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Code: ID0600

A rapid colorimetric test for the differentiation of Listeria monocytogenese from other Listeria species.

INTENDED USE
The Oxoid Biochemical Identification System (O.B.I.S.) mono is a rapid colourimetric test for the determination of D-alanyl aminopeptidase (DALAase). It has been designed for the differentiation of presumptive Listeria species that have been isolated from selective media and plated on to a secondary medium for further biochemical testing.

PRINCIPLE OF THE TEST
The O.B.I.S. mono test offers a rapid screening method for differentiation of Listeria monocytogenes from other Listeria species. This reduces the need for full biochemical identification of all suspect colonies.

Listeria species, with the exception of Listeria monocytogenes, possess the enzyme D-alanyl aminopeptidase^1,2,3.  Oxoid has developed a new system for aminopeptidase testing which uses a non-carcinogenic substrate. This is in response to health concerns associated with amino acid conjugates of ß naphthylamine^4,5 as these are potent carcinogens⁶.

D-alanyl-7-amido-4-methylcoumarin (DALA) is provided as a suspension. An acidic solution of dimethylaminocinnamaldehyde is used as a colour developer. If the substrate is hydrolysed by DALAase, free 7-amino-4-methylcoumarin (7AMC) combines with the developer to produce a purple Schiff’s base⁷.

COMPONENTS OF THE O.B.I.S mono KIT (ID0600M)
Each O.B.I.S mono kit contains the following reagents with enough material for 60 tests:

ID090 O.B.I.S. Reaction Sleeves:-  One pouch containing 30 plastic bags (76mm x 102mm).
ID120 O.B.I.S. mono Buffer:-  One white capped bottle containing 4.5ml of a 0.5% w/v D-alanyl-7-amido-4-methylcoumarin suspension in distilled water.
ID220 O.B.I.S. mono Developing Solution:- One purple capped bottle containing 4.5ml of a 0.5% w/v dimethylamino-cinnamaldehyde in 1M hydrochloric acid.
ID601M O.B.I.S. mono Test Cards:- One pouch containing 10 cards. There are six reaction areas per card, labelled ‘DALA’.

MATERIALS REQUIRED BUT NOT INCLUDED

Sterile plastic disposable inoculating loops
Positive and negative quality control organisms
37 ± 2ºC incubator

PRECAUTIONS
This product is for in vitro diagnostic use.
Do not use O.B.I.S. mono reagents beyond the stated expiry date.
Specimen material may contain pathogenic organisms, handle with appropriate precautions.
The O.B.I.S mono Developing Solution contains acid. Wear suitable personal protective equipment. If the reagents come in to contact with the skin, mucous membranes or eyes, immediately flush the area with water.
Used O.B.I.S. Test Cards and inoculating loops should be disposed of as biohazardous waste. This should be incinerated, or autoclaved at 121°C for at least 15 minutes.

STORAGE AND OPENING
The O.B.I.S. mono kit must be stored at 2°C to 8°C. Allow the pouches to reach room temperature before use to prevent the formation of condensation on the test cards. Open the pouches by cutting at the notch between the end seal and the clip-lock opening.
Remove the number of Test Cards required and reseal the pouch. If fewer tests are required than the number on the Test Card, cut the card and return the unused portion to the pouch. Do not return used Test Cards to the pouch.

When stored as described, O.B.I.S. mono reagents will retain their activity until the expiry date shown on the box.

QUALITY CONTROL PROCEDURE
Each day the kit is used the following procedure should be performed:

Positive control – Use a known DALAase positive strain such as Listeria innocua ATCC® 33090. Follow the method given in the test procedure. Ensure that a purple colour forms within 20 seconds.

Negative control – Use a known DALAase negative strain such as Listeria monocytogenes ATCC® 7644. Follow the method given in the test procedure. Ensure that no purple colour forms within 20 seconds.

SPECIMENS
The test is designed for use from purity plates, not from primary isolation media, as the colonies on primary isolation media are too small to carry out an effective test.

Pick colonies which have typical Listeria morphology from selective Listeria isolation media such as Oxford Agar (CM0856), PALCAM Agar (CM0877) and chromogenic Listeria media, and streak onto a purity plate. O.B.I.S. mono tests can be performed from purity plates recommended by international standards, such as Tryptone Soya Agar (CM0131), Tryptone Soya Yeast Extract Agar (TSA-YE)^8,9 or a recognised chromogenic Listeria medium⁸.

TEST PROCEDURE AND INTERPRETATION OF RESULTS
Important notes:
Before use shake the ID120 O.B.I.S. mono Buffer (white capped bottle) well to suspend the reagent.
Ensure that the culture to be tested is a Gram-positive, catalase-positive, oxidase-negative bacterium.

1. Remove one of the O.B.I.S. mono Test Cards (ID0601M) from the pack.
2. Using a plastic loop, take colonial material equivalent to five colonies (each greater than 1 mm in diameter) from the purity plate.
3. Touch the sample to the reaction zone on the O.B.I.S. mono Test Card. Drag the loop across the diameter of the reaction zone. 
4. Shake the ID120 O.B.I.S. mono Buffer well and dispense one drop on to the inoculated reaction zone. 
5. Place the Test Card in a plastic Reaction Sleeve (ID090), so that the reaction zones are within the sleeve and the portion of the Test card with the Oxoid logo is outside the sleeve. 
6. Place the Test Card and Reaction Sleeve into a 37 ± 2ºC incubator for 10 minutes.
7. Remove from incubator. Dispense one drop of ID220 O.B.I.S. mono Developing Solution onto each of the inoculated reaction zones. 
8. The appearance of a purple colour within 20 seconds, is a positive D alanyl aminopeptidase reaction. A positive reaction indicates the organism is not a Listeria monocytogenes.
   If no colour develops within 20 seconds this is a negative reaction, and indicates the organism is a presumptive Listeria monocytogenes.

LIMITATIONS OF THE TEST
O.B.I.S. mono is intended for the detection of DALAase in Gram-positive, catalase-positive, oxidase-negative short rod shaped bacteria, capable of growing on selective Listeria primary isolation media. It can be used as a screen to differentiate Listeria monocytogenes from other Listeria.
Occasionally aesculin positive Bacillus species may grow on Listeria isolation media. Bacilli are DALAase positive, but the colonies are different from Listeria and bacilli appear as large rods upon Gram staining.

Due to the small size of Listeria colonies on primary isolation media it is not possible to carry out the test directly from these plates. Use of multiple colonies from the primary isolation plate is not reccomended as this may lead to a mixed culture and an incorrect result. International standards recommend sub-culturing presumptive Listeria species on to purity plates TSA (CM131), TSA-YE ^8,9 or a recognised chromogenic Listeria medium⁸.

Occasionally certain Listeria when grown on non-selective media form colonies that are difficult to remove with an inoculating loop. Ensure that there is sufficient colonial material on the inoculating loop before inoculating the reaction area. Failure to pick up sufficient material on the loop will result in a negative DALA test.

O.B.I.S. mono provides presumptive identification of Listeria monocytogenes, but does not replace full biochemical testing.

REFERENCES
1. Kämpfer, P., Böttcher, S., Dott, W., Rüden, H. (1991) Physiological characterization and identification of Listeria species. Zentralblatt für Bakteriologie 275, 423 435.
2. Kämpfer, P. (1992) Differentiation of Corynebacterium spp., Listeria spp., and related organisms by fluorogenic substrates. Journal of Clinical Microbiology 30, 1067 1071.
3. Clark, A.G., McLauchlin, J. (1997) Simple color tests based on an alanyl peptidase reaction which differentiate Listeria monocytogenes from other Listeria species. Journal of Clinical Microbiology 35, 2155-2156.
4. Morgan, J. W. (1987) Evaluation of a rapid method for the determination of L pyrrolidonyl-ß-naphthylamide hydrolysis. Laboratory Medicine 18, 682-683.
5. Bascomb, S. and Manafi, M. (1998) Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic Gram-positive cocci. Clinical Microbiology Reviews 11, 318-340.
6. Anonymous (2000) 2-Naphthylamine. In Croner’s Hazardous Substances: Carcinogens Guide. pp. 2-291. Kingston-upon-Thames: Croner.CCH Group Limited.
7. Druggan, P., Roberts, P.B. and Swaine, D. (1999) A rapid chromogenic method for the differentiation of Citrobacter spp. and Salmonella spp. directly from enteric media. Abstract of the Annual Meeting of the American Society for Microbiology 1999, C444, p.71. Washington: ASM Press.
8. International Standards Organisation (1996) ISO 11290-1, Microbiology of food and animal feeding stuffs – horizontal method for the detection and enumeration of Listeria monocytogenes - part 1: detection method. London: British Standards Institute.
9. FDA (1998) Appendix 3: Trypticase Soy Agar with 0.6% Yeast Extract (TSAYE). In Food and Drug Administration Bacteriological Analytical Manual, 8th edition, revision A ed. Jackson, G. J. pp. 54-55. Gaithersburg: AOAC international.