Module 4
Preparation of solid media for culture and DST
Purpose
To provide you with an understanding of, and the proper skills to
prepare, the different media required for mycobacterial culture
and DST.
Prerequisite
modules
Module 3
Module time
50 minutes + 40 minutes for exercises.
Learning
objectives
At the end of this module you will be able to:
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recognize the different media for mycobacterial 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 media;
check the quality of tubes at the end of the process and store
them properly;
perform the sterility check.
•
Culture media
•
Media preparation
•
Preparation of selective and DST media
•
Quality of media
•
Recognize good- and poor-quality media
•
Design quality control for media preparation
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Content outline
Exercises
Annexes
4.1 Preparation of Löwenstein–Jensen and others egg-based
media.
4.2 Agar-based media
CULTURE MEDIA
The usual bacteriology techniques are not applicable to mycobacterial culture, as M. tuberculosis
grows slowly, with a generation time of 18–24 hours (other bacteria reproduce within minutes).
Moreover, growth requirements are such that M. tuberculosis will not grow on primary isolation on
simple chemically defined media. The only media that allow abundant growth are egg-enriched
media containing glycerol and asparagine, and agar or liquid media supplemented with serum or
bovine albumin.
The following media are used for M. tuberculosis culture:
•
Egg-based solid media:1
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‒
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‒
Löwenstein–Jensen (LJ) medium
Stonebrink medium
Ogawa medium
Modified Ogawa medium (acid-buffered).
Stonebrink medium has the same composition as Löwenstein–Jensen, except that glycerol is
replaced by 0.5% sodium pyruvate.
•
Liquid media (for manual and automated use):
‒
‒
‒
Herman-Kirchner liquid medium
Dubos oleic acid–albumin liquid medium
Middlebrook 7H9 liquid medium.
Each of these media has advantages and disadvantages: solid media require longer (8–12 weeks)
to give a positive culture, but they are less expensive than liquid media and allow the recognition of
the morphology of colonies.
Liquid media are more expensive than solid media and more prone to contamination; they can
easily be spilt (biosafety issue).
The ideal medium should:
‒
‒
‒
‒
be economical and simple to prepare from readily available ingredients;
inhibit the growth of contaminants;
support luxuriant growth of small numbers of bacilli;
have long shelf-life.
Egg-based media should be the first choice for culture of sputum specimens as they meet all the
above-listed requirements. There is growing evidence that the greater sensitivity of liquid media
may give better results with other specimens; however, if liquid media are chosen for M.
tuberculosis culture, inoculation on solid media should also be performed. It is recommended that
both egg-based and liquid media be used for non-repeatable specimens, e.g. cerebrospinal fluid
and biopsy material, but the increase in cost should be considered.
Egg-based media
Egg-based media have several advantages over other media, such as:
‒
‒
‒
‒
‒
‒
‒
1
they are easy to prepare;
the cost is low;
they support good growth of tubercle bacilli;
they can be stored in a refrigerator for several weeks;
there is limited contamination during preparation;
malachite green suppresses the growth of non-mycobacterial organisms;
contamination may involve just a portion of the surface of the media, meaning the culture can
be saved.
Another type of solid medium is agar-based: for further information see Annex 4.2, “Agar-based media”.
The two most important disadvantages are that it takes as long as 8 weeks before cultures become
positive, and preparation of media requires human resources, space and specific equipment, plus
specific organization of the laboratory.
The specificities of egg-based media are that LJ can be used for culture and for DST and, with
pyruvate and without glycerol, it supports the growth of M. bovis.
The Kudoh medium (also called the acid-buffered Ogawa medium) has the advantage that it does
not require a neutralization step during the decontamination procedure; however, it cannot be used
for DST.
Liquid media
The Herman-Kirchner liquid medium is the most useful and least expensive of the liquid media for
culture of tubercle bacilli. It has the additional advantage that it can support a large inoculum.
The Dubos oleic acid–albumin liquid medium is recommended for the cultivation of tubercle bacilli
from cerebrospinal, pleural and peritoneal fluids. It may be prepared from basic ingredients or be
obtained commercially as a ready-to-use base to which sterile albumin or serum is added.
The Middlebrook 7H9 medium may be used for the storage of strains and is used in biochemical
tests for species identification.
Liquid media are the basis for automated systems for M. tuberculosis culture.
The disadvantage of all liquid media is that they are prone to contamination and must be handled
with particular attention to the production of infectious aerosols.
PREPARATION OF SOLID MEDIA FOR CULTURE
Media can be purchased as ready-to-use batches from commercial sources or can be prepared in
the laboratory (which is the less expensive option). If media are to be prepared in the laboratory,
there must be a separate room, dedicated exclusively to media preparation, where no “dirty” items
(e.g. clinical specimens, cultures, waste) are admitted.
Preparing media of good quality requires chemicals of certified purity, clean glassware and freshly
distilled and sterilized water. Certain precautions must be taken to avoid contamination of reagents
and media:
1. Keep the environment as clean as possible. Swab the work surface with a suitable disinfectant
(e.g.70% ethanol) before dispensing sterile reagents and media. Clean the floor with a wet mop
to limit dust.
2. Use sterile glassware and equipment.
3. Use reagent-grade chemicals and reagents unless otherwise specified.
4. Check the temperature of inspissators and hot-air ovens.
5. Follow strict aseptic techniques when preparing media, e.g. flaming flasks and tubes.
6. When preparing egg-based media, carefully clean shells before breaking eggs.
7. Do not overheat media during inspissation.
8. Do not leave prepared media exposed to light (including ultraviolet light); store them in the
refrigerator, in the dark, when not in use.
9. Do not skimp on the volume of medium. Place 6–8 ml of egg medium in each bottle or 20 ml in
each test-tube.
Choice of glassware
The most commonly used containers for egg-based culture media (LJ, Ogawa and Stonebrink) are
reusable glass tubes sealed with cotton wool plugs, stoppers or screw-caps. Universal bottles
sealed with screw-caps are sometimes used; being made of thicker glass, they are sturdier than
most tubes but make the reading of colonies more difficult. The containers should be made of hard,
resistant, borosilicate laboratory glass.
The volume of medium in the container, the surface of the slanted medium and the volume of air
between the medium and the seal of the container are all important factors affecting the growth of
mycobacterial cultures. In choosing the size of culture containers, it should be remembered that
the area of the slope of the medium and the air available within the closed container are both linear
functions of the square of the diameter of the containers. For example, for tubes with diameters of
16 mm and 12 mm, the surface area of the slanted medium of the first is twice that of the second
tube. The length of the medium slant is important – it should not reach the neck of the tube.
Plugs for glassware
Cotton wool plugs have two disadvantages: the smouldering of the burned cotton wool caused by
flaming may consume part of the oxygen in the tube, and the products in the singed cotton wool
may inhibit the growth of tubercle bacilli. Moreover, most fungal contaminations are caused by
cotton wool plugs.
Rubber stoppers should be very carefully chosen, because some inhibit the growth of M.
tuberculosis. It is therefore safer and more convenient to use glass tubes with Teflon-lined plastic
screw-caps. A typical container for culturing mycobacteria on egg-based media is a screw-capped
glass tube with the following measurements: length 150 mm, external diameter 16 mm, wall
thickness 1 mm.
Other suitable containers include:
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plastic (polycarbonate) tubes;
14-ml McCartney bottles;
5-ml bijoux bottles.
Cleaning of glassware
Any material residue, organic or inorganic, that remains on reusable glassware can potentially
inhibit reagent activity as well as bacterial growth. It is therefore recommended that glassware be
scrubbed in hot water, if possible containing a non-ionic detergent, to eliminate residues, rinsed
repeatedly in hot water and finally rinsed in distilled water.
Biological residues (blood, culture media, etc.) on glassware such as pipettes, tubes and conical
flasks can sometimes be difficult to remove. High temperatures (>80 °C dry heat) and strongly
alkaline fluids (pH >12) should be avoided when cleaning. Exceptionally, heavily soiled items can
be cleaned in dichromate solution (100 g of potassium dichromate in 250 ml of concentrated
sulfuric acid and 750 ml of water) for about 4 hours, followed by washing 10 times in soapy water
and rinsing three times with tap and distilled water.
Sterilization
Whenever possible it is recommended that separate autoclaves are used to sterilize “clean” and
contaminated materials. Sterilization of “clean” materials requires 20 minutes at 121 °C; it is
important not to exceed 121 °C because carbohydrates and other thermolabile substances could
be degraded. To sterilize volumes of more than 2 litres, sterilization time should be increased to
30–40 minutes. Sterile materials should be properly labelled and kept separately in a sheltered
place (close and clean cabinet).
Weighing scales
The minimum requirement for weighing is a twin-beam balance that weighs up to 200 g with ±0.1 g
precision. The maximum load of the balance should not be exceeded. Make sure that the balance
is placed on a clean, level, stable, vibration-free and draught-free surface. The balance should be
recalibrated periodically using reference weights.
Preparation of Löwenstein‒Jensen medium
Löwenstein-Jensen (LJ) medium is most widely used for mycobacterial culture and is
recommended by WHO. Different ingredients can favour different growth: LJ containing glycerol
(Stonebrink) favours the growth of M. tuberculosis, LJ without glycerol but with pyruvate
encourages the growth of M. bovis. Both should be used in countries or regions where patients
may be infected with either organism.
A detailed list of ingredients and the procedure for preparing the complete medium are given in
Annex 4.1, “Preparation of Löwenstein-Jensen medium”. The mineral salt solution and the 2%
malachite green solution are commercially available.
The mineralized solution must be sterilized by autoclaving at 121 °C for 30 minutes and then
cooled to room temperature. This solution keeps indefinitely and may be stored in suitable volumes
in the refrigerator.
Fresh hens' eggs are an important ingredient: they must be no more than 7 days old. They must be
cleaned by scrubbing thoroughly in warm water with a plain alkaline soap, rinsed with water and
soaked with 70% ethanol for 15 minutes. To avoid contamination, eggs must be cracked with a
sterile knife into a sterile flask and beaten with a sterile egg whisk or in a sterile blender for 5
minutes: the egg solution must not be sterilized.
For preparing 200 slants, 20–25 eggs are needed, depending on their size.
All the prepared solutions are mixed in a large sterile flask and mixed well: eggs are added by
filtering them through a sterile fabric. Distribute solution in the tubes and coagulate in the
inspissator for no more than 45 minutes to avoid sedimentation of the heavier ingredients.
A number of precautions should be observed when inspissating the prepared medium:
1.
Before loading, heat the inspissator to 80 °C.
2.
Place the bottles in a slanted position (5-10° to the horizontal) in the inspissator.
3.
Coagulate the medium for 45 minutes at 80–85 °C. Since the medium has been prepared with
sterile procedures, this heating is to solidify the medium, not to sterilize it. A relative humidity
of 80% is necessary for good heat transmission.
4.
Do not re-heat: heating for a second or third time has a detrimental effect on the quality of the
medium.
5.
Do not over-heat : the quality of egg-based media deteriorates if coagulation is performed with
excessive heat or for too long. Discolouration of the coagulated medium could indicate
excessive temperature. The appearance of small holes or bubbles on the surface of the
medium also indicates faulty coagulation procedures.
To check sterility, incubate each new batch of media at 35–37 °C for 48 hours. Growth of any
organism indicates poor procedure; the whole batch must be discarded and preparation problems
must be identified and resolved.
Results of the sterility check should be recorded on the appropriate form, such as that shown in
Figure 1; more details are given in Module 11.
Löwenstein–Jensen medium
Batch Volume
no.
(ml)
Date of:
Preparation
or delivery
Start
of
use
Sterility
control
End
of
use
Contamination
Strain/
detected
specimen
(Yes/No)
inoculated
Sensitivity control
Growth detected Growth detected
at 20 days
at 60 days
(colonies)
(colonies)
Control Present Control Present
batch
batch
batch
batch
Fig 1
To avoid the accumulation of expired media, estimate the number of tubes to be prepared or
purchased to cover culture needs for about 2 months of work. The media should be stored in a
cool and humid place, preferably in a refrigerator at 4 °C.
•
Protect any medium from direct light at all times.
•
Place tubes in clean plastic bags or sterile dry containers.
•
Tighten caps of screw-cap tubes before storing.
•
Refrigerate any medium kept for longer than 1 week to minimize dehydration.
•
Discard any medium that shows signs of contamination or dehydration.
LJ medium should not be more than 2 months old!
Preparation of Ogawa medium
The Ogawa medium is an egg-based medium. It is cheaper than LJ medium as it is prepared
without asparagine. Details of the ingredients and procedure for preparing the complete medium
are given in Annex 4.1, “Preparation of Lowenstein–Jensen and others egg-based media”.
All the precautions concerning the quality of eggs, media preparation and inspissation already
described for LJ medium must also be strictly observed for Ogawa medium.
To check sterility, incubate each new batch of media at 35–37 °C for 48 hours. Growth of any
organism indicates poor procedure; the whole batch must be discarded and preparation problems
must be identified and resolved.
After the sterility check, Ogawa medium must be dated and stored in the refrigerator, where it can
be kept for several weeks if the caps are tightly closed to prevent drying out.
Preparation of acid-buffered Ogawa medium
Details of the ingredients and procedure for preparing the acid-buffered Ogawa medium are given
in Annex 4.1. Table 1 compares the ingredients needed for the preparation of LJ and Ogawa
medium
All the precautions concerning the quality of eggs, media preparation and inspissation already
described for LJ medium must be strictly observed for acid-buffered Ogawa medium.
To check sterility, incubate each new batch of media at 35–37 °C for 48 hours. Growth of any
organism indicates poor procedure; the whole batch must be discarded and preparation problems
must be identified and resolved.
After the sterility check, the acid-buffered Ogawa medium must be dated and stored in the
refrigerator, where it can be kept for several weeks if the caps are tightly closed to prevent drying
out.
Table 1. Components of the different egg-based plain media
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
Components
Sodium glutamate
Distilled water up to
Glycerol (ml) or pyruvatea (g)
Egg homogenate
Malachite green (2%)
pH (about)
0.6 g
a Glycerol
in the LJ medium (Stonebrink medium) favours the growth of M. tuberculosis, while LJ medium
without glycerol but containing pyruvate encourages the growth of M. bovis and may be used in areas of high
M. bovis incidence as a second slant for specimens.
PREPARATION OF SELECTIVE AND DST MEDIA
To promote growth of, or to identify, different mycobacterial species or to evaluate drug
susceptibility to M. tuberculosis, LJ media supplemented with different substances and drugs are
used. M. bovis, for example, grows in the presence of pyruvate but not glycerol.
M. tuberculosis does not grow on media containing p-nitrobenzoic acid (PNB) at a concentration of
500 mg/litre (500 µg/ml). This concentration may be reached weighing 250 mg of PNB and
dissolving it in 10 ml propylene glycol in hot water, then mixing 10 ml of this solution with 490 ml of
LJ medium before dispensing.
Selective DST media can be purchased ready to use or can be prepared in the laboratory. In the
latter case, follow the procedure for preparation of culture media already described and add the
selected substances (in correct dilutions) to the complete media before distributing in tubes and
inspissating.
This module gives particular emphasis to various aspects of the preparation of DST media. It is
crucial to ensure proper handling of drug concentrations to guarantee reliable identification of
resistant strains.
DST media
Mass concentrations of drugs to be tested and solvents
Mass concentrations of drugs to be used for susceptibility testing of patients’ isolates and
appropriate solvents are given in Table 2
Table 2. Mass concentrations for drugs to be tested and critical concentration
Test drugs
Designation
Solvent
Final mass concentration in
culture medium (mg/litre)
For quality
Critical
control
concenH37Rv
tration
Abbrev
.
Solvent
Dilution
Isoniazid sulfate
INH
Sterile dw
Sterile dw
0.025•0.05•0.10
0.2
Rifampicin
RMP
DMSOa
dw
2.5•5.0•10.0
40.0
Dihydrostreptomycin
sulfate
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
a If
sodium salt is used, dissolve directly in distilled water.
Please refer to module 10 part 1 and part 2 for further explanations on DST
Preparation of solution of drugs to be tested
Only pure compounds available from the manufacturer should be use for media preparation:
crushed pills or solutions prepared for therapy are not suitable for sensitivity testing. The drugs to
be tested should be stored according to the manufacturer’s instructions in a desiccator at the
appropriate temperature. The amount to be weighed must be calculated according to the activity
(potency) declared by the manufacturer.
Example:
If the activity of dihydrostreptomycin sulfate (Lot no. xyz; activity may change from lot to lot) is
given as 786 mg/g, 1/0.786 = 1.27226 g will have to be weighed to give 1 g of active
dihydrostreptomycin. In other words, any desired weight of dihydrostreptomycin must be
multiplied by a factor of 1.27 to determine the amount to be weighed.
The first dilution of each drug can be stored in – 70°C freezer for subsequent use.
To ensure reliability, all volumes in the dilution schemes should be chosen such that they can be
measured using pipettes or volumetric flasks.
Preparation of culture media containing test substances (test media)
Note: Because of the risk of infection following glass breakage, thick-walled, shock-resistant glass
tubes (or flasks) with a deep (20–30 mm) screw-cap that covers the upper part of the tube should
be used. The screw-cap should allow a certain gas exchange without drying of the medium.
Depending on the number of test-tubes required (for about 4 weeks), a portion of the appropriate
volume of basic culture medium is poured into separate volumetric flasks for each drug and for
each concentration. The exact volume of appropriate drug solution is then added to each
volumetric flask. The two solutions are thoroughly mixed to ensure homogenization. The flasks are
then filled precisely to the maximum volume and once again mixed well to complete
homogenization. The drug-containing medium is then dispensed into sterile, thick-walled, shockresistant glass tubes (or flasks).
Aliquots of 5 ml (some recommend 6 ml) are dispensed into the tubes to be used, which should
have been recently sterilized. All tubes must be marked with the drug name, concentration, and
date of preparation (or lot number).
Control media without drugs should be used, always from the same batch of media.
Two different ways of preparing the test media are currently in use. In Method 1, 1% of drug
solution is added to the basic culture medium – usually without any corrections for the volume
change; in Method 2, 10% of aqueous drug solution is added, in which case, a correction for the
drug volume added has to be made when preparing the basic culture medium. The advantage of
Method 2 is the ease of complete homogenization of the drug component into the egg mass in a
volumetric flask; much greater care is needed when just adding a volume of just 1%.
Method 1
Prepare a standard batch (1620 ml) of LJ basic culture medium according to the SOP “Plain LJ
media”.
•
Isoniazid (INH)
For dry and pure INH, the factor is 1.
Solution I:
Solution II:
Solution III:
10.0 mg INH dissolved in 50 ml distilled water
(200 µg/ml)
2.5 ml Solution I, made up to 25 ml with distilled water (20 µg/ml)
5.0 ml Solution II, made up to 10 ml with distilled water (10 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution II (ml)
Solution III (ml)
Water (ml)
Final volume (ml)
•
0. 2 µg/ml
198
2
–
–
200
0.1 µg/ml
19.8
–
0.2
–
20
0.05 µg/ml
19.8
–
0.10
0.10
20
0.025 µg/ml
19.8
–
0.05
0.15
20
Rifampicin (RMP)
The factor is usually 1 for pure RMP or 1.03 for the sodium salt: check the potency.
Factor 1:
Solution I:
40.0 mg RMP, dissolved in 10.0 ml distilled water
(4000 µg/ml)
Factor 1.03:
Solution I:
Solution II:
41.2 mg RMP, dissolved in 10.0 ml distilled water
(4000 µg/ml)
2.5 ml Solution I, made up to 10.0 ml with distilled water
(1000 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
•
40 µg/ml
198
2
–
200
10 µg/ml
19.8
–
0.2
–
20
5 µg/ml
19.8
–
0.10
0.10
20
2.5 µg/ml
19.8
–
0.05
0.15
20
Dihydrostreptomycin (DSM)
For DSM sulfate, dry and pure, the factor is 1.251. For DSM sulfate, dry and 98% pure (which
is a common value), the factor is 1.277.
Factor 1.251:
Solution I:
Factor 1.277:
12.51 mg DSM dissolved in 25 ml distilled water
(400 µg/ml)
Solution I:
Solution II:
12.77 mg DSM dissolved in 25 ml distilled water
(400 µg/ml)
10 ml Solution I, made up to 20 ml with distilled water (200 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
•
4 µg/ml
198
2
–
–
200
2 µg/ml
19.8
–
0.2
–
20
1 µg/ml
19.8
–
0.10
0.10
20
0.5 µg/ml
19.8
–
0.05
0.15
20
Ethambutol (EMB)
For EMB dihydrochloride, the factor is 1.36.
Factor 1.36:
Solution I:
Solution II:
13.6 mg EMB dissolved in 50 ml distilled water
(200 µg/ml)
5.0 ml Solution I, made up to 20 ml with distilled water (50 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
2 µg/ml
198
2
–
–
200
0.5 µg/ml
19.8
–
0.2
–
20
0.25 µg/ml
19.8
–
0.10
0.10
20
0.125 µg/ml
19.8
–
0.05
0.15
20
Left-over basic culture medium is used for the preparation of control slants for this specific lot.
Method 2
In this method, the volume of aqueous drug solution added is 10% of the total volume (= 162 ml);
this volume therefore has to be subtracted from the 600 ml of water for the preparation of the salt
solution. The amount of ingredients remains unchanged.
Mineral salt solution
Malachite green solution
Homogenized eggs
Total
•
438 ml
20 ml
1000 ml
1458 ml
Isoniazid (INH)
For dry and pure INH, the factor is 1.
Solution I:
Solution II:
Solution III:
10.0 mg INH dissolved in 100 ml distilled water
(100 µg/ml)
1.0 ml Solution I, made up to 50 ml with distilled water (2.0 µg/ml)
5.0 ml Solution II made up to 10 ml with distilled water (1.0 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution II (ml)
0.2 µg/ml
180
20
0.1 µg/ml
18
1
0.05 µg/ml
18
–
0.025 µg/ml
18
–
–
–
200
Solution III (ml)
Water (ml)
Final volume (ml)
•
–
1
20
0.10
0.10
20
0.05
0.15
20
Rifampicin (RMP)
The factor is usually 1 for pure RMP or 1.03 for the sodium salt: check the potency!
Factor 1:
Solution I:
40.0 mg RMP, dissolved in 100.0 ml distilled water
(400 µg/ml)
Factor 1.03:
Solution I:
Solution II:
41.2 mg RMP, dissolved in 100.0 ml distilled water
(400 µg/ml)
2.5 ml Solution I, made up to 10.0 ml with distilled water
(100 µg/ml)
Final concentration in drug media (µg/ml)
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
•
40 µg/ml
180
20
–
–
200
10 µg/ml
18
–
2.0
–
20
5 µg/ml
18
–
1.0
1.0
20
2.5 µg/ml
18
–
0.5
1.5
20
Dihydrostreptomycin (DSM)
For DSM sulfate, dry and pure, the factor is 1.251. For DSM sulfate, dry and 98% pure (which
is a common value), the factor is 1.277.
Factor 1.251:
Solution I:
12.51 mg DSM dissolved in 25o ml distilled water
(40 µg/ml)
Factor 1.277:
Solution I:
Solution II:
12.77 mg DSM dissolved in 250 ml distilled water
(40 µg/ml)
10 ml Solution I, made up to 20 ml with distilled water (20 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
•
4 µg/ml
180
20
–
200
2 µg/ml
18
–
2.0
–
20
Ethambutol (EMB)
For EMB dihydrochloride, the factor is 1.36.
Factor 1.36:
1 µg/ml
18
–
1.0
1.0
20
0.5 µg/ml
18
–
0.5
1.5
20
Solution I:
Solution II:
Solution III:
13.6 mg EMB dissolved in 50 ml distilled water
(200 µg/ml)
5.0 ml Solution I, add up to 50 ml with distilled water (20 µg/ml)
5.0 ml Solution II, made up to 20 ml with distilled water (5 µg/ml)
Final concentration in drug media (µg/ml):
Media (ml)
Solution II (ml)
Solution III (ml)
Water (ml)
Final volume (ml)
2 µg/ml
180
20
–
–
200
0.5 µg/ml
18
–
2.0
–
20
0.25 µg/ml
18
–
1.0
1.0
20
0.125 µg/ml
18
–
0.5
1.5
20
Leftover basic culture medium is diluted with 10% distilled water and used for the preparation of
control slants for this specific lot. Each test tube must be labelled with the date of preparation, the
name of the drug and its concentration.
To check the new batch please proceed as follow:
• Inoculate a DST set consisting of the critical concentration as well as the lower
concentration slants of each antibiotic with the M.tuberculosis H37Rv strain
• The MIC standards for the fully susceptible H37Rv strain are (in mg/ml)
INH 0.06 - RMP 4.0 - DSM 2.0 - EMB 0.5
• H37Rv strain should grow on slants with lower concentrations than MICs and not grow on
MICs or higher concentrations
• If the batch fails the criteria, a new batch has to be produced and all DST of the patient
strains have to repeated with the new batch
• Revise the process to identify problems encountered during the procedure of batch
preparation
DST is fully discussed in module 10
QUALITY OF MEDIA
For mycobacterial culture, as well as for general bacteriology, it is very important to use goodquality media. If the quality of the media is poor, the probability to have false-negative result for M
tuberculosis cultures will increase.
It is the responsibility of laboratory technicians to ascertain the quality of the media. Media should
be discarded if they have the following features:
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bubbles (problems in the coagulation process);
non-homogeneous dye (non-homogeneous melting);
discoloured (problems in the coagulation process);
contamination (problems in sterilization and/or during the procedure);
≥8 weeks old (expired, may give false-negative results);
not properly stored (may give false-negative results).
The dates of preparation of all media and the results of sterility checks and of quality control
checks on media for DST must be recorded in quality assurance documents.
Source material
1. Narvaiz de Kantor I et al. Laboratory services in tuberculosis control. Part III: Culture. Geneva,
World Health Organization, 1998 (WHO/TB/98.258).
2. Kent PT, Kubica GP. Public health mycobacteriology: a guide for the level III laboratory.
Atlanta, GA, U.S. Department of Health and Human Services, Centers for Disease Control,
1985.
3. Collins C, Grange J, Yates M. Organization and practice in tuberculosis bacteriology. London,
Butterworth, 1985.
KEY MESSAGES
 Good-quality media are essential for reliable diagnosis of tuberculosis.
 Liquid media shorten the recovery time but are more prone to contamination than
solid media.
 For drug-containing media, pure drug powders must be used, not drugs for patients’
treatment.
Module 4: Review
Find out how much you have learned by answering these questions.
Describe different media for mycobacteria culture.
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Describe the two methods for adding drug solutions to the media to prepare DST
media.
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How can you recognize poor-quality media?
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