SPECIMEN COLLECTION &

PROCESSING FOR MICROBIAL

DIAGNOSIS

DAKSHINA M NAIR
Ph.D. MEDICAL MICROBIOLOGY
DATE OF CLASS:09/08/24
SPECIMEN COLLECTION:
• Laboratory diagnosis of bacterial infections is useful for the following purposes:
Identification, Treatment and Surveillance purpose.
• Laboratory diagnosis of bacterial infections comprises of several steps—
specimen collection, direct detection, culture, identification and antimicrobial
susceptibility test, serology and molecular methods.
• Specimen collection depends upon the type of infections.
• The proper collection of specimen is of paramount importance for the isolation of
the bacteria in culture.
GENERAL PRINCIPLES
• Standard precautions should be followed for collecting and handling all
specimens.
• Before antibiotics start: Whenever possible, culture specimens should be
collected prior to administration of any antimicrobial agents
• Contamination with indigenous flora should be avoided, especially when
collecting urine and blood culture specimens.
• Avoid contamination by using sterile containers, wear gloves and masks.
• Swabs are though convenient but considered inferior to tissue, aspirate and body
fluids
• Container: Specimens should be collected in sterile, tightly sealed, leak proof,
wide-mouth, screw-capped containers
• Labeling: All specimens must be appropriately labelled with name, age, gender,
treating physician, diagnosis, antibiotic history, type of specimen, and desired
investigation name.
• Rejection: Specimens grossly contaminated or compromised or improperly
labelled may be rejected
• If anaerobic culture is requested, proper anaerobic collection containers with
media should be used.
• Specimen should not be sent in container containing formalin for microbiological
analysis.
SPECIMEN TRANSPORT
• The specimens should reach the laboratory for further processing as soon as
possible after the collection. If required appropriate transport media should be
used.
• For most of the specimens, transport time should not exceed two hours.
However, there are some exceptions:
• Specimens that require an immediate transport ( <15 mins) such as CSF and
body fluids, ocular specimens, tissue specimens, suprapubic aspirate and
bone specimen.
• Urine (midstream) added with preservative (boric acid) is acceptable up to 24
hours, otherwise should be transported within 2 hours
• Stool culture: Stool specimen should be transported within 1 hour, but with
transport medium (Cary-Blair medium) up to 24 hours is acceptable
• Rectal swabs—up to 24 hours is acceptable
• For anaerobic culture: Specimens should be put into Robertson’s cooked meat
broth or any specialized anaerobic transport system and transported immediately
to the laboratory.

Vacutainer swabs

Blood culture bottles

SPECIMEN STORAGE BEFORE PROCESSING
• Most specimens can be stored at room temperature immediately after receipt,
for up to 24 hours.
• However, there are some exceptions:
• Blood cultures—should be incubated at 37°C immediately upon receipt
• Sterile body fluids, bone, vitreous fluid, suprapubic aspirate—should be
immediately plated upon receipt and incubated at 37°C
• Corneal scraping—should be immediately plated at bed-side on to blood agar
and chocolate agar
• Stool culture—can be stored up to 72 hours at 4°C
• Urine (mid-stream and from the catheter), lower respiratory tract specimen,
gastric biopsy (for Helicobacter pylori)—can be stored up to 24 hours at 4°C.
Type of Infections Specimens collected

Bloodstream infection, sepsis Paired blood samples collected aseptically by two step
disinfection ( first with alcohol and then with
chlorhexidine); Adults – 8-10ml and infants – 1-3 ml

Diarrheal diseases Stool, rectal swabs

Meningitis Cerebrospinal fluid (CSF)

Infections in other sterile body parts Sterile body fluids- plural fluid, synovial fluid,
peritoneal fluid

Skin and soft tissue infections Pus/exudates, wound swabs, tissue bits

Anaerobic infections Tissue specimens, blood, bone marrow, sterile body

fluids.

Upper Respiratory infections Throat swab with membrane over the tonsil,
nasopharyngeal swabs
Type of infection Specimens collected

Lower Respiratory infections Sputum, endotracheal aspirate, bronchoalveolar lavage

(BAL).

Pulmonary tuberculosis Sputum, gastric aspirate from infants

Urinary tract infections (UTI) Midstream urine, for catheterized patients collect urine
from catheter tube and not from urobag

Genital infections Urethral swabs, cervical swabs

Eye infection Corneal scrapings, conjunctival swabs

Ear infections Swabs from outer ear, aspirate from inner ear
SAMPLE PROCESSING
• If proper labeling and details of the sample is missing, sample will be rejected.
• Macroscopic examination: The color, consistency and volume of the specimen is
noted.
• Microscopic examination: Gram staining or appropriate stains (eg: Acid fast
staining) are used to visualize the microorganisms.
• Culture: The specimen is inoculated on to appropriate culture medias to promote
the growth of microorganisms. The plates are incubated at appropriate
temperatures for the growth.
Isolation and identification:
• Colony morphology: after incubation, colonies are examined for size, shape and
colour on the culture plates.
• Biochemical tests: certain biochemical tests are performed to confirm the
organisms.
• Molecular testing: it includes PCR/VITEK for precise identification.
• Antimicrobial Susceptibility Testing: to determine the sensitivity or resistance of
bacteria to antibiotics.
• Final reporting will be done after complete identification.