Standard Analytical Protocol for Non-Typhoidal Salmonella in Drinking and Surface Water - USEPA Jeremy Olstadt WI State Laboratory of Hygiene, Madison, WI Background • Prolonged survival in water • 1.2 million illnesses in the US per year (CDC)- most are foodborne • Non-typhoidal Salmonella genus is comprised of Salmonella bongori and Salmonella enterica • 2500 know serotypes – all of which are potential human pathogens • Only a few serotypes cause the majority of illness • Haley (2009) Background • Salmonellosis • Symptoms: diarrhea, vomiting, abdominal cramps • Develops 12-72 hours after infection • Lasts 4-7 days Standard Analytical Procedure - Salmonella • BSL-2 Laboratory • USEPA Method 1682: Salmonella in Sewage Sludge (Biosolids) • Support of EPA Homeland Security Efforts Summary of Method • Identification of Salmonella by: Non-Selective and Selective Media Morphological characteristics Biochemical characteristics Serological characteristics Non-Selective Media – Tryptic Soy Broth XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination XLD TSI Antibody Test Selective Media – MSRV Plate-Modified Semisolid Rappaport- Vassiliadis Agar Quantitative Sample Analysis • 15 Tube MPN (most probable number)Method 3 Rows of 5 tubes 10mL of 3X (TSB), 5ml of 3X(TSB) and 10mL of 1X(TSB) Inoculate 10mL 3X TSB tubes with 20 mL of sample Inoculate 5mL 3X TSB with 10mL of sample Inoculate 10mL of 1X TSB with 1 mL of sample Incubate at 36.0oC for 24+ 2 hours Arrangement of TSB Tubes for initial enrichment Isolation of Salmonella on MSRV Plates • Select TSB tubes exhibiting turbidity • Inoculate MSRV plate with six 30uL drops • Each tube uses a separate MSRV plate • Space drops evenly across plate to prevent overlap of spots • Allow plates to absorb to media for 1 hour • Incubate at 42oC + 0.5oC for 16-18 hours Selective Media – MSRV Plate – Modified Semisolid Rappaport Vassiliadis Agar Isolation on XLD Plates • Examine MSRV plates for motility (whitish halo) • Using an inoculating loop, stab into halo and streak for isolation onto XLD plates • Incubate at 36.0oC + 1.5oC for 18-24 hours • Following incubation look for black and/or pink to red colonies with black center on XLD plates Pure and Mixed Cultures on XLD Pure culture Mixed culture Biochemical Confirmation of Salmonella • Triple Sugar Iron Slant (TSI) • Inoculate a TSI slant with an inoculating needle containing a portion a colony from the XLD plate • Stab into butt of slant and streak the slant • Incubate “loose-capped” at 36.0oC+ 1.5oC for 24+ 2 hours • Positive for Salmonella will have acid(yellow) butt and alkaline (red) slant and possible H2S production XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination Testing XLD TSI Antibody Test Biochemical Confirmation of Salmonella • Lysine Iron Agar (LIA) • Similar to TSI inoculate an LIA slant • Positive Salmonella have alkaline (purple) butt and alkaline (purple) slant and possible H2S production Biochemical Confirmation of Salmonella • Urea Broth • Use a sterile loop to inoculate the broth with a portion of a potential positive colony • Salmonella are urease-negative • If Salmonella, the result will be no color change in urea broth Urea broth Control Proteus Salmonella Serological Analysis - Confirmation • Inoculate a portion of bacteria from the TSI tube into sterile saline • Place two drops onto a slide • Add one drop of antisera to one and one drop of saline to the other • View under magnification for agglutination reaction • Perform a + and – ctrl along side at the same time for comparison XLD Plate, Triple Sugar Iron and Salmonella O antiserum agglutination Testing XLD TSI Antibody Test MPN Methodology • Count the number of positive or confirmed Salmonella positive TSB tubes from the initial step • Apply the number of positive tubes to the MPN table • The volumes analyzed will determine which MPN table you will use • Ex. 20mL,10mL or 1mL vs. 10mL, 1mL or 0.1mL Example MPN Calculation • Three rows of TSB tubes used for 20mL, 10ml and 1mL volumes • Result was turbidity/growth in: – 5 tubes positive containing 20mL of sample – 3 tubes positive containing 10mL of sample – 0 tubes positive containing 1 mL of sample – Designation on chart would be 5-3-0 – 5-3-0 MPN= 0.1151/mL or 11.51/100mL of sample Most Probable Number (MPN) Chart Qualitative Sample Analysis • Samples diluted 1:1 in 2X TSB (Tryptic Soy Broth) • Sample volume of 100mL would use 100mL of 2X TSB • Incubated at 36oC + 1.5oC for 24 + 2 Hours • Followed by isolation using MSRV, XLD, TSI, LIA, Urea Broth and Salmonella O antiserum for confirmation Strengths and Weaknesses of the Method • Strengths – Detects live (potentially infective) cells – Quantitative abilities – Multiple layers of specificity • Selectivity of MSRV • Selectivity of XLD – Distinctive positive reactions • Weakness – TSB permissive growth conditions, need for increased selectivity in initial step – Interference by ubiquitous bacteria and toxic substances – Numerous tubes and plates needed for one quantification – Time to completed test – five days Thanks for your attention • Jeremy Olstadt • Wisconsin State Laboratory of Hygiene • [email protected] • 608 224-6262.