WALLENSTEIN MEDIA

Cat. no. C61 Wallenstein, 20x125mm Tube, 10ml Slant 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Wallenstein Media are recommended for use in the cultivation and isolation of mycobacteria other than tuberculosis. It is particularly useful in the recovery of Mycobacteria avium complex. (3)

SUMMARY

Wallenstein Medium is comprised of an egg yolk medium supplemented with glycerol and malachite green. (4,5) When heated, the egg albumin coagulates, thus providing a solid surface on which mycobacteria can be isolated and cultured. The egg also serves as a source of nitrogen. Glycerol serves as a carbon source and is favorable to the growth of the human type tubercle bacillus while being unfavorable to the bovine type. Malachite green is added as a selective agent, which partially inhibits the growth of other bacteria.

FORMULA

Ingredients per 242ml of deionized water:*

Malachite Green 0.18gm
Glycerol 23.0ml
Egg Yolk Base 735.0ml

* Adjusted and/or supplemented 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), 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: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. Consult listed references for information on specimen collection.1-3,6,7,10)

Method of Use:

1. Inoculate the Wallenstein Media with specimen after decontamination and neutralization, according to test procedures recommended by the Centers for Disease Control (CDC). Consult listed references for methods.(1-3,6,7,10)

2. Incubate medium in a CO2 atmosphere at 35-37ºC. A second set of media should be inoculated for specimens suspected of harboring M. marinum. This second set should be incubated at room temperature (or at 30ºC. if such an incubator is available).(3) Protect from light. Tubed media should be incubated for one week with loosened caps to allow the circulation of CO2 for the initiation of growth. Caps should be tightened after one week in order to prevent dehydration of media, but should be loosened and tightened weekly thereafter to allow for gas exchange.

3. Examine the media within five to seven days, and weekly thereafter for up to eight weeks.

4. Examine plates under light for the appearance of macroscopic growth.

5. Examine tubes under light and magnifying mirror for macroscopic growth. Record and describe colony morphology, rate of growth, and pigmentation on the first day growth is observed.

6. Consult appropriate references for aid in the biochemical identification of acid-fast bacilli.(1,2,6,10)

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth ofMycobacterium species on this medium.(1-3,6,7,10) Examine and record each type of colony morphology, pigment, and growth rate. Biochemical testing is required for definitive identification.

LIMITATIONS

Wallenstein Media require incubation in a 5-10% CO2 atmosphere in order to recover mycobacteria. Mycobacteria, for unknown reasons, are not recovered well from candle extinction jars.(7)

Protect the media from all sources of light, as malachite green is very photo-sensitive.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, decontamination supplies, MycoSeals™ (Cat. no. SS9225), applicator sticks, pipets, incinerator, CO2 incubators, biological safety hoods, and microscopes, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium tuberculosis
H37Ra
ATCC ® 25177

G 21 days 35°C CO 2 ** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium kansasii
Group I
ATCC ® 12478

G 21 days 35°C CO 2 ** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium scrofulaceum
Group II
ATCC ® 19981

G 21 days 35°C CO 2 ** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium intracellulare
Group III
ATCC ® 13950

G 21 days 35°C CO 2 ** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium fortuitum
Group IV
ATCC ® 6841

G 21 days 35°C CO 2 ** Growth; colonies visible in 4 days

User Quality Control

** Atmosphere of incubation is enriched with 5-10% CO2.

PHYSICAL APPEARANCE

Wallenstein Media should appear opaque, and pale green to pale blue in color.

M. tuberculosis growing on LJ Medium

Mycobacterium tuberculosis H37Ra (ATCC ® 25177) colonies growing on Wallenstein Medium (Cat. no. C61). Incubated in CO 2 for 21 days at 35ºC.

M. kansasii Group I growing on LJ Medium

Mycobacterium kansasii Group I (ATCC ® 12478) colonies growing on Wallenstein Medium (Cat. no. C61). Incubated in CO 2 for 21 days at 35ºC.


M. scrofulaceum Group II growing on LJ Medium

Mycobacterium scrofulaceum Group II (ATCC ® 19981) colonies growing on Wallenstein Medium (Cat. no. C61). Incubated in CO 2 for 21 days at 35ºC.

M. intracellulare Group III growing on LJ Medium

Mycobacterium intracellulare Group III (ATCC ® 13950) colonies growing on Wallenstein Medium (Cat. no. C61). Incubated in CO 2 for 21 days at 35ºC.


M. fortuitum Group IV growing on LJ Medium

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Wallenstein Medium (Cat. no. C61). Incubated in CO 2 for 21 days at 35ºC.

LJ Medium

Uninoculated tube of Wallenstein Medium (Cat. no. C61).



REFERENCES

1. 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.

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

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

4. Am. J. Clin. Path.; 11:108, 1941.

5. Laboratory Methods for Clinical and Public Health in Mycobacteriology. 1967. USPHS, CDC Pub. 1547.

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

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

8. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

9. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

10. Vestal, A.L. 1975. Procedures of the Isolation and Identification of Mycobacteria. DHEW (CDC 75-8230). Centers for Diseases Control. Atlanta, GA.

11. Public Health Mycobacteria, A Guide for the Level III Laboratory. 1985. U.S. Dept. of Health & Human Services, Public Health Service, Centers for Disease Control, Atlanta, GA.


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

041816gr