Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Review Date: May 26, 2004 ENTERIC CULTURE MANUAL

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1 Policy #MI/ENT/v02 Page 1 of 1 Section: Subject Title: Table of Contents Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: Review Date: May 26, 2004 FAECES / RECTAL SWABS ENTERIC CULTURE MANUAL TABLE OF CONTENTS Introduction...2 Specimen Collection and Transport...2 Specimen Rejection Criteria...3 Reagents / Materials / Media...3 Procedure...3 Reporting Results...11 DUODENAL OR SMALL BOWEL ASPIRATES / BIOPSIES / SWABS Introduction...13 Specimen Collection and Transport...13 Reagents / Materials / Media...13 Procedure...13 Reporting Results...14 RECTAL / LARGE BOWEL (COLON) BIOPSIES Introduction...15 Specimen Collection and Transport...15 Reagents / Materials / Media...15 Procedure...15 Reporting Results...16 Gastric Aspirates / Biopsies (for Helicobacter pylori)...17 APPENDICES: Appendix I - Reagents / Materials / Media...19 Appendix II - Serological Testing...21 Appendix III - Speciation of Campylobacter...23 Page 1

2 Policy # MI/ENT/01/v02 Page 1of 11 Section: Subject Title: Faeces / Rectal Swab Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: December 5, 2001 I. Introduction FAECES / RECTAL SWABS Acute infectious diarrhea may be caused by a number of different agents including bacteria, viruses and protozoa. The laboratory routinely searches for those bacteria that are most likely to cause diarrhea. Requests for viruses or protozoa will be processed in the Virology section or Parasitology section, respectively. When stool C&S is requested, the specimens will be examined routinely for Salmonella, Shigella, Campylobacter, and E. coli 0157:H7. Upon special request, and if clinically indicated, the laboratory will also culture for the following: Vibrio and Yersinia. For children between one month and 12 years of age (except those in the neonatal intensive care unit), cultures will be routinely set up for Yersinia. All reagents, kits and media MUST be quality controlled before use. All tests must include appropriate controls. (Refer to Quality Control Manual). II. Specimen Collection and Transport A single stool specimen should be collected and transported to the laboratory in Cary-Blair transport medium. When faeces cannot be obtained, a rectal swab is acceptable except for Clostridium difficile toxin assay. The specimen is collected with a sterile swab inserted approximately one inch beyond the anal sphincter and placed in Amies transport medium. If Campylobacter other than C. jejuni/coli is requested, forward the specimen to PHL. Stool specimens for C. difficile toxin assay should be collected in a clean, sterile container (Refer to Virology Manual). Rectal swabs for GC must be sent in Amies transport medium. Specimens for Chlamydia detection or virus isolation should be sent in appropriate transport media (Refer to Virology Manual). Specimens for ova and parasites (O&P) must be collected in SAF (Refer to Parasitology Manual). Page 2

3 Policy # MI/ENT/01/v02 Page 2 of 11 III. Specimen Rejection Criteria Rejection Criteria All formed stools except when S. typhi requested Patient hospitalized for 3 days or more Multiple specimens collected from the same in-patient the same day (only one specimen per patient per test per day is to be processed). Stools from outpatients often arrive in batches and are usually a series taken from separate days. Accession and test the most recent specimen only. Stool sample in Cary-Blair transport medium with yellow indicator indicating failure of the buffering system to maintain a neutral ph. Report Comment Formed stool received. Test cancelled. This specimen was not cultured for community acquired enteric pathogens because the patient has been hospitalised for 3 or more days. Discuss with the Medical Microbiologist if necessary. This specimen has not been processed as a specimen submitted from the same day has already been processed. Multiple specimens received. Only the most recently collected specimen has been processed. This specimen was not processed as the transport medium failed to stabilize the specimen and maintain a neutral ph. Phone ward / physician and document on report. IV. Reagents / Materials / Media Refer to Appendix I. V. Procedure A. Processing of Specimens: a) Direct Examination: Not routinely performed. Upon special request, a Gram stain for faecal leukocytes may be performed. Page 3

4 Policy # MI/ENT/01/v02 Page 3 of 11 b) Culture: Media Incubation MacConkey Agar (MAC) O 2, 35 0 C x hours Hektoen Agar (HEK) O 2, 35 0 C x hours MacConkey Sorbitol Agar (MAC-S) O 2, 35 0 C x hours Campylobacter Agar (CAMPY) Campy Jar 42 0 C x 48 hours Selenite Broth (SEL) 1 O 2, 35 0 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 0 C x 24 hours If Vibrio is requested, add: Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) O 2, 35 0 C x hours Alkaline Phosphate Broth (APB) 2 O 2, 35 0 C x 5-8 hours If Neisseria gonorrhoeae (GC) is requested (rectal swab only), inoculate only: Martin-Lewis Agar (ML) CO 2, 35 0 C x 72 hours If C. difficile toxin assay is requested and appropriate specimen received, forward the specimen to the Virology section (TML) or C. difficile bench (MSH) for testing. Specimen is also set up for Vancomycin Resistant Enterococcus (VRE) screen. Enterococcus Agar (6 µg/ml Vancomycin) O C x 48 hours Notes: 1. Selenite broth is subcultured following overnight incubation onto HEK which is incubated at 35 C in O 2 for hours. Page 4

5 Policy # MI/ENT/01/v02 Page 4 of 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 0 C in O 2 for hours. B. Interpretation of Cultures MACCONKEY/HEKTOEN AGARS Medium MacConkey Agar (MAC) Hektoen Agar (HEK) Suspect colonies Non-Lactose Fermenter (NLF) (colourless or transparent) Green with or without H 2 S Pick one colony of each suspect morphotype and inoculate a urea slant and Trypticase Soy Broth (TSB). Incubate these for a minimum of 3 hours at 35 0 C in O 2. Urea reactions are recorded and the tubes from urea positive isolates are discarded. Isolates with a negative urea test are subcultured from the TSB into TSI, ONPG-PAM and MAC (half plate for purity). Results are read after overnight incubation at 35 0 C in O 2 (See table below). Page 5

6 Policy # MI/ENT/01/v02 Page 5 of 11 Table 1. Characteristic reactions of potential stool pathogens Organism TSI ONPG PPA Motility Indole S. typhi -/ S. arizonae d/+ H 2 S S. paratyphi A -/ Other Salmonella -/+ H 2 S S. sonnei -/ S. dysenteriae -/+ d - - d S. flexneri (1-5) -/ d S. flexneri (type 6) -/ d S. boydii -/ d Y. enterocolitica d/ d 1 may produce small amounts of gas and /or H 2 S 2 occasionally produces H 2 S weakly 3 non-motile at 35 0 C; motile at room temperature 4 may produce a small amount of gas d indicates variable results If results of these tests suggest: Salmonella Shigella Yersinia Perform serotyping (Refer to Appendix II) and Vitek GNI+ If S. typhi, also set up a Vitek GNS-GA. Perform serotyping (Refer to Appendix II), Vitek GNI+ and GNS-GA. Set up Vitek GNI+ and API 20E Notes: 1. Salmonella and Shigella agglutination tests must be performed from a non-selective medium such as TSI. Agglutinations should not be performed from the MAC or HEK plates. Send all Salmonella and Shigella isolates to the Public Health Laboratory (PHL) for further typing and / or identification. Page 6

7 Policy # MI/ENT/01/v02 Page 6 of 11 MACCONKEY WITH SORBITOL AGAR This plate is to be examined for the presence of E. coli 0157:H7. These organisms do not ferment sorbitol and will appear as non-fermenting (colourless) colonies on this medium. Each non-fermenting morphotype is sub-cultured onto a BA and incubated at 35 0 C in O 2 until sufficient growth to perform serologic testing (usually 4 to 24 hours). Using the 0157 latex agglutination test perform serologic testing on suspect colonies growing on the BA plate (Appendix II). Serology positive isolates should then be inoculated to a Vitek GNI+ card. (Check the Vitek card to confirm that the isolate is an E. coli and sorbitol negative. Discard sorbitol-positive isolates). Send the isolate to PHL for confirmation and H typing. CAMPY AGAR Examine after 48 hours incubation. Colonies of Campylobacter are grey or colourless, pinpoint flat or mucoid to convex to spreading across the plate. Perform an oxidase test (strip method) on all suspect morphotypes. If the colonies are oxidase positive perform a Gram stain. Campylobacter will have a typical spiral or gull-winged shaped appearance on Gram stain. Perform catalase and set up susceptibility tests for nalidixic acid (30 µg) and cephalothin (30 µg) on BA (Appendix III). If there are any suspected Campylobacter isolates which do not fit the following reactions, send the organism(s) to PHL for further identification. Test C. jejuni / coli Catalase + Oxidase + Cephalothin 30 µg R Nalidixic acid 30 µg S CIN AGAR Yersinia enterocolitica appears as a small colony with a dark red centre surrounded by a transparent border ( bull s eye ). Any morphotype resembling Y. enterocolitica is set up for Vitek and/or API identification. If a Yersinia species is isolated, consult the microbiologist or charge technologist before reporting. Send the isolate to PHL for confirmation. Page 7

8 Policy # MI/ENT/01/v02 Page 7 of 11 TCBS AGAR After hours incubation, subculture all yellow or blue green colonies to BA and incubate at 35 0 C in O 2 x hours. Perform an oxidase test and Gram stain on all morphotypes growing on BA (Do not perform the oxidase test directly on colonies from the TCBS Agar). Set up a Vitek GNI + card on all oxidase positive gram negative bacilli. MARTIN-LEWIS AGAR 1. Examine the plate after 48 and 72 hours incubation. 2. Perform oxidase test and Gram stain on suspected GC. 3. If there is sufficient growth, perform a Gonogen GC coagglutination test from the primary plates (Refer to Appendix III Genital Manual). 4. Make two CHOC purity plates from suspect GC colonies and incubate in CO 2 at 35 0 C for hours. 5. After hours incubation, do the following: a) Inoculate API NH Strip from the purity plate (Refer to Appendix IV, Genital Manual). b) Perform Gonogen GC coagglutination from the purity plate if unable to perform from the original culture. (Refer to Appendix III, Genital Manual). 6. If after 72 hours incubation, bacterial growth is not typical of GC, flood the plate with oxidase reagent and immediately subculture any oxidase positive colonies onto CHOC. Repeat step 5 on all suspect colonies. 7. GC is positively identified by the reactions set out in Appendix V, Genital Manual. ENTEROCOCCUS AGAR 1. Examine VRE screen plates after 24, 48 and 72 hours incubation. 2. Gram stain any growth on the VRE plate. Subculture all Gram positive cocci to Blood Agar (BA) and incubate in O 2 at 35 0 C x hours prior to identification. 3. Identify E. faecium and E. faecalis as outlined below. Page 8

9 Policy # MI/ENT/01/v02 Page 8 of 11 Figure 1. Work-up of Suspected Vancomycin Resistant Enterococci (VRE) Step 1. Examine the BA plate for purity and colony pigment production (by swab) Discard all yellow-pigmented colonies as Not VRE Set-up PYR (Dry Slide) and 35 o C x hours. POS NEG Not VRE Step 2. Set-up Rapid Xylose (35 o C x 1.5 hours in a water bath) NEG POS Not VRE: E. gallinarum Step If E. faecium is suspected, set-up MIC panel. Interpret biochemical and susceptibility results after 24 hours incubation as outlined in Table If E. faecalis is suspected, set-up Arabinose fermtentation, MGP broth, Ampicillin KB, and BHI vancomycin 6 mg/l screen plate puls growth control plate. For Mount Sinai Hospital patients only, notify Infection Control of negative Xylose test and species of suspected VRE. Step 4. Step 5. Step 6. Read the BHI-6 vancomycin plate at 18, 24 and 48 hours for any growth. If vancomycin-resistant, identify enterococci as outlined in Table 1 below. Notify Infection Control of E. faecalis growing on BHI vancomycin agar and set-up MIC panel. (For Mount Sinai Hospital: do not enter as VRE into LIS until MIC complete). Confirmation of vancomycin-resistance and species identification of enterococci is performed from MIC panels after 24 hours incubation as outlined in Table 2. Page 9

10 Policy # MI/ENT/01/v02 Page 9 of 11 Step If a VRE is identified from the MIC panel: Notify Infection Control and the Ward Set-up 10 ml Brain Heart Infusion broth for Pulse Field Gel Electrophoresis (MSH clients only) Freeze and enter into the Freezer program Table 1. Identification of Vancomycin-Resistant Enterococci Organism Arabinose MGP Ampicillin E. faecium Positive Negative Resistant E. faecalis Negative Negative Susceptible E. gallinarum/casseliflavus Positive Positive Susceptible Salmonella 1. If an isolate is suspected to be Salmonella based on biochemical reactions only, but does not react with any antisera, send the organism to PHL for further testing. Cross-reactions with the Salmonella antisera can occur with some E. coli and other Enterobacteriaceae. Shigella 1. Check for autoagglutination and serotype using all available Shigella antisera (Appendix I). 2. If an isolate is suspected to be Shigella based on biochemical reactions but does not react with any antisera, send the organism to PHL for further testing. Page 10

11 Policy # MI/ENT/01/v02 Page 10 of 11 VI. Reporting Results Telephone all positive reports to ward / physician. Inform infection control of any positive cultures for enteric pathogens from all In-patients. These must be reported to the Medical Officer of Health and flagged in the LIS as Communicable Disease (CD). Negative Report: No Salmonella, Shigella, Campylobacter or E. coli 0157:H7 isolated. Negative Report when Yersinia Culture Performed: No Salmonella, Shigella, Campylobacter, Yersinia or E. coli 0157:H7 isolated. Positive Report: Salmonella species Preliminary report: Final report: Salmonella species. Further identification to follow. Salmonella. Public Health Laboratory Report No.. Shigella species Preliminary report: Final report: Shigella. Serotyping to follow. Shigella, serotype. Public Health Laboratory Report No.. Campylobacter species Final report: Campylobacter jejuni/coli Page 11

12 Policy # MI/ENT/01/v02 Page 11 of 11 E. coli 0157:H7 Preliminary report: Final report: E. coli Further identification to follow. E. coli 0157:H. Public Health Laboratory Report No.. Yersinia species Preliminary report: Final report: Yersinia enterocolitica. Serotyping to follow. Yersina enterocolitica, serotype. Public Health Laboratory Report No.. Vibrio species Negative Report: Preliminary positive report: Final report: Vibrio species not isolated. Vibrio species. Further identification to follow. Vibrio. Public Health Laboratory Report No.. Neisseria gonorrhoeae Negative Report: Positive Report: No Neisseria gonorrhoeae isolated If ML plate is overgrown by swarming Proteus or yeast report ONLY as Unable to rule out Neisseria gonorrhoeae due to bacterial/yeast overgrowth. Neisseria gonorrhoeae isolated (do not quantitate) betalactamase positive or negative (enter beta-lactamase result under breakpoint panel in LIS isolate screen) Page 12

13 Section: Policy # MI/ENT/02/v01 Page 1 of 2 Subject Title: Duodenal or Small Bowel Aspirate / Swab / Biopsy Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: I. Introduction DUODENAL OR SMALL BOWEL ASPIRATE / SWAB / BIOPSY Duodenal and small bowel aspirates and biopsy specimens are processed for O&P only. Swabs are processed for aerobic and anaerobic culture only. Aspirates and biopsy specimens for O & P should be sent immediately to the Parasitology section for processing. If the aspirate is not already in SAF and will not be processed the same day, transfer to SAF and then forward to the Parasitology section for processing. II. Specimen Collection and Transport Aspirates should be collected and transported in a syringe (needle removed) or a clean, sterile container. Biopsy specimens should be collected and transported in a clean, sterile container. Duodenal swabs should be transported in Amies transport medium and a special anaerobic container. III. Reagents / Materials / Media See Appendix I. IV. Procedure A. Processing of Specimens a) Direct Examination: Gram stain not performed. b) Culture: i) Duodenal or Small Bowel Aspirates Duodenal and SB aspirates are processed for O&P only. Specimens should be forwarded to the Parasitology section immediately. If a delay is anticipated and the specimen is not received in SAF, transfer the specimen to SAF. These specimens will not routinely be processed for bacterial culture. Page 13

14 Policy # MI/ENT/02/v01 Page 2 of 2 ii) Duodenal or Small Bowel Swab Media Blood Agar (BA) MacConkey Agar (MAC) Fastidious Anaerobic Agar (BRUC) Kanamycin / Vancomycin Agar (KV) Incubation O 2, 35 0 C 18 x 48 hours O 2, 35 0 C 18 x 24 hours AnO 2, 35 0 C x 48 hours AnO 2, 35 0 C x 48 hours B. Interpretation of cultures Refer to Miscellaneous / Wound Manual. C. Susceptibility Testing Refer to Susceptibility Testing Manual. V. Reporting Results Refer to Miscellaneous / Wound Manual. Page 14

15 Section: Policy # MI/ENT/03/v01 Page 1 of 2 Subject Title: Rectal / Large Bowel (Colon) Biopsies Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: I. Introduction RECTAL / LARGE BOWEL (COLON) BIOPSIES Rectal and Large Bowel (Colon) biopsies are usually collected for investigation of patients with bloody diarrhea. Cytomegalovirus is the most common viral agent associated with this syndrome. Although bacterial agents such as Salmonella, Shigella, E. coli 0157:H7 and others may cause bloody diarrhea, the preferred specimen for detection of these organisms is a stool specimen. However, if requested, bacterial culture will be performed and the specimen will be processed as a stool specimen. A portion of the specimen received in the Microbiology Laboratory should be forwarded to the Virology section for processing. II. Specimen Collection and Transport Specimens are usually collected via a colonoscope or sigmoidoscope and should be transported in a clean sterile container with a small amount of sterile saline or sterile water or viral transport media. III. Reagents / Materials / Media Refer to Appendix I IV. Procedure 1. Processing of Specimens a) Direct Examination: Gram stain not indicated. Page 15

16 Policy # MI/ENT/03/v01 Page 2 of 2 b) Culture: Media MacConkey Agar (MAC) Hekton Agar (HEK) MacConkey Sorbitol Agar (MAC-S) Camyplobacter Agar (CAMPY) Campy Jar Selenite Broth (SEL) Incubation O C x hours O C x hours O C x hours 42 0 C x 48 hours O C x hours V. Reporting Results Refer to Faeces/Rectal Swabs section. VI. References 1. Murray P., Brown E., Pfaller M., Tenover F., Yolken R. Manual of Clinical Microbiology, 7 th Edition, ASM Press, Washington, D.C pp Page 16

17 Section: Policy # MI/ENT/04/v01 Page 1 of 2 Subject Title: Gastric Aspirates / Biopsies (for Helicobacter pylori) Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: I. Introduction GASTRIC ASPIRATES / BIOPSIES (FOR HELICOBACTER PYLORI) H. pylori is implicated in the etiology of some cases of gastritis and peptic ulcers. II. Specimen Collection and Transport This specimen should be collected into a clean sterile container and transported to the laboratory as soon as possible. III. Reagents / Materials / Media Refer to Appendix I. IV. Procedure A. Processing of Specimen: 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) Macerate the remaining tissue for culture: b) Culture: Media Blood Agar (BA) Campylobacter Agar (CAMPY) Incubation Microaerophilic, 36 C x 7 days Microaerophilic, 36 C x 7 days Page 17

18 Policy # MI/ENT/04/v01 Page 2 of 2 B. Interpretation of cultures: a) Examine the direct urea slant after 1 and 4 hours incubation. A positive reaction is presumptive evidence of the presence of H. pylori. b) Examine the plates after 3, 5 and 7 days incubation. Colonies of H. pylori are grey, translucent and small (0.5 to 1.0 mm in diameter). H. pylori is positively identified by the reactions below. Identification of H. pylori: Test Gram stain catalase oxidase urea broth (rapid) Cephalothin 30µg Nalidixic acid 30µg H. pylori Small, gram negative gull-shaped or spiral S (inhibition) R (no zone) C. Susceptibility Testing: Not required. V. Reporting Results Gram Stain: Presence or absence of small, gull-shaped or spiral Gram negative bacilli Culture: Negative Report: Positive Report: "No Helicobacter pylori isolated" "Helicobacter pylori isolated" Page 18

19 Policy # MI/ENT/05/01/v01 Page 1 of 2 Section: Subject Title: Appendix I - Reagents / Materials / Media Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: APPENDIX I REAGENTS / MATERIALS / MEDIA Reagents / Materials / Media : Amies transport medium NCS Diagnostics Blood Agar (BA) - PML Bacteroides Bile Esculin (BBE) Agar Med Prep Campylobacter plate (Campy) Med Prep Cary-Blair transport medium Central Stores Catalase Reagent 3% H 2 O 2 Refer to Media Manual for preparation Cephalothin 30µg disc Oxoid Cefsulodin, Irgasan, Novobiocin agar (CIN) PML E. coli 0157 Latex Agglutination Test Oxoid Enterococcus Agar with Vancomycin (EV) Fastidious Anaerobic Agar (BRUC) Med Prep Gram stain Refer to Media Manual for preparation Hektoen (HEK) Med Prep Kovac s Reagent Refer to Media Manual for preparation MacConkey agar (MAC) - PML MacConkey Sorbitol (MAC-S) - Med Prep Martin-Lewis (ML) - Biomedia Nalidixic acid 30µg disc - Oxoid ONPG-PAM Med Prep Oxidase Strip - API Salmonella antisera Abbott Diagnostic Inc. Salmonella O (A-S) Salmonella H (phase 1 and 2) Salmonella H (phase 2) Salmonella Vi Salmonella 2-O Salmonella 4-O Salmonella 3,10,15,19-O Salmonella 6,7-O Salmonella 8-O Salmonella 9-O Page 19

20 Policy # MI/ENT/05/01/v01 Page 2 of 2 Selenite Broth (SEL) Med Prep Shigella antisera Abbott Diagnostics Inc. S. sonnei (phase 1 and 2) S. flexneri polyvalent S. dysenteriae S. boydii polyvalent 1 S. boydii polyvalent 2 S. boydii polyvalent 3 Trypticase Soy Broth (TSB) Med Prep Triple Sugar Iron (TSI) Med Prep Triphenyl-tetrazolium chloride (TTC) Rapid Xylose Bile Esculinn Agar (BE) PYR (PYR) Page 20

21 Section: Policy # MI/ENT/05/02/v01 Page 1 of 2 Subject Title: Appendix II - Serological Testing Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: Serological Testing APPENDIX II SEROLOGICAL TESTING Serological testing is performed to identify Salmonella, Shigella and E. coli Testing is performed by a slide agglutination test using somatic (O) or flagella (H) antisera. Materials: 1. Glass slides 2. Bacteriology loop 3. Appropriate Salmonella, Shigella or E. coli antisera. (Store refrigerated) 4. Sterile saline Procedure: 1. With a China-marker, divide the slide into two. 2. Put a drop of saline in each section. 3. Using a loop, emulsify the organism in each drop of saline to give a homogenous, fairly dense suspension. 4. Add one drop of undiluted antiserum to one of the suspensions and mix. The other suspension serves as a control. 5. Rock the slide gently and observe for agglutination using indirect lighting over a dark background. Agglutination should be strong and clearly visible within one minute. 6. If there is agglutination in the saline control, the test is invalid. Salmonella spp. If there is little or no H 2 S in the TSI tube and the isolate resembles Salmonella, test with the Vi antiserum first. The antisera are tested in the following order: 1. Polyvalent O (A S) 2. Polyvalent H (phase 1 and 2) 3. Polyvalent H (phase 2) 4. If polyvalent O is positive, then proceed with other O antisera. Page 21

22 Policy # MI/ENT/05/02/v01 Page 2 of 2 5. If the organism does not react with the polyvalent O antiserum, and the isolate is still suspected to be Salmonella due to the biochemical reactions, send to PHL for identification. Shigella spp. 1. Serological confirmation of Shigella isolates is based only on O antigen testing. 2. If the isolate is biochemically a Shigella species, but fails to agglutinate with the grouping sera, test suspect colonies with the following antisera in the following order: a) S. sonnei b) S. flexneri c) S. dysenteriae d) S. boydii 3. Send to PHL for confirmation. E. coli 1. E. coli 0157 antiserum is used. 2. Known positive (LPTP ) and negative (ATCC 25299) must be tested with each batch of testing. Page 22

23 Section: Policy # MI/ENT/05/03/v01 Page 1 of 2 Subject Title: Appendix III - Speciation of Campylobacter Issued by: LABORATORY MANAGER Original Date: March 27, 2000 Approved by: Laboratory Director Revision Date: APPENDIX III SPECIATION OF CAMPYLOBACTER Speciation of Campylobacter spp. using Antimicrobial Agents Speciation is performed using nalidixic acid and cephalothin to determine if the isolate is a Campylobacter jejuni / coli or other Campylobacter species. Materials: 30-ug nalidixic acid disk 30-ug cephalothin disk BA TSB Bacteriology loop Sterile swabs Procedure: 1. Prepare a suspension of the Campylobacter isolate in TSB to match a McFarland standard #2. 2. Inoculate the surface of a BA plate with the broth inoculum using a sterile swab to obtain confluent growth (as for KB sensitivity testing). 3. Place the nalidixic acid and cephalothin disks on the surface of the agar. 4. Incubate at 35 0 C in microaerophilic conditions for hours. Page 23

24 Policy # MI/ENT/05/03/v01 Page 2 of 2 Quality Control: Interpretation Susceptible: Any zone of inhibition Resistant: Growth up to the edge of the disk Test C. jejuni / coli Catalase + Oxidase + Cephalothin 30 µg R Nalidixic acid 30 µg S Note: The results of this test cannot be used for susceptibility reporting. Quality control testing should be performed weekly. Nalidixic acid Cephalothin Campylobacter jejuni - ATCC S R Campylobacter fetus - ATCC R S Reference Karmali, M. A., Antimicrobial Susceptibility of Campylobacter jejuni and Campylobacter fetus subsp. fetus to Eight Cephalosporins with Special Reference to Species Differentiation. Antimicrob. Agents Chemother. 18: Isenberg H.D. Clinical Microbiology Procedure Handbook, Vol. 1, ASM; 1994 p Page 24