CHNOLOGY

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EXERCISE
Widal Test 143
st.
INTRODUCTION
saterial be quantified by means of a titer reading, a method that
Widal test devised in 1896 and named after its inventor, gives the relative quantity or concentration of antibodies
ation Georges-Fernand Widal, a French physician and and so permits different samples to be standardised and
bation bacteriologist. It is an example of tube agglutination test compared. In titer tests, a serum sample is serially dilured
oefore employed in the serological diagnosis of typhoid, an in tubes or a multiple-welled microtiter plate and míxed
enteric fever. It is a presumtive serological test in which with antigen suspension of Salmonella and incubated at
may 37°C in a water bath and observed for agglutination by
ation bacteria causing typhoid are mixed with a serum containing
specific antibodies obtained from infected method. It is examining the bottom of the tubes for sediment (Fig.
one of the most serious of the epidemic enteric infections 143.1). The highest dilution at which agglutination
where contaminated water due to sewage system exists. reaction is visualised is called the antibody titer of the
patient's serum. This is expressed as 1/40, 1/80,1/160 or
Typhoid fever, an enteric fever, is caused by Salmonella
whatever the dilution might be. If the titer is, say 1/640,
rial rvphi (= Salmonella enterica serotype Typhi). Other this is interpreted as 640 antibody units per milliliter of
Salmonella members responsible for causing this fever are
lres Salmonella serotypes Paratyphi A, B, and C. These the patient's serum. In other words, the greater is the
ion organisms possess somatic (O), flagella (H) and capsular concentration of antibodies in that sample, the higher is
(Vi) antigens. Salmonella is a gram-negative, motile, rod its titer. This technique in addition to typhoid has been
shaped facultative anaerobic bacterium with over 2000 adapted to many other diseases.
serovars, and all regarded as human pathogens. REQUIREMENTS
Widal test is a serological identification method for " Patient's serumn (1:10 dilution), known positive
the typhoid pathogens in which the patients serum is
tested for antibodies against H and O antigens of S. typhi "0° (samples can be stored at 4°C for 2 to 15
and S. paratyphi A and B and is used to confirm the days before testing)
" Antigen (1:10 dilution) Salmonella typhi "0"
diagnosis of typhoid fever in the diagnostic laboratory.
(stored at 4°C temperature)
" Saline (0.85% NaCI)
PRINCIPLE Test-tube rack
Salmonella spp. possess somatic (O) and flagella (H) Clean serological tubes (10)
antigens and Widal test detects antibodies in serum against 1-ml pipettes
H and O suspensions of S. typhi and H suspensions of 5-ml pipettes (2)
since S. paratyphi C is
S. paratyphi Aand S. pararyphiBhence Water bath at 37°C
not involved in infection in India, serumn antibodies Defreezer or Refrigerator (storage of antigens and
against this strain are not tested.
The antibodies are serum)
dead
detected by mixing the patients 'serum with Centrifuge
Salmonella cells (antigen) and observed for clumping Glass marking pencil.
37°C.
agglutination after 30 minutes' incubation at
related
Since O antigens in all the three pathogens are PROCEDURE
tested while
to each other, the O antigen of S. (yphi is only " The patient's serum is diluted
with saline (0.85%
paratyphi B
H antigens of S. typhi, S. paratyphi A and S.reaction may NaCI) through a series of tubes as
follows:
are tested individually. An antigen-antibody
 EXERCISE 143: WIDAL

356
LABORATORY
MANUAL OF
MICROBIOLOGY
AND
BIOTECHNOLOGY EXPECTED RE
INTERPRETAT

transferred from tube to tube Apositive agglutin

0.5 ml serum
patient has a discas
increase in th.
daily
on successive dave
Discard
Reaction +++
fever,
woul
of the hand, if
other
antibody titer, or
Procedure
for dilution from day
to day,
of serum typhoid, is prese
In case of
wooly clumps ar
Dilution One ml 1/20 1/40 small granules ar
of patient's
serum positive or negat
Since the :
Addition of 0.5 ml saline in each tube days of illness,
citer rise indicat

Addition of 0.5 ml antigen to each tube

Shaking of the rack to completely

mix the serum and antigen
boaile oicoizodo

dotoil Incubation at 37°C for 18 hours

Centrifugation at 2000 rpm for 7 minutes

b Tubes checked for agglutination

by examining the tubes for sediment

FIGURE 143.1 Procedure of Widal test.

" Arrange ten clean serological test-tubes in the it to tube 10(Fig. 143.1), thus tube 10 has only
front row of a test tube rack and label them one saline but no serum and is used as a negative test
to ten (Fig. 143.1) with a marking pencil. control.
Pipette l ml of the patient's serum into the first Transfer 0.5 ml of Salmonella O antiígen to each
tube (1:10 dilution).
" Add 0.5 ml of saline to each of the remaining
tube, using a fresh 5-ml pipette.
nine tubes (numbered 2-10) using a 5-ml
iShake the rack for completely mixing the antigen
ono and diluted serum.
pipette. " Incubate all the tubes for 18 hours at
Using a 1-ml pipette, transfer 0.5 ml of the serum water bath.
37°Cin a
from tube 1 to tube 2. Mix the contents of the " Centrifuge the tubes at 2000 rpm for 7
tube 2 by carefully drawing the liquid up into minutes.
busl Repeat all the steps separately for H antigens of
the pipette and discharging it slowly back down soig S. typhi, S. paratyphi Aand S. paratyphi B.
into the tube three times.
" Examine each tube for flakes
Repeat this process by transferring 0.5 ml from bols against a black surface and (agelutination)
tube 2 to 3, tube 3 to 4, tube 4 to 5, tube 5 to 6, compare
with negative test control (i.e., tube each tube
tube 6 to 7, tube 7 to 8, tube 8 to 9 and discard ad Record the antibody titer, 10).
the 0.5 ml drawn from tube 9 instead of adding i.e., that highest
dilution in which agglutination is
seen.
ECHNOLOGY XERCISE 143: WIDAL TEST 357

EXPECTED RESULTS AND Slide agglutination method using polyvalent antigen

INTERPRETATION antisera is routinely performed in diagnostic laboratories
in addition to biochemical tests. Salmonella serotype Typhi
Apositive agglutination test gives an indication that the on bismark sulphite agar medium produces circular red/
patient has a disease which may or may not be typhoid. A black colonies that are surrounded by black or brownish
daily increase in the titer, performed on the patient's serum
black zone.
on successive days between lst and 3rd week after onset
Report is given as typhoid + or-, with the antibody
of the fever, would confirm the ryphoid infection. On the titer 160 antibody units/ml of the patient's serum, if the
other hand, if the tests on the patient's serum reveal no antibody titer is, say, 1/160 (Fig. 143.1).
antibody titer, or a lower titer which does not increase
from day to day, indicates that some other disease, than VIVA QUESTIONS
typhoid, is present.
In case of H agglutinins, large loose, cotton and 1. What are the advantages and disadvantages of
wooly clumps are observed while with O agglutinins only Widal test.
fever?
small granules are formed at the bottom of the tube. A 2. What are the causative agents of typhoid
b positive or negative result in a single test is not significant. 3. What is the principal of Widal test?od
Since the antibodies are detected only after 7 to 10 4. What is a titer?
days of illness, widal test should be done later. O antibody 5. What types of colonies are produced by
titer rise indicates recent infection.s) nopEd atzsine Salmonella serotype Typhi.ot otvoina
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