Febrile Agglutinins o shake and mix antigens well before dispensing
Slide screen method
Widal Test
Used in diagnosis of enteric fever: typhoid and
paratyphoid fever caused by Salmonella Typhi and
Salmonella Paratyphi A & B
Antibodies specific to Flagellar Antigen
Somatic Antigen of Salmonella species usually
become detectable in blood seven days after the
onset of infection in individuals infected with typhoid
bass lie ??
this test facilitates quantitative estimation of
antibodies to Salmonella antigens in human serum
by slight and tube agglutination test
Principle: when the colored smooth attenuated
antigen suspensions are mixed and incubated with
patient serum anti Salmonella antibodies present in
the patient's serum reacts with the antigen
suspensions to give agglutination.
Agglutination is a positive test result indicating
presence of anti-salmonella antibodies in the
patient
Agglutination negative test result is indicated by the
absence of agglutination.
Primary sample:
o no special preparation of the patient is required
prior to sample collection by approved
techniques
o collect two milliliters of venous blood in a plain
red topped vacutainer
o do not use hemolyzed and turbid samples
Freshly collected serum is preferable
Samples: Stored at 2-8OC up to 72 hours in delay
testing
Reagents and Consumables:
o Salmonella typhi or antigen suspension\
o Salmonella typhi each antigen suspension
o salmonella para typhi A H antigen suspension
salmonella para typhi B H antigen suspension
o poly specific positive control
o glass light with six reaction circles
o applicator sticks
Equipment
o incubator
o dimer
o 0.1 milliliters and one milliliter serological
pipettes and droppers
o 36 Vidale tubes or con tubes for each sample
o physiological saline
o test tube rack
Test procedure:
o bring reagents and samples to room
temperature before testing
frond
aevangelista
o place one drop of positive control onto a
reaction circle of the glass slide
o place 50 microliters of physiological saline on to
the next reaction circle of the glass slide
o place one drop of
o patient serum to be tested on to each of the
required number of reaction circles
o add one drop of O antigen suspension to the
reaction circles containing positive control and
physiological saline
o add one drop each of the antigen suspensions
that is O, H, AH and BH to the reaction circles
containing the patient's serum
o mix contents of each circle uniformly over the
entire circle with separate mixing sticks
o Rock the slide gently back and forth and observe
for agglutination macroscopically at one minute
Interpretation of results
o observe for agglutination or formation of clumps
in the wells
o Compare it with the positive control.
o Agglutination is a positive test result and
indicates presence of the corresponding
antibody in the patient's serum
o absence of agglutination is a negative test result
and indicates that the corresponding antibody is
not present in the patient serum
o as the test wells show agglutination with O
and AH antigens: corresponding antibodies
namely Salmonella paratyphi O and H
antibodies are present in the patient's serum
hence, the infecting organism is Salmonella
parathyphi A
Slide Semi-Quantitative Method
o recommended for obtaining quick approximate
titers only for the antigens that tested positive in
the slide screening test
o using a pipet place eighty microliters 40
microliters 20 microliters 10 micro liter and 5
micro liter of patient serum to be tested on five
different reaction circles on the glass slide
o the corresponding dilutions obtained will be
1:20, 1:40, 1:80, 1:160, 1:320
o add to each reaction circle a drop of the antigen
which showed agglutination with a test sample
in the screening method
o in our demonstration it will be O and AH
antigens
o using separate mixing sticks, mix the contents of
each circle uniformly over the reaction circles
o Rock the slide gently back and forth
 o Observe for agglutination macroscopically within
one minute
o interpretation of result: agglutination is a
positive test result
o the titer is reported as a reciprocal of the highest
dilution which shows a positive test result
Quantitative tube Method
o take appropriate number of sets
o one set for each antigen suspension of eight con
tubes or test tubes and label them 1 to eight
pipet 1.9 milliliters of physiological saline into
tube number one of all sets to each of the
remaining tubes that is two to eight
o add one milliliters of physiological saline to tube
number one of all sets at 0.1 milliliters of serum
sample to be tested and mix well
o transfer 1 milliliters of the diluted serum sample
from tube number 1 to tube number 2 and mix
well
o transfer 1 milliliter of the dilute serum sample
from tube number 2 to tube number 3 and mix
well
o continue the serial dilution till tube number 7 in
each set
o discard one milliliter of the diluted serum from
tube number 7 of each set
o now the dilutions of the serum sample achieved
from tube number 1 to 7 respectively in each set
is as follows: 1:10, 1:20, 1:40, 1:80, 1:160, 1:320,
1:640
o tube number 8 in all the sets serves as a saline
control to all the tubes that is one to eight of
each set, add one drop of the respective well-
mixed antigen suspensions from the reagent
vials and mix well
o cover and incubate overnight at 37 degree
centigrade
o after incubation dislodge the sedimented button
gently and observe for agglutination
Interpretation of result
o The tube test measures the titer of antibodies
present in the patient's serum
o Titer: is defined as a concentration of an
antibody determined by finding the highest
dilution of serum at which it is still able to cause
agglutination of the antigen
o if the highest dilution of serum is 1 is to x, then
the titer of the antibody will be reported as X
o in this test sample the highest dilution at which
agglutination is visible is 1:80 for O antigen and
1:160 for AH antigen
o no agglutination is observed with H and BH
antigens hence the patient suffers from
Into
aevangelista
salmonella typhi infection with anti or antibody
titer of 80 and anti Ah antibody titer of 160
o titers of TO, TH AH, BH above 1:80 and above:
diagnostically significant
o diagnosis should be conformed with positive
cultures, rapid tests, clinical presentation and
other laboratory tests
Safety precautions
o handle all samples as potentially infectious
o handle all reagents with care and avoid contact
with eye mouth and skin
o do not perform mouth pipetting
o discard used reagents and samples as per
biomedical waste disposal guidelines followed in
your health care setting
Potential sources of variability
o Shake the antigenic suspensions well before use
and ensure the suspension is homogeneous
o a moderate rise in titer of all 3 H agglutinins
occurring simultaneously against all 3 H antigens
is suggestive of recent typhoid and paratyphoid
vaccination
o antibiotic treatment prevents a rise in titer
o a negative result cannot rule out typhoid fever if
the sample has been taken very early in the
course of illness that is earlier than 7 days from
the onset of infection this is because antibodies
have still not reached the detectable limit during
this period diagnosis of typhoid fever can be
more definitively established from increasing
titers of H and O antibodies in successive tests
rather than from a single test result of H and O
titers