Bacte lec 4
- 1. Specimen Collection, Processing and Handling
- 2. KEY POINTS • Collect specimens from site of infection prior to initiation of therapy. • Collect adequate volume of sample for testing required. • For bacterial, fungal and viral cultures submit tissue, fluid/aspirate if possible as these are always superior to a swab specimen.
- 3. KEY POINTS • Use required collection and transport materials to preserve specimen integrity. • Communicate clear orders and source information. • Expedite the transport of specimens to the laboratory and do not allow them to sit in collection areas.
- 4. GENERAL PRINCIPLES • TIMING OF SPECIMEN COLLECTION – Should be collected at a time when the likelihood of recovering the suspected pathogen is greatest. • RECOVERING VIRUSES : greatest in the acute phase of the illness. • RECOVERING BACTERIA : ideally be collected before antimicrobial therapy is started.
- 5. TIMING OF SPECIMEN COLLECTION • ENTERIC INFECTION – Organisms are in greater number during acute/diarrheal stage. • TYPHOID FEVER – Blood culture in the 1st week of infection. – Stool/Urine culture on the 2nd week – Serological tests in the 3rd week
- 6. SPECIMEN VOLUME • MUST BE ADEQUATE for performance of the microbiological studies requested. • IF INSUFFICIENT : notify nurse/physician for additional sample/to prioritize the requests. • IF A SWAB : – use polyester-tipped swab on a plate shaft – avoid calcium alginate swab for viral culture (inactivates herpes simplex virus) • cotton toxic :Neisseria gonorrhoeae • Wood toxic : Chlamydia trachomatis
- 7. SPECIMEN VOLUME • SWAB : not optimal for detection of anaerobes, mycobacteria/fungi, do not use. • An actual tissue sample of fluid aspirate is always superior to a swab specimen for the recovery of pathogenic organisms.
- 8. SPECIMEN COLLECTION • Obtained from the site of infection with minimal contamination from adjacent tissues and organ secretions and collected in a sterile container. • LABEL : name & identification number of the patient, the source of specimen, date and time of collection.
- 9. SPECIMEN TRANSPORT • Placed in a biohazard bag and transported to the laboratory as soon as possible. • IF DELAY IS UNAVOIDABLE : refrigerate to prevent overgrowth of microbiota. – Urine, sputum, other respiratory specimens, stool. – Specimens for detection of Chlamydia trachomatis/viruses. • Cerebrospinal fluid (CSF) and other body fluids, blood, specimens collected for recovery of – Neisseria gonorrhoeae : room tempt., if refrigerated adversely affects recovery of potential pathogens froms these sources.
- 10. UNACCEPTABLE SPECIMENS • SPECIMENS – Received in formalin – In containers from which the sample has leaked – That have been inoculated on to agar plates that have dried out/oudated – Contaminated with barium, dye/oily chemicals • 24hr sputum collection • Foley catheter tips. • Duplicate specimens (except blood cultures) received in a 24hr period • Blood catheter tips submitted for patients w/o concomitant positive blood culture.
- 11. SPECIMEN REJECTED FOR ANAEROBIC CULTURE: • Gastric washings • Urine other than suprapubic aspirate • Stool (except for recovery of Clostridium difficile ) – for epidemiologic studies/ for diagnosis of bacteria associated with food poisoning. • Oropharyngeal specimens (except deep tissue samples obtained during a surgical procedure) • Sputum • Swabs of ileostomy/ colostomy sites. • Superficial skin specimens.
- 12. UNIVERSAL PRECAUTIONS • Must be followed when handling all specimens • APPROPRIATE BARRIERS : to prevent exposure of skin & mucous membranes to the specimen • PPE – Gloves and a lab coat must be worn at all times – Masks, goggles and impermeable gowns/ aprons must be worn in situation which risk of splashes/ droplet formation • Open specimen container in serological safety cabinet.
- 13. REFERRAL TESTING • MUST BE PACKAGED – Shipping specimens/cultures to reference lab – Dangerous good shipping guidelines (IATA website) • SPECIMENS : not more than 40 ml • Cultures of bacteria and fungi – Grown in solid media and tubes • Cap sealed with waterproof tape
- 14. REFERRAL TESTING • Sufficient packing (1°, 2°) – Leak proof – Prevent from breaking – Individually wrapped/separated • Itemized list of contents enclosed, marked/labeled properly • Labeled with an official label – Address, contents, name, telephone number of person responsible for shipping.
- 15. CLINICAL SPECIMENS • BLOOD & BLOOD COMPONENTS • THROAT & NASOPHARYNGEAL DISCHARGES • SPUTUM URINE • CEREBROSPINAL FLUID (CSF) • STOOL • GENITAL DISCHARGE • WOUND DISCHARGE
- 16. BLOOD • Detect blood-borne pathogens • Identify bacteria responsible for – Bacteremia – Sepsis infections of the native and prosthetic valves – Suppurative thrombophlebitis – Mycotic aneurysms – Infections of vascular grafts • Collect before beginning antimicrobial therapy
- 17. BLOOD • When any one/ a combination of the following: – Fever (38°C or greater) – Hypothermia ( 36°C or lower) – Leukocytosis (especially with a left shift) – Granulocytopenia – Hypotension
- 18. BLOOD : SPECIMEN COLLECTION • Phlebotomy technique – Minimize contamination with skin flora – 1st: Skin cleaned with 70% alcohol – 2nd: Apply 1-2% iodine solution, an iodophor/chlorhexidine; dry for 1-2minutes prior to venipuncture.
- 19. BLOOD : APPROPRIATE TIMING FOR DETECTION OF BACTEREMIA & FUNGEMIA • Optimal time to collect : just before a chill (not predictable) • Collected after the onset of fever and chills • Needle and syringe – Without changing needles – Injected directly into bottles of culture media – Inverted several times to mix – Transported to lab at room temp. as soon after collection is possible. – SHOULD NOT BE REFRIGERATED
- 20. BLOOD : SPECIMEN VOLUME • Bacteremia in adults (CFU/ml) = low – 20-30 ml of blood per culture set = strongly recommended. • Infants and children = higher concentration of microorganisms – 1-5 ml of blood per culture = adequate
- 21. BLOOD : SPECIMEN DRAWS • Number of blood specimens = based in the nature of bacteremia – Transient, Intermittent or Continuous – TRANSIENT BACTEREMIA • Follows manipulation of a focus of infection, instrumentation of a contaminated mucosal surface or a surgical procedure in a contaminated site; also in the course of many systemic and localized infections. – INTERMITTENT BACTEREMIA • Associated with an undrained abscess – CONTINUOUS BACTEREMIA • Hallmark of intravascular infection
- 22. BLOOD : SPECIMEN DRAWS • 2-3 20 ml blood samples drawn over 24hr period, aerobic and anaerobic blood culture bottles (sufficient) • 4 blood cultures within 24hr = yielded more • Suggested 30-60 min time interval for the first 2 sets, another 1-2 sets drawn (remaining 24hr) • If antimicrobial therapy is deemed urgent, collect before therapy, 2 separate sites within few minutes.
- 23. BLOOD : SPECIMEN DRAWS • Frequent blood culture contaminants but also true pathogens: – Coagulase – negative Staphylococci – Viridans Streptococci – Corynebacteria – Bacillus spp. , Propionbacterium spp. • 2 sets of blood cultures per febrile episode recommended for differentiation (present in only 1 bottle = contamination)
- 24. RECOVERY OF MICOORGANISMS • Factors that may impede recovery of pathogens: – Antibodies – Complement – Phagocytic WBC – Antimicrobial agents • Approaches : – Dilute blood specimen in broth medium in a 1 : 10 ratio = optimal neutralization of the serum bactericidal activity.
- 25. CONT.. • Incorporating 0.02-0.05% sodium polyethanol sulfonate in the blood culture medium = inhibits – Coagulation – Phagocytosis – Complement activation – Inactivates aminoglycosides • Other methods: – Adding penicillinases to broth media to inactivate penicillins – Using antibiotic – absorbent resins and lysis – centrifugation system
- 26. MANUAL BLOOD CULTURE • CULTURE BOTTLES – Examined daily for 7 days for evidence of growth – Indicated by turbidity, hemolysis, gas production, discrete colonies/combination of these. • If growth + – smear, stain with Gram stain, – read subculture + broth to appropriate agar media • Routine subculture of macrospically negative – broths performed after 6 – 18hrs of inoculation; anaerobic bottle : unnecessary
- 27. CONT.. – ADVANTAGES : low cost, low contamination rate – DISADVANTAGE : time consuming process of routine bottle examination and subculture BIPHASIC SYSTEM – Consists of a broth medium in a bottle to which a chamber containing agar media on a paddle is attached. – Colonies on the agar ate used for identification and susceptibility testing.
- 28. CONT.. LYSIS – CENTRIFUGATION SYSTEM – Consist of a tube containing reagents that inhibit • coagulation and the complement cascade • lyse blood cells • provide a cushion for the microorganisms during centrifugation. – Blood added to tube • Inverted several times, prevent from clotting • Transported ASAP • Centrifuged 30 min at 3000x g • Discard supermatant • Sediment vortexed and plate to agar
- 29. CONT.. – ADVANTAGES : • excellent recovery of Staphylococcus aureus, some Enterobacteriaceae, and fungi (best for molds, Histoplasma capsulatum) • Direct availability of colonies for identification and suspectibility testing • Ability to carry out quantitative cultures • FLEXIBLE : inoculate to special media (Legionella & mycobacteria) – DISADVANTAGES : • Labor – intensive • Less likely recover Steptococcus pneumoniae , Haemophilus influenzae/ anaerobs • Increased risk for contamination
- 30. HANDLING OF SPECIMEN • Specimen can be placed in transport medium: – TRANSGROW : Neiserria gonorrhoeae – AMIES : Fastidious organisms – CARY & BLAIR/STUART’s : Fecal specimen • Urine may be refrigerated but it must be examined within 24hrs • Stool and sputum could also be refrigerated but it must be examined within 2 – 3 hrs.
- 31. • CSF for bacterial culture : – Incubate for not more than 12 hrs. – Stand at room temp not longer than 1 hr. – DO NOT REFRIGERATE : some organisms are sensitive to low temp. • Neisseria meningitidis, Haemophilus influenzae • CSF for viral culture : – Refrigerate immediately – If held for more than 24hrs, freeze specimen at - 70°C
- 32. BLOOD • GENERAL CONSIDERATIONS : – Collect blood during febrile episode – During a chill – TIMING = + BC depends on the pathogenic process of the organism – Obtain 2 simultaneous blood cultures in 2 sites (right and left arm) • Single negative blood culture doe not rule out bacteremia. – Collect blood aseptically – Ideal volume is 10 ml.
- 33. • Use anticoagulant (e.g. SPS) • If Px is on penicillin, administer penicillinase – Not effective against methicillin, cloxacillin, nafcillin • Hold for 2 weeks before reporting for negative – 21 days for Brucella spp. • Indications of (+) blood culture : – Turbidity – Hemolysis – Colony or pellicle formation – Presence of gas or bubble
- 34. • MEDIA USED : – Trypticase Soy Broth – Brain – Heart Infusion – Columbia Broth – Castaneda Broth – Brucella Broth – Thioglycollate Broth • ANTICOAGULANT : – SODIUM POLYANETHOL SULFONASE • Neutralization bactericidal effect of serum • Prevents phagocytosis • Inactivates some antimicrobial agents
- 35. BLOOD COMPONENTS • Organisms found in fatal transfusion reactions are psychrophilic • Gram negative bacilli – Pseudomonas spp. EXCEPT Pseudomonas aeruginosa • PLATELET
- 36. THROAT & NASOPHARYNGEAL • Alpha strep (abundant) but Group A strep.(common pathogen) • Culture must include anaerobic conditions for Beta strep. • MEDIA – Haemophilus influenzae : CAP or BAP w/Staph – Neisseria meningitidis : CAP /Thayer Martin – Bordetella pertussis : Charcoal cephalaxin
- 37. SPUTUM • SUITABLE Sx : – < 10 epithelial cells & >25 pus cells – Early morning sputum • PROCEDURE : – Make a direct smear for GS & AFS – Do concentration technique (ex. NaOH) • Free bacteria by dissolving fats and mucus – Culture
- 38. URINE • Container : sterile wide mouth glass or plastic jar with tight – fitting lids. • Method : – SUPRAPUBIC ASPIRATION (IDEAL) – CATHERTERIZATION/CYSTOSCOPY – CLEAN CATCH MIDSTREAM • Collection : early morning • Processing : within 2grs after collection
- 39. URINE • GS of uncentrifuged Sx for rapid screening of UTI • Presence of numerous squamous cells indicate vaginal/ urethral contamination • PROCEDURE : – Inoculate the Sx in BAP, EMB, Mac using a calibrated loop (0.01 or 0.001 mL) – Incubate overnight – No growth for the 1st 24hrs, continue for 24hrs more before reporting negative.
- 40. Actual # of colonies X Calibrations of loop = # of CFU/mL 100,000 bacterial CFU/mL (indicate infection) • POUR PLATE METHOD : – Prepare 1 : 100 dilution of urine w/ sterile H20 – Transfer 0.1mL of the solution to the Petri dish – Add agar and mix well – Incubate for 18 – 24hrs – Count colonies and multiple by 100 – Report result in bacteria/mL
- 41. CEREBROSPINAL FLUID • PROCEDURE – Centrifuge Sx; make a smear for GS and India ink – Culture Sx • Haemophilus influenzae type B • Neisseria meningiditis • Steptococcus pneumoniae • MEDIA – TSB/thiodlycollate – BAP for Gram (+) cocci – CAP for Gram (-) cocci – EMB/Mac for Gram (-) bacilli
- 42. STOOL CULTURE • Ideal Specimen : – Freshly collected stool on early stage of disease – Rectal swab may be used • Amount : 1-2 grams • Container : Clean, wide mouth with lid • Transport time : 2hrs after collection • Transport medium : Cary Blair
- 43. STOOL CULTURE • PROCEDURE – Put rectal swab in enrichment broth or transport medium – GS is NOT usually done but helps in identifying etiologic agents • Gram + cocci in clusters (Staphylococci) • Gram – comma-shape bacilli (Vibrio) • Gram + bacilli in large numbers • Gram – bacilli
- 44. • 1st day – Inoculate the Sx and incubate it overnight – MEDIA : • Differential (EMB, Mac) • Selective (SSA, HEA, XLD) • Enrichment (Selenite F, APW) • 2nd day – Check diff’l media for LF and NLF – Subculture and do biochemical tests – If there’s no growth, inoculate culture from the enrichment medium into EMB or Mac • 3rd day – Note the patterns of biochemical reactions – If suggestive of Salmonella Shigella, Vibrio DO serological typing – If growth occurs after doing step 2 of 2nd day, subculture into biochemical test media – Incubate overnight and perform first step of day 3.
- 45. EXUDATES & TRANSUDATES • Wounds • Boils • Abscesses • Ulcers • Granules • Rash • Gastric Aspirates • Tissues • Eye discharge • Ear discharge • Effusions • Endocervical • Urethral • Anorectal discharge
- 46. EXUDATES & TRANSUDATES • Discharge/ Fluids : best to aspirate – Dry wound : moisten swab w/ NSS before collecting – Skin lesion : remove crust of pustule/vesicle cap then swab lesion (Tzanck smear) – Endocervical : use swab – Urethra : use swab or scrape mucosa of anterior urethra – Anorectal : insert swab about 4-5cm. Into the anal canal
- 47. COLLECTION • Aspirates : sterile vial • Ulcerative lesions : biopsy w/o preservations • Irrigation, intravenous : sterile vial • Intra-arterial Catheter tips : sterile vial • Swabs : 2 pcs in sterile tube • Fluids : syringe w/ sterile rubber stopper • Corneal scraping : direct inoculation
- 48. GENITAL DISCHARGE • SPECIMEN – Cervical (female) – Urethral (male) – Rectal & throat swabs • STD caused by : – Treponema pallidum – Neisseria gonoreheae – Chlamydia trachomatis – Candida albicans – Gardenerella vaginalis – HSV (Herpes Simplex Virus)
- 49. SUCCESSFUL RECOVERY OF ETIOLOGIC AGENTS DEPENDS ON: • Advance planning • Collection of appropriate and adequate specimen • Correct packaging and rapid transport to the laboratory • Ability of the laboratory to accurately perform the diagnostic tests
- 50. GENERAL GUIDELINES FOR SPECIMEN COLLECTION • Obtain specimens before treatment • Collect material from the appropriate site • Obtain material for culture during the acute stage of the illness • Collect sufficient quantity • Transport the specimen immediately • Label all specimen • Specimens should be accompanied with a request form
- 51. CLINICAL SPECIMEN FOR BACTERIAL CULTURE • Blood • CVD • CSF • Respiratory • Urine • Transudates/Exudate s • Feces/Rectal swab • effussions
- 52. CRITICAL TIME FOR DELIVERY OF SPECIMEN OF THE MICROBIO LAB SPECIMEN DELIVERY TIME Respiratory (sputum, throat culture) 1HR Gastrointestinal (stool, rectal, swab etc.) 1HR Blood culture 1HR Anaerobic 30 MIN Cerebrospinal Fluid (CSF) IMMEDIATELY Other Fluids IMMEDIATELY
- 53. CRITICAL TIME FOR DELIVERY OF SPECIMEN OF THE MICROBIO LAB SPECIMEN DELIVERY TIME Urine 1HR Wound, skin & soft tissue 30MIN Fungal 1HR Mycobacterial 1HR Chlamydia 1HR Except for CSF, body fluids specimens should be delivered w/in 30mins. – 1hr to the lab.
- 56. VARIABLES AFFECTING BLOOD CULTURES • Number of Collections : 2-3 collections/ 24hrs • Volume of Blood YOUNG CHILDREN : 1-2mL in 20mL broth (1:10 to 1:20) ADULTS : 5-10mL in 50mL broth (1:5 to 1:10) • Broth Medium : TSB or BHI • Normal Bactericidal Properties of Blood • Sodium PolyanetholsulFonate (SPS): 0.025% Gelatin : 1% Agar : 0.1%
- 57. COMPONENT OF BLOOD CULTURE BROTH 1 % Gelatin • enhance growth of Neisseria meningitidis 0.1 % Bacto-agar • enhance growth of anaerobic organism • anticoagulant 0.025 % Sodium Polyanetholsulfonate (SPS) • inhibit activity of complement & lysosyme • prevents phagocytosis • inactivates therapeutic concentration of aminoglycosides
- 58. BLOOD CULTURE Incubation Temperature : 35°C – 37°C Incubation Time : 7 DAYS Subculture on BAP & CA : after 14 – 17hrs 3days, 5days, 7days Note: BAP & CA should be incubated at 35°C - 37°C under CO2 enhanced atmosphere.
- 59. COLLECTION & TRANSPORT OF CSF Container Dry Sterile screw caps Volume of sample 3 to 4 mL culture others Transport time immediately w/o delay
- 60. CSF • The specimen should be taken before antibiotics are administered. • Antigen tests may give a specific diagnosis even after antibiotic treatment. • CSF should always be taken from patients with suspected purulent meningitis, regardless of antibiotic treatment.
- 61. CSF SPECIMEN • Volume : 0.5 – 5.0mL • Time of Collection : first week of illness – 1st tube : cell count and differential staining – 2nd tube : Gram’s stain and culture – 3rd tube : protein, glucose and special tests
- 62. Suitability of Sputum for Culture Sputum Classification Based on WBCs & Squamous Epithelial Cell Densities Cell numbers per x 100 (low power) field Group WBCs Epithelial Cells 6 <25 <5 5 >25 <10 4 >25 10-25 3 >25 >25 2 10-25 >25 1 <10 >25 “Only sputum samples in categories 4-6 should be cultured”
- 64. TRANSPORT MEDIUM • Stuart • Amies • Cary Blair