256620

EE Broth Mossel Enrichment
Intended Use
EE Broth Mossel Enrichment is used for selectively enriching and
detecting Enterobacteriaceae, particularly from foods.
Meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)1-3 performance
specifications, where applicable.
Summary and Explanation

EE Broth Mossel Enrichment is prepared according to the formula of Mossel, Visser and Cornelissen.4 The formula contains
dextrose to facilitate growth of most Enterobacteriaceae, thus
ensuring the detection of Salmonella and other lactose-negative
organisms. EE Broth Mossel Enrichment should be used as an
enrichment broth, followed by a selective medium; e.g., Violet
Red Bile Agar.
The enumeration of Enterobacteriaceae is of great concern in
monitoring the sanitary condition of food. Enterobacteriaceae
can be injured in food-processing procedures, which include
exposure to low temperatures, sub-marginal heat, drying, radiation, preservatives or sanitizers.5 Recovery relies on proper resuscitation of damaged cells. EE Broth Mossel Enrichment is used
to detect and enumerate Enterobacteriaceae found per milliliter
or per gram of test sample of food when performing the Most
Probable Number (MPN) technique with pre-enrichment.6,7
EE Broth Mossel Enrichment is listed in the USP as one of the
recommended media for the isolation of bile-tolerant gramnegative bacteria from nonsterile pharmaceutical products.1
Principles of the Procedure

Peptones provide nitrogen, vitamins and amino acids. Dextrose
is a carbon source. Disodium phosphate and monopotassium
phosphate are buffering agents. Brilliant green and oxgall are
selective agents.
Directions for Preparation from

Dehydrated Product
1. Suspend 45 g of the powder in 1 L of purified water.
2. Heat with frequent agitation until dissolved. DO NOT
OVERHEAT. Media is heat sensitive.
3. Dispense into tubes or bottles as required.
4. Heat at 100C in water bath or flowing steam for 30 minutes.
DO NOT AUTOCLAVE.
5. Test samples of the finished product for performance using
stable, typical control cultures.
Sample Collection and Handling

For food samples, follow appropriate standard methods for
details on sample collection and preparation according to sample
type and geographic location.6,7
For pharmaceutical samples, refer to the USP for details on sample collection and preparation for testing of nonsterile products.1
Procedure
For food samples, refer to appropriate standard references for
details on test methods for performing MPN technique with
enrichment using EE Broth Mossel Enrichment.6,7
For pharmaceutical samples, refer to USP General Chapter <62>
for details on the examination of nonsterile products and tests for
isolating Enterobacteriaceae using EE Broth Mossel Enrichment.1
Expected Results
Acid production causes the color of EE Broth Mossel Enrichment to become yellow. A negative reaction results in no color
change and the medium remains green.
Formulae
Difco EE Broth Mossel Enrichment
Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 10.0 g
Dextrose...................................................................... 5.0 g
Disodium Phosphate.................................................... 8.0 g
Monopotassium Phosphate.......................................... 2.0 g
Brilliant Green............................................................ 15.0 mg
Oxgall........................................................................ 20.0 g
BBL EE Broth Mossel Enrichment

Approximate Formula* Per Liter
Pancreatic Digest of Gelatin....................................... 10.0 g
Dextrose...................................................................... 5.0 g
Oxgall........................................................................ 20.0 g
Disodium Phosphate.................................................... 8.0 g
Monopotassium Phosphate.......................................... 2.0 g
Brilliant Green............................................................ 15.0 mg

*Adjusted and/or supplemented as required to meet performance criteria.
Difco & BBL Manual, 2nd Edition
Uninoculated
Tube
Escherichia coli
ATCC 25922
User Quality Control
NOTE: Differences in the Identity Specifications and Cultural Response testing for media offered as both Difco and BBL brands may reflect differences in the
development and testing of media for industrial and clinical applications, per the referenced publications.
Identity Specifications
Identity Specifications
Dehydrated Appearance: Light green, free flowing, homogeneous.
Dehydrated Appearance: Fine, homogeneous and free of extraneous

material.
Solution:
4.5% solution, soluble in purified water. Solution is emerald green, clear.
Prepared Appearance:
Emerald green, clear.
Reaction of 4.5%
Solution at 25C:
7.2 0.2
Solution:
4.5% solution, soluble in purified water. Solution

is medium to dark green with or without a tint
of yellow or blue; clear to slightly hazy.
Prepared Appearance:
Medium to dark green with or without a tint of

yellow or blue; clear to slightly hazy.
Reaction of 4.5%
Solution at 25C:
Cultural Response
Prepare the medium per label directions. Inoculate 9 mL tubes and incubate at 35 2C for 18-24 hours and 48 hours, if necessary.
pH 7.2 0.2
BBL EE Broth Mossel Enrichment (prepared)
Appearance:
Medium to dark green and clear to trace hazy.
Reaction at 25C:
pH 7.2 0.2
ORGANISM
ATCC
INOCULUM CFU
RECOVERY
ACID
Enterobacter
aerogenes
13048
30-100
Good
+ (yellow)
Escherichia coli
25922
30-100
Good
+ (yellow)
Shigella boydii
12030
30-100
Good
Prepare the medium per label directions. Inoculate 10 mL tubes and

incubate at 35 2C for 18-24 hours and 48 hours, if necessary.
Marked to
complete inhibition
ORGANISM
ATCC INOCULUM CFU RECOVERY
Escherichia coli
25922
103-104
Good
+ (yellow)
Pseudomonas
aeruginosa
10145
103-104
Good
Salmonella enterica
subsp. enterica
serotype Typhimurium 14028
103-104
Good
Staphylococcus
aureus
25923
30-100
Cultural Response
Inoculate 100 mL bottles and incubate at 30-35C for 18-24 hours and
48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli ATCC
8739 and incubate at 35-37C for 18-48 hours.

INOCULUM INCUBATION INCUBATION
CFU
TEMP TIME (HOURS) RECOVERY
ORGANISM
ATCC
Escherichia coli 8739
<100
30-35C
24
Growth
<100
35-37C
18-48
Growth
Pseudomonas
aeruginosa
9027
<100
30-35C
24
Growth
Staphylococcus
aureus
6538
>100
30-35C
48
No growth
Shigella sonnei
9290
103-104
Good

ACID
+ (yellow)
to reduced
(yellow green)
Inoculate 100 mL bottles and incubate at 30-35C for 18-24 hours and
48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli
ATCC 8739 and incubate at 35-37C for 18-48 hours.

INOCULUM INCUBATION INCUBATION
CFU
TEMP TIME (HOURS) RECOVERY
ORGANISM
ATCC
<100
30-35C
24
Growth
<100
35-37C
18-48
Growth
Pseudomonas
aeruginosa
9027
<100
30-35C
24
Growth
Staphylococcus
aureus
6538
>100
30-35C
48
No growth
BBL EE Broth Mossel Enrichment (prepared)

Inoculate 90 mL bottles and incubate as directed below.

ORGANISM
ATCC
INOCULUM
CFU
INCUBATION
TEMP
INCUBATION
TIME (HOURS)
RECOVERY
ACID
Enterobacter aerogenes
13048
102-103
35-37C
18-48
Growth
+ (yellow)
Escherichia coli
25922
102-103
35-37C
18-48
Growth
+ (yellow)
Salmonella enterica
subsp. enterica
serotype Typhimurium
13311
102-103
35-37C
18-48
Growth
+ (yellow)
Escherichia coli
8739
10-100
30-35C
18-24
Growth
+ (yellow) to or weak
Pseudomonas aeruginosa
9027
10-100
30-35C
18-24
Growth
N/A
Staphylococcus aureus
6538
102-103
30-35C
48
No growth
References
1. United States Pharmacopeial Convention, Inc. 2008. The United States pharmacopeia 31/The national
formulary 26, Supp. 1, 8-1-08, online. United States Pharmacopeial Convention, Inc., Rockville, Md.
2. European Directorate for the Quality of Medicines and Healthcare. 2008. The European pharmacopoeia, 6th ed., Supp. 1, 4-1-2008, online. European Directorate for the Quality of Medicines and
Healthcare, Council of Europe, 226 Avenue de Colmar BP907-, F-67029 Strasbourg Cedex 1, France.
3. Japanese Ministry of Health, Labour and Welfare. 2006. The Japanese pharmacopoeia, 15th ed.,
online. Japanese Ministry of Health, Labour and Welfare.
4. Mossel, Vissar and Cornelisen. 1963. J. Appl. Bacteriol. 26:444.
5. Hartman and Minnich. 1981. J. Food Prot. 44:385.
6. International Organization for Standardization. 2004 Microbiology of food and animal feeding
stuffs horizontal methods for the detection and enumeration of Enterobacteriaceae Part 1: Detection and enumeration by MPN technique with pre-enrichment. ISO 21528-1, 1st ed., 2004-08-15.
International Organization for Standardization, Geneva, Switzerland.
7. Downes and Ito (ed.). 2001. Compendium of methods for the microbiological examination of foods,
4th ed. American Public Health Association, Washington, D.C.
Availability
COMPF EP ISO JP USP
Cat. No. 256620 Dehydrated 500 g

COMPF EP ISO JP USP
Cat. No. 297005 Dehydrated 500 g

292627 Prepared Bottles, 90 mL (wide mouth)

Pkg. of 10
QC testing performed according to USP/EP/JP performance specifications.

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EE Broth Mossel Enrichment

Summary and Explanation

Principles of the Procedure

Directions for Preparation from

Sample Collection and Handling

BBL EE Broth Mossel Enrichment

*Adjusted and/or supplemented as required to meet performance criteria.

Difco & BBL Manual, 2nd Edition

User Quality Control

Difco EE Broth Mossel Enrichment

BBL EE Broth Mossel Enrichment

Dehydrated Appearance: Light green, free flowing, homogeneous.

Dehydrated Appearance: Fine, homogeneous and free of extraneous

4.5% solution, soluble in purified water. Solution is emerald green, clear.

Emerald green, clear.

4.5% solution, soluble in purified water. Solution

Medium to dark green with or without a tint of

BBL EE Broth Mossel Enrichment (prepared)

Medium to dark green and clear to trace hazy.

Prepare the medium per label directions. Inoculate 10 mL tubes and

ATCC INOCULUM CFU RECOVERY

Escherichia coli 8739

Escherichia coli 8739

Escherichia coli 8739

Escherichia coli 8739

BBL EE Broth Mossel Enrichment (prepared)

Difco & BBL Manual, 2nd Edition

Difco & BBL Manual, 2nd Edition

Cat. No. 256620 Dehydrated 500 g

BBL EE Broth Mossel Enrichment

Cat. No. 297005 Dehydrated 500 g

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