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Stool cultures and C. difficile

17 min

Skills you'll build4 skills
  • Choose stool media and toxin or NAAT tests for the suspected enteric pathogen
  • Identify Enterobacterales species from TSI and other biochemical reactions
  • Tell Vibrio, Aeromonas, and Campylobacter from enteric fermenters in stool culture
  • Tell C. difficile carriage from toxin production using NAAT and toxin results

At the bench: Select specimen preparation, media, and incubation for a culture

Try first

Try first

A diarrheal stool arrives in Cary-Blair for culture. The requisition says the patient ate raw oysters 2 days ago. Your routine stool culture looks for Salmonella, Shigella and Campylobacter. What do you add?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Setting up the enteric culture

Stool for culture is unformed and reaches the laboratory within 2 hours, or travels in Cary-Blair and arrives within 24 hours. Formed stool is rejected for enteric culture and for Clostridioides difficile testing unless the physician documents ileus or possible toxic megacolon. Many laboratories do not run routine enteric culture once a patient has been in hospital for more than 3 days, because community pathogens rarely cause diarrhea that starts that late.1

MediumSelects for
MacConkey, XLD, Hektoen entericLactose and other sugar fermentation and H2S, to detect Salmonella and Shigella
Campylobacter selective agar, 42 °C, microaerophilicC. jejuni and C. coli
Sorbitol-MacConkey (SMAC)O157, which does not ferment sorbitol
TCBSVibrio, on special request
CIN at 25 °CYersinia, on special request

Every stool cultured for enteric pathogens also gets a Shiga toxin immunoassay or stx NAAT, with O157 culture alongside. SMAC alone misses the non-O157 strains that now cause much of the disease.1,2

Identifying the isolate

Colony color narrows the possibilities and does not identify a species. Many organisms share each lactose reaction, and Shigella sonnei can ferment lactose late. The full reaction pattern on triple sugar iron (TSI) agar and other biochemicals, or MALDI-TOF, identifies the isolate. MALDI-TOF libraries do not reliably separate Shigella from E. coli, so supplemental tests complete that identification.3,4

Vibrio, Aeromonas and Campylobacter are oxidase positive. Acid from sugar fermentation on TCBS or MacConkey can make the oxidase test falsely negative, so it is read from blood agar growth.3

C. difficile testing

Test only new, unexplained diarrhea: at least three unformed stools in 24 hours and no laxative in the previous 48 hours.5 Each test answers a different question.

  • Glutamate dehydrogenase (GDH) antigen shows the organism is present. Toxigenic and nontoxigenic strains both make it.
  • NAAT detects the toxin gene, whether or not toxin is being made.
  • The toxin immunoassay detects free toxin in the stool.

Carriage without disease is common. A NAAT-positive, toxin-negative result often reflects carriage, and the report states what was detected.1,5

Follow the bench demonstration

Sources for this step
References
  1. Miller JM, Binnicker MJ, Campbell S, et al. Guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2024 update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. Published online March 5, 2024. doi:10.1093/cid/ciae104
  2. Gould LH, Bopp C, Strockbine N, et al. Recommendations for diagnosis of Shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR Recomm Rep. 2009;58(RR-12):1-14. Accessed September 27, 2026. https://www.cdc.gov/mmwr/preview/mmwrhtml/rr5812a1.htm
  3. Tille PM. Bailey & Scott's Diagnostic Microbiology. 16th ed. Elsevier; 2025.
  4. Association of Public Health Laboratories. Identification Guide: Isolation and Identification of Shigella species and Enteroinvasive Escherichia coli from Culture-Independent Diagnostic Test Positive Specimens. APHL; July 2025. Accessed September 27, 2026. https://www.aphl.org/aboutAPHL/publications/Documents/FS-Shigella-EIEC-CIDT-Guide.pdf
  5. McDonald LC, Gerding DN, Johnson S, et al. Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018;66(7):e1-e48. doi:10.1093/cid/cix1085

Watch one

Read each decision and its explanation.

A stool arrives from a 6-year-old with bloody diarrhea that began 3 days after a visit to a petting farm. It is unformed, in Cary-Blair, and was collected 20 hours ago. The order reads "stool culture".

What do you set up?

  1. Check the specimen: unformed stool in Cary-Blair, received within 24 hours. It is acceptable.

    An unacceptable specimen cannot be cultured, so acceptability is checked first.

  2. Plate the routine media: MacConkey with XLD or Hektoen enteric for Salmonella and Shigella, and Campylobacter selective agar at 42 °C in a microaerophilic atmosphere.

    The routine culture covers the common bacterial causes of community diarrhea.

  3. Add a Shiga toxin immunoassay or stx NAAT on the stool.

    Bloody diarrhea after animal contact fits Shiga toxin-producing E. coli (STEC), and non-O157 strains ferment sorbitol.

  4. Plate SMAC alongside for O157.

    O157 culture gives an isolate for public health, and the toxin test detects every serogroup.

  5. Omit TCBS and CIN.

    No shellfish or pork exposure is recorded, so the special-request media are not needed.

Routine enteric and Campylobacter media, a Shiga toxin immunoassay or stx NAAT, and SMAC for O157.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A colony suspected to be Vibrio grows yellow on TCBS. From which growth is the oxidase test performed?

Incorrect. Acid from sucrose fermentation on TCBS can interfere with the oxidase reaction and give a false-negative result.

Correct. Oxidase is read from growth on a medium without fermentable carbohydrate, such as blood agar, because acid from fermentation on TCBS or MacConkey agar can give a false-negative reaction.

Incorrect. MacConkey agar also contains a fermentable sugar and a pH indicator, and like TCBS it can give an incorrect oxidase result.

Hint
  1. Consider the effect of fermentation on the pH around a colony.
  2. Consider how acid affects the oxidase reaction.

Review Curved and oxidase-positive enteric organisms

Problem 2 of 3 · Hints available

A stool from a patient who meets the testing criteria is glutamate dehydrogenase (GDH)-positive and toxin-negative in a two-step C. difficile algorithm. How is the result interpreted?

Correct. GDH shows C. difficile without showing whether the strain makes toxin, so the discordant pair is arbitrated, for example by NAAT. A NAAT-positive, toxin-negative result often reflects carriage.

Incorrect. GDH is produced by toxigenic and non-toxigenic strains alike, and the toxin assay is negative, so the pair leaves toxin production unresolved.

Incorrect. A positive GDH shows the organism is present. The negative toxin assay leaves toxin production unresolved.

Hint
  1. Consider what each of the two tests detects.
  2. Both toxigenic and nontoxigenic strains produce GDH.

Review Clostridioides difficile and gastrointestinal anaerobes

Problem 3 of 3 · On your own

A colorless colony from MacConkey is oxidase negative. It gives these reactions: TSI alkaline slant over acid butt with H2S, indole negative, urease negative, motile, lysine decarboxylase positive. Which identification do the reactions support?

Many enteric organisms are lactose negative. Shigella makes no H2S and is nonmotile, and this isolate is positive for both.

Named an enteric species from lactose fermentation

Lactose fermentation on MacConkey agar separates pink from colorless colonies. Many species give each pattern, and Shigella sonnei can ferment lactose late, so a species named from the MacConkey reaction can be wrong. Identify the organism from the full biochemical pattern or by MALDI-TOF. E. coli and Shigella need extra tests even then.

P. mirabilis makes H2S and is motile, and it is also urease positive. This isolate is urease negative.

C. freundii shares H2S and motility and is lysine decarboxylase negative. This isolate decarboxylates lysine.

H2S positive, motile, indole negative, urease negative and lysine decarboxylase positive is the typical Salmonella pattern. Serotyping by O and H antigens completes the identification.

Review Enterobacterales

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Ruth Reade, 71, is on day 6 of a hospital stay for a hip fracture.
  • Diarrhea began on day 5, with four unformed stools in the last 24 hours. She has had no laxatives.
  • One stool arrives with orders for a routine stool culture and C. difficile testing.
  • The laboratory runs GDH with toxin immunoassay, and NAAT resolves a discordant pair.
TestResultReference intervalFlag
C. difficile GDH antigenPositiveNegativeNegative
C. difficile toxin A/B immunoassayNegativeNegativeNegative
C. difficile toxin gene NAATPositiveNegativeNegative

Specimen: Not measured on stool. Unformed stool, received 40 minutes after collection

Decision 1 of 2

What happens to the routine stool culture order?

Community enteric pathogens rarely explain diarrhea that starts after 3 days in hospital, and the culture adds cost with little yield.

Her diarrhea began on day 5 in hospital, so the routine culture falls under the laboratory's 3-day rule. C. difficile testing is the appropriate test for hospital-onset diarrhea.

The stool meets the criteria for C. difficile testing. Only the routine culture falls under the 3-day rule.

Review Targeted stool pathogen workup

Decision 2 of 2

How is the C. difficile result reported?

The NAAT shows a strain with the toxin gene. Free toxin was not detected, and this pattern often reflects carriage.

Reported a NAAT-positive, toxin-negative result as infection

A NAAT detects the toxin gene whether or not toxin is being made, and asymptomatic carriage is common. A NAAT-positive, toxin-negative result often reflects carriage, so reporting it as C. difficile infection can label carriage as disease.

GDH and NAAT both show a toxigenic strain is present. Reporting the stool as negative omits that finding.

The report states each finding. The clinician interprets the toxin-negative result together with the patient's illness.

Review Clostridioides difficile and gastrointestinal anaerobes

The testing depends on when and where the diarrhea began. Onset on day 5 in hospital makes C. difficile the test to perform. The negative toxin result then determines how the positive NAAT is reported.

For reference

Sources checked