ARYLSULFATASE BROTH

Cat. no. K93 Arylsulfatase Broth 0.001M, 16x125mm Tube, 2ml 10 or 100 tubes/box
Cat. no. K94 Arylsulfatase Broth 0.003M, 16x125mm Tube, 2ml 10 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Arylsulfatase Broths are chemcally-defined media used in the differentiation of pathogenic mycobacteria based on their ability to produce arylsulfatase.

The 0.001M Arylsulfatase Broth is used in a 3-day test to detect arylsulfatase activity in rapidly-growing mycobacteria. The 0.003M Arylsulfatase Broth is used in a two-week test for the detection of arylsulfatase in slow-growing mycobacteria.

SUMMARY

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

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

FORMULA

Ingredients per 900ml of deionized water:*

Disodium Phosphate 2.5gm
Monopotassium Phosphate 1.0gm
Ammonium Sulfate 0.5gm
Monosodium Glutamate 0.5gm
Sodium Citrate 0.4gm
Magnesium Sulfate 0.05gm
Ferric Ammonium Citrate 0.04gm
Pyridoxine 1.0mg
Zinc Sulfate 1.0mg
Copper Sulfate 1.0mg
Biotin 0.5mg
Calcium Chloride 0.5mg
ADC Enrichment 100.0ml
Tripotassium Phenolphthalein Disulfate**

** Arylsulfatase Broth (0.001M) contains 0.65gm/L Tripotassium Phenolphthalein Disulfate.

** Arylsulfatase Broth (0.003M) contains 1.95gm/L Tripotassium Phenolphthalein Disulfate.

Final pH 6.6 +/- 0.3 at 25ºC.

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

ADC Enrichment
Ingredients per liter of deionized water:*
Bovine Albumin, Fraction V 50.0gm
Dextrose 20.0gm
Sodium Chloride 8.5gm
Catalase 0.03gm

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat 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 the broth with 0.1ml of a 7-day liquid culture or heavily inoculate with organisms cultured on a solid medium.

Incubate the tubes at 35ºC. in an aerobic atmosphere without added CO2. Remove the 0.001M Broth after three days and add no more than six drops of 1M sodium carbonate solution (10.6gm anhydrous Na2 CO3 in 100ml of water), and observe for a color change.

Incubate the 0.003M Broth for two weeks, then remove and add six drops of the 1M sodium carbonate solution.

INTERPRETATION OF RESULTS

A change in the color of the medium to pink or red following the addition of sodium carbonate is a positive reaction. The medium remains colorless if negative. Determine the intensity of the color reaction and record as follows:

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

LIMITATIONS

For identification, the organism must be in pure culture. Morphological, biochemical and /or serological tests should be performed for final identification. Consult appropriate references for detailed information and recommended procedures.(7,8)

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, 1M sodium carbonate solution, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Arylsulfatase Broth Reaction
Mycobacterium fortuitum
ATCC ® 6841
0.001M Pale pink to pink color
Mycobacterium phlei
ATCC ® 11758
0.001M No color reaction
Mycobacterium xenopi
ATCC ® 19250
0.003M Pink to red color
Mycobacterium tuberculosis
ATCC ® 25177
0.003M No color reaction

User Quality Control

PHYSICAL APPEARANCE

Arylsulfatase Broth should appear clear and colorless, with no precipitate.

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 1M 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 1M 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.


080216gr