L22 - Medical Microbiology Laboratory (Salmonella spp.) by Hussein A. Abid

1. Medical Microbiology Laboratory
Gram Negative Bacilli – Enterobacteriaceae
(Salmonella spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. 2
Enterobacteriaceae
 A large Family of aerobic bacterial flora of intestine of
humans and other animals.
 Its members are non-spore forming, non acid-fast,
gram negative bacilli.
 Capsule (variable)
 Motility (variable)
 Aerobic and facultatively anaerobic, grow readily on
ordinary media
 Ferment glucose, reduce nitrates to nitrites, catalase
positive but oxidase negative.

3. 3
CLASSIFICATION ACCORDING TO LACTOSE
FERMENTATION
Lactose Non-fermentative Lactose Fermentative
Salmonella spp.
Shigella spp.
Proteus spp.
Yersinia spp.
E. coli
Klebsiella spp.
Enterobacter spp.
Citrobacter spp.
Culture media used to differentiate lactose fermenter from
lactose non fermenter are:
1. MacConkey’s (Mac) agar medium
2. Eosin Methylene Blue (EMB) agar medium
3. Salmonella-Shigella (SS) agar medium
4. Triple Sugar Iron (TSI) agar medium

4. 4
TAXONOMY
Scientific name Rank
• Enterobacteriaceae Family
• Salmonella Genus
• S. typhi
• S. paratyphi
• S. typhimurium
Species
(medically important spp.)

5. 5
Salmonella spp.
 Gram-negative, motile bacilli (enteric rods), facultative
anaerobes
 Found in the intestines and feces of most birds, reptiles,
and mammals (more than 2,000 species)
 Most Salmonella infections in humans result consumption
of food contaminated with animal feces
 Poultry and eggs are particularly common sources of
Salmonella
 Non-lactose fermenter (NLF), resistant to bile salt,
H2S (+ve)

6. 6
CLINICAL MANIFESTATIONS
 Typhoidal salmonella (enteric fever)
 Typhoid fever: caused by S. typhi
 Paratyphoid fever: mild, caused by S. paratyphi A, B & C
 Non typhoidal salmonella
 Enteritis (gastroenteritis)
 Septicemia (particularly S. choleraesuis, S. typhi, and S. paratyphi).
 Osteomyelitis (infection of bone)
 Localized infections
 Carriers (gall bladder is the reservoir for Salmonella typhi)

7. 7
TYPHOIDAL SALMONELLA
Clinical specimens:
 Blood
 Urine
 Faeces
 Bone marrow
 Bile
 Pus
 CSF

8. 8
TYPHOIDAL SALMONELLA
Ideal specimens:
Specimen Week
Blood for culture First week
Serum for serology (antibodies) Second week
Stool Third week
Urine Fourth week

9. 9
S. typhi (microscopy)
 Gram negative (-ve) plump rods

10. 10
Salmonella (culture)
 MacConkey’s (Mac) agar: medium sized, non-lactose
fermenter colonies.

11. 11
Salmonella (culture)
 Salmonella-Shigella (SS) agar: medium sized 2-4 mm,
shiny white to greyish margin, elevated, round, H2S
production (black centre).

12. 12
Salmonella (culture)
 Xylose Lysine Deoxycholate (XLD) agar: Salmonella
colonies are identified easily by the production of H2S
(black center colonies).

13. 13
Salmonella (blood culture)
 Volume of blood: 10 to 15 mL from adults and adolescents, 2
to 4 mL in children.
 Ratio of blood to bile broth: 1:10
 Or add saponin to brain-heart infusion (BHI) broth with 0.05%
SPS.
 Inoculate the blood immediately
 Transport immediately, never store under 15 ºC.
 Incubate as soon as possible
 When blood culture bottles are not available, direct plating of
blood buffy coat from 5 to 10ml sterile heparinized blood onto
Columbia agar plates containing 0.05% saponin is
recommended.

14. 14
Salmonella (blood culture)

15. 15
Salmonella (blood culture)
 Check for turbidity and evidence of growth after 1, 2, 3
and 7 days.
 Bottles showing signs of growth – Do culture on solid
media
 Sub-culturing done in MacConkey, XLD and Blood agar
 On day 7, all the bottles sub-cultured before being
discarded as negative.

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BIOCHEMICAL TESTS
1. Catalase: positive (+ve)
2. Oxidase: negative (-ve)
3. Motility: motile
4. Triple Sugar Iron (TSI):
5. Indole, MR, VP, Citrate (IMViC):
6. Urease test: positive (-ve)
7. Api 20E: 6704752
• S. typhi: K/A, gas (-), H2S (+)
• Others: K/A, gas (+), H2S (+)
• S. typhi: – + – –
• S. paratyphi A: – + – +

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BIOCHEMICAL TESTS

18. 18
SEROLOGICAL TESTS
1- Agglutination test
 known sera + unknown culture
 clumping within few minutes (+)
 rapid preliminary identification of cultures
2- Tube Dilution Agglutination test (Widal Test)
 test to detect antibodies formed vs O and H antigen
 rise in antibody titers, serum obtained at intervals of 7-
10 days

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WIDAL TEST
1- Tools, reagents & consumables
2- Qualitative (slide) test
3- Quantitative (slide) test
4- Quantitative (tube) test
5- Limitations

20. 20
TOOLS, CONSUMABLES AND REAGENTS
Widal test kit
Test tubesNormal saline
Marker pen
MicropipetteMicropipette tip
Stick

21. 21
QUALITATIVE SLIDE TEST PROCEDURE
1. Bring all reagents to room temperature and mix well.
2. Add 1 drop of test sample (25 µL) into each reaction circle labeled
as O, H, AH, BH according to given antigen solution.
3. Add 1 drop of positive control (25 µL) into the circle marked as PC
and 1 drop of negative control (25 µL) into the reaction circle marked
as NC.
4. Add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’,
Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’ to circles
labeled as O, H, AH, BH respectively in which test samples has been
added.
5. Mix it thoroughly with the aid of applicator stick and rotate the slide
gently.
6. Observe for agglutination.

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QUALITATIVE SLIDE TEST RESULTS
• Positive Test: Agglutination within a minute
• Negative Test: No agglutination

23. 23
QUANTITATIVE SLIDE TEST PROCEDURE
1. Bring all reagents to room temperature and mix well.
2. Dispense one drop of saline into the first reaction circle
and then place 5, 10, 20, 40, 80 µL of the test sample on
the remaining circles.
3. Add a drop of the antigen, which showed agglutination
with the test sample in the screening (qualitative)
method, to each circle.
4. Mix the contents of each circle with the aid of applicator
stick and rotate the slide gently.
5. Observe for agglutination.

24. 24
QUANTITATIVE SLIDE TEST RESULTS
 The antibody titer of the test sample is its highest dilution
that gives a visible agglutination.
 80 µL corresponds to 1 in 20 dilution, 40 µL to 1 in 40, 20
µL to 1 in 80, 10 µL to 1 in 160 and 5 µL corresponds to 1
in 320 titer.
 Agglutinin titer greater than 1:80 is considered as
significant infection and low titers indicate absence of
infection.

25. 25
QUANTITATIVE TUBE TEST PROCEDURE
1. Bring all reagents to room temperature and mix well.
2. Prepare 4 sets of test tubes for individual antigen. Each set contains
1- 8 tubes.
3. Add 1.9 mL of 0.85% sterile saline to tube No. 1 of each antigen set.
4. To tube No. 2-8 of all sets add 1 mL of physiological saline.
5. To tube No. 1 of all sets add 0.1 mL of test sample to be tested and
mix well.
6. Transfer 1 mL of the diluted serum sample from tube No. 1 to tube
No. 2 and mix well.
7. Transfer 1 mL of the diluted serum sample from tube No. 2 to tube
No. 3 and mix well. Continue this serial dilution till tube No. 7 in each
set of antigen.

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QUANTITATIVE TUBE TEST PROCEDURE
8. Discard 1.0 ml of the diluted serum from tube No.7 of each set.
9. So the dilutions of the serum sample from tube No. 1 to 7 respectively in
each antigen set are 1:20, 1:40,1:80, 1:160, 1: 320, 1:640, 1: 1280.
10. Tube No. 8 is negative control with 0.85% sterile saline.
11. To one set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘O’
antigen.
12. In second set i.e. from tube no.1- 8 add 50 µl of Salmonella typhi ‘H’
antigen.
13. Respectively for third and fourth sets, add Salmonella paratyphi ‘AH’ and
Salmonella paratyphi ‘BH’ to all tubes from 1-8.
14. Mix well, cover and incubate these tubes overnight at 37 ºC
(approximately 18 hours).
15. After incubation dislodge the sediment and observe for agglutination.

27. 27
QUANTITATIVE TUBE TEST PROCEDURE

28. 28
QUANTITATIVE TUBE TEST RESULTS
 The antibody titer of the test sample is its highest dilution
that gives a visible agglutination.
 Agglutinin titer greater than 1:80 is considered as
significant infection and low titers indicate absence of
infection.

29. 29
QUANTITATIVE TUBE TEST RESULTS

30. 1. Tests done within 7 days of illness and after 4 weeks are
usually negative.
2. The local titer of the place should be known for the results
interpreted correctly.
3. This test (quantitative) is highly time consumable.
4. Previous typhoid vaccination may contribute to elevated
agglutinins in the non-infected population.
5. Other infections of non-enteric salmonella infection such as
Typhus, Immunological disorders, chronic liver disease may
cause false positive reaction.
6. Cross reaction between malaria parasites and salmonella
antigens may cause false positive Widal agglutination test
30
LIMITATIONS OF WIDAL TEST

31.  Enzyme immunoassay (EIA) and enzyme-linked
immunosorbent assay (ELISA) have been developed for
Salmonella, and simple-to-use lateral flow test strips using
immunochromatographic technology have also been developed
into commercial products by a number of manufacturers.
 Molecular methods are also available, notably DNA
hybridization and PCR assays for the identification of
Salmonella enterica. However, these are generally designed for
use as part of a method for rapid detection and screening rather
than for confirmation.
31
OTHER METHODS FOR DETECTION
AND CONFIRMATION