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

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

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 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

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 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

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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

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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

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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.

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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.

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 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

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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.

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 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

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TYPES
tubed as
deeps
• semi-quantitative catalase test
Gruft
• Addition of penicillin, nalidixic acid, rna
• Isolation and cultivation of mycobacteria
with
Iron
• to determine iron uptake for differentiation and
identification of mycobacteria
with
Pyruvic
Acid
• enrichment medium used for enhanced growth of
tubercle bacilli
with 5%
sodium
chloride
• used to characterize certain strains of
mycobacteria

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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.

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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

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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

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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% CO2
atmosphere in order to recover mycobacteria.
Mycobacteria, for unknown reasons, are not recovered
well from candle extinction jars.

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 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

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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

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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

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