Lowenstein Jensen (LJ) Media Composition, Preparation, Uses

Lowenstein Jensen (LJ) Media

Lowenstein Jensen Medium (LJ Medium) is a highly selective medium.

Solid media that are used to isolate as well as cultivation of Mycobacteria can be egg-based or Agar-based.
Egg-based media comprise whole eggs or egg yolks potatoes, salts, potato flour and glycerol, and are cured through inspissation. The egg-based media are LowensteinJensen Medium is the most frequently employed.

Lowenstein Jensen (LJ) Medium was initially developed by Lowenstein and contains Congo red and malachite Green dyes.
Jensen altered Lowensteins Medium by changing its citrate and phosphate content by removing the congo red dye, and by increasing the malachite-green concentration.
Gruft changed Lowenstein Jensen (LJ) Medium by adding two antimicrobics in order to improve the selectivity.

This medium helps in development of broad Mycobacteria species and is also suitable to test for niacin.

Lowenstein Jensen (LJ) Media Principle

L-Asparagine as well as Potato Flour are rich in nutrients and nitrogen. Monopotassium Phosphate and Magnesium Sulfate both enhance the growth of living organisms and act as buffers. Malachite green blocks the development of the majority of contaminants that are able to survive the decontamination process and also encourages Mycobacteria growth. Egg Suspension contains essential fatty acids as well as protein to help Mycobacteria’s metabolism. If heated the egg albumin will coagulate creating an ideal surface for the inoculation. Glycerol is an energy source for carbon and can be beneficial to the growth of the human tubercle bacillus, but not to bovine tubercle.

In the Gruft method Penicillin and Nalidixic acid, along with malachite green stop the development of the majority of contaminants that remain after decontamination of specimen. It also encourages the earliest development of mycobacteria. It acts as a stimulant and helps to improve Mycobacteria’s isolation rate.

Lowenstein Jensen (LJ) Media Composition and Ingredients

Mineral salt solution

Ingredients	Amount
Potassium dihydrogen phosphate anhydrous (KH2PO4)	2.4 g
Magnesium sulphate anhydrous: (MgSo4.7H20)	0.24 g
Magnesium citrate	0.6 g
Asparagine	3.6 g
Glycerol (reagent grade)	12 ml
Distilled water	600 ml

Mix the components in the correct orderin distilled water through heating. Autoclave at 121°C for thirty minutes in order to clean. Cool to ambient temperature. The solution is stored for a long time and can be stored in adequate quantities inside the refrigerator.

Malachite green solution 2%

Ingredients	Amount
Malachite green dye	2.0 g
Distilled water	100 ml

*Dissolve the dye into the distillate water completely. Sort it out and place at room temperature in the freezer.

Homogenized whole eggs

Take a fresh (those do not exceed 7 days old) eggs from a hen

Cleanse the eggs by rubbing thoroughly with a toothbrush in soap and water.
Allow the eggs to soak at least 30 minutes in soapy water.
Wash eggs thoroughly in running water. Soak eggs in 70 percent ethanol for 15 minutes.

Prior to handling the eggs, scrub them dry and rinse your hands with disinfectant.
Crack the eggs using the beaker’s edge into a sterilized flask, and beat them with a sterilized blender in 30 second intervals to a minute.

Lowenstein Jensen (LJ) Media Preparation

Aseptically collect the following reagents within the flask, which is large and sterile, and mix them thoroughly:

Ingredients	Amount
Mineral salt solution	600ml
Malachite green	20 ml
Homogenised eggs (25-30 eggs, depending on size)	1000ml

The egg medium in its entirety is divided into 6-8ml portions within sterile, universal bottles, or bottles for culture (14 Ml or 28ml) with caps are shut and then inspissated immediately to avoid the sedimentation of the heavier ingredients.

The majority of cultures are produced in bottles rather than in Petri dishes due to the length of time needed for incubation. The use of bottles reduces the possibility of contamination as well as drying of the media (if they are securely shut).

Coagulation of medium

The inspissator should be heated to 85 degrees Celsius to accelerate the increase in the temperature prior to loading.

Set the bottles in a slanted orientation inside the inspissator. Then, make the medium coagulate in 50 minute increments, at a temperature of 85degC (since the medium was made with sterile precautions, the purpose of this heating is to harden this medium and is not clean it).

The egg’s quality gets worse when coagulation happens at a high temperature or over a long period of time. The discoloration of the coagulated media could be caused by excessive temperatures. The appearance of tiny bubbles or holes at the top of the medium could be a sign of a malfunctioning coagulation procedure.

Sterility check of Lowenstein Jensen (LJ) Media

After inspissation of the entire media batch in the media bottles must be kept at 35degC to 37degC for 24 hours for a test for sterility of the bacterium.

After 24 hours, 5 percent of the slopes must be randomly picked and incubated for 14 days in order to test for fungal sterilization.
In both cases, the contamination rate shouldn’t exceed 10 %.

Storage

The LJ medium must be date and kept with the batch number in the refrigerator. It will last up to 4 weeks in the event that the caps are kept tightly shut to stop drying the medium.

Inoculation and Incubation of Lowenstein Jensen (LJ) Media

Two slopes LJ medium are recommended on each sample (an additional slope should be inoculated with pyruvate is recommended in M. bovis-endemic areas).

Take out the condensed moisture prior to the inoculation.
Each slope should be inoculated by inoculating each slope with 0.2-0.4 milliliters (2-4 drops, or 2 – 4 loopfuls) of the sediment that has been centrifuged and spread over the entire surface.

Keep the cultures incubating at 35 to 37 degrees Celsius until the growth is visible or the cultures are discarded as negative after 8 weeks.

Result Interpretation of Lowenstein Jensen (LJ) Media

The culture should be read between 5-7 days after the vaccination and every week after that for up to 8 weeks.
The most common colonies are pigmented, rough, and dry on medium LJ.

The green hue that the material has is caused due to malachite green , which acts as one of the agents selective that prevent the growth of many other contaminants.
Rapid growers can mature their colonies in 7 days. slow growers take longer than 7 days to mature colonies.
Pigment production:

White, cream or buff = Nonchromogenic (NC)
Lemon, yellow, orange, red = Chromogenic (Ch)

Colony Characteristic of Different Microorganisms on Lowenstein Jensen (LJ) Media

Organism	Colony Characteristic
Mycobacterium avium	smooth, nonpigmented colonies
Mycobacterium gordonae	smooth, yellow, orange colonies
Mycobacterium kansasii	photochromogenic, smooth to rough
Mycobacterium smegmatis	wrinkled,creamy white colonies
M. tuberculosis	granular, rough, warty, dry friable colonies

Examination Schedule

Every culture should be examined for 72 hours following the inoculation to ensure that the liquid has evaporated completely, to seal the caps tight to stop drying out of the media and to identify contaminants. After that, the cultures are reviewed every week, or when this isn’t operationally feasible, at least three times, namely

After one week, it is possible to identify mycobacteria rapidly growing that could be confused with M. tuberculosis.
After 3-4 weeks, you can detect positive culture from M. tuberculosis, as well with other mycobacteria that slow-growing which could be harmless saprophytes or pathogens
After 8 weeks, it is possible to identify very slow-growing mycobacteriasuch as M. tuberculosis. Before concluding the culture is negative.

Lowenstein Jensen (LJ) Media Uses

It is used to aid in the treatment of Mycobacterial infections.
It is used to test susceptibility to antibiotics in isolates.
It can also be used to distinguish the different mycobacterium species (by colony appearance and the rate of growth, biochemical characteristics, and microscopy).

Limitations of Lowenstein Jensen (LJ) Media

It is suggested that serological or biochemical tests be conducted on colonies of pure culture for full confirmation of the species.
LJ Media require incubation in an atmosphere of 5-10% CO2 to help recover Mycobacteria. Mycobacteria, for a variety of reasons, do not recover efficiently from candle extinction containers.
Results of negative culture cannot rule out mycobacteria-related active infections.

Because of the variation in nutrition Certain strains could be discovered that do not grow well or are unable to develop in this medium. More tests are needed to confirm the presence for Mycobacterium spp.
The media must be shielded from any light source because malachite is extremely photosensitive.

References

https://en.wikipedia.org/wiki/L%C3%B6wenstein%E2%80%93Jensen_medium

Laboratory services in Tuberculosis Control, Part III: Culture, World Health Organization
https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/LJMedia.htm
http://himedialabs.com/td/m162.pdf