1. Development of Compact Dry SL for Salmonella detection
S.Mizuochi１）,H.Teramura１）,S.Nirazuka１）,H.Kodaka１）,K.Inoue２）、K.Tamura３）
1)Research Dept., Nissui Pharmaceutical, 2) Saitama Pref. Ranzango Hp.,
3)National Institute of Infectious Diseases

2. Background
　Normally, Salmonella detection from food sample is processed these following steps, Pre-culture, Selective enrichment culture, Isolation and Confirmation/Identification. There are strong customer demands to improve the test procedure for more simple and rapid one, if it could have an equivalent result.
　We develop simple and easy medium for Salmonella detection (Compact Dry SL, hereinafter CDSL) that detects Salmonella by combination of biochemical reaction, specific enzyme reaction and detection of motility of Salmonella. CDSL is screening method for detecting Salmonella which inoculate pre-culture sample as a specimen.
　Herewith we report CDSL evaluation results compared with conventional culture methods using meats on the market,
① growth and color forming used by pure culture strains
② recovery of Salmonella from food materials
③ comparison (sensitivity/specificity/agreement) between conventional method and CDSL

3. Package, CDSL

4. Comparison of operation between conventional method and CDSL
Conventional CDSL
Day 1 Specimen　25g Specimen 25g Pre-culture Pre-culture
(BPW/EEM) (BPW/EEM)
↓ ↓
Day 2　 Selective Enrichment Culture CDSL
(TT/RV/SBG/SC)
Day 3　 Isolation Presume, presence of Salmonella
(MLCB/DHL/XLD/Rambach) Isolation
Day 4　 Presume, presence of Salmonella Confirmation/Identification
Confirmation/Identification (Biochemical/Serological Tests)
(Biochemical/Serological Tests)　　

5. Operating procedure of CDSL (1)
Pre-culture Medium
（BPW,EEM)
Sample 25g
Pre-incubation
20-24 hours at 35℃
Inoculate 0.1mL of Pre-culture solution
on CDSL
(approx. 1cm far from the edge of plate) SL ×

6. Operating procedure of CDSL (2)×
Drop 1mL of sterilized water at the opposite point
where specimen dropped. ↓
Confirm water diffused all over the plate and
turn over the plate capped.
Incubate 20 – 24 hours at ℃.
Interpretation

7. Test Principle and Features of Compact Dry SL
Base medium: DHL agar
Additives: Pigment / Magnesium chloride / Novobiocin / Other additives
Test principle:
1) Medium alkalization that changes medium color from blue-purple to yellow by Salmonella’s lysine decarboxylase ability.
2) Colony color will be greening caused by decomposition of chromogenic substrate with specific enzyme on Salmonella.
3) Salmonella produces hydrogen (not all strains) sulfide which generate black colonies.
4) Detect motility of Salmonella.
Features:
1) CDSL detects Salmonella one day earlier than conventional culture methods.
2) Isolated colonies on the plate can be fished for further identification tests.
3) Ready to use and portable plate which no need to special preparation.

8. Chicken (42℃, 24 hours incubation)
Positive reaction
Motility, Yellow sheet, Greening colony,
Black colonies are generated by hydrogen sulfide.
Negative reaction
No color change
Red – Red purple

9. CDSL reactions (1) Standard strains

10. CDSL reactions (2) Gram Negative Bacteria rather than Salmonella

11. CDSL reactions (3) Salmonella spp.- 1

12. CDSL reactions (3) Salmonella spp.- 2

13. Recovery of Salmonella from inoculated Food samples (1)
Method Flow Chart
Food Sample 25g + Buffer Peptone Water 225mL
Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth
Inoculation: 0.1 mL mL mL
Incubate 24 hours at 42C°
Confirmation: TSI agar / LIM Medium
Biochemical Tests: PYR / NPA
Serological Test
Dispense 9mL each into sterile test tube
Food SPC
Ground Chicken Breast x CFU/mL
Dried whole Egg x CFU/mL
BPW
Dilute S. Enteritidis (5.1 x 108 CFU/mL) from 10-1 to 10-8 Dilute and inoculate 1mL each into above tube
Confirmation of typical Salmonella colonies
MLCB Rambach XLD

14. Recovery of Salmonella from inoculated Food samples (1)

15. Recovery of Salmonella from inoculated Food samples (2) (Sensitivity test for pre-cultured sample)
Method Flow Chart
Chicken Breast 25g
(SPC 8.3 x 103 CFU/g)
Inoculate to below media (incubate 24 hours at 42C°)
Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth
　　Inoculation: 0.1 mL mL mL
Confirmation: TSI agar / LIM Medium
Biochemical Tests: PYR / NPA
Serological Test
Inoculate 1.0mL of Pre-cultured Salmonella Typhimurium 4-207B in TSB (over night) to each food sample
(Controlled 101 to 104 CFU/mL)
Confirmation of typical Salmonella colonies
MLCB Rambach XLD
(Incubate 24 hours at 35C°)
Egg Yolk 25g
(SPC < 10 CFU/g)
Chicken Breast Egg Yolk
EEM 225mL (Incubate 24 hours at 35C°) BPW 225mL

16. Recovery of Salmonella from inoculated Food samples (2) (Sensitivity test from pre-cultured sample)

17. Comparison of Salmonella detection by methods from Selective Enrichment Media by meat products (1)
Method Flow Chart
Food Specimen 25g + EEM Broth 225mL (Meat products, 60 samples)
Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate Broth
Inoculation: 0.1 mL mL mL mL
Incubate 24 hours at 42C°
MLCB agar / Rambach agar / XLD agar
Incubate 24 hours at 35C°
Confirmation: TSI agar / LIM Medium
Biochemical Tests: PYR / NPA
Serological Test
Pre-Enrichment culture <24 hours at 35C°>

18. Comparison of Salmonella detection by methods from
Selective Enrichment Media by meat products (2)
Name of Product
(selected unintentionally)
Number of specimen
Compact Dry SL Positives
DIASALM Positives
RV Positives
TT Positives
Chicken (breast/ground) 8
Chicken (dark/sliced) 4
Chicken (breast)
Chicken (breast white) 6 3
Chicken (leg dark) 2
Chicken (ground)
Chicken (wing)
Chicken (skin)
Chicken (gizzard)
Chicken (steamed)
Bacon
Sausage
Processed Ham
Fried Chicken (Frozen)
Japanese Style Fried Chicken (Frozen)
TOTAL 60 36 34 30 32

19. Comparison of Salmonella detection methods from
Selective Enrichment Media by meat products (3)
Comparison of Sensitivity, Specificity and Agreement between Compact Dry SL and Conventional method
CDSL
DIASALM
CDSL vs DIASALM
Sensitivity 34 / 34 x 100 = 100%
Specificity 24 / 26 x 100 = %
Agreement 58 / 60 x 100 = %
CDSL
R V
CDSL vs RV Broth
Sensitivity 30 / 30 x 100 = 100%
Specificity 24 / 30 x 100 = 80%
Agreement 54 / 60 x 100 = 90%
CDSL
T T
CDSL vs Tetrathionate Broth
Sensitivity 32 / 32 x 100 = 100%
Specificity 24 / 28 x 100 = %
Agreement 56 / 60 x 100 = %

20. Conclusion
All Salmonella samples are being positive on Compact Dry SL. Several high concentration of Proteus and Pseudomonas strains showed “yellowed” reaction, but other Gram negatives were negative or inhibited its growth.
Sensitivity and specificity of CDSL were higher than conventional methods when detected Salmonella from meat products on the market.
Preparation of medium is not necessary for CDSL and the presumption of test result may receive one day earlier than conventional enrichment culture methods that will be useful for food manufacturers.