Module 4
Preparation of solid media
for culture and DST
1
Learning objectives
At the end of this module you will be able to:
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

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recognize the different media for mycobacteria
culture and explain their advantages/disadvantages;
prepare all the reagents, including drug solutions,
for preparation of media for culture and DST;
prepare and dispense the culture medium;
check the quality of tubes at the end of the process
and store them properly;
perform the sterility check.
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Content outline
• Description of culture media
• Preparation of plain culture media
• Preparation of selective and drugcontaining media
• Quality of media
3
Culture media
Two main groups:
• Egg-based media
– Löwenstein–Jensen medium
– Kudoh modified Ogawa medium (acid-buffered)
• Liquid media
– Herman-Kirchner liquid medium
– Dubos oleic acid–albumin liquid medium
– Middlebrook 7H9 liquid medium
4
Culture media
The ideal medium should:
– be economical and simple to prepare;
– inhibit the growth of contaminants;
– support luxuriant growth of small
numbers of bacilli;
– have long shelf-life.
5
Egg-based media: advantages
•
•
•
•
•
•
Easy to prepare.
Cheap.
Support good growth of tubercle bacilli.
Long shelf-life (several weeks at 4 ºC).
Limited contamination during preparation.
Malachite green minimizes the growth of nonmycobacterial organisms.
• Contamination may not cover all the surface of
the medium.
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Egg-based media: disadvantages
• Long time (up to 8 weeks) for evident
growth.
• Human resources, space, specific
equipment needed.
• Possible problems in obtaining eggs of
good quality, genuine inspissator.
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Specificities of egg-based media
• Löwenstein–Jensen (LJ):
– use for culture and DST;
– with pyruvate and without glycerol for M. bovis.
• Kudoh medium (acid-buffered Ogawa):
– no need for neutralization/centrifugation during
decontamination procedure;
– cannot be used for DST.
8
Liquid media
• Middlebrook 7H9 and modified versions
• Commercially available
• Automated systems available
9
Liquid media: advantages
• Shorter recovery time:
– culture solid: 16 days for smear-positive, 29 days for
smear-negative (on average);
– culture liquid: 8 days for smear-positive, 16 days for
smear-negative (on average).
• Increased sensitivity compared with solid media,
especially for:
– cerebrospinal fluid
– pleural fluid
– biopsies.
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Liquid media: disadvantages
• Prone to contamination
• Biosafety issue
• Cost
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Solid vs liquid media
Solid
Liquid
Cost
+
+++
Contamination
+
++
Semiquantitative results
+
Not possible
AFB + colonies
AFB
+
++
+++
+
Morphology
Biohazard
Isolation time
Use of combination of liquid and solid media increases sensitivity
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Plain egg-based media
preparation
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Media preparation room
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General precautions
1.
2.
3.
4.
5.
6.
7.
8.
9.
Keep the environment as clean as possible.
Use sterile glassware and equipment.
Use high-grade chemicals and reagents unless
otherwise specified.
Check temperature of inspissators.
Follow strict aseptic techniques when preparing
media, e.g. flaming flasks and tubes.
Clean and disinfect shells before breaking eggs.
Do not overheat media during inspissation.
Do not leave prepared media exposed to light.
Do not skimp on the volume of medium.
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Choice of glassware
• Reusable glass tubes sealed with screwcaps and made from resistant borosilicate
laboratory glass
• 14-ml McCartney bottles
• 5-ml bijou bottles
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Cleaning of glassware
•
•
•
•
Brush glassware in hot water.
Eliminate residue.
Rinse repeatedly in hot water.
Rinse in distilled water.
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Egg-based media
1. Prepare mineral salt solution.
2. Prepare malachite green solution, 2%.
Note: 1 and 2 are commercially available.
3. Homogenize whole eggs (20–25 eggs for 200
tubes).
4. Prepare complete medium.
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Egg-based media composition
Components
LJ, modified
Ogawa
Ogawa modified (Kudoh)
Monopotassium dihydrophosphate
(KH2PO4), anhydrous
2.4 g
6g
12 g
Magnesium sulfate (MgSO4 ·7H2O)
0.24 g
–
Magnesium citrate
0.6 g
–
L-Asparagine
3.6 g
–
–
6g
3g
600 ml
600 ml
600 ml
12 ml or 7.2 g
36 ml
24 ml
1000 ml
1200 ml
1200 ml
20 ml
36 ml
24 ml
6.8
6.8
6.4
Sodium glutamate
Distilled water up to
Glycerol (ml) or pyruvatea (g)
Egg homogenate
Malachite green (2%)
pH (about)
0.6 g
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Egg preparation
1. Wash eggs with soap and water.
2. Soak eggs in 70% ethanol for 15
minutes.
3. Filter whipped eggs through
sterile gauze fabric.
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Media preparation
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Coagulation of medium
1. Before loading, heat the inspissator to 80 ºC.
2. Place the bottles in a slanted position.
3. Coagulate the medium for 45 minutes at
80–85 ºC in 80% relative humidity.
4. Do not heat further.
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Pay attention!
• Quality of egg-based media deteriorates
when coagulation is done at too high a
temperature or for too long.
• Discolouration of coagulated medium may be
due to excessive temperature or prolonged
heating time.
• Small holes or bubbles on the surface of the
medium indicate faulty coagulation
procedures.
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Quality control and storage
Sterility check
• Incubate the whole media batch at 35–37 ºC for
48 hours. Discard if there is any growth.
Storage
• Date media and store in the refrigerator.
• Media will keep for several weeks if caps are
tightly closed to prevent drying out.
• Slants should not be older than 2 months.
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Media log-sheet – first part
Preparation: SALT SOLUTION
Operator's name:
Date of preparation:
Quantity
(ml or g)
Reagents
Manufacturer
Reference
Batch
number
Expiry
date
Monopotassium
dihydrophosphate (KH2PO4),
anhydrous.
Magnesium sulfate
(MgSO4 ·7H2O)
Magnesium citrate
L-Asparagine
Sodium glutamate
Glycerol (ml) or pyruvate* (g)
Distilled water up to
Autoclave cycle
Operator's name
Date:
Time (min)
Temperature (ºC)
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Media log-sheet – second part
Preparation: Second step
Preparation date:
Operator’s name:
Reagents
Manufacturer
Reference
Batch number
Quantity
(ml or g)
Malachite green
Eggs
Number of eggs:
Inspissator
cycle
Date:
Operator's name:
Time (min)
Temperature
(ºC)
Sterility check
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Preparation of selective and
DST media
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Selective media
• LJ with pyruvate, without glycerol:
M. bovis
• LJ with p-nitrobenzoic Acid (PNB):
M. tuberculosis does not grow
Follow the procedure for preparation of culture media
already described and add the selected substances
(in correct dilutions) to the complete media before
they are distributed into tubes and inspissated.
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Mass concentrations for drugs
and critical concentration
Test drugs
Designation
Abbrev.
Isoniazid
Solvent
Final mass concentration in
culture medium (mg/litre)
Solvent
Dilution
For quality
control
H37Rv (MTB
control, strain)
Critical
concentration
INH
Sterile dw
Sterile dw
0.025•0.05•0.10
0.2
Rifampicin
RMP
DMSO
Sterile dw
2.5•5.0•10.0
40.0
Dihydrostreptomycin
DSM
Sterile dw
Sterile dw
0.5•1.0•2.0
4.0
Ethambutol
EMB
Sterile dw
Sterile dw
0.125•0.25•0.5
2.0
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Procedure
• Follow the procedure for preparation of
culture media.
• Add the selected substances (in solution)
to the complete media before distribution
into tubes and inspissation.
• Follow the procedure for inspissation and
all the precautions already described.
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Drugs
• Concentration is crucial for reliable DST.
• Use pure drug powders, not drugs for
patients’ treatment.
Amount to be weighed = 1/potency
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Preparation
• Method 1
Add 1% of drug solution to the basic culture
medium. No further correction for the final total
volume.
• Method 2
Add 10% of aqueous drug solution and adjust to
the final total volume of medium prepared.
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Method 1
Prepare a standard batch (1620 ml) of LJ basic culture
medium according to the SOP “Plain LJ media”.
Isoniazid (INH) potency factor 1:
Solution I: 10.0 mg INH dissolved in 50 ml distilled water
(200 µg/ml)
Solution II: 2.5 ml Sol. I made up to 25 ml with distilled water (20 µg/ml)
Solution III: 5.0 ml Sol. II made up to 10 ml with distilled water
(10
µg/ml)
0. 2 µg/ml
0.1 µg/ml
0.05 µg/ml
0.025 µg/ml
198
19.8
19.8
19.8
Solution II (ml)
2
–
–
–
Solution III (ml)
–
0.2
0.10
0.05
Water (ml)
–
–
0.10
0.15
200
20
20
20
Media (ml)
Final volume (ml)
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Method 2
• Aqueous drug solution: 10 % of total volume
(= 162 ml for 1620 ml of final volume of the batch).
• Subtract this volume water from the 600 ml of water
used to prepare the salt solution.
Mineral salt solution
438 ml
Malachite green solution
20 ml
Homogenized eggs
1000 ml
Total
1458 ml
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Method 2
Isoniazid (INH): factor 1.0
Solution I: 10.0 mg INH dissolved in 100 ml distilled water (100 µg/ml)
Solution II: 1.0 ml Sol. I made up to 50 ml with distilled water (2.0 µg/ml)
Solution III: 5.0 ml Sol. II made up to 10 ml with distilled water (1.0 µg/ml)
0.2 µg/ml
0.1 µg/ml
0.05 µg/ml
0.025 µg/ml
Media (ml)
180
18
18
18
Solution II (ml)
20
1
–
–
Solution III (ml)
–
–
0.10
0.05
Water (ml)
–
1
0.10
0.15
200
20
20
20
Final volume (ml)
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Quality control – DST
• Slants should not be older than 4 weeks
• Inoculate 2 DST sets ( one with critical concentration
and one with the lower concentration slants of each
antibiotic) with the M. tuberculosis H37Rv strain (fully
susceptible).
• The MIC standards for the fully susceptible H37Rv are
(mg/ml): INH 0.06, RMP 4.0, DSM 2.0, EMB 0.5
• H37Rv should grow only on slants with lower
concentration then than MIC
• If the batch fails the criteria, it should be discarded and
a new batch should be prepared and tested
• Reasons for failure should be discussed and examined
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Poor-quality media
•
•
•
•
•
Bubbles
Non-homogeneous dye solution
Discolouration
Contamination
≥8 weeks old (>4 weeks for drugcontaining media)
• Improper storage
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True and false exercise
1. Use of a combination of liquid and solid
media increases sensitivity of culture.
2. Overheating of media during
inspissation guarantees sterility of the
slants.
3. Discolouration of media is an indication
of a poor-quality medium.
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Module review: take-home messages
 Good-quality media are essential for reliable
diagnosis of tuberculosis.
 Liquid media shorten the recovery time but are
more susceptible to contamination.
 For preparation of drug-containing media, pure
drug powders must be used, not the drugs used
for treatment of patients.
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Self-assessment
• Describe the different media used for
mycobacterial culture.
• Describe the two options for adding drug
solutions to the media to prepare DST media.
• How can you recognize poor-quality media?
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