SODIUM CARBONATE, 2N

Cat. no. Z102 Sodium Carbonate, 2N 15ml

INTENDED USE

Hardy Diagnostics Sodium Carbonate, 2N is recommended for use with Arylsulfatase Broth (Cat. no. K93 and K94) to aid in differentiation of pathogenic mycobacteria based on their ability to produce arylsulfatase.

SUMMARY

The arylsulfatase enzyme hydrolyzes the bond between a sulfate group and an aromatic ring structure. Phenolphthalein is liberated by this enzymatic process and the presence of this is indicated by a red color change when Sodium Carbonate, 2N is added.

Arylsulfatase is produced by many mycobacterial species in varying concentrations. (1-3) The ability to produce a detectable level of arylsulfatase is a biochemical characteristic used in the differentiation of some Mycobacterium species. (4-8)

The test is performed by inoculating a broth containing tripotassium phenolphthalein disulfate with a Mycobacterium isolate. (7) If arylsulfatase is produced, it splits the phenolphthalein substrate, releasing free phenolphthalein, which turns pink to red when alkali is added to the medium.

The 0.001M Arylsulfatase Broth is used for rapidly-growing arylsulfatase producers, such as Mycobacterium fortuitum and Mycobacterium chelonae .

The 0.003M Arylsulfatase Broth is used for slow-growing mycobacteria such as Mycobacterium szulgai , Mycobacterium triviale , Mycobacterium xenopi , Mycobacterium tuberculosis and Mycobacterium flavescens . (4,7)

REAGENT FORMULA

Ingredients per 100ml of deionized water:*

Sodium Carbonate 10.6gm

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. away from direct light. The reagent should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: These media are not suitable for use directly with clinical specimens or other sources containing mixed microbial flora. Consult appropriate references for more information. (4-8)

Organisms to be cultivated must first be isolated in pure culture on appropriate medium.

Inoculate Arylsulfatase Broth (Cat. no. K93 and K94) per manufacturers instructions.

Incubate the tubes at 35ºC. in an aerobic atmosphere without increased CO 2 . Remove the 0.001M broth after three days and add no more than six drops of 2N Sodium Carbonate solution and observe for a color change.

Incubate the 0.003M broth for two weeks, then remove and add six drops of the 2N Sodium Carbonate solution.

INTERPRETATION OF RESULTS

An immediate pink to red color change after addition of Sodium Carbonate, 2N is a positive reaction.

No color change after the addition of Sodium Carbonate, 2N is a negative reaction.

Positive results may be compared with a set of color standards and the intensity of the color recorded from +/- to 5+. The intensity of the color reaction can be recorded as follows:

Color reaction Score
No color change (-)
Pale Pink (1+)
Pink (2+)
Light Red (3+)
Red (4+)
Deep Red (5+)

LIMITATIONS

Always test a few tubes of the prepared, uninoculated substrate medium for free phenolphthalein by adding a few drops of 2N Sodium Carbonate. Free phenolphthalein in the media can lead to false-positive results.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerator, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms
Rate of Bacterial Growth
Arylsulfatase
Broth
Reaction
Mycobacterium fortuitum
ATCC ® 6841
Rapid: Three Days
0.001M
Positive: Pale pink to pink color
Mycobacterium phlei
ATCC ® 11758
Rapid: Three Days
0.001M
Negative: No color reaction
Mycobacterium xenopi
ATCC ® 19250
Slow: Two Weeks
0.003M
Positive: Pink to red color
Mycobacterium tuberculosis
ATCC ® 25177
Slow: Two Weeks
0.003M
Negative: No color reaction

User Quality Control

PHYSICAL APPEARANCE

Sodium Carbonate, 2N should appear clear and colorless.

Positive

Mycobacterium fortuitum (ATCC ® 6841) growing in 0.001M Arylsulfatase Broth (Cat. no. K93). Incubated aerobically for three days at 35ºC. Subsequent to incubation, six drops of 2N sodium carbonate solution (Cat. no. Z102) were added to the tube. The pink color change was indicative as positive for the production of arylsulfatase.

Negative

Mycobacterium tuberculosis (ATCC ® 25177) growing in 0.001M Arylsulfatase Broth (Cat. no. K93). Incubated aerobically for three days at 35ºC. Subsequent to incubation, six drops of 2N sodium carbonate solution (Cat. no. Z102) were added to the tube. No pink color change was indicative as negative for the production of arylsulfatase.

REFERENCES

1. Kubica, G.P., and A.L. Ridgon. The Arylsulfatase activity of acid-fast bacilli. III. Preliminary investigation of rapidly growing acid-fast bacilli. Am. Rev. Respir. Dis.; 83:737-740.

2. Kubica, G.P., and A.L. Ridgon. The Arylsulfatase activity of acid-fast bacilli. I. Investigation of stock cultures of acid-fast bacilli. Am. Rev. Respir. Dis.; 83:728-732.

3. Whitehead, J.E.M., et al. Arylsulfatase Activity of Mycobacteria. J. Path. Bact.; 65:451-460.

4. 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.

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

6. Washington, J.A., (ed.). Laboratory Procedures in Clinical Microbiology, 2nd ed. Springer-Verlag, New York, NY.

7. Vestal, A.L. 1975. "Procedures for the Isolation and Identification of Mycobacteria", DHEW Publication No. (CDC) 75-8230. U.S. Department of Health, Education, and Welfare. Center for Disease Control, Atlanta, GA.

8. Sommers, H.M. and J.P. Russell. 1967. Clinically Significant Mycobacteria: Their Recognition and Identification. American Society of Clinical Pathologists, Chicago, IL.

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

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

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


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

041816gr