Overview
Salmonella is a genus of Gram-negative bacilli responsible for enteric fever (typhoid and paratyphoid), gastroenteritis (food poisoning), and septicaemia. Salmonella typhi, the causative agent of typhoid fever, remains one of the most important human bacterial pathogens in endemic regions.
Salmonellae are divided into an enteric fever group (exclusively human parasites) and a food poisoning group (primarily animal parasites that can infect humans). This lesson covers morphology, cultural and biochemical characteristics, antigenic structure, pathogenesis, laboratory diagnosis including blood/stool/urine culture, and the Widal reaction.
Learning Objectives
After this lesson you will be able to…- Describe the morphology of Salmonella.
- Discuss the cultural characteristics of Salmonella.
- Explain the biochemical reactions of Salmonella.
- Demonstrate and interpret the Widal reaction.
Clinical Story
Why This MattersA 22-year-old returning traveller presents with a week of stepwise rising fever, relative bradycardia, abdominal discomfort and rose spots on the trunk. The physician suspects typhoid fever and orders a blood culture in the first week of illness — when bacteraemia is most detectable — while planning a Widal test as supportive evidence. The lab team must isolate the organism from bile broth, confirm it as Salmonella typhi biochemically, and report promptly, since delayed diagnosis risks intestinal perforation.
Core Concepts
Salmonellae are Gram-negative rods, motile with peritrichate flagella (except S. gallinarum-pullorum). They are divided into two clinically important groups: the enteric fever group (S. typhi and S. Paratyphi A, B, C — exclusively or primarily human parasites) and the food poisoning group (animal parasites that may infect humans and cause gastroenteritis).
Salmonellae are aerobic and facultatively anaerobic, growing readily on simple media over pH 6–8 and temperature 15–41°C (optimum 37°C). Colonies are large, circular, smooth and colourless on MacConkey and Deoxycholate Citrate Agar (DCA) due to absence of lactose fermentation. Selenite F broth and Tetrathionate broth are the standard enrichment media used to boost recovery from stool samples containing mixed flora.
Salmonellae ferment glucose, mannitol and maltose with acid and gas — except S. typhi, which is anaerogenic (no gas produced). Lactose, sucrose and salicin are not fermented, and indole is not produced. They are MR positive, VP negative, and citrate positive. The bacilli are killed at 55°C in one hour or 60°C in 15 minutes; boiling, chlorination and pasteurisation destroy them, though they may survive weeks in polluted water and months in ice.
Salmonellae possess three key antigens: H antigen (flagellar, heat-labile protein, destroyed by boiling or alcohol but not formaldehyde), O antigen (somatic, a phospholipid-protein-polysaccharide complex identical with endotoxin, unaffected by boiling, alcohol or weak acids), and Vi antigen (surface/capsular antigen associated with virulence). Classification uses the Kauffman-White scheme, based on O and H antigen agglutination, with serological groups designated 1, 2, 3, etc. Kauffman's biochemical classification divides Salmonella into four subgenera by host range and clinical relevance.
Infection is acquired by ingesting contaminated food; bacilli attach to ileal microvilli, penetrate the submucosa, and are phagocytosed by polymorphs and macrophages — their ability to resist intracellular killing and multiply within cells is a direct measure of virulence. They spread via mesenteric lymph nodes and the thoracic duct into the bloodstream (bacteraemia), then multiply abundantly in the gall bladder (bile is a good culture medium) and are discharged into the intestine, involving Peyer's patches and ileal lymphoid follicles, which may ulcerate and cause intestinal perforation or haemorrhage. Incubation period is usually 7–14 days (range 3–56 days).
Diagnosis relies on isolation of bacilli and demonstration of antibodies. Blood culture is positive mostly in the first week — 5–10 mL blood is inoculated into 0.5% bile broth, subcultured onto MacConkey agar; S. typhi is anaerogenic while paratyphoid bacilli produce gas. Faeces culture uses MacConkey, DCA and Wilson-Blair media (S. typhi forms large black colonies on Wilson-Blair due to H₂S production; S. paratyphi A produces green colonies). Urine culture is generally positive in the second/third weeks. The Widal reaction measures H and O agglutinins using Dreyer's tube (H agglutination — loose cotton-woolly clumps) and Felix tube (O agglutination — disc-like pattern) against patient serum dilutions.
Laboratory Principle
The Widal reaction is a serological agglutination test based on the principle that antibodies (agglutinins) produced against Salmonella H (flagellar) and O (somatic) antigens during infection will cause visible clumping when mixed with the corresponding standardised antigen suspension. H antigen agglutination produces loose, fluffy, cotton-wool-like clumps because flagella interlock loosely, while O antigen agglutination produces a tighter, compact, disc-like deposit because somatic antigens on the cell wall bind more rigidly — this visually distinct clumping pattern allows the two antibody types to be read and titrated separately in serial serum dilutions.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| Bile Broth (0.5%) | Ox bile-based enrichment broth | Blood culture enrichment for Salmonella | 2–8°C |
| Selenite F Broth | Selenite-based selective enrichment | Enrichment of Salmonella from stool | 2–8°C |
| Tetrathionate Broth | Tetrathionate-based selective enrichment | Enrichment of Salmonella from stool | 2–8°C |
| Wilson-Blair Medium | Bismuth sulphite agar | Selective medium — black colonies for S. typhi | 2–8°C, protect from light |
| Deoxycholate Citrate Agar (DCA) | Selective/differential medium | Isolation of Salmonella from faeces | 2–8°C |
| Salmonella O and H Antisera/Antigens | Standardised suspensions | Widal reaction and slide agglutination | 2–8°C |
Step-by-Step Procedure
Collect 5–10 mL blood aseptically and inoculate into 50–100 mL of 0.5% bile broth; incubate overnight at 37°C.
Subculture onto MacConkey agar; pick pale, non-lactose-fermenting colonies for biochemical testing and motility.
Plate fresh stool directly onto MacConkey, DCA and Wilson-Blair media; incubate at 37°C for 18–24 hours and examine for pale colonies (MacConkey/DCA) or large black colonies (Wilson-Blair, S. typhi).
Test motility, indole (negative), urease (negative), and fermentation of glucose, mannitol, maltose (positive, S. typhi anaerogenic) but not lactose or sucrose.
Emulsify growth from an agar slope in saline and test against polyvalent and group-specific O/H antisera, always including a saline control for autoagglutination.
Mix serial serum dilutions with H and O antigens in Dreyer's and Felix tubes respectively; incubate overnight at 37°C in a water bath and read agglutination titres against saline controls.
Flow Diagram
Quality Control
Include known S. typhi and S. paratyphi reference strains as positive controls, and a non-Salmonella Enterobacteriaceae as a negative control, with each new batch of Wilson-Blair, DCA and bile broth media. Run known positive and negative sera with every Widal test batch, always including a saline autoagglutination control.
Participate in EQAS programmes for enteric fever serology and culture-based diagnosis, given the significant public health reporting implications of confirmed typhoid cases, and periodically cross-check Widal titre interpretation against culture-confirmed cases to monitor local baseline titres.
Reference Values
Key Diagnostic Parameters⚠️ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Positive blood culture with biochemically confirmed S. typhi | Diagnostic of active typhoid fever | Begin appropriate antibiotic therapy; notify public health authority |
| Rising Widal titre on paired sera (acute and convalescent) | Supportive evidence of recent/current Salmonella infection | Correlate with clinical picture; single titre alone is not conclusive |
| Large black colonies on Wilson-Blair medium | Suggestive of S. typhi (H₂S production) | Confirm biochemically and serologically before final report |
| Green colonies on Wilson-Blair medium | Suggestive of S. paratyphi A | Confirm biochemically and serologically before final report |
| Positive stool culture in asymptomatic individual | Possible chronic carrier state | Consider public health follow-up, especially in food handlers |
Common Errors & How to Avoid Them
Cause: A single elevated titre can result from past infection, vaccination, or cross-reaction, not necessarily current disease.
Prevention: Whenever possible, demonstrate a rising titre between acute and convalescent paired sera, and always correlate serology with culture and clinical findings.
Cause: Bacteraemia is most detectable in the first week; sensitivity drops significantly in later weeks as the organism localises to the gall bladder and intestine.
Prevention: Collect blood cultures as early as possible in the febrile illness, and consider stool/urine culture in later weeks when blood culture yield falls.
Cause: Assuming all Salmonella produce gas from glucose fermentation can lead to misidentification, since S. typhi is uniquely anaerogenic.
Prevention: Remember S. typhi does not produce gas from sugar fermentation, unlike S. paratyphi and most other Salmonella serotypes, and use this as a specific identification clue.
Laboratory Tips from the Bench
When enteric fever is suspected, always collect at least 5–10 mL of blood for culture — smaller volumes significantly reduce the sensitivity of bile broth culture due to the naturally low bacterial load in blood.
If first bile broth subculture is negative, don't give up — repeat subculture every other day and only declare the culture negative after a full 10 days of incubation, since bacterial numbers can be very low.
Remember 'Typhi is Tranquil' — S. typhi is anaerogenic (produces no gas from sugar fermentation), unlike the more 'active' gas-producing paratyphoid bacilli.
Important Notes
Intestinal perforation and haemorrhage are life-threatening complications of untreated or late-diagnosed enteric fever, arising from ulceration of Peyer's patches and ileal lymphoid follicles — early diagnosis and treatment are essential to prevent this.
The Widal test has significant limitations, including cross-reactivity with other Enterobacteriaceae and variability due to prior vaccination or endemic background titres — it should always be interpreted alongside clinical findings and, where possible, culture confirmation.
Interactive Quiz
Test Your KnowledgeFlashcards
Tap to flipClick or tap any card to reveal the answer. Use arrow keys to navigate in single-card mode.
Clinical Case Study
Apply Your Knowledge7-day history of stepwise rising fever, relative bradycardia, generalised malaise, abdominal discomfort, and a faint rose-coloured maculopapular rash on the trunk noted on examination.
The pale non-lactose-fermenting colonies, indole-negative and anaerogenic biochemical profile, positive O antiserum agglutination, and a documented 4-fold rise in Widal O titre between acute and convalescent sera together confirm Salmonella typhi infection, consistent with the classic clinical presentation of typhoid fever.
- →Blood culture in the first week of fever gives the highest diagnostic yield.
- →A single Widal titre is not diagnostic; a rising titre on paired sera is more meaningful.
- →Anaerogenic sugar fermentation is a key biochemical clue distinguishing S. typhi from paratyphoid strains.
Frequently Asked Questions
A single titre can be elevated due to past infection, prior vaccination, or cross-reaction with other Enterobacteriaceae sharing similar antigens, so a rising titre between acute and convalescent paired sera provides much stronger evidence of current infection.
Bile is actually a good culture medium for Salmonella, allowing the organism to multiply abundantly within the gall bladder during enteric fever, which is why bile broth is used to enhance blood culture sensitivity and why chronic gall bladder carriage can occur.
The enteric fever group (S. typhi, S. Paratyphi A/B/C) are primarily or exclusively human pathogens transmitted person-to-person via contaminated food/water, while the food poisoning group are primarily animal parasites (zoonotic) that can secondarily infect humans through contaminated animal products.
Quick Revision
10-Minute ReviewKey Takeaways
- Salmonella typhi causes typhoid fever, a serious systemic infection with potential for intestinal perforation.
- S. typhi's anaerogenic fermentation pattern is a key biochemical identifier distinguishing it from other Salmonella.
- Blood culture timing matters — sensitivity is highest in the first week of illness.
- The Widal reaction is supportive, not definitive, and is best interpreted using paired rising titres.
- The H, O and Vi antigen system underlies both Salmonella classification and vaccine development (Vi vaccine).
Competency Checklist
Track Your MasteryReferences
- National Institute of Open Schooling. Microbiology — Lesson 23: Salmonella.
- Ananthanarayan R, Paniker CKJ. Textbook of Microbiology. Universities Press.
- World Health Organization. Typhoid fever fact sheet and diagnostic guidelines.