LOWENSTEIN-JENSEN MEDIUM WITH 5% SODIUM CHLORIDE

Cat. no. C29 Lowenstein-Jensen with 5% NaCl, 20x125mm Tube,
10ml Slant
20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Lowenstein-Jensen with 5% NaCl is used to test Mycobacterium species for salt tolerance.

SUMMARY

The original formulation of Lowenstein-Jensen media was developed by Lowenstein who incorporated congo red and malachite green to inhibit unwanted bacteria. (9,10) The present formulation, a glycerated egg-based medium, is based upon Jensen's modification. Jensen's version eliminates congo red and uses a moderate concentration of malachite green to prevent growth of the majority of contaminants surviving decontamination of the specimen. This formulation also encourages the earliest possible growth of mycobacteria.

When heated, the egg albumin coagulates, thus providing a solid surface for inoculation. Nitrogen, fatty acids, and proteins are supplied by egg and asparagine. 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 acts as an inhibitory agent toward microorganisms other than mycobacteria. (11)

The addition of 5% NaCl to the Lowenstein-Jensen formulation allows differentiation of slowly growing mycobacteria from rapidly growing mycobacteria based on salt tolerance. This medium supports the growth of most rapid growers as well as the slower growing Mycobacterium triviale . Some strains of Mycobacterium flavescens may also grow. Distinction between other members of the Mycobacterium fortuitum complex from Mycobacterium chelonae subsp. chelonae is facilitated by the inability of the latter to grow on this salt containing medium. (15)

FORMULA

Ingredients per liter of deionized water:*

Sodium Chloride 50.0gm
Potato Flour 30.0gm
Asparagine 3.6gm
Monopotassium Phosphate 2.4gm
Magnesium Sulfate 0.24gm
Magnesium Citrate 0.6gm
Malachite Green 0.4gm
Glycerol 7.5ml
Egg Base 625.0ml

* 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, 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

Method of Use:

1. From a pure culture, prepare a suspension that is equivalent to a 1.0 McFarland Standard. (15)

2. Inoculate the Lowenstein-Jensen with NaCl slant with 100ul from the suspension. (15)

3. Incubate medium at 35ºC. in ambient air. (15)

4. Examine the media weekly for up to four weeks.

INTERPRETATION OF RESULTS

A positive test for sodium chloride tolerance is the growth of 50 or more colonies on the slant. A negative test is the appearance of less than 50 colonies. (15)

LIMITATIONS

Protect the media from all sources of light, as malachite green is very photosensitive.

Selective media often inhibit, to some extent, specific strains of organisms for which they are designed to select.

The color of LJ media may range from a pale-green to a dark blue-green. Do not use media that has turned yellow, as it will interfere with the interpretation of the pigmentation of mycobacteria. Most contaminating bacteria will turn the media blue.

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Mycobacterium fortuitum
Group IV
ATCC ® 6841

** 21 days 35°C Aerobic Growth; 50 or more colonies
Mycobacterium kansasii
Group I
ATCC ® 12478

** 21 days 35°C Aerobic Inhibited
Mycobacterium scrofulaceum
Group II
ATCC ® 19981

** 21 days 35°C Aerobic Inhibited
Mycobacterium intracellulare
Group III
ATCC ® 13950

** 21 days 35°C Aerobic Inhibited
Mycobacterium tuberculosis
H37Ra
ATCC ® 25177

** 21 days 35°C Aerobic Inhibited

User Quality Control

** Inoculate LJ with 5% NaCl with 100ul of a suspension equivalent to a 1.0 McFarland Standard.

PHYSICAL APPEARANCE

Lowenstein-Jensen Medium with 5% NaCl should appear opaque, and pale green in color.

M. fortuitum Group IV growing on LJ Medium w/ 5% NaCl

Mycobacterium fortuitum Group IV (ATCC ® 6841) colonies growing on Lowenstein-Jensen Medium with 5% NaCl (Cat. no. C29). Incubated aeobically for 21 days at 35ºC.

LJ Medium w/ 5% NaCl

Mycobacterium kansasii Group I (ATCC ® 12478) growth inhibited on Lowenstein-Jensen Medium with 5% NaCl (Cat. no. C29). Incubated aerobically for 21 days at 35ºC.

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. Cohn, M.L., et al. 1968. Am. Rev. Respir. Dis.; 98:295.

5. Gruft, H. 1965. J. Bacteriol.; 90:829.

6. IIsenberg, 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. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

9. Lowenstein, E. 1933. Ann. Inst. Pasteur.; 50:161.

10. Lowenstein, E. 1931. Zentralbl. Bakteriol. Parasetenkd. Infektionskr.; 120:127.

11. Jensen, F. 1932. Zentralbl. Bakteriol. Parasetenkd. Infektionskr., Abt. 1 Orig.; 125:222.

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

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

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

15. Conville, P.S. and F.G. Witebsky. 1998. J. Clinical Micro.; 36:6.


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031016gr