Research Note

Evaluation of a Modified Buffered Peptone Water Enrichment


For use with the VIP for EHEC in Ground Beef
Linda Mui, Andrew Lienau, Stephanie Leung and Robin Forgey
BioControl Systems, Inc. , 12822 SE 32nd Street, Bellevue, WA 98005

Abstract

A rapid method to detect EHEC in foods using the VIP for EHEC was previously reported ( 1,2).
This method, which has been approved as an AOAC Official Method consists of an overgnight (18-28 hours)
enrichment protocol using Modified Trypticase Soy Broth (mTSB) and Novobiocin. The ground beef industry has
need for a faster preliminary screening enrichment protocol because of short shelf life and an accelerated
distribution network found in the industry. Reported herein is an accelerated, 8 hour enrichment protocol with a
detection limit of <1 CFU/g of E.coli O157:H7. This enrichment protocol represent a significant departure from
the Official Method but affords the benefits of rapid determinations for time sensitive samples. Ground beef
samples were inocalated with E.coli O157:H7 isolates at three different levels, low medium and high. The
inoculated samples were enriched in modiffed Buffered Peptone Water (3.4) supplemented with acriflavin,
cefsoludin and vancomycin (mBPW + acv) for eight hours at 42 ºC. After incubation 0.1 ml of each enriched
sample was placed into the VIP unit and results were recorded after 10 minutes. A total of 65 samples were tested
and confirmed with complete correlation between the VIP for EHEC using the accelerated, 8 hour enrichment and
the reference cultural method.

The AOAC Official Method for detection of E.coli
O157:H7 using the VIP for EHEC consists of an 18-28h
enrichment in mTSB and Novobiocin.
The ojective of the study was to evaluate the effectiveness
of a reduced enrichment time on the ability to detect E.coli
O157: H7 in ground beef using the Vip for EHEC. The
USDA cultural method (5) was chosen as the reference
methodology.
Materials and Methods
Inoculation of Samples. A total of 65 ground beef samples
were tested, 60 inoculated and 5 uninoculated control
samples. The ground beef was purchased at the wholesale
level. The samples were inoculated with E.coli O157:H7
that had previously been isolated from ground beef. The
culture was enriched overnight in Brain Heart infusion at
35 ºC. Serial dilutions of the overnight enrichment were
made in Butterfield´s Buffered Phosphate Diluent and the
appropriate volume was mixed into the ground beef. The
beef was inoculated at three different levels, low medium
and high. Twenty samples were inoculated for each level.
The low level was designed to approximate 1-10 cfu/25g,
the medium level to approximate 10-75 cfu/25g. and the
high level to approximate greater than 100 cfu/25g. The
inoculated beef was refrigerated overnight prior to running
the samples.
Contamination levels were determined by running MPNs
using the United States Department of Agriculture
(USDA) cultural methods as outlined in Figure1. For the
VIP for EHEC, inoculated samples were enriched by
weighing a 25g sample into 225ml of prewarmed
mBPW+acv.
Sample were incubated at 42 ºC without shaking for
eight hours after mixing. Positive and negative media
controls were also included. After incubation, 0,1 ml of
each enriched sample was placed into the VIP unit
according to the manufacturer´s directions for use. Results
were recorded after ten minutes.
Confirmation of Samples. Both positive and negative
samples were confirmed via the USDA cultural method.
Serial dilutions of the enriched samples were spread plated
on MacConkey Sorbitol Agar with BCIG (MSA/BCIG)
and incubated at 42 ºC overnight. Typical colonies were
selected from each sample and tranferred to Eosin
Methylene Blue Agar (EMB) and Phenol Red Sorbitol
MUG Agar (PRS/MUG). Colonies that displayed the
typical reactions on these two agars were then identifield
using the API 20E.
(BioMerieux) and serotyped for O157:H7 agglutination
(Difco).
Results and Discussion, Results are summarized in Table
1. In the low level, the VIP identified seventeen
presumptive positive samples and three negative samples,
all of which verified by plating and cultural confirmation.
The three negative samples did not exhibit O157 colonies
on isolation agars, in the medium and high levels, all
samples were positive the VIP and confirmed by plating.
There were no false positive or false negative samples.
The VIP for EHEC and the cultural method produced the
same results on each sample.
5- Okrend, A,. and B.E. Rose. 1989. USDA-FSIS
Laboratory Comunication No. 38, Revision 3,
U.S. Department of Agriculture, Food Safety and
inspection Service, Wasghington DC.
Fig. 1. USDA Method for E.coli O157:H7
Add Sample to modified EC Broth
with Novobicion
incubate 20-24h at 35 ºC

Table 1.
Number of positive samples on VIP for EHEC using 8-
hour Enrichment Protocol
Inoculation MPN/g Positive Confirmed
Level by VIP Positivo
Uninoculated <0.003 0 0
Spread plate serial dilutions
Low 0.38 17 17
Onto MSA/B-CIG
Medium 2.4 20 20 Incubate 18-24h at 42 ºC
High >11 20 20

The VIP for EHEC has been previously studied using a

different enrichment protocol that requires an 18-hour
enrichment (1,2). Based on these studies, the VIP for
EHEC was granted AOAC Official Method status
(methods 996.09). The 8-hour enrichment protocol Pick at least 12 Typical
mentioned above is a significant deviation from the Colonies to EMB & PRS/MUG
AOAC Official Method, and therefore, is not considered
Incubate 18-24h at 35 ºC
an Official Method. Howerer, it may be useful as a
Modified Buffered Peptone (mBPW)
preliminary screening procedure for time sensintive
samples where immediate determinations are necessary.
The data from this study indicates that the 8-hour
enrichment may detect E.coli O157:H7 in ground beef

References
Run biochemicals
1- Feldsine, P.T Falbo-Nelson , M.T., S. Brunelle Using API20E
and Forgey, R.L, 1997. Visual.
Immunoprecipitase Assay (VIP) for Detection
Enterohemohagic Escherichia coli (EHEC)
O157:H7 in Selected Foods: Collaborative Study.
J. AOAC Int´l. 80 517-529.
2- Feldsine, P.T Forgey, R.L. Falbo-Nelson, M.T.
and S.Brunelle. 1997. Escherichia coli O157:H7
Visual Immunoprecipitate Assay: A Comparative
Validation Study. J. AOAC Int´l. 80:43:83.
3- Restaino, L 1996. Accentuate the positive. Food
Quality 2.68-70
4- Restaino, L, Castillo, H.J.; Stewart, D., and M.L.
Tortorelio. 1996. Antibody direct apiflourescent
filter technique and immunomagnetic separation
for 10h screening and 24h confirmation of
Escherichia coli O157:H7 in beef. J. Food Safety
Prot. 59:1072-1075.
 Modified Buffered Peptone (mBPW)

Peptone 10g
NaCI 5g
Na2 PO4 3.6g
KH2PO4 1.5g
Casamino Acids 5g
Yeast Extarct 6g
Lactose 10g
Distilled water* 1000ml

* pH 7.2 ± 0.2 prior to addition of the above ingredients

Add all of the above ingredients to one liter of purified water. Mix well to dissolve.
Dispense in the desired aliquots and autoclave at 121 ºC for 15 minutes. After cooling,
add antibiotic stock solutions in the following amounts per liter of media:

Acriflavin 0.1ml
Cefsulodin 0.1ml
Vancomycin 0.08ml

Preparation of Antibiotic Stock Solutions:

Prepare a stock solution of each antibiotic (acriflavin, cefsulodin and vancomycin) by dissolving
1000mg of each antibiotic in a separate tube containing 10ml of distilled water. Filter sterilize the
solution can be kept for several months in foil wrapped tubes at 4 ºC.