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TML/MSH Department of Microbiology Laboratory Policy & Procedure Manual

Policy QPCMI06003.02 of 40

Section: Microbiology Lab Specimen Management Manual

Subject Title: Specimen Processing Procedure

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

TORONTO MEDICAL LABORATORIES/MOUNT SINAI HOSPITAL, DEPARTMENT OF MICROBIOLOGY

NOTE: This is a CONTROLLED document. Any documents appearing in paper form that are not stamped in red "MASTER COPY" are not controlled and should be checked against the document (titled as above) on the server prior to use.

T:\Microbiology\New Manual\Live Manual\Quality Manual\Process Control\Specimen Processing Procedure QPCMI06003.doc

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








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








Blood Culture








Enterics Faeces/Rectal Swabs

a) Direct Examination: Not routinely performed. For special request, prepare a Gram stain for faecal leukocytes.

b) Culture:

Media Incubation MacConkey Agar (MAC) O2, 35oC x 18 - 24 hours

Hektoen Agar (HEK) O2, 35oC x 18 - 24 hours MacConkey Sorbitol Agar (SMAC) O2, 35oC x 18 - 24 hours Campylobacter Agar (CAMPY) Campy Jar 42oC x 48 hours Selenite Broth (SEL) 1 O2, 35oC x 12-18 hours

If Yersinia is requested or patient is >1 month–12 years old (except for NICU), add: Cefsulodin Irgasan Novobiocin Agar (CIN) O2, 30oC x 48 hours If Vibrio is requested, add: Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) O2, 35oC x 18 - 24 hours Alkaline Phosphate Broth (APB) 2 O2, 35oC x 5 - 8 hours If Pleiomonas or Aeromonas is requested, add: Blood Agar (BA) O2, 35oC x 24 hours If Neisseria gonorrhoeae (GC) is requested (rectal swab only), inoculate only: Martin-Lewis Agar (ML) CO2 , 35oC 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) O2 35oC x 48 hours








Notes: 1. Subculture Selenite broth following overnight incubation onto HEK. Incubate the sub-cultured HEK at 35?C in O2 for 18 - 24 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 35oC in O2 for 18 – 24 hours.

Rectal/Large Bowel (Colon) Biopsies

a) Direct Examination: Gram stain not indicated. b) Culture:

Media Incubation MacConkey Agar (MAC) Hekton Agar (HEK) MacConkey Sorbitol Agar (SMAC) Camyplobacter Agar (CAMPY) Selenite Broth (SEL)

O2 35oC x 18 – 24 hours O2 35oC x 18 – 24 hours O2 35oC x 18 – 24 hours Campy Jar 42oC x 48 hours O2 35oC x 18 – 24 hours








Genital Specimens

Cervical Swabs a) Direct Examination: Not indicated. b) Culture:

Media Incubation Martin-Lewis Agar (ML) CO2, 35oC 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. b) Culture:

Media Incubation

Colistin Nalidixic Acid Agar (CNA) Todd Hewitt Broth for Group B Strep. (TH)

O2, 35oC x 48 hours O2, 35oC 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.








Vaginal Swab for Culture a) Direct Examination: not required

b) Culture:

Media Incubation Colistin Nalidixic Acid Agar (CNA) Todd Hewitt Broth for Group B Strep(TH)

CO2 , 35oC x 48 hours O2, 35oC x 24 hours

Martin-Lewis Agar (ML) (if requested) CO2 , 35oC x 72 hours MacConkey (MAC) (for <12 years old) CO2 , 35oC x 48 hours

Urethral Swab a) Direct Examination: Gram stain - Quantitate the presence of pus cells and intracellular gram negative diplococci. b) Culture:

Media Incubation Martin-Lewis Agar (ML) CO2 , 35oC x 72 hours

Penis Swab

a) Direct Examination: Gram stain. b) Culture:

Media Incubation BA MAC CNA ML*

CO2 , 35oC, x 48 hours O2, 35oC, x 48 hours O2, 35oC, x 48 hours CO2 , 35oC, 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.








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. b) Culture:

Media Incubation Blood Agar (BA)* CO2, 35oC x 48 hours

Martin-Lewis Agar (ML)a CO2, 35oC x 72 hours MacConkey Agar (MAC)* O2, 35oC x 48 hours *Use a 10 ? l disposable culture loop to inoculate media aUse a swab to inoculate media

aDilute specimen 1:2 using sterile saline before inoculating ML agar








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:

Media Incubation

Blood Agar (BA) Chocolate Agar (CHOC) Martin-Lewis Agar (ML) MacConkey Agar (MAC) Fastidious Anaerobic Agar (BRUC)* Kanamycin-Vancomycin Agar (KV)* Fastidious Anaerobic Broth (THIO)*

CO2 , 35oC x 48 hours CO2 , 35oC x 48 hours CO2 , 35oC x 72 hours O2, 35oC x 48 hours AnO2, 35oC x 48 hours AnO2, 35oC x 48 hours O2, 35oC 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 -70oC freezer for minimum of 3 months.

Genital Ulcer Swab Refer to Ministry of Health Specimen Collection Guide








Intra-Uterine Device

a) Direct Examination: Gram stain of secretions. Examine for the presence of branching gram positive bacilli suggestive of Actinomyces species.

b) Culture: Not indicated.








Infection Control Screening

Methicillin Resistant Staphylococcus aureus (MRSA) Screen

a) Direct Examination: Not indicated

b) Culture: Routine Screening:

Media Incubation Mannitol Salt Agar + 10 mg/L Cefoxitin (MSFOX)* O2, 35oC 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:

Media Incubation MRSA Broth O2, 35oC 4 hrs or overnight on shaker then subculture from MRSA Broth to: Mannitol Salt Agar + 10 mg/L Cefoxitin (MSFOX) O2, 35oC x 48 h

Vancomycin Resistant Enterococcus (VRE) Screen a) Direct Examination: Not indicated

b) Culture in non-outbreak setting:

Media Incubation

¼ mEnterococcus agar + 6mg/L vancomycin (EV) O2, 35oC x 72h








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:

Media Incubation

i) Before 10 am, place each rectal swab into:

3 mL Brain Heart Infusion broth (BHIB) in a 10 mL tube O2 at 35oC x 5 hrs minimum on shaker

ii) Between 3-4pm on same day, subculture swab to:

CNA + 6mg/L vancomycin and Amphotericin B (CNAV6) O2 at 35oC x 48h Streak for single colonies Add 30? g vancomycin disc to main inoculum








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 28oC.

v) For special requests, send skin, hair and nail specimens to mycology for processing.








Table 1 - PROCESSING AND INCUBATION OF SPECIMENS FOR MYCOLOGY SPECIMEN FS IMA EBM BEAA SAB G MYCOSEL Incubation4

O2, 28oC

Bone Marrow5 X X X X 4 weeks Brain Biopsy/Abscess X X X 4 weeks Sterile Fluids:

CSF1 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 Effluent2 X X X 3 weeks Aqueous/Vitreous X X X 3 weeks

Eye/Corneal Ulcer X X 3 weeks Blood Culture3, 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/Wound2 X X X X 3 weeks Autopsy X X X 3 weeks Respiratory Secretions:

Sputum2 X X X X 4 weeks Tracheal Aspirate2 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 Nose2 X X 3 weeks Mastoid X X X 3 weeks Ear X X X 3 weeks Urine2 X X 3 weeks Hair, Skin & Nail on special requests

X X 3 weeks

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








FS Fungal Stain IMA Inhibitory Mould Agar with Chloramphenicol IMAO Inhibitory Mould Agar with Chloramphenicol, sterile olive oil to be overlaid at

Mycology bench. BEAA Blood Agar with Gentamicin, Chloramphenicol, Cyclohexamide, Egg Albumin and

Avidin EBM Esculin Base Medium SAB G S Agar with Gentamicin MYCOSEL contains Chloramphenicol and Cyclohexamide








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.








7) Culture:

Media Incubation Blood Agar (BA) Haemophilus Isolation Medium (HI) MacConkey Agar (MAC) Inihibitory Mold Agar (IMA) * Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)*

CO2, 35oC x 48 hours CO2, 35oC x 48 hours CO2, 35oC x 48 hours O2, 28oC x 4 weeks O2, 28oC x 4 weeks O2, 28oC 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) O2, 35oC x 5 days Keep the BA, HI and MAC plates CO2, 35oC x 5 days If Nocardia is requested, add: Sodium Pyruvate Agar (PYRA)

O2, 35oC x 4 weeks

* Forward inoculated fungal media to Mycology Section for incubation and work-up.








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 and 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.


7) Culture: Media Incubation Blood Agar (BA) Haemophilus Isolation Medium (HI)

CO2, 35oC x 48 hours CO2, 35oC x 48 hours








Media Incubation MacConkey Agar (MAC) Inihibitory Mold Agar (IMA) * Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)*

CO2, 35oC x 48 hours O2, 28oC x 2 weeks O2, 28oC x 2 weeks O2, 28oC 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) O2, 35oC x 5 days Keep the BA, HI and MAC plates CO2, 35oC x 5 days If Nocardia is requested, add: Sodium Pyruvate Agar (PYRA)

O2, 35oC 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.








a) Direct Examination: Not indicated. b) Culture:

Media Incubation Blood Agar (BA) CO2, 35oC x 48 hours Haemophilias Isolation Medium (HI) CO2, 35oC x 48 hours MacConkey Agar (MAC) CO2, 35oC 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) O2, 35oC x 5 day Keep the BA, HI and MAC plates CO2, 35oC x 5 days

Epiglottal Swabs

a) Direct examination: Not indicated

b) Culture: Medium Incubation Blood Agar (BA) CO2, 35oC x 48 hours Haemophilus Isolation Medium (HI) CO2, 35oC 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.


Macerate (using a hand grinder) the remaining biopsy tissue using sterile, saline as the

diluent.








b) Culture:

Media Incubation Blood Agar (BA) Chocolate Agar (CHOC) MacConkey Agar (MAC) Fastidious Anaerobe Agar (BRUC) Fastidious Anaerobic Broth (THIO) Inhibitory Mold Agar (IMA) * Esculin Base Medium (EBM) * Blood Egg Albumin Agar (BEAA) *

CO2, 35oC x 48 hours CO2, 35oC x 48 hours O2, 35oC x 48 hours AnO2, 35oC x 48 hours

O2, 35oC x 48 hours

O2, 28oC x 4 weeks O2, 28oC x 4 weeks O2, 28oC 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) O2, 350C x 5 days Keep the BA, HI and MAC plates for 5 days If Nocadia is requested, add: Sodium Pyruvate Agar (PYRA) O2, 35oC 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 b) Culture: Not indicated.

Nasal Swabs

a) Direct Examination: Not indicated.








b) Culture:

Media Incubation __________________________________________________________________ If C&S is requested: Colistin-Nalidixic Agar (CNA) CO2, 35oC x 48 hours

If N. meniningitidis is requested, add: Martin-Lewis Agar (ML) CO2, 35oC x 72 hours

If MRSA is requested: Mannitol Salt Agar with Oxacillin (MSAOX) O2, 35oC 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

b) Culture:

Media Incubation Blood Agar (BA) CO2, 35oC x 48 hours Haemophilus Isolation Medium (HI) CO2, 35oC x 48 hours If Actinomyces requested or suggested on Gram stain, add: Fastidious Anaerobic Agar (BRUC) AnO2, 35oC x 7 days Kanamycin / Vancomycin Agar (KV) AnO2, 35oC x 7 days








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.

b) Culture: Media Incubation Blood Agar (BA) CO2, 35oC x 48 hours Haemophilus Isolation Medium (HI) CO2, 35oC x 48 hours MacConkey Agar (MAC) CO2, 35oC x 48 hours

Inhibitory Mold agar (IMA)* O2, 28oC x 4 weeks Esculin Base Medium (EBM)* O2, 28oC x 4 weeks

If anaerobic culture requested, add: Kanamycin Vancomycin Agar (KV) AnO2, 35oC x 48 hours Fastidious Anaerobic Agar (BRUC) AnO2, 35oC x 48 hours Fastidious Anaerobic Broth (THIO) O2, 35oC 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.








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. b) Culture:

Media Incubation Blood Agar (BA) Haemophilus Isolation Medium (HI) MacConkey Agar (MAC)

CO2, 35oC x 48 hours CO2, 35oC x 48 hours CO2, 35oC x 48 hours








If B. cepacia is requested or specimen is from a patient with Cystic Fibrosis, add: OF base, colistin, bacitracin & lactose Agar (OCBL) O2, 35oC x 5 days Keep the BA, HI and MAC plates CO2, 35oC x 5 days If Nocardia culture is requested, add: Sodium Pyruvate Agar (PYRA)

O2, 35oC x 4 weeks

If fungal culture is requested, add: Inhibitory Mold Agar (IMA)* Esculin Base Medium (EBM)* Blood Egg Albumin Agar (BEAA)*

O2, 28oC x 4 weeks O2, 28oC x 4 weeks O2, 28oC 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.

b) Culture:

Media Incubation Blood Agar (BA) AnO2, 35oC x 18-24 hours If Neisseria gonorrhoeae requested, add: Martin-Lewis Agar (ML)

CO2, 35oC 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.








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 35oC for 1 – 4 hours. iii) Macerate the remaining tissue for culture: b) Culture:

Media Incubation 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

b) Culture:

Media Incubation Blood Agar (BA) Chocolate Agar (CHOC) MacConkey Agar (MAC)

CO2 , 35oC x 48 hours CO2 , 35oC x 48 hours O2, 35oC x 48 hours








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 depar tments for more

specimen. Consult a Microbiologist if the sterility of the extra specimen is questionable.








5. 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: Media Incubation

Blood Agar CO2, 35oC x 4 days Chocolate Agar CO2, 35oC x 4 days

Fastidious Anaerobic Broth O2, 35oC x 4 days

If fungus or cryptococcus is requested, add: Inhibitory Mould Agar2 O2, 28oC x 3 weeks Esculin Base Medium2 O2, 28oC x 3 weeks 1Forward inoculated fungal media to Mycology section for incubation and work-up.








Other Sterile Fluids – Amniotic, Pleural (Thoracentesis/Empyema), Peritoneal (Ascites), Synovial (Jo int), 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: Media Incubation Blood Agar (BA) CO2, 35oC x 4 days

Chocolate Agar (CHOC) CO2, 35oC x 4 days

Fastidious Anaerobic Agar (BRUC) AnO2, 35oC x 48 hours

Fastidious Anaerobic Broth (THIO) O2, 35oC x 4 days








If fungus is requested, add: Inhibitory Mould Agar (IMA) 1 O2, 28oC x 3 weeks Esculin Base Medium (EBM) 1 O2, 28oC x 3 weeks

1 Forward inoculated fungal media to Mycology section for incubation and work-up.








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 4oC 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 : Media Incubation

If specimen is clear: Bact/Alert bottles* Processed as per blood culture protocol If specimen is cloudy: Blood Agar (BA) CO2, 35oC x 4 days Chocolate Agar (CHOC) CO2, 35oC x 4 days MacConkey Agar (MAC) O2, 35oC 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.








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.

b) Culture:

Media Incubation 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)

O2, 35oC x 5 days CO2, 35oC x 4 days CO2, 35oC x 4 days CO2, 35oC x 4 days O2, 35oC x 4 days








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.

b) Culture: Media Incubation Blood Agar (BA) CO2, 35oC x 4 days

Chocolate Agar (CHOC) CO2, 35oC x 4 days

Fastidious Anaerobic Agar (BRUC) AnO2, 35oC x 48 hours

Kanamycin / Vancomycin Agar (KV) AnO2, 35oC x 48 hours Fastidous Anaerobic Broth (THIO) O2, 35oC x 4 days

Inhibitory Mould Agar (IMA)1 O2, 28oC x 4 weeks Esculin Base Medium (EBM)1 O2, 28oC x 4 weeks Blood Egg Albumin Agar (BEAA)1 O2, 28oC x 4 weeks 1Forward inoculated fungal media to the Mycology Section for incubation and

work-up.








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. b) Culture: Media Incubation Blood Agar (BA) CO2, 35oC x 4 days Chocolate Agar (CHOC) CO2, 35oC x 4 days FAN Aerobic Blood Culture bottle (FO2) in BacT/Alert 35oC x 5 days








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.

b) Culture:

Mix the urine specimen and dip a sterile calibrated 0.001 mL or 0.01 mL loop vertically into the sample. Streak the loop down the centre of the plate and then cross-streaked at a 90o 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








TABLE 1. Specimen type, inoculum, media and incubation conditions.

Media Incubation Voided urine : use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC)

O2 35oC x 18 – 24 hours O2 35oC x 18 – 24 hours

In and Out catheter/catheter insertion urine : use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC)


Aseptically collected urine: use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC)


Suprapubic Bladder Aspirate: use 0.001 mL loop Blood Agar (BA) MacConkey Agar (MAC) Pre-reduced Fastidious Anaerobic Agar (BRUC)

O2 35oC x 48 hours O2 35oC x 48 hours AnO2 35oC x 48 hours

Segmented urines labeled as VB1, VB2, VB3: use both 0.01 mL and 0.001 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 0.001 mL loops (2 sets) Blood Agar (BA) MacConkey Agar (MAC) Label the plates with the inoculum volume.


Urine for routine fungus : use 0.001 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)

O2 35oC x 18 – 24 hours Room temperature x 18-24 hours

O2 35oC x 18 – 24 hours








Media Incubation 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

O2 28oC x 3 weeks. O2 28oC 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.








Wounds/Tissues/Aspirates

Duodenal or Small Bowel Aspirate/Swab/Biopsy

a) Direct Examination: Gram stain not performed.

b) Culture:

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

Media Incubation Blood Agar (BA) O2, 35oC x 18 - 48 hours MacConkey Agar (MAC) O2, 35oC x 18 - 24 hours Fastidious Anaerobic Agar (BRUC) AnO2, 35oC x 48 hours Kanamycin / Vancomycin Agar (KV) AnO2, 35oC x 48 hours

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