COLLECTION, TRANSPORT

and
PROCESSING of SPECIMEN
Julia Aira Marie C. Sanchez, RMT
Bacteriology, Section-In-Charge
TriCity Medical Center Inc.
LEARNING OBJECTIVES:

• At the end of this session, you should be able to learn:

1. The general guidelines used for collection of specimen
2. The ideal clinical specimen for aerobic culture
3. The guidelines for transport of clinical specimen for
aerobic culture.
GENERAL SPECIMEN SELECTION AND
COLLECTION GUIDELINES
• Collect specimen before antibiotic treatment
• Collect from appropriate site
• Practice proper and aseptic collection technique
• Ensure sufficient quantity
• Follow recommended time of transport
• Label specimen accordingly
• Accompany specimen with complete request form
CLINICAL SPECIMENS FOR AEROBIC
CULTURE
BLOOD
(Including Bone Marrow Aspirate)
When is blood culture requested?
Acute illnesses
Fever of unknown origin
Acute infective endocarditis
Suspected bacterial endocarditis
Still symptomatic ≥ 3 days after the 1st collection. Collection is done before
the next introduction of the antibiotics (must coordinate with the doctor and
the nurse).
BLOOD COLLECTION GUIDELINES

1. Wear proper PPEs.

2. Disinfect the culture bottle.
2.1 Apply sterile cotton balls with 70% isopropyl alcohol or
chlorhexidine wipes to rubber stopper and wait 1 min or let the cotton stay
until inoculation.
3. Venipuncture:
3.1 Palpate vein before disinfection of venipuncture site.
3.2 Cleanse the skin with 70% isopropyl alcohol.
(if no alcohol pad is available, use sterilized cotton balls w/ 70% isopropyl
alcohol)
BLOOD COLLECTION GUIDELINES

3.3 Disinfect the skin concentrically, with povidone-iodine (30 secs-

1 min.)
or chlorhexidine, 30 secs. (not for infants < 2 mos.)
3.4 Allow the disinfectant to dry. DO NOT PALPATE VEIN at this
point without sterile glove. (disinfect the gloves with alcohol if there is
a need to palpate again)
3.5 Collect blood the required amount of blood. (see next slide)
3.6 After venipuncture, remove iodine from the skin with alcohol.
BLOOD COLLECTION GUIDELINES
4. Inoculation into BCB
4.1 Remove the cotton from the rubber septum of the BCB
and make sure that it is dry before inoculation.
4.2 Without changing needle, directly inject blood into
culture bottle
*Note: Do not replace needle, Hepatitis or HIV infections
can be acquired from accidental needlesticks
4.3 Mix gently by swirling the BCB.
COLLECTION METHOD
BLOOD

• CULTURE MEDIA: BAP, CAP, MAC

• INCUBATION TEMPERATURE: 35˚C to
37˚C (BAP, CA: 5% CO2; MAC: ambient
air)
• INCUBATION PERIOD: 7 DAYS
• SUBCULTURE: 24 hours (preliminary
result), 3 days, 5 days, 7 days (final
result)
• MICROSCOPY: Gram stain from
BCB/BHIB (as requested)
COLLECTION METHOD
 CEREBROSPINAL FLUID
• 1st vial - protein and glucose and • What if the volume collected is
special tests insufficient?

• 2nd vial or last vial - Gram stain

and culture

• 3rd vial - cell count and

differential staining
CEREBROSPINAL FLUID
• VOLUME: 0.5 – 5 mL (sterile container)
• TIME OF COLLECTION: Within the 1st week of
illness.
• CULTURE MEDIA: BAP, CA, MAC
(BAP, CA: 5% CO2 MAC: ambient air)
• ENRICHMENT BROTH: BHI (incubate 24 hrs. then
subculture)
• TRANSPORT MEDIUM: Trans Isolate Medium
(optional)
• SUBCULTURE: 24 hours (preliminary result)
• INCUBATION PERIOD: 3 days (final result)
• MICROSCOPY: Gram stain and India ink (routine)
CEREBROSPINAL FLUID
OTHER BODY FLUIDS
• What and Where to collect?
1. Synovial fluid – from joints
2. Pleural fluid – from pleural cavity (space between lungs and
inner chest wall)
3. Peritoneal fluid – from abdominal cavity
4. Pericardial fluid –from pericardial sac
5. Hydrocoele – from testicles
OTHER BODY FLUIDS
• CULTURE MEDIA: BAP, CA, MAC
• INCUBATION TEMPERATURE: 35˚C to 37˚C
(BAP, CA: 5% CO2; MAC: ambient air)
• ENRICHMENT BROTH: BHI (incubate 24 hrs.
then subculture)
• SUBCULTURE: 24 hours (preliminary result)
• INCUBATION PERIOD: 3 days (final result)
• MICROSCOPY: Gram stain (routine)
TISSUE
• TRANSPORT DEVICE: Sterile, screw-capped container
• CULTURE MEDIA: BAP, CA, MAC
• ENRICHMENT BROTH: Thioglycollate/BHI
• INCUBATION TEMPERATURE: 35˚C to 37˚C (BAP, CA: 5% CO2;
MAC: ambient air)
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain (routine)
* Note: Add sterile saline to keep tissue moist
TISSUE
• Preparation of tissue for culture:
1. Place the tissue in a sterile petri dish
2. Slice the tissue into small pieces
3. Place the sliced tissue in sterile tissue homogenizer
(or use sterile mortar and pestle)
4. Add 2-3 drops of NSS or TSB/BHI
5. Macerate
6. Remove the plunger, add 3-5 drops or more, mix.
7. Inoculate: BHI, BAP, CA, MAC, GS
TISSUE
URINE
Methods of Collection:
1. Clean catch midstream
1.1 Females: wash with soap and water or use soap pad and rinse
with moistened pad, including the folds of the skin.
1.2 Males: simple cleansing of the urethral meatus
2. Cytoscopy or Catherization
2.1 cleanse the urethral opening with soap and water.
2.2 Rinse area with gauze pad aseptically, insert catheter and
allow 15 ml to pass, then collect urine in a sterile container.
URINE
3. Indwelling catheter
3.1 disinfect the catheter collection port with 70% alcohol.
3.2 Clamp catheter below port and allow urine to collect in tubing
for 10-20 min.
3.3 Use needle and syringe to aseptically collect 5-10 mL of urine.
3.4 Transfer to a sterile container (only for symptomatic).
4. Suprapubic aspiration: for small children and neonates
*TRANSPORT DEVICE: sterile, wide mouth, leak-proof container.
URINE
URINE
• CULTURE MEDIA: BAP, MAC
• INCUBATION TEMPERATURE: 35˚C to 37˚C
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain (as requested)
* calibrated loop to be used for inoculation (continuous streaking)
URINE
EXUDATES
Collection Guidelines:

• DISINFECTION: Remove surface exudate by wiping with sterile

saline or 70% alcohol
 OPEN WOUND: aspirate or swab deep into the lesion to
firmly sample the lesion’s “fresh border”
 CLOSED WOUND: aspirate

WARNING: DO NOT USE SYRINGE FOR TRANSPORT!!!

EXUDATES
• CULTURE MEDIA: BAP, MAC, CAP
• ENRICHMENT BROTH:
Thioglycollate
• INCUBATION TEMPERATURE:
35˚C to 37˚C
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain
(routine)
WOUND DISCHARGE / ASPIRATES from
ABSCESS
URETHRAL / ENDOCERVICAL/VAGINAL/
PENILE / ANORECTAL DISCHARGE
• Collection Methods:
URETHRAL / ENDOCERVICAL/VAGINAL/
PENILE / ANORECTAL DISCHARGE
• COLLECTION METHOD: 2 Swabs
• CULTURE MEDIA: BAP, CAP, MAC,
MTM
• INCUBATION TEMPERATURE: 35˚C to
37˚C (BAP, CA, MTM: 5% CO2; MAC:
ambient air)
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain (routine)
URETHRAL / ENDOCERVICAL/VAGINAL/
PENILE / ANORECTAL DISCHARGE
EAR/ EYE DISCHARGE
• Collection Method
I. INNER EAR DISCHARGE: aspirate (if ear drum is intact) or swab
II. OUTER EAR DISCHARGE: moistened swab
III. EYE DISCHARGE: moistened swab (conjunctiva)
scrapings (corneal scrapings)
aspirate (vitreous fluid)
Note: collection will be done by the physician and inoculate directly on a plated
media.

WARNING: DO NOT USE SYRINGE FOR TRANSPORT!!!

EAR/ EYE DISCHARGE
• CULTURE MEDIA: BAP, GBA, BCA,
MAC
• ENRICHMENT BROTH:
Thioglycollate
• INCUBATION TEMPERATURE: 35˚C
to 37˚C
(BAP, CA, GBA: 5% CO2; MAC:
ambient air)
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain (routine)
UPPER RESPIRATORY TRACT:
NASOPHARYNGEAL SWAB (NPS)/ ASPIRATE (NPA)

 Bordetella Pertussis/B. parapertussis

Collection:
1. Gently elevate the tip of the nose.
2. Gently insert a small swab (with flexible
handle, i.e. Dacron or Rayon) into a nostril
straight back along the nasal septum until a
distinct feel of resistance - indicates that the
posterior nares has been reached.
NASOPHARYNGEAL SWAB/ASPIRATE
(NPS/NPA)
3. Rotate swab slowly for 5 sec. to absorb secretions.
4. Place in a Regan Lowe Transport medium
5. Cut the handle (plastic or wire) with a sterilized
scissors to fit into the container.
NASOPHARYNGEAL SWAB/ASPIRATE
(NPS/NPA)
 To consider B. pertussis/B. parapertussis
• TYPE OF SWAB: Dacron or Rayon swabs (2)
• CULTURE MEDIA: Regan Lowe
• TRANSPORT MEDIUM: Regan Lowe
Transport Medium
• INCUBATION TEMPERATURE: 35˚C to 37˚C
(moist environment)
• INCUBATION PERIOD: 7 days
• MICROSCOPY: NOT DONE
NASOPHARYNGEAL SWAB/ASPIRATE
(NPS/NPA)
NASAL SWAB or NOSE SWAB
MRSA (carrier)
• Collection:
1. Insert a swab, premoistened
with sterile NSS, approx. 1-2 cm
into the nares.
2. Rotate the swab slowly against
the nasal mucosa.
NASAL SWAB or NOSE SWAB
NASAL SWAB or NOSE SWAB
To consider MRSA:
• TYPE OF SWAB: Cotton swab
• CULTURE MEDIA: BAP
• TRANSPORT MEDIUM: Amies TM (general)
• INCUBATION TEMPERATURE: 35˚C to 37˚C
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: NOT ROUTINELY DONE
THROAT SWAB
• How to Collect Throat Swab?
Corynebacterium diphtheriae
 Streptococcus pyogenes (and other pyogenic spp.)
Neisseria gonorrhoeae
MRSA (carrier)
• Collection:
1. Instruct the patient to tilt the head back and breathe deeply.
2. Depress tongue with a tongue depressor.
3. Instruct the patient to say “ah”
4. Using a sterile swab, firmly rub the tonsils and posterior
pharynx, and other inflamed areas or any purulent area.
THROAT SWAB
Pseudomembrane
• Collection:
1. Collect piece/s of pseudomembrane or
2. Swab the inner portion of the pseudomembrane
• TRANSPORT MEDIUM: Amies TM
• CULTURE MEDIA: BAP,
CTBA (Cystine Tellurite Blood Agar)
• INCUBATION TEMPERATURE: 35˚C to 37˚C + 5% CO2
• INCUBATION PERIOD: 48 hours
• MICROSCOPY: Gram stain/Methylene Blue stain
THROAT SWAB
THROAT SWAB
LOWER RESPIRATORY TRACT
SPUTUM /ENDOTRACHEAL ASPIRATE /
TRACHEAL ASPIRATE
1. Bronchoalveolar lavage or wash
1.1 collect in sputum/mucus trap

2. Bronchial brush
2.1 Place brush in a sterile container
with1 mL sterile NSS
LOWER RESPIRATORY TRACT
SPUTUM /ENDOTRACHEAL ASPIRATE /
TRACHEAL ASPIRATE
3. Sputum, Expectorated - early morning collection
3.1 brushing the teeth and gargle or rinse with water
3.2 Instruct patient to cough deeply
3.3 Collect in a sterile container

Note: Do not collect specimen after meals at least 30 min before

collection
Suitability Criteria for Culture:
LOWER RESPIRATORY TRACT
SPUTUM /ENDOTRACHEAL ASPIRATE /
TRACHEAL ASPIRATE
• TRANSPORT DEVICE: sterile, leak-proof
container
• CULTURE MEDIA: BAP, GBA, BCA, MAC
• INCUBATION TEMPERATURE: 35˚C to SBAP BCA
37˚C (BAP, BCA, GBA: 5% CO2; MAC:
ambient air)
• MICROSCOPY: Gram stain (routine)
• INCUBATION PERIOD: 48 hours
MAC GBA
LOWER RESPIRATORY TRACT
SPUTUM /ENDOTRACHEAL ASPIRATE /
TRACHEAL ASPIRATE
STREAKING TECHNIQUE
REFERENCES
• Research Institute of Tropical Medicine
• Manual of Clinical Microbiology, 11th Edition, 2015. J. H.
Jorgensen, M.A. Pfaller,K.C. Carroll, G. Funke, M.L. Landry, S.S.
Richter, D.W. Warnock
• Koneman’s Color Atlas and Textbook of Diagnostic Microbiology,
6th Edition 20 . W.W. Winn Jr., S. Allen, W. Janda, E. Koneman, G.
Procop, P. Schreckenberger, G. Woods
THANK YOU!