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OXOID BIOCHEMICAL IDENTIFICATION SYSTEM - PYR

Code: ID0580

The Oxoid Biochemical Identification System (O.B.I.S.) - PYR is a rapid colorimetric test for the determination of PYRase activity in Streptococci and Citrobacter spp.

Principal of the Test
PYRase activity distinguishes Group A streptococci and enterococci from other streptococcal groups including Group D streptococci (previously called faecal streptococci)^1,2, Thus offering a rapid diagnostic alternative to time-consuming culture methods. In addition, the same enzymatic activity may be used to aid differentiation of Salmonella spp. from Citrobacter spp. and other Enterobacteriaceae ^3,4.

The PYRase test has traditionally been based on the use of a b-naphthylamide peptide. This is a potent carcinogen ⁵. Oxoid has developed a new system using a non-carcinogenic substrate, in response to associated health concerns.

The O.B.I.S. - PYR test uses Test Cards impregnated with L-pyroglutamic acid 7-amino-4-methyl-coumarin (7AMC) and dimethylamino-cinnamaldehyde for the detection of PYRase activity. The enzymatic hydrolysis of this substrate by enterococci, Group A streptococci and Citrobacter spp. produces a purple colour following the addition of the Developing Solution ⁶.

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

ID581 O.B.I.S. Test Cards:-1 Pouch containing 10 Cards and a moisture absorbent sachet. There are 6 reaction areas on each card. 60 tests in total. Each Test Card is impregnated with 7AMC
ID200 Developing Solution:- 1 dropper bottle containing 7ml of 1 M hydrochloric acid 0.5% w/v dimethylamino-cinnamaldehyde
ID100 Buffer Solution:- 1 dropper bottle containing 7ml of Phosphate Buffered Saline (PBS)
Instruction Leaflet

Materials required but not provided
Mixing sticks or microbiological loop.

Precautions
This product is for in vitro diagnostic use only.
Do not use O.B.I.S. PYR reagents beyond the stated expiry date.
Specimen material may contain pathogenic organisms, handle with appropriate precautions.
The Developing Solution (ID200) contains an acid. 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 Test Cards and mixing sticks should be disposed of as biohazardous waste and incinerated or autoclaved for 15 minutes at 121°C.

Storage and Opening
This Kit must be stored at 2-8°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 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, 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.

Positive control - Use a known pyroglutamyl aminopeptidase positive strain such as Enterococcus faecalis ATCC® 29212, Streptococcus pyogenes ATCC® 19615 or 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 PYRase negative strain such as Streptococcus agalactiae ATCC® 13813 or Salmonella enteriditis ATCC® 13076. Follow the method given in the test procedure. Ensure that no purple colour forms within 20 seconds.

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

Specimen Collection and Preparation
For details of specimen collection and treatment a standard text book should be consulted ⁷.

When identifying enterococci and Group A streptococci, fresh primary or secondary cultures grown overnight on non selective media such as blood agar give best results. Colonies tested must be Gram-positive cocci and catalase-negative. In case of insufficient growth, a subculture should be performed.

When identifying Citrobacter spp. from Salmonella spp. and Enterbacteriaceae, colonies from non-selective media such as (XLD Medium, MLCB Agar, Desoxycholate Citrate Agar, Salmonella Shigella Agar, Brilliant Green Agar or Hektoen Enteric Agar may be tested. Colonies should be Gram-negative, oxidase-negative and urease-negative ⁷.

Test Procedure 

1. Apply one suspect colony (0.5mm or larger) onto the test area (enough to make a visible smear)
2. Moisten test area with 1 drop of Buffer
3. Incubate the inoculated Test Card at room temperature (15-30°C) for 5 minutes
4. Dispense 1 drop of Developing solution onto the test area. Development of a vivid purple colour on and around the colonies within 20 seconds confirms PYRase activity.

Reading and Interpretation of Results
Positive Result
A positive result is indicated by the development of a vivid purple colour in the inoculated portion of the test area within a 20 second period following addition of Developing Solution.

Negative Result
A negative result is indicated by lack of colour development in the inoculated portion of the test area within a 20 second period following addition of Developing Solution.

EXPECTED RESULTS

Limitations of the Test
O.B.I.S.-PYR is intented for the detection of PYRase activity in Gram-positive, catalase negative cocci and Citrobacter spp. Less commonly encountered isolates of lactococci and aerococci may be PYRase positive. The confirmation of enterococci or Group A streptococci can be achieved by serological grouping with a suitable test, e.g. Oxoid Streptococcal Grouping Kit (DR0595), Oxoid Strep Plus (DR0575) or Oxoid Dryspot Streptococcal Grouping Kit (DR400).
Some strains of Enterobacter cloacae are PYRase negative.
The reactions with O.B.I.S. - PYR are a marker for enzyme activity and atypical strains may occasionally occur.
A slight colour change may develop with negative reactions. This is restricted to the immediate site of inoculation. Refer to the positive and negative controls to aid interpretation.
Incubation beyond 20 seconds following addition of the Developing Solution may produce non-specific colour reactions. It is important therefore that the test is read as indicated.

References
1. Ellener, P.D., Williams, D. A., Hosmer, M. E. and Cohenford, M. (1985). Preliminary evaluation of rapid colorimetric method for the presumptive identification of Group A streptococci and enterococci. J. Clin. Microbiol. 22. 880-881.
2. Facklam, R. R., Padula, J. F., Thacker, L. G., Wortham, E. C. and Sconyers, B. J. (1974). Presumptive identification of Group A,B and D streptococci. Appl. Microbiol. 27. 107-109
3. 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.
4. Bennett, A. R., MacPhee, S., Betts, R. and Post, D. (1999). Use of enzyme pyrrolidonyl peptidase (Oxoid PYR test) to distinguish Citrobacter from Salmonella isolated from foods. Letters in Applied Microbiology. 28. 175-178.
5. 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.
6. Data on file at Oxoid.
7. Faklam, R. R. and Washing, J. A. (1991). Streptococci and Aerococci pp. 238-258, In Lunette, I. H. Balles, A., Hausler (Jr), W. J. and Shadomy, H. J. (eds) Manual of Clinical Microbiology, 5^th edition. Amer. Soc. For Microb., Washington D.C.