Module 4 Preparation of solid media for culture and dst
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Module 4Preparation of solid media for culture and DST
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:
Stonebrink medium has the same composition as Löwenstein–Jensen, except that glycerol is replaced by 0.5% sodium pyruvate.
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:
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:
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.
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:
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.
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.
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:
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
Fig 1
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.
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
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.
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
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, shock-resistant 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%.
Prepare a standard batch (1620 ml) of LJ basic culture medium according to the SOP “Plain LJ media”.
For dry and pure INH, the factor is 1. Solution I: 10.0 mg INH dissolved in 50 ml distilled water (200 µg/ml) Solution II: 2.5 ml Solution I, made up to 25 ml with distilled water (20 µg/ml) Solution III: 5.0 ml Solution II, made up to 10 ml with distilled water (10 µg/ml) Final concentration in drug media (µg/ml):
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: 41.2 mg RMP, dissolved in 10.0 ml distilled water (4000 µg/ml) Solution II: 2.5 ml Solution I, made up to 10.0 ml with distilled water (1000 µg/ml) Final concentration in drug media (µg/ml):
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 µg/ml) Factor 1.277: Solution I: 12.77 mg DSM dissolved in 25 ml distilled water (400 µg/ml) Solution II: 10 ml Solution I, made up to 20 ml with distilled water (200 µg/ml) Final concentration in drug media (µg/ml):
For EMB dihydrochloride, the factor is 1.36. Factor 1.36: Solution I: 13.6 mg EMB dissolved in 50 ml distilled water (200 µg/ml) Solution II: 5.0 ml Solution I, made up to 20 ml with distilled water (50 µg/ml) Final concentration in drug media (µg/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 438 ml Malachite green solution 20 ml Homogenized eggs 1000 ml Total 1458 ml
For dry and pure INH, the factor is 1. Solution I: 10.0 mg INH dissolved in 100 ml distilled water (100 µg/ml) Solution II: 1.0 ml Solution I, made up to 50 ml with distilled water (2.0 µg/ml) Solution III: 5.0 ml Solution II made up to 10 ml with distilled water (1.0 µg/ml) Final concentration in drug media (µg/ml):
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: 41.2 mg RMP, dissolved in 100.0 ml distilled water (400 µg/ml) Solution II: 2.5 ml Solution I, made up to 10.0 ml with distilled water (100 µg/ml) Final concentration in drug media (µg/ml)
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: 12.77 mg DSM dissolved in 250 ml distilled water (40 µg/ml) Solution II: 10 ml Solution I, made up to 20 ml with distilled water (20 µg/ml) Final concentration in drug media (µg/ml):
For EMB dihydrochloride, the factor is 1.36. Factor 1.36: Solution I: 13.6 mg EMB dissolved in 50 ml distilled water (200 µg/ml) Solution II: 5.0 ml Solution I, add up to 50 ml with distilled water (20 µg/ml) Solution III: 5.0 ml Solution II, made up to 20 ml with distilled water (5 µg/ml) Final concentration in drug media (µg/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:
INH 0.06 - RMP 4.0 - DSM 2.0 - EMB 0.5
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 good-quality 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 Another type of solid medium is agar-based: for further information see Annex 4.2, “Agar-based media”. Yüklə 213,53 Kb. Dostları ilə paylaş:
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