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HiPer® Widal Test Teaching Kit (Slide Test)

Product Code: HTI017

Number of experiments that can be performed: 20

Duration of Experiment: 1hour

Storage Instructions:
 The kit is stable for 12 months from the date of manufacture
 Store all the antigen solutions, Test sample, positive and negative
control at 2-8oC
 Other kit contents can be stored at room temperature (15-25oC)

Registered Office
HiMedia Laboratories Pvt Ltd.
WHO Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Fax: 6147 1920
15
GMP Thane, (West) 400604, Maharashtra, INDIA. Web: www.himedialabs.com
CERTIFIED Customer Care No.: 00-91-22-6116 9797 Email : info@himedialabs.com
Tel: 00-91-22-6147 1919, 6903 4800 mb@himedialabs.com

The information contained herein is believed to be accurate and complete. However no warranty or guarantee whatsoever is made
or is to be implied with respect to such information or with respect to any product, method or apparatus referred to herein
 Index

Sr. No. Contents Page No.

1 Aim 3

2 Introduction 3

3 Principle 3

4 Kit Contents 4

5 Materials Required But Not Provided 4

6 Storage 4

7 Important Instructions 4

8 Procedure 5

9 Observation and Result 6

10 Interpretation 6

11 Troubleshooting Guide 6

2
Aim:
To detect the presence of Salmonella genus which causes enteric or Typhoid Fever by using qualitative slide
agglutination test.

Introduction:
Widal test is a serological method to diagnose enteric fever or typhoid which is caused by the infection with
pathogenic microorganisms like Salmonella typhi, Salmonella paratyphi A, B and C. This method of
diagnostic test is based upon a visible agglutination reaction either in a test tube or on a slide between
antibodies of patient serum and antigens specifically prepared from Salmonella sp.

Principle:
Enteric fever or typhoid is a life threatening disease which usually occurs due to the infection of pathogenic
microorganisms, e.g. Salmonella typhi, Salmonella paratyphi A, B and C. These microorganisms are
transmitted to human body through food and drinks contaminated with fecal matter. Early diagnosis and
treatment for this fever are essential to avoid serious clinical complications. During the course of infection
antibodies are produced against Salmonella antigens. Widal test, a serological method for the detection of
Salmonella sp., was developed by F Widal in 1896. During this test a visible agglutination is formed due to
the reaction in a test tube or on a slide between antibodies present in the infected person's blood sample
and specific antigens of S. typhi and S. paratyphi. For the slide agglutination test, stained Salmonella
antigens are used to detect the presence of specific agglutinin in the patient’s serum. The slide agglutination
test is used as a primary screening procedure.
The organisms causing enteric fever possesses two major antigens namely somatic antigen, O and a flagellar
antigen, H along with another surface antigen, Vi. During infection antibodies are produced in patient’s sera
against Salmonella typhi O and H and Salmonella paratyphi AH and BH antigens. During infection antibodies
are produced in patient’s sera against these antigens. Antigens specifically prepared from this organism are
used in the agglutination test to detect the presence of antibodies in patients’ sera which are elucidated in
response to infection by these bacteria. There are some agglutinins that are produced in the patient’s serum
during the fever period, which react with somatic antigen O of Salmonella typhi, A or B of Salmonella
paratyphi and then with flagellar antigen H which is common in most of the Salmonella species. In this test
four specific antigen suspensions are used e.g. Salmonella typhi ( H antigen), Salmonella typhi (O antigen),
Salmonella paratyphi - A and Salmonella paratyphi - B. If agglutination occurs with O antigen then it is
considered positive for Salmonella typhi. If agglutination occurs in A or B antigen then it is confirmed as
positive for Salmonella paratyphi. Agglutination will occur in H antigen circle for all the cases of antigens
like O, A, and B. Salmonella species are characterized by three antigens present on the cell, as shown in
figure 1.

Vi O
Anti- Ant-
gen gen H Antigen

Fig 1: Antigenic structure of Salmonella

3
O Antigen: This is a somatic antigen and is present on the outer membrane of the cell. Its specificity is
determined by the nature of the repeating units in the outer O-polysaccharide chain. Somatic antigens are
heat stable, alcohol resistant and forms compact and granular clumps when mixed with O antisera.
Vi antigens: This is a virulence antigen which is a capsular polysaccharide that overlays the O antigen. This
capsule is not necessary for infection but it increases the infectivity by making it less detectable by the
body’s immune system. It is heat labile and can be detected using Vi antisera. Vi antigen can interfere with
O antigen testing.
H Antigens: This is a heat labile flagellar antigen which is inactivated both by boiling and alcohol. H antigens
rapidly form fluffy clumps when treated with the corresponding antisera. H antigen induces rapid formation
of corresponding antibodies as it is strongly immunogenic.
Widal Test Teaching Kit (Slide Test) utilizes the principle for rapid slide agglutination to detect the presence
of Salmonellatyphi and paratyphi. The kit gives direct results in form of visible agglutination.

Kit Contents:

Table 1: Enlists the materials provided in this kit with their quantity and recommended storage

Sr. No. Quantity Storage

Product Code Materials Provided
20 expts
1 TKC347 Salmonella typhi ‘O’ Antigen 1.6 ml 2-80C
2 TKC348 Salmonella typhi ‘H’ Antigen 1.6 ml 2-80C
3 TKC349 Salmonella paratyphi ‘AH’ Antigen 1.6 ml 2-80C
4 TKC350 Salmonella paratyphi ‘BH’ Antigen 1.6 ml 2-80C
5 TKC351 Positive control 0.6 ml 2-80C
6 TKC352 Negative control 0.6 ml 2-80C
7 TKC383 Test Serum Sample 2.4 ml 2-80C
8 TKC353 Slide for Widal 1 No. RT
9 TKC125 Disposable Mixing Sticks 120 Nos. RT

Materials Required But Not Provided:

Micropipettes, Tips, Gloves and Masks.

Storage:

HiPer® Widal Test Teaching Kit (Slide Test) is stable for 12 months from the date of manufacture without
showing any reduction in performance. On receipt, store the Antigen solutions, Positive control and
Negative control at 2-8oC.

Important Instructions:

1. Widal Slide Test Kit should be used according to the kit instructions.
2. Allow all reagents to reach room temperature before use.
3. Do not dilute any of the kit reagents.
4. Do not inter mix the reagents.
5. Do not freeze any of the kit reagents.
6. Ensure the slide is clean and dry prior to use.
7. 4 the reagents.
Wear masks and gloves while handling
Procedure:

1. Before starting the experiment, bring all reagents to room temperature and mix well.
2. Mark the circles of slides as PC (Positive control), NC (Negative control), O, H, AH, BH as per antigen
solutions used for testing (as shown in figure 2).
3. Add a drop (25 µl) of positive control into the circle marked as PC of given slide.
4. Then add 25 µl of negative control into the reaction circle marked as NC.
5. Add 25 µl of test sample into each reaction circle labeled as O, H, AH, BH according to given antigen
solution.
6. Add 25 µl of Antigen solution of Salmonella typhi 'H' into PC and NC circle each. Mix well using new
mixing stick for each circle.
7. To circles labeled as O, H, AH, BH in which test samples have been added, add antigen solutions of
Salmonella typhi 'O', Salmonella typhi 'H', Salmonella paratyphi 'AH' and Salmonella paratyphi 'BH',
respectively.
8. Mix the content of each reaction circle uniformly with separate mixing stick.
9. Rock the slide gently (approximately for one minute) and observe for agglutination.

Note: After usage, wash the slide with appropriate disinfectant and let it dry. The slide is reusable.

Negative control + Test sample + Test sample +

'H' Antigen 'O' Antigen 'BH' Antigen

NC O BH

PC H AH

Positive control + Test sample + Test sample +

'H' Antigen ‘H’ Antigen ‘AH’ Antigen

Fig 2: Diagrammatic representation of Widal Slide test

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 Observation and Result:

After mixing the test sample with Antigen Solution, Positive control, Negative control separately observe
for the agglutination reaction.

Fig 3: Agglutination reaction for different antigen solutions with Test sample

Well labeled as ‘pc’: Positive control + Salmonella typhi ‘H’ Antigen

Well labeled as ‘nc’: Negative control + Salmonella typhi ‘H’ Antigen
Well No. 1: Test Sample + Salmonella typhi ‘H’ Antigen
Well No. 2: Test Sample + Salmonella typhi ‘O’ Antigen
Well No. 3: Test Sample + Salmonella typhi ‘AH’ Antigen
Well No. 4: Test Sample + Salmonella typhi ‘BH’ Antigen

Interpretation:

During the infection of Salmonella, the human body responds to the antigenic stimulus and as a result corresponding
antibody are produced. When the test sample is treated with colored and attenuated Salmonella antigen suspensions,
the antibodies present in the sample react with the antigen suspension to give clearly visible agglutination which can be
seen through naked eye. Therefore, formation of agglutination indicates positive result which means the presence of
anti-salmonella antibodies in the test sample. No agglutination is a negative test result which indicates the absence of
anti-salmonella antibodies.

Please refer disclaimer Overleaf.

6
 Troubleshooting Guide:

Sr. Problem Possible Cause Solution

No.
False positive The reagents were Ensure that the reagents are added properly
result mixed with each other onto the respective well without mixing with
1
other reagent.
Incubated for a longer The results should be read within the time
time period mentioned in the brochure
No agglutination The reagents are not Ensure that the antigen solutions, positive and
2 observed stored under proper negative control are stored in refrigerator
conditions (2-8oC)

Technical Assistance:

At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of
technical assistance mail at mb@himedialabs.com

8°C Storage temperature

2°C

Do not use if package is

damaged

HiMedia Laboratories Private Limited,

Reg. Off: Plot No. C-40, Road No. 21Y,
MIDC, Wagle Industrial Area, Thane,
(West) 400604, Maharashtra, INDIA.
Web: www.himedialabs.com

01/2027

PIHTI017_O/0124 HTI017-09

Disclaimer :
User must ensure suitability of the product(s) in their application prior to use. Products conform solely to the information contained in this and
other related HiMedia™ publications. The information contained in this publication is based on our research and development work and is to the
best of our knowledge true and accurate. HiMedia™ Laboratories Pvt Ltd reserves the right to make changes to specifications and information
related to the products at any time. Products are not intended for human or animal or therapeutic use but for laboratory,diagnostic, research or
further manufacturing use only, unless otherwise specified. Statements contained herein should not be considered as a warranty of any kind,
expressed or implied, and no liability is accepted for infringement of any patents.

HiMedia Laboratories Pvt. Ltd. Reg.office : Plot No. C-40, Road No. 21Y, MIDC, Wagle Industrial Area, Thane, (West) 400604, Maharashtra, INDIA. Customer
Care No.: 00-91-22-6116 9797 Tel: 00-91-22-6147 1919, 6903 4800 Email: techhelp@himedialabs.com Website: www.himedialabs.com