PRECIPITATIO
NBY HABSHIBA WILLIAM
RAMSNEHI COLLEGE OF NURSING
VENEREAL DISEASE RESEARCH LABORATORY
TEST (VDRL)
It is the most widely used precipitation (flocculation) test used in
diagnosis of venereal disease syphilis caused by a spirochete,
[Link]. It is a simple & rapid test requiring very small quantity of
serum.
Procedure: In this test 0.05 ml of inactivated serum (56°C for 30
minutes) of patient is taken in a special slide with rings or depressions
of 14/16 mm diameter each & depth of 1.75 mm. One drop of antigen
(cardiolipin) is added with syringe delivering 60 drops in 1 ml. The slide
is rotated manually or in VDRL rotator at 180 revolutions per minute
for 4 minutes & the Ag-Ab reaction is studied under low power
objective of microscope.
Results: Presence of clumps indicate positive test where as uniformly
Advantages: VDRL is a standard test for use with CSF. It is a
screening test & is positive in 70% of primary & 98% of secondary
syphilis. The test is negative in persons with late syphilis.
Disadvantages: VDRL has one disadvantage as it gives false
biological reaction in conditions like leprosy, hepatitis, malaria,
vaccination, pregnancy etc. Positive VDRL test reverts to negative, 6-
24 months after effective treatment of early syphilis.
AGGLUTINATION
When a particulate antigen (Bacteria, RBC's) is mixed with its
corresponding Ab in presence of electrolytes (NaCl) at a suitable
temperature (37°C) & pH (7.4), the particles are clumped or
agglutinated. IgM Ab's agglutinate better than IgG antibody.
TECHNIQUES OF AGGLUTINATION TEST:
The test may be carried out in slide as well as in tube:
1. Slide agglutination test (Micro agglutination): It is widely
used method to detect unknown Ag (Bacteria, RBC's in blood
grouping etc.) On a clean glass slide a drop of antigen (made in
saline) is mixed with a drop of antiserum. The reaction occurs
immediately. Clumps are formed in few minutes (Fig.3.6a).
2. Tube agglutination tests (Macro agglutination): It is a
quantitative test used to determine or estimate the titer of Ab & to
confirm the result of slide agglutination test. It is routinely used for
serological diagnosis of typhoid, paratyphoid, brucellosis & typhus
fevers (Fig.3.6b).
WIDAL TEST
Widal test is an agglutination test employed in the serological diagnosis of enteric
fever (typhoid & paratyphoid) caused by [Link] & [Link] (A & B). The specific
antibodies are usually detectable in patient's blood at the end of first week of
infection. The test is named after a French physician & bacteriologist Georges
Fernand Isidore Widal who first developed this test for diagnosis of typhoid.
Principle: The serum of a patient is tested for 'O' & 'H' antibodies by using antigenic
suspensions [Link] 'O'(somatic Ag) & [Link] 'H' (flagellar Ag) respectively. For
paratyphoid testing, the antigen suspensions used are [Link] 'AH' & [Link]
'BH'.
Preparation of antigens: [Link] 'O' antigen & [Link] 'H' antigen are prepared
from [Link] 901 strain. 'O' antigen for [Link] A & B are not taken as they cross
Methods: Widal test may be conducted in two ways;
1. WIDAL (slide agglutination/micro agglutination)
TEST:
Procedure:
1. In each four rings of the slide, labeled as 'O','H','AH' & 'BH' add one drop
of serum.
2. Add corresponding antigen drop (one) in each ring of the glass plate.
3. Mix the antigen suspension and the diluted serum drop in each ring by
using separate applicator stick.
4. Slowly rock and tilt the glass plate and observe for 3 minutes.
5. Record the degree of agglutination as 4+, 3+, 2+, 1+ or no
agglutination (negative) Fig.3.7a.
2. WIDAL (tube agglutination /macro agglutination)
TEST:
Procedure: In this test two types of tubes are used:
a. Round-bottomed Felix tubes.
b. Conical-bottomed Dreyer's tubes.
1. For each serum sample, arrange 4 rows (2 rows of Felix tubes & 2
rows of Dreyer's tubes) of 6 tubes each in a widal rack.
2. Take 6 tubes in other rack for preparation of master dilution.
a. Keep 7ml normal saline in tube 1 & 3.5 ml in other 5 tubes.
b. Add 0.5 ml of test serum in tube 1 & mix well.
c. Take 3.5 ml from tube 1 & transfer to tube 2. Mix well & continue
3. Transfer 0.5 ml from master dilution tube to each of the
corresponding vertical rows in test rack. Place 0.5 ml normal
saline in each of tubes in 7th row to serve as control.
4. Add 0.5 ml of [Link] O antigen to each of 7 tubes in first
horizontal row.
5. Add 0.5 ml of S. typhi H antigen to each of 7 tubes in second
horizontal row.
6. Add 0.5 ml of [Link] A(H) antigen to each of 7 tubes in
third row.
7. Add 0.5 ml of [Link] B(H) antigen to each of 7 tubes in fourth
row to obtain dilution of 1:30, 1:60, 1:120, 1:240, 1:480 & 1:960.
8. Shake the rack well to mix & incubate the rack in
thermostatically controlled water bath maintained at
37°C for overnight incubation.
Reading: The control tubes must be examined first,
where they should show no agglutination. The
agglutination of 'O 'antigen appears as a 'matt' or
'carpet' or 'granular disc- like' pattern at the bottom of
[Link] 'O' antigen containing tube. The agglutination of
'H' antigen appears as loose, wooly or cottony
Interpretation: The highest dilution of serum that produces a positive
agglutination is taken as titer.
1. Agglutination appears by the end of first week. The titers increase during
second, third & fourth week after which it gradually declines.
2. Single test is usually of not much value. Demonstration of rising titer of
antibody by testing two or more sera specimen is more meaningful than
single test.
3. Patients already treated with antibiotics may not show any rise in titer,
instead there may be fall in titer.
4. Patients who have received vaccine against salmonella may give false
positive reaction.
5. Individuals who had suffered from enteric fever in past sometimes develop
anti-salmonella antibodies during an unrelated or closely related infection.
This is called anamnestic response with temporary rise in 'H' titer only.
Uses of agglutination reaction:
1. Identification of bacteria
2. Serological diagnosis of infection e.g. widal
for typhoid & paratyphoid.
3. Haemagglutination test e.g., Paul Bunnel,
Rose Waaler test.
THANK
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