Module 4

Preparation of solid media for culture and DST

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Learning objectives

At the end of this module you will be able to:

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 drug-

containing media• Quality of media

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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

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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.

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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 non-

mycobacterial 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.

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Liquid media

• Middlebrook 7H9 and modified versions

• Commercially available

• Automated systems available

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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

Morphology AFB + colonies AFB

Biohazard + ++

Isolation time +++ +

Use of combination of liquid and solid media increases sensitivity12

Plain egg-based media preparation

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Media preparation room

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General precautions1. Keep the environment as clean as possible.

2. Use sterile glassware and equipment.

3. Use high-grade chemicals and reagents unless otherwise specified.

4. Check temperature of inspissators.

5. Follow strict aseptic techniques when preparing media, e.g. flaming flasks and tubes.

6. Clean and disinfect shells before breaking eggs.

7. Do not overheat media during inspissation.

8. Do not leave prepared media exposed to light.

9. Do not skimp on the volume of medium.

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Choice of glassware

• Reusable glass tubes sealed with screw-caps 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

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Components LJ, modified Ogawa Ogawa modified (Kudoh)

Monopotassium dihydrophosphate (KH2PO4), anhydrous

2.4 g 6 g 12 g

Magnesium sulfate (MgSO4 ·7H2O) 0.24 g –

Magnesium citrate 0.6 g – 0.6 g

L-Asparagine 3.6 g –

Sodium glutamate – 6 g 3 g

Distilled water up to 600 ml 600 ml 600 ml

Glycerol (ml) or pyruvatea (g) 12 ml or 7.2 g 36 ml 24 ml

Egg homogenate 1000 ml 1200 ml 1200 ml

Malachite green (2%) 20 ml 36 ml 24 ml

pH (about) 6.8 6.8 6.4

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 partPreparation: SALT SOLUTION

Operator's name:

Date of preparation:

Reagents

Quantity (ml or g)

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 cycleDate:

Operator's name

Time (min) Temperature (ºC)

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Media log-sheet – second part

Preparation: Second step Preparation date:Operator’s name:

Reagents Quantity

(ml or g)

Manufacturer Reference Batch number

Malachite green

Eggs Number of eggs:

Inspissator cycleDate:

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 Solvent Final mass concentration in culture medium (mg/litre)

Designation Abbrev. Solvent Dilution For quality control

H37Rv (MTB control, strain)

Critical concen-tration

Isoniazid 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

Dihydro-streptomycin

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 1Prepare 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

Media (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

Final volume (ml) 200 20 20 2033

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 mlHomogenized eggs 1000 mlTotal 1458 ml

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Method 2

Isoniazid (INH): factor 1.0Solution 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

Final volume (ml) 200 20 20 20

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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 examined36

Poor-quality media

• Bubbles

• Non-homogeneous dye solution

• Discolouration

• Contamination

• ≥8 weeks old (>4 weeks for drug-containing 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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