ID HIPPURATE TEST TECHNICAL INFORMATION   ID- HIPPURATE CAT # 60040 INTENDED USE The Gibson ID- Hippurate kit is intended for use as a rapid colorimetric test for the detection of hippurate hydrolysis by microbial enzymes. SUMMARY AND EXPLANATION The differentiation of microorganisms on the basis of hippurate hydrolysis is applicable to a variety of species. Streptococcus agalactiae (group B)            can be difterentiated from other beta-h emolytic streptococci on the basis of this test. Hydrolysis of hippurate is also a distinguishing characteristic of Gardnerella vaginalis. Also, Campylobacterjejuni can be separated from other campylobacter species by this test. PRINCIPLES Glycine and sodium benzoate are produced upon the hydrolysis of sodium hippurate by the enzyme hippuricase. Ninhydrin reagent is used to         detect the glycine end product. The formation of a purple color is positive for glycine. MATERIALS PROVIDED Each Gibson Rapid Hippurate kit contains the following reagents sufficient for 10 tests: 10-Test Vials with paper disc Formula: Sodium Hippurate 150 gms Phosphate Buffer 1000 mIs 1- 3m1 bottle Ninhydrin Reagent Formula: Ninhydrin 35gms Butanol 500mls Acetone 500mls 1-3m1 bottle Deionized Water PRECAUTIONS: This test is intended for use by those trained in appropriate laboratory and bacteriological procedures. This test is for IN          VITRO DIAGNOSTIC USE only. Precautions should be taken against the dangers of microbiological hazards. Specimens, containers, media,               and test vials should be sterilized after use. Reagents should not come into contact with skin, eyes, or clothing. Do not inhale or ingest reagents.             In case of an accident, seek medical attention immediately. STORAGE INSTRUCTIONS: The ID- Hippurate kit must be stored at 2-8C to prevent breakdown of the substrates and reagents. Avoid          exposing other cups to fluctuations in temperature. Do not use this product if the expiration date has passed. EVIDENCE OF DETERIORATION: Materials provided should not be used if the expiration date has passed. If any deficiencies are              noted with this product notify the manufacturer. SPECIMEN COLLECTION Information on specimen collection and procedures for isolation and purification can be found in standard reference texts. PROCEDURES (1) MATERIALS REQUIRED BUT NOT PROVIDED: The standard clinical microbiological equipment such as loop, burner, deionized         water and incubator are needed for procedures involving the use of this product. Other materials required include the following: American Type     Culture Collection (ATCC) strains- Streptococcus agalactiae (13813) and Streptococcus pyogenes (19615). Use one vial for each specimen tested. 1. Remove cap from test vial. 2. Add 6 drops of deionized water to each vial. 3. Select 4-5 well isolated, morphologically similar colonies and inoculate the test vial. 4. Mix until a cloudy suspension of the test organism is produced. 5. Replace cap and incubate at 37C for 2 hours. 6. Remove cap and add 6 drops of the ninhydrin reagent. Do not shake vial. 7. Replace cap and re-incubate test cup at 37C for 10 minutes. Do not incubate longer than 1/2 hour: false positives may occur. INTERPRETATION USER QUALITY CONTROL Daily quality control should be performed in accordance with proper laboratory procedures using organisms that will produce known positive and negative reactions. Follow steps under PROCEDURE section. If quality control results are false clinical results should not be recorded. Notify the manufacturer if proper results are not obtained. The following American Type Culture Collection strains are recommended: Streptococcus agalactiae ATCC 13813 -- Purple color change (Positive) Streptococcus pyogenes ATCC 19615 -- No color change (Negative) LIMITATIONS OF PROCEDURE Avoid exposing unused vials to fluctuations in temperature. Occasional strains of C. jejuni may be hippurate negative.(2-3) Nearly all (99%)              group B streptococci, whether they are beta-hemolytic or non-hemolytic, hydrolyze hippurate and therefor react positively.(4) Group D streptococci     may also react positively but are distinguished by the Bile Esculin test. Beta hemolytic streptococci other than group B do not hydrolyze hippurate. (5) REFERENCES 1. Facklam, R. Streptococci and Aerococci. In: Len nette, Balows, Hausler, and Truant, Manual of Clinical Microbiology, 3rd ed., ASM, Washington, D.C. 1980. 2. Morris, G.K., M.R. El Sherbeeny, C.M. Patton, H. Kodaka, G.L. Lombard, P. Edmonds, D.G. Hollis, and D.J. Brenner. 1985. Comparison       of four hippurate hydrolysis methods for identification of thermophilic Campylobacter spp. J. Clin. Microbiol. 22:714-718. 3. Totten, P.A., C.M. Patton, F.C. Tenover, T.J. Barrett, W.E. Stamm, A.G. Steigerwalt, J.Y. Lin, K.K. Holmes, and D.J. Brenner. 1987. Prevalence and characterization of hippuratenegative Campylobacter jejuni in King County, Washington. J. Clin. Micrbiol. 25:1747-1752. 4. Hwang, M.N., and G.M. Ederer, 1975. J. Clin. Microbiol. 1:114-115. 5. Lennette, Balows, Hausler, Shadomy. 1985. Manual of clinical microbiology. ASM

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