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OXOID BIOCHEMICAL IDENTIFICATION SYSTEM
(O.B.I.S.) SALMONELLA

Code: ID0570

The Oxoid Biochemical Identification System (O.B.I.S.) Salmonella Test is a rapid colorimetric test for the determination of pyroglutamyl aminopeptidase (PYRase) and nitrophenylalanine deaminase (NPA) activity.

Principal of the Test
The O.B.I.S. salmonella Test offers a rapid screening method to distinguish Salmonella spp. from those organisms exhibiting similar colonial appearance on common selective Salmonella media. This reduces the need for a full biochemical identification of suspect colonies.

Identification of Salmonella relies on primary isolation of the organism on selective enteric media. However there are several other genera among the Enterobacteriaceae also capable of growth on such media and which can have similar colony morphology to salmonellae. The lack of PYRase and NPA activity in Salmonella spp. can be used to differentiate them from Citrobacter spp. Which possess PYRase activity ^1,2,3 and Proteus, Morganella and Providencia spp. Which have NPA activity ⁴.

Oxoid has developed a new system for PYRase testing ⁵, using a non-carcinogenic substrate in response to health concerns associated with the use of b -naphthylamide (a potent carcinogen) ⁶. The PYRase area on the O.B.I.S. salmonella Test Card is impregnated with L-pyroglutamic acid 7-amino-4-methylcoumarin (7AMC) ⁵. Dimethylaminocinnamaldehyde is used as a colour development reagent. The enzymatic hydrolysis of the substrate produces a purple colour on addition of the O.B.I.S. PYR Developing Solution ⁵.

The NPA area on the O.B.I.S. Salmonella Test Card is impregnated with nitrophenylalanine. Deaminatin of the reagent is shown by an orange-brown colour when the O.B.I.S. NPA Developing Soloution (0.25M sodium hydroxide) is added.

Components of the O.B.I.S. Salmonella Kit
Each O.B.I.S. Salmonella Kit contains the following reagents sufficient for 60 tests:

ID571 O.B.I.S. Salmonella Test Cards:- 2 Zip Lock Pouches, each containing 10 Cards and a desiccant. There are 3 tests on the card each consisting of 2 reaction areas.
Each reaction area designated as PYR is impregnated with 1% w/v L-pyroglutamic acid 7-amino-4-methylcoumarin (7 AMC).
Each reaction area designated as NPA is impregnated with 5% w/v nitrophenylalanine.
ID200 O.B.I.S. PYR Developing Solution:- 1 dropper bottle containing 7ml of 1M hydrochloric acid 0.5% w/v dimethylamino-cinnamaldehyde
ID210 O.B.I.S. NPA Developing Solution:- 1 dropper bottle containing 7ml of 0.25M sodium hydroxide
ID100 O.B.I.S. Buffer Solution:- 1 dropper bottle containing 7ml of Phosphate Buffered Saline (PBS).
Instruction Leaflet.

Materials required but not provided

Microbiological Loop
Positive and negative quality control organisms

Precautions
This product is for in vitro diagnostic use only.
Do not use O.B.I.S. salmonella reagents beyond the stated expiry date.
Specimen material may contain pathogenic organisms, handle with appropriate precautions.
The O.B.I.S. PYR Developing Solution (ID200) contains an acid. The O.B.I.S. NPA Developing Solution (ID210) contains a weak base. Avoid direct contact by wearing suitable protective equipment. If the reagents come into contact with the skin, mucous membranes or eyes immediately wash the area thoroughly with plenty of water.
Used O.B.I.S. Test Cards and microbiological loops should be disposed of as biohazardous waste and incinerated or autoclaved for 15 minutes at 121°C.

Storage and Opening
The O.B.I.S. Salmonella Kit must be stored at 2-8°C. Allow the pouches to reach room temperature before use to prevent the formation of condesation on the Test Cards. Open the pouches by cutting at the notch between the end seal and the zip lock opening. Once opened, remove the number of Test Cards required for immediate testing (testing within 10 minutes) and reseal the pouch straight away. If fewer tests are reqiured, cut the Test Card and return the unused portions to the pouch. Do not return used portions to the pouch as they will be contaminated. When stored as indicated, O.B.I.S. Salmonella reagents will retain their activity until the expiry date shown on the box.

Quality Control Procedure
Each day the Kit is used the following control procedures should be performed.

PYRase Test

Positive control - Use a known pyroglutamyl aminopeptidase positive strain such as Citrobacter freundii ATCC® 8090. Follow the method given in the test procedure. Ensure that a purple colour forms within 20 seconds.

Negative control - Use a known pyroglutamyl aminopeptidase negative strain such as Salmonella enteriditis ATCC® 13076. Follow the method given in the test procedure. Ensure that no purple colour forms within 20 seconds.

NPA Test

Positive control - Use a known phenylalanine deaminase positive strain such as Proteus mirabilis ATCC® 43071. Follow the method given in the test procedure. Ensure that an orange-brown colour forms within 20 seconds.

Negative control - Use a known phenylalanine deaminase negative strain such as Citrobacter freundii ATCC® 8090 or Salmonella enteriditis ATCC® 13076. Follow the method given in the test procedure. Ensure that no orange-brown colour forms within 20 seconds.

Do not use the reagents if the reactions with control organisms are incorrect.

Specimens
Colonies with typical Salmonella morphology on the following media may be tested : Brilliant Green Agar (Modified) (CM0329), MLCB Agar (CM0783), XLD Medium (CM0469), SS Agar (CM0099), SS Agar Modified (CM0533), Hektoen Enteric Agar (CM0419), Desoxycholate Citrate Agar (CM0035) of Bismuth Sulphite Agar (CM0201). Colonies tested should be Gram-negative and Oxidase negative.
Plates should be tested a maximum of one hour after being removed from the incubator.

Test Procedure and Interpretation of Results

Important Note: nitrophenylalanine impregnated on the NPA Test area is light sensitive. Exposure to light for more than 1 hour must be avoided.

1. Select one suspect colony (0.5mm or larger). Apply the colony to the PYR and NPA Test areas (sufficient colony should be applied to each area to make a visible smear)
2. Moisten both Test areas with 1 drop of Buffer solution (ID100).
3. Incubate the inoculated Test Card at room temperature (15-30°C) for 5 minutes.
4. Dispense 1 drop of O.B.I.S. PYR Developing Solution (ID200) to the PYR Test area. Development of a vivid purple colour confirms PYRase activity.
5. Dispense 1 drop of the O.B.I.S. NPA Developing Solution (ID210) to the test area. Development of an orange-brown colour on and around the smeared colony within 20 seconds confirms NPA activity.

Interpretation chart
Typical organism reactions

Trial Results
Results from pure cultures

*One Citrobacter strain showing a negative PYRase reaction. This was confirmed as a Citrobacter youngae thus showing a truly negative result.
In trail of 470 faecal samples O.B.I.S. Salmonella gave the following results:

Sensitivity 100%
Specificity 81.5% - 95.4%*
* Depending on the plating medium used.

Limitations of the Test
O.B.I.S. Salmonella is intented for the detection of PYRase and NPA activity in Gram -negative, Oxidase negative micro-organisms. It can be used as a screen to differentiate Salmonella spp. from Citrobacter, Proteus, Providencia and Morganella spp. from the following selective enteric media: Brilliant Green Agar (Modified) (CM0329), MLCB Agar (CM0783), XLD Medium (CM0469), SS Agar (CM0099), SS Agar Modified (CM0533), Hektoen Enteric Agar (CM0419), Desoxycholate Citrate Agar (CM0035) of Bismuth Sulphite Agar (CM0201).
A few isolates of H₂S-positive Escherichia coli may appear as salmonellas on MLCB Agar. These organisms will show the same results as Salmonella spp. on the O.B.I.S. Salmonella Test as they lack both PYRase and NPA activity.
The neutral red in Desoxycholate Citrate Agar CM0035 may produce a pink colour on the O.B.I.S.Test Card, to avoid any confusion with the positive purple reaction in the PYR test the result should be compared to a positive control strain.
Escherichia coli and indole-positive Proteus spp. obtained from media with a high tryptophan content may generate a blue-green colour development on PYR Test area. This is a negative result.
Citrobacter youngae may produce a PYRase-negative result and show the same result as Salmonella spp. in the Test.
O.B.I.S. Salmonella provides a reliable presumptive identification of salmonellae, but does not replace the need for full biochemical testing.

References
1. Chagla, A. H., Borczyk, A. A., Aldom, J. E., Dalla Rosa, S. and Cole, D. D. (1993). Evaluation of the L-Pyrrolidonyl-b-Napthylamide hydrolosis test for differentiation of members of the families Enterbacteriaceae and Vibrionaceae. J. Clin. Microbiol. 31. 1946-1948.
2. Mulczyk, M. and Szewczuk, A. (1970). Pyrrolionyl Peptidase in Bacteria: A New Colorimetric Test for Differentiation of Entertobacteriaceae. J. Gen. Microbiol. 61 9-13.
3. Inoue, K., Miki, K., Tamura, K. and Sakazaki, R. (1996). Evaluation of L-Pyrrolionyl Peptidase Paper Strip test for Differentiation of Members of the Family Entertobacteriaceae, Particularly Salmonella spp. J. Clin. Microbiol. 34. 1811-1812.
4. Giammanco, G., Pignato, S. and Agodi, A. (1985). A Simple Chromogenic Test For Rapid Screening of Proteus and Providencia Bacteria. Microbiolgica. 8. 395-397.
5. Druggan, P., Roberts, P. and Swaine, D. (1999). A Rapid Chromogenic Method for the Differentiation of Citrobacter spp. and Salmonella spp. Directly from Enteric Media. Abstr. C-444 p71 Abstr. 99^th Annual Meet. Am. Soc. Microbiol. 1999.
6. Bascomb, S. and Manafi, M. (1998). Use of Enzyme tests in the Characterization and Identification of Aerobic and Facultatively Anaerobic Gram-Positive Cocci. Clinical Microbiological Reviews. 11. 318-340.

may produce a PYRase-negative result and show the same result as