SPECIMEN COLLECTION IN THE MICROBIOLOGY LAB

• Standard Operating Procedures in the Microbiology Lab

  1. Specimen collection

  2. Macroscopic description and direct microscopic examination (DME)

  3. Inoculate culture media

  4. Positive culture

  5. Specie ID

  6. AST

  7. Communication of laboratory findings
• Blood culture is performed to determine the etiologic agent of septicemia
• Types of specimen for blood culture

  • Blood
• Criteria for specimen rejection for blood culture

  • Blood collected using anticoagulants other than sodium polyanetholsulfonate 0.025%
  • When venipuncture procedure did not adhere to proper aseptic technique
  • Prolonged standing of blood for > 2 hours
• Common agents of infective endocarditis

  • Viridans streptococci
  • Enterococci
  • Streptococcus bovis
  • Staphylococcus aureus
  • Staphylococci (coagulase-negative)
  • Enterobacteriaceae
  • Pseudomonas spp. (Usually seen in drug users)
  • Haemophilus aphrophilus
  • Unusual gram neg bacilli e.g. Actinobacillus, Cardiobacterium, Eikenella, Coxiella burnetti
• Common agents of IV catheter-associated bacteremia

  • Staphylococcus epidermidis
  • Staphylococci (coagulase-negative)
  • Staphylococcus aureus
  • Enterobacteriaceae
  • Pseudomonas aeruginosa
  • Candida spp.
  • Corynebacterium spp.
  • Other gram neg rods
• Venipuncture for blood culture collection

  1. Review request form and identify the patient

  2. Palpate for the vein with a gloved finger

  3. Cleanse the skin with 70% ethanol or isopropyl alcohol

  4. Cleanse again with 2% chlorhexidine (preferred) or 2% iodine

  5. Initiate venipuncture via closed evacuated tube system using the winged infusion set(preferred) or open system (syringe method)

  6. Inoculate the blood culture bottles with the sample if blood is collected via open system then transport the bottles to the lab ASAP
• Blood culture bottles

  • No need to change syringe needles when inoculating BD Bactec bottle
  • Do not refrigerate blood culture bottles
  • Incubate the bottles for 7 days before reporting a negative result
• Best practice for blood culture

  • Volume of blood sample: Adults 3-10 ML, Pediatric patients 0.5-5ml
  • Frequency of sample collection: 2-4 blood samples drawn at different time intervals but not more than 3 draws within a 24 hour period
  • The collection of a single sample is discouraged because the volume of blood cultured is not sufficient for detecting some infections
• Anticoagulant

  0.025-0.05% sodium polyanethol sulfonate
• BD Bactec resin media


  • Polymeric adsorbent resin
  • Cation exchange resin
  • Increases microbial recovery and time to detection by neutralizing antibiotic effect, increasing surface of attachment for bacteria to grow, mechanical action to break up RBC and WBC to release intracellular bacteria, binding blood components that target bacterial cells, binding toxic substances from cellular growth
• Automated blood culture bottles are used for blood culture
• CSF culture is performed to diagnose bacterial or fungal meningitis
• Types of specimen for CSF culture

  • Cerebrospinal fluid
• Criteria for specimen rejection for CSF culture

  • CSF collected in a non-sterile container
  • Unlabeled containers
  • Specimen is collected > 30 mins
  • CSF sample is collected from patients undergoing antimicrobial therapy
• Common agents of bacterial meningitis

  • Haemophilus influenzae
  • Neisseria meningitidis
  • Streptococcus pneumoniae
  • Group A and Group B Streptococci
  • Enterobacteriaceae
  • Listeria monocytogenes
• Common agents of viral meningitis

  • Coxsackieviruses A and B
  • Herpes Simplex Virus
  • Arboviruses
  • Mumps virus
• Common agents of meningitis of parasitic origin

  • Naegleria fowleri
  • Toxoplasma gondii
  • Entamoeba histolytica
  • Strongyloides stercoralis
• Common agents of chronic meningitis

  • M. tuberculosis
  • Cryptococcus neoformans
  • Coccidoides immitis
  • Histoplasma capsulatum
  • Blastomyces sp.
  • Candida sp.
  • Nocardia
  • Actinomyces
• CSF specimen collection

  1. Only physicians can collect CSF

  2. Done via lumbar tap or spinal tap between L3 and L4 vertebrae

  3. Disinfect skin with iodine or chlorhexidine before puncture

  4. Collect 3 tubes
• Quantity of CSF

  Min. 5-10 ml
• Allowable transport time for CSF

  Less than 15 mins and processed immediately
• Storage of CSF


  Room temperature. Do not refrigerate.
• Specimen processing for CSF


  1. Cytocentrifugation at 1500g for 15 mins

  2. Decant the supernatant into a sterile tube leaving approximately 0.5 ml of fluid to suspend the sediment

  3. Supernatant can be used for antigen testing for rapid diagnostic tests that detects polysaccharide capsular antigen of H. influenzae, N. meningitidis, and S. pneumoniae

  4. The supernatant should be kept even if it has o use

  5. Make a smear by putting a drop of CSF on the center of the slide and allow it to dry

  6. Heat fix the smear or use methanol to fix
• Tubes for CSF specimen

  • Tube 1 - Chemistry & Immunology Dept.
  • Tube 2 - Microbiology Dept.
  • Tube 3 - Hematology Dept.
• Direct microscopic examination of CSF

  1. Perform Gram stain to report for the presence of erythrocytes, inflammatory cells, and bacteria

  2. Perform Methylene blue stain (rapid)

  3. Perform India Ink for the detection of Cryptococcus neoformans

  4. Perform wet mount to observe for motile amoeba causing Primary Amoebic Meningoencephalitis (PAM)
• India ink positive


  • Naegleria fowleri
• Wet mount is used to diagnose Primary Amoebic Meningoencephalitis (PAM)
• Tube 2 - Microbiology Department

  • For culture and sensitivity test
• Tube 3 - Hematology Department

  • For total and differential WBC count which is essential to differentiate bacterial vs. non-bacterial meningitis
• Normal CSF glucose

  50-75 mg/dl
• Normal CSF proteins

  20-40 mg/dl
• Bacterial meningitis

  Decreased CSF glucose, Increased CSF proteins
• Viral meningitis

  Normal or slightly increased CSF glucose, Normal CSF proteins
• CSF culture


  1. Inoculate BA,CA,MAC, EMB, THIOGLYCOLATE BROTH using several drops of the resuspended sediment

  2. Incubate plates at 37oC for at least 72 hrs

  3. Incubate broth at room temp for 5 days
• Results of microscopy and all positive cultures are reported immediately to the physician
• Throat culture is performed for indications like tonsillitis, pharyngitis, epiglottitis, gonorrhea, diphtheria, and Vincent's angina
• Common pathogenic bacteria from throat swab

  • Group A beta hemolytic Streptococci
  • Klebsiella pneumoniae
  • Bacteroides spp. and other anaerobes
  • Corynebacterium diphtheriae
  • Neisseria gonorrheae
  • Haemophilus influenzae
  • Bordetella pertussis
• How to collect throat swab for culture

  1. Identify the patient

  2. Prepare necessary materials

  3. Using a tongue depressor, introduce the swab between the tonsils without touching the buccal cavity. Let the patient say "aaaah"

  4. Swab back and forth across the posterior pharynx, both tonsils, any areas of inflammation, exudation, or ulceration

  5. Place the swab in a transport medium and send to the lab ASAP

  6. Transport time: < 2 hrs at RT

  7. Storage: < 24 hrs, RT
• Culture media for throat swab

  • BA - stab
  • CA - incubate in candle jar with 5 % CO2 at 35oC
  • MAC or EMB
  • THIOGLYCOLATE BROTH
  • MODIFIED THAYER MARTIN (MTM)