SAMPLING AND ANAYSIS FOR
MICROBIAL CONTAMINAINTION OF
This module will cover…
• Outbreak case studies of four major
types dairy product pathogens
• Sampling for each of those pathogens
• Sample analysis using approved
• Resolution of the outbreak
This case history and lab methods
relate to Salmonella Enteritidis
Late September 1994: Minnesota Dept. of Health detects
increase in the number of Salmonella Enteritidis infections
in residents in southern Minnesota.
National Outbreak of Salmonellosis
outbreaks are typically
associated with direct
or indirect fecal
contamination of food
There are more then
one million cases of
year in the U.S.
Most infections involve
diarrhea (+/- blood and
In severe cases
bacteria may cross into
• Salmonella is found in GI tracts of
humans, animals and birds and
can survive long periods in the
• Salmonella Enteritidis (SE) is one
of over 2,500 different serotypes
• SE outbreaks are most frequently
associated with improperly
cooked eggs or chicken.
October 5 - 6: Fifteen patients having consumed
Schwan’s ice cream were at 10 fold greater increase in
risk of infection over controls.
Initial Case-Control Study
Starting with home
delivery of ice cream,
Schwan’s grew to be
the largest direct-to-
home food delivery
provider in the US.
Background on the Schwan’s Company
October 6: MN Dept. of Health informed the company
that 67 people had been infected with Salmonella
Enteritidis and a strong statistical link existed between
the patients and consumption of Schwan’s ice cream.
Immediately plant inspections commenced and product
and environmental testing was initiated.
Public Health Actions
The company immediately halted production & sale of
dozens of ice-cream products and initiated a nation-wide
The company initiated a paid advertising campaign asking
people not to eat Schwan's ice cream products.
The company initiated a free hot line and medical testing.
One week after the
isolated from ice
It was later
Culturing Ice Cream Products
Official FDA Culture Method for
Following that we will return with
the results of the outbreak
Milk Testing for Salmonella –
Turn around time:
4-6 days for negative
6-9 days for positive
Sample received from inspectors
Chain of Custody Form
Submission Form Signed and Dated
225mL of Lactose broth
Sterile disposable beaker
Aseptically add 25mL of sample
to 225mL of Lactose broth. Mix.
Let sit at room temperature for 1
hour. After 1 hour, adjust pH, if
necessary, to 6.8 ± 0.2 with
sterile 1 N NaOH or 1 N HCl.
Incubate Lactose broth
aerobically at 35-37°C for 22-26
After incubation, Transfer 0.1mL mixture to 10mL Rappaport-
Vassiliadis (RV) medium and another 1mL mixture to 10mL
tetrathionate (TT) broth. Vortex. Incubate RV medium 24 ± 2 h at
42 ± 0.2°C (circulating, thermostatically-controlled, water bath).
Incubate TT broth 24 ± 2 h at 43 ± 0.2°C (circulating,
thermostatically-controlled, water bath)
After incubation: Mix (vortex, if tube) and
streak 3 mm loopful (10µL) incubated TT
broth and RV broth onto separate bismuth
sulfite (BS) agar, XLD agar, and Hektoen
enteric (HE) agar. Incubate plates at 35-
37°C for 22-26 hours. Examine the plates
for the presence of suspect Salmonella
XLD agar: appear as pink colonies with
or without black centers
HE agar: appear as blue-green to blue
colonies with or without black centers
BS agar: appear as brown, gray, or black
colonies; sometimes they have a metallic
Typical Salmonella biochemical reactions
Salmonella serotyping confirmation
Tube agglutination to determine
Return to the results of the
• No defects were noted during inspection of the central
ice cream manufacturing plant in Marshall MN.
• Ice cream was manufactured from pasteurized premixes
purchased from two outside suppliers.
• The tanker trucks transporting the premix were also
used to carry non-pasteurized liquid eggs, oils, corn and
Investigation of Ice Cream Production
• SOPs called for cleaning and sanitizing after
transporting liquid eggs but documentation was absent.
• “To save time drivers could elect to bypass the cleaning
procedure after unloading eggs.” (NEJM: May 16, 1996)
• Samples from egg farms served by the trucking
company yielded the same phage type as the outbreak
Investigation of Trucking Company
• There is sufficient evidence to conclude the outbreak
resulted when pasteurized ice cream pre-mix was
contaminated during transport in tankers previously used
to haul raw liquid eggs.
• 41 of 48 states receiving the product reported cases.
• Based on an attack rate calculated in Minnesota victims
and the amount of ice cream shipped nationally,
researchers estimated that 224,000 people were infected.
• This represents the largest Salmonella Enteritidis
outbreak by a common vehicle recognized in the U.S.
Scope of the Outbreak
• In February 1995 a settlement covering some 100,000
people was reached.
• Individual compensation ranged from $80 up to $75,000,
depending on the severity of a victim's illness.
Company Changes in Processing
• Schwan’s contracted so that only dedicated tankers
hauled only pasteurized pre-mix.
• Schwan’s added facilities so that pre-mix could be re-
pasteurized upon arrival.
• Schwan’s increased QA testing and sensitivity.
• These actions served as education for other companies.
At the time the company’s actions (voluntary recall before
the product bacterial isolation, paid advertising warnings)
were viewed as unconventional, but have since been
imitated by other companies facing recalls.
Legacy of the Outbreak
This concludes the presentation on
the investigation, sampling and
analysis of Salmonella dairy