Professional Documents
Culture Documents
Prerequisite
modules
Module 3
Module time
Learning
objectives
Culture media
Media preparation
Quality of media
Content outline
Exercises
Annexes
CULTURE MEDIA
The usual bacteriology techniques are not applicable to mycobacterial culture, as M. tuberculosis
grows slowly, with a generation time of 1824 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
Stonebrink medium has the same composition as LwensteinJensen, except that glycerol is
replaced by 0.5% sodium pyruvate.
Liquid media (for manual and automated use):
Each of these media has advantages and disadvantages: solid media require longer (812 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:
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:
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 acidalbumin 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.
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:
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
3040 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 LwensteinJensen medium
Lwenstein-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 Lwenstein-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, 2025 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.
2.
Place the bottles in a slanted position (5-10 to the horizontal) in the inspissator.
3.
Coagulate the medium for 45 minutes at 8085 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 3537 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.
LwensteinJensen medium
Batch Volume
no.
(ml)
Date of:
Sterility
Sensitivity control
co
ntr
ol
Preparation
or delivery
Start
of
use
End
of
use
Contamination
Strain/
detected
specimen
(Yes/No)
inoculated
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.
Refrigerate any medium kept for longer than 1 week to minimize dehydration.
To check sterility, incubate each new batch of media at 3537 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)
2.4 g
6g
12 g
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
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.
DST media
Isoniazid sulfate
Solvent
Abbrev.
Solvent
Dilution
INH
Sterile dw
Sterile dw
Rifampicin
RMP
DMSO
Dihydrostreptomycin
sulfate
DSM
Ethambutol
EMB
dw
2.55.010.0
40.0
Sterile dw
Sterile dw
0.51.02.0
4.0
Sterile dw
Sterile dw
0.1250.250.5
2.0
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 manufacturers 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 70C 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 (2030 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:
0. 2 g/ml
198
2
200
0.1 g/ml
19.8
0.2
20
0.05 g/ml
19.8
0.10
0.10
20
0.025 g/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:
(4000 g/ml)
Factor 1.03:
Solution I:
Solution II:
40 g/ml
198
2
200
10 g/ml
19.8
0.2
20
5 g/ml
19.8
0.10
0.10
20
2.5 g/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:
(400 g/ml)
Factor 1.277:
Solution I:
Solution II:
4 g/ml
198
2
200
2 g/ml
19.8
0.2
20
1 g/ml
19.8
0.10
0.10
20
0.5 g/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:
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
2 g/ml
198
2
200
0.5 g/ml
19.8
0.2
20
0.25 g/ml
19.8
0.10
0.10
20
0.125 g/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:
0.2 g/ml
180
20
0.1 g/ml
18
1
0.05 g/ml
18
0.025 g/ml
18
200
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:
(400 g/ml)
Factor 1.03:
Solution I:
Solution II:
Media (ml)
Solution I (ml)
Solution II (ml)
Water (ml)
Final volume (ml)
40 g/ml
180
20
200
10 g/ml
18
2.0
20
5 g/ml
18
1.0
1.0
20
2.5 g/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:
(40 g/ml)
Factor 1.277:
Solution I:
Solution II:
4 g/ml
180
20
200
2 g/ml
18
2.0
20
Ethambutol (EMB)
For EMB dihydrochloride, the factor is 1.36.
Factor 1.36:
1 g/ml
18
1.0
1.0
20
0.5 g/ml
18
0.5
1.5
20
Solution I:
Solution II:
Solution III:
Media (ml)
Solution II (ml)
Solution III (ml)
Water (ml)
Final volume (ml)
2 g/ml
180
20
200
0.5 g/ml
18
2.0
20
0.25 g/ml
18
1.0
1.0
20
0.125 g/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:
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
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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