Widal test Widal test Introduction It is a

Widal test

Widal test Introduction Ø It is a serological approach for the diagnosis of typhoid and paratyphoid fever in clinical laboratory. Ø This is a test for the measurement of H and O agglutinins of typhoid and paratyphoid bacilli in patient’s sera.

Materials Widal test 1. Sera from suspected patients 2. Antigens Suspension of S. typhi "O" antigen, TO Suspension of S. typhi "H" antigen; TH Suspension of S. paratyphi A "H" antigen, PAH Suspension of S. schottmuelleri "H" antigen, PBH Suspension of S. hirschfeldii "H" antigen, PCH 3. Normal saline (0. 9% Na. Cl) 4. Test tubes and pipettes

Widal test Procedures 1 TO TH PAH PBH PCH 2 3 4 5 6

Widal test —two folds serial dilution of the serum Tubes NS (ml) 1 2 3 4 5 6 0. 5 0. 5 ↘ Serum (ml) 0. 5 (1: 10) 1: 20 1: 40 0. 5 Antigen (ml) dilution 1: 40 0. 5 ↘ 0. 5 1: 80 1: 160 1: 320 ↘ 0. 5 ml discarded 1: 320 0. 5 1: 640 negative control overnight in a 37℃ waterbath

Widal test observation of the results Ø H agglutination: loose clump, like cotton or wool Ø O agglutination: compact film, scattering at the bottom of the tubes

Widal test ØAgglutination titer is determined as the highest dilution of serum which can cause ++ bacteria agglutination. 1 2 3 4 5 6 TO ++++ ++ ++ + - - TH ++++ ++ ++ - - PAH ++ + - - PBH - - - PCH - - - 1: 40 1: 80 1: 160 1: 320 1: 640 Negative control

Widal test Interpretation of results O<1: 80, TH<1: 160, PH<1: 80 Normal value O ≥ 1: 80 & TH ≥ 1: 160 or O ≥ 1: 80 & PH ≥ 1: 80 Typhoid fever Paratyphoid fever O ≥ 1: 80 & H <1: 160 or O ≥ 1: 80 & PH <1: 80 Early infection or other salmonella infections O <1: 80 & H ≥ 1: 160 or O <1: 80 & PH ≥ 1: 80 Vaccination or nonspecific memory reaction

Experiment Isolation and Identification of Enterobacteriaceae

The common procedures of the Isolation and Identification of Enterobacteriaceae Specimen (feces or rectal swab) ↓ Differential or selective culture media (such as EMB agar) ↓ Colonies Lactose (-) lactose (+) ↓ Double sugar iron slant Other biochemical reactions (Such as urea slant) slide agglutination tests

5 students/group § Materials and Methods • Bacterial strains Slant culture of E. coli Slant culture of Proteus vulgaris Slant culture of S. typhi Slant culture of S. dysenteriae Slant culture of Enterobacter. aerogen

¡ Culture medium and inoculation methods Culture Medium Prior Color Inoculation Method EMB agar Dark red Four-area streak Indole broth Pale yellow Liquid inoculation Double sugar iron slant Red Streak slant surface and stab into it Urea slant Pale yellow Streak slant surface Citrate slant Green Streak slant surface Note: Each bacterial strain will be inoculated into all the five kinds of culture media. After inoculation, incubate the plates and tubes overnight at 37℃.

Four-area streak dilution technique 1/10 1/5 1/4 EMB agar plate

¡ Culture medium and inoculation methods Culture Medium Prior Color Inoculation Method EMB agar Dark red Four-area streak Indole broth Pale yellow Liquid inoculation Double sugar iron slant Red Streak slant surface and stab into it Urea slant Pale yellow Streak slant surface Citrate slant Green Streak slant surface Note: Each bacterial strain will be inoculated into all the five kinds of culture media. After inoculation, incubate the plates and tubes overnight at 37℃.

Liquid Inoculation

¡ Culture medium and inoculation methods Culture Medium Prior Color Inoculation Method EMB agar Dark red Four-area streak Indole broth Pale yellow Liquid inoculation Double sugar iron slant Red Streak slant surface and stab into it Urea slant Pale yellow Streak slant surface Citrate slant Green Streak slant surface Note: Each bacterial strain will be inoculated into all the five kinds of culture media. After inoculation, incubate the plates and tubes overnight at 37℃.

Streak slant surface and stab into

¡ Culture medium and inoculation methods Culture Medium Prior Color Inoculation Method EMB agar Dark red Four-area streak Indole broth Pale yellow Liquid inoculation Double sugar iron slant Red Streak slant surface and stab into it Urea slant Pale yellow Streak slant surface Citrate slant Green Streak slant surface Note: Each bacterial strain will be inoculated into all the five kinds of culture media. After inoculation, incubate the plates and tubes overnight at 37℃.

Slant inoculation

5 students/group （lab report) 1 2 E. coli Proteus EMB Indole broth 3 S. typhi Double sugar iron 4 S. dysenteriae Urea slant 5 E. aerogen Citrate slant

Observe the results (1) Eosin-Methylene Blue Agar (EMB) Reagents: Eosin and Methylene-blue Carbohydrate Source: Lactose Principle: a precipitate can be formed at acidic p. H Purpose: differentiate lactose fermenters from non-lactose fermenters. Interpretation: Lactose fermenter: purple Lactose non-fermenter: colorless

EMB agar E. coli: black and typically have a metallic green sheen negative

EMB agar E. coli ( ++ ) Proteus S. typhi (-) S. dysenteriae E. aerogens (-) (+)

Observe the results (2) Idole Broth purpose: Distinguish Enterobacteriaseae based on the ability to produce indole from tryptophan. principle: tryptophanase indole Kovac’s reagent Interpretation Positive Test - red ring Negative Test - no color development rosindole (red )

Idole Broth positive negative

Idole Broth E. coli (+) Proteus S. typhi (+) (-) S. dysenteriae E. aerogens (+)

(3) Double suger iron slant main component: Glucose: 0. 1% Lactose: 1% Phenol red: as an indicator Ferrous sulfate(Fe. SO 4) : Fe. SO 4+H 2 S →Fe. S(black)+H 2 SO 4 purpose: It provides information about carbohydrate fermentation. Principle:

Only glucose fermenting Slant: oxidized →neutral →red Butt: not oxidized →yellow

Lactose fermenting yellow both on the slant and in the butt

H 2 S production Fe. SO 4+H 2 S Fe. S(black)+H 2 SO 4

Gas production

Double suger iron slant

Double suger iron slant E. coli Slant yellow butt Proteus S. typhi S. dysenteriae red yellow E. aerogens red yellow Gas (+) (-) (-) (+) H 2 S (-) (+) (-)

Observe the results (4) Citrate Slant purpose: Determine if organism can use citrate as its sole metabolic (carbon) energy source. reagents: bromothymol blue (a p. H indicator) sodium citrate (the sole source of carbon) principle: sodium citrate citrase Interpretation: Positive: blue Negative: green sodium carbonate

Citrate Slant

Citrate Slant E. coli Proteus S. typhi S. dysenteriae E. aerogens (-) (-) (+)

Observe the results (5) Urea slant purpose: determine if organism can break down urea or not. reagents: urea, phenol red principle: if urease is present, urea is broken down to ammonia causing rise to p. H Interpretation: Positive: red Negative: pale yellow

Urea slant

Urea slant E. coli Proteus (-) (+) S. typhi S. dysenteriae E. aerogens (-) (+)

slide agglutination test Procedures 1) Divide the slide into two equal parts 2) Add one drop of NS on the left side, and then on the right side 3) Emulsify S. typhi in the left side，and S. dysenteriae in the right side. 4) Add one drop of diagnostic sera for S. typhi on the left side, and then on the right side 5) Gently shake the slide and observe the results