Stool Culture

Acute infectious diarrhea may be caused by a number of different agents including bacteria, viruses and protozoa. The laboratory routinely searches for bacteria that are most likely to cause diarrhea.

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 Vibrio spp. and Yersinia enterocolitica.

See Stool Culturing from notes section.

setup

Rejection time

72 hours

Specific rejection criteria

Rejection Criteria Report / Comment
All formed stools except when S. typhi(typhoid fever) requested “Formed stool received. Test cancelled.”
Patient hospitalized for 3 days or more Refer for C. difficile toxin testing and VRE screen “Patient has been hospitalized for 3 days or more. Because infectious diarrhea caused by normal bacterial and protozoal pathogens is extremely unlikely to be acquired in hospital, ova and parasite and culture examination will not be performed. If the sample is diarrheal a C. difficile cytotoxin assay will be performed. If you feel a nosocomial outbreak is occurring please contact the Infection Control Officer. If there are unusual circumstances warranting a culture or ova and parasite exam please contact the microbiology laboratory.”
Multiple specimens collected from the same in-patient the same day “This specimen has not been processed as a specimen collected on the same day has already been processed.”
CDT requested on formed stool “Specimen consisted of FORMED STOOL which indicates that C. difficile testing is not warranted. If examination seems clinically indicated, please contact the microbiology laboratory.”

Phone ward / physician and document on report if specimen rejected.

Container

Enteric Pathogen Transport

Specimen Holding Temperature

4°C

Direct Examination

Not routinely performed. Upon special request, a Gram stain for fecal leukocytes may be performed.

Culture

MacConkey Agar (MAC) O2 35°C x 18 – 24 hours
Hektoen Agar (HEK) O2 35°C x 18 – 24 hours
MacConkey Sorbitol Agar (SMAC) O2 35°C x 18 – 24 hours
Campylobacter Agar (CAMP) Campy Jar 42°C x 72 hours

If Yersinia is requested add:

MacConkey (MAC) at 25°C O2 25°C x 48 hours

If Vibrio is requested, add:

Thiosulphate Citrate Bile Salt Sucrose Agar (TCBS) O2 35°C x 18 – 24 hours

If C. difficile toxin assay is requested and appropriate specimen received, forward for testing. Specimen is also set up for Vancomycin Resistant Enterococcus (VRE) screen.

Enterococcus Agar (6 µg/ml Vancomycin) O2 35°C x 72 hours

If N. gonorrhoeae(GC) is requested (rectal swab only) inoculate only:

Vancomycin Colistin Nystatin Agar (VCN) CO2 35°C x 72 hours

Followup

Medium Suspect Colonies
MacConkey agar (MAC) Non-Lactose Fermenter (NLF) (colourless or transparent)
Hektoen Agar (HEK) Green with or without H2S

Pick two colonies of each suspect morphotype and inoculate a urea slant and Triple Sugar Iron (TSI) slant and a Heart Infusion Agar slant (HIA). Results are read after overnight incubation at 35°C in O2 (See table below).

Table 1 Characteristic reactions of potential stool pathogens

Organism TSI Urea
Salmonella typhi Alk/Acid 1. Neg
Salmonella arizonae Var./Acid H2S Neg
Salmonella paratyphi A Alk/Acid 2. Neg
Other Salmonella Alk/Acid H2S Neg
Shigella spp. Alk/Acid No Gas 3. Neg
Y. enterocolitica Var./Acid +/- (slow)
  1. may produce small amounts of gas and /or H2S
  2. occasionally produces H2S weakly
  3. S. flexneri(type 6) may produce a small amount of gas“Var.” indicates variable results

If results of these tests suggest:

  • Salmonella – Perform serotyping, ID and Sens
  • Shigella – ID and Sens
  • Yersinia – ID and Sens and Motility (TSB) at 25º and 35º C

Salmonella agglutination tests must be performed from a non-selective medium such as TSI or HIA. Agglutinations should not be performed from the MAC or HEK plates.

If an isolate is suspected to be Salmonella based on biochemical reactions only, but does not react with any antisera, send to reference lab for further testing. Cross-reactions with the Salmonella antisera can occur with some E. coli and other Enterobacteriaceae.

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.

If sorbitol non-fermenting colonies are observed pick 4 colonies to HIA slants, incubate overnight and perform E. coli 0157:H7 latex antiserum testing. Set up ID and sens. on latex positives.

Examine after 72 hours incubation. Colonies of Campylobacter are grey or colourless, pinpoint flat or mucoid to convex to spreading across the plate.

Perform a Gram stain on all suspect colony types. If they have the typical spiral or gull-winged shaped appearance on Gram stain perform an oxidase test. Campylobacter spp. are oxidase positive.

Do hippurate and catalase tests on suspect isolates.

After 18 – 24 hours incubation, subculture all yellow or blue green colonies to BA and incubate at 35°C in O2 x 18-24 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 ID on all oxidase positive Gram-negative bacilli.

  • Examine the plate after 48 and 72 hours incubation.
  • Perform oxidase test and Gram stain on suspected GC.
  • If there is sufficient growth, directly inoculate an API NH
  • If there is not sufficient growth make two CHOC purity plates from suspect GC colonies and incubate in CO2 at 35°C for 18-24 hours.
  • After 18-24 hours incubation, inoculate API NH Strip from the purity plate.
  • Examine after 18 – 24 hours incubation. Suspect morphotypes will appear as small, flat, colourless or pale pink colonies.
  • Inoculate TSI and Urea Slant and if typical inoculate ID and motility test at 25° C (+) and 35° C (-).
  • Select pink or magenta/red colonies on M-Enterococcus agar (VRE agar from PHL) incubated a full 24 hours and do Gram stain. Incubate plates for 72 hours before reporting as negative.
  • If Gram positive cocci, subculture to BA to obtain isolated colonies.
  • Set up Methyl alpha-D-Glucopyranoside (MGP) sugar at 35° C and TTC motility medium at 30° C to rule out E. gallinarumE. casseliflavus and E. flavescens. These three species may have elevated MICs to vancomycin (usually approx. 8 mg/L) but are not true VREs. They may grow on the screen plates and must be distinguished from E. faecalisand E. faecium – the two species of interest.

Motility (with TTC dye) and pigment production

  • Negative – possible True VRE
  • Positive – E. gallinarumE. casseliflavus or E. flavescens

MGP

  • Negative (PURPLE) – possible True VRE
  • Positive (YELLOW) – E. gallinarum or E. casseliflavus

NOTE When set up early both the motility and MGP may be positive within 5 hours. If not they must be incubated for 24 hours.

Further Workup on Motility Negative / MGP Negative Isolates

ID and sensitivity

Reporting

All significant isolates from enteric samples have Public Health Implications

  • Telephone all positive reports to ward / physician.
  • Inform Infection Control of any positive cultures for enteric pathogens from all In-patient and ER patients.
  • Record information in Significant Isolates Log Book.

“No Salmonella, Shigella, Campylobacter or E. coli 0157:H7 isolated.”

Salmonella

Preliminary report: “Salmonella species isolated. Further report to follow.”

Final Report: “Salmonella XXXX isolated.” Edit significant Isolates log book.

Shigella

Preliminary report: “Shigella species isolated. Further report to follow”

Final report: “Shigella XXXX isolated”. Edit significant isolates log book.

Campylobacter Preliminary report: “Campylobacter species isolated. Further report to follow.”

Final report: “Campylobacter XXXX isolated.”

E. coli O157:H7

Preliminary report: “E. coli O157 H7 presumptively identified. Further report to follow.”

Final report: “E. coli O157 H7 identified.”

Yersinia Negative report: “No Yersinia species isolated”.

Final report: “Yersina enterocolitica isolated”.

Vibrio

Negative Report: “No Vibrio species isolated”.

Preliminary report: “Vibrio species isolated. Further report to follow”.

Final report: “Vibrio XXXX isolated.”

Neisseria gonorrhoeae

Negative Report: “No Neisseria gonorrhoeae isolated” If VCN plate is overgrown by swarming Proteus or yeast report ONLY “Unable to rule out Neisseria gonorrhoeae due to bacterial/yeast overgrowth.”

Positive Report: “Neisseria gonorrhoeae isolated” (do not quantitate) “beta-lactamase positive” or “beta-lactamase negative”.