Experiment 05Clinical SerodiagnosisBacterial Agglutination
Widal Tube Agglutination Titration for Serodiagnosis of Enteric (Typhoid & Paratyphoid) Fever
Lab Duration: 24 Hours Incubation • Serial Tube Dilution
🎯 Aim: To perform the quantitative Widal tube agglutination assay using serial dilutions of patient serum against standardized killed bacterial suspensions of Salmonella enterica serovars Typhi (somatic O and flagellar H antigens) and Paratyphi (AH and BH antigens), determine antibody titers, and clinically interpret acute versus convalescent phases of enteric fever.
🔬 Principle & Serological Antigens
The Widal test, developed by Georges-Fernand Widal in 1896, is a tube agglutination test that measures circulating agglutinating serum antibodies (agglutinins) against specific antigenic preparations of Salmonella enterica serovars:
- Somatic O-Antigen (TO): Lipopolysaccharide (LPS) endotoxin component of the bacterial outer cell envelope. Because it is non-flagellar, O-agglutination produces a compact, granular, chalky precipitate that is difficult to dislodge upon shaking, settling in flat-bottomed Felix tubes. Antibodies against TO appear first (end of the 1st week of fever) and decline rapidly after recovery. An O-titer $ge 1:160$ strongly indicates active acute infection.
- Flagellar H-Antigen (TH, AH, BH): Heat-labile flagellin protein. Because flagella interlock loosely, H-agglutination produces a loose, cotton-wool-like, fluffy floccose precipitate that forms rapidly and settles in conical-bottomed Dreyer tubes. Antibodies against TH appear later (2nd week of illness) and persist for months to years, often indicating past infection, endemic exposure, or TAB vaccination.
Diagnostic Criteria:
In endemic regions, a single baseline cutoff titer of $ge 1:160$ for TO and $ge 1:160$ for TH is considered clinically suspicious. However, the definitive gold standard confirmation is demonstrating a four-fold or greater rise in antibody titer between paired serum specimens collected 7 to 10 days apart (acute vs. convalescent phase).
📋 Step-by-Step Tube Dilution Protocol
- Tube Rack Setup: Set up 4 rows of 8 small test tubes ($75 times 12,text{mm}$ Dreyer or Kahn tubes) labeled: Row 1 = *S. Typhi* O (TO); Row 2 = *S. Typhi* H (TH); Row 3 = *S. Paratyphi* AH; Row 4 = *S. Paratyphi* BH.
- Master Serial Dilution: Pipette $1.9,text{mL}$ of sterile physiological saline ($0.85%,text{NaCl}$) into Tube 1, and $1.0,text{mL}$ of saline into Tubes 2 through 8 for each row. Add $0.1,text{mL}$ of patient serum to Tube 1 (yielding an initial $1:20$ dilution). Mix thoroughly, transfer $1.0,text{mL}$ to Tube 2 ($1:40$), mix and transfer $1.0,text{mL}$ to Tube 3 ($1:80$), continuing serial doubling dilutions through Tube 7 ($1:1280$). Discard $1.0,text{mL}$ from Tube 7. Tube 8 receives NO serum and serves as the negative antigen control.
- Antigen Addition: Add 1 drop ($~50,mutext{L}$) of standardized killed antigen suspension to all 8 tubes of the corresponding row:
Row 1 = TO antigen; Row 2 = TH antigen; Row 3 = AH antigen; Row 4 = BH antigen.
(Final serum dilutions become $1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560$). - Incubation: Shake the racks to mix. Incubate in a water bath at $37^circtext{C}$ for 4 hours (or at $50^circtext{C}$ for 2 hours), then transfer to $4^circtext{C}$ refrigerator overnight for complete sedimentation.
- Reading Agglutination: Examine tubes against a black background using indirect light:
- $++++$ : Complete agglutination; 100% clearing of supernatant with heavy precipitate.
- $+++$ : 75% clearing of supernatant with visible precipitate.
- $++$ : 50% clearing with moderate precipitate (endpoint titer definition).
- $-$ : Uniformly turbid suspension; smooth round button of unagglutinated bacteria.
The titer is defined as the highest serum dilution showing at least $50%$ ($++$) agglutination.
