ENVR 133, ENVIRONMENTAL HEALTH MICROBIOLOGY, SPRING
Detection and Quantitation of Salmonella in Water and Wastewater
INTRODUCTION
The Salmonellae are an important genus of enteric bacterial pathogens that can be
transmitted by fecally contaminated water and food. Until recently, Direct detection and
quantitation of pathogens in water, wastes and food was not done routinely because of the
general limitations of searching for selected pathogens instead of the more prevalent and
abundant fecal indicators and because of the more complex technical requirements and
other limitations of the detection methods. However, detection of pathogens is now
considered useful and even essential in some circumstances. For example, the quality of
biosolids (treated sewage sludge) is based in part on the presence of Salmonella. Food
safety regulations now require Salmonella monitoring in poultry and meat products.
When investigating waterborne and foodborne outbreaks of enteric disease, it is helpful if
the pathogen infecting humans can also be found in the incriminated water or food.
A variety of methods have been used over the years to detect and quantify Salmonellae in
water, food and other environmental samples. As yet, no single isolation method has
emerged as the best in all respects. The Salmonellae are closely related and
physiologically similar to many other Enterobacteriaceae that are often non-pathogenic.
It is difficult to maximize salmonella recovery and minimize the growth of these more
plentiful other kinds of enteric bacteria. However, the methods now used are considered
relatively sensitive and able to achieve salmonella recovery despite the presence of other
non-Salmonellla bacteria during the detection and recovery processing steps.
Typically, the approach used for salmonella isolation involves the following steps:
pre-enrichment in a non-selective broth culture medium,
enrichment in a selective broth medium,
isolation of presumptive salmonella in the enrichment by plating on differential and
selective solid (agar) solid media to get discrete colonies,
confirmation of colony isolates by biochemical characterization,
and verification by serological identification and or molecular analysis.
Various combinations of pre-enrichment, enrichment, isolation media and incubation
temperatures have been reported. Therefore, the choice of a method is somewhat
arbitrary unless comparative studies are done using the sample types of interest.
In addition to merely detecting Salmonella in environmental samples, the concentration
of salmonella can be estimated by most probable number (MPN) methods. This is done
by separately enriching different quantities (volumes) of test sample, sometimes in
replicate. Analogous to the MPN methods for coliforms and other indicator bacteria, the
salmonella concentration in the test sample can be estimated from the number and size
(volume or mass) of confirmed positive sample portions.
PURPOSE
In this lab exercise we will attempt to detect and quantify salmonella concentrations in
raw and treated sewage using pre-enrichment, enrichment and isolation media now
commonly employed for their detection on water and wastes.
MATERIALS
Samples: Raw sewage and treated sewage and upstream and downstream samples from
Morgan Creek receiving water.
Pre-enrichment medium: buffered peptone water.
Enrichment medium: Rappaport-Vassiliadis broth.
Colony isolation medium: Salmonella-Shigella agar
Purification and storage medium: tryptic soy agar slants.
Confirmatory biochemical tests: Enterotube biochemical test kit
Work in pairs of two students
Obtain samples of raw or treated sewage and a water sample (up- or down-stream as
directed by the instructor. Mix the sample 25 times before removing aliquots.
For up- and down-stream and treated sewage effluent samples, add 100 ml and 10 ml of
sample to another 1 liter and 100 ml of BPW, respectively. For raw sewage, add 100, 10
ml, and 1 ml of sample to 1 liter, 100ml and 10 ml of BPW, respectively.
Incubate samples at 37oC overnight (preferably <18 hr.). After incubation, transfer 1 ml
of each pre-enrichment volume to 10 ml of each R-V medium as directed by the
instructor. Incubate R-V enrichments at 43 oC for 24 hours.
Streak from each enrichment onto SS agar and incubate overnight at 41oC and look for
typical salmonella colonies.
Appearance
Likely Bacterium
Colorless and translucent
Salmonella and Shigella
Translucent with black center
Salmonella and Proteus
Ping to red
E. coli
Pink to white, cream colored, opaque or mucoid
Non-E. coli coliforms
The number of colonies picked from initial SS agar plates depends on the number
available, the amount of confirmatory media available and your ambition. Pick at least
two typical colonies from each type of plate for each sample dilution, if they are present.
Pick presumptive salmonella colonies, re-streak on SS-agar and incubate overnight at
37oC.
Observe for typical Salmonella colonies on the re-streak plates.
Pick these colonies, streaked onto tryptic soy agar slants and incubate overnight at 37oC.
Then, store slants of presumptive Salmonella at 4oC for further biochemical analysis in a
future lab period.
Salmonella typhimurium (left)
Salmonella Dublin (right)