Presentation on theme: "Development of Compact Dry SL for Salmonella detection"— Presentation transcript:
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 strains② recovery of Salmonella from food materials③ comparison (sensitivity/specificity/agreement) between conventional method and CDSL
4 Comparison of operation between conventional method and CDSL Conventional CDSLDay 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) IsolationDay 4 Presume, presence of Salmonella Confirmation/IdentificationConfirmation/Identification (Biochemical/Serological Tests)(Biochemical/Serological Tests)
5 Operating procedure of CDSL (1) Pre-culture Medium（BPW,EEM)Sample 25gPre-incubation20-24 hours at 35℃Inoculate 0.1mL of Pre-culture solutionon CDSL(approx. 1cm far from the edge of plate)SL×
6 Operating procedure of CDSL (2) ×Drop 1mL of sterilized water at the opposite pointwhere specimen dropped.↓Confirm water diffused all over the plate andturn over the plate capped.Incubate 20 – 24 hours at ℃.Interpretation
7 Test Principle and Features of Compact Dry SL Base medium: DHL agarAdditives: Pigment / Magnesium chloride / Novobiocin / Other additivesTest 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 reactionMotility, Yellow sheet, Greening colony,Black colonies are generated by hydrogen sulfide.Negative reactionNo color changeRed – Red purple
13 Recovery of Salmonella from inoculated Food samples (1) Method Flow ChartFood Sample 25g + Buffer Peptone Water 225mLCompact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis BrothInoculation: 0.1 mL mL mLIncubate 24 hours at 42C°Confirmation: TSI agar / LIM MediumBiochemical Tests: PYR / NPASerological TestDispense 9mL each into sterile test tubeFood SPCGround Chicken Breast x CFU/mLDried whole Egg x CFU/mLBPWDilute S. Enteritidis (5.1 x 108 CFU/mL) from 10-1 to 10-8 Dilute and inoculate 1mL each into above tubeConfirmation of typical Salmonella coloniesMLCB 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 ChartChicken 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 mLConfirmation: TSI agar / LIM MediumBiochemical Tests: PYR / NPASerological TestInoculate 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 coloniesMLCB Rambach XLD(Incubate 24 hours at 35C°)Egg Yolk 25g(SPC < 10 CFU/g)Chicken Breast Egg YolkEEM 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 ChartFood Specimen 25g + EEM Broth 225mL (Meat products, 60 samples)Compact Dry SL DIASALM (MERCK) Rappaport-Vassiliadis Broth Tetrathionate BrothInoculation: 0.1 mL mL mL mLIncubate 24 hours at 42C°MLCB agar / Rambach agar / XLD agarIncubate 24 hours at 35C°Confirmation: TSI agar / LIM MediumBiochemical Tests: PYR / NPASerological TestPre-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 specimenCompact Dry SL PositivesDIASALM PositivesRV PositivesTT PositivesChicken (breast/ground)8Chicken (dark/sliced)4Chicken (breast)Chicken (breast white)63Chicken (leg dark)2Chicken (ground)Chicken (wing)Chicken (skin)Chicken (gizzard)Chicken (steamed)BaconSausageProcessed HamFried Chicken (Frozen)Japanese Style Fried Chicken (Frozen)TOTAL6036343032
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 methodCDSLDIASALMCDSL vs DIASALMSensitivity 34 / 34 x 100 = 100%Specificity 24 / 26 x 100 = %Agreement 58 / 60 x 100 = %CDSLR VCDSL vs RV BrothSensitivity 30 / 30 x 100 = 100%Specificity 24 / 30 x 100 = 80%Agreement 54 / 60 x 100 = 90%CDSLT TCDSL vs Tetrathionate BrothSensitivity 32 / 32 x 100 = 100%Specificity 24 / 28 x 100 = %Agreement 56 / 60 x 100 = %
20 ConclusionAll 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.