Policy QPCMI Page 1 of 40

Transcription

1 Policy QPCMI Page 1 of 40 Prepared by: QA Committee Original Date: October 21, 2004 Issued by: Laboratory Manager Revision Date: April 10, 2006 Approved by: Laboratory Director Annual Review Date: April 10, 2006 Table of Contents Blood Culture...3 Enterics...4 Faeces/Rectal Swabs...4 Rectal/Large Bowel (Colon) Biopsies...5 Genital Specimens...6 Cervical Swabs...6 Group B Streptococcus Screen...6 Vaginal Swab for Vaginitis/Vaginosis Screen...6 Vaginal Swab for Culture...7 Urethral Swab...7 Penis Swab...7 Seminal Fluid...8 Endometrial Swabs, Biopsies and Curettings, Placenta Swab/Tissue,...9 Products of Conception, Endometrial/Uterine, Cul de sac/transvaginal,...9 Fallopian Tube, Tubo-Ovarian Swabs or Aspirates...9 Genital Ulcer Swab...9 Intra-Uterine Device...10 Infection Control Screening...11 Methicillin Resistant Staphylococcus aureus (MRSA) Screen...11 Vancomycin Resistant Enterococcus (VRE) Screen...11 Mycology...14 Respiratory Specimens...17 Bronchoalveolar Lavage (BAL), Bronchoscopy Aspirates/Washings Routine...17 Routine Lung Transplant Bronchoscopy Specimen...19 CMV Surveillance Bronchoscopy Specimens...20

2 Policy QPCMI Page 2 of 40 Bronchial Brush Specimens...20 Epiglottal Swabs...21 Open Lung/Transthoracic Needle/Transbronchial Lung Biopsies/Lung Aspirates...21 Mouth Swabs...22 Nasal Swabs...22 Nasopharyngeal Swabs/Auger Suctions for Bordetella pertussis...23 Oral Abscess Swabs...23 Sinus/Antral Specimens...24 Sputum including Endotracheal tube and Tracheotomy Specimens...24 Throat Swabs...26 Gastric Aspirates/Biopsies (for Helicobacter pylori)...27 Gastric Aspirates/Swabs from Neonates or Stillborn...27 Sterile Fluids...28 Cerebrospinal Fluids...28 Other Sterile Fluids Amniotic, Pleural (Thoracentesis/Empyema), Peritoneal (Ascites), Synovial (Joint), Pericardial, Tympanocentesis, Intraocular, Hydrocele Fluids etc...30 Peritoneal Dialysis Effluent...32 Predialysis Fluid...33 Bone Marrow (Aspirates or biopsies)...34 Blood, Platelets, & Other Transfusion Products...35 Urines...36 Wounds/Tissues/Aspirates...39 Duodenal or Small Bowel Aspirate/Swab/Biopsy...39 Record of Edited Revisions...40

3 Policy QPCMI Page 3 of 40 Blood Culture

4 Policy QPCMI Page 4 of 40 Enterics Faeces/Rectal Swabs a) Direct Examination: Not routinely performed. For special request, prepare a Gram stain for faecal leukocytes. MacConkey Agar (MAC) O 2, 35 o C x hours Hektoen Agar (HEK) O 2, 35 o C x hours MacConkey Sorbitol Agar (SMAC) O 2, 35 o C x hours Campylobacter Agar (CAMPY) Campy Jar 42 o C x 48 hours Selenite Broth (SEL) 1 O2, 35 o C x hours If Yersinia is requested or patient is >1 month 12 years old (except for NICU), add: Cefsulodin Irgasan Novobiocin Agar (CIN) O 2, 30 o C x 48 hours If Vibrio is requested, add: Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) O 2, 35 o C x hours Alkaline Phosphate Broth (APB) 2 O 2, 35 o C x 5-8 hours If Pleiomonas or Aeromonas is requested, add: Blood Agar (BA) O 2, 35 o C x 24 hours If Neisseria gonorrhoeae (GC) is requested (rectal swab only), inoculate only: Martin-Lewis Agar (ML) CO 2, 35 o C x 72 hours If C. difficile toxin assay is requested and appropriate specimen received, forward the specimen to the Virology section for testing. Specimen is also set up for Vancomycin Resistant Enterococcus (VRE) screen: Enterococcus Agar (6? g/ml Vancomycin) O 2 35 o C x 48 hours

5 Policy QPCMI Page 5 of 40 Notes: 1. Subculture Selenite broth following overnight incubation onto HEK. Incubate the sub-cultured HEK at 35?C in O 2 for hours. 2. Subculture APB to TCBS Agar after 5-8 hours incubation. Planter has to notify the technologist on the Enteric bench at the time of processing. Incubate the TCBS Agar at 35 o C in O 2 for hours. Rectal/Large Bowel (Colon) Biopsies a) Direct Examination: Gram stain not indicated. MacConkey Agar (MAC) Hekton Agar (HEK) MacConkey Sorbitol Agar (SMAC) Camyplobacter Agar (CAMPY) Selenite Broth (SEL) O 2 35 o C x hours O 2 35 o C x hours O 2 35 o C x hours Campy Jar 42 o C x 48 hours O 2 35 o C x hours

6 Policy QPCMI Page 6 of 40 Genital Specimens Cervical Swabs a) Direct Examination: Not indicated. Martin-Lewis Agar (ML) CO 2, 35 o C x 72 hours If Group B streptococcus is requested, refer to the Group B streptococcus screen section. Group B Streptococcus Screen a) Direct Examination: Not indicated. Colistin Nalidixic Acid Agar (CNA) Todd Hewitt Broth for Group B Strep. (TH) O 2, 35 o C x 48 hours O 2, 35 o C x 24 hours Vaginal Swab for Vaginitis/Vaginosis Screen a) Direct Examination: i. Wet mount: To be set up immediately. Gently press the swab into a drop of sterile saline on a slide. Place a cover slip on the slide and examine under the microscope using the 40X objective. Examine for the presence of Trichomonas vaginalis.. ii. Gram stain: Examine for the presence of yeast, clue cells and organisms associated with bacterial vaginosis.

7 Policy QPCMI Page 7 of 40 Vaginal Swab for Culture a) Direct Examination: not required Colistin Nalidixic Acid Agar (CNA) Todd Hewitt Broth for Group B Strep(TH) Martin-Lewis Agar (ML) (if requested) MacConkey (MAC) (for <12 years old) CO 2, 35 o C x 48 hours O 2, 35 o C x 24 hours CO 2, 35 o C x 72 hours CO 2, 35 o C x 48 hours Urethral Swab a) Direct Examination: Gram stain - Quantitate the presence of pus cells and intracellular gram negative diplococci. Martin-Lewis Agar (ML) CO 2, 35 o C x 72 hours Penis Swab a) Direct Examination: Gram stain. BA CO 2, 35 o C, x 48 hours MAC O 2, 35 o C, x 48 hours CNA O 2, 35 o C, x 48 hours ML* CO 2, 35 o C, x 72 hours *Occasionally, urethral swabs may be labelled as penile swabs. Neiserria gonorrhoeae culture is set up for this reason. The ML should be inoculated by rotating the swab in a Z streak manner. The inoculum is then streaked by the ISOplater to obtain discrete colonies. Examine ML plates at 48 and 72 hours.

8 Policy QPCMI Page 8 of 40 Seminal Fluid Set up all specimens for culture and sensitivity testing as outlined below. After inoculating the culture media below, forward the remainder of the specimen to the Virology lab for molecular detection of GC and CT. a) Direct Examination: Gram stain. Blood Agar (BA)* CO 2, 35 o C x 48 hours Martin-Lewis Agar (ML) a CO 2, 35 o C x 72 hours MacConkey Agar (MAC)* O 2, 35 o C x 48 hours *Use a 10? l disposable culture loop to inoculate media a Use a swab to inoculate media a Dilute specimen 1:2 using sterile saline before inoculating ML agar

9 Policy QPCMI Page 9 of 40 Endometrial Swabs, Biopsies and Curettings, Placenta Swab/Tissue, Products of Conception, Endometrial/Uterine, Cul de sac/transvaginal, Fallopian Tube, Tubo-Ovarian Swabs or Aspirates a) Preparation of specimen for culture: 1. If tissue is received, aseptically macerate the tissue with the use of a tissue grinder or stomacher. 2. If STAT TB is requested and approved by the Microbiologist, prepare a slide for Acid Fast Bacilli (FLUOROCHROME STAIN for MYCOBACTERIUM). A portion of the specimen should be forwarded to the Public Health Laboratory (PHL) for processing. 3. If TB culture is requested, send half of the specimen to PHL. b) Direct examination: Gram stain. c) Culture: Blood Agar (BA) CO 2, 35 o C x 48 hours Chocolate Agar (CHOC) CO 2, 35 o C x 48 hours Martin-Lewis Agar (ML) CO2, 35 o C x 72 hours MacConkey Agar (MAC) O 2, 35 o C x 48 hours Fastidious Anaerobic Agar (BRUC)* AnO 2, 35 o C x 48 hours Kanamycin-Vancomycin Agar (KV)* AnO 2, 35 o C x 48 hours Fastidious Anaerobic Broth (THIO)* O 2, 35 o C x 48 hours * If tissue/aspirate or anaerobic swab is received, add BRUC, KV and THIO. For tissues and biopsies, freeze remaining tissue in the -70 o C freezer for minimum of 3 months. Genital Ulcer Swab Refer to Ministry of Health Specimen Collection Guide

10 Policy QPCMI Page 10 of 40 Intra-Uterine Device a) Direct Examination: Gram stain of secretions. Examine for the presence of branching gram positive bacilli suggestive of Actinomyces species. Not indicated.

11 Policy QPCMI Page 11 of 40 Infection Control Screening Methicillin Resistant Staphylococcus aureus (MRSA) Screen a) Direct Examination: Not indicated Routine Screening: Mannitol Salt Agar + 10 mg/l Cefoxitin (MSFOX)* O 2, 35 o C x 48 h * If multiple swabs from a single patient are received individually, then process as separate specimens. If multiple swabs from a single patient are received as a "bundle" with a single label and order number, then process all swabs in the bundle on a single MSFOX plate. Outbreak Screening: MRSA Broth O 2, 35 o C 4 hrs or overnight on shaker then subculture from MRSA Broth to: Mannitol Salt Agar + 10 mg/l Cefoxitin (MSFOX) O 2, 35 o C x 48 h Vancomycin Resistant Enterococcus (VRE) Screen a) Direct Examination: Not indicated b) Culture in non-outbreak setting: ¼ menterococcus agar + 6mg/L vancomycin (EV) O 2, 35 o C x 72h

12 Policy QPCMI Page 12 of 40 Note: In the event of an outbreak investigation, the above protocol (b) may not apply. Process specimens as per outbreak protocol (c) below only on prior arrangement by Infection Control or Microbiologist. All swabs will arrive in the laboratory by 9a.m. on Monday or Tuesday only so that they may be processed as a batch. c) Culture in outbreak setting: i) Before 10 am, place each rectal swab into: 3 ml Brain Heart Infusion broth (BHIB) in a 10 ml tube O 2 at 35 o C x 5 hrs minimum on shaker ii) Between 3-4pm on same day, subculture swab to: CNA + 6mg/L vancomycin and Amphotericin B (CNAV6) O 2 at 35 o C x 48h Streak for single colonies Add 30? g vancomycin disc to main inoculum

13 Policy QPCMI Page 13 of 40

14 Policy QPCMI Page 14 of 40 Mycology 1. All specimens, except for skin scrapings, nail and hair (see below) are processed initially in the general planting section of the Microbiology Laboratory. Requests for skin scrapings, nails and hair should be forwarded to the Public Health Lab (PHL) for processing. Fungal smears and inoculated culture plates should be forwarded to the Mycology section for staining, interpretation, incubation and work-up. 2. Fungal smears: i) At least one, and where applicable, two smears from each specimen should be prepared and forwarded to the mycology section. One smear is for fungal staining and the second is held in reserve for special stains (if indicated). ii) In the planting area, the smears should be fixed in 99.9% methanol for 5 minutes before staining. iii) Fungal smears should be stained using Fungi-Fluor? (Refer to Staining Methods). iv) For special requests, send skin, hair and nail specimens to mycology for processing. 3. Culture: i) Refer to Bacteriology manual and/or Table 1 below, for appropriate media to be inoculated depending on specimen type. Between 0.1and 0.5 ml of fluid specimens is recommended for each media. ii) Tissue specimens should be mixed with filtered sterile saline and homogenized in the Stomacher or hand grinder before inoculating the culture media. NB: If a Zygomycete is suspected, DO NOT grind the specimen or put it into the Stomacher. Instead, cut the tissue with a sterile scalpel and use sterile forceps to inoculate the media. iii) Sterile body fluids and urine specimens should be centrifuged at 3500 rpm x 20 min prior to inoculating them. The supernate should be removed leaving sufficient volume to resuspend the sediment and inoculate the appropriate number of culture media. iv) Inoculated culture plates should be held unsealed overnight at room temperature to dry the plates. The following morning, all plates should be placed in a plastic bag and sent to Mycology to be sealed with Parafilm to prevent contamination and incubated at 28 o C. v) For special requests, send skin, hair and nail specimens to mycology for processing.

15 Policy QPCMI Page 15 of 40 Table 1 - PROCESSING AND INCUBATION OF SPECIMENS FOR MYCOLOGY SPECIMEN FS IMA EBM BEAA SAB G MYCOSEL 4 O 2, 28 o C Bone Marrow 5 X X X X 4 weeks Brain Biopsy/Abscess X X X 4 weeks Sterile Fluids: CSF 1 X X 3 weeks Pleural Fluid X X X 3 weeks Synovial Fluid X X X 3 weeks Pericardial/Ascitic Fluid X X X 3 weeks Peritoneal/Dialysis Effluent 2 X X X 3 weeks Aqueous/Vitreous X X X 3 weeks Eye/Corneal Ulcer X X 3 weeks Blood Culture 3, 5 X X X 4 weeks Lung Biopsy X X X X 4 weeks Tissue Biopsy X X X X 4 weeks Lymph Node X X X X 4 weeks Skin Biopsy X X X 3 weeks Clot/Thrombus X X X 3 weeks Bone/Joint X X X 4 weeks Heart Valves X X X 4 weeks Intravascular Grafts X X X 4 weeks Abscess/Pus/Wound 2 X X X X 3 weeks Autopsy X X X 3 weeks Respiratory Secretions: Sputum 2 X X X X 4 weeks Tracheal Aspirate 2 X X X X 4 weeks Bronchial Washing X X X X 4 weeks BAL (Routine) X X X X 4 weeks BAL(Lung Transplant Screen) X X X 2 weeks Sinus/Antral Wash X X X X 2 weeks Nose 2 X X 3 weeks Mastoid X X X 3 weeks Ear X X X 3 weeks Urine 2 X X 3 weeks Hair, Skin & Nail on special X X 3 weeks requests 1. Cryptococcal Antigen in the Planting Area 2. Only if Fungal Culture is specifically requested 3. Collected in "Isolator 10" Tube 4. If dimorphic fungus is rquested, regardless of site, media should be incubated for 6 weeks. 5. For Malassezia request, incubate IMA And IMAO for one week Only

16 Policy QPCMI Page 16 of 40 FS IMA IMAO BEAA EBM SAB G MYCOSEL Fungal Stain Inhibitory Mould Agar with Chloramphenicol Inhibitory Mould Agar with Chloramphenicol, sterile olive oil to be overlaid at Mycology bench. Blood Agar with Gentamicin, Chloramphenicol, Cyclohexamide, Egg Albumin and Avidin Esculin Base Medium S Agar with Gentamicin contains Chloramphenicol and Cyclohexamide

17 Policy QPCMI Page 17 of 40 Respiratory Specimens Bronchoalveolar Lavage (BAL), Bronchoscopy Aspirates/Washings Routine All specimens should be processed for C&S, fungus and TB. Additional processing should be performed only if requested. Bronchial specimens from the same patient should be pooled together and processed as a single sample. 1) If the specimen volume is less than the amount the requested tests, add sterile saline to meet the required volume for the tests requested. (See step 5 for required volumes) Proceed to step 4. 2) Centrifuge the entire specimen at 3500 rpm for 20 minutes. 3) Remove the supernatant in excess of 13 ml to a labelled sterile container. 4) Vortex to mix well. 5) Split the specimen into samples: - 2 ml for C & S and fungus - If PCP is requested, forward 2 ml to Virology for processing - 5 ml for TB (send to PHL) - 2 ml for Virology (if requested) - 1 ml for Legionella (if requested, send to PHL) - 1 ml for Chlamydia pneumoniae (if requested, send to PHL) 6) Direct Examination: Prepare 3 smears for: i) Gram stain Examine for presence of pus cells, epithelial cells and organisms. Report commensal flora, listing specific organism only if predominant. NB: If yeast is predominant organism seen, then report. If yeast is seen mixed with other organisms and is not the predominant organism, then report as commensal flora without specifically commenting on the presence of yeast. ii) Fungifluor stain Forward slide to Mycology Section for staining and interpretation. iii) Extra smear held in Mycology Section for special stains.

18 Policy QPCMI Page 18 of 40 7) Culture: Blood Agar (BA) Haemophilus Isolation Medium (HI) MacConkey Agar (MAC) Inihibitory Mold Agar (IMA) * Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)* CO 2, CO2, CO 2, O 2, O 2, O 2, 35 o C x 48 hours 35 o C x 48 hours 35 o C x 48 hours 28 o C x 4 weeks 28 o C x 4 weeks 28 o C x 4 weeks If B. cepacia is requested or specimen is from a patient with Cystic Fibrosis, add: OF Base, Colistin, Bacitracin & Lactose Agar (OCBL) O 2, 35 o C x 5 days Keep the BA, HI and MAC plates CO 2, 35 o C x 5 days If Nocardia is requested, add: Sodium Pyruvate Agar (PYRA) O 2, 35 o C x 4 weeks * Forward inoculated fungal media to Mycology Section for incubation and work-up.

19 Policy QPCMI Page 19 of 40 Routine Lung Transplant Bronchoscopy Specimen All specimens should be processed for C&S and fungus. Additional processing should be performed only if requested. Bronchial specimens from the same patient should be pooled together a nd processed as a single sample EXCEPT specimen from Trillium Gift of Life Donor (identified by MRN prefixed with DONOR ) 1) If the specimen volume is less than 10 ml, add sterile saline to a final volume of 10 ml. Proceed to step 4. 2) Centrifuge the entire specimen at 3500 rpm for 20 minutes. 3) Remove the supernatant in excess of 13 ml to a labelled sterile container. 4) Vortex to mix well. 5) Split the specimen into samples: - 2 ml for C & S and fungus - If PCP is requested forward 2 ml to Virology for processing - 5 ml for TB if requested, (send to PHL) - 2 ml for Virology - 1 ml for Legionella (if requested, send to PHL) - 1 ml for Chlamydia pneumoniae (if requested, send to PHL) 6) Direct Examination: Prepare 3 smears for: i) Gram stain Examine for presence of pus cells, epithelial cells and organisms. Report commensal flora, listing specific organism only if predominant. NB: If yeast is predominant organism seen, then report. If yeast is seen mixed with other organisms and is not the predominant organism, then report as commensal flora without specifically commenting on the presence of yeast. ii) Fungifluor stain Forward slide to Mycology Section for staining and interpretation. iii) Extra smear held in Mycology Section for special stains. 7) Culture: Blood Agar (BA) Haemophilus Isolation Medium (HI) CO 2, 35 o C x 48 hours CO 2, 35 o C x 48 hours

20 MacConkey Agar (MAC) Policy QPCMI Page 20 of 40 CO 2, 35 o C x 48 hours Inihibitory Mold Agar (IMA) * Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)* O 2, O 2, O 2, 28 o C x 2 weeks 28 o C x 2 weeks 28 o C x 2 weeks If B. cepacia is requested or specimen is from a patient with Cystic Fibrosis, add: OF Base, Colistin, Bacitracin & Lactose Agar (OCBL) O 2, 35 o C x 5 days Keep the BA, HI and MAC plates CO 2, 35 o C x 5 days If Nocardia is requested, add: Sodium Pyruvate Agar (PYRA) O 2, 35 o C x 4 weeks * Forward inoculated fungal media to Mycology Section for incubation and work-up. CMV Surveillance Bronchoscopy Specimens Specimens for routine CMV surveillance require CMV culture only. Refer specimens to Virology immediately upon receipt. Bronchial Brush Specimens All brushes received in small cap (bijou) bottles in 1 ml Ringer's lactate solution should be processed "STAT" because colony counts may change if processing is delayed. If the brush is received in <1 ml of fluid, add sufficient Ringer's lactate solution to make up 1 ml. Document in the Test Comment field of the LIS as Brush received in wrong volume of fluid. If a dry brush is received, add 1 ml of Ringer's lactate solution into the vial. Document in the Test Comment field of the LIS as Dry brush received. Mix the vial well by vortexing vigorously. Measure 0.01 ml of the sample (using a calibrated urine loop) and inoculate onto routine media for culture of aerobic bacteria.

21 Policy QPCMI Page 21 of 40 a) Direct Examination: Not indicated. Blood Agar (BA) CO 2, 35 o C x 48 hours Haemophilias Isolation Medium (HI) CO 2, 35 o C x 48 hours MacConkey Agar (MAC) CO 2, 35 o C x 48 hours If B. cepacia is required or specimen is from a patient with Cystic Fibrosis, add: Of Base, Colistin, Bacitracin & Lactose Agar (OCBL) O 2, 35 o C x 5 day Keep the BA, HI and MAC plates CO 2, 35 o C x 5 days Epiglottal Swabs a) Direct examination: Not indicated Medium Blood Agar (BA) Haemophilus Isolation Medium (HI) CO2, 35 o C x 48 hours CO 2, 35 o C x 48 hours Open Lung/Transthoracic Needle/Transbronchial Lung Biopsies/Lung Aspirates a) Direct examination: Using clean, sterile slides make 3 touch preparation smears from a cut surface of the biopsy for: i) Gram stain: Examine for presence of organisms and cells. ii) Fungifluor stain: Forward to Mycology Section for staining and interpretation. iii) Extra smear held in Mycology Section for special stains. Macerate (using a hand grinder) the remaining biopsy tissue using sterile, saline as the diluent.

22 Blood Agar (BA) Chocolate Agar (CHOC) MacConkey Agar (MAC) Fastidious Anaerobe Agar (BRUC) Fastidious Anaerobic Broth (THIO) Policy QPCMI Page 22 of 40 CO 2, 35 o C x 48 hours CO 2, 35 o C x 48 hours O 2, 35 o C x 48 hours AnO 2, 35 o C x 48 hours O 2, 35 o C x 48 hours Inhibitory Mold Agar (IMA) * Esculin Base Medium (EBM) * Blood Egg Albumin Agar (BEAA) * O2, 28 o C x 4 weeks O 2, 28 o C x 4 weeks O 2, 28 o C x 4 weeks If B. cepacia is requested or the specimen is from a patient with Cystic Fibrosis, add: OF Base, Colistin, Bacitracin & Lactose Agar (OCBL) O 2, 35 0 C x 5 days Keep the BA, HI and MAC plates for 5 days If Nocadia is requested, add: Sodium Pyruvate Agar (PYRA) O 2, 35 o C x 4 weeks * Forward inoculated fungal cultures to Mycology for incubation and work-up. Mouth Swabs Applies to both C&S and Fungus test requests: a) Direct Examination: Gram stain Not indicated. Nasal Swabs a) Direct Examination: Not indicated.

23 Policy QPCMI Page 23 of 40 If C&S is requested: Colistin-Nalidixic Agar (CNA) CO 2, 35 o C x 48 hours If N. meniningitidis is requested, add: Martin-Lewis Agar (ML) CO 2, 35 o C x 72 hours If MRSA is requested: Mannitol Salt Agar with Oxacillin (MSAOX) O 2, 35 o C x 48 hours Nasopharyngeal Swabs/Auger Suctions for Bordetella pertussis The specimen should be forwarded to the Public Health Laboratory (PHL) for processing. Oral Abscess Swabs If an anaerobic swab is received and Actinomyces is not requested, process for aerobic culture only. Do not process for anaerobic culture. Document in the TEST Comment field in the LIS as Inappropriate specimen for anaerobic culture, therefore not processed a) Direct Examination: Gram stain Blood Agar (BA) Haemophilus Isolation Medium (HI) CO 2, 35 o C x 48 hours CO 2, 35 o C x 48 hours If Actinomyces requested or suggested on Gram stain, add: Fastidious Anaerobic Agar (BRUC) Kanamycin / Vancomycin Agar (KV) AnO 2, 35 o C x 7 days AnO 2, 35 o C x 7 days

24 Policy QPCMI Page 24 of 40 Sinus/Antral Specimens a) Direct examination: i) Gram stain - Examine for and quantitate the presence of pus cells and organisms. ii) Fungifluor - Forward to Mycology Section for staining and interpretation. Blood Agar (BA) CO 2, 35 o C x 48 hours Haemophilus Isolation Medium (HI) CO 2, 35 o C x 48 hours MacConkey Agar (MAC) CO 2, 35 o C x 48 hours Inhibitory Mold agar (IMA)* O 2, 28 o C x 4 weeks Esculin Base Medium (EBM)* O 2, 28 o C x 4 weeks If anaerobic culture requested, add: Kanamycin Vancomycin Agar (KV) AnO2, 35 o C x 48 hours Fastidious Anaerobic Agar (BRUC) AnO 2, 35 o C x 48 hours Fastidious Anaerobic Broth (THIO) O 2, 35 o C x 48 hours *Forward inoculated fungal media to Mycology section for incubation and work-up. Sputum including Endotracheal tube and Tracheotomy Specimens Notes: Flush small volumes of endotracheal tube (ETT) secretions from suction tubings of neonates with a small volume of sterile trypticase soy broth (TSB) into a sterile container before processing as an unscreened sputum specimen. An ETT tip from an adult is an unacceptable specimen and will not be processed. Notify the ward immediately and issue a report stating ETT tips are not acceptable for culture. For requests for viruses, forward sample to the Virology section for processing.

25 Policy QPCMI Page 25 of 40 Requests for STAT Ziehl-Neelsen acid-fast bacilli (AFB) staining must be approved by a medical microbiologist or infection control practitioner before processing in-house. A portion of the sample should also always be forwarded to the Public Health Laboratory for processing. If mycobacteria (TB) is requested, forward a portion of the specimen to the Public Health Laboratory (PHL) for processing. If Legionella is requested, forward a portion of the specimen to the Public Health Laboratory (PHL) for processing. a) Direct Examination: i) Gram Stain Sputum is always contaminated to some degree with oropharyngeal organisms. Consequently, a screening procedure for routine culture is required to exclude grossly contaminated specimens or saliva. DO NOT screen PMH patients, endotracheal tube (ETT) aspirates, suctioned samples or any specimens requesting Mycobacterium tuberculosis (TB) only or fungus culture only. Screening Procedure Select the most purulent portion of the specimen for Gram staining and culture. Scan the smear under low power (10X magnification) as soon as possible and examine for epithelial cells. ii) Approved requests for STAT acid fast stain: Direct smear from an unconcentrated specimen. iii) Fungus requests: Prepare smear for Fungifluor and forward to Mycology Section for staining and interpretation. Blood Agar (BA) Haemophilus Isolation Medium (HI) MacConkey Agar (MAC) CO 2, CO 2, CO 2, 35 o C x 48 hours 35 o C x 48 hours 35 o C x 48 hours

26 Policy QPCMI Page 26 of 40 If B. cepacia is requested or specimen is from a patient with Cystic Fibrosis, add: OF base, colistin, bacitracin & lactose Agar (OCBL) O 2, 35 o C x 5 days Keep the BA, HI and MAC plates CO 2, 35 o C x 5 days If Nocardia culture is requested, add: Sodium Pyruvate Agar (PYRA) O 2, 35 o C x 4 weeks If fungal culture is requested, add: Inhibitory Mold Agar (IMA)* Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)* O 2, O 2, O 2, 28 o C x 4 weeks 28 o C x 4 weeks 28 o C x 4 weeks * Forward inoculated fungal media to Mycology section for incubation and work-up. Throat Swabs a) Direct Examination: Not indicated for Group A streptococcus, Neisseria gonorrhoeae or Neisseria meningitidis. If yeast (thrush) is suspected / requested: Gram stain. Examine for presence of pseudohyphae and/or budding yeast. If Vincent s angina is suspected / requested: Gram stain. Examine for presence of spirochetes and/or fusiform bacilli and pus cells. Blood Agar (BA) AnO 2, 35 o C x hours If Neisseria gonorrhoeae requested, add: Martin-Lewis Agar (ML) CO 2, 35 o C x 72 hours If Corynebacterium diphtheriae requested, forward swab to Public Health Laboratory (PHL) for processing. Note: The ML plate is inoculated by rolling the swab in a Z pattern over the medium followed by cross streaking with a sterile loop over the entire plate. Set up anaerobic jar at 1600 hours and at the end of the evening shift.

27 Policy QPCMI Page 27 of 40 Gastric Aspirates/Biopsies (for Helicobacter pylori) a) Direct Examination: i) Use one half of the tissue to prepare a smear for Gram stain and to directly inoculate a urea slant. ii) Incubate the urea slant at 35 o C for 1 4 hours. iii) Macerate the remaining tissue for culture: Blood Agar (BA) Campylobacter Agar (CAMPY) Microaerophilic, 35 C x 7 days Microaerophilic, 35 C x 7 days Gastric Aspirates/Swabs from Neonates or Stillborn a) Direct Examination: Gram Stain Blood Agar (BA) Chocolate Agar (CHOC) MacConkey Agar (MAC) CO 2, 35 o C x 48 hours CO 2, 35 o C x 48 hours O 2, 35 o C x 48 hours

28 Policy QPCMI Page 28 of 40 Sterile Fluids Cerebrospinal Fluids NB: If only VDRL is requested, the specimen will NOT be cultured. All other requests require C&S. These specimens must be processed immediately. 1. Examine the fluid for colour, xanthochromia, turbidity and the presence of visible blood or clots. Record the observations in the LIS under COMMENT in the SOURCE screen. 2. If any of the following is requested: Cryptococcus or cryptococcal antigen, India ink preparation or Fungal culture perform a cryptococcal antigen test on the supernatant or from the original specimen (see Technical Manual) 3. If bacterial antigens are requested, this test is no longer available. If >1 ml of specimen is received: 4. Make a direct cytospin smear using 2-3 drops of unspun CSF. 5. Centrifuge the remaining specimen at 3500 rpm x 20 minutes. 6. Transfer the supernatant to a sterile tube which can be used for biochemical, viral, molecular, Cryptococcal antigen or serological tests if required. Label clearly remaining supernatant and refrigerate. 7. Use the sediment to inoculate the culture media listed below. Label clearly any remaining sediment and refrigerate. 8. If Mycobacteria (TB) is requested, reconstitute sediment with 1 ml of supernatant and label as such. Forward the specimen to the Public Health Laboratory (PHL) for processing. If <1 ml of specimen is received: 4. DO NOT centrifuge the specimen. Consider calling other departments for more specimen. Consult a Microbiologist if the sterility of the extra specimen is questionable.

29 Policy QPCMI Page 29 of If more than one test is ordered, call the physician to prioritize the test ordered. 6. For routine C&S, prepare a Gram smear using a drop of CSF and then directly inoculate the culture media listed below. 7. If Mycobacteria (TB) is requested, forward a portion of the specime n to the Public Health Laboratory (PHL) for processing. 8. If virology is requested, forward a portion of the specimen to the Virology Section for processing. 9. If a swab is received, a. in the Order/Entry Specimen QA Comment field of the LIS as UNST b. in the Result Comment field enter Swab received insufficient specimen. A negative result may not rule out infection. Process the swab as per routine. A. Direct Examination: Gram stain Make 2 smears if the specimen is grossly bloody. B. Culture: Blood Agar CO 2, 35 o C x 4 days Chocolate Agar CO2, 35 o C x 4 days Fastidious Anaerobic Broth O 2, 35 o C x 4 days If fungus or cryptococcus is requested, add: Inhibitory Mould Agar 2 O 2, 28 o C x 3 weeks Esculin Base Medium 2 O 2, 28 o C x 3 weeks 1 Forward inoculated fungal media to Mycology section for incubation and work-up.

30 Policy QPCMI Page 30 of 40 Other Sterile Fluids Amniotic, Pleural (Thoracentesis/Empyema), Peritoneal (Ascites), Synovial (Joint), Pericardial, Tympanocentesis, Intraocular, Hydrocele Fluids etc NB: Chest tube drainage will be treated as a wound specimen (Refer to Wound/Tissues/Aspirates Culture Manual). 1. If a clotted specimen is received, emulsify the coagulated material using a sterile swab or loop. 2. If >1 ml of fluid is received, centrifuge the specimen (3500 rpm x 20 minutes). If <1 ml of specimen is received, do NOT centrifuge the specimen. 3. Transfer the supernatant into a sterile container and refrigerate until the final report is issued. Label the container as supernatant. 4. Use the sediment to prepare the smears and inoculate the culture media. If <1 ml of specimen is received, prepare smears and inoculate culture media directly. 5. If viral culture is requested, forward a portion of the specimen to the Virology Section for processing. 6. If Mycobacteria (TB) is requested, forward a portion of the specimen to the Public Health Laboratory (PHL) for processing. 7. If a swab is received, a. In the Order/Entry Specimen QA Comment field of the LIS as UNST b. In the Result Comment field enter Swab received insufficient specimen. A negative result may not rule out infection. Process the swab as per routine. A. Direct Examination: Gram stain Calcofluor white stain (If fungus is requested). B. Culture: Blood Agar (BA) CO 2, 35 o C x 4 days Chocolate Agar (CHOC) CO 2, 35 o C x 4 days Fastidious Anaerobic Agar (BRUC) AnO 2, 35 o C x 48 hours Fastidious Anaerobic Broth (THIO) O 2, 35 o C x 4 days

31 Policy QPCMI Page 31 of 40 If fungus is requested, add: Inhibitory Mould Agar (IMA) 1 O 2, 28 o C x 3 weeks Esculin Base Medium (EBM) 1 O 2, 28 o C x 3 weeks 1 Forward inoculated fungal media to Mycology section for incubation and work-up.

32 Policy QPCMI Page 32 of 40 Peritoneal Dialysis Effluent NB: No more than one dialysis fluid per patient should be processed every other day. If a bag of cloudy fluid is received after a clean one is processed, culture and sensitivity is always done. A portion of the fluid should be sent to Hematology for cell count if requested. 1) Examine the bags noting the colour and turbidity of the fluid. Record the observations in the LIS under COMMENT in the SOURCE screen. 2) Shake bag well to mix. Working at the sink, disinfect the port with 70% ethanol and let stand for several minutes. Open clamp and run off some of the dialysate. 3) Fill a red top vacutainer tube with approximately 5 ml of dialysate and send to Hematology for cell count. 4) Fill three 50 ml sterile plastic centrifuge tubes with dialysate. Centrifuge two of the tubes (3500 rpm x 20 minutes). Store the third centrifuge tube at 4 o C for at least 7 days. Discard the bag after collecting dialysate. 5) Decant the supernatant leaving approximately 10 ml in each tube. Vortex well to resuspend the sediment and pool the two tubes into one. Prepare Gram smear and inoculate culture media as outlined below. a) Direct Examination: i) If specimen is cloudy: Gram stain ii) If specimen is clear: Gram stain is not needed. b) Culture : If specimen is clear: Bact/Alert bottles* Processed as per blood culture protocol If specimen is cloudy: Blood Agar (BA) CO 2, 35 o C x 4 days Chocolate Agar (CHOC) CO2, 35 o C x 4 days MacConkey Agar (MAC) O 2, 35 o C x 4 days BacT/Alert bottles* Processed as per blood culture protocol *Inoculate both an aerobic and anaerobic bottle with 8 to 9 ml of fluid each.

33 Policy QPCMI Page 33 of 40 Predialysis Fluid 1. Examine the fluid noting the colour and turbidity. Record the observations in the LIS under COMMENT in the SOURCE screen. 2. Centrifuge specimens (3500 rpm x 20 minutes). 3. Decant supernatant leaving approximately 2-3 ml. Vortex well to resuspend the sediment. a) Direct Examination: i) If specimen is cloudy: Gram stain ii) If specimen is clear: No Gram stain is needed. If specimen is clear: Fastidious Anaerobic Broth (THIO) If specimen is cloudy: Blood Agar (BA) MacConkey Agar (MAC) Chocolate Agar (CHOC) Fastidious Anaerobic Broth (THIO) O 2, 35 o C x 5 days CO 2, 35 o C x 4 days CO 2, 35 o C x 4 days CO 2, 35 o C x 4 days O 2, 35 o C x 4 days

34 Policy QPCMI Page 34 of 40 Bone Marrow (Aspirates or biopsies) 1. If <1 ml is received, sterile saline may be added to a final volume of 1 ml so that the media listed below can be inoculated. 2. If a clotted specimen is received, emulsify the coagulated material using a sterile swab or loop. 3. A portion of ALL specimens should be forwarded to the Public Health Laboratory (PHL) for Mycobacteria (TB) culture. a) Direct Examination: Gram stain. Calcofluor white stain. Blood Agar (BA) CO 2, 35 o C x 4 days Chocolate Agar (CHOC) CO2, 35 o C x 4 days Fastidious Anaerobic Agar (BRUC) AnO 2, 35 o C x 48 hours Kanamycin / Vancomycin Agar (KV) AnO 2, 35 o C x 48 hours Fastidous Anaerobic Broth (THIO) O 2, 35 o C x 4 days Inhibitory Mould Agar (IMA) 1 O 2, 28 o C x 4 weeks Esculin Base Medium (EBM) 1 O 2, 28 o C x 4 weeks Blood Egg Albumin Agar (BEAA) 1 O 2, 28 o C x 4 weeks 1 Forward inoculated fungal media to the Mycology Section for incubation and work-up.

35 Policy QPCMI Page 35 of 40 Blood, Platelets, & Other Transfusion Products Both a sample of the transfusion product from the bag and a segment from the tubing should be processed. With aseptic technique, use a needle and syringe to take a sample from each. Enter the order into the LIS twice: one for the tubing and the second for the bag. a) Direct Examination: Not indicated. Blood Agar (BA) CO 2, 35 o C x 4 days Chocolate Agar (CHOC) CO 2, 35 o C x 4 days FAN Aerobic Blood Culture bottle (FO2) in BacT/Alert 35 o C x 5 days

36 Policy QPCMI Page 36 of 40 Urines a) Direct Examination: Gram stain: Not routinely performed. If specifically requested, perform Gram stain directly on unspun specimen. Fungal stain: Not routinely performed. If dimorphic fungus or cryptococcus specifically requested, prepare 2 slides with spun urine (3500 rpm x 15 min.). Send unstained slides to mycology for staining and interpretation. Eosinophil stain: Not routinely performed. If requested, prepare smear by cytospin method and fix in absolute alcohol. Stain slide as per Technical manual. Mix the urine specimen and dip a sterile calibrated ml or 0.01 ml loop vertically into the sample. Streak the loop down the centre of the plate and then crossstreaked at a 90 o angle to the inoculum. (See diagram below). If using the Autostreaker, inoculate the plate with a small streak using a sterile calibrated loop (See diagram below). The amount of urine inoculated is dependent on the type of urine specimen that is processed. (See Table below) i) Autostreaker ii) Manual inoculation

37 Policy QPCMI Page 37 of 40 TABLE 1. Specimen type, inoculum, media and incubation conditions. Voided urine: use ml loop Blood Agar (BA) MacConkey Agar (MAC) In and Out catheter/catheter insertion urine : use ml loop Blood Agar (BA) MacConkey Agar (MAC) Aseptically collected urine: use ml loop Blood Agar (BA) MacConkey Agar (MAC) O 2 O 2 O 2 O 2 O 2 O 2 35 o C x hours 35 o C x hours 35 o C x hours 35 o C x hours 35 o C x hours 35 o C x hours Suprapubic Bladder Aspirate: use ml loop Blood Agar (BA) MacConkey Agar (MAC) Pre-reduced Fastidious Anaerobic Agar (BRUC) O 2 O 2 AnO 2 35 o C x 48 hours 35 o C x 48 hours 35 o C x 48 hours Segmented urines labeled as VB1, VB2, VB3: use both 0.01 ml and ml loops (2 sets) Blood Agar (BA) MacConkey Agar (MAC) Label the plates with the inoculum volume. Prostatic Fluid labeled as EPS sent together with VB1, 2 or 3 or labeled as Seminal Fluid sent together with VB1, 2 or 3: use both 0.01 ml and ml loops (2 sets) Blood Agar (BA) MacConkey Agar (MAC) Label the plates with the inoculum volume. O 2 O 2 O2 O 2 35 o C x hours 35 o C x hours 35 o C x hours 35 o C x hours Urine for routine fungus: use ml loop Blood Agar (BA) First day: Second day: Hi-light the label on the BA plate to note that the plate has to be held for the second day. MacConkey Agar (MAC) O 2 35 o C x hours Room temperature x hours O 2 35 o C x hours

38 Policy QPCMI Page 38 of 40 Urine for Cryptococcus or specific filamentous fungi: centrifuge urine at 3500 rpm, for 15 minuntes EBM IMA Inoculate 1 drop of sediment per plate. Forward plates to Mycology O 2 O 2 28 o C x 3 weeks. 28 o C x 3 weeks. Note: For specimens processed after 1600 hours, place the plates in a separate basket in the incubator. Special Requests : Eosinophil Stain Not routinely performed. If requested, prepare smear by cytospin method and fix in absolute alcohol. Stain slide as per Technical manual. Bacterial Latex Agglutination Not done due to poor sensitivity and specificity. Anaerobes Appropriate specimen is bladder suprapubic aspirate see above Table. Process these specimens immediately. If requested on other urines, consult the Medical Microbiologist. Chlamydia Detection Send the specimen to Virology immediately. Legionella Antigen Detection Send the specimen to the Provincial Health Laboratory. Leptospira Detection Send the specimen to the Provincial Health Laboratory. Cryptococcus/Systemic Fungi See above Table. TB Culture Send the specimen to the Provincial Health Laboratory. Viral Culture Send the specimen to Virology immediately. Parasitology Schistosomiasis Send the specimen to Parasitology immediately.

39 Policy QPCMI Page 39 of 40 Wounds/Tissues/Aspirates Duodenal or Small Bowel Aspirate/Swab/Biopsy a) Direct Examination: Gram stain not performed. i) Duodenal or Small Bowel Aspirates Process Duodenal and Small Bowel aspirates for O&P only. Forward the specimen to the Parasitology section immediately. If a delay is anticipated and the specimen is not received in SAF, transfer the specimen to SAF. DO NOT process these specimens routinely for bacterial culture unless requested. ii) Duodenal or Small Bowel Swab Blood Agar (BA) MacConkey Agar (MAC) Fastidious Anaerobic Agar (BRUC) Kanamycin / Vancomycin Agar (KV) O2, 35 o C x hours O 2, 35 o C x hours AnO 2, 35 o C x 48 hours AnO 2, 35 o C x 48 hours