Module 4 Preparation of solid media for culture and DST

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.

Content outline Description of culture media Preparation of plain culture media Preparation of selective and drug- containing media Quality of media

Culture media Two main groups: Egg-based media Liquid 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

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.

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.

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.

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.

Liquid media Middlebrook 7H9 and modified versions Commercially available Automated systems available

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.

Liquid media: disadvantages Prone to contamination Biosafety issue Cost

Use of combination of liquid and solid media increases sensitivity 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 sensitivity

Plain egg-based media preparation

Media preparation room

General precautions 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.

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

Cleaning of glassware Brush glassware in hot water. Eliminate residue. Rinse repeatedly in hot water. Rinse in distilled water.

Egg-based media Prepare mineral salt solution. Prepare malachite green solution, 2%. Note: 1 and 2 are commercially available. Homogenize whole eggs (20–25 eggs for 200 tubes). Prepare complete medium.

Egg-based media composition 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 L-Asparagine 3.6 g Sodium glutamate 3 g Distilled water up to 600 ml Glycerol (ml) or pyruvatea (g) 12 ml or 7.2 g 36 ml 24 ml Egg homogenate 1000 ml 1200 ml Malachite green (2%) 20 ml pH (about) 6.8 6.4

Egg preparation Wash eggs with soap and water. Soak eggs in 70% ethanol for 15 minutes. Filter whipped eggs through sterile gauze fabric.

Media preparation

Coagulation of medium Before loading, heat the inspissator to 80 ºC. Place the bottles in a slanted position. Coagulate the medium for 45 minutes at 80–85 ºC in 80% relative humidity. Do not heat further.

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.

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.

Media log-sheet – first part Preparation: 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 cycle Date: Operator's name Time (min) Temperature (ºC)

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 cycle Date: Operator's name: Time (min) Temperature (ºC) Sterility check

Preparation of selective and DST media

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.

Mass concentrations for drugs and critical concentration Test drugs Solvent Final mass concentration in culture medium (mg/litre) Designation Abbrev. Dilution For quality control H37Rv (MTB control, strain) Critical concen-tration Isoniazid INH Sterile dw 0.025•0.05•0.10 0.2 Rifampicin RMP DMSO 2.5•5.0•10.0 40.0 Dihydro-streptomycin DSM 0.5•1.0•2.0 4.0 Ethambutol EMB 0.125•0.25•0.5 2.0

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.

Amount to be weighed = 1/potency Drugs Concentration is crucial for reliable DST. Use pure drug powders, not drugs for patients’ treatment. Amount to be weighed = 1/potency

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.

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 Media (ml) 198 19.8 Solution II (ml) 2 – Solution III (ml) 0.2 0.10 0.05 Water (ml) 0.15 Final volume (ml) 200 20

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

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 Solution II (ml) 20 1 – Solution III (ml) 0.10 0.05 Water (ml) 0.15 Final volume (ml) 200

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

Poor-quality media Bubbles Non-homogeneous dye solution Discolouration Contamination ≥8 weeks old (>4 weeks for drug-containing media) Improper storage

True and false exercise Use of a combination of liquid and solid media increases sensitivity of culture. Overheating of media during inspissation guarantees sterility of the slants. Discolouration of media is an indication of a poor-quality medium.

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.

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?