Clinical Bacteriology_LEC

College of Medical Technology

SPECIMEN HANDLING, TRANSPORT AND PROCESSING - swabs: 1 for culture, 1 for direct microscopy

General Guidelines for Specimen Collection 7. For lesions, wounds and abscesses
1. Specimens should be collected during - The specimen is collected from the
the acute (early) phase of an illness (or advancing margin of the lesion and
within 2-3 days for viral infections) and should be collected by needle
before antibiotics are administered aspiration rather than by swabs
- Why? Because these antibiotics will - Place in sterile tube
interfere in the isolation of - Avoid normal flora
organisms; it inhibits the growth of ● Dacron/polyester swab
organisms. - Using cotton swabs can be
Why the acute phase? toxic to bacteria because of
- Because in this phase, organisms excessive fatty acids.
are actively dividing
8. Aspirated specimen must be placed into
2. Select the correct anatomic site for a sterile tube or transport vial and not
collection of the specimen squirted onto a swab

3. Before collection, the site should be 9. Label the specimen accurately with the
cleansed properly specific anatomic site and the patient
- To prevent contamination of the demographic profile
sample with our normal flora;
normal flora might interfere in the 10. Transport the specimen to the
identification and isolation of laboratory promptly or make provisions
organisms to store the specimen in an
environment that will not degrade the
4. The specimen collected should be a suspected organism(s)
representation of the infected/diseased
Area

5. The quantity of the specimen should be

sufficient enough for diagnostic testing

6. Swabs generally are poor specimens

(except nares and throat specimens) if
tissue or needle aspirates can be
obtained
- swabs are never appropriate for
anaerobic culture
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Specimen containers and other materials for

collection SELF STUDY: TABLE 6.1 – SPECIMEN
1. Specimens for microbiology cultures COLLECTION GUIDELINES (Mahon, p.108)
should be collected in sterile containers.
STool collection
Stool specimens can be collected in
clean, non sterile containers
- Best way to separate or isolate
pathogenic from non-pathogenic
organisms is to use a selective
medium – contains inhibitors that
inhibits the normal flora
- Negative result not sufficient to exclude
bacteria or parasite
- Need of 3 specimen
Preservative: Buffered Formalin & Polyvinyl alcohol
w/ zinc
- Refrigerate 2 hrs delay

2. Swabs for specimens from URT,

external ear, eye and genital tract
- Swabs are not recommended for
routine collection because they are
easily contaminated and can
become dried out
*Dacron or calcium alginate is
Preferred

Patient Preparation
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- Needle through the abdomen into bladder

to collect urine sample
- Bladder must be full before performing
procedure
- Tested for anaerobic organism submitted in
syringe
- For pediatric practice
Procedure: Disinfect the skin. Needle aspiration
above the symphysis pubis through the abdominal
wall to the full bladder.

INDWELLING CATHETER OR FOLEY CATHETER

- Develop bacteriuria that can cause
predispose them to severe infection

SPECIMEN REJECTION
● Identification of a mislabeled specimen
or requisition should not be done over
● Provide patients with thorough the telephone
instructions on how to collect the ● The term wound is not an appropriate
sample (ex. Urine, stool, sputum) specimen, and the exact anatomic site
Urine Collection must be provided
Best procedure: catheter and/or mid-stream clean ● All rejected specimens require a phone
catch (because it prevents the call to the person in charge of collecting
contaminants) the specimen
- Least invasive ● Specimens that are impossible to
- First morning urine is advisable recollect or that would require the
- Urine remained 4 hrs in the bladder patient to undergo another invasive
decreases chance of false negative procedure (bone marrow, spinal fluid,
Straight Catheterized Urine or surgery) may need to be processed
- Slightly invasive regardless of the situation
- Collection of bladder urine
- Risk of introducing urethral organisms to REASONS FOR SPECIMEN REJECTION:
the bladder route UTI 1. The information on the label does not
Procedure: Cleanse urethral area. Insert the match the information on the
catheter into the bladder. Pass 15mL urine then requisition slip
collect remainder 2. The specimen is transported at the
improper temperature
Suprapubic bladder aspiration 3. The specimen has not been transported
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College of Medical Technology

in the proper medium Specimen Transport

4. The quantity of specimen is insufficient 1. Ideally, specimen should be transported
for testing (QNS) to the laboratory within 30 minutes of
collection
5. The transport of specimen exceeds 2 For CSF samples, it should be
hours post collection and the specimen transported within 15 minutes
has not been preserved properly For anaerobic bacteria, transport
- If exceeds the 2 hour, there will be should not take more than 10 Minutes
increased count of bacteria

Why do we need to transport anaerobic bacteria

6. The specimen is preserved in a fixative
within 10 minutes? Why should CSF samples be
(formalin) – it will kill the bacteria
transported within 15 minutes?
- Specimens should be transported within a
7. The specimen has been reserved for
certain amount of time because adverse
anaerobic culture from a site known to
environmental changes in oxygen, pH, and
have anaerobes as a part of the normal
temperature can prevent the recovery of
flora (vagina, mouth)
certain microorganisms and allow
overgrowth of others.
8. The specimen has already dried up
- CSF should be transported within 15
when transported to the laboratory
minutes to preserve the integrity of the
- Prone in swab testing
specimen and not delay the laboratory
processing and reporting of results (i.e.
9. Processing a specimen with a
Gram stain).
questionable medical value

2. All specimen containers should be leak

10. More than one specimen from the
proof
same source (except blood) and from
3. All specimens should be transported
the same patient was submitted on the
within sealable, leak-proof, plastic bags;
same day
specimen bags should be marked with
biohazard label
11. A single swab was submitted with
multiple requests for various organisms
4. Use of appropriate special preservatives
or holding media for transport of
12. Expectorated sputum in which the
specimen delayed for more than 30
Gram stain reveals <25 WBCs and >10
minutes is important in ensuring
epithelial cells (saliva)
organism viability
Accept: >25 WBCs and <10
Changes in temperature: Neisseria meningitidis and
epithelial cells
Haemophilus
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Changes in pH: Shigella spp. - Secondary packaging should be water tight

- Third packaging should be rigid
Transport Media
Stuart’s medium – is the most commonly used; SPECIMEN PRESERVATION
semi-solid medium that prevents oxidation and 1. Preservatives
drying during transport. ● Boric acid – maintains the appropriate
- Upper respiratory tract specimens and fecal colony counts (urine) at room
specimens temperature for 24 hours;
- For fastidious organism bacteriostatic property
Urine preservative tablet
Amie’s medium – commonly used for throat, vaginal, also contains boric acid as a preservative.
wound and skin swabs. - dissolve in the urine if refrigeration is not
- Charcoal contained possible after 2 hours.
- For Neisseria gonorrhea and Bordetella
pertussis Stool specimens for C. difficile toxin assay
should be collected without a preservative
Remel Cary Blair medium – a semisolid medium for and can be refrigerated; if more than 48
stool and rectal swabs. hours, it should be at 70°C
- Transport for gram negative and anaerobe
bacteria 2. Transport or holding media
- has an inorganic phosphate buffer for
sodium glycerophosphate. 3. Charcoal
to absorb fatty acids present
Transgrow – used for fastidious bacteria in the specimen that could kill fastidious
organisms (Neisseria gonorrheae and
JEMBEC system – contains selective agar and a Bordetella pertussis)
carbon dioxide generating tablet. Buffered glycerol-saline
- For transport of Neisseria gonorrhea and is a common stool transport medium, but is
Moraxella spp. not preferred because it is toxic to enteric
- Contained Sodium Bicarbonate and Sodium bacteria like Vibrio and Campylobacter
Citrate
4. Anticoagulants
5. The specimen should be transported to Are used to prevent clotting of
the laboratory immediately after blood, bone marrow and synovial
collection fluid
- Shipping of infectious substances: - 0.025% (w/v) Sodium Polyanethol
there should be at least triple Sulfonate (SPS) – is used for blood
package culture media because Neisseria
- Primary receptacle spp and some anaerobic bacteria
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College of Medical Technology

are sensitive to higher concentration digested and decontaminated can be

refrigerated and processed once a day
Importance of 0.025% SPS:
1. To inhibit phagocytosis and LEVELS OF SPECIMEN PRIORITIZATION
complement activation Always prioritize clinical bacteriology culture!!
2. To neutralize bactericidal effect of (to prevent contamination)
serum
3. To neutralize aminoglycoside antibiotics
Heparin – for mycobacteria and
viral cultures; it may inhibit growth
of Gram-positive bacteria and yeast
4. Use for bone marrow or synovial fluid
5. Not used: citrate and EDTA
6. Higher concentrations can inhibit N
gonorrhoeae

Blood culture bottles

Mostly with ARD (Antimicrobial Removal device) Direct Microscopic Examination
- Penicillinase :penicillin ● It is usually not performed on throat,
- Magnesium sulfate: tetracycline nasopharyngeal or stool specimens
- P-Aminobenzoic acid: sulfonamide - Because they contain normal flora;
direct microscopy examination’s
Specimen Prioritization purpose is to give us an initial result
● When a specimen is received with about the culture
multiple requests but the amount of
specimen is insufficient, the clinician ● Sputum can be rejected if it represents
should prioritize the testing saliva and not lower respiratory tract
secretions by quantitation of white
● When multiple specimens arrive at the blood cells or squamous epithelial cells
same time, priority should be given to MACROSCOPIC
those that are most critical (CSF, Tissue, - SWAB OR ASPIRATE
blood and sterile fluids (no normal - STOOL CONSISTENCY (FORMED OR LIQUID,
flora)) BLOODY OR MUCUS)
● Urine, throat, sputum, stool or wound - VOLUME
drainage specimens can be saved later - FLUID: CLEAR OR CLOUDY
● AFB, viral and fungal specimens – batch MICROSCOPIC
processing - SPUTUM
● Specimens for AFB that need to be
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College of Medical Technology

- GIVE TECHNOLOGIST AND PHYSICIAN OF Serum for serological studies –

INFECTIOUS PROCESS frozen up to 1 week at -20°C
- GUIDE FOR WORK UP - Tissues or specimen for long storage = -70°C
- DICTATE THE NEED FOR NON ROUTINE OR
ADDITIONAL TESTING ● Sterile body fluids, pus, urine and
Notes to remember: sputum are inoculated directly onto
● Most routine bacterial cultures are held selected media
from 48 – 72 hours
● Cultures for anaerobes and broth ● Large volumes of sterile body fluids
cultures may be held for 5 – 7 days (peritoneal, pleural, continuous
● Unusual organisms may require special ambulatory peritoneal dialysis) are
medium or conditions beyond the routine concentrated to increase the recovery
of bacteria
SPecimen Storage
● If specimens cannot be processed ● Specimens received on swabs can be
immediately after receiving it in the inoculated directly to culture media
laboratory, the must be stored at:
● Decontamination – Legionella or
1. Refrigerator temperature - 4°C Mycobacteria (purpose: to remove
Specimens: Urine (24 hrs), stool, viral normal flora)
specimens, sputum, catheters and swabs
BACT ALERT 3D 240
- Automated Blood Culture System
For stool, if delay is no longer than
designed to do blood culture
2 hours, the specimen can be added
- For both aerobic and anaerobic
to Cary-Blair transport media
cultures
- Capacity: 240 blood culture bottles
Specimens suspected of containing
- 24 hours continuous scanning of
anaerobic bacteria should never be
bottles for growth
stored in the refrigerator
Advantage: quick identification of
bacterial growth in the bottles
2. Incubator temperature - 35°C
- Quality control incorporated
CSF should always be kept at this
Principle: It detects growth of bacteria through the
temperature at 6 hrs
colorimetric detection of CO2 produced by growing
- 3 collection tubes CHEM, MICRO and
organisms.The culture bottle itself contains 40 mL
HEMA
of media as well as an internal sensor that detects
3. Ambient (room) temperature – 22
CO2 as an indicator for microbial growth.
to 25°C
4. Freezer temperature - -20°C or -
70°C