8.1 Enteric Pathogens: Culture and Colony Morphology
Key Takeaways
- Routine stool screens use MacConkey plus HE or XLD at 35–37 °C; add Campy-BAP at 42 °C in a microaerophilic atmosphere, CIN at 25–30 °C for Yersinia enterocolitica, and TCBS plus alkaline peptone water for Vibrio.
- Salmonella and Shigella are oxidase-negative non-lactose fermenters; Salmonella is usually H2S-positive on HE/XLD, while Shigella is H2S-negative, nonmotile, and green or red without a black center.
- Campylobacter, Vibrio, Aeromonas, and Plesiomonas are oxidase-positive; never read oxidase from TCBS or other dye-containing selective agar—subculture to blood agar first.
- Campylobacter jejuni is a curved gull-wing Gram-negative rod that grows as gray spreading colonies on Campy-BAP at 42 °C, not on MacConkey or HE incubated in air at 35 °C.
- Do not report “normal fecal flora” or “no enteric pathogens” unless the indicated screening plates were inoculated; missing Campy-BAP, CIN, or TCBS means those organisms were never excluded.
8.1 Enteric Pathogens: Culture and Colony Morphology
Quick Answer: Plate stool to MacConkey and HE or XLD at 35–37 °C, Campy-BAP at 42 °C microaerophilic, CIN at 25–30 °C when Yersinia is in the differential, and TCBS after alkaline peptone water when Vibrio is possible. Salmonella and Shigella are oxidase-negative non-lactose fermenters; Campylobacter, Vibrio, Aeromonas, and Plesiomonas are oxidase-positive. Do not report “normal flora” if screening plates were not used.
Stool is the opposite of CSF: it is never sterile. Outline II.F Gastrointestinal of the M(ASCP) Examination Content Guideline (revised September 25, 2025) still sits inside Analytic Procedures for Bacteriology (45–55% of the exam), but the analytic job is different. You are not proving sterility. You are finding a short list of major enteric pathogens in a background of Enterobacterales, enterococci, anaerobes, and yeast. The organisms named on the outline are Salmonella spp., Shigella spp., toxigenic Escherichia coli, Campylobacter spp., Vibrio spp., Yersinia enterocolitica, Aeromonas spp., and Plesiomonas shigelloides. Toxigenic E. coli (STEC) is finished on SMAC, toxin assays, and serotyping in Section 8.2; this section is the colony morphology and culture engine those assays still depend on.
Accept fresh unformed stool or stool in Cary-Blair transport. Dry rectal swabs and specimens sitting unpreserved at room temperature for hours lose Shigella and Campylobacter first. Do not refrigerate raw stool for prolonged bacterial culture if a transport medium is available; Cary-Blair is the exam default. Formed stool can be cultured when the order is appropriate, but it is a poor specimen for acute infectious diarrhea and is rejected for Clostridioides difficile testing (Section 8.2).
Primary plates and the “normal flora” rule
A defensible enteric screen always includes a differential Gram-negative plate and a Salmonella/Shigella selective plate, plus Campylobacter selective agar. Special requests add Yersinia and Vibrio media. Blood agar is useful for oxidase, hemolysis (Aeromonas), and a nonselective look, but blood agar plus MacConkey is not an enteric pathogen screen.
| Medium | Incubation | What it selects or shows | Colony the exam wants |
|---|---|---|---|
| MacConkey agar | 35–37 °C, air or CO2 | Bile salts and crystal violet; lactose + neutral red | Salmonella/Shigella colorless (NLF); most E. coli pink (LF) |
| Hektoen enteric (HE) | 35–37 °C | Lactose/sucrose/salicin plus H2S indicators | Salmonella blue-green with black center; Shigella green; fermenters orange-salmon |
| Xylose lysine deoxycholate (XLD) | 35–37 °C | Xylose, lysine, lactose/sucrose, H2S | Salmonella red with black center; Shigella red without H2S; E. coli yellow |
| Campy-BAP (CVA or similar) | 42 °C, microaerophilic (~5% O2, 10% CO2, 85% N2), 48–72 h | Antibiotics suppress flora | C. jejuni gray, moist, spreading along the streak |
| CIN agar | 25–30 °C, 48 h | Cefsulodin-irgasan-novobiocin; mannitol | Y. enterocolitica bull’s-eye (red center, colorless rim) |
| TCBS agar | 35 °C | High pH, bile, sucrose | V. cholerae yellow (sucrose+); V. parahaemolyticus green (sucrose−) |
| Alkaline peptone water | 35 °C, pH ~8.4–8.6, then subculture | Enrichment for Vibrio | Broth film subbed to TCBS after 6–8 h |
Do not report “normal fecal flora” when screening plates are not used. That sentence is an outline-level reporting rule, not a courtesy. If Campy-BAP was never incubated microaerophilic at 42 °C, you did not look for Campylobacter. If CIN was not set up, you did not exclude Y. enterocolitica. If seafood, cholera-belt travel, or watery rice-water stool never triggered alkaline peptone water and TCBS, you did not exclude Vibrio. MacConkey-only workups also fail Salmonella/Shigella because colorless colonies are buried in other non-lactose fermenters and there is no H2S cue. Report pathogens recovered or no Salmonella, Shigella, or Campylobacter isolated (and name any extra screens you actually performed). Do not invent a flora comment that implies a complete hunt.
Lactose, H2S, and the oxidase split
On MacConkey, non-lactose fermenters are colorless. Salmonella and Shigella are NLF (S. sonnei is a delayed lactose fermenter and can look pale-pink late). Most commensal E. coli colonies are lactose-positive and pink; that is why MacConkey alone is a weak Salmonella/Shigella screen and why HE/XLD exist.
H2S on HE or XLD is the fastest Salmonella clue: green or red colony with a black center. Proteus and Citrobacter can mimic that black center; urea-positive colonies are Proteus, not Salmonella. Shigella is H2S-negative.
Oxidase splits the outline list in half:
- Oxidase-negative: Salmonella, Shigella, Y. enterocolitica, toxigenic E. coli.
- Oxidase-positive: Campylobacter, Vibrio, Aeromonas, Plesiomonas shigelloides.
Perform oxidase from blood agar or another nonselective plate. Selective dyes (TCBS, HE, MacConkey) cause false reactions. Plesiomonas is the oxidase-positive member of the Enterobacterales that the exam still treats with the Vibrio/Aeromonas look-alikes.
Salmonella and Shigella on the plate
Salmonella spp. grow as NLF colonies on MacConkey and as green-to-blue-green (HE) or red (XLD) colonies with H2S. Most gastroenteritis serotypes are motile, urease-negative, and produce gas. TSI is typically K/A, gas, H2S. Indole is negative. Lysine decarboxylase is usually positive. Selenite or gram-negative broth can enrich low numbers, then you subculture back to HE/XLD. S. Typhi is the exception pattern: scant gas, a thin “mustache” of H2S, citrate-negative, and often a blood-culture diagnosis more than a heavy stool growth. Reptiles, poultry, eggs, and peanut-butter outbreaks are transmission stories; the bench identification is still NLF + H2S + oxidase-negative + urea-negative.
Shigella spp. are NLF, nonmotile, H2S-negative, and usually gas-negative. On HE they are green; on XLD they are red. TSI is K/A, no gas, no H2S—the same skeleton as a non-H2S Salmonella, which is why motility, lysine, and serogrouping matter. S. sonnei (group D) is the usual U.S. isolate and is ONPG-positive; S. flexneri (group B) is common worldwide; S. dysenteriae (group A) is the most severe. Shigella is fragile. Plate promptly and do not dismiss a few green colonies as “just flora.”
Exam scenario. Colorless MacConkey colonies, black-centered HE colonies, oxidase-negative, urea-negative: work up Salmonella and send the isolate for serotyping. Green HE colonies, no H2S, nonmotile, TSI K/A: work up Shigella. Do not report either as “NLF, not further identified.”
Campylobacter, Vibrio, Yersinia, Aeromonas, and Plesiomonas
Campylobacter jejuni is the highest-volume bacterial stool pathogen in many U.S. laboratories and the organism you will miss if you skip Campy-BAP. It does not grow on MacConkey or HE in ambient air at 35 °C. Incubate Campy-BAP microaerophilic at 42 °C for 48 hours (hold 72 hours). A candle jar is not microaerophilic enough. Colonies are gray, moist, and run along the streak line. The Gram stain is a curved, poorly staining, gull-wing rod; many benches use carbol-fuchsin or basic fuchsin as the counterstain so the cells are visible. Wet mounts show darting motility. The isolate is oxidase-positive and catalase-positive; C. jejuni is hippurate-positive, which separates it from C. coli. Nalidixic-acid susceptible / cephalothin resistant is the classic disk pattern for C. jejuni. C. fetus is a blood-culture organism that prefers 37 °C and does not require 42 °C; do not force every campylobacter onto the stool 42 °C story.
Vibrio spp. need a clinical trigger: raw oysters, seawater, cholera-endemic travel, or massive watery diarrhea. Enrich in alkaline peptone water, then plate TCBS. V. cholerae and V. alginolyticus ferment sucrose and are yellow; V. parahaemolyticus is sucrose-negative and green; V. vulnificus is variable on TCBS but is the wound/sepsis oyster organism in patients with liver disease. All are oxidase-positive from blood agar. V. cholerae and V. mimicus grow without added salt; the halophiles (V. parahaemolyticus, V. vulnificus) need NaCl. The string test (0.5% sodium deoxycholate) is positive for V. cholerae. Vibriostatic agent O/129 helps separate Vibrio (usually susceptible) from Aeromonas (resistant).
Yersinia enterocolitica is a small NLF coccobacillus that prefers CIN agar at 25–30 °C. Colonies are bull’s-eye: a red mannitol-fermenting center with a translucent rim. It is oxidase-negative and urease-positive, motile at 25 °C and nonmotile at 37 °C—the temperature motility split the exam loves. Cold enrichment is historical; CIN at room-to-cool incubation is the practical method. Pork, chitterlings, and winter pediatric mesenteric adenitis that mimics appendicitis are the clinical frame. The organism can also seed cold-stored blood products, but that is a transfusion story, not a stool-plate story.
Aeromonas spp. (A. hydrophila and relatives) grow on MacConkey, often beta-hemolytic on blood agar, oxidase-positive, and glucose-fermenting (they are not Pseudomonas). Many are indole-positive and grow in 0% NaCl. Freshwater wounds and diarrhea after untreated water are classic. Plesiomonas shigelloides is oxidase-positive, ferments inositol, is usually O/129 susceptible, and can cross-react with Shigella antiserum—confirm with oxidase and biochemicals before you call Shigella. Both genera are freshwater/seafood organisms and will be missed if the bench throws away every oxidase-positive Gram-negative rod from stool as “contaminant Pseudomonas.”
Exam scenario. Gray colonies only on Campy-BAP after 48 hours at 42 °C, gull-wing Gram-negative rods, oxidase-positive, hippurate-positive: C. jejuni. Yellow TCBS colonies after alkaline peptone water, oxidase-positive, growth in 0% NaCl: V. cholerae until serogrouped. Bull’s-eye CIN colonies at 28 °C, urease-positive, motile at 25 °C only: Y. enterocolitica. Oxidase-positive, indole-positive rod from a lake-water diarrhea specimen that is O/129 resistant: Aeromonas, not Shigella.
A stool specimen is plated to MacConkey, XLD, and Campy-BAP. After 48 hours, gray moist colonies are present only on Campy-BAP. The isolate is a curved Gram-negative rod that is oxidase-positive. Which incubation condition recovered this organism?
Colorless colonies on MacConkey agar produce blue-green colonies with black centers on Hektoen enteric agar. The isolate is oxidase-negative and urease-negative. The most likely enteric pathogen is:
A laboratory inoculates stool only to blood agar and MacConkey agar, incubates both at 35 °C in air, and reports “normal fecal flora.” Why is that report not acceptable on the M(ASCP) enteric workup?