RAPID'Listeria spp.

RAPID'Listeria spp.

Catalog# Description
Dehydrated base
3564744 500 g

Supplements
3564745 Supplement 1 (Freeze dried, 10 vials)
3564746 Supplement 2 (Liquid, 10 vials)

FIELD OF APPLICATION
Selective chromogenic medium for identification and enumeration of Listeria spp. in human food products and
environmental samples.

PRINCIPLE
Identification of Listeria spp. using RAPID’Listeria spp. medium is based on the detection of β-D-glucosidase activity
by a chromogenic substrate. Listeria colonies are blue to bluish-green.
The medium is made selective by the combined action of lithium chloride and an antibiotic mixture.

VALIDATIONS
The RAPID’Listeria spp method has been certified for the detection of Listeria spp. by NF Validation and AOAC Research
Institute.

NF VALIDATION by AFNOR Certification:

Target Certificate references Scope Reference method Validation Protocol

All human food products and

Listeria NF EN ISO NF EN ISO
production environmental
spp 11290‐1/2017 16140-2 (2016)
BRD 07/12 – 12/06 samples

AOAC Performance Tested by AOAC Research Institute:

Target Reference method Scope Certificate references Validation protocol

USDA Microbiology
Stainless steel, plastic,
Laboratory 080701
Listeria spp. ceramic, and sealed AOAC-RI
Guidebook Chatper
concrete
8.11

STANDARDS
 ISO 11290-1:2017 - Microbiology of the food chain -- Horizontal method for the detection and enumeration of
Listeria monocytogenes and of Listeria spp. -- Part 1: Detection method
 ISO 11290-2:2017 - Microbiology of the food chain -- Horizontal method for the detection and enumeration of
Listeria monocytogenes and of Listeria spp. -- Part 2: Enumeration method
 USDA Microbiology Laboratory Guidebook, Chapter 8.11 Isolation and Identification of Listeria
monocytogenes from Meat, Poultry, Egg and Environmental Samples.

STORAGE / SHELF-LIFE / BATCH

- Dehydrated: + 15° to 25°C, in carefully sealed package, in a cool, dry and dark place
- Agar base in bottle, Supplement 1 & 2 and Pre-poured: + 2° to 8°C in a dark place
- Plate prepared from the dehydrated: 1 week at + 2-8°C, in carefully sealed package, in a dry and dark place
- Expiration date and batch number are shown on the package

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THEORETICAL FORMULA

Peptone 20 g Silica 20 g
Yeast extract 1g Growth activators 2g
Sodium pyruvate 2g Chromogenic mixture 75 mg
Iron ammonium citrate 0.5 g Antibiotic mixture 40 mg
Maltose 1g Agar 12 g
Sodium chloride (NaCl) 4g Distilled water 1000 ml
Lithium chloride (LiCl) 10.5 g
Final pH (25°C) = 7.0-7.5

PREPARATION OF COMPLETE MEDIUM

 Always shake bottle before use
 Dissolve 71.5 g of powder (3564744) in 950 L of distilled water and mix until a homogenous suspension is
obtained
 Sterilize in the autoclave at 121°C for 15 min. Cool to 47-50°C
 For 1 L of medium, aseptically add two flasks of supplement 1 (3564745), each reconstituted with 25 ml of sterile
water. Mix well
 For 1 L of medium, add two 5-ml flasks of supplement 2 (3564746). Mix well.
 Verify the pH (7 - 7.5)
 Pour into sterile Petri dishes

Note 1: Reconstitution concentration: 71.5 g /L. 500 g of powder makes 7 liters of medium

PRODUCTS AND MATERIALS REQUIRED (Not supplied and non-exhaustive list)

Equipment and material:
 Scales  Sterile Petri dishes (Ø 90 mm)  Water-bath
 Sterile weighing bags  Sterile pipettes  Incubators or incubation room
 Mill  Sterile spreaders  All usual laboratory equipment
 Stirrer-homogenizer  Sterile Pasteur pipette

Sample preparation:
Tryptone Salt diluent: Fraser ½ broth:
3555754 9 ml x 25 tubes 3555797 6 x 225 ml bottles
3555756 90 ml x 6 bottles 3555794 4 x 3 L bags
3564544 500 g 3564604 Dehydrated base 500 g
3555796 4 x 3 L bags 3564616 Supplement

Confirmation:
RAPID’L.mono agar: PALCAM: iQ-Check Kits:
3563694 20 dishes x 90 mm 3564754 Dehydrated 500g 3578113 Listeria spp.
3563964 120 dishes x 90 mm 3564752 Supplement
3555294 Ready-to-use pack
3564293 Dehydrated base 500 g
3564294 Supplement 1
3564746 Supplement 2

PROTOCOL
• Detection of Listeria spp.
 Preparation of samples
- Dilute n g or n ml of sample in 9 x n ml of Fraser ½ broth
- Incubate at 30 ± 1°C for 24 ± 2 hr

Note 2:
 After incubation, the enrichment broth may be stored at 2-8°C for 72 hr, before the inoculation
 In the context of NF Validation mark, no samples of over 25 g were tested

 Isolation and Incubation

- At the end of incubation, remove 0.1 ml of Fraser ½ broth using a sterile pipette and place drops on the outside edge
of half of the surface of the RAPID’Listeria spp. (diagram 1)
- Using a sterile swab, spread over half the surface by means of to-and-fro movements (diagram 2)

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- Using a sterile Pasteur pipette, isolate on the other half of the agar surface (diagram 3) by spreading the deposit in
relatively close streaks over the whole dish, starting from the edge of the previous spread
- Incubate the upturned dishes at 37 ± 1°C for 24 ± 2 hr

Starting point
Swab
Diagram 1: Diagram 2: Diagram 3:
Ending point

0.1 ml deposit Starting point

Ending point
Pasteur pipette
READING AND CONFIRMATIONS
• Reading
- Take a reading of the Listeria spp. after 24 ± 2 hr of incubation. Listeria spp. appear as characteristic blue to bluish
green colonies on RAPID’Listeria spp..

Note 3:
 It is possible to take the reading up until 48 hr of incubation of the RAPID’Listeria spp. agar
 After incubation, RAPID’Listeria spp. agar plates may be kept in the cold 5 ± 3°C for 72 hr before reading and
confirmation if applicable
 If characteristic colonies are present on RAPID’Listeria spp., confirmation and identification of the species
involved can be performed

• Confirmation
In the context of NF Validation mark, perform confirmation on at least one characteristic colony.

- Using the conventional tests described in the standardised ISO reference methods (with purification step)
- Using nucleic probes as described in ISO 7218 standard, using isolated colonies (with or without purification step), for
example: iQ-Check® Listeria spp PCR method (3578113)
- Repicking and spotting of at least one colony isolated from RAPID’Listeria spp. onto RAPID’L.mono agar, together
with a Gram test and a catalase test. Up to 15 colonies can be confirmed on a single plate of RAPID’L.mono agar
- Repicking and spotting at least one isolated colony on a PALCAM agar plate. Up to 15 colonies can be confirmed on
a single PALCAM agar plate
- Using any other NF Validation certified method based on a different principle from that of RAPID’Listeria spp. The
validated second method protocol should be respected in its entirety, i.e. all stages preceding the intermediary stage
from which confirmation is sought must be common to the two methods.

Note 4:
 In the event of discrepant results (positive using the RAPID’Listeria spp. method but unconfirmed by one of the
methods described above), the laboratory shall use whatever means are necessary to ensure that the result
delivered is valid

 Expression of results/Calculations
Please refer to ISO 11290-1 and 2, and ISO 7218 standards

PRECAUTIONS
 Usual precautions for handling potentially contaminated products in a microbiology laboratory must be observed
 As part of the NF validation, the enumeration of Listeria spp. has not been tested
 Some Bacillus pumilus strains can give non typical dark blue colonies
 Before using RAPID’Listeria spp. plates, allow drying at 25-50°C according to ISO standard 7218, until droplets
disappear from the surface of the medium. However, avoid prolonged drying so as not to modify the efficiency of the
medium.
 Respect Good Laboratory Practices (EN ISO 7218)
 See SDS for Product Safety Information, www.bio-rad.com

TECHNICAL SUPPORT IN THE UNITED STATES

In the United States, for technical assistance please call (800) 4BIORAD. Select option 2 for technical support. To
place an order, please call (800) 4BIORAD and press option 1 for customer care.

QUALITY CONTROL
Every product manufactured and marketed by Bio-Rad is subject to a quality assurance procedure at all stages, from
reception of raw materials through to marketing of the finished products. Each batch of finished product undergoes

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 RAPID'Listeria spp.
quality control and is marketed only if it satisfies the acceptability criteria. Documentation relative to the production and
quality control of each batch is kept on file.

QUALITY AND PERFORMANCE OF THE TEST

See quality certificate available on www.bio-rad.com/certificate (Catalog#/ref# and Lot# number are required)

KEY WORDS
RAPID’Listeria spp. / Listeria / Listeria monocytogenes / Detection / Enumeration / Food products / Environment /
Fraser / Glucosidase / Chromogenic / Medium

BIBLIOGRAPHY
• OTTAVIANI F., OTTAVIANI M., AGOSTI M. (1997b): Differential agar medium for Listeria monocytogenes. Quimper

Froid Symposium Proceedings p6. ADRIA Quimper France. 16-18 June 1997.

For more information about Bio-Rad Food Testing products, visit our website: www.bio-rad.com/foodscience

© 2018 Bio-Rad Laboratories, Inc. Page 4 of 4 Bulletin 7244EN Rev 2 01-July-2019