RAPID ANGINOSUS ID KIT

Cat. no. Z14 Rapid Anginosus ID Kit 10 tests/kit
Each kit contains:
Z14A - Rapid Arginine, 13x100mm Tube, 0.5ml
Z14B - Rapid VP, 13x100mm Tube, 0.5ml

10 tubes
10 tubes

Cat. no. R14 Rapid VP, 13x100mm Tube, 0.5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Rapid Anginosus ID Kit is used to detect arginine decarboxylase activity and perform the Voges-Proskauer test, in as little as four hours, to assist in the identification of the Streptococcus anginosus group (formerly S. milleri). Rapid VP is also useful for differentiating coagulase-positive strains of Staphylococcus spp.

SUMMARY

Hardy Diagnostics' Rapid Anginosus ID Kit can be used to identify streptococcal isolates suspected of belonging to the anginosus group (S. anginosus, S. constellatus, S. intermedius).

Infection by Streptococcus anginosus group (formerly known asStreptococcus milleri) can cause endocarditis, CNS infections and abscesses, bacteremia, oral infections, neonatal sepsis, deep-seated sepsis, intra-abdominal infections, pyogenic liver abscesses, deep-tissue abscesses, appendicitis, and pulmonary infections.(1-3) S. anginosus can also cause soft tissue infections, which become severe in patients that are intravenous-drug users or have uncontrolled diabetes.(3) Early detection and treatment of S. anginosus group is essential, and with Hardy Diagnostics' Rapid Anginosus ID Kit, results can be obtained in as little as four hours.

Conventional methods for detecting arginine hydrolysis require an extended period of incubation. The tests generally involve the degradation of arginine, resulting in an increase in pH and indicated by the development of a purple color. Tests are usually incubated for a period of up to seven days. An overlay of mineral oil acts as a barrier to oxygen and prevents alkalinization of the surface of the medium.

Voges and Proskauer, in 1898, first observed the production of a red color after the addition of potassium hydroxide to cultures grown on specific media.(6) Harden later revealed that the development of the red color was a result of acetyl-methyl carbinol production.(7) In 1936 Barrit made the test more sensitive by adding alpha-naphthol to the medium before adding potassium hydroxide.(8)

The Voges-Proskauer test identifies bacteria that are able to metabolize pyruvic acid to form acetyl-methyl carbinol (acetoin). This end product, in the presence of atmospheric oxygen and 40% potassium hydroxide is converted to diacetyl. Diacetyl, under the catalytic action of alpha-naphthol and creatine, is converted into a pink-red complex.(3) This is a positive Voges-Proskauer (VP) test reaction. The conventional Voges-Proskauer test tube method requires an incubation of up to 72 hours.

The Voges-Proskauer test is also useful for differentiating coagulase-positive species S. aureus (positive VP) from S. hyicus and S. intermedius (negative VP).(4)

Rapid Arginine consists of peptones and yeast extract which supply nitrogenous and other nutrients necessary for bacterial growth. Bromcresol purple is a pH indicator. If the amino acid arginine is hydrolyzed, the pH of the medium shifts upward resulting in a color change. This reaction requires an anaerobic environment, which can be created by a mineral oil overlay.

Rapid VP consists of peptones and glucose (which supply nitrogenous and other nutrients necessary for bacterial growth) in a phosphate-buffered solution. A color change results from the above described reaction between acetoin and diacetyl.

FORMULA

Ingredients per liter of deionized water:*

Rapid Arginine (Z14A):
L-Arginine 10.0gm
Meat Peptone 5.0gm
Yeast Extract 3.0gm
Bromcresol Purple 0.01gm

Final pH 5.5 +/- 0.2 at 25ºC.

Rapid VP (R14 and Z14B):
Dipeptone 7.0gm
Dextrose 5.0gm
Potassium Phosphate 5.0gm

Final pH 6.9 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is temperature sensitive; protect from excessive heat and moisture.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organisms. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Method of use:
(For Cat. no. R14 follow the directions for the Rapid VP only)

1. A suspect colony should be grown on a non-selective media, such as Chocolate Agar, Cat. no. E14 or Blood Agar, Cat. no. A10, however Chocolate Agar will yield higher growth of Streptococcus spp. and is recommended.

2. From this 18-24 hour old pure culture plate, heavily inoculate (greater than a McFarland Latex #4, Cat. no. ML4) one tube of Rapid Arginine and one tube of Rapid VP.

3. Overlay the inoculated Rapid Arginine broth with at least 0.5ml of Sterile Mineral Oil (Cat. no. Z80).

4. Incubate aerobically at 35-37 degrees C. for four hours.

VOGES-PROSKAUER TEST performed AFTER incubation

5. Add three drops of 5% alpha-naphthol (Voges-Proskauer Reagent A, Cat. no. Z91) to the Rapid VP tube and agitate tube to mix.

6. Add one drop of 40% KOH (Voges-Proskauer Reagent B, Cat. no. Z92) to the Rapid VP tube and agitate tube to mix.

7. Allow tube to remain undisturbed at room temperature for 15-30 minutes.

INTERPRETATION OF RESULTS

ARGININE HYDROLYSIS

For reference, compare an uninoculated reference tube with inoculated tube.

Positive reaction: development of a purple color. Must be darker than the uninoculated reference tube.

Negative reaction: no development of color (remains pale yellow to light gray).

VOGES-PROSKAUER TEST

A positive VP test is demonstrated by the development of a pink-red color on the surface of the medium 15-30 minutes after the addition of the reagents.

A negative VP test is demonstrated by the appearance of a yellow color on the surface of the medium. Development of a copper-like color is also interpreted as negative.

PRESUMPTIVE IDENTIFICATION BASED ON RESULTS OBTAINED WITH RAPID ANGINOSUS ID KIT

Viridans Streptococcal Group Arginine VP
Anginosus group +
purple
+
pink-red
Mitis group V
purple/yellow
-
yellow
Mutans, Salivarius, and Bovis groups -
yellow
+
pink-red

LIMITATIONS

In addition to the Streptococcus anginosus group, S. porcinus (rarely isolated from humans), and S. ratti are also positive for both arginine hydrolysis and the Voges-Proskauer test, thus it is recommended that further biochemical tests be performed on pure cultures for complete identification. For more information, see appropriate references.(3-6) Acidification of fermentation broth with sorbitol (Cat. no. Y93 or Y114) is useful as S. porcinus and S. ratti are positive while the S. anginosus group is negative.

Some organisms that are capable of producing acetyl methyl carbinol (acetoin) produce false-negative VP reactions when incubated for 48-72 hours. Rapid VP should not exhibit false-negative VP reactions because of the heavy suspension and short incubation time (as little as four hours).

When adding the VP reagents to the suspension, it is important that the alpha-naphthol be added first and the KOH added second. A change in the order may produce invalid test results.

False-positive VP results may occur if VP tests are read beyond one hour following the addition of reagents.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, Blood Agar (Cat. no. A10), other culture media, swabs, applicator sticks, incinerators, Sterile Mineral Oil (Cat. no. Z80), Saline, 0.85% (Cat. no. K248 or R45), Voges-Proskauer Reagent A (Cat. no. Z91), Voges-Proskauer Reagent B (Cat. no. Z92), McFarland Latex #4 (Cat. no. ML4), pipets, and incubators, etc. are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Reaction
Time Temperature Atmosphere Arginine Voges-Proskauer
Streptococcus agalactiae
ATCC® 12386**
E 4hr 35°C Aerobic Positive
(purple)
Negative
(yellow)
Streptococcus salivarius
ATCC® 13419**
E 4hr 35°C Aerobic Negative
(yellow)
Positive
(pink-red)
Streptococcus anginosus
ATCC® 33397**
E 4hr 35°C Aerobic Positive
(purple)
Positive
(pink-red)

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

Rapid Arginine should appear clear, and pale yellow.
Rapid VP should appear clear, and light amber in color.

Z14A Rapid Arginine

Z14A - Rapid Arginine tube
LEFT: Streptococcus anginosus (ATCC® 33397)
Positive reaction at four hours.
RIGHT: Streptococcus salivarius (ATCC® 13419)
Negative reaction at four hours.



Z14B Rapid VP

Z14B - Rapid VP tube
LEFT: Streptococcus anginosus (ATCC® 33397)
Positive reaction at four hours.
RIGHT: Streptococcus agalactiae (ATCC® 12386)
Negative reaction at four hours.



REFERENCES

1. Ruoff, K.L., 1988. Streptococcus anginosus ("Streptococcus milleri"): The Unrecognized Pathogen. Clin. Microbiol. Rev.; Vol. 1, p. 102-108.

2. Whitworth, J.M., 1991. Use of Rapid Carbohydrate Utilisation Test for Identifying " Streptococcus milleri Group". J. Clin. Pathol.; Vol. 44, p. 329-333.

3. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology. J.B. Lippincott Company, Philadelphia, PA.

4. Versalovic, J., et al. Manual of Clinical Microbiology. American Society for Microbiology, Washington, D.C.

5. Tille, P.M., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

6. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

7. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

8. Voges, O. and B. Proskauer. 1898. Zeit. Hyg.; 28:20-32.

9. Harden, A. 1906. Proc. Roy. Soc., (London); 77:424-425.


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

032316gr