Lowenstein jensen medium

The presentation tells details of the Lowenstein-Jensen medium a selective medium for mycobacterium.

1. 01/10/2018 1 Löwenstein –Jensen medium Presented By- Mr. Sushant Balasaheb Jadhav M. Tech. Pharma . Biotech. Roll No. – 18PBT206

2. 01/10/2018 2 Lowenstein-Jensen (LJ)  is the selective medium which is used for the cultivation and isolation of  Mycobacterium species. It  was developed by  Lowenstein  who incorporated congo red and malachite green to inhibit unwanted bacteria . 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 . Löwenstein –Jensen medium

3. 01/10/2018 3 Jensen’s modification formulation also encourages the earliest possible growth of mycobacteria . When grown on LJ medium,  M. tuberculosis  appears as brown, granular colonies (sometimes called " buff, rough and tough "). The medium must be incubated for a significant length of time , usually four weeks , due to the slow doubling time of  M. tuberculosis  (15–20 hours) compared with other bacteria . Löwenstein –Jensen medium

4. 01/10/2018 4 Composition of LJ Medium Ingredients Amount Potato Flour (Potato Starch) 30.0 gm L-Asparagine 3.6 gm Monopotassium Phosphate 2.4 gm Magnesium Citrate 0.6 gm Malachite Green 0.4 gm Magnesium Sulfate 0.24 gm Glycerol 12 ml Egg suspension 1000 ml Distilled Water 600 ml

5. 01/10/2018 5 The original formulation included starch , which was later found to be unnecessary, so omitted . Low levels of  penicillin  and nalidixic acid  are also present in LJ medium to inhibit growth of Gram-positive and Gram-negative bacteria, to limit growth to  Mycobacterium  species only. Presence of malachite green in the medium inhibits most other bacteria . It is disinfected and solidified by a process of inspissation. Composition of LJ Medium

6. 01/10/2018 6 Composition of LJ Medium Presence of glycerol enhances the growth of  M. tuberculosis . If the slopes are made on test tubes, they must be stored in cold and used within a month . For cultivation of M. bovis , glycerol is omitted and  sodium pyruvate is added . The medium appears green, opaque, and opalescent .

7. 01/10/2018 7 Preparation of LJ Medium Dissolve 37.3 gm of the medium in 600 ml of distilled water containing 12 ml of glycerol . Heat if necessary to dissolve the medium completely . Autoclave at 121°C for 15 minutes . Prepare 1000 ml of a uniform suspension of fresh eggs under aseptic conditions. Avoid whipping air into suspension during the collection and mixing .

8. 01/10/2018 8 Aseptically mix the 1000 ml of egg suspension with 600 ml of the sterile Lowenstein-Jensen Medium cooled to 50 – 60°C , avoiding air bubbles . Dispense the finished medium into sterile screw-cap test tubes . Place the tubes in a slanted position and heat at 85°C for 45 minutes. Preparation of LJ Medium

9. 01/10/2018 9 Principle of LJ Medium L-Asparagine  and  Potato Flour  are sources of nitrogen and vitamins.  Monopotassium Phosphate  and  Magnesium Sulfate  enhance organism growth and act as buffers . Malachite green,  prevent the growth of the majority of contaminants surviving decontamination of the specimen while encouraging the growth of Mycobacteria.

10. 01/10/2018 10 Egg Suspension  provide fatty acids and protein required for the metabolism of mycobacteria . When heated, the  egg albumin  coagulates, thus providing a solid surface for inoculation . 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. Principle of LJ Medium

11. 01/10/2018 11 TYPES

12. 01/10/2018 12 Colony Morphology on LJ Medium Cultures should be read within 5 to 7 days after inoculation and once a week thereafter for up to 8 weeks . Typical non pigmented, rough, dry colonies are seen on LJ medium. The green color of the medium is due to the presence of malachite green which is one of the selective agents.

13. 01/10/2018 13 Uses For diagnosis of mycobacterial infections For testing antibiotic susceptibility of isolates For differentiating different species of  Mycobacterium  (by colony morphology, growth rate, biochemical characteristics, and microscopy ) Distinctive clusters of colorless  Mycobacterium tuberculosis

14. 01/10/2018 14 Mycobacterium tuberculosis Mycobacterium bovis Eugonic , rough tough and buff Dysgonic Aerobic Microaerophillic Glycerol enhancement + Glycerol enhancement - Pyruvate enhancement + Pyruvate enhancement - Niacin production + Niacin production - Uses

15. 01/10/2018 15 Limitations of LJ Medium It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification . LJ Media require incubation in a 5-10% CO 2 atmosphere in order to recover mycobacteria. Mycobacteria, for unknown reasons, are not recovered well from candle extinction jars .

16. 01/10/2018 16 Negative culture results do not rule-out active infection by mycobacteria . Due to nutritional variation , some strains may be encountered that grow poorly or fail to grow on this medium. Further tests are necessary for confirmation of  Mycobacterium  spp. Limitations of LJ Medium

17. 01/10/2018 17 Alternative culture media While the LJ medium is the most popular means of culturing mycobacteria , as recommended by the International Union against Tuberculosis , several alternative media have been investigated Solid media Egg-based – Petragnani medium and Dorset medium Blood-based – Tarshis medium Serum-based – Loeffler medium Potato-based – Pawlowsky medium Liquid media Dubos' medium Proskauer and Beck's medium Sula's medium Sauton's medium

18. 01/10/2018 18 Rapid detection techniques The chief limitation of culture-based techniques is the time it takes to culture positivity, which can be several months . Several new molecular technologies have emerged in recent years to secure more speedy confirmation of diagnosis. Polymerase chain reaction GeneXpert MTB/RIF Loop-mediated isothermal amplification

19. 01/10/2018 19 THANK YOU