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.

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
Difco™ EE Broth Mossel Enrichment
Dehydrated Appearance: Light green, free flowing, homogeneous.
Solution:                       4.5% solution, soluble in purified water. Solution
                                   is emerald green, clear.
Prepared Appearance:    Emerald green, clear.
Reaction of 4.5%
Solution at 25°C:           7.2 ± 0.2
Cultural Response
Difco™ EE Broth Mossel Enrichment
Prepare the medium per label directions. Inoculate 9 mL tubes and incubate at 35 ± 2°C for 18-24 hours and 48 hours, if necessary.

Inoculate 100 mL bottles and incubate at 30-35°C for 18-24 hours and 48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli ATCC 8739 and incubate at 35-37°C for 18-48 hours.

Identity Specifications
BBL™ EE Broth Mossel Enrichment
Dehydrated Appearance: Fine, homogeneous and free of extraneous material.
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 25°C:            pH 7.2 ± 0.2
BBL™ EE Broth Mossel Enrichment (prepared)
Appearance:                 Medium to dark green and clear to trace hazy.
Reaction at 25°C:          pH 7.2 ± 0.2
Cultural Response
BBL™ EE Broth Mossel Enrichment
Prepare the medium per label directions. Inoculate 10 mL tubes and incubate at 35 ± 2°C for 18-24 hours and 48 hours, if necessary.

Inoculate 100 mL bottles and incubate at 30-35°C for 18-24 hours and 48 hours, if necessary. Inoculate a 20 mL tube with Escherichia coli ATCC 8739 and incubate at 35-37°C for 18-48 hours.

BBL™ EE Broth Mossel Enrichment (prepared)
Inoculate 90 mL bottles and incubate as directed below.

Formula

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
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.

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 100°C 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 samplecollection 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.

† QC testing performed according to USP/EP/JP performance specifications.

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