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 RappaportVassiliadis 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
olstadjm@slh.wisc.edu
608 224-6262