is
  generated using a commercially available campy
  pack to consume O2 and ?CO2.
• They do not compete well with indigenous
  microbiota and require a selective medium. Most
  of these media use a combination of antibiotics
  as selective agents
• Vancomycin inhibit Gram positives
• Trimethoprim inhibit swarming Proteus
• Amphotericin B inhibit fungi
• Cefoperazone inhibit enteric Gram negative rods
  and Pseudomonas

75
Culturing enterohemorrhagic E. coli EHEC

• Various strains of intestinal pathogenic E. coli
  have caused sporadic cases of GI infections
  worldwide. These strains are referred to by
  various names (enteropathogenic, enterotoxigenic,
  enteroinvasive, enterohemorrhagic).
• Enterohemorrhagic E. coli (EHEC) came to the
  forefront in the US in 1988 when it was
  contracted by several people who ate undercooked
  hamburgers from Jack In The Box, a NW
  restaurant chain. This strain was eventually
  designated E. coli 0157-H7, a serotype
  designation.
• Many people became severely ill with painful
  explosive bloody diarrhea. Some people developed
  acute renal failure and ischemic bowel disease,
  and a few died.
• EHEC is a potentially deadly strain of E. coli
  due to the production of a potent toxin known as
  verotoxin (VTEC) or shiga-like toxin (STEC).
  EHEC infection most often comes from meat (beef)
  contaminated with the organism from the animals
  feces. The pathology of this organism will be
  discussed later.

76
continued

• EHEC is detectable on a selective/differential
  medium, Sorbitol MacConkey (SMAC). EHEC forms
  colorless colonies on SMAC where other E. coli
  colonies are red.
• Commercially available ELISA tests can be used to
  confirm presumptive EHEC isolates.
• The LGH microbiology lab uses EHEC to report
  these enteropathogenic E. coli

77
Fecal specimens C.difficile

• Diarrhea that develops during a stay in the
  hospital (ie. is nosocomial) is rarely caused by
  the same organism associated with fecal-oral or
  food born disease.
• Patients who develop diarrhea thee or more days
  after initial hospitalization should not be
  cultured for enteric pathogens unless one of
  their stool specimens has shown to be negative
  for the Clostridium difficile cytotoxin (i. e.
  have a negative C.diff test).
• Nosocomial diarrhea is almost always associated
  with C.difficile in hospitalized patients who are
  receiving or have had antibiotic treatment
  antibiotic associated pseudomembranous colitis.
  
• This antibiotic therapy eradicates a persons
  normal intestinal microbiota giving the pathogen
  an opportunity to proliferate.

78
continued

• Pseudomembranous colitis is characterized by the
  formation of a membrane-like structure in the
  intestine. C. difficile produced a toxin that
  results in damage to, and sloughing of layers of
  the intestinal mucosa forming this
  pseudomembrane.
• Clostridium difficile is resistant to many
  antibiotics
• The most common method for detecting C. difficile
  in feces is an ELISA test for antigen. Again,
  cases of C. difficile are usually nosocomial and
  suspected when hospitalized patients develop
  diarhea after antibiotic treatment.

79
Fecal specimen culture methods

• Vibrio, Aeromonas, and Pleisiomonas
• Vibrio cholera, Vibrio parahaemolyticus,
  Aeromonas sp, and Pleisiomonas sp. are found only
  sporadically as enteric pathogens in most
  developed countries
• Most strains of Vibrio cholera have been
  associated with epidemics and pandemics
• Sheeps blood agar will grow the infrequently
  isolated enteric pathogens Vibrio, Aeromonas, and
  Pleisiomonas
• There are selective media available for each of
  these pathogens but it is not practical to use
  them routinely due to their low incidence
• A major characteristic that differentiates
  Vibrio, Aeromonas, and Pleisiomonas from most
  enteric Gram negative rods is the production of
  cytochrome oxidase oxidase positive
• Colonies of oxidase positive Gram negative rods
  that grow on SBA from fecal samples much be
  tested to rule out Vibrio, Aeromonas, and
  Pleisiomonas

80
Fecal specimen culture methods

• Detecting Yersinia entercolitica
• Y. entercolitica is unique among enteric
  pathogens in that its optimum growth temperature
  is room temperature
• There are selective/differential media for Y.
  enterocolitica but it is not practical to use
  them routinely due to their low incidence
• Y. enterocolitica grows as tiny pinpoint lactose
  negative colonies on MacConkey incubated at 37oC
  for 24 hr.
• If these plates are incubated at room temperature
  an additional 24h period, colonies of Y.
  enterocolitica will enlarge considerably.
• Bacteria exhibiting this behavior are further
  identified to rule out Yersinia.

81
Fecal Specimens

• Staphylococcus aureus, Salmonella enteritidis and
  Bacillus cereus are relatively common causes of
  food poisoning in the USA
• Symptoms of most bacterial food poisoning
  pathogens occur within 72 hours of ingestion and
  include nausea, vomiting, and abdominal cramps
• Except for the culturing of Salmonella, diagnosis
  of food poisoning is best done by examining the
  food product directly for the bacterial toxins

82
Fecal Specimens upper GI tract

• Gastritis is an inflammation of the stomach or
  upper GI tract
• Upper GI infection symptoms generally include
  anorexia, nausea, occasional vomiting and
  abdominal pain, but NOT profuse diarrhea as is
  characteristic of a lower GI infection.
• Upper GI specimens for culture are rare
  (including H. pylori) and are limited to
  situations where diagnosis by other means is not
  possible.
• Bacteria agents responsible for acute toxic food
  poisoning can be recovered from vomit samples.
• Gastric biopsies are becoming more common
  (usually a biopsy taken in surgery with the aid
  of a gastroscope) for recovery of Helicobacter
  pylori, although other methods such as
  histological examination, urease enzyme tests,
  DNA testing for the urease gene, or serology
  tests are usually used.

83
Fecal Specimens upper GI tract

• Helicobacter pylori is the most common cause of
  gastritis in patients who produce a normal amount
  stomach hydrochloric acid
• H. pylori produces a very potent urease that can
  be rapidly detected when the biopsy specimen is
  placed in a urea substrate containing a pH
  indicator.
• Urease hydrolyzes urea yielding ammonia ammonia
  is highly alkaline and will quickly change the
  color of certain pH indicators.

84
Urine Specimens UTIs

• 80 of UTIs in people ages 2-65 originate from
  endogenous fecal organisms, mostly from E. coli.
  
• UTIs are fairly common among females of all ages
  due to the short exposed urethral design. Sexual
  intercourse, contraceptives, and pregnancy
  predispose women to UTIs.
• 1 of all neonates develop UTI associated
  bacteremia (bacteriuria) with the incidence being
  higher in boys ???
• Childhood bacteriuria can result in kidney
  damage, and frequent episodes are suggestive of
  underlying structural abnormality.
• After age 65 the incidence in men dramatically
  increases and the incidence in women increases
  only moderately
• Infections of older men are usually associated
  with prostate disease, a very common occurrence
  in this age group

85
UTIs

• The higher incidence of UTIs in hospitalized
  patients is often associated with UT
  manipulations such as prostate examination,
  urinary catheterization and instrumentation
• UTI pathogens can ascend from the perineum to the
  urethra (urethritis), bladder (cystitis) or
  ureters (ureteritis), or ultimately to the kidney
  to cause pyelonephritis.
• Lower UTIs, urethritis and cystitis, are not
  associated with long term disabilities. Upper
  UTIs, ureteritis and especially pyelonepritis,
  are more severe and are associated with long term
  renal dysfunction.

86
UTI symptoms

• Neonates and children younger than 2 years of age
  with UTIs usually present with nonspecific
  symptoms including failure to thrive, vomiting,
  and fever
• Symptoms of lower UTIs occur with the same
  frequency in older children and adults dysuria,
  frequency of urination, urgency of urination,
  small amounts of turbid urine with each void, and
  occasional suprapubic tenderness
• Upper UTIs present the same symptoms as lower
  UTIs (a source of confusion) as well as flank
  pain and fever
• A majority women over 65 years old with UTIs do
  not have symptoms other than turbid urine.

87
UTI etiology

• E. coli is the leading cause of community
  acquired and nosocomial UTIs in most cases (see
  exception below). Most uropathogenic E. coli (
  other enterics) have fillamentous protein
  adhesins that allow specific attachment to UT
  epithelial cells a virulence factor.
• Other enteric Gram (-) rods that frequently cause
  community acquired UTIs include Klebsiella
  pneumonia, various species of Enterobacter, and
  Proteus sp. (mostly P. mirabilis)
• Gram positive bacteria associated with community
  acquired UTIs include Enterococcus and
  Staphylococcus aureus
• Staphylococcus saprophyticus is the leading cause
  of lower and upper UTIs in college age women, but
  rarely causes infections in other groups
• Other prominent etiologies in hospitalized
  patients include K. pneumoniae, P. mirabilis,
  Pseudomonas aeruginosa, Enterococcus, S. aureus,
  coagulase (-) Staphylococcus (mostly S.
  epidermidis) and Candida albicans

88
Urine Specimen culture methods

• Unlike most other clinical specimens, the culture
  of urine must be done quantitatively presence
  of a UTI correlates with the number of bacteria
  in the specimen (CFU/mL).
• ?105 CFU/ml of a pure culture from catheterized
  urine was established in the 1950s to correlate
  with asymptomatic pyelonephritis in women. Later
  it was determined that most people with
  symptomatic pyelonephritis and cystitis will also
  have colony counts of ? 105 CFU/ml
• Colony counts lt 105 CFU/ml may correlate with
  UTIs in certain circumstances such as occurs when
  bacteria that cause urethritis get trapped
  within the urethra, or when cells are
  significantly clumped, each leading to colony
  counts much lower than 105 CFU/ml, even far lt103
  CFU/ml
• Studies show that symptomatic females with
  urethritis associated with a single species and a
  colony count of 103 CFU/ml or greater should be
  treated with antibiotics.

89
Urine culture plating

• Two procedures can be used to do urine colony
  counts serial dilution of the urine using
  diluents and pour plates and the calibrated loop
  method
• The calibrated loop method is a much simpler than
  the calibrate loop method
• Perhaps it is not as accurate but it is
  sufficiently accurate to be useful for diagnosing
  UTIs
• In either case there are a few media that are
  used.
• MacConkey agar or equivalent is always included
  since the vast majority of UTIs are caused by
  Gram-negative rods.
• SBA is included as well to increase recovery.
  Hemolysis data may also prove useful in
  presumptive ID.

90
Urine plating calibrated loop method

• Mix specimen thoroughly, then insert a sterile
  0.01 or 0.001 calibrated loop vertically into the
  urine until the loop is completely submerged.
• Place the filled loop flat onto the agar and drag
  a 1 streakdirectly across the agar bisecting it
  into equal halves.
• Streak perpendicular to the 1 streak in a way
  that spreads the specimen thoroughly over the
  entire plate surface.
• Repeat in the opposite direction.

91
Urine plating calibrated loop method

• Plating 0.01mL of urine is equivalent to making a
  1100 dilution. Colony counts from a 0.01mL loop
  must be multiplied by 100 to give CFU/mL values.
• For statistical validity, the number of colonies
  on a given quantitative culture plate should be
  between 25 and 250.
• The reporting unit is CFU/mL and not colonies
  per mL

92
Urine plating dilution method

• Making serial dilutions of urine in sterile water
  (?) and then plating a measured volume allows for
  a very accurate colony count.
• Even though this method is very accurate it is
  tedious to perform.
• The kind of accuracy this method allows is not
  required for urinary colony counts and the
  calibrated loop method is easier to perform and
  the results are clinically relevant

93
Urine Specimen collection - catheter

• Properly collected catheterized urine rarely is
  contaminated with indigenous microbes
• However, the process of catheterizing is a risk
  factor for causing UTIs and is not recommended
  for routine sampling
• If the patient is going to be catheterized for
  some other purpose or has an indwelling urinary
  catheter, this can provide an excellent specimen
  for culture
• Urine taken from an indwelling catheter must not
  be taken from the collecting bag
• The specimen should be taken aseptically with a
  needle and syringe from the catheter port after
  disinfecting it with alcohol.
• Again, this is not recommended for routine
  samples. The clean catch method is the routine
  method of choice.

94
Urine Specimen collection clean catch

• It is now known that clean-catch counts are
  comparable to catheterized specimen counts,
  although they are more susceptible to
  contamination with indigenous microbes.
• The urethral opening is colonized with a variety
  of indigenous microbes, some of which are UTI
  pathogens. These microbes can contaminate urine
  samples and cause misdiagnosis similar to the
  case with saliva in sputum.
• Collection of mid-stream urine prevents much of
  the problem of contamination with indigenous
  microbes.
• Clean-catch applies to urine that does not make
  contact with any body surface after exiting the
  urethral opening.
• This is easy for males, but often requires
  instruction from trained personnel for a good
  sample from a female.
• These samples are best taken first thing in the
  morning.

95
Urine Specimens

• Urine specimens must be processed within 2 hrs of
  collection, or otherwise be refrigerated.
  Refrigerated specimens can be kept up to 24hr
  without deterioration
• Suprapubic aspiration (SPA), another method of
  urine specimen collection, is reserved almost
  exclusively for neonates, small children and
  occasionally adults in whom clean catch midstream
  urine has failed to establish a diagnosis
• Suprapubic aspirated urine is the only type of
  urine specimen suitable for anaerobic culture,
  although UTIs due to anaerobic bacteria are very
  rare.
• Suprapubic aspiration is done by qualified
  medical personnel, usually a physician, and is
  only used when absolutely necessary due to the
  associated risk. However SPA samples are the
  type least likely to be contaminated.

96
Urine Specimens

• SPA
• The skin directly over the bladder is
  anesthetized and disinfected
• Then a needle is inserted through the skin and a
  syringe is used to aspirate 10mL urine for
  culture
• Cytoscopy
• Cystoscopy is a procedure that uses a fiber-optic
  device to visualize the urinary tract
• During this exam urine specimens can be collected
  from the bladder or ureters for culture. These
  are processed as catheterized specimens

97
Urine specimens

• Properly collected, freshly voided urine from a
  noninfected individual processed immediately
  should have colony counts ? 103 CFU/ml, most
  likely far below this value.
• A given UTI is usually caused by a single species
  of microbe, however in patients with an
  indwelling catheter a UTI can be caused by two or
  rarely three different species
• If gt 3 organisms grow in a urine specimen (other
  than those taken by catheterization), no matter
  what the colon count is, this is considered
  evidence of contamination, and further processing
  is not warranted
• If 2 different organisms grow at a colony count
  of ? 105 CFU/ml each, both are considered to be
  etiologic agents of the UTI
• Any number of organisms in catheterized,
  cystoscopy, or suprapubic aspirated urines, is
  presumed to be of clinical significance

98
Urine Specimens direct exam

• Direct microscopic examination of urine for
  culture is not routinely performed in most
  clinical labs
• Occasionally a physician will request a Gram
  stain when anticipating the immediate
  administration of antimicrobial therapy
• A drop of unspun urine is placed on a microscope
  slide, allowed to dry, fixed, and Gram stained
• If the number of microbial cells averages one or
  more per oil immersion field, this correlates
  rather well with approximately 105 CFU/mL

99
Cerebrospinal Fluid (CSF)

• Meningitis is inflammation of the meninges. A
  number of pathogens cause various meningitic
  syndromes.
• Acute meningitis is a medical emergency - it can
  be associated with high morbidity and mortality
  if not diagnosed and treated early in the course
  of the disease
• Acute meningitis is commonly caused by only a few
  rapid growing bacteria that cause inflam