1. Module 4 Preparation of solid media for culture and DST

2. 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.

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

4. 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

5. 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.

6. 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.

7. 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.

8. 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.

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

10. 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.

11. Liquid media: disadvantages
Prone to contamination
Biosafety issue
Cost

12. 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

13. Plain egg-based media preparation

14. Media preparation room

15. 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.

16. 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

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

18. 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.

19. 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

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

21. Media preparation

22. 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.

23. 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.

24. 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.

25. 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)

26. 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

27. Preparation of selective and DST media

28. 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.

29. 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

30. 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.

31. 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

32. 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.

33. 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

34. 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 ml
Malachite green solution ml
Homogenized eggs ml
Total ml

35. 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

36. 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

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

38. 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.

39. 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.

40. 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?