STREP ID TRIPLATE

INTENDED USE Remel Strep ID Triplate is comprised of solid media recommended for use in qualitative procedures for the differentiation and presumptive identification of streptococci and enterococci.

SUMMARY AND EXPLANATION Section I contains the substrate PYR (L-pyroglutamic acid-β-naphthylamide) for the detection of pyrrolidoxyl peptidase. In 1981, Godsey, Schulman, and Eriquez described a test for L-pyrrolidine hydrolysis to identify group A streptococci and enterococci.1 In 1982, Facklam et al. reported that the PYR test could replace the bacitracin and 6.5% salt tolerance tests to presumptively identify group A streptococci and enterococci.2 Section II contains Tryptic Soy Agar supplemented with 5% sheep blood. Streptococcal isolates are grouped according to their hemolytic pattern on Sheep Blood Agar.3 The sheep blood also provides the erythrocytes needed for performing the CAMP test which presumptively identifies group B streptococci.4 Section III contains Bile Esculin Agar which provides a reliable means of identifying group D streptococci and enterococci. Rochaix first demonstrated the value of esculin hydrolysis in the identification of enterococci.5 Meyer and Schonfeld found that 61 of 62 strains of enterococci hydrolyzed esculin in a bile containing medium.6 Facklam and Moody tested 700 strains of streptococci representing all known serological groups on a bile esculin medium developed by Swan and determined that 100% of the group D streptococci were bile esculin positive.7,8

PRINCIPLE In Section I, group A streptococci and enterococci can be differentiated from other streptococci by their ability to hydrolyze L-pyroglutamic acid-β-naphthylamide (PYR). PYR serves as the substrate for detection of pyrrolidoxyl peptidase. Following hydrolysis of PYR by the peptidase, the resulting β-naphthylamine produces a red color upon the addition of N,N-dimethylaminocinnamaldehyde. Section II contains Tryptic Soy Agar as a base medium. This medium is highly nutritious due to casein and soy peptones which supply organic nitrogen, amino acids, and peptides. The sodium chloride maintains osmotic equilibrium. Sheep blood is incorporated in the medium to detect the hemolytic reactions of streptococci. The CAMP test is performed in Section II by making a single streak of the test isolate perpendicular to a streak of a beta lysin-producing strain or Beta Lysin Disk. Group B streptococci produce a protein-like compound called CAMP factor that acts synergistically with beta lysin to produce an arrowhead or crescent-shaped area of complete lysis. Section III contains Bile Esculin Agar which will presumptively identify group D streptococci and enterococci. These organisms hydrolyze esculin, resulting in the production of esculetin and dextrose. Esculetin reacts with the ferric ions in the medium to form a brown-black complex which surrounds the colonies. Bile is incorporated in the medium as a selective agent.

REAGENTS (CLASSICAL FORMULAE)* Section I (PYR Agar): Casein Peptone...... 15.0 g L-Pyroglutamic Acid-β-naphthylamide...... 0.1 g Soy Peptone...... 5.0 g Agar...... 15.0 g Sodium Chloride...... 5.0 g Demineralized Water...... 1000.0 ml

Section II (Tryptic Soy Agar w/ Sheep Blood): Casein Peptone...... 15.0 g Sheep Blood...... 5 % Soy Peptone...... 5.0 g Agar...... 15.0 g Sodium Chloride...... 5.0 g Demineralized Water...... 1000.0 ml pH 7.3 ± 0.2 @ 25°C

Section III (Bile Esculin Agar): Oxgall (Bile)...... 40.0 g Esculin...... 1.0 g Gelatin Peptone ...... 5.0 g Ferric Citrate...... 0.5 g Beef Extract...... 3.0 g Agar...... 15.0 g Demineralized Water...... 1000.0 ml pH 6.6 ± 0.2 @ 25°C

*Adjusted as required to meet performance standards.

PROCEDURE 1. Implement appropriate procedures to verify that the test isolate is a streptococci or an enterococci. 2. Select 3-4 well-isolated colonies from a pure, 18-24 hour culture. Streak the colonies across the agar surface of Sections I and III. 3. In Section II, perform the test for CAMP factor according to one of the following methods: a. Inoculate the agar with a streak of β-lysin-producing S. aureus (e.g., S. aureus ATCC® 33862). Follow with a streak of the test isolate perpendicular to the S. aureus and leave a 1-2 mm space in between. b. Place a Beta Lysin Disk (REF R21120) on the uninoculated medium. Follow with a streak of the test isolate perpendicular to the disk and leave a 1-2 mm space in between. 4. Incubate the plate aerobically at 35-37°C for 18-24 hours. 5. Following incubation, add 1-2 drops of PYR Reagent (REF R21258) to the PYR Agar (Section I) and observe for a red color development. 6. Observe Section II for the formation of an arrowhead or crescent-shaped area of complete lysis at the juncture of the test isolate and the S. aureus or the Beta Lysin Disk. 7. Observe Section III for a brown to black color development in the area surrounding growth.

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INTERPRETATION OF THE TEST Section I (PYR Test): Positive Test - Red color development within 1 minute after addition of PYR Reagent Negative Test - Yellow or slightly orange color development after addition of PYR Reagent

Section II (CAMP Test): Positive Test - Arrowhead or crescent-shaped zone of at the juncture of the test isolate and the S. aureus culture or Beta Lysin Disk Negative Test - Absence of enhanced hemolysis at the juncture of the test isolate and the S. aureus or Beta Lysin Disk

Section III (Esculin Hydrolysis): Positive Test - Brown-black color development of the medium Negative Test - No blackening of the medium

QUALITY CONTROL All lot numbers of Strep ID Triplate have been tested using the following quality control organisms and have been found to be acceptable. Testing of control organisms should be performed in accordance with established laboratory quality control procedures. If aberrant quality control results are noted, patient results should not be reported.

RESULTS CONTROL INCUBATION PYR CAMP ESCULIN pyogenes ATCC® 19615 Aerobic, 24 h @ 35 - 37°C + - - ATCC® 12386 Aerobic, 24 h @ 35 - 37°C - + - Streptococcus gallolyticus ATCC® 9809 Aerobic, 24 h @ 35 - 37°C - - + faecalis ATCC® 29212 Aerobic, 24 h @ 35 - 37°C + - +

LIMITATIONS 1. Do not incubate this plate in a CO2 atmosphere. Some group A streptococci may give a false-positive CAMP test if incubated in CO2. 2. Streptococci which cannot be grouped by this presumptive methodology should be tested by more complete biochemical tests or by serological typing. 3. It is imperative that all reactions be interpreted in conjunction with the hemolytic reaction of the organism.

BIBLIOGRAPHY 1. Godsey, J., R. Schulman, and L. Eriquez. 1981. Abstract #C84. Abstracts of the General Meeting of the American Society for Microbiology. ASM, Washington, D.C. 2. Facklam, R.R., L.G. Thacker, B. Fox, and L. Eriquez. 1982. J. Clin. Microbiol. 15:987-990. 3. Baron, E.J. and S.M. Finegold. 1986. Bailey and Scott’s . 7th ed. Mosby, St. Louis, MO. 4. Christie, R., N.E. Atkins, and E. Munch-Peterson. 1944. Aust. J. Exp. Biol. 22:197-200. 5. Rochaix, A. 1924. Cr. Soc. Biol. 90:771-772. 6. Meyer, V.R. and H. Schonfeld. 1926. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 99:402-418. 7. Facklam, R.R. and M.D. Moody. 1969. Bacteriol. Proc. Abstract M33. 8. Swan, A. 1954. J. Clin. Pathol. 7:160-163. 9. Facklam, R.R. 1974. and Identification of Streptococci. U.S. Dept. of Health, Education & Welfare. Public Health Service. CDC, Atlanta, GA.

Refer to the front of Remel Technical Manual of Microbiological Media for General Information regarding precautions, product storage and deterioration, specimen collection, storage and transportation, materials required, quality control, and limitations.

ATCC® is a registered trademark of American Type Culture Collection.

IFU 2382, Revised November 15, 2007 Printed in U.S.A.

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