Coll Trans Micro Specimens


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  • Coll Trans Micro Specimens

    1. 1. Principles of collection and submission of culture specimens to Microbiology lab Dr Kamran Afzal FCPS Microbiology
    2. 2. Introduction <ul><li>Use of even the most precise and exhaustive lab exam with inadequate clinical materials may produce grossly misleading results </li></ul><ul><li>It is necessary to teach medical assistants and discuss with clinicians the importance of proper collection and submission of lab specimens </li></ul><ul><li>Obtain material that matches to the desired analysis and is properly collected </li></ul><ul><li>Samples are often collected by the nurses; doctors are responsible for initiating treatment </li></ul>
    3. 3. <ul><li>Lack of knowledge that </li></ul><ul><ul><li>Body is colonised with microbial flora and ‘pathogen’ can be found as a part of normal colonizing flora </li></ul></ul><ul><ul><li>Unable to differentiate between ‘colonisation’ and ‘infection’ </li></ul></ul><ul><ul><li>Often positive ‘Bacteriology culture report’ is treated than patient </li></ul></ul><ul><li>Faulty collection and transportation often results in failure to isolate the causative organism </li></ul><ul><li>Inappropriate result / report </li></ul><ul><li>As a result inappropriate / excessive antibiotics are prescribed </li></ul>
    4. 4. <ul><li>Information derived from the results has impact on </li></ul><ul><ul><li>Diagnosis of infectious diseases </li></ul></ul><ul><ul><li>Antibiotic prescribing </li></ul></ul><ul><ul><li>Formulation of local antibiotic policy </li></ul></ul><ul><ul><li>Public health impact e.g., food handlers </li></ul></ul><ul><ul><li>Infection Control measures e.g., MRSA </li></ul></ul><ul><ul><li>Target for reduction of C difficile , MSSA and MRSA bacteraemias </li></ul></ul><ul><li>Specific procedures in collecting specimens for culture will certainly improve the quality of services of Microbiology Department </li></ul>
    5. 5. Gen considerations - collection <ul><li>Specimen obtained before start of antibiotic therapy </li></ul><ul><li>If started, inform lab about antibiotics </li></ul><ul><li>Clean skin with antiseptic using enough mechanical friction for cleansing </li></ul><ul><li>70% Ethyl alcohol: 2 min, 2% Iodine: 1 min, Povidone-Iodine: 1 min </li></ul><ul><li>Instruct patient appropriately where needed (sputum and urine) </li></ul><ul><li>Collect sufficient quantity, sterile container </li></ul>
    6. 6. <ul><li>Proper labeling </li></ul><ul><ul><li>Patient’s name, test type, date, clinical info </li></ul></ul><ul><li>Preferably collect in working hours </li></ul><ul><li>Collect specimen as early as possible in acute phase, these agents tend to disappear rapidly after the onset of symptoms </li></ul><ul><li>All specimens for viral culture to be frozen and stored at -70 o C </li></ul><ul><li>Collect acute and convalescent serum specimens along with specimen for viral isolation </li></ul>
    7. 7. Blood Culture Specimens <ul><li>Collect very carefully because misleading results may be obtained due to contaminants </li></ul><ul><li>Common contaminants are S. epidermidis , Micrococcus and diphtheroids from skin </li></ul><ul><li>Wash hands with soap and water, wear sterile gloves </li></ul><ul><li>Cleanse the skin over venepuncture site in a 5 cm circle with 70% Alcohol, leave for 2 min </li></ul><ul><li>Apply Povidone-Iodine in circles, leave for 1 min </li></ul>
    8. 8. <ul><li>Apply tourniquet, relocate the vein but don’t touch it </li></ul><ul><li>Again remove the Iodine with 70% Alcohol following the pattern of application </li></ul><ul><li>Insert the needle into vein and withdraw blood </li></ul><ul><li>Change needle before injecting the blood into culture bottle </li></ul><ul><li>Clean rubber bung of bottle with Iodine and inject an amount of blood equal to 10% of vol of medium (30ml=3ml blood, 50ml=5ml blood) </li></ul><ul><li>Clean the site with 70% Alcohol again </li></ul>
    9. 9. <ul><li>Blood culture at the peak of temp has got NO advantageous value, as at that time, chances of dead organisms (endotoxin) is more than alive organisms </li></ul><ul><li>If antibiotics have been given, collect blood when the conc. is expected to be lowest in blood </li></ul><ul><li>Sensitivity of a blood culture is directly related to the volume of blood submitted. Two blood culture sets (aerobic and anaerobic bottles) before administration of antibiotics is 98% sensitive </li></ul><ul><ul><li>(J. Clin. Microbiol. 1998 36: 657-661) </li></ul></ul>
    10. 10. <ul><li>TRANSPORTATION </li></ul><ul><li>Never refrigerate the inoculated blood culture medium. If there is some delay, keep it at room temp, but preferably at 37 o C </li></ul><ul><li>Keep the container upright </li></ul><ul><li>Protect from extreme heat and cold </li></ul>
    11. 11. Cerebrospinal Fluid Specimens <ul><li>CSF must always be collected by an experienced MO </li></ul><ul><li>Wash the site of CSF collection, e.g. at back of lumbar region , with Povidone-Iodine thoroughly twice or thrice in a circular fashion starting from the centre of that circle </li></ul><ul><li>Cleanse the Iodine with 70% alcohol in the same manner as that of iodine </li></ul>
    12. 12. <ul><li>A sterile wide bore needle is inserted between the third and fourth lumbar vertebrae and CSF is allowed to drip into a sterile container (about 1 ml) </li></ul><ul><li>Some times when CSF is being taken in emergency, there is no sterile container available; in that case the CSF can be collected in the sterile disposable syringe as such </li></ul>
    13. 13. <ul><li>Lab staff should be advised before a lumbar puncture is performed so that they are prepared to receive and examine the specimen immediately </li></ul><ul><li>If CSF is to be collected for RE simultaneously, then always get the separate container for that collection </li></ul><ul><li>If viral infection is suspected, then also collect faeces or a rectal swab, a nose swab and a throat swab in addition to the CSF </li></ul><ul><li>For virus isolation, collect 0.5-1.0 ml of CSF in a dry, sterile, leak-proof container </li></ul>
    14. 14. <ul><li>TRANSPORTATION </li></ul><ul><li>Don’t keep the CSF specimen in the refrigerator, even if there is some delay in sending the specimen to laboratory </li></ul><ul><li>Always send the specimen immediately to the laboratory </li></ul><ul><li>Unlike CSF collected for bacterial culture, CSF for virus culture should be refrigerated immediately after collection. This specimen should be transported to the lab in an insulated cold box </li></ul>
    15. 15. Pus / Pus Swabs <ul><li>Whenever possible collect pus rather than pus swabs </li></ul><ul><li>Surroundings and superficial part of a wound should be cleansed with sterile DW and dry sterile swab </li></ul><ul><li>Pus from an abscess is best collected at the time the abscess is incised or drained or rather it has ruptured spontaneously </li></ul><ul><li>If pus is oozing from a site, the superficial pus should be cleaned with dry sterile swab and discarded, then pus can be collected by pressing the surrounding area </li></ul>
    16. 16. <ul><li>As far as possible, a specimen from a wound should be collected before an aseptic dressing is applied </li></ul><ul><li>Using a sterile technique, aspirate up to 5 ml of pus. Transfer it to a sterile leak proof container, if not available, pus can be sent in a syringe as such </li></ul><ul><li>Label the specimen and deliver it to the lab with the request form with appropriate clinical notes </li></ul><ul><li>Always send pus (not swab) for anaerobic culture </li></ul>
    17. 17. <ul><li>TRANSPORTATION </li></ul><ul><li>Deliver the specimen as soon as possible </li></ul><ul><li>If specimen is to be transported, put the swab in an Amie’s tpt medium and make a smear for Gram / ZN staining and fix in absolute alcohol </li></ul><ul><li>If collected in tpt medium, deliver within 6 hrs </li></ul><ul><li>Smear slides for Gram / ZN stain can be sent to the lab, wrapped in simple clean paper </li></ul>
    18. 18. Urine Specimens <ul><li>Whenever possible, collect the MIDSTREAM urine specimen </li></ul><ul><li>Advise the patient to collect the specimen as: </li></ul><ul><li>MALE </li></ul><ul><ul><li>Wash the genital area thoroughly with soap and water </li></ul></ul><ul><ul><li>Start urination in toilet, after a few moments, place a sterile open-mouthed container into the stream, withdraw the container before the stream ends </li></ul></ul><ul><ul><li>Place and tighten the cap securely </li></ul></ul>
    19. 19. <ul><li>FEMALE </li></ul><ul><ul><li>Wash the genital area thoroughly with soap and water </li></ul></ul><ul><ul><li>With two fingers of one hand, separate the two labia. With the other hand, rinse the area with water from front to back </li></ul></ul><ul><ul><li>Start urination in the toilet, the urine should not touch the skin and it should flow directly down </li></ul></ul><ul><ul><li>Place a sterile open-mouthed container into the stream, withdraw the container before the stream ends </li></ul></ul><ul><ul><li>Place and tighten the cap securely </li></ul></ul>
    20. 20. <ul><li>INFANTS AND THOSE WHO DON’T COOPERATE </li></ul><ul><ul><li>Attachable sterile plastic bags may be used after washing the genital area </li></ul></ul><ul><ul><li>If the patient has not voided urine, the attached bag can be removed </li></ul></ul><ul><ul><li>Patient should be rescrubbed and new collection device attached </li></ul></ul><ul><ul><li>Alternatively, urine can be collected other methods </li></ul></ul><ul><ul><ul><li>Supra-pubic tapping, catheterization or supra-pubic aspiration by a medical officer </li></ul></ul></ul>
    21. 21. Faecal Specimens <ul><li>Rectal swabs are often helpful in identifying the cause of acute bacterial diarrhea when specimen cannot be collected readily </li></ul><ul><li>Swabbing lesions of the wall of the rectum or sigmoid colon during proctoscopy or sigmoidoscopy is much more likely to be productive than is blind rectal swabbing </li></ul><ul><li>Faeces should be passed directly into a clean, waxed cardboard container that is fitted with a tight cover </li></ul>
    22. 22. <ul><li>Specimens may be rendered unsatisfactory by residual soap, detergent or disinfectant in the bedpan or may be contaminated with urine </li></ul><ul><li>Faeces passed can then be transferred to another container which may have transport medium </li></ul><ul><li>The transferred specimen should include any pus, blood or mucus that may have been passed </li></ul><ul><li>If salmonellae, shigellae, yersinia and campylobacter are suspected, the specimen of faeces can be put up into Cary Blair transport medium and if cholera is suspected, specimen is put up into 10 ml sterile APW </li></ul>
    23. 23. <ul><li>TRANSPORTATION </li></ul><ul><li>The specimen should be delivered to the laboratory as soon as possible </li></ul><ul><li>If salmonella, shigella and yersinia are suspected stool should be obtained in Cary-Blair medium and conveyed to the laboratory within 48 hrs </li></ul><ul><li>If campylobacter is suspected, the specimen should be sent to the laboratory within 6 hrs in Cary-Blair medium </li></ul><ul><li>If cholera is suspected, specimen in APW should be sent to the laboratory within 8 hrs </li></ul>
    24. 24. Sputum Specimens <ul><li>Give the patient a dry, wide mouthed, leak proof container </li></ul><ul><li>Early morning specimen is preferred </li></ul><ul><li>Patient should cough deeply to produce a sputum specimen </li></ul><ul><li>For MTB culture, collect 3 x fresh, early morning specimens, keep in a refrigerator to be pooled or processed individually </li></ul><ul><li>In un-cooperative patients, MTB may be recovered from gastric aspiration in the ward </li></ul>
    25. 25. <ul><li>FOR TUBERCULOSIS: </li></ul><ul><ul><li>3 x consecutive early morning specimens collected in sterile container, provided at least 90 ml urine is passed each time </li></ul></ul><ul><ul><li>Alternatively, 24 hr urine collected in a container with 1.8% boric acid as a preservative </li></ul></ul>
    26. 26. Throat/Nasal Swabs Specimens <ul><li>Taken under direct visualization with good lighting </li></ul><ul><li>Areas of exudation, membrane formation or inflammation are choice sites, otherwise rub the tonsillar crypts </li></ul><ul><li>For Bordetella pertussis , collect nasopharyngeal or per nasal swab. Inform lab in advance to prepare fresh culture media for this organism </li></ul><ul><li>Per nasal swab is also used to collect specimens from nasal carriers ( S. aureus ) </li></ul>
    27. 27. <ul><li>For viral agents, instruct patient to gargle with nutrient broth </li></ul><ul><li>For M. leprae , nasal swab or nasal washing is used </li></ul><ul><li>TRANSPORTATION </li></ul><ul><li>Streptococcal pharyngitis or Diphtheria </li></ul><ul><ul><li>Stuart tpt medium, silica gel </li></ul></ul><ul><li>Whooping cough </li></ul><ul><ul><li>Bordetella tpt medium </li></ul></ul><ul><li>Viral infection </li></ul><ul><ul><li>VTM, but if delay, keep at -70 0 C </li></ul></ul>
    28. 28. <ul><li>Clean the EAM with a dry swab and remove any discharge present at or near it </li></ul><ul><li>Use auditory apparatus with light if required </li></ul><ul><li>Collect or aspirate the discharge in container </li></ul><ul><li>If pus cannot be obtained, collect specimen on a dry cotton wool swab </li></ul><ul><li>For diagnosis of external otitis, clean the ear with a mild gemicide (1:1000 Benzalkonium chloride) to reduce the contaminating skin flora </li></ul>Ear Specimens
    29. 29. <ul><li>Collected by a MO </li></ul><ul><li>Using a dry, sterile cotton wool swab, collect the discharge, or retract the lower lid </li></ul><ul><li>Aspiration of fluid from the orbit is contraindicated in case of orbital cellulitis </li></ul><ul><li>Chlamydia trachomatis </li></ul><ul><ul><li>Make a smear for Giemsa stain </li></ul></ul><ul><ul><li>Anaesthetize the conjunctiva with eye drops and obtain conjunctival scrapings with a spatula </li></ul></ul><ul><li>Acanthamoeba </li></ul><ul><ul><li>Obtain corneal scrapings in the lab and examine for motility immediately under the microscope </li></ul></ul>Eye Specimens
    30. 30. <ul><li>TRANSPORTATION </li></ul><ul><ul><li>Eye specimens should be cultured immediately as the natural secretions of the eye contain antibacterial enzyme </li></ul></ul><ul><ul><li>If there is delay, put it in Amies tpt medium and send it to the lab within 6 hrs </li></ul></ul><ul><ul><li>Herpes simplex virus and Adenovirus </li></ul></ul><ul><ul><ul><li>Corneal material - VTM </li></ul></ul></ul>
    31. 31. Dental / Oral Specimens <ul><li>For RCT, the tooth is isolated by means of a rubber dam </li></ul><ul><li>A sterile field is established, the tooth is swabbed with 70% alcohol and after the root canal is exposed, a sterile paper point is inserted, removed and placed into a tpt medium (Amie’s or Stuart’s tpt media) </li></ul><ul><li>Or, needle aspiration can be used if there is sufficient material </li></ul>
    32. 32. <ul><li>Abscess – clean its surface thoroughly with sterile saline / DW, aspirate with a sterile D / syringe </li></ul><ul><li>To increase the chances of isolation of anaerobes, pus specimen is always better than a pus swab </li></ul><ul><li>Immediate transportation to the lab </li></ul><ul><li>If there is delay, and only pus swab could be obtained, put it in Amie’s tpt medium or Robertson Cooked Meat Medium (RCM) </li></ul>
    33. 33. General considerations – Transportation <ul><li>Outer surface of the container should not be soiled with the specimen </li></ul><ul><li>Sufficient absorbent material should be present between the container and outer box </li></ul><ul><li>Container must be free from cracks, cap should be leak-proof </li></ul><ul><li>Keep a register for all the specimens sent </li></ul><ul><li>Some specimens require to be sent in ‘transport media’ (Amie’s, Stuart’s, VTM etc) </li></ul>
    34. 34. <ul><li>Use preferably plastic/disposable container </li></ul><ul><li>In some cases, if there’s a delay, the specimen should be kept at 4 0 C (urine), or 37 0 C (CSF) </li></ul><ul><li>An appropriately filled request form should accompany the specimen, which should be able to guide the microbiologist to select appropriate culture media </li></ul>
    35. 35. Transport Media <ul><li>AIM </li></ul><ul><ul><li>To reduce the effect of desiccation on the swab </li></ul></ul><ul><ul><li>To dilute the effects of inhibitory substances on swab or in the clinical material itself </li></ul></ul><ul><ul><li>To reduce the over growth of commensals </li></ul></ul>
    36. 36. Cary-Blair Tpt Medium <ul><ul><li>Has a pH of 8.4 </li></ul></ul><ul><ul><li>Contains Sod Thioglycollate, buffer, NaCl, Agar, CaCl 2 , DW </li></ul></ul><ul><ul><li>Shelf life of 6 months </li></ul></ul><ul><li>Uses </li></ul><ul><ul><li>Useful for preservation of ‘viability of enteric pathogens’ </li></ul></ul><ul><ul><li>Also a good tpt medium for ‘ Yersinia pestis ’ </li></ul></ul><ul><ul><li>(Plague bacillus) </li></ul></ul>
    37. 37. Amies Tpt Medium <ul><ul><li>Has a pH of 7.4 </li></ul></ul><ul><ul><li>Contains Sod Thioglycollate, buffer, KCl, Agar, CaCl 2 , MgCl 2, DW </li></ul></ul><ul><ul><li>Shelf life of 9 months </li></ul></ul><ul><li>Uses </li></ul><ul><ul><li>Useful for tpt of anaerobes </li></ul></ul><ul><ul><li>Also a good tpt medium for urethral and genital area specimens ( N. gonorrhoeae ), and sputum for culture </li></ul></ul>
    38. 38. Stuart Tpt Medium <ul><li>Uses </li></ul><ul><ul><li>Tpt of urethral and genital area specimens </li></ul></ul><ul><ul><li>Tpt of sputum for culture </li></ul></ul><ul><ul><li>Tpt of throat swab for </li></ul></ul><ul><ul><ul><li>Corynebacterium diphtheriae </li></ul></ul></ul><ul><ul><ul><li>Streptococcus pyogenes </li></ul></ul></ul>
    39. 39. APW <ul><li>pH 8.6 – 9.0 </li></ul><ul><li>Shelf life 2 yrs </li></ul><ul><li>Uses </li></ul><ul><ul><li>Tpt of Vibrio cholerae and other vibrios </li></ul></ul>
    40. 40. VTM <ul><li>Contains Hank’s balanced salt solution, bovine albumin, phenol red, nystatin, penicillin, streptomycin and buffer </li></ul><ul><li>pH 7.0 </li></ul><ul><li>Uses </li></ul><ul><ul><li>Any specimen suspected to contain viral pathogens </li></ul></ul>
    41. 41. Cultures That Should Include a Gram Stain <ul><li>CSF or sterile body fluid </li></ul><ul><li>Eye </li></ul><ul><li>Purulent discharge </li></ul><ul><li>Sputum, transtracheal aspirate or BAL </li></ul><ul><li>All surgical specimens </li></ul><ul><li>Tissue </li></ul><ul><li>Urethral exudates (intracellular gonococcus) </li></ul><ul><li>Vaginal / endo-cervical specimens </li></ul><ul><li>Wounds </li></ul>
    42. 42. <ul><li>Unlabeled or improperly labeled specimen </li></ul><ul><li>Prolonged storage or transport </li></ul><ul><li>Improper or damaged container </li></ul><ul><li>Specimen received in fixative </li></ul><ul><li>Oropharyngeal contaminated sputum </li></ul><ul><li>Specimens unsuitable for culture request </li></ul><ul><ul><li>Urine from Foley’s catheter </li></ul></ul><ul><li>Criteria for rejection must be readily available in the lab and laboratory specific </li></ul>Criteria For Rejection of Microbiological Specimens
    43. 44. Tables
    44. 45. Principle #1: The specimen must be collected with a minimum of contamination as close to site of infection as possible (cont.)
    45. 46. Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest
    46. 47. Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest (cont)
    47. 48. Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests
    48. 49. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
    49. 50. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
    50. 51. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
    51. 52. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
    52. 53. Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms (Cont)
    53. 54. Suggested Transport Media – General Comments
    54. 55. Principle #5: Collect all microbiology test samples prior to the institution of antibiotics
    55. 56. Principle #6: The specimen container must be properly labeled and sealed prior to transport
    56. 57. Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport Minimize transport time and maximize use of transport media as much as possible
    57. 58. Environmentally Fragile Organisms QA monitor??
    58. 59. Principle #8: Special handling/Collection instruction must be followed
    59. 60. Principle #8: Special handling/Collection instruction must be followed (Cont.) <ul><li>First, communicate with those that are doing collections. </li></ul><ul><li>Collection instructions are written and available. </li></ul><ul><li>Get involved with nursing orientation/education days and ask to have the instructions given out; poster board learning; quiz or competencies. </li></ul><ul><li>Talk to providers when there are problems with specimen collection; they sometimes do not know they could do it better. </li></ul>
    60. 61. Principle #9: Improper specimen Collected for Ordered Test