HIPPURATE 
K310- 50 tests,
K1310- 28 tests 

DISCUSSION: 
Key Hippurate tablets are used to test for sodium hippurate hydrolysis. Glycine, an end product of hippurate hydrolysis, reacts with ninhydrin to produce a dark blue color.  This is valuable when testing Streptococci and Gardnerella.  Most Group B streptoccoci will hydrolyze hippurate. Although some Group D enterocicci also hydrolyze hippurate, they attack esculin and are rarely beta hemolytic. 

MSDS: 
Each tablet contains 4 mg. of sodium hippurate and inert fillers which are  not known at this time to be hazardous. Ninhydrin reagent contains 50 mg. of ninhydrin in 0.4 ml. of solution with Dimethyl sulfoxide (DMSO). DMSO is harmful if inhaled, ingested, or absorbed through the skin, and may cause allergic reactions. In case of contact, flush eyes and skin with large amounts of water and remove any contaminated clothing. DMSO reacts violently with a large number of chemicals.  Never add this reagent to anything except as recommended. 

MATERIALS REQUIRED:
Hippurate tablets require fresh 24 hour growth on media appropriate for the specimen.  Consult a clinical microbiology manual for recommendations.  The following items are required but not provided: 
small test tubes (e.g. 12 x 75) (provided with K1310) 
Inoculating Loop 
Distilled water, pH 7.0-7.2 
Concentrated Ninhydrin solution (Cat. # K475) 

NINHYDRIN REAGENT: 
Ninhydrin reagent K475 is sold separately. Prepare the Ninhydrin reagent by adding 0.4 ml. of the solution to 5 ml. of isopropyl alcohol.  The diluted reagent has the same expiration date as the concentrate.  Ninhydrin will stains hands and clothing so handle appropriately.

STORAGE:     
Store tightly covered in a dry place at room temperature.

PROCEDURE:  
(1)Place 0.4 ml. (about 10 drops) of distilled water in a small test tube. 
(2)Inoculate heavily with 8-10 isolated colonies, mixing well. The suspension should be milky and opaque.  Avoid picking up agar with the loop. 
(3)Add one Key Hippurate Tablet and incubate for at least 6 hours at 37oC.
(4) After incubation, add 0.2 ml. of the diluted ninhydrin reagent and reincubate for 10 minutes. Observe for color change.
K1310 is provided with the tablet already in the tube. 

INTERPRETATION: 
The appearance of an intense blue to blue/black color within 10 minutes is a positive test.  A faint color should be disregarded.  If results are inconclusive, the test must be redone with a longer incubation period.  Reincubation of the already completed test will not give a more conclusive result.

QUALITY CONTROL:
Known positive (Group B strep) and negative (Hippurate negative Enterococcus) organisms should be run with each test group.  Dispose of all used material in a manner appropriate for biohazardous material.  

REFERENCES: 
(1) Manual of Clinical Microbiology, 5th Edition, Chapter 122, "Reagents and Stains" and Chapter 48 "Gardnerella and other ill-defined Genera". (2) Bailey and Scott's Diagnostic Microbiology, Seventh Edition, Chapter 25 Streptococci".