Cat. no. K1532 Pro Disc 50 per package


Enzymatic hydrolysis of L-proline-naphthylamide releases free-naphthylamine which is detected and shown by the color change after adding PEP reagent. This test is especially of use in screening for Clostridium difficile from CCFA. A negative test indicates the organism cannot be Clostridium difficile. The test is also of value when used with other tests in the speciation of anaerobes, both gram-positive and gram-negative, and certain other organisms such as enterics.


The discs contain L-proline-naphthylamide: naphthylamides have been identified as possible carcinogens however when used as directed the discs present no hazard. PEP reagent contains hydrochloric acid, is poisonous, mildly corrosive, and stains clothing and hands. Handle with care. Consult poison control center if ingested.



1. Place 1 disc onto a slide or into a small test tube. Moisten slightly with distilled water. (Do not over saturate.)

2. Inoculate heavily with a loopful of organism from a fresh pure 24 hour culture plate or slant.

3. Wait 2 minutes (at room temperature) then add 1 drop of PEP reagent. Wait 1-2 minutes for color to develop. Do not read after more than 2 minutes.


The appearance of a dark pink to red color is positive. Indole reactions of organisms grown on any media containing tryptophane (e.g. blood agar) may interfere but will still produce predictable reactions. From such media, positive PRO reactions will range from dark pink or red (PRO+/indole-) to very dark blue or purple (PRO+/indole+). Green or turquoise (indole+), and yellow (indole-) are both PRO negative.


Store discs and reagent tightly covered with desiccant in a dry place at 2-8 degrees C. Discs do not have to be warmed before use.


Each lot should be tested with known positive and negative organisms. Some suggested strains are Clostridium difficile ATCC® 9689 (positive) and Clostridium perfringens ATCC® 13124 (negative). Positive PRO and Indole may be shown by testing with Clostridium sordelliiATCC® 9714. Dispose of all used material in a manner appropriate for biohazardous material.

Pro Discs

Showing positive (left disc) and negative (right disc) reactions on Pro Disc (Cat. no. K1532).
The discs were moistened with deionized water and growth from 24 hour cultures of Clostridium difficile (ATCC® 9689) and Clostridium perfringens (ATCC® 13124) was applied to the left and right discs, respectively. The discs were incubated at room temperature for two minutes. After the incubation process, one drop of PEP reagent was added to each disc. The dark pink color development within two minutes was indicative of a positive PRO reaction.


1. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

2. Kilian, M and Bulow, P. 1976. "Rapid Diagnosis of Enterobacteriaceae", Acta Path. Microbio.; Scan, Sect B, 84:245-251.

3. Wadsworth. 1993. "Glucosidase tests", Anaerobic Bacteriology Manual, 5th ed., p. 152.

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