1.
Escherichia coli (focus: ETEC, EHEC, UPEC)
Sample Collection
• Stool → for diarrheagenic E. coli (ETEC, EHEC)
• Urine (midstream clean catch) → for UTI
• Blood → in sepsis or neonatal meningitis
Transport
• Use Cary-Blair medium for stool if delay >2 hours
• Urine: transport within 2 hours or refrigerate
• Blood: blood culture bottle (aerobic)
Culture
• MacConkey agar → pink colonies (lactose fermenter)
• EMB agar → green metallic sheen
• Sorbitol MacConkey agar → EHEC O157:H7 appears colorless
Microscopy
• Gram stain: Gram-negative rods (pink)
• Not diagnostic alone — used to rule out contamination
Specific/Confirmatory Tests
• Indole test: positive
• TSI: A/A, gas production
• Serotyping: O157:H7 detection for EHEC
• ELISA/PCR for enterotoxins (ST, LT) or Shiga-like toxin
• Uropathogenic E. coli: tested for hemolysin production, P fimbriae
2. Shigella spp.
Sample Collection
• Fresh stool sample or rectal swab during acute illness
• Avoid if diarrheal phase has subsided (shedding drops)
Transport
• Cary-Blair transport medium if >2 hours delay
• Avoid buffered glycerol saline (inhibits Shigella)
Culture
• MacConkey agar: pale colonies (NLF)
• XLD agar: red colonies (non-H2S)
• SS agar: colorless colonies
• Selenite F broth: enrichment
Microscopy
• Stool wet mount: many PMNs, RBCs
• No motility
• Trophozoites absent (rule out amoebiasis)
Specific/Confirmatory Tests
• Slide agglutination with Shigella antisera (A–D groups)
• Serotyping: S. dysenteriae type 1 (Shiga toxin producer)
• PCR/ELISA: Shiga toxin genes (Stx1/Stx2)
3. Vibrio cholerae
Sample Collection
• Watery stool during acute diarrheal phase
• Rectal swab in Cary-Blair if liquid stool unavailable
Transport
• Cary-Blair medium; keep cool, process within 6 hours
Culture
• Alkaline Peptone Water (APW): enrichment
• TCBS agar: yellow colonies (sucrose fermenting)
• Blood agar: β-hemolytic, but not specific
Microscopy
• Gram stain: comma-shaped gram-negative rods
• Darting motility on wet mount
• String test: mucolytic string on mixing with sodium deoxycholate
Specific/Confirmatory Tests
• Oxidase positive
• Agglutination with O1 or O139 antisera
• Cholera red reaction (lab-based nitrosoindole test)
• PCR/ELISA for cholera toxin genes
4. Campylobacter jejuni
Sample Collection
• Fresh stool sample (ideally within 2 hours)
• Blood if systemic symptoms (rare)
Transport
• Special transport media: Campy-thioglycollate, dry ice for longer transit
• Maintain microaerophilic environment
Culture
• Skirrow’s medium, Campy-BAP
• Incubate at 42°C, microaerophilic (5% O₂, 10% CO₂)
• Growth in 48–72 hours
Microscopy
• Gram stain: small curved/comma-shaped gull-wing GNRs
• Darting motility seen in fresh wet mounts
Specific/Confirmatory Tests
• Oxidase +, Catalase +
• Hippurate hydrolysis + (for C. jejuni only)
• Antigen detection ELISA, PCR, and stool latex agglutination kits
5. Clostridium species
Clostridium perfringens
Sample Collection
• Tissue (necrotic), wound exudate, food remnants, feces
Transport
• Anaerobic transport media (e.g., Robertson cooked meat medium)
Culture
• Anaerobic blood agar → large colonies
• Double zone of hemolysis (α-toxin + θ-toxin)
Microscopy
• Gram-positive rods, boxcar-shaped, few/no spores
• No leukocytes in food poisoning samples
Specific Tests
• Nagler’s reaction: lecithinase on egg yolk agar
• Reverse CAMP test: enhanced hemolysis with Group B Strep
• Toxin detection (α-toxin) by ELISA/PCR
Clostridium difficile
Sample Collection
• Unformed stool sample only (never test formed stool)
• Colon biopsy (rare)
Transport
• Process quickly or store at 2–8°C, within 2 hours
Culture
• CCFA (Cycloserine-Cefoxitin-Fructose Agar) under anaerobic conditions
• Smells like horse stable
Microscopy
• Gram-positive rods, spore-forming, not specific
• Colonoscopy shows pseudomembranes
Specific Tests
• GDH antigen (sensitive) + Toxin A/B EIA or PCR (specific)
• Cell cytotoxicity assay (gold standard but rarely done)
• NAAT (nucleic acid amplification tests): most accurate
