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LISTERIA IDENTIFICATION SYSTEM 12L

CODE: MB1128

Intended use
The Microbact™ Listeria 12L system is intended to be used for the identification of Listeria spp. isolated from clinical, food and food related samples.
The Microbact™ Listeria 12L system is a standardised micro substrate system designed to simulate conventional biochemical substrates used for the identification of Listeria spp ^1,2. Each identification strip consists of 12 tests, (11 sugar utilisation tests plus a rapid haemolysis test). The reactions occurring during the incubation period are demonstrated through either a colour change in the sugar utilisation tests or in the lysis of sheep red blood cells in the haemolysis test.
The Microbact™ Listeria 12L may be used as a rapid 4 hour incubation test when an inoculum equivalent to a MacFarland 0.5 Standard is prepared, or as an 18-24 hour incubation test when inoculated from a single colony.
After incubation, the reactions are read visually and interpreted using the Microbact™ Computer Aided Identification Package.

Principle of test
Organism identification is based on pH change and substrate utilisation as established by published reference methodologies ^1,2. Refer to the table of reactions below for the substrates in each well, specific reaction principles and colour changes. Tests can be read at 4 and 24 hours.

The following species can be identified using the Microbact™ Listeria 12L System

Warnings and precutions
1. These strips are intended for in vitro use only; for use only by qualified laboratory personnel using aseptic techniques and established precautions against microbiological hazards.
Qualified laboratory personnel shall:
- be employed to work in a laboratory capable of handling Category 2 pathogens
- understand basic microbiological procedures including sterile and aseptic techniques
- be trained on handling the strips, preparing them and reading the results. The level of academic qualification is not paramount as the kit is designed to be simple to use.
2. Used materials must be autoclaved, incinerated or immersed in germicide before disposal.
3. DO NOT incubate 12L strips in a CO2 incubator as as substrates and/or enzyme reactions could be adversely affected, giving false reactions.
4. DO NOT use the Haemolysin Reagent if it appears to be contaminated (very dark or haemolysed).

Storage instructions
Store at 2–8°C. The test strips are stable until the expiry date specified if kept unopened in the foil envelopes at this temperature.
Once opened the Haemolysin Reagent may be used, using aseptic technique, until the expiry date shown on the bottle.

Kit Components:
1 x Holding Tray
20 x Report Forms
20 x Test strips
20 x Suspending Media

Materials required but not provided:
Haemolysin Reagent - MB1249A
MicrobactTM Computer Aided Identification Package.
Inoculating Loop.
Incubator (35°C ± 2°C).
Sterile Pipettes.

Set up proceedure
This test may be performed on colonies isolated from a wide range of selective and non-selective media.

Preparation of the inoculum
Before testing, isolates should be checked to ensure that they belong to the genus Listeria (Oxoid Listeria Test Kit, motile at 25°C, non-motile at 37°C, catalase-positive, oxidase-negative, short Gram-positive bacilli)
1. 4 Hour test
Pick 4-5 colonies from an 18-24 hour culture and emulsify in suspending medium. Mix thoroughly to prepare a homogeneous suspension.
2. 18- 24 Hour test
Pick a single isolated colony from an 18-24 hour culture and emulsify in suspending medium. Mix thoroughly to prepare a homogeneous suspension.
Inoculation
1. Remove a test strip from its foil pouch and place in the holding tray provided.
2. Warm the haemolysin reagent to room temperature.
3. Remove the lid from the test strip.
4. Using a sterile Pasteur pipette place 4 drops (approximately 100µl) of the bacterial suspension into each well.
5. Add 1 drop of haemolysin reagent to well number 12.
6. Replace the lid.
7. Place 1 drop of the inoculum onto an appropriate non-selective medium for a purity check. Incubate at 35°C ± 2°C for 24 hours. Check for purity.
Incubation
Incubate inoculated strip(s) at 35°C ± 2°C for 4 hours or 18-24 hours depending on the inoculum prepared. (See section entitled Preparation of the Inoculum).
Note: If reactions cannot be interpreted with confidence after 4 hours incubation, replace the strip(s) in the incubator. Reactions may be read after further incubation.
Reading of the test strip(s)
Remove the strip(s) from the incubator.
Remove the lid.
Record all test results onto the report form. For assistance in interpreting the results refer to the Table of Reactions.
Interpretation
Compare the test reactions obtained with the predicted results in the Data Table provided.
Example:

Microbact™ code = 5657

Computer aided identification package
A four digit octal code has been adopted for the Microbact™ Listeria 12L, each group of 3 reactions producing a single digit of the four digit code. Using the results obtained, the indices of the positive reactions are circled. The sum of these indices in each group of three reactions forms the code number.
The Microbact™ Computer Aided Identification Package or the Listeria Profile Register provided may then be consulted for the identification choices.

Quality control
The overall performance of the system should be monitored by testing appropriate control strains. The following organisms are recommended for independent laboratory assessment.

Limitations
1. The Microbact™ Listeria 12L, Listeria identification system is designed to identify bacteria belonging to the genus Listeria. It cannot be used to identify organisms belonging to other genera.
2. Specimens or samples may contain a mixture of species. This may result in mixed cultures being used to prepare the inoculum when 4 hour identifications are attempted. If the results achieved using this heavy inoculum do not produce a reaction pattern clearly representative of a single species:
Check the purity plate. A horse blood agar plate may be of assistance.
Check catalase (+), oxidase (- ) and Gram morphology (Gram-positive bacillus).
Repeat the test using a single colony.
Note: All Listeria spp. should be positive for esculin, arabitol and trehalose.

Performance characteristics
In house studies have shown that identification to the species level can be achieved with 92% of isolates after 4 hours incubation and 100% of isolates after 24 hours incubation when results are interpreted using the Computer Aided Identification Package. (see below)

Compiled data from various studies³ :

(a) One strain identified as Listeria ivanovii by genotypic methods yielded a biochemical profile for Listeria seeligeri using biochemical tests including another commercial identification kit and traditional biochemical tests.
(b) Two strains deposited as Listeria grayi yielded biochemical profiles for Listeria innocua using the 12L and another commercial biochemical identification system.

REPRODUCIBILITY CHARACTERISTICS (Data on file at Oxoid):

Over a 14 month period 10 batches of Microbact™ 12L strips were tested for reproducibility using 6 Listeria species. As the table shows, identification both within and between batches was 100% reproducible for all Listeria species.

References
1. USDA, FSIS Method for the isolation and identification of Listeria monocytogenes, from processed meat and poultry products.
2. AS 1766.1.15(INT) 1991, TC 34/SC5 N307, FDA Bacteriological Analytical Manual, 7th Edn (1992) 141 162.
3. CCFRA report on file at Oxoid Ltd.