Specimen Collection

• Tube with cotton-wool plug

  Shelf life: 3 weeks
• Tube with loose cap

  Shelf life: 2 weeks
• Container with screw cap

  Shelf life: 3 months
• Petri dishes, if sealed in plastic bags

  Shelf life: 4 weeks
• Loose cap is often used for liquid culture in tubes because they avoid accidental spill or loss of fluid unlike cotton-wool plugs that may absorb fluid upon contact
• Common Specimen for Bacterial Culture


  • Sputum
  • Blood
  • Urine
  • Stool
• Causes for Meningitis by Age Group


  • Neonates: Streptococcus agalactiae
  • Children: Haemophilus influenzae
  • Adult: Neisseria meningitidis
  • Elderly: Streptococcus pneumoniae
• Gross colony characteristics


  • Hemolysis
  • Size
  • Form/Margin
  • Elevation
  • Color
  • Density
  • Consistency
  • Pigment
  • Odor
• Hemolysis


  Lysis of the red blood cells surrounding or underneath the colony caused by enzymatic or toxic activity of bacteria
• S. galactiae


  Group B b-hemolytic Streptococci
• Hemolysis is grossly observed by putting the plate against a light. Clear areas should be visible, indicating hemolysis
• S. pyogenes


  Viridans-group Streptococci
• Inhibitors in MacConkey Agar


  • Crystal violet
  • Bile salts
  • Sodium Azide
• Size


  • Large
  • Medium
  • Small
  • Pinpoint
• General Guidelines for Specimen Collection


  • Specimen should be collected at the correct anatomic site and should be representative of the infection
  • Specimen should be collected at the acute phase of illness (2-3 days if viral infection)
  • Specimen should be collected before the initiation of antibiotic therapy
  • Specimen Preservation
  • Timing of Collection should be considered
  • Specimen should be collected under sterile, aseptic conditions to avoid contamination with normal flora
  • Calcium alginate, Dacron or Rayon swabs can be used for specimen collection for bacterial culture
  • The specimen must be labeled accurately with patient information, date, and the specific anatomic site
  • The specimen must be taken to the laboratory and examined promptly (within 2 hours)
  • All appropriate specimens should have direct microscopic examination
  • Always talk to the requesting physician or colleagues before discarding any specimen
  • Urine cultures and tissues from burn victims are plated quantitatively
• Semi-quantitative (General Isolation) Streak


  Generally used for the determination of urinary tract infection through quantitative measurement of the number of bacteria present in the urine
• Form or Margin


  • Filamentous
  • Irregular
  • Smooth
  • Rough
  • Rhizoid
• Filamentous margin is expected only in Bacillus anthracis, characteristic medusa head colonies in Blood Agar Plate
• Elevation


  • Flat
  • Raised
  • Convex or dome
  • Umbilicate
  • Umbonate
• Quantitative Streak

  No Urinary Tract Infection: Normal Flora @ <150,000 CFU/mL

  With Urinary Tract Infection: Normal Flora @ >150,000 CFU/mL + Staphylococcus saprophyticus @ =/>10,000 CFU/mL
• CFU


  Colony Forming Units
• The typical elevation is flat and convex
• For the inoculation of urine specimen for bacterial culture, a calibrated loop is utilized. Depending on the calibration, one inoculum contains 1 – 10 uL of urine
• Density


  • Transparent
  • Translucent
  • Opaque
• Specimen Storage Temperature Conditions


  • Refrigerator Temperature (4*C): Urine, stool, sputum, swabs, and viral specimens

  Incubator Temperature (35*C): CSF
• Swarming bacteria


  Characteristic of Proteus mirabilis and Proteus vulgaris, inhibited by the addition of Trimethoprim sulfamethoxazole
• Storage Requirement prior to Specimen Processing


  • Plate as soon as possible: Body fluids, Bone specimens, Gastric biopsy, Suprapubic aspirate of urine, Prostatic specimens

  24 hours at room temperature: Abscess, Outer ear specimens, Upper Respiratory Tract Specimens, Tissue Specimens, Genital Tract Specimens

  6 hours at room temperature: Inner Ear Specimens

  Urethral specimens – place immediately upon receipt in JEMBEC at 35*C

  Indefinitely at room temperature: Hair, Nail, Skin scrapings

  6 hours at 35*C: Blood, Bone Marrow, Corneal Scrapings

  24 hours at 4*C (refrigerator): CSF, Lower Respiratory Tract Specimens, Clean-voided midstream urine, Straight catheter urine

  72 hours at 4*C (refrigerator): Rectal Swab, Stool Culture
• Consistency


  • Brittle
  • Creamy
  • Dry
  • Waxy
• Pigment


  Inherent characteristic of a specific organism, e.g. Pseudomonas aeruginosa (green), Serratia marcescens (red), Staphylococcus aureus (yellow/gold), Chromobacterium violaceum (purple)
• Odor


  • Staphylococcus aureus (old sock)
  • Pseudomonas aeruginosa (grape-like)
  • Proteus mirabilis (putrid)
  • Nocardia (freshly lawned)
  • Haemophilus (mousy/mouse nest)
• Transport Medium (Holding Media)


  • Cary Blair: For stool pathogens

  Stuart's: Virus transport media

  Amies: Respiratory pathogens

  Transgrow: For Neisseria

  JEMBEC: For Neisseria gonorrhoeae

  Todd Hewitt: Streptococcus agalactiae
• Reasons for Specimen Rejection


  • The information on the label does not match the information on the requisition slip

  The specimen is transported at an improper temperature

  The transported specimen has not been placed in the proper medium

  The quantity of specimen is not sufficient (QNS) for testing

  The transport specimen exceeds the two hours post collection period

  The specimen is preserved in a fixative (formalin)

  The specimen has already dried up upon transporting to the laboratory

  The specimen is leaking

  More than one specimen from the same source (except for blood) and from the same patient is submitted on the same day

  Expectorate sputum in which the stain reveals <25 WBCs and >10 epithelial cells
• Bartlett's Criteria


  WBC/PMNs: <10 (score 0), 10-25 (<25) (score 1), >25 (score 2)

  Epithelial Cell: <10 (score 0), 10-25 (<25) (score -1), >25 (score -2)

  Accept: PMNs >25, Ep <10
• Specimen Prioritization Levels


  • Level 1 - Critical/Invasive: Amniotic fluid, blood, brain, CSF, serous fluids

  Level 2 - Unpreserved: Bone, feces, sputum, tissue, other body fluids not listed in level 1

  Level 3 - Quantitation required: Catheter tip, urine, tissue

  Level 4 - Preserved: Urine, feces, and swabs in holding media

  
• Specimen Types


  • Blood

  Cerebrospinal fluid (CSF)

  Gastrointestinal Tract Specimens

  Urinary Tract Specimens

  Respiratory Tract Specimens - Sputum

  Genitourinary Specimen

  Wounds and Aspirate
• Blood


  • Blood is normally sterile; used to evaluate bacteremia, septicemia, and fever of unknown origin

  The venipuncture site should be disinfected with 70% alcohol and povidone iodine

  Blood should be drawn during the febrile episode, or 30 – 45 minutes before fever spikes

  Povidone iodine should be in contact with the skin for 30 – 60 seconds

  One set of culture bottle should be drawn: one for aerobic culture (10-15 mL adults, 2.5-5 mL children) and one for anaerobic culture (10-15 mL adults, 2.5-5 mL children)

  Concentration of SPS should NOT exceed 0.025% w/v

  Heparin can also be used as an anticoagulant

  For cases of Brucella, cultures should be incubated for 3 weeks

  Signs of growth: Hemolysis, Gas bubbles, Turbidity
• Cerebrospinal fluid (CSF)


  • Collected through lumbar puncture or spinal tap

  CSF is normally sterile; used to evaluate meningitis

  Disinfect the skin before aspiration

  Required volume upon collection depends on the clinical area needed: 1st tube - clinical chemistry/serology, 2nd tube - microbiology for culture, 3rd tube - clinical hematology for cell count, 4th tube - additional tests for microbiology

  Primary isolation medium: Chocolate Agar

  Minimum quantity: Bacteria - >1 mL, Fungi - >2 mL, AFB - >2 mL, Virus - >1 mL
• Gastrointestinal Tract Specimens


  • Stool is the specimen of choice for detecting gastrointestinal pathogens

  Rectal swab is collected by inserting a swab 2.5 cm past anal sphincter

  Overgrowth of pathogens by normal flora should be prevented, selective media can be used

  Patient should excrete directly to the container

  Specimen should not be taken from the toilet bowl

  Specimen should not be contaminated with urine or water
• Urinary Tract Specimens


  • Includes midstream clean catch urine, catheterized urine, and suprapubic aspirate

  Ideally, first morning urine is used since it is more concentrated

  In midstream clean catch, the patient is instructed to cleanse the genital

  Catheterized urine may be collected by inserting catheter into the bladder, the allow passage of first 15 mL

  Suprapubic aspirate is collected via needle aspiration

  Inoculate urine in plating media using calibrated loop (1 uL or 10 uL) in order to perform colony count

  Colony forming units / mL is calculated by dividing the number of colonies counted, by the volume capacity of loop in mL
• Respiratory Tract Specimens - Sputum


  • Ideally collected in the morning; rinse mouth or gargle with water then instruct patient to deep cough and expectorate into a sterile container

  Three morning sputum specimens for acid fast smears; morning specimens are preferred since they are more concentrated

  Should be gram stained and checked for acceptability using Bartlett's criteria