Lowenstein Jensen Medium Base w/o Starch

Intended Use

Recommended for drug resistance testing of Mycobacteria in accordance with WHO.

Composition**

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 7.24 grams in 600 ml purified/distilled water containing 12 ml glycerol (for bovine bacteria or other glycerophobic organisms, addition of glycerol is not desirable). Heat if necessary to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Meanwhile prepare 1000 ml of whole egg emulsion collected aseptically. Add and mix egg emulsion base gently to obtain uniform mixture. Distribute in sterile screw capped tubes. Arrange tubes in a slanted position. Coagulate and inspissate the medium in an inspissator, water bath or autoclave at 85°C for 45 minutes.

Principle And Interpretation

The original LJ medium was formulated by Lowenstein (1) and modified by Jensen (2) and Gruft (3,4) with addition of two antimicrobial agents. Lowenstein Jensen Medium Base w/o starch is recommended for resistance testing by WHO. Lowenstein-Jensen (L-J) Medium without potato starch with drugs incorporated before inspissation is the modification of the International Union Against Tuberculosis (IUAT) (5,6,7).

Malachite green prevents growth of the majority of contaminants that survived the decontamination procedures for the specimen, thus encouraging earliest possible growth of Mycobacteria. Do not add glycerol to the medium if bovine or other glycerophobic strains are to be cultured (8). Malachite green serves as an inhibitor and also as a pH indicator. Formation of blue zone indicates a decrease in pH by gram-positive contaminants (e.g. Streptococci) and yellow zones indicate dye destruction by gram-negative bacilli. Proteolytic contaminants cause localized or complete digestion of medium. Hardy et al (9) recommended each specimen to be inoculated and incubated in triplicate, so as

a. To identify saprophytes at room temperature (25°C).

b. To identify presence or absence of pigmentation by photochromogenes and scotochromogenes at 35°C alternately in light and dark as per the type of organism.

Routinely, cultivation is carried out aerobically at 35°C.

Refer appropriate references for standard test procedures of decontamination and isolation (5,10,11,12,13).

Type of specimen

Clinical samples : Sputum

Specimen Collection and Handling

For clinical samples: Refer appropriate references for standard test procedures of decontamination and isolation (10-15). After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions

In Vitro diagnostic use only. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.

Limitations

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user’s unique requirement.

Performance and Evaluation

Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.

Quality Control

Appearance Greenish blue to peacock blue homogeneous free flowing powder

Colour and Clarity of prepared medium The mixture of sterile basal medium and whole egg emulsion, when inspissated, coagulates to yield pale bluish green coloured opaque, smooth slants

Cultural Response Cultural characteristics observed in presence of 5-10% Carbon dioxide (CO2), with added egg emulsion base, after an incubation at 35-37°C for 2-4 weeks.

Storage and Shelf Life

Store below 10-30°C in a tightly closed container and the prepared medium at 2-8°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle inorder to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within stated expiry period.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (10-15).

Reference

1. Lowenstein E., 1931, Zentralb. Bacteriol., Parasitenked. Infekitonskr. Abt. I. Orig., 120:127.
2. Jensen K. A., 1932, Zentralb. Bacteriol., Parasitenked. Infekitonskr. Abt. I. Orig., 125:222.
3. Gruft, 1963, Am. Rev. Respir. Dis., 88:412.
4. Gruft, 1971, Health Lab. Sci., 8:79.
5. International Union Against Tuberculosis and Lung Disease. Public Health Service National Tuberculosis Reference Laboratory and the National Laboratory Network. Minimum reqirements. Role and operation in low-income country, Paris 1998.
6. Jensen K.A. Towards, a standardization of Laboratory methods. Second report of the Sub committee of laboratory methods of the IUAT. BILL Int Union Tuberc. 1955 : 25 (1-2):89-104.
7. World Health Organization laboratory services in tuberculosis control, part III culture. Geneva 1998: Publication No.WHO/TB/98:258.
8. MacFaddin J. F., 1985, Media for Isolation-Cultivation-Identification-Maintenance of Medical Bacteria, Vol. 1, Williams and Wilkins, Baltimore.
9. Hardy A. V. et al, 1958, Am. J. Publ. Hlth., 48 (1):754.
10. Cernoch P., Enns R., Saubolle M. and Wallace R., 1994, Cumitech, 16A, Laboratory Diagnosis of the Mycobacterioses coord , Ed., Weissfeld, ASM, Washington, D. C.
11. Forbes B. A., Sahm A. S. and Weissfeld D. F., Bailey & Scotts Diagnostic Microbiology, 10th Ed., 1998, Mosby, Inc.,St. Louis, Mo.
12. Isenberg, (Ed.), 1992, Clinical Microbiology Procedures Handbook, Vol. I, ASM, Washington, D. C.
13. Kent P. T and Kubica G. P., 1985, Public Health Mycobacteriology: A Guide to the level III Laboratory, USDHHS, Centers for Disease Control, Atlanta, Ga.
14. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock., D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
15. Murray P. R., Baron E. J., Jorgensen J. H., Pfaller M. A., Yolken R. H., (Eds.), 8th Ed., 2003, Manual of clinical Microbiology, ASM, Washington, D.C.