LOWENSTEIN JENSEN DEEP

Cat. no. C27 Lowenstein Jensen Medium, 20x125mm Tube, 5ml Deep 20 tubes/box

INTENDED USE

Hardy Diagnostics Lowenstein Jensen Deep is recommended for use in the semiquantitative catalase test to aid in differentiation 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. (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. A tubed deep of Lowenstein Jensen Media was developed by Wayne and is useful for the classification of mycobacteria.

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. (5)

Acid-fast bacilli produce catalase, an intracellular, soluble enzyme. This enzyme is capable of splitting hydrogen peroxide into water and oxygen. The production of gas, apparent by the presence of oxygen bubbles, indicates catalase activity. The semiquantitative catalase test divides the mycobacteria into two groups, those that produce less than 45mm of bubbles and those that produce more than 45mm of bubbles. M. kansasii, M. simiae , most scotochromogens, the non-photochromogenic saprophytes and the rapid growers usually produce more than 45mm of bubbles. M. tuberculosis, M. marinum, M. avium complex, M. xenopi and M. gastri are among the mycobacteria that produce less than 45mm of bubbles. (2,3,8)

FORMULA

Ingredients per liter of deionized water:*

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

* 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. (1-3,6,7,12)

Method of Use:

1. Inoculate the medium with either 0.1ml of a 7-day liquid culture or a loopful of growth from an actively growing slant.

2. Inoculate test organism(s) as well as a strong catalase producer ( M. kansasii ) and a weak catalase producer ( M. intracellulare ) as controls.

3. Incubate tubes aerobically or in 5-10% CO 2 , with loose caps, at 35-37ºC. for two weeks.

4. Prepare a fresh 1:1 mixture of 10% Tween 80 and 30% hydrogen peroxide.

5. Add 1ml of the Tween ® 80-peroxide mixture to each culture.

6. Allow the tubes to stand at room temperature for 5 minutes before measuring the height of the columns of bubbles. Record, in millimeters, the height of the column of bubbles above the medium surface.

LIMITATIONS

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

INTERPRETATION OF RESULTS

An uninoculated tube of medium should be used as a negative control.

In the semiquantitative catalase test, most mycobacteria fall into two groups: (2,3,6,7,12)

1. Column of bubbles >45mm height above medium surface

M. chelonae M. scrofulaceum M. simiae
M. fortuitum M. ulcerans M. triviale
M. gordonae M. terrae complex M. smegmatis
M. kansasii M. marinum

2. Column of bubbles <45mm in height above medium surface

M. avium complex M. bovis M. gastri
M. haemophilum M. intracellulare M. malmoense
M. tuberculosis M. xenopi

Refer to listed references for additional information. (2,3,6,7,12)

MATERIALS REQUIRED BUT NOT PROVIDED

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

QUALITY CONTROL

Lowenstein Jensen Deeps are tested for growth performance only.

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

PHYSICAL APPEARANCE

Lowenstein Jensen Media should appear opaque, and pale green in color.

M. tuberculosis growing on Lowenstein Jensen Deep

Mycobacterium tuberculosis H37Ra (ATCC ® 25177) growing on Lowenstein Jensen Deep (Cat. no. C27). Incubated in CO 2 for 21 days at 35ºC.

M. kansasii growing on Lowenstein Jensen Deep

Mycobacterium kansasii Group I (ATCC ® 12478) growing on Lowenstein Jensen Deep (Cat. no. C27). Incubated in CO 2 for 21 days at 35ºC.



M. scrofulaceum growing on Lowenstein Jensen Deep

Mycobacterium scrofulaceum Group II (ATCC ® 19981) growing on Lowenstein Jensen Deep (Cat. no. C27). Incubated in CO 2 for 21 days at 35ºC.

M. intracellulare growing on Lowenstein Jensen Deep

Mycobacterium intracellulare Group III (ATCC ® 13950) growing on Lowenstein Jensen Deep (Cat. no. C27). Incubated in CO 2 for 21 days at 35ºC.



M. fortuitum growing on Lowenstein Jensen Deep

Mycobacterium fortuitum Group IV (ATCC ® 6841) growing on Lowenstein Jensen Deep (Cat. no. C27). Incubated in CO 2 for 21 days at 35ºC.



bubbles > 45mm

Semiquantitative Catalase Test for Mycobacterium fortuitum ATCC ® 6841. Incubated in CO 2 for two weeks at 35ºC. in a Lowenstein-Jensen Deep (Cat. no. C27). Equal parts of 10% Tween ® 80 (Cat. no. Z101) and 30% hydrogen peroxide were mixed and a 1mL aliquot was added to the tube. The tube was allowed to stand at room temperature for five minutes before the bubbles were measured.

bubbles > 45mm

Close up of the tube on the left. Showing that the height of the bubbles was greater than 45mm.

bubbles < 45mm

Semiquantitative Catalase Test for Mycobacterium intracellulare ATCC ® 13950. Incubated in CO 2 for two weeks at 35ºC. in a Lowenstein-Jensen Deep (Cat. no. C27). Equal parts of 10% Tween ® 80 (Cat. no. Z101) and 30% hydrogen peroxide were mixed and a 1mL aliquot was added to the tube. The tube was allowed to stand at room temperature for five minutes before the bubbles were measured.

bubbles < 45mm

Close up of the tube on the left. Showing that the height of the bubbles was less than 45mm.

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

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

12. 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.
Tween is a registered trademark of ICI Americas, Inc.

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