Professional Documents
Culture Documents
Purpose To provide you with an understanding of, and the proper skills to prepare, the different media required for mycobacterial culture and DST. Module 3 50 minutes + 40 minutes for exercises. At the end of this module you will be able to: 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. Exercises Annexes 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
Content outline
4.1 Preparation of LwensteinJensen 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 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 LwensteinJensen (LJ) medium Stonebrink medium Ogawa medium Modified Ogawa medium (acid-buffered).
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): Herman-Kirchner liquid medium Dubos oleic acidalbumin liquid medium Middlebrook 7H9 liquid medium.
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: 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: 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 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: 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 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. 3. Before loading, heat the inspissator to 80 C. Place the bottles in a slanted position (5-10 to the horizontal) in the inspissator. 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. Do not re-heat: heating for a second or third time has a detrimental effect on the quality of the medium. 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.
4. 5.
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 co ntr ol Preparation or delivery Start of use End of use Contamination Strain/ detected specimen (Yes/No) inoculated Growth detected Growth detected at 20 days at 60 days (colonies) (colonies) Control Present Control Present batch batch batch batch Sensitivity control
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 LowensteinJensen 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 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, 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 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
Components Monopotassium dihydrophosphate (KH2PO4), anhydrous Magnesium sulfate (MgSO4 7H2O) Magnesium citrate L-Asparagine Sodium glutamate Distilled water up to Glycerol (ml) or pyruvatea (g) Egg homogenate Malachite green (2%) pH (about)
a
LJ, modified 2.4 g 0.24 g 0.6 g 3.6 g 600 ml 12 ml or 7.2 g 1000 ml 20 ml 6.8
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
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
Sterile dw DMSO
a
Sterile dw Sterile dw
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: 10.0 mg INH dissolved in 50 ml distilled water (200 g/ml) 2.5 ml Solution I, made up to 25 ml with distilled water (20 g/ml) 5.0 ml Solution II, made up to 10 ml with distilled water (10 g/ml)
Rifampicin (RMP) The factor is usually 1 for pure RMP or 1.03 for the sodium salt: check the potency. Factor 1: Solution I: Factor 1.03: Solution I: Solution II: 41.2 mg RMP, dissolved in 10.0 ml distilled water (4000 g/ml) 2.5 ml Solution I, made up to 10.0 ml with distilled water (1000 g/ml) 40.0 mg RMP, dissolved in 10.0 ml distilled water (4000 g/ml)
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 25 ml distilled water (400 g/ml)
Factor 1.277: Solution I: Solution II: 12.77 mg DSM dissolved in 25 ml distilled water (400 g/ml) 10 ml Solution I, made up to 20 ml with distilled water (200 g/ml)
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 g/ml) 5.0 ml Solution I, made up to 20 ml with distilled water (50 g/ml)
Media (ml) Solution I (ml) Solution II (ml) Water (ml) Final volume (ml)
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 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 g/ml) 1.0 ml Solution I, made up to 50 ml with distilled water (2.0 g/ml) 5.0 ml Solution II made up to 10 ml with distilled water (1.0 g/ml) 438 ml 20 ml 1000 ml 1458 ml
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: Factor 1.03: Solution I: Solution II: 41.2 mg RMP, dissolved in 100.0 ml distilled water (400 g/ml) 2.5 ml Solution I, made up to 10.0 ml with distilled water (100 g/ml) 40.0 mg RMP, dissolved in 100.0 ml distilled water (400 g/ml)
Media (ml) Solution I (ml) Solution II (ml) Water (ml) Final volume (ml)
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: Solution I: Solution II: 12.77 mg DSM dissolved in 250 ml distilled water (40 g/ml) 10 ml Solution I, made up to 20 ml with distilled water (20 g/ml) 12.51 mg DSM dissolved in 25o ml distilled water (40 g/ml)
Ethambutol (EMB) For EMB dihydrochloride, the factor is 1.36. Factor 1.36:
13.6 mg EMB dissolved in 50 ml distilled water (200 g/ml) 5.0 ml Solution I, add up to 50 ml with distilled water (20 g/ml) 5.0 ml Solution II, made up to 20 ml with distilled water (5 g/ml)
Media (ml) Solution II (ml) Solution III (ml) Water (ml) Final volume (ml)
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: 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. _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________
Describe the two methods for adding drug solutions to the media to prepare DST media. _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________
How can you recognize poor-quality media? _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________