LOWENSTEIN JENSEN (LJ) MEDIA

Cat. no. C21 Lowenstein Jensen, 20x125mm Tube, Slant 20 tubes/box
Cat. no. X22 Lowenstein Jensen, 50ml Hardy Flask™, Slant 20 flasks/box
Cat. no. C23 LJ, Gruft Modification, 20x125mm Tube, Slant 20 tubes/box
Cat. no. X23 LJ, Gruft Modification, 50ml Hardy Flask™, Slant 20 flasks/box
Cat. no. C25 LJ, Selective, 20x125mm Tube, Slant 20 tubes/box
Cat. no. C28 LJ with Pyruvate, 20x125mm Tube, Slant 20 tubes/box
Cat. no. X19 LJ with Pyruvate, 50ml Hardy Flask™, Slant 20 flasks/box
Cat. no. C37 LJ with Iron, 20x125mm Tube, Slant 20 tubes/box
Cat. no. X21 LJ with Iron, 50ml Hardy Flask™, Slant 20 flasks/box

INTENDED USE

Hardy Diagnostics Lowenstein Jensen Media are recommended for use in the cultivation and isolation of Mycobacterium species.

SUMMARY

The original formulation of Lowenstein Jensen media was developed by Lowenstein who incorporated congo red and malachite green to inhibit unwanted bacteria.(8,9) 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.(14) Malachite green acts as an inhibitory agent toward microorganisms other than mycobacteria.(6)

The Lowenstein Jensen, Gruft Modification formula is the same as the LJ formulation except for the addition of penicillin, nalidixic acid and ribonucleic acid (RNA).(4) Nalidixic acid and penicillin inhibit most of the gram-negative bacteria. RNA serves as a growth stimulant for the increased isolation rate of mycobacteria.

Lowenstein Jensen, Selective media, in addition to LJ ingredients, contains cycloheximide, lincomycin, and nalidixic acid. Cycloheximide suppresses saprophytic fungi while lincomycin inhibits gram-positive bacteria.

Lowenstein Jensen with Pyruvate incorporates pyruvic acid into the LJ basal medium to stimulate the growth of M. bovis and mycobacteria spp. other than M. tuberculosis.

Lowenstein Jensen with Iron is used to determine iron uptake as a means of differentiation between slow and rapid growing Mycobacterium species.(3,7) This is done by using an aqueous solution of 20% ferric ammoninum citrate. The ability of certain species, such as M. fortuitum, to take up soluble iron salts from the culture media results in the growth of colonies with a rusty brown color.

FORMULA

Ingredients per 367.5ml of deionized water:*

Lowenstein Jensen Medium:

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

Lowenstein Jensen, Gruft Modification is the same as LJ Medium with the addition of:

Nalidixic Acid 56.0mg
Penicillin 52.8mg
Ribonucleic Acid 0.05mg

Lowenstein Jensen, Selective is the same as LJ Medium with the addition of:

Nalidixic Acid 35.0mg
Lincomycin 2.0mg
Cycloheximide 0.4gm

Lowenstein Jensen with Pyruvate is the same as LJ Medium with the addition of:

Pyruvic Acid 2.5mg

Lowenstein Jensen with Iron is the same as LJ Medium with the addition of:

Ferric Ammonium Citrate 10.0gm

Final pH 7.2 +/- 0.3 at 25°C (except Cat. Nos. C28 and X19 for which the final pH is 7.1 +/- 0.3 at 25°C).

* 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

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.(1,3,5,7,11,15)

Method of Use:

1. Inoculate the Lowenstein Jensen Media with specimen after decontamination and neutralization, according to test procedures recommended by the Centers for Disease Control. Consult listed references for methods.(1,3,5,7,11,15)

2. Incubate medium in a CO2 atmosphere at 35-37ºC. 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.

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 on the first day growth is observed.

6. Consult appropriate references for recording the number of colonies and for aid in the biochemical identification of acid-fast bacilli.(1,5,11,15)

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth of Mycobacterium species on this medium.(1,3,5,7,11,15) Examine and record each type of colony morphology, pigment, and growth rate. Biochemical testing is required for definitive identification.

LIMITATIONS

Lowenstein Jensen 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.(2)

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, slides, decontamination supplies, applicator sticks, pipets, incinerators, CO2 incubators, microscopes, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
LJ Media and LJ Media with Iron:
Mycobacterium tuberculosis
H37Ra
ATCC® 25177
G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium kansasii
Group I
ATCC® 12478
G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium scrofulaceum
Group II
ATCC® 19981
G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium intracellulare
Group III
ATCC® 13950
G 21 days 35°C CO2** Growth; colonies seen in 2 weeks, mature in 3 weeks
Mycobacterium fortuitum
Group IV
ATCC® 6841
G 21 days 35°C CO2** Growth; colonies visible in 4 days
Additionally, the following organisms are tested on LJ, Gruft Modification and LJ, Selective:
Escherichia coli
ATCC® 25922
B 24hr 35°C Aerobic Partial to complete inhibition
Staphylococcus aureus
ATCC® 25923
B 24hr 35°C Aerobic Partial to complete inhibition

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

User Quality Control

PHYSICAL APPEARANCE

All Lowenstein Jensen Media should appear opaque, and pale green in color. LJ Media with iron may contain a precipitate settled at the bottom of the flask or tube.

M. tuberculosis growing on LJ Medium

Mycobacterium tuberculosis H37Ra (ATCC® 25177) colonies growing on Lowenstein Jensen Medium (Cat. no. C21). Incubated in CO2 for 21 days at 35ºC.

M. kansasii Group I growing on LJ Medium

Mycobacterium kansasii Group I (ATCC® 12478) colonies growing on Lowenstein Jensen Medium (Cat. no. C21). Incubated in CO2 for 21 days at 35ºC.




M. scrofulaceum Group II growing on LJ Medium

Mycobacterium scrofulaceum Group II (ATCC® 19981) colonies growing on Lowenstein Jensen Medium (Cat. no. C21). Incubated in CO2 for 21 days at 35ºC.

M. intracellulare Group III growing on LJ Medium

Mycobacterium intracellulare Group III (ATCC® 13950) colonies growing on Lowenstein Jensen Medium (Cat. no. C21). Incubated in CO2 for 21 days at 35ºC.




M. fortuitum Group IV growing on LJ Medium

Mycobacterium fortuitum Group IV (ATCC® 6841) colonies growing on Lowenstein Jensen Medium (Cat. no. C21). Incubated in CO2 for 21 days at 35ºC.

LJ Medium

Uninoculated tube of Lowenstein Jensen Medium (Cat. no. C21).


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

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

4. Gruft, H. 1965. J. Bacteriol., 90:829.

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

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

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

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

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

10. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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

12. 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.

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

14. Schaeffer, W.B. 1952. Growth requirements of dysgonic and eugonic strains of Mycobacterium tuberculosis var bovis. J. Exp. Med. 96(3): 207-219.

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

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

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