Standard Operating Procedure

Subject Culture: Routine Stool

Index Number Lab-3105
Section Laboratory
Subsection Microbiology
Category Departmental
Contact Sarah Stoner
Last Revised 1/17/2017

References
Required document for Laboratory Accreditation by the College of American Pathologists.

Applicable To
Employees of the Gundersen Health Systems laboratories.

Detail
This document establishes guidelines for isolation and identification of stool pathogens.

PRINCIPLE:
Gastroenteritis can be caused by bacteria, parasites or viruses. Physician input can help the lab
determine which tests are appropriated for detecting the etiological agent. When only a routine fecal
culture is requested, however, the lab should search for the most common or more easily detected
bacterial agents of diarrhea. Our laboratory looks for Salmonella, Shigella, E.coli O157:H7 and
Campylobacter.

CLINICAL SIGNIFICANCE:
Salmonella, Shigella, Campylobacter and E.coli O157:H7 are the most prevalent stool pathogens isolated
by our laboratory. They are implicated as agents of foodborne illness and isolates need to be reported
to the state for epidemiological purposes. Salmonella outbreaks have been associated with raw eggs,
reptiles, and poor hand washing. E.coli O157:H7 outbreaks have been traced to hamburger, raw milk,
sausage, roast beef, un-chlorinated municipal water, apple cider, raw vegetables, salads, and
mayonnaise. E.coli O157:H7 spreads easily from person to person because the infectious dose is low:
outbreaks associated with person to person spread have occurred in schools, long term care institutions,
families, and day-care facilities. Campylobacter has been associated with poultry products and
contaminated water.

SPECIMEN:
Refer to Lab-1215 Microbiology Specimen Collection and Transport.
Fresh stool received within 1-2 hours of passage.
1. Stool in Cary-Blair transport media (store at 4-6°C).
2. Routine bacterial fecal cultures should not be performed on patients hospitalized for > 3 days.
The exception to this is a hospital outbreak.

REAGENTS/MATERIALS:
1. BBL Mac Conkey II with sorbitol (commercial) SMAC for E.coli O157:H7.
2. Hektoen Agar Plate (commercial).

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 Standard Operating Procedure

3. Mac Conkey Agar Plate (commercial).

4. Campylobacter Agar Plate (commercial).
5. TSI slant
6. KIA slant
7. LIA slant
8. Urea slant
9. Blood Agar Plate (BAP)
10. VITEK2 GNI, AST-GN66 (Salmonella and Shigella only), GPI and AST-GP67 cards
11. 0.9% sterile saline
12. Salmonella serotyping antisera
13. Shigella serotyping antisera
14. RIM Test Kit

EQUIPMENT/INSTRUMENTATION: N / A

QUALITY CONTROL:
1. Refer to Lab-3257 User Quality Assurance Procedure for Culture Media.
2. The stock isolate of Campylobacter jejuni is subbed daily to BAP, for each jar in use. This serves
as a growth indicator to ensure that the correct atmospheric conditions were used during
incubation.

Implementation
Inoculate the following media: SMAC, Hektoen, Mac Conley and Campylobacter agar. If blood or mucus
is present in the stool, use this portion to inoculate the plates. If a gram stain is requested make a
smear also.
1. Streak plates for isolation.
2. Incubation:
a. SMAC, HEK and MAC agars should be incubated in the non-CO2 incubator at 35 to
37°C. Hold plates for 48 hours before discarding.
b. Media for isolation of Campylobacter spp. should be incubated in a microaerobic
atmosphere (5% oxygen) at 42°C for 72 hours before discarding.

PROCEDURAL NOTES:
Routine stool cultures do not include screening for Yersinia. If Yersinia is incidentally recovered on
routine cultures, work up, and report to physician. (Physicians should order a culture for Yersinia if
suspected.)

CALCULATIONS: N/A

INTERPRETATION OF GROWTH ON CULTURE MEDIA FOR STOOL SAMPLES:

Medium Pathogen Colony Morphology Identification Procedure
Mac Conkey Colorless or transparent TULN (see chart) and BAP
SMAC Colorless or transparent TULN plus sorbitol, dulcitol and
BAP (refer to identification
scheme for E.coli O157:H7)

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 Standard Operating Procedure

Hektoen Blue or blue green with or
without black centers
Campy plate Grey to pinkish, flat to mucoid to
convex to spreading all over
plate
TULN (see chart) and BAP
Gram stain, Wet Prep and
Oxidase (see Table 1)

ENTERIC SCREENING:
1. Suspicious colonies should be set up to the following tubes and plate.
a. TSI: stab the butt and streak the slant
b. KIA: stab the butt and streak the slant
c. LIA: stab the butt x 2 and streak the slant
d. Urea: streak the slant
e. BAP (to be used for indole, oxidase, PYRr, serotyping and gni/gns if necessary)
If the SMAC plate has a large number of clear colonies of the same morphologic
colony type, you can do a RIM test from the SMAC plate and do not do a TULN. If
positive sub to a BAP to get a pure isolate for the gni card or API strip. Refer to Lab-
3106 RIM Test for E.coli O157:H7
2. Incubate tubes overnight at 35-37°C (non-CO2 incubator)
3. Determine if the organism is a potential pathogen by comparing reactions and doing quick
tests. Follow the flow chart provided.
4. Suggestive screening results should have complete biochemical identification.
a. Vitek 2 gni
b. Serology – Refer to Lab-3326 Serotyping Salmonella; Lab-3328 Serotyping Shigella;
Lab-3106 RIM Test for E.coli O157
5. Do sensitivities on Salmonella and Shigella species.
6. Confirmed pathogens should be called to the RN or doctor.
7. Streak a nutrient slant x 2. Send all stool pathogens to the state lab for food borne illness
surveillance. (Wisconsin residents to Wisconsin State Lab, Minnesota residents to MN State
Lab. Iowa want Salmonella and Shigella, but not Campylobacter.)

CAMPYLOBACTER SPECIES
1. Examine plates at 24, 48 and 72 hours for characteristic colonies, which can range from flat,
spreading colonies that cover the entire surface of the plate to very small, convex,
translucent colonies. Colony color can range from gray to yellowish or pinkish.
2. Presumptive identification
a. Gram stain shows small, slightly convex, gram negative rods that are “s” or seagull
shape.
b. Wet prep shows darting or tumbling motility.
c. Oxidase positive.
d. Catalase positive.
3. Definitive identification
a. Hippurate test: C.jejuni is the only Campylobacter that is Hippurate positive. Report
any Hippurate negative isolate as Campylobacter species.

E.COLI O157:H7

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 Standard Operating Procedure

1. If the TULN is interpreted as E.coli, the sorbitol is (-) and the dulcitol is (+), proceed with RIM
test. If the SMAC plate has a large number of clear colonies of the same morphologic colony
type, you can do a RIM test from the SMAC plate and do not do a TULN. If positive, sub to a BAP
to a pure isolate for the gni or API strip. Refer to Lab-3106 RIM Test for E.coli O157.
2. Isolate must be confirmed to be an E.coli before sending out as E.coli O157. Set up to gni card
or API strip to confirm.
3. When interpreting the API strip, pay particular attention to the raffinose, lysine, ornithine,
arginine and sorbitol. 90% of E.coli O157 will have the following results:
Raffinose (+), Lysine (+), Ornithine (+), Arginine (-) and Sorbitol (-)

DIFFERENTIAL CHART: TULN

Reading TSI, LIA, Urea, KIA (On suspected Salmonella and Shigella – non-lactose fermenting colonies)
Non- TSI H2S LIA H2S UREA KIA
Pathogens
Citrobacter A/A + or K/A + - or A/A
K/A trace - trace K/A
Klebsiella K/A - K/A - Trace A/A
Enterobacter Or or Or Or
Serratia A/A K/K + K/A
Proteus K/A +/- K/A -/+ + K/A
R/A
Providencia K/A - R/A - - K/A
E.coli K/A - K/K - - A/A
A/A K/A K/A

Pathogens TSI H2S LIA H2S Urea KIA

other than
E.coli O157
and
Camylobacter
Salmonella or K / A Trace or K/K + - K/A
Edwardsiella +
Arizona sp. A/A + K/K + - K / A or
A/A
Possible K/A - K/A - - K/A
Shigella
Yersinia A/A - K/A - - or K with acid
enterocolitica Trace (yellow
Or orange
Salmonella A/A - K/A - - color)
paratyphi K/A K/A
Refer to Lab-3105.1 Stool Culture Work Aide for additional guidance from the Clinical Microbiology
Procedure Handbook. Copy is in the drawer by the R3 desk.

REPORTING:

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1. A preliminary culture report will be sent out at 24 hours. Additional preliminary reports should
be sent out on anything significant after 24’.
2. A final culture report will be sent out when all identifying tests are completed. A final report
negative report will be sent out as: No Salmonella, Shigella, E.coli O157:H7 or Campylobacter
isolated. Notify microbiology at ext. 53138 if other pathogens are suspected.
3. The following are considered pathogen in a stool culture:
a. Campylobacter
b. Salmonella
c. Shigella
d. Yersinia enterocolitica (Dr. must specifically request)
e. E.coli O157:H7
f. Vibrio (Dr. must specifically request)

LIMITATIONS: N/A

REVIEW AND CHANGES:

This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Medical Director or designated
person. Changes require retyping the document or form and review by the Medical Director.

REFERENCES:
1. “Essential Procedures for Clinical Microbiology”, Henry D. Isenberg, 1998, pp. 90-94.
2. “Clinical Microbiology Procedures Handbook”, Henry D. Isenberg, 1992, pp.1.10.1 – 1.10.23 (
3. “Identification of Enterobacteriacae”, P.R. Edwards, W.H. Ewing, 3rd Edition, Burgess
Publishing Company, 1972.
4. Enteric Bacteriology laboratories, DHEW-PSH-CDC, Atlanta, GA, July 1973
5. Wisconsin State Lab of Hygiene, Bacteriology, May 2004
6. “Clinical Microbiology Procedures Handbook”, Henry D. Isenberg, 2004, Figure 3.8.1-2
Flowchart for identification of stool pathogens from routine stool cultures.

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